Cocaine modulates allosteric D2-σ1 receptor-receptor interactions on dopamine and glutamate nerve terminals from rat striatum.

Science.gov (United States)

Beggiato, Sarah; Borelli, Andrea Celeste; Borroto-Escuela, Dasiel; Corbucci, Ilaria; Tomasini, Maria Cristina; Marti, Matteo; Antonelli, Tiziana; Tanganelli, Sergio; Fuxe, Kjell; Ferraro, Luca

2017-12-01

The effects of nanomolar cocaine concentrations, possibly not blocking the dopamine transporter activity, on striatal D 2 -σ 1 heteroreceptor complexes and their inhibitory signaling over Gi/o, have been tested in rat striatal synaptosomes and HEK293T cells. Furthermore, the possible role of σ 1 receptors (σ 1 Rs) in the cocaine-provoked amplification of D 2 receptor (D 2 R)-induced reduction of K + -evoked [ 3 H]-DA and glutamate release from rat striatal synaptosomes, has also been investigated. The dopamine D 2 -likeR agonist quinpirole (10nM-1μM), concentration-dependently reduced K + -evoked [ 3 H]-DA and glutamate release from rat striatal synaptosomes. The σ 1 R antagonist BD1063 (100nM), amplified the effects of quinpirole (10 and 100nM) on K + -evoked [ 3 H]-DA, but not glutamate, release. Nanomolar cocaine concentrations significantly enhanced the quinpirole (100nM)-induced decrease of K + -evoked [ 3 H]-DA and glutamate release from rat striatal synaptosomes. In the presence of BD1063 (10nM), cocaine failed to amplify the quinpirole (100nM)-induced effects. In cotransfected σ 1 R and D 2L R HEK293T cells, quinpirole had a reduced potency to inhibit the CREB signal versus D 2L R singly transfected cells. In the presence of cocaine (100nM), the potency of quinpirole to inhibit the CREB signal was restored. In D 2L singly transfected cells cocaine (100nM and 10μM) exerted no modulatory effects on the inhibitory potency of quinpirole to bring down the CREB signal. These results led us to hypothesize the existence of functional D 2 -σ 1 R complexes on the rat striatal DA and glutamate nerve terminals and functional D 2 -σ 1 R-DA transporter complexes on the striatal DA terminals. Nanomolar cocaine concentrations appear to alter the allosteric receptor-receptor interactions in such complexes leading to enhancement of Gi/o mediated D 2 R signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Manipulation of isolated brain nerve terminals by an external magnetic field using D-mannose-coated γ-Fe2O3 nano-sized particles and assessment of their effects on glutamate transport.

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Borisova, Tatiana; Krisanova, Natalia; Borуsov, Arsenii; Sivko, Roman; Ostapchenko, Ludmila; Babic, Michal; Horak, Daniel

2014-01-01

The manipulation of brain nerve terminals by an external magnetic field promises breakthroughs in nano-neurotechnology. D-Mannose-coated superparamagnetic nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) salts followed by oxidation with sodium hypochlorite and addition of D-mannose. Effects of D-mannose-coated superparamagnetic maghemite (γ-Fe2O3) nanoparticles on key characteristics of the glutamatergic neurotransmission were analysed. Using radiolabeled L-[(14)C]glutamate, it was shown that D-mannose-coated γ-Fe2O3 nanoparticles did not affect high-affinity Na(+)-dependent uptake, tonic release and the extracellular level of L-[(14)C]glutamate in isolated rat brain nerve terminals (synaptosomes). Also, the membrane potential of synaptosomes and acidification of synaptic vesicles was not changed as a result of the application of D-mannose-coated γ-Fe2O3 nanoparticles. This was demonstrated with the potential-sensitive fluorescent dye rhodamine 6G and the pH-sensitive dye acridine orange. The study also focused on the analysis of the potential use of these nanoparticles for manipulation of nerve terminals by an external magnetic field. It was shown that more than 84.3 ± 5.0% of L-[(14)C]glutamate-loaded synaptosomes (1 mg of protein/mL) incubated for 5 min with D-mannose-coated γ-Fe2O3 nanoparticles (250 µg/mL) moved to an area, in which the magnet (250 mT, gradient 5.5 Т/m) was applied compared to 33.5 ± 3.0% of the control and 48.6 ± 3.0% of samples that were treated with uncoated nanoparticles. Therefore, isolated brain nerve terminals can be easily manipulated by an external magnetic field using D-mannose-coated γ-Fe2O3 nanoparticles, while the key characteristics of glutamatergic neurotransmission are not affected. In other words, functionally active synaptosomes labeled with D-mannose-coated γ-Fe2O3 nanoparticles were obtained.

Curcumin improves synaptic plasticity impairment induced by HIV-1gp120 V3 loop

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Ling-ling Shen

2015-01-01

Full Text Available Curcumin has been shown to significantly improve spatial memory impairment induced by HIV-1 gp120 V3 in rats, but the electrophysiological mechanism remains unknown. Using extracellular microelectrode recording techniques, this study confirmed that the gp120 V3 loop could suppress long-term potentiation in the rat hippocampal CA1 region and synaptic plasticity, and that curcumin could antagonize these inhibitory effects. Using a Fura-2/AM calcium ion probe, we found that curcumin resisted the effects of the gp120 V3 loop on hippocampal synaptosomes and decreased Ca 2+ concentration in synaptosomes. This effect of curcumin was identical to nimodipine, suggesting that curcumin improved the inhibitory effects of gp120 on synaptic plasticity, ameliorated damage caused to the central nervous system, and might be a potential neuroprotective drug.

Strontium, barium, and manganese metabolism in isolated presynaptic nerve terminals

International Nuclear Information System (INIS)

Rasgado-Flores, H.; Sanchez-Armass, S.; Blaustein, M.P.; Nachshen, D.A.

1987-01-01

To gain insight into the mechanisms by which the divalent cations Sr, Ba, and Mn affect neurotransmitter release from presynaptic nerve terminals, the authors examined the sequestration of these cations, ion comparison to Ca, by mitochondrial and nonmitochondrial organelles and the extrusion of these cations from isolated nerve terminals. Sequestration was studied in synaptosomes made leaky to small ions by treatment with saponin; efflux was examined in intact synaptosomes that were preloaded with the divalent cations by incubation in depolarizing (K rich) media. The selectivity sequence for ATP-dependent mitochondrial uptake that they observed was Mn>>Ca>Sr>>Ba, whereas that for the SER was Ca ≥ Mn>Sr>>Ba. When synaptosomes that were preloaded with divalent cations were incubated in Na- and Ca-free media, there was little efflux of 45 Ca, 133 Ba, 85 Sr, or 54 Mn. When the incubation was carried out in media containing Na without Ca, there was substantial stimulation of Ca and Sr efflux, but only slight stimulation of Ba or Mn efflux. In Na-free media, the addition of 1 mM Ca promoted the efflux of all four divalent cations, probably via Ca-divalent cation exchange. In summary, the sequestration and extrusion data suggest that, with equal loads, Mn will be buffered to the greatest extent, whereas Ba will be least well buffered. These results may help to explain why Mn has a very long-lasting effect on transmitter release, while the effect of Sr is much briefer

Traumatic stress causes distinctive effects on fear circuit catecholamines and the fear extinction profile in a rodent model of posttraumatic stress disorder.

Science.gov (United States)

Lin, Chen-Cheng; Tung, Che-Se; Lin, Pin-Hsuan; Huang, Chuen-Lin; Liu, Yia-Ping

2016-09-01

Central catecholamines regulate fear memory across the medial prefrontal cortex (mPFC), amygdala (AMYG), and hippocampus (HPC). However, inadequate evidence exists to address the relationships among these fear circuit areas in terms of the fear symptoms of posttraumatic stress disorder (PTSD). By examining the behavioral profile in a Pavlovian fear conditioning paradigm together with tissue/efflux levels of dopamine (DA) and norepinephrine (NE) and their reuptake abilities across the fear circuit areas in rats that experienced single prolonged stress (SPS, a rodent model of PTSD), we demonstrated that SPS-impaired extinction retrieval was concomitant with the changes of central DA/NE in a dissociable manner. For tissue levels, diminished DA and increased NE were both observed in the mPFC and AMYG. DA efflux and synaptosomal DA transporter were consistently reduced in the AMYG/vHPC, whereas SPS reduced NE efflux in the infralimbic cortex and synaptosomal NE transporter in the mPFC. Furthermore, a lower expression of synaptosomal VMAT2 was observed in the mPFC, AMYG, and vHPC after SPS. Finally, negative correlations were observed between retrieval freezing and DA in the mPFC/AMYG; nevertheless, the phenomena became invalid after SPS. Our results suggest that central catecholamines are crucially involved in the retrieval of fear extinction in which DA and NE play distinctive roles across the fear circuit areas. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

[Effect of baicalin on ATPase and LDH and its regulatory effect on the AC/cAMP/PKA signaling pathway in rats with attention deficit hyperactivity disorder].

Science.gov (United States)

Zhou, Rong-Yi; Wang, Jiao-Jiao; You, Yue; Sun, Ji-Chao; Song, Yu-Chen; Yuan, Hai-Xia; Han, Xin-Min

2017-05-01

To study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD). A total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA. Compared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (PATPase activity (PATPase activity (PATPase activity (PATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

The SNAP-25 gene is associated with cognitive ability: evidence from a family-based study in two independent Dutch cohorts

NARCIS (Netherlands)

Gosso, M.F.; de Geus, E.J.C.; van Belzen, MJ; Polderman, T.J.C.; Heutink, P.; Boomsma, D.I.; Posthuma, D.

2006-01-01

The synaptosomal-associated protein of 25 kDa (SNAP-25) gene plays an integral role in synaptic transmission, and is differentially expressed in the mammalian brain in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra and cerebellar granular cells. Recent studies have suggested

Use of 3H-muscimol for GABA receptor studies

International Nuclear Information System (INIS)

Snodgrass, S.R.

1978-01-01

It is stated that gamma aminobutyric acid (GABA) is a major transmitter in the mammalian central nervous system and studies of synaptic receptors for neurotransmitters have been useful in many areas of neuropharmacology. Although GABA receptors can be studied using 3 H-GABA itself, a ligand which does not bind to GABA uptake sites would be valuable for autoradiography and for other studies of receptor function. Muscimol (3-hydroxy-5-aminomethly-isoxazole) is a naturally occurring GABA analogue found in Amanita muscaria. It seems to enter the brain after peripheral injection. Evidence is here presented of the binding of 3 H-muscimol by brain tissue. The ability of muscimol to alter evoked release of GABA by synaptosomes was also of muscimol to alter evoked release of GABA by synaptosomes was also used to verify the ability of muscimol to alter the function of GABA neurones. (author)

Adaptation of Microplate-based Respirometry for Hippocampal Slices and Analysis of Respiratory Capacity

Science.gov (United States)

Schuh, Rosemary A.; Clerc, Pascaline; Hwang, Hyehyun; Mehrabian, Zara; Bittman, Kevin; Chen, Hegang; Polster, Brian M.

2011-01-01

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O2 consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O2 consumption. As a first evaluation of the technique, we compared whole slice bioenergetics to previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O2 consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O2 consumption that was well-coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared to previous measurements in cells or synaptosomes, although enhanced slightly (to ~150% of basal respiration) by the acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O2 electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to efficiently measure O2 consumption from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology. PMID:21520220

Acacetin inhibits glutamate release and prevents kainic acid-induced neurotoxicity in rats.

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Tzu-Yu Lin

Full Text Available An excessive release of glutamate is considered to be a molecular mechanism associated with several neurological diseases that causes neuronal damage. Therefore, searching for compounds that reduce glutamate neurotoxicity is necessary. In this study, the possibility that the natural flavone acacetin derived from the traditional Chinese medicine Clerodendrum inerme (L. Gaertn is a neuroprotective agent was investigated. The effect of acacetin on endogenous glutamate release in rat hippocampal nerve terminals (synaptosomes was also investigated. The results indicated that acacetin inhibited depolarization-evoked glutamate release and cytosolic free Ca(2+ concentration ([Ca(2+]C in the hippocampal nerve terminals. However, acacetin did not alter synaptosomal membrane potential. Furthermore, the inhibitory effect of acacetin on evoked glutamate release was prevented by the Cav2.2 (N-type and Cav2.1 (P/Q-type channel blocker known as ω-conotoxin MVIIC. In a kainic acid (KA rat model, an animal model used for excitotoxic neurodegeneration experiments, acacetin (10 or 50 mg/kg was administrated intraperitoneally to the rats 30 min before the KA (15 mg/kg intraperitoneal injection, and subsequently induced the attenuation of KA-induced neuronal cell death and microglia activation in the CA3 region of the hippocampus. The present study demonstrates that the natural compound, acacetin, inhibits glutamate release from hippocampal synaptosomes by attenuating voltage-dependent Ca(2+ entry and effectively prevents KA-induced in vivo excitotoxicity. Collectively, these data suggest that acacetin has the therapeutic potential for treating neurological diseases associated with excitotoxicity.

Regulation of somatostatin release in the nervous system of the rat

International Nuclear Information System (INIS)

Sheppard, M.

1979-08-01

This thesis represents the work done to study the release of somatostatin from the rat central nervous system in vitro, providing some evidence for a physiological role for somatostatin. Somatostatin was measured by a sensitive and specific radioimmunoassay devoloped in the laboratory. Chapter 2 reviews the literature on hypothalamic peptides and control of an anterior pituitary function, somatostatin, other central nervous system peptides and neurosecretion. Chapter 3 describes the central nervous system tissue dissection technique, the radioimmunoassay for somatostatin and the tissue levels of somatostatin immunoreactivity in different areas of the central nervous system. Chapter 4 deals with the release of immunoreactive somatostatin from incubated rat hypothalamus in vitro and the influence of other hormones and neuropeptides on this release. Chapter 5 describes the preparation of isolated nerve endings (synaptosomes) from four different areas of rat brain, the localisation of somatostatin to the synaptosome fraction of brain homogenates and the release of somatostatin from these synaptosomes. Chapter 6 deals with the release of somatostatin from incubated rat spinal cord in vitro. Chapter 7 presents the results of the characterisation of released immunoreactive material, the technique utilised being serial dilution of immunoreactive material and comparison to the standard curve, Sephadex gel chromatography, affinity chromatography, and the effect of released immunoreactive somatostatin on growth hormone release from perifused hemipituitaries in vitro, i.e. biological activity. Chapter 8 provides a summary of the main conclusions reached in this study and is followed by the Appendix describing chemical and biochemical methods, histological techniques, and statistical methods

Characterization and regulation of [3H]-serotonin uptake and release in rodent spinal

International Nuclear Information System (INIS)

Stauderman, K.A.

1986-01-01

The uptake and release of [ 3 H]-serotonin were investigated in rat spinal cord synaptosomes. In the uptake experiments, sodium-dependent and sodium-independent [ 3 H]-serotonin accumulation processes were found. Sodium-dependent [ 3 H]-serotonin accumulation was: linear with sodium concentrations up to 180 mM; decreased by disruption of membrane integrity or ionic gradients; associated with purified synaptosomal fractions; and reduced after description of descending serotonergic neurons in the spinal cord. Of the uptake inhibitors tested, the most potent was fluoxetine (IC 50 75 nM), followed by desipramine (IC 50 430 nM) and nomifensine (IC 50 950 nM). The sodium-independent [ 3 H]-serotonin accumulation process was insensitive to most treatments and probably represents nonspecific membrane binding. Thus, only sodium-dependent [ 3 H]-serotonin uptake represents the uptake process of serotonergic nerve terminals in rat spinal cord homogenates. In the release experiments, K + -induced release of previously accumulated [ 3 H]-serotonin was Ca 2+ -dependent, and originated from serotonergic synaptosomes. Exogenous serotonin and 5-methyoxy-N,N-dimethyltryptamine inhibited [ 3 H]-serotonin release in a concentration-dependent way. Of the antagonists tested, only methiothepin effectively blocked the effect of serotonin. These data support the existence of presynaptic serotonin autoreceptors on serotonergic nerve terminals in the rat spinal cord that act to inhibit a voltage and Ca 2+ -sensitive process linked to serotonin release. Alteration of spinai cord serotonergic function may therefore be possible by drugs acting on presynaptic serotonin autoreceptors in the spinal cord

Neuroprotective effects of the novel glutamate transporter inhibitor (-)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic acid, which preferentially inhibits reverse transport (glutamate release) compared with glutamate reuptake

DEFF Research Database (Denmark)

Colleoni, Simona; Jensen, Anders Asbjørn; Landucci, Elisa

2008-01-01

on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest...

Glutamate dehydrogenase is essential to sustain neuronal oxidative energy metabolism during stimulation

DEFF Research Database (Denmark)

Hohnholt, Michaela C; Andersen, Vibe H; Andersen, Jens V

2017-01-01

by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during...

Acetyl-L-carnitine improves aged brain function.

Science.gov (United States)

Kobayashi, Satoru; Iwamoto, Machiko; Kon, Kazuo; Waki, Hatsue; Ando, Susumu; Tanaka, Yasukazu

2010-07-01

The effects of acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, on memory and learning capacity and on brain synaptic functions of aged rats were examined. Male Fischer 344 rats were given ALCAR (100 mg/kg bodyweight) per os for 3 months and were subjected to the Hebb-Williams tasks and AKON-1 task to assess their learning capacity. Cholinergic activities were determined with synaptosomes isolated from brain cortices of the rats. Choline parameters, the high-affinity choline uptake, acetylcholine (ACh) synthesis and depolarization-evoked ACh release were all enhanced in the ALCAR group. An increment of depolarization-induced calcium ion influx into synaptosomes was also evident in rats given ALCAR. Electrophysiological studies using hippocampus slices indicated that the excitatory postsynaptic potential slope and population spike size were both increased in ALCAR-treated rats. These results indicate that ALCAR increases synaptic neurotransmission in the brain and consequently improves learning capacity in aging rats.

A critical role for protein degradation in the nucleus accumbens core in cocaine reward memory.

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Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

2013-04-01

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization.

Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux

DEFF Research Database (Denmark)

Binda, Francesca; Dipace, Concetta; Bowton, Erica

2008-01-01

of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated...

Insulin prevents mitochondrial generation of H₂O₂ in rat brain.

Science.gov (United States)

Muller, Alexandre Pastoris; Haas, Clarissa Branco; Camacho-Pereira, Juliana; Brochier, Andressa Wigner; Gnoatto, Jussânia; Zimmer, Eduardo Rigon; de Souza, Diogo Onofre; Galina, Antonio; Portela, Luis Valmor

2013-09-01

The mitochondrial electron transport system (ETS) is a main source of cellular ROS, including hydrogen peroxide (H₂O₂). The production of H₂O₂ also involves the mitochondrial membrane potential (ΔΨm) and oxygen consumption. Impaired insulin signaling causes oxidative neuronal damage and places the brain at risk of neurodegeneration. We evaluated whether insulin signaling cross-talks with ETS components (complexes I and F₀F₁ATP synthase) and ΔΨm to regulate mitochondrial H₂O₂ production, in tissue preparations from rat brain. Insulin (50 to 100 ng/mL) decreased H₂O₂ production in synaptosomal preparations in high Na(+) buffer (polarized state), stimulated by glucose and pyruvate, without affecting the oxygen consumption. In addition, insulin (10 to 100 ng/mL) decreased H₂O₂ production induced by succinate in synaptosomes in high K(+) (depolarized state), whereas wortmannin and LY290042, inhibitors of the PI3K pathway, reversed this effect; heated insulin had no effect. Insulin decreased H₂O₂ production when complexes I and F₀F₁ATP synthase were inhibited by rotenone and oligomycin respectively suggesting a target effect on complex III. Also, insulin prevented the generation of maximum level of ∆Ψm induced by succinate. The PI3K inhibitors and heated insulin maintained the maximum level of ∆Ψm induced by succinate in synaptosomes in a depolarized state. Similarly, insulin decreased ROS production in neuronal cultures. In mitochondrial preparations, insulin neither modulated H2O2 production or oxygen consumption. In conclusion, the normal downstream insulin receptor signaling is necessary to regulate complex III of ETS avoiding the generation of maximal ∆Ψm and increased mitochondrial H2O2 production. Copyright © 2013 Elsevier Inc. All rights reserved.

Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido 3H GBR-12935

International Nuclear Information System (INIS)

Berger, S.P.; Martenson, R.E.; Laing, P.; Thurkauf, A.; Decosta, B.; Rice, K.C.; Paul, S.M.

1991-01-01

A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido 3 H GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido 3 H GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of 3 H GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido 3 H GBR-12935 binding to rat striatal membranes. These data suggest that 3-azido 3 H GBR-12935, like other diphenylpiperazines such as 3 H GBR-12935 and 3 H GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido 3 H GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido 3 H GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido 3 H GBR-12935 is glycosylated

Role of platelet-activating factor in long-term potentiation of the rat medial vestibular nuclei.

Science.gov (United States)

Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1998-06-01

In rat brain stem slices, we investigated the role of platelet activating factor (PAF) in long-term potentiation (LTP) induced in the ventral part of the medial vestibular nuclei (MVN) by high-frequency stimulation (HFS) of the primary vestibular afferent. The synaptosomal PAF receptor antagonist, BN-52021 was administered before and after HFS. BN-52021 did not modify the vestibular potentials under basal conditions, but it reduced the magnitude of potentiation induced by HFS, which completely developed after the drug wash-out. The same effect was obtained by using CV-62091, a more potent PAF antagonist at microsomal binding sites, but with concentrations higher than those of BN-52021. By contrast both BN-52021 and CV-6209 had no effect on the potentiation once induced. This demonstrates that PAF is involved in the induction but not in the maintenance of vestibular long-term effect through activation of synaptosomal PAF receptors. In addition, we analyzed the effect of the PAF analogue, 1-O-hexadecyl-2-O- (methylcarbamyl)-sn-glycero-3-phosphocoline (MC-PAF) and the inactive PAF metabolite, 1-O-hexadecyl-sn-glycero-3-phosphocoline (Lyso-PAF) on vestibular responses. Our results show that MC-PAF, but not Lyso-PAF induced potentiation. This potentiation was prevented by D,L-2-amino 5-phosphonopentanoic acid, suggesting an involvement of N-methyl-D-aspartate receptors. Furthermore, under BN-52021 and CV-6209, the MC-PAF potentiation was reduced or abolished. The dose-effect curve of MC-PAF showed a shift to the right greater under BN-52021 than under CV-6209, confirming the main dependence of MC-PAF potentiation on the activation of synaptosomal PAF receptors. Our results suggest that PAF can be released in the MVN after the activation of postsynaptic mechanisms triggering LTP, and it may act as a retrograde messenger which activates the presynaptic mechanisms facilitating synaptic plasticity.

Region-Specific Defects of Respiratory Capacities in the Ndufs4(KO Mouse Brain.

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Ernst-Bernhard Kayser

Full Text Available Lack of NDUFS4, a subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase, causes Leigh syndrome (LS, a progressive encephalomyopathy. Knocking out Ndufs4, either systemically or in brain only, elicits LS in mice. In patients as well as in KO mice distinct regions of the brain degenerate while surrounding tissue survives despite systemic complex I dysfunction. For the understanding of disease etiology and ultimately for the development of rationale treatments for LS, it appears important to uncover the mechanisms that govern focal neurodegeneration.Here we used the Ndufs4(KO mouse to investigate whether regional and temporal differences in respiratory capacity of the brain could be correlated with neurodegeneration. In the KO the respiratory capacity of synaptosomes from the degeneration prone regions olfactory bulb, brainstem and cerebellum was significantly decreased. The difference was measurable even before the onset of neurological symptoms. Furthermore, neither compensating nor exacerbating changes in glycolytic capacity of the synaptosomes were found. By contrast, the KO retained near normal levels of synaptosomal respiration in the degeneration-resistant/resilient "rest" of the brain. We also investigated non-synaptic mitochondria. The KO expectedly had diminished capacity for oxidative phosphorylation (state 3 respiration with complex I dependent substrate combinations pyruvate/malate and glutamate/malate but surprisingly had normal activity with α-ketoglutarate/malate. No correlation between oxidative phosphorylation (pyruvate/malate driven state 3 respiration and neurodegeneration was found: Notably, state 3 remained constant in the KO while in controls it tended to increase with time leading to significant differences between the genotypes in older mice in both vulnerable and resilient brain regions. Neither regional ROS damage, measured as HNE-modified protein, nor regional complex I stability, assessed by blue native

Pharmacological effects of two cytolysins isolated from the sea ...

Indian Academy of Sciences (India)

Prakash

2009-12-07

Dec 7, 2009 ... [3H] in rat brain synaptosomes without causing them to swell (Migues et al.1999) ... and resistances ranging from 10 to 20 MΩ as previously described (Garateix et ... amplitude of depolarization induced by the application of. Ach was ... percentage of change with respect to control conditions ±. SEM and they ...

Differential effects of benzodiazepines on phospholipid methylation in hippocampus and cerebellum of rats

Energy Technology Data Exchange (ETDEWEB)

Tacconi, M.T.; Salmona, M.

1988-01-01

To elucidate the relationship between the occupancy of BDZ binding sites and phospholipid methylation in brain, the authors examined phosphatidylethanolamine-N-methyltransferase (PEMT) activity in synaptosomes of rat hippocampi and cerebella in the presence of BDZ ligands with different modes of action. We found that Ro 5-4864, a specific ligand for peripheral type receptors, increased PL methylation in hippocampal and cerebellar synaptosomes. This effect was directly related to receptor occupancy, since the specific antagonist PK11195 inhibited the rise in PEMT activity induced by Ro 5-4864. Clonazepam, on the other hand, tended to reduce PL production in cerebellum and hippocampus except for hiccocampal (/sup 3/H)-phosphatidyl-N-monomethylethanolamine which was elevated by 40 to 70% at doses ranging from 10/sup -9/ to 10/sup -6/M. When equimolar concentrations of the antagonist Ro 15-1788 were given in association the clonazepam-induced phosphatidyl-N-monomethylethanolamine increase was reduced by 70%. These data support the involvement of structural and functional membrane alterations in the action of BDZ. 20 references, 2 figures, 2 tables.

Microwave radiation, in the absence of hyperthermia, has no detectable effect on synapsin I levels or phosphorylation

International Nuclear Information System (INIS)

Browning, M.D.; Haycock, J.W.

1988-01-01

Recent reports have indicated that microwave radiation can produce effects on a variety of cell types in vitro. To determine whether microwave radiation might be neurotoxic, the effects of microwave radiation on synapsin I have been examined. Synapsin I is a neuron-specific phosphoprotein that is present in all neurons, where it is localized to the presynaptic terminal and is associated with synaptic vesicles. O'Callaghan and Miller have demonstrated that studies of such neuron-specific proteins can provide reliable indices of neurotoxicity. We have used a radioimmunoassay for synapsin I to determine whether microwave irradiation has any effect on the levels of synapsin I. Neither acute nor chronic exposure to microwave irradiation had any detectable effect on synapsin I levels. We have also examined the calcium-dependent phosphorylation of synapsin I in synaptosomes isolated from rats that had been subjected to microwave radiation. The phosphorylation of synapsin I in synaptosomes reflects numerous components of the presynaptic aspect of neuronal transmission. At intensities below that required to produce mild hyperthermia, no effects of microwave irradiation were seen on synapsin I phosphorylation

Synaptosomal ecto-5'-nucleotidases activity modulation after ionizing irradiation

International Nuclear Information System (INIS)

Drakulic, D.; Stanojevic, I.; Petrovic, S.; Velickovic, N.; Horvat, A.

2009-01-01

Adenine nucleotides, such as ATP and adenosine are involved in the regulation of variety of physiological processes in the central nervous system (CNS), including development and tissue remodeling following trauma, stroke, ischemia or neurodegenerative disorders. Ecto-5'- nucleotidase (ecto-5'-NT), membrane enzyme, catalyzes the last step of extracellular nucleotide degradation and it is responsible for purinergic signaling modulation and termination. In order to investigate if ionizing irradiation could modulate CNS purinergic signalization in synaptic plasma membranes (SPM) the activity of ecto-5'-NT was monitored after whole-body acute irradiation with low (0,5 Gy) or therapeutic (2 Gy) doses, 1h, 24h and 72h after irradiating juvenile (15-day old), prepubertal (30 days), pubertal (60 days) and adult (90-day old) female rats. Results suggest that acute irradiation could modulate activity of the enzymes that are necessary for purinergic signal termination depended of dose and time after irradiation, as well as brain development stage. (author) [sr

Mechanism of toxicity of MPTP: A cause of Parkinsonism in human beings

International Nuclear Information System (INIS)

Denton, T.L.

1988-01-01

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found in 1983 to cause a syndrome virtually identical to Parkinson's Disease in humans and other primates. The symptoms, as in idiopathic Parkinson's syndrome, are due to destruction of dopaminergic neurons in the pars compacta of the substantia nigra resulting in depletion of dopamine in the basal ganglia. The mechanism of toxicity was investigated with a dopamine containing cell line, PC12, a MPTP resistant variant (MPTP r ), and synaptosomes from the striate cortex of mice, rats, guinea pigs and a monkey. The mechanism of acute effects was studied with membrane preparations from human and rat striate cortex. MPTP displaced [ 3 H]haloperidol from binding sites in human and rat striate cortex, but could not displace [ 3 H]flupenthixol, suggesting that MPTP is a D2 receptor ligand of equivalent potency in both species. MPTP was a competitive inhibitor of uptake of [ 3 H]dopamine in PC12 but did not accumulate in PC12 or in synaptosomes of rat, guinea pig, mouse or monkey striate cortex. 100 uM MPTP depleted catecholamine levels in PC12 cells by about 50%, without killing

Development of paradigm for the study of amino acid neurotransmitter release in human autopsy brain samples

International Nuclear Information System (INIS)

Kuo, K.-W.; Dodd, P.R.

2001-01-01

Full text: This study attempted to establish a release protocol to characterize both the vesicular and cytoplasmic components of amino acid transmitter release in human synaptosomes. Experiments with rat synaptosomes showed that, with depolarizing concentrations of K + ions, vesicular release could be successfully differentiated from cytoplasmic release for preloaded L-[ 3 H ]glutamate and [ 14 C ]GABA. However, human tissue studies did not give clear-cut results. Experiments were carried out to optimize the release paradigm as well as to improve the vesicular uptake of labeled transmitters. A 'pulse- chase' protocol, with an unlabelled D-aspartate chase, was performed in human tissue samples in order to enhance the L-[ 3 H ] glutamate release signal derived from exocytosis by removing the cytoplasmic pool of L-[ 3 H ] glutamate first. However, the results showed that total release was not enhanced effectively in comparison with the non-pulse-chase protocol. In brief, the pulse-chase protocol did not build up the vesicular pool of L-[ 3 H ]glutamate, though the cytoplasmic L- [ 3 H ] glutamate pool was effectively depressed by D-aspartate. Further studies applied 4- aminopyridine (4-AP) to trigger release, to circumvent the problem of the reversal of plasma membrane transporters caused by raised K + ion concentrations. The results showed that the application of 4-AP elicited the release of amino acid transmitters from rat synaptosomes, but failed to produce successful release signals in the human tissue experiments. Our findings suggest that the vesicular compartment may be impaired by freezing and affected by post-mortem delay (PMD). Rat studies showed that the freezing step had a major effect on Ca 2+-dependent release, as less L- [3 H ]glutamate and [ 14 C ]GABA were released from the frozen rat tissue preparations. Moreover, there was an indication of a decline in L-[ 3 H ]glutamate release with increasing PMD. Copyright (2001) Australian Neuroscience Society

Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse

OpenAIRE

Zhang, Ye; Sloan, Steven A.; Clarke, Laura E.; Caneda, Christine; Plaza, Colton A.; Blumenthal, Paul D.; Vogel, Hannes; Steinberg, Gary K.; Edwards, Michael S. B.; Li, Gordon; Duncan, John A.; Cheshier, Samuel H.; Shuer, Lawrence M.; Chang, Edward F.; Grant, Gerald A.

2015-01-01

The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains, and to maintain these cells in serum-free cultures. We found that human astrocytes have similar abilities to murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to exist...

Impact of Combined Prenatal Ethanol and Prenatal Stress Exposures on Markers of Activity-Dependent Synaptic Plasticity in Rat Dentate Gyrus

OpenAIRE

Staples, Miranda C.; Porch, Morgan W.; Savage, Daniel D.

2014-01-01

Prenatal ethanol exposure and prenatal stress can each cause long-lasting deficits in hippocampal synaptic plasticity and disrupt learning and memory processes. However, the mechanisms underlying these perturbations following a learning event are still poorly understood. We examined the effects of prenatal ethanol exposure and prenatal stress exposure, either alone or in combination, on the cytosolic expression of activity-regulated cytoskeletal (ARC) protein and the synaptosomal expression o...

International Conference on Partitioning in Aqueous Two-Phase Systems in Biochemistry Cell Biology and Biotechnology (7th) Held in New Orleans, Louisiana on June 2-7, 1991.

Science.gov (United States)

1991-11-30

and Virus Like Particles (VLPs, cloned in yeast presently being used for the development of an AIDS vaccine). This paper will describe partition...PARTITIONING OF CEREBROCORTICAL SYNAPTOSOMES M. J. L6pez.P6rez Departamento de Bioquimica y Biologia Molecular y Celular . Facultad de Veterinaria...Pascual, T. Muifio-Blanco, J. A. Cebriin-Pdrez and M. J. L6pez-Pdrez. Departamento de Bioqufmica y Biologia Molecular y Celular . Facultad de

Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

International Nuclear Information System (INIS)

King, B.F.; Somani, S.M.

1987-01-01

The distribution of 3 H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3 H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3 H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27,474 +/- 2825 DPM/gm at 60 min (P < .05). The cytosol contains the highest RA: 223,341 +/- 21,044 DPM/gm at 30 min which declined to 53,475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of the organelle. 21 references, 1 figure, 2 tables

Binding assays for the quantitative detection of P. brevis polyether neurotoxins in biological samples and antibodies as therapeutic aids for polyether marine intoxication. Annual report, 1 December 1987-30 November 1988

Energy Technology Data Exchange (ETDEWEB)

Baden, D.G.

1988-12-15

The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) can be detected using two separate types of specific binding reaction. Using tritiated PbTx-3 as a specific probe for binding to voltage-dependent sodium channels in rat brain synaptosomes or to specific polyclonal antibodies, binding equilibria and displacement by unlabeled brevetoxins were compared. Labeled toxin can be displaced in a competitive manner by any of the other 5 naturally-occurring toxins; the quantitative displacement ability of each appears to reflect individual potency in fish bioassay. A comparison of ED50 in Radioimmunoassay and ED50 in synaptosome binding assay indicates that the former assay is useful for detection of toxins which possess the structural backbone of PbTx-3, the immunizing hapten. Thus, the two assays have quantitative applicability; the sodium channel with respect to potency and the antibodies with respect to structure. Microtiter plate assays utilizing each specific brevetoxin binding component and enzyme-linked toxin hapten have been successful and indicate a general applicability of colorimetric prototypes. There, is however, considerable manipulation required to decrease non-specific binding of the hydrophobic toxin-enzyme complex to the plates. Preliminary studies aimed at producing monoclonal antibodies have been explored using brevetoxins linked to keyhole limpet hemocyanin.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Science.gov (United States)

Simor, Attila; Györffy, Balázs András; Gulyássy, Péter; Völgyi, Katalin; Tóth, Vilmos; Todorov, Mihail Ivilinov; Kis, Viktor; Borhegyi, Zsolt; Szabó, Zoltán; Janáky, Tamás; Drahos, László; Juhász, Gábor; Kékesi, Katalin Adrienna

2017-03-01

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain. Copyright © 2017 Elsevier Inc. All rights reserved.

The influence of L-DOPA on the accumulation of lipid peroxidation products in some brain structures affected by radiation

International Nuclear Information System (INIS)

Babaev, R.A.; Kocharli, R.Kh.; Akhmedova, G.Sh.; Gasanova, A.A.; Babaev, Kh.F.

1990-01-01

A study was made of the effect of L-DOPA on the dynamics of changes in lipid peroxidation products (LPP) and the content of various types of SH-groups in certain brain structures (oblongata, cerebellum, visual and sensorimotor cortex) and their synaptosomal fractions upon irradiation. The preadministration of L-DOPA to irradiated rats inhibited LPP accumulation, prevented the decrease in the content of various types of thiols and thus exerted an antioxidant effect

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

International Nuclear Information System (INIS)

Lin, Tzu-Yu; Lu, Cheng-Wei; Wang, Chia-Chuan; Lu, Jyh-Feng; Wang, Su-Jane

2012-01-01

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K + channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca 2+ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] C ), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca v 2.2 (N-type) and Ca v 2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na + /Ca 2+ exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca 2+ entry and ERK/synapsin I signaling pathway. -- Highlights: ► Hispidulin inhibited glutamate release from rat cerebrocortical synaptosomes. ► This action did

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

Energy Technology Data Exchange (ETDEWEB)

Lin, Tzu-Yu [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei, 22060, Taiwan (China); Department of Mechanical Engineering, Yuan Ze University, Taoyuan, 320, Taiwan (China); Lu, Cheng-Wei [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei, 22060, Taiwan (China); Wang, Chia-Chuan; Lu, Jyh-Feng [School of Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang Dist., New Taipei, 24205, Taiwan (China); Wang, Su-Jane, E-mail: med0003@mail.fju.edu.tw [Graduate Institute of Basic Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang Dist., New Taipei, 24205, Taiwan (China); School of Medicine, Fu Jen Catholic University, No.510, Zhongzheng Rd., Xinzhuang Dist., New Taipei, 24205, Taiwan (China)

2012-09-01

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K{sup +} channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca{sup 2+} ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca{sup 2+} concentration ([Ca{sup 2+}]{sub C}), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca{sub v}2.2 (N-type) and Ca{sub v}2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na{sup +}/Ca{sup 2+} exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca{sup 2+} entry and ERK/synapsin I signaling pathway. -- Highlights: ► Hispidulin inhibited glutamate release from rat

Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings.

Science.gov (United States)

Musante, Veronica; Neri, Elisa; Feligioni, Marco; Puliti, Aldamaria; Pedrazzi, Marco; Conti, Valerio; Usai, Cesare; Diaspro, Alberto; Ravazzolo, Roberto; Henley, Jeremy M; Battaglia, Giuseppe; Pittaluga, Anna

2008-09-01

The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [3H]d-aspartate ([3H]D-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1-100microM) potentiated the K+(12mM)-evoked [3H]D-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [3H]D-ASP exocytosis when applied at 0.3microM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30-100microM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [3H]D-ASP exocytosis caused by 0.3microM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50microM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3microM 3,5-DHPG failed to facilitate the K+-evoked [3H]D-ASP overflow from mGlu5 receptor knockout (mGlu5-/-) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50microM 3,5-DHPG failed to affect the [3H]D-ASP exocytosis from cortical synaptosomes obtained from crv4/crv4 and mGlu5-/-mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the "high affinity" binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the "low affinity" binding sites.

[{sup 14}C]Serotonin uptake and [O-methyl-{sup 11}C]venlafaxine kinetics in porcine brain

Energy Technology Data Exchange (ETDEWEB)

Smith, D.F. E-mail: dfsmith@inet.uni2.dk; Hansen, S.B.; Oestergaard, L.; Gee, A.D.; Danielsen, E.; Ishizu, K.; Bender, D.; Poulsen, P.H.; Gjedde, A

2001-08-01

As part of our program of developing PET tracers for neuroimaging of psychotropic compounds, venlafaxine, an antidepressant drug, was evaluated. First, we measured in vitro rates of serotonin uptake in synaptosomes prepared from selected regions of porcine brain. Then, we determined the pharmacokinetics of venlafaxine, [O-methyl-{sup 11}C]-labeled for PET. Synaptosomal studies showed that the active uptake of [{sup 14}C]5-HT differed markedly between brain regions, with highest rates in hypothalamus, raphe region, and thalamus, and lowest rates in cortex and cerebellum. PET studies showed that the unidirectional rate of uptake of [O-methyl-{sup 11}C]venlafaxine from blood to brain was highest in the hypothalamus, raphe region, thalamus and basal ganglia and lowest in the cortex and cerebellum. Under normal physiological conditions, the capillary permeability-surface area (PS) product for [O-methyl-{sup 11}C]venlafaxine could not be estimated, because of complete flow-limitation of the cerebral uptake. Nevertheless, a correlation occurred between the apparent partition volume of the radiotracer and the rate of active uptake of 5-HT in selected regions of the porcine brain. During hypercapnia, limitations of blood-brain transfer were observed, giving PS-products for water that were only ca. 50% higher than those of venlafaxine. Thus, under normal physiological conditions, the rate of uptake of venlafaxine from blood into brain is completely flow-limited.

[Detection of ciguatoxins: advantages and drawbacks of different biological methods].

Science.gov (United States)

Boydron-Le Garrec, Raphaële; Benoit, Evelyne; Sauviat, Martin-Pierre; Laurent, Dominique

2005-01-01

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.

Functional roles of DNA polymerases β and γ

International Nuclear Information System (INIS)

Huebscher, U.; Kuenzle, C.C.; Spadari, S.

1979-01-01

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC2.7.7.7)β and γ were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. uv irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase β which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase γ was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that under these conditions, DNA replication occurs in miatochondria, and exploiting the fact that DNA polymerase γ is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase γ in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase α, we conclude that DNA polymerase α is mainly responsible for DNA replication in nuclei, DNA polymerase β is involved in nuclear DNA repair, and DNA polymerase γ is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase α in DNA repair or for DNA polymerase β in DNA replication cannot be excluded

Activation analysis study on subcellular distribution of trace elements in human brain tumor

International Nuclear Information System (INIS)

Zheng Jian; Zhuan Guisun; Wang Yongji; Dong Mo; Zhang Fulin

1992-01-01

The concentrations of up to 11 elements in subcellular fractions of human brain (normal and malignant tumor) have been determined by a combination of gradient centrifugation and INAA methods. Samples of human brain were homogenized in a glass homogenizer tube, the homogenate was separated into nuclei, mitochondrial, myelin, synaptosome fractions, and these fractions were then analyzed using the INAA method. The discussions of elemental subcelleular distributions in human brain malignant tumor are presented in this paper. (author) 11 refs.; 2 figs.; 4 tabs

Presynaptic muscarinic receptors: Change of sensitivity during long-term drug treatment

International Nuclear Information System (INIS)

Marchi, M.; Raiteri, M.

1986-01-01

The authors investigate some of the characteristics of auto- and heteroreceptors from different brain areas in male rats; their alteration in sensitivity following chronic drug treatment is monitored. The synaptosomes were prelabeled with tritium-choline or tritium-dopamine and the release of tritium-acetylcholine and tritium-DA was studied in superfusion. It is shown that the difference in susceptibility between auto- and heteroreceptors with respect to changes of sensitivity may represent a further criterion to discriminate between muscarinic receptor subtypes

Effects on the sodium channel of some new cardiotonic drugs: the 4-, 5-, and 6-pyridyl-2(1H)-quinolone derivatives

International Nuclear Information System (INIS)

Grima, M.; Beguin, M.F.; Millanvoye-Van Brussel, E.M.; Decker, N.; Schwartz, J.

1988-01-01

To study the action of some new cardiotonic drugs, the 4-, 5-, and 6-pyridyl-2(1H)-quinolone series, on the fast Na+ channel, we compared the effects of eight compounds of this series and milrinone on 22 Na uptake in rat brain synaptosomes and in rat heart muscle cells in culture. The action of tetrodotoxin, a specific Na+ channel blocker, on the positive inotropic effect of these compounds on guinea pig atria was also examined. The new positive inotropic agents enhance 22 Na uptake in synaptosomes in a dose-dependent manner. The activities, expressed as percentage of the maximum activity of protoveratrine B, a classic Na+ channel agonist, reached 70% for milrinone, 60% for compound 7, 57% for compound 6, and less than 50% for the other drugs. For compound 8, but not for milrinone, it was possible to observe a stimulatory effect of the 22 Na uptake on heart muscle cells in culture. Tetrodotoxin (1 and 100 microM) inhibited the stimulatory effects of the inotropic drugs on both preparations. The positive inotropic activities of protoveratrine B, milrinone, and compounds 5 and 8, in guinea pig atria, were inhibited by tetrodotoxin. The affinity and the activity of the other compounds were unchanged in the presence of tetrodotoxin. Our results showed that the stimulation of Na+ influx through the fast Na+ channel might represent a part of the mechanism of action of the inotropic effect of some new cardiotonic drugs

Effects on the sodium channel of some new cardiotonic drugs: the 4-, 5-, and 6-pyridyl-2(1H)-quinolone derivatives

Energy Technology Data Exchange (ETDEWEB)

Grima, M.; Beguin, M.F.; Millanvoye-Van Brussel, E.M.; Decker, N.; Schwartz, J.

1988-09-01

To study the action of some new cardiotonic drugs, the 4-, 5-, and 6-pyridyl-2(1H)-quinolone series, on the fast Na+ channel, we compared the effects of eight compounds of this series and milrinone on /sup 22/Na uptake in rat brain synaptosomes and in rat heart muscle cells in culture. The action of tetrodotoxin, a specific Na+ channel blocker, on the positive inotropic effect of these compounds on guinea pig atria was also examined. The new positive inotropic agents enhance /sup 22/Na uptake in synaptosomes in a dose-dependent manner. The activities, expressed as percentage of the maximum activity of protoveratrine B, a classic Na+ channel agonist, reached 70% for milrinone, 60% for compound 7, 57% for compound 6, and less than 50% for the other drugs. For compound 8, but not for milrinone, it was possible to observe a stimulatory effect of the /sup 22/Na uptake on heart muscle cells in culture. Tetrodotoxin (1 and 100 microM) inhibited the stimulatory effects of the inotropic drugs on both preparations. The positive inotropic activities of protoveratrine B, milrinone, and compounds 5 and 8, in guinea pig atria, were inhibited by tetrodotoxin. The affinity and the activity of the other compounds were unchanged in the presence of tetrodotoxin. Our results showed that the stimulation of Na+ influx through the fast Na+ channel might represent a part of the mechanism of action of the inotropic effect of some new cardiotonic drugs.

Agmatine Prevents Adaptation of the Hippocampal Glutamate System in Chronic Morphine-Treated Rats.

Science.gov (United States)

Wang, Xiao-Fei; Zhao, Tai-Yun; Su, Rui-Bin; Wu, Ning; Li, Jin

2016-12-01

Chronic exposure to opioids induces adaptation of glutamate neurotransmission, which plays a crucial role in addiction. Our previous studies revealed that agmatine attenuates opioid addiction and prevents the adaptation of glutamate neurotransmission in the nucleus accumbens of chronic morphine-treated rats. The hippocampus is important for drug addiction; however, whether adaptation of glutamate neurotransmission is modulated by agmatine in the hippocampus remains unknown. Here, we found that continuous pretreatment of rats with ascending doses of morphine for 5 days resulted in an increase in the hippocampal extracellular glutamate level induced by naloxone (2 mg/kg, i.p.) precipitation. Agmatine (20 mg/kg, s.c.) administered concurrently with morphine for 5 days attenuated the elevation of extracellular glutamate levels induced by naloxone precipitation. Furthermore, in the hippocampal synaptosome model, agmatine decreased the release and increased the uptake of glutamate in synaptosomes from chronic morphine-treated rats, which might contribute to the reduced elevation of glutamate levels induced by agmatine. We also found that expression of the hippocampal NR2B subunit, rather than the NR1 subunit, of N-methyl-D-aspartate receptors (NMDARs) was down-regulated after chronic morphine treatment, and agmatine inhibited this reduction. Taken together, agmatine prevented the adaptation of the hippocampal glutamate system caused by chronic exposure to morphine, including modulating extracellular glutamate concentration and NMDAR expression, which might be one of the mechanisms underlying the attenuation of opioid addiction by agmatine.

ASIC2 Subunits Target Acid-Sensing Ion Channels to the Synapse via an Association with PSD-95

OpenAIRE

Zha, Xiang-ming; Costa, Vivian; Harding, Anne Marie S.; Reznikov, Leah; Benson, Christopher J.; Welsh, Michael J.

2009-01-01

Acid-sensing ion channel-1a (ASIC1a) mediates H+-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not co-immunoprecipitate with PSD-95. We found that ASIC2...

Expression of presynaptic markers in a neurodevelopmental animal model with relevance to schizophrenia

DEFF Research Database (Denmark)

Karlsen, Anna S; Kaalund, Sanne Simone; Møller, Morten

2013-01-01

Administration of N-methyl-D-aspartate receptor antagonist phencyclidine (PCP) to rat pups at postnatal day (PND) 7, 9, and 11 [neonatal PCP (neoPCP) model] induces cognitive deficits similar to those observed in schizophrenia. Expression of presynaptic SNARE protein, synaptosomal......-associated protein of 25 kDa (Snap25), has been shown to be downregulated in postmortem brains from patients with schizophrenia. The present study was designed to investigate the long-term effects of neoPCP administration on expression of presynaptic markers altered in schizophrenia. Using radioactive in...

Regulatory changes in presynaptic cholinergic function assessed in rapid autopsy material from patients with Alzheimer disease: Implications for etiology and therapy

International Nuclear Information System (INIS)

Slotkin, T.A.; Seidler, F.J.; Crain, B.J.; Bell, J.M.; Bissette, G.; Nemeroff, C.B.

1990-01-01

Brain regions from patients with or without Alzheimer disease (AD) were obtained within 2 hr of death and examined for indices of presynaptic cholinergic function. Consistent with loss of cholinergic projections, cerebral cortical areas involved in AD exhibited decreased choline acetyltransferase activity. However, remaining nerve terminals in these regions displayed marked up-regulation of synaptosomal high affinity [ 3 H]choline uptake, a result indicative of relative cholinergic hyperactivity. As choline uptake is also rate-limiting in acetylcholine biosynthesis, these findings have implications for both therapy and identification of causes contributing to neuronal death in AD

Estradiol receptors mediate estradiol-induced inhibition of mitochondrial Ca^{2+} efflux in rat caudate nucleus and brain stem

OpenAIRE

PETROVIC, SNJEZANA; MILOSEVIC, MAJA; RISTIC-MEDIC, DANIJELA; VELICKOVIC, NATASA; DRAKULIC, DUNJA; GRKOVIC, IVANA; HORVAT, ANICA

2015-01-01

Our earlier studies found that in vitro estradiol modulates mitochondrial Ca2+ transport in discrete brain regions. The present study examined the role of estradiol receptors (ERs) in estradiol-induced inhibition of Ca^{2+} efflux from synaptosomal mitochondria isolated from rat caudate nuclei and brain stems. Radioactively labeled CaCl_2 (0.6?0.75 µCi ^45CaCl_{2}) was used for Ca^{2+} transport monitoring. The results revealed that in the presence of ER antagonist 7\\alpha,17ß-[9[(4,4,5,5,5-...

Diet fat alters synaptosomal phosphatidylethanolaminemethyl-transferase activity and phosphatidylcholine synthesis in brain

International Nuclear Information System (INIS)

Hargreaves, K.M.; Clandinin, M.T.

1986-01-01

Phosphatidylcholine (PC) can be synthesized via three routes, each having potentially different metabolic fates. One route for PC synthesis is methylation of phosphatidylethanolamine (PE). To examine if dietary fat affects membrane PE composition and phosphatidylethanolaminemethyltransferase (PEMT) activity, male weanling rats were fed semi-purified diets containing 20% (w/w) fat of differing fatty acid composition for 24 days. Microsomal and synaptic plasma membranes were isolated and phospholipid composition analyzed. PEMT activity was measured by incorporation of the methyl group from 3 H-S-adenosylmethionine into PE. Polyunsaturated diets high in omega 6 fatty acids produce a high ratio of omega 6/omega 3 fatty acids in synaptic plasma membranes. Dietary omega 3 and omega 6 fatty acid levels are reflected in membrane phospholipid content of 22:6(3), 20:4(6), 22:4(6) and 22:5(6). Diet-induced increase in these longer chain homologues of omega 6 and omega 3 fatty acids and a high ratio of omega 6/omega 3 fatty acids in PE are both associated with increased PEMT activity. These results suggest that diet-fat induced change in fatty acid composition of membrane PE results in transition in PEMT activity and synthesis of PC in brain, by providing preferred species of PE for methylation

Toward development of an in vitro model of methamphetamine-induced dopamine nerve terminal toxicity.

Science.gov (United States)

Kim, S; Westphalen, R; Callahan, B; Hatzidimitriou, G; Yuan, J; Ricaurte, G A

2000-05-01

To develop an in vitro model of methamphetamine (METH)-induced dopamine (DA) neurotoxicity, striatal synaptosomes were incubated at 37 degrees C with METH for different periods of time (10-80 min), washed once, then tested for DA transporter function at 37 degrees C. METH produced time- and dose-dependent reductions in the V(max) of DA uptake, without producing any change in K(m). Incubation of synaptosomes with the DA neurotoxins 1-methyl-4-phenyl-pyridinium ion, 6-hydroxydopamine, and amphetamine under similar conditions produced comparable effects. In contrast, incubation with fenfluramine, a serotonin neurotoxin, did not. METH-induced decreases in DA uptake were selective, insofar as striatal glutamate uptake was unaffected. Various DA transporter blockers (cocaine, methylphenidate, and bupropion) afforded complete protection against METH-induced decreases in DA uptake, without producing any effect themselves. METH's effects were also temperature dependent, with greater decreases in DA uptake occurring at higher temperatures. Tests for residual drug revealed small amounts (0.1-0.2 microM) of remaining METH, but kinetic studies indicated that decreases in DA uptake were not likely to be due to METH acting as a competitive inhibitor of DA uptake. Decreases in the V(max) of DA uptake were not accompanied by decreases in B(max) of [(3)H]WIN 35,428 binding, possibly because there is no mechanism for removing damaged DA nerve endings from the in vitro preparation Collectively, these results give good support to the development of a valid in vitro model that may prove helpful for elucidating the mechanisms underlying METH-induced DA neurotoxicity.

Effect of high fat diets on the NTPDase, 5'-nucleotidase and acetylcholinesterase activities in the central nervous system.

Science.gov (United States)

Kaizer, Rosilene Rodrigues; Spanevello, Rosélia Maria; Costa, Eduarda; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

2018-02-01

High fat diets are associated with the promotion of neurological diseases, such as Alzheimer disease (AD). This study aim investigate the high fat diets role to promotion of AD using as biochemistry parameter of status of central nervous system through the NTPDase, 5'-nucleotidase and acetylcholinesterase (AChE) activities in brain of young rats. The intake of high fat diets promotes an inhibition of purinergic and cholinergic functions, mainly in the long-term exposure to saturated and saturated/unsaturated diets. The AChE activity was decreased to supernatant and synaptosomes tissues preparations obtained from cerebral cortex in average of 20%, to both groups exposed to saturated and saturated/unsaturated diets, when compared to the control group. Very similar results were found in hippocampus and cerebellum brain areas. At same time, the adenine nucleotides hydrolysis in synaptosomes of cerebral cortex were decreased to ATP, ADP and AMP after the long-term exposure to high fat diets, as saturated and saturated/unsaturated. The inhibition of ATP hydrolysis was of 26% and 39% to saturated and saturated/unsaturated diets, respectively. ADP hydrolysis was decreased in 20% to saturated diet, and AMP hydrolysis was decreased in 25% and 33% to saturated and saturated/unsaturated diets, respectively, all in comparison to the control. Thus, we can suggest that the effects of high diets on the purinergic and cholinergic nervous system may contribute to accelerate the progressive memory loss, to decline in language and other cognitive disruptions, such as AD patients presents. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

Science.gov (United States)

Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

2016-05-01

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

Discovery and characterization of cnidarian peptide toxins that affect neuronal potassium ion channels.

Science.gov (United States)

Castañeda, Olga; Harvey, Alan L

2009-12-15

Peptides have been isolated from several species of sea anemones and shown to block currents through various potassium ion channels, particularly in excitable cells. The toxins can be grouped into four structural classes: type 1 with 35-37 amino acid residues and three disulphide bridges; type 2 with 58-59 residues and three disulphide bridges; type 3 with 41-42 residues and three disulphide bridges; and type 4 with 28 residues and two disulphide bridges. Examples from the first class are BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS (or kaliseptine) from Anemonia sulcata (now A. viridis). These interfere with binding of radiolabelled dendrotoxin to synaptosomal membranes and block currents through channels with various Kv1 subunits and also intermediate conductance K(Ca) channels. Toxins in the second class are homologous to Kunitz-type inhibitors of serine proteases; these toxins include kalicludines (AsKC 1-3) from A. sulcata and SHTXIII from S. haddoni; they block Kv1.2 channels. The third structural group includes BDS-I, BDS-II (from A. sulcata) and APETx 1 (from Anthropleura elegantissima). Their pharmacological specificity differs: BDS-I and -II block currents involving Kv3 subunits, while APETx1 blocks ERG channels. The fourth group comprises the more recently discovered SHTX I and II from S. haddoni. Their channel blocking specificity is not yet known but they displace dendrotoxin binding from synaptosomal membranes. Sea anemones can be predicted to be a continued source of new toxins that will serve as molecular probes of various K(+) channels.

Fisetin as a caloric restriction mimetic protects rat brain against aging induced oxidative stress, apoptosis and neurodegeneration.

Science.gov (United States)

Singh, Sandeep; Singh, Abhishek Kumar; Garg, Geetika; Rizvi, Syed Ibrahim

2018-01-15

In the present study, attempts have been made to evaluate the potential role of fisetin, a caloric restriction mimetic (CRM), for neuroprotection in D-galactose (D-gal) induced accelerated and natural aging models of rat. Fisetin was supplemented (15mg/kg b.w., orally) to young, D-gal induced aged (D-gal 500mg/kg b.w subcutaneously) and naturally aged rats for 6weeks. Standard protocols were employed to measure pro-oxidants, antioxidants and mitochondrial membrane potential in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuronal, aging as well as inflammatory marker genes. We have also evaluated apoptotic cell death and synaptosomal membrane-bound ion transporter activities in brain tissues. Our data demonstrated that fisetin significantly decreased the level of pro-oxidants and increased the level of antioxidants. Furthermore, fisetin also ameliorated mitochondrial membrane depolarization, apoptotic cell death and impairments in the activities of synaptosomal membrane-bound ion transporters in aging rat brain. RT-PCR data revealed that fisetin up-regulated the expression of autophagy genes (Atg-3 and Beclin-1), sirtuin-1 and neuronal markers (NSE and Ngb), and down-regulated the expression of inflammatory (IL-1β and TNF-α) and Sirt-2 genes respectively in aging brain. The present study suggests that fisetin supplementation may provide neuroprotection against aging-induced oxidative stress, apoptotic cell death, neuro-inflammation, and neurodegeneration in rat brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.

Science.gov (United States)

Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A

2013-08-01

Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.

Characterization, localization and function of pertussis toxin-sensitive G proteins in the nervous systems of Aplysia and Loligo

International Nuclear Information System (INIS)

Vogel, S.S.

1989-01-01

The author has characterized pertussis toxin-sensitive G proteins in the nervous systems of the gastropod mollusc Aplysia and the cephalopod Loligo using [ 32 P]ADP-ribosylation and immunoblotting with G protein specific antisera. As in vertebrates, this class of G protein is associated with membranes and enriched in nervous tissue in Aplysia. Analysis of dissected Aplysia ganglia reveal that it is enriched in neuropil, a region containing most of the central nervous system synapses. Because both Aplysia and Loligo synaptosomes are enriched in pertussis toxin-sensitive G proteins, it is likely that they are found in synaptic terminals. Fractionation of Aplysia synaptosomes into membrane and vesicle fractions reveals that, although the majority of G protein is recovered in the plasma membrane fraction, a small proportion is recovered in the vesicle fraction. He shows that G proteins are on intracellular membranes by ADP-ribosylating extruded axoplasm with pertussis toxin. A plausible explanation for vesicular localization of G protein in axoplasm is that G proteins are transported to terminals on vesicles. He has shown, using ligature experiments with Aplysia connectives and temperature block experiments in the giant axon of Loligo, that G proteins move by anterograde fast axonal transport. Injection of pertussis toxin into the identified Aplysia neuron L10 blocks histamine-induced presynaptic inhibition of transmitter release. This suggests that pertussis toxin sensitive G proteins play a role in modulating transmitter release at synaptic terminals. In the giant synapse of Loligo, he presents preliminary data that demonstrates that the activation of G proteins in the presynaptic terminal results in decreased transmitter release

Synaptic vesicle dynamic changes in a model of fragile X.

Science.gov (United States)

Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

2016-01-01

Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

Effect of hypoxia on the incorporation of [2-3H] glycerol and [1-14C[-palmitate into lipids of various brain regions

International Nuclear Information System (INIS)

Alberghina, M.; Giuffrida, A.M.

1981-01-01

The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired. The in vivo incorporation of [2-3H] glycerol and [1-14C] palmitate into lipids of microsomes, mitochondria, myelin, and synaptosomes, purified form cerebral hemispheres, was significantly lower in the hypoxic animals than in the controls. The same effect was observed on the incorporation of labeled precursors into lipids of mitochondria purified from cerebellum and brainstem. In particular, the labeling of th major phospholipids present - ie, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) - in the mitochondria of the three brain regions examined decreased after hypoxic treatment

Temporal characteristics of stress-induced decrease in benzodiazepine reception in C57BL/6 and BALB/c mice.

Science.gov (United States)

Yarkova, M A; Seredenin, S B

2014-10-01

We studied the duration of the drop of specific (3)H-flunitrazepam binding by synaptosomal membranes from the brain of C57Bl/6 and BALB/c mice after open-field and "contact with predator" tests. It was found that reduced benzodiazepine reception in BALB/c mice after open-field test persisted for 1.5 h, but no changes of this parameter were found in C57Bl/6 mice. After contact with predator, the binding capacity of the benzodiazepine site of GABAA receptor was reduced for 8 h in BALB/c mice and for 24 h in C57Bl/6 mice.

Decreased number of interneurons and increased seizures in neuropilin 2 deficient mice: implications for autism and epilepsy.

Science.gov (United States)

Gant, John C; Thibault, Oliver; Blalock, Eric M; Yang, Jun; Bachstetter, Adam; Kotick, James; Schauwecker, Paula E; Hauser, Kurt F; Smith, George M; Mervis, Ron; Li, YanFang; Barnes, Gregory N

2009-04-01

Clinically, perturbations in the semaphorin signaling system have been associated with autism and epilepsy. The semaphorins have been implicated in guidance, migration, differentiation, and synaptic plasticity of neurons. The semaphorin 3F (Sema3F) ligand and its receptor, neuropilin 2 (NPN2) are highly expressed within limbic areas. NPN2 signaling may intimately direct the apposition of presynaptic and postsynaptic locations, facilitating the development and maturity of hippocampal synaptic function. To further understand the role of NPN2 signaling in central nevous system (CNS) plasticity, structural and functional alterations were assessed in NPN2 deficient mice. In NPN2 deficient mice, we measured seizure susceptibility after kainic acid or pentylenetetrazol, neuronal excitability and synaptic throughput in slice preparations, principal and interneuron cell counts with immunocytochemical protocols, synaptosomal protein levels with immunoblots, and dendritic morphology with Golgi-staining. NPN2 deficient mice had shorter seizure latencies, increased vulnerability to seizure-related death, were more likely to develop spontaneous recurrent seizure activity after chemical challenge, and had an increased slope on input/output curves. Principal cell counts were unchanged, but GABA, parvalbumin, and neuropeptide Y interneuron cell counts were significantly reduced. Synaptosomal NPN2 protein levels and total number of GABAergic synapses were decreased in a gene dose-dependent fashion. CA1 pyramidal cells showed reduced dendritic length and complexity, as well as an increased number of dendritic spines. These data suggest the novel hypothesis that the Sema 3F signaling system's role in appropriate placement of subsets of hippocampal interneurons has critical downstream consequences for hippocampal function, resulting in a more seizure susceptible phenotype.

Effects of antibiotics on uptake of calcium into isolated nerve terminals

International Nuclear Information System (INIS)

Atchison, W.D.; Adgate, L.; Beaman, C.M.

1988-01-01

The goal of the present study was to determine whether several antibiotics which are known to block neuromuscular transmission would impair depolarization-dependent and/or -independent uptake of calcium into isolated nerve terminals prepared from forebrain synaptosomes of rats by conventional methods. Antibiotics tested for potential block of Ca++ uptake included the aminoglycosides neomycin and streptomycin, the lincosamide clindamycin, oxytetracycline and polymyxin B. Drugs were applied in concentrations ranging from 1 to 1000 microM. Uptake of 45Ca was determined during depolarization induced by an elevated K+ concentration (77.5 mM). Influxes of 45Ca during 1 and 10 sec of depolarization were used to assess Ca++ uptake via a fast, inactivating path and total uptake, respectively. Uptake of 45Ca during 10 sec of depolarization into synaptosomes which were previously depolarized for 10 sec in the presence of 77.5 mM K+ but in the absence of external Ca++ was used to measure uptake during a slow, noninactivating path. Total depolarization-dependent uptake of 45Ca was depressed significantly by all antibiotics tested except oxytetracycline; however, the various agents differed with respect to their efficacy and potency as blockers of Ca influx. The fast component of uptake, which is thought to be associated with neurotransmitter release, was decreased significantly by all antibiotics. Neomycin and polymyxin were the most potent and most effective at lowering fast phase 45Ca influx; streptomycin, was intermediate in effectiveness whereas clindamycin and oxytetracycline were only effective at concentrations greater than or equal to 100 microM. Only clindamycin, streptomycin and polymyxin B caused significant reductions in the slow phase of 45Ca uptake

Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.

Science.gov (United States)

Baumann, Michael H; Bulling, Simon; Benaderet, Tova S; Saha, Kusumika; Ayestas, Mario A; Partilla, John S; Ali, Syed F; Stockner, Thomas; Rothman, Richard B; Sandtner, Walter; Sitte, Harald H

2014-05-01

Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [(3)H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [(3)H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.

Expression of the Ly-6 family proteins Lynx1 and Ly6H in the rat brain is compartmentalized, cell-type specific, and developmentally regulated

DEFF Research Database (Denmark)

Thomsen, Morten Skøtt; Cinar, Betül; Jensen, Majbrit Myrup

2014-01-01

regarding the distribution and developmental regulation of these proteins in the brain. We use protein cross-linking and synaptosomal fractions to demonstrate that the Ly-6 proteins Lynx1 and Ly6H are membrane-bound proteins in the brain, which are present on the cell surface and localize to synaptic...... demonstrate that Lynx1 and Ly6H are expressed in cultured neurons, but not cultured micro- or astroglial cultures. In addition, Lynx1, but not Ly6H was detected in the CSF. Finally, we show that the Ly-6 proteins Lynx1, Lynx2, Ly6H, and PSCA, display distinct expression patterns during postnatal development...

The human immunodeficiency virus-1 protein Tat and its discrete fragments evoke selective release of acetylcholine from human and rat cerebrocortical terminals through species-specific mechanisms.

Science.gov (United States)

Feligioni, Marco; Raiteri, Luca; Pattarini, Roberto; Grilli, Massimo; Bruzzone, Santina; Cavazzani, Paolo; Raiteri, Maurizio; Pittaluga, Anna

2003-07-30

The effect of the human immunodeficiency virus-1 protein Tat was investigated on neurotransmitter release from human and rat cortical nerve endings. Tat failed to affect the release of several neurotransmitters, such as glutamate, GABA, norepinephrine, and others, but it evoked the release of [3H]ACh via increase of cytosolic [Ca2+]. In human nerve terminals, the Tat effect partly depends on Ca2+ entry through voltage-sensitive Ca2+ channels, because Cd2+ halved the Tat-evoked release. Activation of group I metabotropic glutamate receptors (mGluR) and mobilization of Ca2+ from IP3-sensitive intraterminal stores are also involved, because the Tat effect was prevented by mGluR antagonists 2-methyl-6-(phenylethynyl)pyridine hydrochloride and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester and by the IP3 receptor antagonists heparin and xestospongin C. Furthermore, the group I selective mGlu agonist (RS)-3,5-dihydroxyphenylglycine enhanced [3H]ACh release. In rat nerve terminals, the Tat-evoked release neither depends on external Ca2+ ions entry nor on IP3-mediated mechanisms. Tat seems to cause mobilization of Ca2+ from ryanodine-sensitive internal stores because its effect was prevented by both 8-bromo-cyclic adenosine diphosphate-ribose and dantrolene. The Tat-evoked release from human synaptosomes was mimicked by the peptide sequences Tat 32-62, Tat 49-86, and Tat 41-60. In contrast, the Tat 49-86 and Tat 61-80 fragments, but not the Tat 32-62 fragment, were active in rat synaptosomes. In conclusion, Tat elicits Ca2+-dependent [3H]ACh release by species-specific intraterminal mechanisms by binding via discrete amino acid sequences to different receptive sites on human and rat cholinergic terminals.

Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants.

Science.gov (United States)

Gross, Joshua D; Kaski, Shane W; Schroer, Adam B; Wix, Kimberley A; Siderovski, David P; Setola, Vincent

2018-02-01

Regulators of G protein signaling are proteins that accelerate the termination of effector stimulation after G protein-coupled receptor activation. Many regulators of G protein signaling proteins are highly expressed in the brain and therefore considered potential drug discovery targets for central nervous system pathologies; for example, here we show that RGS12 is highly expressed in microdissected mouse ventral striatum. Given a role for the ventral striatum in psychostimulant-induced locomotor activity, we tested whether Rgs12 genetic ablation affected behavioral responses to amphetamine and cocaine. RGS12 loss significantly decreased hyperlocomotion to lower doses of both amphetamine and cocaine; however, other outcomes of administration (sensitization and conditioned place preference) were unaffected, suggesting that RGS12 does not function in support of the rewarding properties of these psychostimulants. To test whether observed response changes upon RGS12 loss were caused by changes to dopamine transporter expression and/or function, we prepared crude membranes from the brains of wild-type and RGS12-null mice and measured dopamine transporter-selective [ 3 H]WIN 35428 binding, revealing an increase in dopamine transporter levels in the ventral-but not dorsal-striatum of RGS12-null mice. To address dopamine transporter function, we prepared striatal synaptosomes and measured [ 3 H]dopamine uptake. Consistent with increased [ 3 H]WIN 35428 binding, dopamine transporter-specific [ 3 H]dopamine uptake in RGS12-null ventral striatal synaptosomes was found to be increased. Decreased amphetamine-induced locomotor activity and increased [ 3 H]WIN 35428 binding were recapitulated with an independent RGS12-null mouse strain. Thus, we propose that RGS12 regulates dopamine transporter expression and function in the ventral striatum, affecting amphetamine- and cocaine-induced increases in dopamine levels that specifically elicit acute hyperlocomotor responses.

Purification of a neurotoxin from the venom of the 'armed spider' and synthesis of a radioactive probe

International Nuclear Information System (INIS)

Santos, Raquel Gouvea; Renterghem, Catherine Van; Mori, Yasuo; Martin-Moutot, Nicole; Mansuelle, Pascal; Sampieri, Francois; Seagar, Michel; Lima, Maria Elena de

2002-01-01

The venom of the 'armed spider', a South American spider from the genus Phoneutria contains several toxins that exert important biological effects. ω-Phonetoxin IIA (ω-PtxIIA) is a potent neurotoxin from this venom evoking flaccid paralysis and death after intracerebro-ventricular injection in mouse. This toxin blocks HVA calcium channels. Calcium channels have been shown to be important targets in neurological pathophysiology like ataxia and migraine. In order to shed more light on the mechanism of action of w-PtxIIA, we have purified this toxin to synthesize a radioactive probe. Crude venom was fractionated in three steps of reversed-phase liquid chromatography (RP-HPLC). Spectrometric analysis of the purified fraction (causing flaccid paralysis) was recorded on a MALDI-TOF Perseptive Voyager Elite spectrometer and a 8363 kDa peptide was detected. After amino acid sequencing the purity of the peptide was confirmed and it was identified as ω-PtxIIA. ω-PtxIIA was radiolabeled with 125 I using the lactoperoxidase as a oxidizing agent. The stability of the probe synthesized was verified by the binding studies on rat brain synaptosomal membranes. The specific and saturable binding of the 125 I-ω-PtxIIA to the membranes indicate that the iodine introduced in the toxin molecule did not interfere with its activity. Native ω-PtxIIA competed with the 125 I-ω-PtxIIA bound to the specific sites on synaptosomes (IC 50 = 0.54 n M). In this paper we described a new and simpler purification protocol of the ω-PtxIIA and synthesized a probe that proved to be useful to study the mechanism of action of 125 I-ω-PtxIIA. Selective toxins for calcium channel are very useful tools to the study of some neuronal pathophysiology. (author)

Purification of a neurotoxin from the venom of the 'armed spider' and synthesis of a radioactive probe

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Santos, Raquel Gouvea [INSERM, Marseille (France). Lab. Canaux Ioniques]|[Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Renterghem, Catherine Van; Mori, Yasuo; Martin-Moutot, Nicole; Mansuelle, Pascal; Sampieri, Francois; Seagar, Michel [INSERM, Marseille (France). Lab. Canaux Ioniques; Cordeiro, Marta Nascimento; Diniz, Carlos Ribeiro [FUNED - Fundacao Ezequiel Dias, Belo Horizonte, MG (Brazil); Lima, Maria Elena de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Lab. de Venenos e Toxinas Animais

2002-07-01

The venom of the 'armed spider', a South American spider from the genus Phoneutria contains several toxins that exert important biological effects. {omega}-Phonetoxin IIA ({omega}-PtxIIA) is a potent neurotoxin from this venom evoking flaccid paralysis and death after intracerebro-ventricular injection in mouse. This toxin blocks HVA calcium channels. Calcium channels have been shown to be important targets in neurological pathophysiology like ataxia and migraine. In order to shed more light on the mechanism of action of w-PtxIIA, we have purified this toxin to synthesize a radioactive probe. Crude venom was fractionated in three steps of reversed-phase liquid chromatography (RP-HPLC). Spectrometric analysis of the purified fraction (causing flaccid paralysis) was recorded on a MALDI-TOF Perseptive Voyager Elite spectrometer and a 8363 kDa peptide was detected. After amino acid sequencing the purity of the peptide was confirmed and it was identified as {omega}-PtxIIA. {omega}-PtxIIA was radiolabeled with {sup 125} I using the lactoperoxidase as a oxidizing agent. The stability of the probe synthesized was verified by the binding studies on rat brain synaptosomal membranes. The specific and saturable binding of the {sup 125} I-{omega}-PtxIIA to the membranes indicate that the iodine introduced in the toxin molecule did not interfere with its activity. Native {omega}-PtxIIA competed with the {sup 125} I-{omega}-PtxIIA bound to the specific sites on synaptosomes (IC{sub 50}= 0.54 n M). In this paper we described a new and simpler purification protocol of the {omega}-PtxIIA and synthesized a probe that proved to be useful to study the mechanism of action of {sup 125} I-{omega}-PtxIIA. Selective toxins for calcium channel are very useful tools to the study of some neuronal pathophysiology. (author)

Regulation of angiotensin II-induced neuromodulation by MARCKS in brain neurons.

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Lu, D; Yang, H; Lenox, R H; Raizada, M K

1998-07-13

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)beta subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCbeta subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II-stimulated accumulation of TH and DbetaH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DbetaH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148-165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DbetaH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCbeta and its redistribution from varicosities to neurites is important in Ang II-induced synaptic accumulation of TH, DbetaH, and NE. They suggest that a coordinated stimulation of transcription of TH, DbetaH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCbeta-MARCKS pathway are key in persistent

Synaptic protein changes after a chronic period of sensorimotor perturbation in adult rats: a potential role of phosphorylation/O-GlcNAcylation interplay.

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Fourneau, Julie; Canu, Marie-Hélène; Cieniewski-Bernard, Caroline; Bastide, Bruno; Dupont, Erwan

2018-05-28

In human, a chronic sensorimotor perturbation (SMP) through prolonged body immobilization alters motor task performance through a combination of peripheral and central factors. Studies performed on a rat model of SMP have shown biomolecular changes and a reorganization of sensorimotor cortex through events such as morphological modifications of dendritic spines (number, length, functionality). However, underlying mechanisms are still unclear. It is well known that phosphorylation regulates a wide field of synaptic activity leading to neuroplasticity. Another post-translational modification that interplays with phosphorylation is O-GlcNAcylation. This atypical glycosylation, reversible and dynamic, is involved in essential cellular and physiological processes such as synaptic activity, neuronal morphogenesis, learning and memory. We examined potential roles of phosphorylation/O-GlcNAcylation interplay in synaptic plasticity within rat sensorimotor cortex after a SMP period. For this purpose, sensorimotor cortex synaptosomes were separated by sucrose gradient, in order to isolate a subcellular compartment enriched in proteins involved in synaptic functions. A period of SMP induced plastic changes at the pre- and postsynaptic levels, characterized by a reduction of phosphorylation (synapsin1, AMPAR GluA2) and expression (synaptophysin, PSD-95, AMPAR GluA2) of synaptic proteins, as well as a decrease in MAPK/ERK42 activation. Expression levels of OGT/OGA enzymes was unchanged but we observed a specific reduction of synapsin1 O-GlcNAcylation in sensorimotor cortex synaptosomes. The synergistic regulation of synapsin1 phosphorylation/O-GlcNAcylation could affect presynaptic neurotransmitter release. Associated with other pre- and postsynaptic changes, synaptic efficacy could be impaired in somatosensory cortex of SMP rat. Thus, synapsin1 O-GlcNAcylation/phosphorylation interplay also appears to be involved in this synaptic plasticity by finely regulating neural activity

Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein

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Johns Roger A

2008-10-01

Full Text Available Abstract Postsynaptic density (PSD-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence.

Sex-specific differences in mitochondria biogenesis, morphology, respiratory function, and ROS homeostasis in young mouse heart and brain.

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Khalifa, Abdel Rahman M; Abdel-Rahman, Engy A; Mahmoud, Ali M; Ali, Mohamed H; Noureldin, Maha; Saber, Saber H; Mohsen, Mahmoud; Ali, Sameh S

2017-03-01

Sex-specific differences in mitochondrial function and free radical homeostasis are reported in the context of aging but not well-established in pathogeneses occurring early in life. Here, we examine if sex disparity in mitochondria function, morphology, and redox status starts early and hence can be implicated in sexual dimorphism in cardiac as well as neurological disorders prevalent at young age. Although mitochondrial activity in the heart did not significantly vary between sexes, female brain exhibited enhanced respiration and higher reserve capacity. This was associated with lower H 2 O 2 production in female cardiac and brain tissues. Using transmission electron microscopy, we found that the number of female cardiac mitochondria is moderately greater (117 ± 3%, P  = 0.049, N  = 4) than male's, which increased significantly for cortical mitochondria (134 ± 4%, P  = 0.001, N  = 4). However, male's cardiac mitochondria exhibited fragmented, circular, and smaller mitochondria relative to female's mitochondria, while no morphologic sex-dependent differences were observed in cortical mitochondria. No sex differences were detected in Nox2 and Nox4 proteins or O 2 -consuming/H 2 O 2 -producing activities in brain homogenate or synaptosomes. However, a strong trend of increased EPR-detected NOX superoxide in male synaptosomes hinted at higher superoxide dismutase activity in female brains, which was confirmed by two independent protocols. We also provide direct evidence that respiring mitochondria generally produce an order-of-magnitude lower reactive oxygen species (ROS) proportions than currently estimated. Our results indicate that sex differences in mitochondrial biogenesis, bioenergetics, and morphology may start at young age and that sex-dependent SOD capacity may be responsible for differences in ROS homeostasis in heart and brain. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological

Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants.

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Laura Musazzi

2010-01-01

Full Text Available Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release.Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated, and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486. On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats. Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability.Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress

Methamphetamine protects against MPTP neurotoxicity in C57BL mice.

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Sziráki, I; Kardos, V; Patthy, M; Pátfalusi, M; Budai, G

1994-01-14

Methamphetamine (5 mg/kg) administered 30 min prior to each injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (3 x 30 mg/kg, at 24 h intervals) prevents the reduction of striatal levels of dopamine and its metabolites in C57BL mice. Methamphetamine and amphetamine inhibit the uptake of 1-methyl-4-phenylpyridinium (MPP+) by striatal synaptosomes of rats. A 30-min post-treatment with methamphetamine or amphetamine also prevents the MPTP-induced dopamine depletion, suggesting that their protective effect is related to the blockade of MPP+ uptake into dopaminergic neurons. Since amphetamine and methamphetamine are themselves neurotoxins at higher doses, this work demonstrated the protection against the actions of one neurotoxin by the administration of another.

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

International Nuclear Information System (INIS)

Crofton, K.M.; Reiter, L.W.; Mailman, R.B.

1987-01-01

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on 3 H-flunitrazepam (FLU), 3 H-muscimol (MUS), and ( 35 S-t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with 3 H-FLU and 3 H-MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of ( 35 S-TBPS at concentrations as high as 50 pM. Type II pyrethroids inhibited ( 35 S-TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 pM. The data presented suggest that the interaction of Type II pyrethroids with the GABA receptor-ionophore complex is restricted to a site near the TBPS/picrotoxinin binding site

Interactions of alaproclate, a selective 5HT-uptake blocker, with muscarinic receptors: in vivo and in vitro studies

International Nuclear Information System (INIS)

Danielsson, E.; Bartfai, T.; Nordstrom, O.; Ogren, S.O.; Unden, A.

1986-01-01

Cholinergic mechanisms play an important role in higher brain functions such as learning or memory. It is hoped that drugs which improve cholinergic transmission would be therapeutically effective in senile dementia. The results of biochemical studies on the interaction of alaproclate with the muscarinic system are summarized. Tritium-4-N-methylpiperidinylbenzilate and methyl tritium-choline chloride were used in the studies. Salivation and hypothermia were studied in adult male mice following injection of alaproclate preceding the injection of oxotremorine by 30 min. Salivation, tremor and hypothermia caused by oxotremorine were found to be centrally mediated muscarinic responses. Tritium-ACh release was studied from synaptosomes. The metabolites of alaproclate, alanine and 2(4 chlorophenyl) 1-1 dimethylethanol, do not produce tremor

5-(2-Aminopropyl)benzofuran and phenazepam demonstrate the possibility of dependence by increasing dopamine levels in the brain.

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Cha, Hye Jin; Lee, Kwang-Wook; Eom, Jang-Hyeon; Kim, Young-Hoon; Shin, Jisoon; Yun, Jaesuk; Han, Kyoungmoon; Kim, Hyung Soo

2016-10-01

Although 5-(2-aminopropyl)benzofuran (5-APB) and 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam) are being used as recreational drugs, research on their dependence liability or mechanisms of action is lacking. The present study aimed to evaluate the behavioral effects and dependence liability of these drugs using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques were used to assess the substance-induced alterations in synaptosome-released dopamine. While both of the tested substances elicited increases in conditioned place preference and dopamine, neither of them facilitated self-administration, suggesting that 5-APB and phenazepam have rewarding effects, rather than reinforcing effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

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Bossi, Simone; Musante, Ilaria; Bonfiglio, Tommaso; Bonifacino, Tiziana; Emionite, Laura; Cerminara, Maria; Cervetto, Chiara; Marcoli, Manuela; Bonanno, Giambattista; Ravazzolo, Roberto; Pittaluga, Anna; Puliti, Aldamaria

2018-01-01

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1 crv4/crv4 ) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1 crv4 and Grm5 ko mice to generate double mutants (Grm1 crv4/crv4 Grm5 ko/ko ) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1 crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1 crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1 crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1 crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Hydrogen Sulfide Ameliorates Homocysteine-Induced Alzheimer's Disease-Like Pathology, Blood-Brain Barrier Disruption, and Synaptic Disorder.

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Kamat, Pradip K; Kyles, Philip; Kalani, Anuradha; Tyagi, Neetu

2016-05-01

Elevated plasma total homocysteine (Hcy) level is associated with an increased risk of Alzheimer's disease (AD). During transsulfuration pathways, Hcy is metabolized into hydrogen sulfide (H2S), which is a synaptic modulator, as well as a neuro-protective agent. However, the role of hydrogen sulfide, as well as N-methyl-D-aspartate receptor (NMDAR) activation, in hyperhomocysteinemia (HHcy) induced blood-brain barrier (BBB) disruption and synaptic dysfunction, leading to AD pathology is not clear. Therefore, we hypothesized that the inhibition of neuronal NMDA-R by H2S and MK801 mitigate the Hcy-induced BBB disruption and synapse dysfunction, in part by decreasing neuronal matrix degradation. Hcy intracerebral (IC) treatment significantly impaired cerebral blood flow (CBF), and cerebral circulation and memory function. Hcy treatment also decreases the expression of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) in the brain along with increased expression of NMDA-R (NR1) and synaptosomal Ca(2+) indicating excitotoxicity. Additionally, we found that Hcy treatment increased protein and mRNA expression of intracellular adhesion molecule 1 (ICAM-1), matrix metalloproteinase (MMP)-2, and MMP-9 and also increased MMP-2 and MMP-9 activity in the brain. The increased expression of ICAM-1, glial fibrillary acidic protein (GFAP), and the decreased expression of vascular endothelial (VE)-cadherin and claudin-5 indicates BBB disruption and vascular inflammation. Moreover, we also found decreased expression of microtubule-associated protein 2 (MAP-2), postsynaptic density protein 95 (PSD-95), synapse-associated protein 97 (SAP-97), synaptosomal-associated protein 25 (SNAP-25), synaptophysin, and brain-derived neurotrophic factor (BDNF) showing synapse dysfunction in the hippocampus. Furthermore, NaHS and MK801 treatment ameliorates BBB disruption, CBF, and synapse functions in the mice brain. These results demonstrate a neuro-protective effect of H2S over Hcy

Synaptic dysbindin-1 reductions in schizophrenia occur in an isoform-specific manner indicating their subsynaptic location.

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Konrad Talbot

Full Text Available BACKGROUND: An increasing number of studies report associations between variation in DTNBP1, a top candidate gene in schizophrenia, and both the clinical symptoms of the disorder and its cognitive deficits. DTNBP1 encodes dysbindin-1, reduced levels of which have been found in synaptic fields of schizophrenia cases. This study determined whether such synaptic reductions are isoform-specific. METHODOLOGY/PRINCIPAL FINDINGS: Using Western blotting of tissue fractions, we first determined the synaptic localization of the three major dysbindin-1 isoforms (A, B, and C. All three were concentrated in synaptosomes of multiple brain areas, including auditory association cortices in the posterior half of the superior temporal gyrus (pSTG and the hippocampal formation (HF. Tests on the subsynaptic tissue fractions revealed that each isoform is predominantly, if not exclusively, associated with synaptic vesicles (dysbindin-1B or with postsynaptic densities (dysbindin-1A and -1C. Using Western blotting on pSTG (n = 15 and HF (n = 15 synaptosomal fractions from schizophrenia cases and their matched controls, we discovered that synaptic dysbindin-1 is reduced in an isoform-specific manner in schizophrenia without changes in levels of synaptophysin or PSD-95. In pSTG, about 92% of the schizophrenia cases displayed synaptic dysbindin-1A reductions averaging 48% (p = 0.0007 without alterations in other dysbindin-1 isoforms. In the HF, by contrast, schizophrenia cases displayed normal levels of synaptic dysbindin-1A, but 67% showed synaptic reductions in dysbindin-1B averaging 33% (p = 0.0256, while 80% showed synaptic reductions in dysbindin-1C averaging 35% (p = 0.0171. CONCLUSIONS/SIGNIFICANCE: Given the distinctive subsynaptic localization of dysbindin-1A, -1B, and -1C across brain regions, the observed pSTG reductions in dysbindin-1A are postsynaptic and may promote dendritic spine loss with consequent disruption of auditory information

Taurine effects on 45Ca2+ transport in retinal subcellular fractions

International Nuclear Information System (INIS)

Pasantes-Morales, H.; Ademe, R.M.; Lopez-Colome, A.M.

1979-01-01

The effect of taurine on 45 Ca 2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45 Ca 2+ uptake was observed in retinal fractions incubated for 1-5 min in a Krebs-bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45 Ca 2+ uptake; in the crude synaptosomal fraction, taurine reduced 45 Ca 2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45 Ca 2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role of taurine in the regulation of calcium fluxes in the retina. (Auth.)

Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

International Nuclear Information System (INIS)

Pollack, A.E.; Wooten, G.F.

1987-01-01

The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

Cerebroprotective activity of U-50488H: Relationship to interactions with excitatory amino acids and calcium

International Nuclear Information System (INIS)

Camacho Ochoa, M.

1987-01-01

The mechanism underlying the anticonvulsant and cerebroprotective activity of U-50488H was evaluated using 45 Ca ++ uptake in rat Ficoll purified synaptosomes, ( 3 H)-2-deoxyglucose uptake in selected mouse brain regions, ( 3 H)kainic acid binding to mouse forebrain synaptic membranes and incidence of KA-induced lesions in the CA3 region of the mouse hippocampus. U-50488H causes reduction in K + -evoked 45 Ca ++ uptake. These effects are comparable to those of the calcium channel blockers verapamil and nifedipine and seem to be related to calcium dependent mechanisms. Changes in saturability, specificity and dissociation constant values of kainic acid receptor binding were demonstrated in the presence of U-50488H at concentrations similar to those used in 45 Ca ++ uptake studies and in the presence of calcium and chloride ions

Characterization of [125I]omega-conotoxin binding to brain N calcium channels and (-)[3H] desmethoxyverapamil binding to novel calcium channels in osteoblast-like osteosarcoma cells

International Nuclear Information System (INIS)

Wagner, J.A.

1987-01-01

This dissertation provides molecular evidence for a diversity of Ca 2+ channels in neuronal and non-neuronal tissues. First, I demonstrated specific, reversible, saturable binding sites for omega [ 125 I]conotoxin GVIA (omega[ 125 I]CTX) in rat brain and rabbit sympathetic ganglion. Omega [ 125 I]CTX binding has a unique pharmacology, ion selectivity, and anatomical distribution in rat brain. Omega [ 125 I]CTX binding was solubilized, retaining an appropriate pharmacology and ion selectivity. Omega[ 125 I]CTX binding may be associated with a Ca 2+ channel because the K/sub D/ of omega [ 125 I]CTX is similar to the IC 50 of inhibition of depolarization-induced 45 Ca 2+ flux into rat brain synaptosomes. Specific (-)[ 3 H]desmethoxyverapamil ((-)[ 3 H]DMV) binding sites were demonstrated on osteoblast-like osteosarcoma cell membranes

Individually reared rats

International Nuclear Information System (INIS)

Kraeuchi, K.; Gentsch, C.; Feer, H.

1981-01-01

The influence of social isolation in rats on postsynaptic alpha 1 - and beta-adrenergic receptors, on the cAMP generating system and on the presynaptic uptake mechanism in the central noradrenergic system was examined in different brain regions. Rearing rats in isolation from the 19th day of life for 12 weeks leads in all regions to a general tendency for a reduction in 3 H-DHA binding, to an enhanced 3 H-WB4101 binding and to a decreased responsiveness of the noradrenaline sensitive cAMP generating system. These changes reach significance only in the pons-medulla-thallamusregion. Isolated rats showed an increased synaptosomal uptake of noradrenaline, most pronounced and significant in the hypothalamus. Our data provide further support for a disturbance in central noradrenergic function in isolated rats. (author)

Effect of runway training on rat brain tyrosine hydroxylase: differential effect of continuous and partial reinforcement schedules.

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Boarder, M R; Feldon, J; Gray, J A; Fillenz, M

1979-12-01

Previous experiments have implicated ascending noradrenergic systems in the development of the behavioural responses to different patterns of reward. In this report food deprived male Sprague--Dawley rats were trained to run a straight alley for good reward on a continuous reinforcement (CRF) or a partial reinforcement (PRF) schedule. Tyrosine hydroxylase measured in a partially solubilized preparation from hippocampus and hypothalamus at the end of acquisition was not different from controls, indicating that enzyme induction does not occur during either training schedules. However, hippocampal synaptosomal tyrosine hydroxylation rates from the CRF group was significantly higher than from either the PRF group or the handled controls. This indicates that at the end of the acquisition schedule the noradrenergic projection to hippocampus was more active in the CRF group than with the PRF group or the handled control.

Beta amyloid differently modulate nicotinic and muscarinic receptor subtypes which regulate in vitro and in vivo the release of glycine in the rat hippocampus

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Stefania eZappettini

2012-07-01

Full Text Available Using both in vitro (hippocampal synaptosomes in superfusion and in vivo (microdialysis approaches we investigated whether and to what extent β amyloid peptide 1-40 (Aβ 1-40 interferes with the cholinergic modulation of the release of glycine (GLY in the rat hippocampus. The nicotine-evoked overflow of endogenous GLY in hippocampal synaptosomes in superfusion was significantly inhibited by Aβ 1-40 (10 nM while increasing the concentration to 100 nM the inhibitory effect did not further increase. Both the Choline (Ch (α7 agonist; 1 mM and the 5-Iodo-A-85380 dihydrochloride (5IA85380, α4β2 agonist; 10 nM-evoked GLY overflow were inhibited by Aβ1-40 at 100 nM but not at 10nM concentrations. The KCl evoked [3H]GLY and [3H]Acetylcholine (ACh overflow were strongly inhibited in presence of oxotremorine; however this inhibitory muscarinic effect was not affected by Aβ1-40. The effects of Aβ1-40 on the administration of nicotine, veratridine, 5IA85380 and PHA 543613 hydrochloride (PHA543613 (a selective agonist of α7 subtypes on hippocampal endogenous GLY release in vivo were also studied. Aβ 1-40 significantly reduced (at 10 μM but not at 1 μM the nicotine evoked in vivo release of GLY. Aβ 1-40 (at 10 μM but not at 1 μM significantly inhibited the PHA543613 (1 mM-elicited GLY overflow while was ineffective on the GLY overflow evoked by 5IA85380 (1 mM. Aβ 40-1 (10 μM did not produce any inhibitory effect on nicotine evoked GLY overflow both in the in vitro and in vivo experiments. Our results indicate that a the cholinergic modulation of the release of GLY occurs by the activation of both α7 and α4β2 nicotinic ACh receptors (nAChRs as well as by the activation of inhibitory muscarinic ACh receptors (mAChRs and b Aβ 1-40 can modulate cholinergic evoked GLY release exclusively through the interaction with α7 and the α4β2 nAChR nicotinic receptors but not through mAChR subtypes.

Ciproxifan, a histamine H{sub 3} receptor antagonist and inverse agonist, presynaptically inhibits glutamate release in rat hippocampus

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Lu, Cheng-Wei; Lin, Tzu-Yu [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan (China); Chang, Chia-Ying [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Department of Chemistry, Fu Jen Catholic University, No. 510, Chung-Cheng Road, Hsin-Chuang District, New Taipei City 24205, Taiwan (China); Huang, Shu-Kuei [Department of Anesthesiology, Far-Eastern Memorial Hospital, Pan-Chiao District, New Taipei City 22060, Taiwan (China); Wang, Su-Jane, E-mail: med0003@mail.fju.edu.tw [School of Medicine, Fu Jen Catholic University, No. 510, Chung-Cheng Rd., Hsin-Chuang, New Taipei 24205, Taiwan (China); Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan City, Taiwan (China)

2017-03-15

Ciproxifan is an H{sub 3} receptor antagonist and inverse agonist with antipsychotic effects in several preclinical models; its effect on glutamate release has been investigated in the rat hippocampus. In a synaptosomal preparation, ciproxifan reduced 4-aminopyridine (4-AP)-evoked Ca{sup 2+}-dependent glutamate release and cytosolic Ca{sup 2+} concentration elevation but did not affect the membrane potential. The inhibitory effect of ciproxifan on 4-AP-evoked glutamate release was prevented by the Gi/Go-protein inhibitor pertussis toxin and Ca{sub v}2.2 (N-type) and Ca{sub v}2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was not affected by the intracellular Ca{sup 2+}-release inhibitors dantrolene and CGP37157. Furthermore, the phospholipase A{sub 2} (PLA{sub 2}) inhibitor OBAA, prostaglandin E{sub 2} (PGE{sub 2}), PGE2 subtype 2 (EP{sub 2}) receptor antagonist PF04418948, and extracellular signal-regulated kinase (ERK) inhibitor FR180204 eliminated the inhibitory effect of ciproxifan on glutamate release. Ciproxifan reduced the 4-AP-evoked phosphorylation of ERK and synapsin I, a presynaptic target of ERK. The ciproxifan-mediated inhibition of glutamate release was prevented in synaptosomes from synapsin I-deficient mice. Moreover, ciproxifan reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that ciproxifan, acting through the blockade of Gi/Go protein-coupled H{sub 3} receptors present on hippocampal nerve terminals, reduces voltage-dependent Ca{sup 2+} entry by diminishing PLA{sub 2}/PGE{sub 2}/EP{sub 2} receptor pathway, which subsequently suppresses the ERK/synapsin I cascade to decrease the evoked glutamate release. - Highlights: • Ciproxifan presynaptically reduces glutamate release in the hippocampus in vitro. • Decrease in voltage-dependent Ca{sup 2+} influx is involved. • A role for the PLA{sub 2}/PGE{sub 2}/EP{sub 2} pathway in the action of

Four weeks' inhalation exposure of Long Evans rats to 4-tert-butyltoluene: Effect on evoked potentials, behaviour and brain neurochemistry

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Lam, Henrik Rye; Ladefoged, Ole; Østergaard, Grete

2000-01-01

Long-lasting central nervous system (CNS) neurotoxicity of 4-tert-butyltoluene (TBT) has been investigated using electrophysiology, behaviour, and neurochemistry in Long Evans rats exposed by inhalation to 0, 20, or 40 p.p.m. TBT 6 hr/day, 7 days/week for 4 weeks. Flash evoked potentials...... and somatosensory evoked potentials were not affected by TBT In Auditory Brain Stem Response there was no shift in hearing threshold, but the amplitude of the first wave was increased in both exposed groups at high stimulus levels. Three to four months after the end of exposure, behavioural studies in Morris water...... maze and eight-arm maze failed to demonstrate any TBT induced effects. Exposure was followed by a 5 months exposure-free period prior to gross regional and subcellular (synaptosomal) neurochemical investigations of the brain. TBT reduced the NA concentration in whole brain minus cerebellum...

The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes

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Boström, Pontus; Andersson, Linda; Vind, Birgitte

2010-01-01

/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results......OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known...... association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control...

Oridonin Attenuates Synaptic Loss and Cognitive Deficits in an Aβ1-42-Induced Mouse Model of Alzheimer's Disease.

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Sulei Wang

Full Text Available Synaptic loss induced by beta-amyloid (Aβ plays a critical role in the pathophysiology of Alzheimer's disease (AD, but the mechanisms underlying this process remain unknown. In this study, we found that oridonin (Ori rescued synaptic loss induced by Aβ1-42 in vivo and in vitro and attenuated the alterations in dendritic structure and spine density observed in the hippocampus of AD mice. In addition, Ori increased the expression of PSD-95 and synaptophysin and promoted mitochondrial activity in the synaptosomes of AD mice. Ori also activated the BDNF/TrkB/CREB signaling pathway in the hippocampus of AD mice. Furthermore, in the Morris water maze test, Ori reduced latency and searching distance and increased the number of platform crosses in AD mice. These data suggest that Ori might prevent synaptic loss and improve behavioral symptoms in Aβ1-42-induced AD mice.

2-Amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid

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Johansen, Tommy N; Janin, Yves L; Nielsen, Birgitte

2002-01-01

In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S......)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA...... a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific...

Synaptosomal-associated protein 25 (Snap-25) gene polymorphism frequency in fibromyalgia syndrome and relationship with clinical symptoms.

Science.gov (United States)

Balkarli, Ayse; Sengül, Cem; Tepeli, Emre; Balkarli, Huseyin; Cobankara, Veli

2014-05-31

SNAP-25 protein is contributory to plasma membrane and synaptic vesicle fusions that are critical points in neurotransmission. SNAP-25 gene is associated with behavioral symptoms, personality and psychological disorders. In addition, SNAP-25 protein can be related to different neurotransmitter functions due to its association with vesicle membrane transition and fusion. This is important because neurologic, cognitive, and psychologic disorders in fibromyalgia syndrome (FMS) can be related to this function. This relationship may be enlightening for etiopathogenesis of FMS and treatment approaches. We aimed to study a SNAP-25 gene polymorphism, which is related to many psychiatric diseases, and FMS association in this prospective study. We included 71 patients who were diagnosed according to new criteria and 57 matched healthy women in this study. Both groups were evaluated regarding age, height, weight, BMI, education level, marital and occupational status. A new diagnosis of FMS was made from criteria scoring, SF-36, Beck depression scale, and VAS that were applied to the patient group. SNAP-25 gene polymorphism and disease activity score correlations were compared. Mean age was 38±5,196 and 38.12±4.939 in patient and control groups, respectively (p=0.542). No significant difference was found between groups regarding age, height, weight, BMI, education level, marital or occupational status (p > 0.05). Ddel T/C genotype was significantly higher in the patient group (p = 0.009). MnlI gene polymorphism did not show a correlation with any score whereas a significant correlation was found between Ddel T/C genotype and Beck depression scale and VAS score (p < 0.05). FMS etiopathogenesis is not clearly known. Numerous neurologic, cognitive and psychological disorders were found during studies looking at cause. Our study showed increased SNAP-25 Ddel T/C genotype in FMS patients compared to the control group, which is related to behavioral symptoms, personality and psychological disorders in FMS patients.

Developmental and Diurnal Expression of the Synaptosomal-Associated Protein 25 (Snap25) in the Rat Pineal Gland

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Karlsen, Anna S; Rath, Martin Fredensborg; Rohde, Kristian

2013-01-01

pineal gland but the biological importance of this is unknown. In this study, we demonstrate a high expression of mRNA encoding Snap25 in all parts of the rat pineal complex, the superficial-, and deep-pineal gland, as well as in the pineal stalk. Snap25 showed a low pineal expression during embryonic...... stages with a strong increase in expression levels just after birth. The expression showed no day/night variations. Neither removal of the sympathetic input to the pineal gland by superior cervical ganglionectomy nor bilateral decentralization of the superior cervical ganglia significantly affected...

Release of [3H]-monoamines from superfused rat striatal slices by methylenedioxymethamphetamine (MDMA)

International Nuclear Information System (INIS)

Levin, J.A.; Schmidt, C.J.; Lovenberg, W.

1986-01-01

MDMA is a phenylisopropylamine which is reported to have unique behavioral effects in man. Because of its structural similarities to the amphetamines the authors have compared the effects of MDMA and two related amphetamines on the spontaneous release of tritiated dopamine (DA) and serotonin (5HT) from superfused rat striatal slices. At concentrations of 10 -7 - 10 -5 M MDMA and the serotonergic neurotoxin, p-chloroamphetamine, were equipotent releasers of [ 3 H]5HT being approximately 10x more potent than methamphetamine. However, methamphetamine was the more potent releaser of [ 3 H]DA by a factor of approximately 10x. MDMA-induced release of both [ 5 H]5HT and [ 3 H]DA was Ca 2+ -independent and inhibited by selective monoamine uptake blockers suggesting a carrier-dependent release mechanism. Synaptosomal uptake experiments with (+)[ 3 H]MDMA indicated no specific uptake of the drug further suggesting the effect of uptake blockers may be to inhibit the carrier-mediated export of amines displaced by MDMA

Amphetamine-metabolites of deprenyl involved in protection against neurotoxicity induced by MPTP and 2'-methyl-MPTP.

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Sziráki, I; Kardos, V; Patthy, M; Pátfalusi, M; Gaál, J; Solti, M; Kollár, E; Singer, J

1994-01-01

The ability of 1-deprenyl to protect against the parkinsonian effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been attributed to the inhibition of conversion of MPTP to MPP+ (1-methyl-4-phenylpyridinium) catalyzed by MAO-B. We report here that deprenyl-treatment in mice has an additional neuroprotective element associated with the rapid metabolization of 1-deprenyl to 1-methamphetamine and 1-amphetamine. 1-Methamphetamine and 1-amphetamine inhibit MPP(+)-uptake into striatal synaptosomes prepared from rats. Post-treatment by 1-deprenyl, 1-methamphetamine, 1-amphetamine (at times when MPTP is no longer present in the striatum of mice) protects against neurotoxicity in C57BL mice by blocking the uptake of MPP+ into dopaminergic neurons, and even against the neurotoxicity induced by 2'CH3-MPTP, which is partly bioactivated by MAO-A. These findings may have clinical implications since deprenyl has recently been found to delay the progression of Parkinson's disease.

Use of 3h-γ-aminobutyric acid for transport studies with isolated nerve-terminals from rat brain

International Nuclear Information System (INIS)

Halvarsson, G.B.; Karlsson, I.; Sellstroem, A.

1985-01-01

Isolated synaptosomes were used to study the problem of net accumulation of neurotransmitters. The time-course and the kinetics of exogenous and endogenous GABA transport were studied by liquid-scintillation counting and HPLC-amino acid analysis respectively. Different pools of GABA were suggested by a 6-fold difference in tissue-to-medium-ratio of endogenous vs. exogenous GABA. Net accumulation, exchange and net efflux of GABA was found to be a function of the GABA concentration in the incubation medium. The K/sub m/s for net accumulation and for 3 H-GABA accumulation were 2.68 +/- 1.16 and 6.19 +/- 1.26 μM respectively, whereas the V/sub max/s were 5.9 +/- 4.9 and 134 +/- 13 pmol/mg w.w min respectively. This means that the transport studies which use exogenous substances (e.g. 3 H-GABA) considerably overestimate the transport by overlooking the magnitude of the counter transport. 22 references, 5 figures, 2 tables

Ascorbic acid and striatal transport of [3H]1-methyl-4-phenylpyridine (MPP+) and [3H]dopamine

International Nuclear Information System (INIS)

Debler, E.A.; Hashim, A.; Lajtha, A.; Sershen, H.

1988-01-01

The inhibition of uptake of [ 3 H]dopamine and [ 3 H]1-methyl-4-phenylpyridine (MPP + ) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of [ 3 H]MPP + uptake. No inhibition of [ 3 H]dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC 50 3 H]dopamine and [ 3 H]MPP + transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors except 4-phenylpyridine and lobeline, which are moderate inhibitors of both [ 3 H]dopamine and [ 3 H]MPP + uptake. These similarities in potencies are in agreement with the suggestion that [ 3 H]MPP + and [ 3 H] are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on [ 3 H]MPP + transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event

Isoguvacine binding, uptake, and release: relation to the GABA system

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White, W F; Snodgrass, S R

1983-06-01

Isoguvacine (1,2,3,6-tetrahydropyridine-4-carboxylic acid) is a GABA (gamma-aminobutyric acid) agonist with limited conformational flexibility. In these studies we investigated the binding, uptake, and release of (3H) isoguvacine by use of tissue preparations of rat CNS, comparing the results with similar studies of (3H)GABA. The results from these investigations indicate that isoguvacine binds to membrane preparations of rat forebrain with pharmacological characteristics similar to the post-synaptic GABA recognition site; that it is transported into synaptosomal preparations by an uptake system similar to the high-affinity GABA uptake system; and that recently accumulated isoguvacine is released in a Ca2+-dependent manner and by heteroexchange with external GABA. The ability of isoguvacine and gamma-hydroxybutyric acid to decrease the K+-stimulated Ca2+-dependent release process was also investigated. The results indicate that isoguvacine interactions have many of the biochemical features of GABA synaptic function, isoguvacine being, however, less potent than GABA.

Excitatory amino acid receptor ligands: resolution, absolute stereochemistry, and enantiopharmacology of 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid

DEFF Research Database (Denmark)

Johansen, T N; Ebert, B; Bräuner-Osborne, Hans

1998-01-01

(RS)-2-Amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (Bu-HIBO, 6) has previously been shown to be an agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors and an inhibitor of CaCl2-dependent [3H]-(S)-glutamic acid binding (J. Med. Chem. 1992, 35, 3512......-3519). To elucidate the pharmacological significance of this latter binding affinity, which is also shown by quisqualic acid (3) but not by AMPA, we have now resolved Bu-HIBO via diastereomeric salt formation using the diprotected Bu-HIBO derivative 11 and the enantiomers of 1-phenylethylamine (PEA). The absolute...... equipotent as inhibitors of CaCl2-dependent [3H]-(S)-glutamic acid binding, neither enantiomer showed significant affinity for the synaptosomal (S)-glutamic acid uptake system(s). AMPA receptor affinity (IC50 = 0.48 microM) and agonism (EC50 = 17 microM) were shown to reside exclusively in the S...

Inhibition of calmodulin - regulated calcium pump activity in rat brain by toxaphene

International Nuclear Information System (INIS)

Trottman, C.H.; Moorthy, K.S.

1986-01-01

In vivo effects of toxaphene on calcium pump activity in rat brain synaptosomes was studied. Male Sprague-Dawley rats were dosed with toxaphene at 0,25,50, and 100 mg/kg/day for 3 days and sacrificed 24 h after last dose. Ca 2+ -ATPase activity and 45 Ca uptake were determined in brain P 2 fraction. Toxaphene inhibited both Ca 2+ -ATPase activity and 45 Ca 2+ uptake and the inhibition was dose dependent. Both substrate and Ca 2+ activation kinetics of Ca 2+ -ATPase indicated non-competitive type of inhibition as evidenced by decreased catalytic velocity but not enzyme-substrate affinity. The inhibited Ca 2+ -ATPase activity and Ca 2+ uptake were restored to normal level by exogenously added calmodulin which increased both velocity and affinity. The inhibition of Ca 2+ -ATPase activity and Ca 2+ uptake and restoration by calmodulin suggests that toxaphene may impair active calcium transport mechanisms by decreasing regulator protein calmodulin levels

Cholecystokinin-8 suppressed /sup 3/H-etorphine binding to rat brain opiate receptors

Energy Technology Data Exchange (ETDEWEB)

Wang, X.J.; Fan, S.G.; Ren, M.F.; Han, J.S.

1989-01-01

Radioreceptor assay (RRA) was adopted to analyze the influence of CCK-8 on /sup 3/H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 suppressed the binding of /sup 3/H-etorphine. This effect was completely reversed by proglumide at 1/mu/M. Rosenthal analysis for saturation revealed two populations of /sup 3/H-etorphine binding sites. CCK-8 inhibited /sup 3/H-etorphine binding to the high affinity sites by an increase in Kd and decrease in Bmax without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 was also completely reversed by proglumide at 1/mu/M. Unsulfated CCK-8 produced only a slight increase in Kd of the high affinity sites without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

Naturally occurring compounds affect glutamatergic neurotransmission in rat brain.

Science.gov (United States)

Martini, Lucia Helena; Jung, Fernanda; Soares, Felix Antunes; Rotta, Liane Nanci; Vendite, Deusa Aparecida; Frizzo, Marcos Emilio dos Santos; Yunes, Rosendo A; Calixto, João Batista; Wofchuk, Susana; Souza, Diogo O

2007-11-01

Natural products, including those derived from plants, have largely contributed to the development of therapeutic drugs. Glutamate is the main excitatory neurotransmitter in the central nervous system and it is also considered a nociceptive neurotransmitter, by acting on peripheral nervous system. For this reason, in this study we investigated the effects of the hydroalcoholic extracts from Drymis winteri (polygodial and drimanial), Phyllanthus (rutin and quercetine), Jathopha elliptica (jatrophone), Hedyosmum brasiliense (13HDS), Ocotea suaveolens (Tormentic acid), Protium kleinii (alphabeta-amyrin), Citrus paradise (naringin), soybean (genistein) and Crataeva nurvala (lupeol), described as having antinociceptive effects, on glutamatergic transmission parameters, such as [(3)H]glutamate binding, [(3)H]glutamate uptake by synaptic vesicles and astrocyte cultures, and synaptosomal [(3)H]glutamate release. All the glutamatergic parameters were affected by one or more of these compounds. Specifically, drimanial and polygodial presented more broad and profound effects, requiring more investigation on their mechanisms. The putative central side effects of these compounds, via the glutamatergic system, are discussed.

Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men

DEFF Research Database (Denmark)

Vigelsø Hansen, Andreas; Gram, Martin; Wiuff, Caroline

2016-01-01

by aerobic training in young and older men. METHODS: 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus...... lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl...... GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. CONCLUSION: Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

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Jensen, Kathrine L; Runegaard, Annika H; Weikop, Pia

2017-01-01

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson's disease, attention...... therapeutic targets for these diseases. Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT......) functionality(5). When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue...

Protective Effects Induced by Microwave-Assisted Aqueous Harpagophytum Extract on Rat Cortex Synaptosomes Challenged with Amyloid β-Peptide.

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Ferrante, Claudio; Recinella, Lucia; Locatelli, Marcello; Guglielmi, Paolo; Secci, Daniela; Leporini, Lidia; Chiavaroli, Annalisa; Leone, Sheila; Martinotti, Sara; Brunetti, Luigi; Vacca, Michele; Menghini, Luigi; Orlando, Giustino

2017-08-01

Harpagophytum procumbens is a plant species that displays anti-inflammatory properties in multiple tissues. The iridoid glycosides arpagoside, harpagide, and procumbide appear to be the most therapeutically important constituents. In addition, harpagoside treatment exerted neuroprotective effects both in vitro and in vivo. Considering these findings, the aim of the present work is to explore the possible protective role of the previously described microwave-assisted aqueous extract of H. procumbens on rat hypothalamic (Hypo-E22) cells, and in rat cortex challenged with amyloid β-peptide (1-40). In this context, we assayed the protective effects induced by H. procumbens by measuring the levels of malondialdehyde, 3-hydroxykynurenine (3-HK), brain-derived neurotrophic factor, and tumor necrosis factor-α, 3-HK. Finally, we evaluated the effects of H. procumbens treatment on cortex levels of dopamine, norepinephrine, and serotonin. H. procumbens extract was well tolerated by Hypo-E22 cells and upregulated brain-derived neurotrophic factor gene expression but down-regulated tumor necrosis factor-α gene expression. In addition, the extract reduced amyloid β-peptide stimulation of malondialdehyde and 3-HK and blunted the decrease of dopamine, norepinephrine, and serotonin, in the cortex. In this context, our work supports further studies for the evaluation and confirmation of Harpagophytum in the management of the clinical symptoms related to Alzheimer's disease. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Correlation between (3H)dopamine specific uptake and (3H)GBR 12783 specific binding during the maturation of rat striatum

International Nuclear Information System (INIS)

Bonnet, J.J.; Costentin, J.

1989-01-01

The development of the specific uptake of dopamine in the rat striatum during the early postnatal period is compared with the ontogenetic changes of the specific binding of ( 3 H)GBR 12783 to the site of uptake inhibition. During maturation, the increase in the specific binding of ( 3 H)GBR 12783 parallels the increase in the specific uptake of dopamine. ( 3 H)GBR 12783 specific binding sites increase in number from day 1 postpartum until 40 days, when they reach the adult level. In 40 day-old rats, the weight of the striatum represents 80% of adult values. The affinity of ( 3 H)GBR 12783 for the inhibition site is similar in membrane preparations obtained from 6 day-old pups and adults; this results in a same ability of the inhibitor to block the specific uptake of dopamine into synaptosomes obtained from pups or adult rats. These data support the hypothesis of the existence of a single molecular entity including both the inhibition site and the carrier itself

Pharmacological analysis of calcium antagonist receptors

International Nuclear Information System (INIS)

Reynolds, I.J.

1987-01-01

This work focuses on two aspects of the action of calcium antagonist drugs, namely, the interaction of drugs with receptors for verapamil-like calcium antagonists, and the interactions of drugs with voltage-sensitive calcium fluxes in rat brain synaptosomes. From binding studies I have found that the ligand of choice for labeling the verapamil receptor is (-)[ 3 H]desmethoxy-verapamil. This drug labels potently, reversibly and stereoselectively two receptors in membranes prepared from rat brain and rabbit skeletal muscle tissues. In equilibrium studies dihydropyridine calcium antagonists interact in a non-competitive fashion, while many non-DHPs are apparently competitive. In-depth kinetic studies in skeletal muscle membranes indicate that the two receptors are linked in a negative heterotropic fashion, and that low-affinity binding of (-) [ 3 H]desmethoxy-verapamil may be to the diltiazem receptor. However, these studies were not able to distinguish between the hypothesis that diltiazem binds to spatially separate, allosterically coupled receptors, and the hypothesis that diltiazem binds to a subsite of the verapamil receptor

Lycopene depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase C in rat cerebrocortical nerve terminals.

Science.gov (United States)

Lu, Cheng-Wei; Hung, Chi-Feng; Jean, Wei-Horng; Lin, Tzu-Yu; Huang, Shu-Kuei; Wang, Su-Jane

2018-05-01

Lycopene is a natural dietary carotenoid that was reported to exhibit a neuroprotective profile. Considering that excitotoxicity and cell death induced by glutamate are involved in many brain disorders, the effect of lycopene on glutamate release in rat cerebrocortical nerve terminals and the possible mechanism involved in such effect was investigated. We observed here that lycopene inhibited 4-aminopyridine (4-AP)-evoked glutamate release and intrasynaptosomal Ca 2+ concentration elevation. The inhibitory effect of lycopene on 4-AP-evoked glutamate release was markedly reduced in the presence of the Ca v 2.2 (N-type) and Ca v 2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was insensitive to the intracellular Ca 2+ -release inhibitors dantrolene and CGP37157. Furthermore, in the presence of the protein kinase C inhibitors GF109203X and Go6976, the action of lycopene on evoked glutamate release was prevented. These results are the first to suggest that lycopene inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic Ca 2+ entry and protein kinase C activity.

Distribution of [{sup 3}H]diadenosine tetraphosphate binding sites in rat brain

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Miras-Portugal, M.T. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain); Palacios, J.M. [Laboratorios Almirall, Research Center, Cardener 68, 08024 Barcelona (Spain); Torres, M. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain); Cortes, R. [Departamento de Neuroquimica, Centro de Investigacion y Desarrollo, CSIC Jordi Girona 18-26, 08034 Barcelona (Spain); Rodriguez-Pascual, F. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain)

1997-01-06

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [{sup 3}H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation.The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain

International Nuclear Information System (INIS)

Miras-Portugal, M.T.; Palacios, J.M.; Torres, M.; Cortes, R.; Rodriguez-Pascual, F.

1997-01-01

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [ 3 H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation.The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

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Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Time-related sex differences in cerebral hypoperfusion-induced brain injury

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Stanojlović Miloš

2014-01-01

Full Text Available Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments, should be tested at this time point. [Projekat Ministarstva nauke Republike Srbije, br. 173044 i br. 41014

Methamphetamine and inflammatory cytokines increase neuronal Na+/K+-ATPase isoform 3: relevance for HIV associated neurocognitive disorders.

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Gurudutt Pendyala

Full Text Available Methamphetamine (METH abuse in conjunction with human immunodeficiency virus (HIV exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS, collectively termed HIV Associated Neurocognitive Disorders (HAND. Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV differing by one regimen, METH treatment, we identified the neuron specific Na(+/K(+-ATPase alpha 1 isoform 3 (ATP1A3 to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse.

Chronic Cerebral Ischaemia Forms New Cholinergic Mechanisms of Learning and Memory

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E. I. Zakharova

2010-01-01

Full Text Available The purpose of this research was a comparative analysis of cholinergic synaptic organization following learning and memory in normal and chronic cerebral ischaemic rats in the Morris water maze model. Choline acetyltransferase and protein content were determined in subpopulations of presynapses of “light” and “heavy” synaptosomal fractions of the cortex and the hippocampus, and the cholinergic projective and intrinsic systems of the brain structures were taken into consideration. We found a strong involvement of cholinergic systems, both projective and intrinsic, in all forms of cognition. Each form of cognition had an individual cholinergic molecular profile and the cholinergic synaptic compositions in the ischaemic rat brains differed significantly from normal ones. Our data demonstrated that under ischaemic conditions, instead of damaged connections new key synaptic relationships, which were stable against pathological influences and able to restore damaged cognitive functions, arose. The plasticity of neurochemical links in the individual organization of certain types of cognition gave a new input into brain pathology and can be used in the future for alternative corrections of vascular and other degenerative dementias.

Synaptosomal Protein of 25 kDa (Snap25 Polymorphisms Associated with Glycemic Parameters in Type 2 Diabetes Patients

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Nasser M. Al-Daghri

2016-01-01

Full Text Available A possible role of Snap25 polymorphisms in type 2 diabetes mellitus (T2DM was evaluated by analyzing three SNPs within intron 1 in a region known to affect the gene expression in vitro. Genomic DNA from 1019 Saudi individuals (489 confirmed T2DM and 530 controls was genotyped for SNPs rs363039, rs363043, and rs363050 in Snap25 using the TaqMan Genotyping Assay. Significantly higher levels of fasting glucose and HbA1c were detected in T2DM patients carrying the rs363050 (AG/GG genotypes compared to the (AA genotype (f=4.41, df=1, and p=0.03 and f=5.31, df=1, and p=0.03, resp.. In these same patients, insulin levels were significantly decreased compared to the (AA individuals (f=7.29, df=1, and p=0.009. Significant associations were detected between rs363050 (AG/GG genotypes and increasing fasting glucose levels (p=0.01 and OR: 1.05, HbA1c levels (OR: 5.06 and p=0.02, and lower insulinemia (p=0.03 and OR: 0.95 in T2DM patients. The minor Snap25 rs363050 (G allele, which results in a reduced expression of Snap25, is associated with altered glycemic parameters in T2DM possibly because of reduced functionality in the exocytotic machinery leading to suboptimal release of insulin.

β-Endorphin biotransformation in brain: Formation of γ-endorphin by a synaptosomal plasma membrane associated endopeptidase distinct from cathepsin D

NARCIS (Netherlands)

Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de

1980-01-01

cSPM preparations of rat brain contain a peptidase activity which generates γ-endorphin from β-endorphin. Some properties of this enzyme were studied and compared with those of cathepsin D. Maximal accumulation of γ-endorphin upon digestion of β-endorphin with a cSPM preparation was found at neutral

Cytokines and cytokine networks target neurons to modulate long-term potentiation.

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Prieto, G Aleph; Cotman, Carl W

2017-04-01

Cytokines play crucial roles in the communication between brain cells including neurons and glia, as well as in the brain-periphery interactions. In the brain, cytokines modulate long-term potentiation (LTP), a cellular correlate of memory. Whether cytokines regulate LTP by direct effects on neurons or by indirect mechanisms mediated by non-neuronal cells is poorly understood. Elucidating neuron-specific effects of cytokines has been challenging because most brain cells express cytokine receptors. Moreover, cytokines commonly increase the expression of multiple cytokines in their target cells, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Here, we review evidence on both direct and indirect-mediated modulation of LTP by cytokines. We also describe novel approaches based on neuron- and synaptosome-enriched systems to identify cytokines able to directly modulate LTP, by targeting neurons and synapses. These approaches can test multiple samples in parallel, thus allowing the study of multiple cytokines simultaneously. Hence, a cytokine networks perspective coupled with neuron-specific analysis may contribute to delineation of maps of the modulation of LTP by cytokines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

International Nuclear Information System (INIS)

Saporito, M.S.; Warwick, R.O. Jr.

1989-01-01

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B decreased K + evoked 3 H-5-HT release from superfused HYP slices by 25%. Bacitracin, a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K + evoked 3 H-5-HT release. Phosphoramidon (PAN, 10 μM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K + evoked 3 H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 μM), enhanced both BN and NM-C inhibition of 3 H-5-HT release. Bestatin (BST, 10 μM) had no effect on BN or NM-C inhibitory activity on 3 H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3 H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3 H-5-HT uptake

Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

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Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

2009-01-01

AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

ETUDE FONCTIONNELLE DES SYSTEMES DE CAPTURE SYNAPTOSOMALE ET VESICULAIRE DE DOPAMINE

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M SLIMANI

2001-06-01

Full Text Available L‘injection stéréotaxique unilatérale dans la substance noire de la 6 hydroxy-dopamine ( 6OH-DA se traduit par une chute  parallèle  de  la   capture de dopamine tritiée au  niveau  synaptosomale de  l'ordre  de -70 % et dans les préparations vésiculaires de l'ordre de -69 %, comparées aux préparations issues de Rats non lésés. Ces résultats montrent que ces deux préparations synaptosomales et vésiculaires sont bien d'origine dopaminergique. D'autre part, une étude comparative de l'effet de quelques agents pharmacologiques (β carbolines, imipraminiques, amphétamine et les inhibiteurs purs de la capture synaptosomale sur la capture synaptosomale et vésiculaire de la dopamine tritiée in-vitro, montre qu'ils agissent comme de puissants inhibiteurs. Nous montrons également que le transporteur vésiculaire diffère du transporteur synaptosomal par sa stéréospécificité et sa sensibilité aux agents pharmacologiques.

Molecular bases of protective immune responses against botulinum neurotoxin A--how antitoxin antibodies block its action.

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Atassi, M Zouhair; Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger

2007-01-01

In studies from this laboratory, we localized the regions on the H chain of botulinum neurotoxin A (BoNT/A) that are recognized by anti-BoNT/A antibodies (Abs) and block the activity of the toxin in vivo. These Abs were obtained from cervical dystonia patients who had been treated with BoNT/A and had become unresponsive to the treatment, as well as blocking Abs raised in mouse, horse, and chicken. We also localized the regions involved in BoNT/A binding to mouse brain synaptosomes (snp). Comparison of spatial proximities in the three-dimensional structure of the Ab-binding regions and the snp binding showed that except for one, the Ab-binding regions either coincide or overlap with the snp regions. It should be folly expected that protective Abs when bound to the toxin at sites that coincide or overlap with snp binding would prevent the toxin from binding to nerve synapse and therefore block toxin entry into the neuron. Thus, analysis of the locations of the Ab-binding and the snp-binding regions provides a molecular rationale for the ability of protecting Abs to block BoNT/A action in vivo.

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

International Nuclear Information System (INIS)

Schulz, Joseph R.; Vega-Beltran, Jose L. de la; Beltran, Carmen; Vacquier, Victor D.; Darszon, Alberto

2004-01-01

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca 2+ and Na + influx and K + and H + efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba 2+ , and has a PK + /PNa + selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization

Reconstitution of high-affinity opioid agonist binding in brain membranes

Energy Technology Data Exchange (ETDEWEB)

Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

1991-03-15

In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

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Ewan West

2015-06-01

Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

Modification by oxazepam of the diurnal variations in brain 125I-melatonin binding sites in sham-operated and pinealectomized rats

International Nuclear Information System (INIS)

Anis, Y.; Zisapel, N.; Nir, I.; Schmidt, U.

1992-01-01

Sham-operated and pinealectomized male rats were maintained at 14 h light: 10 h dark cycles (lights-on 5.00 h) and injected daily, for 14 days, with oxazepam or vehicle. 125 I-melatonin binding was recorded in synaptosomes prepared at 10.00, 18.00, and 24.00 h from the hypothalamus, hippocampus and medulla-pons of the rats. In the sham-operated, vehicle treated rats, specific 125 I-melatonin binding in all brain areas studied was higher at 18.00 h, whereas in the oxazepam-treated animals, binding was higher at 24.00 h than at the other times tested. In the pinealectomized, vehicle-treated rats, the binding recorded at 18.00 h in all three brain areas, was lower than at the other times of day tested. Oxazepam treatment decreased 125 I-melatonin binding at 24.00 h in the hippocampus and medulla-pons of the pinealectomized rats and did not significantly affect the binding in the hypothalamus. These results indicate the ability of oxazepam, pinealectomy and their combination, to manipulate the diurnal variations in 125 I-melatonin binding sites in the rat brain

Depletion of rat cortical norepinephrine and the inhibition of [3H]norepinephrine uptake by xylamine does not require monoamine oxidase activity

International Nuclear Information System (INIS)

Dudley, M.W.

1988-01-01

Inhibition of monoamine oxidase A through pretreatment of rats with clorgyline or the pro-drug MDL 72,394 did not block the amine-depleting action of xylamine. Xylamine treatment resulted in a loss of approximately 60% of the control level of norepinephrine in the cerebral cortex. A 1-hr pretreatment, but not a 24-hr pretreatment, with the monoamine oxidase B inhibitor, L-deprenyl, prevented the depletion of norepinephrine by xylamine. In addition, pretreatment with MDL 72,974, a monoamine oxidase B inhibitor without amine-releasing or uptake - inhibiting effects, did not prevent cortical norepinephrine levels. Inhibition of monoamine oxidase by either MDL 72,974 or MDL 72,394 did not prevent the inhibition of [ 3 H]norepinephrine uptake into rat cortical synaptosomes by xylamine. These data indicate that monoamine oxidase does not mediate the amine-releasing or uptake inhibiting properties of xylamine. The protection afforded by L-deprenyl following a 1-hr pretreatment most probably was due to accumulation of its metabolite, L-amphetamine, which would inhibit the uptake carrier. A functional carrier is required for depletion since desipramine administered 1 hr prior to xylamine, was also able to prevent depletion of norepinephrine

Brain uptake of pipecolic acid, amino acids, amines following intracarotid injection in the mouse

International Nuclear Information System (INIS)

Nishio, H.; Giacobini, E.

1981-01-01

The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI of D,L-[3H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid, L-proline, and piperidine show a significant inhibitory effect. Amont the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations

Shp2 in Forebrain Neurons Regulates Synaptic Plasticity, Locomotion, and Memory Formation in Mice

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Kusakari, Shinya; Saitow, Fumihito; Ago, Yukio; Shibasaki, Koji; Sato-Hashimoto, Miho; Matsuzaki, Yasunori; Kotani, Takenori; Murata, Yoji; Hirai, Hirokazu; Matsuda, Toshio; Suzuki, Hidenori

2015-01-01

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation. PMID:25713104

Thiamin deficiency on fetal brain development with and without prenatal alcohol exposure.

Science.gov (United States)

Kloss, Olena; Eskin, N A Michael; Suh, Miyoung

2018-04-01

Adequate thiamin levels are crucial for optimal health through maintenance of homeostasis and viability of metabolic enzymes, which require thiamine as a co-factor. Thiamin deficiency occurs during pregnancy when the dietary intake is inadequate or excessive alcohol is consumed. Thiamin deficiency leads to brain dysfunction because thiamin is involved in the synthesis of myelin and neurotransmitters (e.g., acetylcholine, γ-aminobutyric acid, glutamate), and its deficiency increases oxidative stress by decreasing the production of reducing agents. Thiamin deficiency also leads to neural membrane dysfunction, because thiamin is a structural component of mitochondrial and synaptosomal membranes. Similarly, in-utero exposure to alcohol leads to fetal brain dysfunction, resulting in negative effects such as fetal alcohol spectrum disorder (FASD). Thiamin deficiency and prenatal exposure to alcohol could act synergistically to produce negative effects on fetal development; however, this area of research is currently under-studied. This minireview summarizes the evidence for the potential role of thiamin deficiency in fetal brain development, with or without prenatal exposure to alcohol. Such evidence may influence the development of new nutritional strategies for preventing or mitigating the symptoms of FASD.

SNAP-25 in hippocampal CA3 region is required for long-term memory formation

International Nuclear Information System (INIS)

Hou Qiuling; Gao Xiang; Lu Qi; Zhang Xuehan; Tu Yanyang; Jin Meilei; Zhao Guoping; Yu Lei; Jing Naihe; Li Baoming

2006-01-01

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593-1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation

Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood.

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Kojima, N; Hanamura, K; Yamazaki, H; Ikeda, T; Itohara, S; Shirao, T

2010-01-13

Dendritic spines are postsynaptic structures at excitatory synapses that play important roles in synaptic transmission and plasticity. Dendritic spine morphology and function are regulated by an actin-based cytoskeletal network. Drebrin A, an adult form of drebrin, is an actin-binding protein in dendritic spines, and its decrease is purportedly concerned with synaptic dysfunction in Alzheimer's disease. Rapid conversion of drebrin E, an embryonic form of drebrin, to drebrin A occurs in parallel with synaptic maturation. To understand the physiological role of drebrin isoform conversion in vivo, we generated knockout mice in which a drebrin A-specific exon was deleted from the drebrin gene. Drebrin A-specific knockout (DAKO) mice expressed drebrin E, which substituted for drebrin A. Subcellular fractionation experiment indicated that cytosolic form of drebrin was increased in the brains of DAKO mice. Furthermore, drebrin accumulation in synaptosomes of DAKO mice was much higher than that of wild-type (WT) mice. DAKO mice were viable and showed no apparent abnormalities in their gross brain morphology and general behaviors. However, DAKO mice were impaired in a context-dependent freezing after fear conditioning. These data indicate that drebrin A plays an indispensable role in some processes of generating fear learning and memory.

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation.

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Pardo, Beatriz; Rodrigues, Tiago B; Contreras, Laura; Garzón, Miguel; Llorente-Folch, Irene; Kobayashi, Keiko; Saheki, Takeyori; Cerdan, Sebastian; Satrústegui, Jorgina

2011-01-01

The glutamate-glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate-glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo (13)C nuclear magnetic resonance labeling with (13)C(2)acetate or (1-(13)C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

Neuropeptide Y binding sites in rat brain identified with purified neuropeptide Y-I125

International Nuclear Information System (INIS)

Walker, M.W.; Miller, R.J.

1986-01-01

Neuropeptide Y (NPY) is a widely distributed neuronally localized peptide with 36 amino acids, 5 of which are tyrosines. The authors wished to investigate the properties of specific receptors for NPY. They therefore labeled the tyrosines with I125 using chloramine T and then purified the peptide using HPLC. A single mono-iodinated species of NPY which yielded > 85% specific binding in rat forebrain synaptosomes was selected as the ligand for all subsequent experiments. A time course of binding showed that equilibrium conditions were reached in 60 minutes at 21 0 C. Scatchard plots revealed a single class of binding sites with a Kd and a Bmax of 3 x 10-10 M and 28 pmol/mg, respectively. Competition binding with unlabeled NPY showed 50% displacement of bound ligand at 1 x 10-10 M NPY. Competition binding with rat pancreatic polypeptide (RPP), a homologous peptide possessing little NPY-like activity, showed 50% displacement of bound ligand at 2 x 10 -7 M RPP. No binding was observed on F-11 or PC12 neuronal cell lines, or on HSWP fibroblast cells. They conclude that NPY-I125 purified to homogeneity with HPLC is a highly selective ligand for NPY receptor sites. They are currently investigating such sites in brain, gut, and other tissues

Botulinum toxin's axonal transport from periphery to the spinal cord.

Science.gov (United States)

Matak, Ivica; Riederer, Peter; Lacković, Zdravko

2012-07-01

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application. Copyright © 2012 Elsevier Ltd. All rights reserved.

Excitant amino acid projections from rat amygdala and thalamus to nucleus accumbens

International Nuclear Information System (INIS)

Robinson, T.G.; Beart, P.M.

1988-01-01

High affinity uptake of D-[ 3 H]aspartate, [ 3 H]choline and [ 3 H]GABA was examined in synaptosomal-containing preparations of rat nucleus accumbens septi 7 to 10 days after unilateral or bilateral N-methyl-D-aspartate lesions confined to the parataenial nucleus of the thalamus or the basolateral nucleus of the amygdala. Uptake of both D-[ 3 H]aspartate and [ 3 H]choline was significantly reduced (11% and 14% less than control, respectively) by unilateral lesion of the thalamus, whereas [ 3 H]GABA uptake was unaffected. Bilateral thalamic lesions significantly reduced D-[ 3 H]aspartate uptake (11% less than control) into homogenates of the nucleus accumbens, whilst [ 3 H]GABA uptake was unaltered. D-[ 3 H]aspartate uptake was significantly reduced (26% less than control) following unilateral lesion of the amygdala, whereas both [ 3 H]GABA and [ 3 H]choline uptake were unaffected. Bilateral amygdaloid lesions significantly increased D-[ 3 H]aspartate uptake (39% greater than control), whilst uptake of [ 3 H]GABA was not affected. The results implicate glutamate and/or aspartate as putative neurotransmitters in afferent projections from the basolateral amygdala and the parataenial thalamus to the nucleus accumbens. Thalamic afferents to the nucleus accumbens may also utilize acetylcholine as their transmitter

AHR-11797: a novel benzodiazepine antagonist

International Nuclear Information System (INIS)

Johnson, D.N.; Kilpatrick, B.F.; Hannaman, P.K.

1986-01-01

AHR-11797(5,6-dihydro-6-methyl-1-phenyl- 3 H-pyrrolo[3,2,1-ij]quinazolin-3-one) displaced 3 H-flunitrazepam (IC 50 = 82 nM) and 3 H-Ro 15-1877 (IC 50 = 104 nM) from rat brain synaptosomes. AHR-11797 did not protect mice from seizures induced by maximal electroshock or subcutaneous Metrazol (scMET), nor did it induce seizures in doses up to the lethal dose. However, at 31.6 mg/kg, IP, it significantly increased the anticonvulsant ED 50 of chlordiazepoxide (CDPX) from 1.9 to 31.6 mg/kg, IP. With 56.7 mg/kg, IP, of AHR-11797, CDPX was inactive in doses up to 100 mg/kg, IP. AHR-11797 did not significantly increase punished responding in the Geller and Seifter conflict procedure, but it did attenuate the effects of diazepam. Although the compound is without anticonvulsant or anxiolytic activity, it did have muscle relaxant properties. AHR-11797 blocked morphine-induced Straub tail in mice (ED 50 = 31 mg/kg, IP) and it selectively suppressed the polysnaptic linguomandibular reflex in barbiturate-anesthetized cats. The apparent muscle relaxant activity of AHR-11797 suggests that different receptor sites are involved for muscle relaxant vs. anxiolytic/anticonvulsant activities of the benzodiazepines

Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain

International Nuclear Information System (INIS)

Worley, P.F.; Baraban, J.M.

1987-01-01

The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using [ 3 H]batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of [ 3 H]BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of [ 3 H]BTX-B binding sites in forebrain sections also agrees with biochemical determinations. Autoradiographic localizations indicate that [ 3 H]BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations

In vitro evaluation of antioxidant and neuroprotective effects of curcumin loaded in Pluronic micelles

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Cvetelina Gorinova

2016-09-01

Full Text Available Curcumin is a polyphenolic substance with attractive pharmacological activities (e.g. antioxidant, anti-inflammatory, anticancer. Incorporation of curcumin in polymeric micelles could overcome the problems associated with its instability and low aqueous solubility. The aim of this study was to load curcumin in polymeric micelles based on Pluronic® P 123 or Pluronic® F 127 triblock copolymers and evaluate the antioxidant and neuroprotective effects after micellization. The micelles were prepared and loaded with curcumin by applying the dissolution method. Higher encapsulation efficiency was observed in the micelles formulated with Pluronic® P 123. These micelles were characterized with small size and narrow size distribution. The effects of micellar curcumin were investigated in two in vitro models. First, the capacity of micellar curcumin to inhibit iron/ascorbic acid-induced lipid peroxidation in rat liver microsomes was evaluated. Micellar curcumin and free drug showed similar inhibition of lipid peroxidation. Second, micellar curcumin and free curcumin showed protective potential in a model of 6-hydroxydopamine induced neurotoxicity in rat brain synaptosomes. The results from both methods indicated preservation of antioxidant and neuroprotective activity of curcumin in micelles. The small micellar size, high loading capacity and preservation of antioxidant activity of curcumin into Pluronic micelles, suggested their further evaluation as a curcumin delivery system.

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse.

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Masakazu Kataoka

Full Text Available Synaptosomal-associated protein of 25 kDa (SNAP-25 is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC phosphorylates Ser(187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187 of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

International Nuclear Information System (INIS)

Asermely, K.E.; O'Neill, J.J.

1986-01-01

The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37 0 C in buffers containing tritium choline (5 μM 0.124 μC/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10 -5 M compared with 50% inhibition on brain synaptosomes. At 5 x 10 -4 M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K + -ion (25 mM) were unsuccessful; tissue 3 H-choline uptake appeared to oscillate over a 30 min period

Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

Energy Technology Data Exchange (ETDEWEB)

Asermely, K.E.; O' Neill, J.J.

1986-03-01

The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37/sup 0/C in buffers containing tritium choline (5 ..mu..M 0.124 ..mu..C/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10/sup -5/ M compared with 50% inhibition on brain synaptosomes. At 5 x 10/sup -4/ M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K/sup +/-ion (25 mM) were unsuccessful; tissue /sup 3/H-choline uptake appeared to oscillate over a 30 min period.

Cleavage of SNAP25 and its shorter versions by the protease domain of serotype A botulinum neurotoxin.

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Rahman M Mizanur

Full Text Available Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25 and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of kcat, Km, and catalytic efficiency kcat/Km. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.

Synthesis and evaluation of racemic [11C]NS2456 and its enantiomers as selective serotonin reuptake radiotracers for PET

International Nuclear Information System (INIS)

Smith, D.F.; Bender, D.; Marthi, K.; Cumming, P.; Hansen, S.B.; Peters, D.; Oestergaard Nielsen, E.; Scheel-Krueger, J.; Gjedde, A.

2001-01-01

Positron emission tomography (PET) radiotracers are needed for quantifying serotonin uptake sites in the living brain. Therefore, we evaluated a new selective serotonin reuptake inhibitor, NS2456, to determine whether it is suited for use in PET. Racemic NS2456 [(1RS,5SR)-8-methyl-3-[4-trifluoromethoxyphenyl]-8-azabicyclo [3.2.1]oct-2-ene] and its N-demethylated analog, racemic NS2463, selectively inhibited serotonin uptake in rat brain synaptosomes; their IC 50 values were 3000-fold lower for [ 3 H]serotonin than for either [ 3 H]dopamine or [ 3 H]noradrenaline. The enantiomers of NS2463 were also potent inhibitors of serotonin uptake in vitro, but they failed to show stereoselectivity. Racemic NS2463 as well as its enantiomers were radiolabelled by N-methylation with C-11, yielding [ 11 C]NS2456 for use in PET of the living porcine brain. The compounds crossed the blood-brain barrier rapidly and accumulated preferentially in regions rich in serotonin uptake sites (e.g., brainstem, subthalamus and thalamus). However, their binding potentials were relatively low and no stereoselectivity was found. Thus, neither racemic [ 11 C]NS2456 nor its [ 11 C]-labelled enantiomers are ideal for PET neuroimaging of neuronal serotonin uptake sites

Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+, K+-ATPase activity

International Nuclear Information System (INIS)

Angel, I.; Hauger, R.L.; Luu, M.D.; Giblin, B.; Skolnick, P.; Paul, S.M.

1985-01-01

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 0 C) resulted in a time-dependent decrease in specific (+)-[ 3 H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[ 3 H]amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[ 3 H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[ 3 H]amphetamine binding, suggesting the involvement of Na + , K + -ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na + ,K + -ATPase activity and the number of specific high-affinity binding sites for [ 3 H]ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-[ 3 H]amphetamine and [ 3 H]ouabain binding. These data suggest that the (+)-[ 3 H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na + ,K + -ATPase activity, and the latter may be involved in the glucostatic regulation of appetite

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

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Takagi, Hiroki; Seta, Yuji; Kataoka, Shinji; Nakatomi, Mitsushiro; Toyono, Takashi; Kawamoto, Tatsuo

2018-03-10

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

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Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Dose calcium channel blocker verapamil decrease urinary VMA levels in sympathoadrenal hyperactive patients with posttraumatic stress disorder?

Institute of Scientific and Technical Information of China (English)

Munawar Alam Ansari; Shahida PAhmed; Zahida Memon

2008-01-01

Objective:The majority of the patients with posttraumatic stress disorders (PTSD)embrace augmented urina-ry flow of Vanillylmandelic Acid (VMA)than normal subjects owing to superior sympathetic doings,which steer to cardiovascular catastrophe.Urinary flow of VMA was evaluated as sympathoadrenal bustle marker in patients with posttraumatic stress disorder.Calcium ion shows a noteworthy dependability in nervousness owing to its special effects on brain synaptosomes.So this study was conducted to explore the effects of Verapamil on sympathoadrenal motion in patients with PTSD.Methods:Placebo controlled clinical tryout was conducted. At first hundred (100)PTSD patients were chosen and enrolled in the study,from department of Psychological Medicine Dow University of Health Sciences,Karachi.Verapamil 120 mg/day was specified in divided doses to group-I (n =50)patients and group-II (n =50)patients received placebo therapy on a daily basis for nine weeks.Each and every patient was monitored weekly,all the way through extent of study.Results:Under-neath the posttraumatic stress disorder,urinary excretion of VMA was greater.Calcium channel blocker vera-pamil additionally abolished the embellished retort in urinary flow of VMA appreciably in patients with PTSD. Conclusion:Verapamil was experiential to be exceedingly effectual treatment.It reduces VMA levels in u-rine,and on the whole cardiovascular threat in PTSD patients.

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

International Nuclear Information System (INIS)

Singer, H.S.; Coyle, J.T.; Frangia, J.; Price, D.L.

1981-01-01

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release of L-[3H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinity glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process for L-[3H]glutamate resulted in elevation of Vmax with no significant alteration in KT as compared to age-matched controls. Depolarization-induced release of L-[3H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat

Antidiabetic and Neuroprotective Effects of Trigonella Foenum-graecum Seed Powder in Diabetic Rat Brain

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P. Kumar

2012-01-01

Full Text Available Trigonella foenum-graecum seed powder (TSP has been reported to have hypoglycemic and hyperinsulinemic action. The objective of the study was to examine the antidiabetic and neuroprotective role of TSP in hyperglycemiainduced alterations in blood glucose, insulin levels and activities of membrane linked enzymes (Na+K+ATPase, Ca2+ATPase, antioxidant enzymes (superoxide dismutase, glutathione S-transferase, calcium (Ca2+ levels, lipid peroxidation, membrane fluidity and neurolipofuscin accumulation in the diabetic rat brain. Female Wistar rats weighing between 180 and 220 g were made diabetic by a single injection of alloxan monohydrate (15 mg/100 g body weight, diabetic rats were given 2 IU insulin, per day with 5% TSP in the diet for three weeks. A significant increase in lipid peroxidation was observed in diabetic brain. The increased lipid peroxidation following chronic hyperglycemia was accompanied with a significant increase in the neurolipofuscin deposition and Ca2+ levels with decreased activities of membrane linked ATPases and antioxidant enzymes in diabetic brain. A decrease in synaptosomal membrane fluidity may influence the activity of membrane linked enzymes in diabetes. The present study showed that TSP treatment can reverse the hyperglycemia induced changes to normal levels in diabetic rat brain. TSP administration amended effect of hyperglycemia on alterations in lipid peroxidation, restoring membrane fluidity, activities of membrane bound and antioxidant enzymes, thereby ameliorating the diabetic complications.

Overexpression of parkin in rat nigrostriatal dopamine system protects against methamphetamine neurotoxicity

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Liu, Bin; Traini, Roberta; Killinger, Bryan; Schneider, Bernard; Moszczynska, Anna

2013-01-01

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum, sparing other striatal terminals and cell bodies. We previously detected a deficit in parkin after binge METH in rat striatal synaptosomes. Parkin is an ubiquitin-protein E3 ligase capable of protecting dopamine neurons from diverse cellular insults. Whether the deficit in parkin mediates the toxicity of METH and whether parkin can protect from toxicity of the drug is unknown. The present study investigated whether overexpression of parkin attenuates degeneration of striatal dopaminergic terminals exposed to binge METH. Parkin overexpression in rat nigrostriatal dopamine system was achieved by microinjection of adeno-associated viral transfer vector 2/6 encoding rat parkin (AAV2/6-parkin) into the substantia nigra pars compacta. The microinjections of AAV2/6-parkin dose-dependently increased parkin levels in both the substantia nigra pars compacta and striatum. The levels of dopamine synthesizing enzyme, tyrosine hydroxylase, remained at the control levels; therefore, tyrosine hydroxylase immunoreactivity was used as an index of dopaminergic terminal integrity. In METH-exposed rats, the increase in parkin levels attenuated METH-induced decreases in striatal tyrosine hydroxylase immunoreactivity in a dose-dependent manner, indicating that parkin can protect striatal dopaminergic terminals against METH neurotoxicity. PMID:23313192

Radioimmunoassay for Lys8, Asn9, neurotensin 8-13: tissue and subcellular distribution of immunoreactivity in chickens

International Nuclear Information System (INIS)

Carraway, R.E.; Ruane, S.E.; Ritsema, R.S.

1983-01-01

A sensitive and specific radioimmunoassay (RIA) for Lys8, Asn9, neurotensin 8-13 (LANT-6) has been developed which utilizes 125I-labeled LANT-6 and rabbit antisera raised towards conjugates of synthetic LANT-6 and bovine thyroglobulin. The antiserum described (TG-22) allows the detection of ca 100 fmol of LANT-6 and crossreacts less than 0.01% with chicken or bovine NT. Dose-response relationships for the native (chicken) and synthetic peptides were indistinguishable. Using this assay the distribution of immunoreactive LANT-6 (iLANT-6) through various tissues of the chicken was studied and compared with that of chicken NT (iNT) determined by RIA. Both iNT and ILANT-6 were found primarily in the brain and gastrointestinal tract, however, their regional distributions were found to differ. Subcellular distribution studies in homogenates of chicken brain indicated that both iNT and iLANT-6 were associated with synaptosome-like and vesicle-like particles. In homogenates of small intestine, pancreas and colon iNT and iLANT-6 appeared to be within osmotically sensitive, sedimentable particles. Analyses using high pressure liquid chromatography established that chicken iLANT-6 co-eluted with the synthetic peptide and that similar substances were present in extracts of rat brain and intestine. These results are consistent with ''messenger' roles for these peptides

Neurochemical, pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids

International Nuclear Information System (INIS)

Sharif, N.A.; Roberts, P.J.

1984-01-01

Specific binding of L-[ 3 H]glutamate ([ 3 H]Glu) and L[ 3 H]Asp) to cerebellar membranes represented a time-, temperature-, pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K/sub off//K/sub on/ . K/sub d/) for [ 3 H]Glu binding to this fraction indicated high-affinity (433 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [ 3 H]Glu and [ 3 H]Asp as receptors with differential pharmacological specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37 degrees C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents and L-Glu (50-1000 microM) provided protection against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8-12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8-12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously

Structure Activity Relationship of Brevenal Hydrazide Derivatives

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Allan Goodman

2014-03-01

Full Text Available Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.

Effect of ouabain on the gamma-[3H]aminobutyric acid uptake and release in the absence of Ca(+)+ and K(+)-depolarization

International Nuclear Information System (INIS)

Santos, M.S.; Goncalves, P.P.; Carvalho, A.P.

1990-01-01

The effect of ouabain on the uptake of tritiated [ 3 H]GABA and on its release in the absence of Ca(+)+ was studied in brain cortex synaptosomes. Ouabain, in the absence of Ca(+)+ and K(+)-depolarization, induces the release of [ 3 H]GABA with half-maximal effect occurring at a concentration of about 7 X 10(-6) M. Parallel measurements of the effects of ouabain on the [ 3 H]GABA uptake and the Na+,K(+)-adenosine triphosphatase activity show that ouabain inhibits both mechanisms and that the half-maximal effect also occurs at about the same ouabain concentration. Although [ 3 H]GABA release is stimulated by ouabain, it appears that the inhibition of [ 3 H]GABA uptake is due to a direct effect on the uptake mechanism, inasmuch as the initial velocity of the process is inhibited by ouabain. Ouabain requires extracellular Na+ for [ 3 H]GABA release and for membrane depolarization and, in the absence of Na+, ouabain does not cause either [ 3 H]GABA release or membrane depolarization. No significant changes in the Na+ gradients occur under conditions which permit release of [ 3 H]GABA, but the Na+,K(+)-adenosine triphosphatase activity is inhibited, which may be responsible for membrane depolarization, which in turn may cause [ 3 H]GABA release or inhibit its uptake

The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.

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Chang-Gyu Hahn

Full Text Available Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1 and the pH based differential extraction of synaptic membranes (Methods 2 and 3. All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

Identification of fibroblast growth factor receptor 3 (FGFR3 as a protein receptor for botulinum neurotoxin serotype A (BoNT/A.

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Birgitte P S Jacky

Full Text Available Botulinum neurotoxin serotype A (BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206 to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs, making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3 as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

Science.gov (United States)

Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

2013-01-01

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

Synaptic proteome changes in the superior frontal gyrus and occipital cortex of the alcoholic brain.

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Etheridge, Naomi; Lewohl, Joanne M; Mayfield, R Dayne; Harris, R Adron; Dodd, Peter R

2009-06-24

Cognitive deficits and behavioral changes that result from chronic alcohol abuse are a consequence of neuropathological changes which alter signal transmission through the neural network. To focus on the changes that occur at the point of connection between the neural network cells, synaptosomal preparations from post-mortem human brain of six chronic alcoholics and six non-alcoholic controls were compared using 2D-DIGE. Functionally affected and spared regions (superior frontal gyrus, SFG, and occipital cortex, OC, respectively) were analyzed from both groups to further investigate the specific pathological response that alcoholism has on the brain. Forty-nine proteins were differentially regulated between the SFG of alcoholics and the SFG of controls and 94 proteins were regulated in the OC with an overlap of 23 proteins. Additionally, the SFG was compared to the OC within each group (alcoholics or controls) to identify region specific differences. A selection were identified by MALDI-TOF mass spectrometry revealing proteins involved in vesicle transport, metabolism, folding and trafficking, and signal transduction, all of which have the potential to influence synaptic activity. A number of proteins identified in this study have been previously related to alcoholism; however, the focus on synaptic proteins has also uncovered novel alcoholism-affected proteins. Further exploration of these proteins will illuminate the mechanisms altering synaptic plasticity, and thus neuronal signaling and response, in the alcoholic brain.

Metabolism of γ-hydroxyl-[1-14C] butyrate by rat brain: relationship to the Krebs cycle and metabolic compartmentation of amino acids

International Nuclear Information System (INIS)

Doherty, J.D.; Roth, R.H.

1978-01-01

Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1- 14 C] butyric acid (GHB) or [1- 14 C] succinate. The loss of isotope (as 14 CO 2 ) was similar for both substances. The [1- 14 C] GHB metabolites lost 75% of the label and the [1- 14 C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1- 14 C] GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the α-amino group. These results also indicate that the label from [1- 14 C] GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radio-labelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments prefentially metabolize GHB. It was found that for [1- 14 C] GHB the ratio was 4.20 +- 0.18 (S.E. for n = 7) and for [1- 14 C] succinate the ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed. (author)

Low-Frequency rTMS Ameliorates Autistic-Like Behaviors in Rats Induced by Neonatal Isolation Through Regulating the Synaptic GABA Transmission

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Tao Tan

2018-02-01

Full Text Available Patients with autism spectrum disorder (ASD display abnormalities in neuronal development, synaptic function and neural circuits. The imbalance of excitatory and inhibitory (E/I synaptic transmission has been proposed to cause the main behavioral characteristics of ASD. Repetitive transcranial magnetic stimulation (rTMS can directly or indirectly induce excitability and synaptic plasticity changes in the brain noninvasively. However, whether rTMS can ameliorate autistic-like behaviors in animal model via regulating the balance of E/I synaptic transmission is unknown. By using our recent reported animal model with autistic-like behaviors induced by neonatal isolation (postnatal days 1–9, we found that low-frequency rTMS (LF-rTMS, 1 Hz treatment for 2 weeks effectively alleviated the acquired autistic-like symptoms, as reflected by an increase in social interaction and decrease in self-grooming, anxiety- and depressive-like behaviors in young adult rats compared to those in untreated animals. Furthermore, the amelioration in autistic-like behavior was accompanied by a restoration of the balance between E/I activity, especially at the level of synaptic transmission and receptors in synaptosomes. These findings indicated that LF-rTMS may alleviate the symptoms of ASD-like behaviors caused by neonatal isolation through regulating the synaptic GABA transmission, suggesting that LF-rTMS may be a potential therapeutic technique to treat ASD.

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

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Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

2017-07-04

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

Neuronal Nicotinic Receptors as New Targets for Amphetamine-Induced Oxidative Damage and Neurotoxicity

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Elena Escubedo

2011-06-01

Full Text Available Amphetamine derivatives such as methamphetamine (METH and 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy” are widely abused drugs in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS production seems to be one of the main causes. Recent research has demonstrated that blockade of a7 nicotinic acetylcholine receptors (nAChR inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, a7 nAChR antagonists (methyllycaconitine and memantine attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to a7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on a7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on a7 and heteromeric nAChR populations have been found.

Evaluation of the binding of the radiolabeled antidepressant drug, 18F-fluoxetine in the rodent brain: an in vitro and in vivo study

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Mukherjee, Jogeshwar; Das, Malay K.; Yang Zhiying; Lew, Robert

1998-01-01

We have developed 18 F-fluoxetine as a radiotracer analog of the antidepressant drug fluoxetine (Prozac). In vitro saturation experiments of 18 F-fluoxetine were carried out on rat midbrain tissue and citalopram was used for measuring nonspecific binding. A saturation curve for the binding of 18 F-fluoxetine was not obtained. Even when fluoxetine (10 μM) was used for measurements of nonspecific binding, a saturation curve was difficult to obtain. Other compounds, such as deprenyl, clorgyline, amphetamine, and reserpine were also not able to reduce the binding of 18 F-fluoxetine. Ex vivo autoradiographic experiments with 18 F-fluoxetine did not reveal any specific uptake in various brain regions. In vivo administration of 18 F-fluoxetine in rats showed similar uptake in all the brain regions with little regional selectivity. A subcellular analysis of rat brain tissue after intravenous (IV) administration of 18 F-fluoxetine indicated significant amounts of binding in mitochondria and synaptosomes. In summary, in vitro experiments with 18 F-fluoxetine indicate little specific binding. Binding to the serotonin transporter was not identifiable. High nonspecific binding of the tracer resulting from its subcellular nature in the brain masks the ability to detect binding to the serotonin uptake sites in vivo. These findings indicate that a large portion of the binding of 18 F-fluoxetine in rat brains is subcellular and clears slowly out of the cells. Other sites, such as monoamine oxidase, may also play a significant role in the action of fluoxetine

Differential sensitivity of long-sleep and short-sleep mice to high doses of cocaine.

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de Fiebre, C M; Ruth, J A; Collins, A C

1989-12-01

The cocaine sensitivity of male and female long-sleep (LS) and short-sleep (SS) mice, which have been selectively bred for differential ethanol-induced "sleep-time," was examined in a battery of behavioral and physiological tests. Differences between these two mouse lines were subtle and were seen primarily at high doses. At high doses, SS mice were more sensitive than LS mice, particularly to cocaine-induced hypothermia; however, significant hypothermia was not seen except at doses which were very near to the seizure threshold. During a 60-min test of locomotor activity, LS mice showed greater stimulation of Y-maze activity by 20 mg/kg cocaine than SS mice. Consistent with the finding of subtle differences in sensitivity to low doses of cocaine. LS and SS mice did not differ in sensitivity to cocaine inhibition of synaptosomal uptake of [3H]-dopamine, [3H]-norepinephrine or [3H]-5-hydroxytryptamine. However, consistent with the finding of differential sensitivity to high doses of cocaine, SS mice were more sensitive to the seizure-producing effects of the cocaine and lidocaine, a local anesthetic. It is hypothesized that the differential sensitivity of these mouse lines to high doses of cocaine is due to differential sensitivity to cocaine's actions on systems that regulate local anesthetic effects. Selective breeding for differential duration of alcohol-induced "sleep-time" may have resulted in differential ion channel structure or function in these mice.

Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs

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Westmoreland Susan V

2010-02-01

Full Text Available Abstract Background The trace amine associated receptor family is a diverse array of GPCRs that arose before the first vertebrates walked on land. Trace amine associated receptor 1 (TAAR1 is a wide spectrum aminergic receptor that acts as a modulator in brain monoaminergic systems. Other trace amine associated receptors appear to relate to environmental perception and show a birth-and-death pattern in mammals similar to olfactory receptors. Results Across mammals, avians, and amphibians, the TAAR1 gene is intact and appears to be under strong purifying selection based on rates of amino acid fixation compared to neutral mutations. We have found that in dogs it has become a pseudogene. Our analyses using a comparative genetics approach revealed that the pseudogenization event predated the emergence of the Canini tribe rather than being coincident with canine domestication. By assessing the effects of the TAAR1 agonist β-phenylethylamine on [3H]dopamine uptake in canine striatal synaptosomes and comparing the degree and pattern of uptake inhibition to that seen in other mammals, including TAAR1 knockout mice, wild type mice and rhesus monkey, we found that the TAAR1 pseudogenization event resulted in an uncompensated loss of function. Conclusion The gene family has seen expansions among certain mammals, notably rodents, and reductions in others, including primates. By placing the trace amine associated receptors in an evolutionary context we can better understand their function and their potential associations with behavior and neurological disease.

Compensatory molecular and functional mechanisms in nervous system of the Grm1(crv4) mouse lacking the mGlu1 receptor: a model for motor coordination deficits.

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Rossi, Pia Irene Anna; Musante, Ilaria; Summa, Maria; Pittaluga, Anna; Emionite, Laura; Ikehata, Masami; Rastaldi, Maria Pia; Ravazzolo, Roberto; Puliti, Aldamaria

2013-09-01

The metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, the only members of group I mGlu receptors, are implicated in synaptic plasticity and mechanisms of feedback control of glutamate release. They exhibit nearly complementary distributions throughout the central nervous system, well evident in the cerebellum, where mGlu1 receptor is most intensely expressed while mGlu5 receptor is not. Despite their different distribution, they show a similar subcellular localization and use common transducing pathways. We recently described the Grm1(crv4) mouse with motor coordination deficits and renal anomalies caused by a spontaneous mutation inactivating the mGlu1 receptor. To define the neuropathological mechanisms in these mice, we evaluated expression and function of the mGlu5 receptor in cerebral and cerebellar cortices. Western blot and immunofluorescence analyses showed mGlu5 receptor overexpression. Quantitative reverse transcriptase-polymerase chain reaction results indicated that the up-regulation is already evident at RNA level. Functional studies confirmed an enhanced glutamate release from cortical cerebral and cerebellar synaptosomes when compared with wild-type that is abolished by the mGlu5 receptor-specific inhibitor, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). Finally, acute MPEP treatment of Grm1(crv4/crv4) mice induced an evident although incomplete improvement of motor coordination, suggesting that mGlu5 receptors enhanced activity worsens, instead of improving, the motor-coordination defects in the Grm1(crv4/crv4) mice.

Analeptic activity produced by TRH microinjection into basal forebrain area of the rat

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Horita, A.; Carino, M.A.; Lai, H.

1986-01-01

Earlier, Kalivas and Horita demonstrated that the analeptic effect of TRH was mediated in part by cholinergic neurons in the medial septum-diagonal band of Broca (MS-DBB). Since the MS-DBB constitutes part of the cholinergic basal forebrain system, the present study investigated whether the area designated as the n. basalis of Meynert (NBM) was also sensitive to TRH in producing an antipentobarbital effect. Saline or TRH (0.5 μl) was microinjected via bilateral stainless steel cannulae implanted stereotaxically into the NBM using the coordinates of Wenk et al. Accuracy of cannula placement was confirmed by histological examination. Rats treated with PB (40 mg/kg, i.p.) lost their righting reflex for 130 +/- 28 min. Intrabasalis injection of TRH (but not saline) in doses of 0.1-1.0 μg exerted analeptic activity as follows: 0.1 μg = 81 +/- 21 min; 0.5 μg = 65 +/- 19 min; 1.0 μg = 45 +/- 10 min. All of these doses exerted significant shortening of narcosis duration of pentobarbitalized rats. The analeptic effect of TRH was blocked by atropine pretreatment, indicating that it was mediated via cholinergic mechanisms. High affinity, sodium-dependent 3 H-choline uptake by cortical synaptosomes prepared from these animals was also increased by TRH. These results suggest that the cholinergic neurons of NBM are highly sensitive to TRH and contributes to the analeptic effect of TRH

Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse

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Engy A. Abdel-Rahman

2016-01-01

Full Text Available Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with H2O2 generation in freshly isolated synaptosomes using high resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that although synaptic mitochondria exhibit substantially higher respiratory activities (8–82-fold greater than NOX oxygen consumption depending on mitochondrial respiratory state, NADPH-dependent oxygen consumption is associated with greater H2O2 production (6-7-fold higher NOX-H2O2. We also show that, in terms of the consumed oxygen, while synaptic mitochondria “leaked” 0.71%±0.12 H2O2 during NAD+-linked resting, 0.21%±0.04 during NAD+-linked active respiration, and 0.07%±0.02 during FAD+-linked active respiration, NOX converted 38%±13 of O2 into H2O2. Our results indicate that NOX rather than mitochondria is the major source of synaptic H2O2. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons.

Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.

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Hoerder-Suabedissen, Anna; Korrell, Kim V; Hayashi, Shuichi; Jeans, Alexander; Ramirez, Denise M O; Grant, Eleanor; Christian, Helen C; Kavalali, Ege T; Wilson, Michael C; Molnár, Zoltán

2018-05-30

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

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Padma Singh

Full Text Available Botulinum neurotoxins (BoNTs, etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylaminomethyl-8-quinolinol; NSC 84096 to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC(50 values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A.

Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington's Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid.

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Vodicka, Petr; Mo, Shunyan; Tousley, Adelaide; Green, Karin M; Sapp, Ellen; Iuliano, Maria; Sadri-Vakili, Ghazaleh; Shaffer, Scott A; Aronin, Neil; DiFiglia, Marian; Kegel-Gleason, Kimberly B

2015-01-01

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HD gene, which encodes the protein Huntingtin. Huntingtin associates with membranes and can interact directly with glycerophospholipids in membranes. We analyzed glycerophospholipid profiles from brains of 11 month old wild-type (WT) and Q140/Q140 HD knock-in mice to assess potential changes in glycerophospholipid metabolism. Polar lipids from cerebellum, cortex, and striatum were extracted and analyzed by liquid chromatography and negative ion electrospray tandem mass spectrometry analysis (LC-MS/MS). Gene products involved in polar lipid metabolism were studied using western blotting, immuno-electron microscopy and qPCR. Significant changes in numerous species of glycerophosphate (phosphatidic acid, PA) were found in striatum, cerebellum and cortex from Q140/Q140 HD mice compared to WT mice at 11 months. Changes in specific species could also be detected for other glycerophospholipids. Increases in species of lyso-PA (LPA) were measured in striatum of Q140/Q140 HD mice compared to WT. Protein levels for c-terminal binding protein 1 (CtBP1), a regulator of PA biosynthesis, were reduced in striatal synaptosomes from HD mice compared to wild-type at 6 and 12 months. Immunoreactivity for CtBP1 was detected on membranes of synaptic vesicles in striatal axon terminals in the globus pallidus. These novel results identify a potential site of molecular pathology caused by mutant Huntingtin that may impart early changes in HD.

Ginsenoside Rg1 Ameliorates Behavioral Abnormalities and Modulates the Hippocampal Proteomic Change in Triple Transgenic Mice of Alzheimer’s Disease

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Lulin Nie

2017-01-01

Full Text Available Alzheimer’s disease (AD is one of the most common neurodegenerative diseases, so far, there are no effective measures to prevent and cure this deadly condition. Ginsenoside Rg1 (Rg1 was shown to improve behavioral abnormalities in AD; however, the potential mechanisms remain unclear. In this study, we pretreated 7-month-old 3xTg-AD mice for 6 weeks with Rg1 and evaluated the effects of Rg1 on the behaviors and the protein expression of hippocampal tissues. The behavioral tests showed that Rg1 could improve the memory impairment and ameliorate the depression-like behaviors of 3xTg-AD mice. Proteomic results revealed a total of 28 differentially expressed hippocampal proteins between Rg1-treated and nontreated 3xTg-AD mice. Among these proteins, complexin-2 (CPLX2, synapsin-2 (SYN2, and synaptosomal-associated protein 25 (SNP25 were significantly downregulated in the hippocampus of 3xTg-AD mice compared with the WT mice, and the treatment of Rg1 modulated the expression of CPLX2 and SNP25 in the hippocampus of 3xTg-AD mice. The expression of CPLX2, SYN2, and SNP25 was further validated by Western blot analysis. Taken together, we concluded that Rg1 could be a potential candidate drug to improve the behavioral deficits in AD via modulating the expression of the proteins (i.e., CPLX2, SYN2, and SNP25.

Leftward shift in the voltage-dependence for Ca2+ currents activation induced by a new toxin from Phoneutria reidyi (Aranae, Ctenidae) venom.

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Vieira, L B; Pimenta, A M C; Richardson, M; Bemquerer, M P; Reis, H J; Cruz, J S; Gomez, M V; Santoro, M M; Ferreira-de-Oliveira, R; Figueiredo, S G; Snutch, T P; Cordeiro, M N

2007-02-01

Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis.

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Winkle, Cortney C; Hanlin, Christopher C; Gupton, Stephanie L

2016-03-16

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

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D. Olivera-Severo

2006-07-01

Full Text Available Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Evaluation of the binding of the radiolabeled antidepressant drug, {sup 18}F-fluoxetine in the rodent brain: an in vitro and in vivo study

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Mukherjee, Jogeshwar E-mail: jogeshwar_mukherjee@ketthealth.com; Das, Malay K.; Yang Zhiying; Lew, Robert

1998-10-01

We have developed {sup 18}F-fluoxetine as a radiotracer analog of the antidepressant drug fluoxetine (Prozac). In vitro saturation experiments of {sup 18}F-fluoxetine were carried out on rat midbrain tissue and citalopram was used for measuring nonspecific binding. A saturation curve for the binding of {sup 18}F-fluoxetine was not obtained. Even when fluoxetine (10 {mu}M) was used for measurements of nonspecific binding, a saturation curve was difficult to obtain. Other compounds, such as deprenyl, clorgyline, amphetamine, and reserpine were also not able to reduce the binding of {sup 18}F-fluoxetine. Ex vivo autoradiographic experiments with {sup 18}F-fluoxetine did not reveal any specific uptake in various brain regions. In vivo administration of {sup 18}F-fluoxetine in rats showed similar uptake in all the brain regions with little regional selectivity. A subcellular analysis of rat brain tissue after intravenous (IV) administration of {sup 18}F-fluoxetine indicated significant amounts of binding in mitochondria and synaptosomes. In summary, in vitro experiments with {sup 18}F-fluoxetine indicate little specific binding. Binding to the serotonin transporter was not identifiable. High nonspecific binding of the tracer resulting from its subcellular nature in the brain masks the ability to detect binding to the serotonin uptake sites in vivo. These findings indicate that a large portion of the binding of {sup 18}F-fluoxetine in rat brains is subcellular and clears slowly out of the cells. Other sites, such as monoamine oxidase, may also play a significant role in the action of fluoxetine.

Monamine oxidase inhibitors: current and emerging agents for Parkinson disease.

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Fernandez, Hubert H; Chen, Jack J

2007-01-01

Monoamine oxidase type B (MAO-B) is the predominant isoform responsible for the metabolic breakdown of dopamine in the brain. Selective inhibition of brain MAO-B results in elevation of synaptosomal dopamine concentrations. Data have been reported regarding the selective MAO-B inhibitors, rasagiline and selegiline, for the symptomatic treatment of Parkinson disease (PD). Selegiline has demonstrated efficacy as monotherapy in patients with early PD (Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism study), but evidence of selegiline efficacy as adjunctive treatment in levodopa-treated PD patients with motor fluctuations is equivocal. A new formulation of selegiline (Zydis selegiline) has been evaluated in 2 small, placebo-controlled studies as adjunctive therapy to levodopa. The Zydis formulation allows pregastric absorption of selegiline, minimizing first-pass metabolism, and thereby increasing selegiline bioavailability and reducing the concentration of amphetamine metabolites. Rasagiline is a selective, second-generation, irreversible MAO-B inhibitor, with at least 5 times the potency of selegiline in vitro and in animal models. Rasagiline has demonstrated efficacy in 1 large, randomized, double-blind, placebo-controlled trial (TVP-1012 in Early Monotherapy for Parkinson's Disease Outpatients) as initial monotherapy in patients with early PD, and in 2 large, controlled trials (Parkinson's Rasagiline: Efficacy and Safety in the Treatment of "Off," Lasting Effect in Adjunct Therapy With Rasagiline Given Once Daily) as adjunctive treatment in levodopa-treated PD patients with motor fluctuations. Unlike selegiline, rasagiline is an aminoindan derivative with no amphetamine metabolites. A randomized clinical trial is underway to confirm preclinical and preliminary clinical data suggesting rasagiline has disease-modifying effects.

Progress and Promise of Attention-Deficit Hyperactivity Disorder Pharmacogenetics

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Froehlich, Tanya E.; McGough, James J.; Stein, Mark A.

2010-01-01

One strategy for understanding variability in attention-deficit hyperactivity disorder (ADHD) medication response, and therefore redressing the current trial-and-error approach to ADHD medication management, is to identify genetic moderators of treatment. This article summarizes ADHD pharmacogenetic investigative efforts to date, which have primarily focused on short-term response to methylphenidate and largely been limited by modest sample sizes. The most well studied genes include the dopamine transporter and dopamine D4 receptor, with additional genes that have been significantly associated with stimulant medication response including the adrenergic α2A-receptor, catechol-O-methyltransferase, D5 receptor, noradrenaline (norepinephrine) transporter protein 1 and synaptosomal-associated protein 25 kDa. Unfortunately, results of current ADHD pharmacogenetic studies have not been entirely consistent, possibly due to differences in study design, medication dosing regimens and outcome measures. Future directions for ADHD pharmacogenetics investigations may include examination of drug-metabolizing enzymes and a wider range of stimulant and non-stimulant medications. In addition, researchers are increasingly interested in going beyond the individual candidate gene approach to investigate gene-gene interactions or pathways, effect modification by additional environmental exposures and whole genome approaches. Advancements in ADHD pharmacogenetics will be facilitated by multi-site collaborations to obtain larger sample sizes using standardized protocols. Although ADHD pharmacogenetic efforts are still in a relatively early stage, their potential clinical applications may include the development of treatment efficacy and adverse effect prediction algorithms that incorporate the interplay of genetic and environmental factors, as well as the development of novel ADHD treatments. PMID:20088618

RANTES modulates the release of glutamate in human neocortex.

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Musante, Veronica; Longordo, Fabio; Neri, Elisa; Pedrazzi, Marco; Kalfas, Fotios; Severi, Paolo; Raiteri, Maurizio; Pittaluga, Anna

2008-11-19

The effects of the recombinant chemokine human RANTES (hRANTES) on the release of glutamate from human neocortex glutamatergic nerve endings were investigated. hRANTES facilitated the spontaneous release of d [(3)H]D-aspartate ([(3)H]DASP-) by binding Pertussis toxin-sensitive G-protein-coupled receptors (GPCRs), whose activation caused Ca(2+) mobilization from inositol trisphosphate-sensitive stores and cytosolic tyrosine kinase-mediated phosphorylations. Facilitation of release switched to inhibition when the effects of hRANTES on the 12 mM K(+)-evoked [(3)H]D-ASP exocytosis were studied. Inhibition of exocytosis relied on activation of Pertussis toxin-sensitive GPCRs negatively coupled to adenylyl cyclase. Both hRANTES effects were prevented by met-RANTES, an antagonist at the chemokine receptors (CCRs) of the CCR1, CCR3, and CCR5 subtypes. Interestingly, human neocortex glutamatergic nerve endings seem to possess all three receptor subtypes. Blockade of CCR1 and CCR5 by antibodies against the extracellular domain of CCRs prevented both the hRANTES effect on [(3)H]D-ASP release, whereas blockade of CCR3 prevented inhibition, but not facilitation, of release. The effects of RANTES on the spontaneous and the evoked release of [(3)H]D-ASP were also observed in experiments with mouse cortical synaptosomes, which may therefore represent an appropriate animal model to study RANTES-induced effects on neurotransmission. It is concluded that glutamate transmission can be modulated in opposite directions by RANTES acting at distinct CCR receptor subtypes coupled to different transduction pathways, consistent with the multiple and sometimes contrasting effects of the chemokine.

Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

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Sikorra, Stefan; Litschko, Christa; Müller, Carina; Thiel, Nadine; Galli, Thierry; Eichner, Timo; Binz, Thomas

2016-01-29

Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular basis of immunogenicity to botulinum neurotoxins and uses of the defined antigenic regions.

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Atassi, M Z

2015-12-01

Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

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Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A; Ravindranath, Vijayalakshmi

2017-12-01

Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APP Swe /PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

Effects of surgical and chemical lesions on neurotransmitter candidates in the nucleus accumbens of the rat

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Walaas, I; Fonnum, F

1979-01-01

The origin of fibers containing different neurotransmitter candidates in the nucleus accumbens of rat brain has been studied with surgical and chemical lesion techniques. Destruction of the medial forebrain bundle decreased the activity of aromatic amino acid decarboxylase by 80% in the nucleus. Cutting of the fornix or a hemitransection decreased the high affinity uptake of glutamate by 45% and the endogenous level of glutamate by 33%. The high affinity uptake of glutamate was concentrated in the synaptosomal fraction and the decrease after the lesion was most pronounced in this fraction. Restricted lesions indicated that fibers in the fimbria/fornix coming from the subiculum were responsible for this part of the glutamate uptake in the nucleus. Local injection of kainic acid into the nucleus was accompanied by a 75% decrease in choline acetyltransferase and a 35% decrease in acetylcholineserase activities, a 70% decrease in glutamate decarboxylase activity and a 60% decrease in the high affinity uptake of ..gamma..-aminobutyrate, a 45% decrease in high affinity glutamate uptake, and no change in aromatic amino acid decarboxylase activity. Performing a lesion of the fornix after kainic acid injection led to an 85% decrease in high affinity glutamate uptake, without further affecting the other neuronal markers. The results indicate that all aminergic fibers to the nucleus accumbens are ascending in the medial forebrain bundle, that the subiculum-accumbens fibers are glutamergic and the nucleus also contains intrinsic glutamergic or aspartergic cells. Cholinergic and ..gamma..-aminobutyrate-containing cells are wholly intrinsic to the nucleus.

Functional Coding Variation in Recombinant Inbred Mouse Lines Reveals Novel Serotonin Transporter-Associated Phenotypes

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Carneiro, Ana [Vanderbilt University; Airey, David [University of Tennessee Health Science Center, Memphis; Thompson, Brent [Vanderbilt University; Zhu, C [Vanderbilt University; Rinchik, Eugene M [ORNL; Lu, Lu [University of Tennessee Health Science Center, Memphis; Chesler, Elissa J [ORNL; Erikson, Keith [University of North Carolina; Blakely, Randy [Vanderbilt University

2009-01-01

The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, SLC6A4) figures prominently in the etiology or treatment of many prevalent neurobehavioral disorders including anxiety, alcoholism, depression, autism and obsessive-compulsive disorder (OCD). Here we utilize naturally occurring polymorphisms in recombinant inbred (RI) lines to identify novel phenotypes associated with altered SERT function. The widely used mouse strain C57BL/6J, harbors a SERT haplotype defined by two nonsynonymous coding variants (Gly39 and Lys152 (GK)). At these positions, many other mouse lines, including DBA/2J, encode Glu39 and Arg152 (ER haplotype), assignments found also in hSERT. Synaptosomal 5-HT transport studies revealed reduced uptake associated with the GK variant. Heterologous expression studies confirmed a reduced SERT turnover rate for the GK variant. Experimental and in silico approaches using RI lines (C57Bl/6J X DBA/2J=BXD) identifies multiple anatomical, biochemical and behavioral phenotypes specifically impacted by GK/ER variation. Among our findings are multiple traits associated with anxiety and alcohol consumption, as well as of the control of dopamine (DA) signaling. Further bioinformatic analysis of BXD phenotypes, combined with biochemical evaluation of SERT knockout mice, nominates SERT-dependent 5-HT signaling as a major determinant of midbrain iron homeostasis that, in turn, dictates ironregulated DA phenotypes. Our studies provide a novel example of the power of coordinated in vitro, in vivo and in silico approaches using murine RI lines to elucidate and quantify the system-level impact of gene variation.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

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Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

2017-10-27

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds.

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Ohtubo, Yoshitaka; Iwamoto, Masafumi; Yoshii, Kiyonori

2012-06-01

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Neurobehavioral toxicology of pyrethroid insecticides

International Nuclear Information System (INIS)

Crofton, K.M.

1986-01-01

Pyrethroid insecticides are classified as either Type I or Type II based upon in vivo toxic signs, and neurophysiological and biochemical data. Both axonal sodium channels and the γ-aminobutyric acid (GABA) receptor complex have been proposed as the major site of action of the Type II pyrethroids. This investigation characterized the behavior and biochemical effects of low dosages of pyrethroids in rats. Type I and II pyrethroids were tested for effects on figure-eight maze activity and the acoustic startle response (ASR). All compounds decreased figure-eight maze activity. Interactions of Type I and II pyrethroids with the three major binding sites on the GABA complex were determined in vivo. Radioligand binding experiments assessed in vitro interactions of pyrethroids with the three major GABA-complex binding sites. None of the pyrethroids competed for [ 3 H]-muscimol or [ 3 H]-flunitrazepam binding. Only Type II pyrethroids inhibited binding of [ 35 S]-t-butylbicyclophosphorothionate (TBPS) in cortical synaptosome preparations with K/sub i/ values of 5 to 10 μM. The [ 35 S]-TBPS data implicate the TBPS/picrotoxinin binding site in the mechanism of Type II pyrethroid toxicity. The results of these experiments support the classification of pyrethroids into two classes, and demonstrate the utility of the figure-eight maze and the ASR in studies to elucidate neurotoxic mechanisms. The interaction of the Type II pyrethroids is probably restricted to the TBPS/picrotoxinin binding domain on the GABA complex as shown by both the in vivo and in vitro studies

Behavioral studies with anxiolytic drugs. IV. Serotonergic involvement in the effects of buspirone on punished behavior of pigeons

International Nuclear Information System (INIS)

Witkin, J.M.; Mansbach, R.S.; Barrett, J.E.; Bolger, G.T.; Skolnick, P.; Weissman, B.

1987-01-01

Interactions of the nonbenzodiazepine anxiolytic, buspirone, with serotonin (5-HT) were studied using behavioral and neurochemical procedures. Punished responding was studied in pigeons as this behavior is a generally acknowledged preclinical predictor of anxiolytic activity and because buspirone increases punished responding of pigeons with greater potency and efficacy than in other species. Keypeck responses were maintained under either fixed-interval or fixed-ratio schedules of food presentation; every 30th response produced a brief electric shock and suppressed responding (punishment). Buspirone (0.1-5.6 mg/kg i.m.) produced dose-related increases in punished responding which reached a maximum at 1 mg/kg. A serotonin agonist, MK-212 (0.01 mg/kg), antagonized whereas the 5-HT antagonist, cyproheptadine (0.01 mg/kg), potentiated the effects of buspirone without having behavioral effects of their own. The characteristics of [ 3 H]-5-HT binding in pigeon brain membranes were similar to results reported in mammalian brain. Neither buspirone, MJ-13805 (gepirone, a related analog), nor MJ-13653 (a buspirone metabolite), significantly affected [ 3 H]-5-HT binding and none of the compounds appreciably inhibited uptake of [ 3 H]-5-HT into pigeon cerebral synaptosomes. Hill coefficients significantly less than unity for all drugs except 5-HT suggested multiple serotonergic binding sites for buspirone and analogs. Buspirone and MJ-13805 (1 nM) inhibited [ 3 H]ketanserin binding (a measure of 5-HT2 binding sites) in pigeon cerebrum with Ki values above 10(-6) M. The number of [ 3 H]ketanserin binding sites was estimated to be 109 fmol/mg of protein in pigeon cerebrum compared to 400 fmol/mg of protein in rat cerebrum

5-Iodo-2-aminoindan, a nonneurotoxic analogue of p-iodoamphetamine

International Nuclear Information System (INIS)

Nichols, D.E.; Johnson, M.P.; Oberlender, R.

1991-01-01

A rigid analogue, 5-iodo-2-aminoindan (5-IAI), of the serotonin neurotoxic halogenated amphetamine p-iodoamphetamine (PIA) was pharmacologically evaluated for production of serotonin neurotoxicity. A comparison was also made between 5-IAI and PIA in the two-lever drug discrimination paradigm in rats trained to discriminate saline from 3,4-methylenedioxymethamphetamine (MDMA) or saline from the alpha-ethyl homologue of MDMA, MBDB. PIA and 5-IAI were both behaviorally active, and fully substituted in both groups of animals, but were considerably less potent than p-chloroamphetamine (PCA). PIA had about twice the potency of PCA as an inhibitor of 3 H-5-HT uptake in rat brain cortical synaptosomes, while 5-IAI was only about 75% as potent as PCA in this assay. A single 40 mg/kg dose of PIA resulted in a 40% reduction of 5-HT and 5-HIAA levels and in the number of 5-HT uptake sites in rat cortex at one week sacrifice. The same dose of 5-IAI with one week sacrifice led to about a 15% decrease in 5-HIAA levels and number of 5-HT uptake sites, but only the latter was statistically significant. In rat hippocampus, PIA gave significant decreases in all serotonin markers examined, while 5-IAI slightly but significantly decreased only 5-HT levels. Neither compound produced any change in catecholamine or catecholamine metabolite levels. The results confirm earlier reports of the selective serotonin neurotoxicity of PIA, which is less severe than that of PCA, and also demonstrate that its rigid analogue 5-IAI does not appear to cause significant serotonin deficits in the rat

Proteomic analysis of adrenocorticotropic hormone treatment of an infantile spasm model induced by N-methyl-D-aspartic acid and prenatal stress.

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Jing Wang

Full Text Available Infantile spasms is an age-specific epileptic syndrome associated with poor developmental outcomes and poor response to nearly all traditional antiepileptic drugs except adrenocorticotropic hormone (ACTH. We investigated the protective mechanism of ACTH against brain damage. An infantile spasm rat model induced by N-methyl-D-aspartate (NMDA in neonate rats was used. Pregnant rats were randomly divided into the stress-exposed and the non-stress exposed groups, and their offspring were randomly divided into ACTH-treated spasm model, untreated spasm model, and control groups. A proteomics-based approach was used to detect the proteome differences between ACTH-treated and untreated groups. Gel image analysis was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric protein identification and bioinformatics analysis. Prenatal stress exposure resulted in more severe seizures, and ACTH treatment reduced and delayed the onset of seizures. The most significantly up-regulated proteins included isoform 1 of tubulin β-5 chain, cofilin-1 (CFL1, synaptosomal-associated protein 25, malate dehydrogenase, N(G,N(G-dimethylarginine dimethylaminohydrolase 1, annexin A3 (ANXA3, and rho GDP-dissociation inhibitor 1 (ARHGDIA. In contrast, tubulin α-1A chain was down-regulated. Three of the identified proteins, ARHGDIA, ANXA3, and CFL1, were validated using western blot analysis. ARHGDIA expression was assayed in the brain samples of five infantile spasm patients. These proteins are involved in the cytoskeleton, synapses, energy metabolism, vascular regulation, signal transduction, and acetylation. The mechanism underlying the effects of ACTH involves the molecular events affected by these proteins, and protein acetylation is the mechanism of action of the drug treatment.

CDKL5 knockout leads to altered inhibitory transmission in the cerebellum of adult mice.

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Sivilia, S; Mangano, C; Beggiato, S; Giuliani, A; Torricella, R; Baldassarro, V A; Fernandez, M; Lorenzini, L; Giardino, L; Borelli, A C; Ferraro, L; Calzà, L

2016-06-01

Mutations in the X-linked cyclin-dependent kinase-like 5 gene (CDKL5) are associated to severe neurodevelopmental alterations including motor symptoms. In order to elucidate the neurobiological substrate of motor symptoms in CDKL5 syndrome, we investigated the motor function, GABA and glutamate pathways in the cerebellum of CDKL5 knockout female mice. Behavioural data indicate that CDKL5-KO mice displayed impaired motor coordination on the Rotarod test, and altered steps, as measured by the gait analysis using the CatWalk test. A higher reduction in spontaneous GABA efflux, than that in glutamate, was observed in CDKL5-KO mouse cerebellar synaptosomes, leading to a significant increase of spontaneous glutamate/GABA efflux ratio in these animals. On the contrary, there were no differences between groups in K(+) -evoked GABA and glutamate efflux. The anatomical analysis of cerebellar excitatory and inhibitory pathways showed a selective defect of the GABA-related marker GAD67 in the molecular layer in CDKL5-KO mice, while the glutamatergic marker VGLUT1 was unchanged in the same area. Fine cerebellar structural abnormalities such as a reduction of the inhibitory basket 'net' estimated volume and an increase of the pinceau estimated volume were also observed in CDKL5-KO mice. Finally, the BDNF mRNA expression level in the cerebellum, but not in the hippocampus, was reduced compared with WT animals. These data suggest that CDKL5 deletion during development more markedly impairs the establishment of a correct GABAergic cerebellar network than that of glutamatergic one, leading to the behavioural symptoms associated with CDKL5 mutation. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Fluorinated phenmetrazine "legal highs" act as substrates for high-affinity monoamine transporters of the SLC6 family.

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Mayer, Felix P; Burchardt, Nadine V; Decker, Ann M; Partilla, John S; Li, Yang; McLaughlin, Gavin; Kavanagh, Pierce V; Sandtner, Walter; Blough, Bruce E; Brandt, Simon D; Baumann, Michael H; Sitte, Harald H

2018-05-15

A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market and adverse clinical effects have been reported. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine (IC 50 values 80 μM). Experiments directed at identifying transporter-mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na + /H + ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. Hence, this study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at catecholamine transporters implicated in abuse and addiction. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.' Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

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Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

2018-03-09

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

The subcellular and organ distribution and natural form of histidyl-proline diketopiperazine in rat brain determined by a specific radioimmunoassay.

Science.gov (United States)

Yanagisawa, T; Prasad, C; Peterkofsky, A

1980-11-10

Histidyl-proline diketopiperazine is produced in brain as a product of the metabolism of thyrotropin-releasing hormone. A number of the previously observed central nervous system and pituitary activities resulting from an exposure to thyrotropin-releasing hormone appear to involve the conversion of the releasing factor to the cyclic dipeptide. In the present study, the development of a rabbit antiserum that is highly specific for histidyl-proline diketopiperazine is described; the antiserum has essentially no capability to bind thyrotropin-releasing hormone or a number of other related peptides. The antibody can also distinguish between the natural form of the cyclic dipeptide and a diastereomer containing D-proline. A procedure for extraction, with high yield, of histidyl-proline diketopiperazine from brain is described. With the aid of the specific antiserum it was found that the preponderance of the cyclic dipeptide in rat brain is bound to high molecular weight material, mainly in the range of Mr = 70,000; histidyl-proline diketopiperazine can be disassociated from this material by boiling in salt/methanol solution. The concentration of the dipeptide in rat brain is in the range of 275 to 565 pmol/brain, approximately 2.5 times the concentrations determined for thyrotropin-releasing hormone (113 to 210 pmol/brain). A study of the subcellular distribution of histidyl-proline diketopiperazine and thyrotropin-releasing hormone suggests that the releasing factor is concentrated in synaptosomal vesicles while the diketopiperazine is not. A determination of the regional distribution of thyrotropin-releasing hormone and histidyl-proline diketopiperazine indicated that both peptides are found in highest concentrations in pituitary and hypothalamus, but are detectable in other areas of brain as well.

Protein-energy malnutrition developing after global brain ischemia induces an atypical acute-phase response and hinders expression of GAP-43.

Science.gov (United States)

Smith, Shari E; Figley, Sarah A; Schreyer, David J; Paterson, Phyllis G

2014-01-01

Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.

Synaptic Interactome Mining Reveals p140Cap as a New Hub for PSD Proteins Involved in Psychiatric and Neurological Disorders

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Annalisa Alfieri

2017-06-01

Full Text Available Altered synaptic function has been associated with neurological and psychiatric conditions including intellectual disability, schizophrenia and autism spectrum disorder (ASD. Amongst the recently discovered synaptic proteins is p140Cap, an adaptor that localizes at dendritic spines and regulates their maturation and physiology. We recently showed that p140Cap knockout mice have cognitive deficits, impaired long-term potentiation (LTP and long-term depression (LTD, and immature, filopodia-like dendritic spines. Only a few p140Cap interacting proteins have been identified in the brain and the molecular complexes and pathways underlying p140Cap synaptic function are largely unknown. Here, we isolated and characterized the p140Cap synaptic interactome by co-immunoprecipitation from crude mouse synaptosomes, followed by mass spectrometry-based proteomics. We identified 351 p140Cap interactors and found that they cluster to sub complexes mostly located in the postsynaptic density (PSD. p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell-cell junction organization. Gene co-expression data further support convergent functions: the p140Cap interactors are tightly co-expressed with each other and with p140Cap. Importantly, the p140Cap interactome and its co-expression network show strong enrichment in genes associated with schizophrenia, autism, bipolar disorder, intellectual disability and epilepsy, supporting synaptic dysfunction as a shared biological feature in brain diseases. Overall, our data provide novel insights into the molecular organization of the synapse and indicate that p140Cap acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders.

A comparison of the effects on central 5-HT function of sibutramine hydrochloride and other weight-modifying agents

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Heal, D J; Cheetham, S C; Prow, M R; Martin, K F; Buckett, W R

1998-01-01

Effects on 5-HT function of sibutramine and its active metabolites, BTS 54 354 and BTS 54 505, were compared with fluoxetine, (+)-fenfluramine and (+)-amphetamine.In vitro sibutramine weakly inhibited [3H]-5-HT uptake into brain synaptosomes. BTS 54 354, BTS 54 505 and fluoxetine were powerful [3H]-5-HT uptake inhibitors, whereas (+)-fenfluramine and (+)-amphetamine were very much weaker. Conversely, whilst sibutramine, its metabolites and fluoxetine did not release [3H]-5-HT from brain slices at ⩽10−5M, (+)-fenfluramine and (+)-amphetamine concentration-dependently increased [3H]-5-HT release.Sibutramine and fluoxetine had no effect on 5-hydroxytryptophan (5-HTP) accumulation in either frontal cortex or hypothalamus at doses Sibutramine (10 mg kg−1 i.p.) and fluoxetine (10 mg kg−1 i.p.) produced slow, prolonged increases of extracellular 5-HT in the anterior hypothalamus. In contrast, (+)-fenfluramine (3 mg kg−1 i.p.) and (+)-amphetamine (4 mg kg−1 i.p.) induced rapid, short-lasting increases in extracellular 5-HT.Only (+)-fenfluramine (10 mg kg−1) altered 5-HT2A receptors in rat frontal cortex when given for 14 days, producing a 61% reduction in receptor number and a 18% decrease in radioligand affinity.These results show that sibutramine powerfully enhances central 5-HT function via its secondary and primary amine metabolites; this effect, like that of fluoxetine, is almost certainly mediated through 5-HT uptake inhibition. By contrast, (+)-fenfluramine enhances 5-HT function predominantly by increasing 5-HT release. (+)-Amphetamine, though weaker than (+)-fenfluramine, also enhances 5-HT function by release. PMID:9786502

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

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Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissociable roles of dopamine and serotonin transporter function in a rat model of negative urgency.

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Yates, Justin R; Darna, Mahesh; Gipson, Cassandra D; Dwoskin, Linda P; Bardo, Michael T

2015-09-15

Negative urgency is a facet of impulsivity that reflects mood-based rash action and is associated with various maladaptive behaviors in humans. However, the underlying neural mechanisms of negative urgency are not fully understood. Several brain regions within the mesocorticolimbic pathway, as well as the neurotransmitters dopamine (DA) and serotonin (5-HT), have been implicated in impulsivity. Extracellular DA and 5-HT concentrations are regulated by DA transporters (DAT) and 5-HT transporters (SERT); thus, these transporters may be important molecular mechanisms underlying individual differences in negative urgency. The current study employed a reward omission task to model negative urgency in rats. During reward trials, a cue light signaled the non-contingent delivery of one sucrose pellet; immediately following the non-contingent reward, rats responded on a lever to earn sucrose pellets (operant phase). Omission trials were similar to reward trials, except that non-contingent sucrose was omitted following the cue light prior to the operant phase. As expected, contingent responding was higher following omission of expected reward than following delivery of expected reward, thus reflecting negative urgency. Upon completion of behavioral training, Vmax and Km were obtained from kinetic analysis of [(3)H]DA and [(3)H]5-HT uptake using synaptosomes prepared from nucleus accumbens (NAc), dorsal striatum (Str), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC) isolated from individual rats. Vmax for DAT in NAc and for SERT in OFC were positively correlated with negative urgency scores. The current findings suggest that mood-based impulsivity (negative urgency) is associated with enhanced DAT function in NAc and SERT function in OFC. Copyright © 2015 Elsevier B.V. All rights reserved.

Levels of glutamate, aspartate, GABA, and taurine in different regions of the cerebellum after x-irradiation-induced neuronal loss

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Rea, M.A.; McBride, W.J.; Rohde, B.H.

1981-01-01

The levels of glutamate (Glu), aspartate (Asp), gamma-amino-n-butyric acid (GABA), and taurine (Tau) were determined in the cortex, molecular layer, and deep nuclei of cerebella of adult rats exposed to X-irradiation at 12-15 days following birth (to prevent the acquisition of late-forming granule cells; 12-15x group) and 8-15 days following birth (to prevent the acquisition of granule and stellate cells; 8-15x group). Also, the levels of the four amino acids were measured in the crude synaptosomal fraction (P2) isolated from the whole cerebella of the control, 12-15x, and 8-15x groups. The level of Glu was significantly decreased by (1) 6-20% in the cerebellar cortex; (2) 15-20% in the molecular layer; and (3) 25-50% in the P2 fraction of the X-irradiated groups relative to control values. The content of Glu in the deep nuclei was not changed by X-irradiation treatment. Regional levels of Asp were unchanged by X-irradiation, while its level in P2 decreased by 15-30% after treatment. The levels of GABA and Tau in the molecular layer, deep nuclei, or P2 were not changed in the experimental groups. However, there was a 15% increase in the levels of GABA and Tau in the cerebellar cortex of the 8-15x group relative to control values. The data support the proposed role of glutamate as the excitatory transmitter released from the cerebellar granule cells but are inconclusive regarding a transmitter role for either Tau or GABA from cerebellar stellate cells

A Subset of Palisade Endings Only in the Medial and Inferior Rectus Muscle in Monkey Contain Calretinin

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Lienbacher, Karoline; Ono, Seiji; Fleuriet, Jérome; Mustari, Michael; Horn, Anja K. E.

2018-01-01

Purpose To further chemically characterize palisade endings in extraocular muscles in rhesus monkeys. Methods Extraocular muscles of three rhesus monkeys were studied for expression of the calcium-binding protein calretinin (CR) in palisade endings and multiple endings. The complete innervation was visualized with antibodies against the synaptosomal-associated protein of 25 kDa and combined with immunofluorescence for CR. Six rhesus monkeys received tracer injections of choleratoxin subunit B or wheat germ agglutinin into either the belly or distal myotendinous junction of the medial or inferior rectus muscle to allow retrograde tracing in the C-group of the oculomotor nucleus. Double-immunofluorescence methods were used to study the CR content in retrogradely labeled neurons in the C-group. Results A subgroup of palisade and multiple endings was found to express CR, only in the medial and inferior rectus muscle. In contrast, the en plaque endings lacked CR. Accordingly, within the tracer-labeled neurons of the C-group, a subgroup expressed CR. Conclusions The study indicates that two different neuron populations targeting nontwitch muscle fibers are present within the C-group for inferior rectus and medial rectus, respectively, one expressing CR, one lacking CR. It is possible that the CR-negative neurons represent the basic population for all extraocular muscles, whereas the CR-positive neurons giving rise to CR-positive palisade endings represent a specialized, perhaps more excitable type of nerve ending in the medial and inferior rectus muscles, being more active in vergence. The malfunction of this CR-positive population of neurons that target nontwitch muscle fibers could play a significant role in strabismus.

Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects.

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Carpenter, Colleen; Zestos, Alexander G; Altshuler, Rachel; Sorenson, Roderick J; Guptaroy, Bipasha; Showalter, Hollis D; Kennedy, Robert T; Jutkiewicz, Emily; Gnegy, Margaret E

2017-09-01

Amphetamines (AMPHs) are globally abused. With no effective treatment for AMPH addiction to date, there is urgent need for the identification of druggable targets that mediate the reinforcing action of this stimulant class. AMPH-stimulated dopamine efflux is modulated by protein kinase C (PKC) activation. Inhibition of PKC reduces AMPH-stimulated dopamine efflux and locomotor activity. The only known CNS-permeant PKC inhibitor is the selective estrogen receptor modulator tamoxifen. In this study, we demonstrate that a tamoxifen analog, 6c, which more potently inhibits PKC than tamoxifen but lacks affinity for the estrogen receptor, reduces AMPH-stimulated increases in extracellular dopamine and reinforcement-related behavior. In rat striatal synaptosomes, 6c was almost fivefold more potent at inhibiting AMPH-stimulated dopamine efflux than [ 3 H]dopamine uptake through the dopamine transporter (DAT). The compound did not compete with [ 3 H]WIN 35,428 binding or affect surface DAT levels. Using microdialysis, direct accumbal administration of 1 μM 6c reduced dopamine overflow in freely moving rats. Using LC-MS, we demonstrate that 6c is CNS-permeant. Systemic treatment of rats with 6 mg/kg 6c either simultaneously or 18 h prior to systemic AMPH administration reduced both AMPH-stimulated dopamine overflow and AMPH-induced locomotor effects. Finally, 18 h pretreatment of rats with 6 mg/kg 6c s.c. reduces AMPH-self administration but not food self-administration. These results demonstrate the utility of tamoxifen analogs in reducing AMPH effects on dopamine and reinforcement-related behaviors and suggest a new avenue of development for therapeutics to reduce AMPH abuse.

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

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Carles Gil

Full Text Available Epsilon toxin (Etx is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3-phosphate and phosphatidylinositol (5-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

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Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

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Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-09-01

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Selective labeling of serotonin uptake sites in rat brain by [3H]citalopram contrasted to labeling of multiple sites by [3H]imipramine

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D'Amato, R.J.; Largent, B.L.; Snowman, A.M.; Snyder, S.H.

1987-01-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes [ 3 H]citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for [ 3 H]citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of [ 3 H]citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of [ 3 H]citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of [ 3 H]imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific [ 3 H]imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of [ 3 H]imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in [ 3 H]citalopram and serotonin-sensitive [ 3 H]imipramine binding with only a small effect on serotonin-insensitive [ 3 H]imipramine binding. The dissociation rate of [ 3 H]imipramine or [ 3 H]citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive [ 3 H]imipramine high affinity binding sites closely resembles that of [ 3 H]citalopram binding

Autoradiographic localization of 3H-paroxetine-labeled serotonin uptake sites in rat brain

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De Souza, E.B.; Kuyatt, B.L.

1987-01-01

Paroxetine is a potent and selective inhibitor of serotonin uptake into neurons. Serotonin uptake sites have been identified, localized, and quantified in rat brain by autoradiography with 3H-paroxetine; 3H-paroxetine binding in slide-mounted sections of rat forebrain was of high affinity (KD = 10 pM) and the inhibition affinity constant (Ki) values of various drugs in competing 3H-paroxetine binding significantly correlated with their reported potencies in inhibiting synaptosomal serotonin uptake. Serotonin uptake sites labeled by 3H-paroxetine were highly concentrated in the dorsal and median raphe nuclei, central gray, superficial layer of the superior colliculus, lateral septal nucleus, paraventricular nucleus of the thalamus, and the islands of Calleja. High concentrations of 3H-paroxetine binding sites were found in brainstem areas containing dopamine (substantia nigra and ventral tegmental area) and norepinephrine (locus coeruleus) cell bodies. Moderate concentrations of 3H-paroxetine binding sites were present in laminae I and IV of the frontal parietal cortex, primary olfactory cortex, olfactory tubercle, regions of the basal ganglia, septum, amygdala, thalamus, hypothalamus, hippocampus, and some brainstem areas including the interpeduncular, trigeminal, and parabrachial nuclei. Lower densities of 3H-paroxetine binding sites were found in other regions of the neocortex and very low to nonsignificant levels of binding were present in white matter tracts and in the cerebellum. Lesioning of serotonin neurons with 3,4-methylenedioxyamphetamine caused large decreases in 3H-paroxetine binding. The autoradiographic distribution of 3H-paroxetine binding sites in rat brain corresponds extremely well to the distribution of serotonin terminals and cell bodies as well as with the pharmacological sites of action of serotonin

Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

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Odeon, María Mercedes; Andreu, Marcela; Yamauchi, Laura; Grosman, Mauricio; Acosta, Gabriela Beatriz

2015-01-01

Postnatal stress alters stress responses for life, with serious consequences on the central nervous system (CNS), involving glutamatergic neurotransmission and development of voluntary alcohol intake. Several drugs of abuse, including alcohol and cocaine, alter glutamate transport (GluT). Here, we evaluated effects of chronic postnatal stress (CPS) on alcohol intake and brain glutamate uptake and transporters in male adolescent Wistar rats. For CPS from postnatal day (PD) 7, pups were separated from their mothers and exposed to cold stress (4 °C) for 1 h daily for 20 days; controls remained with their mothers. Then they were exposed to either voluntary ethanol (6%) or dextrose (1%) intake for 7 days (5-7 rats per group), then killed. CPS: (1) increased voluntary ethanol intake, (2) did not affect body weight gain or produce signs of toxicity with alcohol exposure, (3) increased glutamate uptake by hippocampal synaptosomes in vitro and (4) reduced protein levels (Western measurements) in hippocampus and frontal cortex of glial glutamate transporter-1 (GLT-1) and excitatory amino-acid transporter-3 (EAAT-3) but increased glutamate aspartate transporter (GLAST) levels. We propose that CPS-induced decrements in GLT-1 and EAAT-3 expression levels are opposed by activation of a compensatory mechanism to prevent excitotoxicity. A greater role for GLAST in total glutamate uptake to prevent enlarged extracellular glutamate levels is inferred. Although CPS strongly increased intake of ethanol, this had little impact on effects of CPS on brain glutamate uptake or transporters. However, the impact of early life adverse events on glutamatergic neurotransmission may underlie increased alcohol consumption in adulthood.

Type III Cells in Anterior Taste Fields Are More Immunohistochemically Diverse Than Those of Posterior Taste Fields in Mice.

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Wilson, Courtney E; Finger, Thomas E; Kinnamon, Sue C

2017-10-31

Activation of Type III cells in mammalian taste buds is implicated in the transduction of acids (sour) and salty stimuli. Several lines of evidence suggest that function of Type III cells in the anterior taste fields may differ from that of Type III cells in posterior taste fields. Underlying anatomy to support this observation is, however, scant. Most existing immunohistochemical data characterizing this cell type focus on circumvallate taste buds in the posterior tongue. Equivalent data from anterior taste fields-fungiform papillae and soft palate-are lacking. Here, we compare Type III cells in four taste fields: fungiform, soft palate, circumvallate, and foliate in terms of reactivity to four canonical markers of Type III cells: polycystic kidney disease 2-like 1 (PKD2L1), synaptosomal associated protein 25 (SNAP25), serotonin (5-HT), and glutamate decarboxylase 67 (GAD67). Our findings indicate that while PKD2L1, 5-HT, and SNAP25 are highly coincident in posterior taste fields, they diverge in anterior taste fields. In particular, a subset of taste cells expresses PKD2L1 without the synaptic markers, and a subset of SNAP25 cells lacks expression of PKD2L1. In posterior taste fields, GAD67-positive cells are a subset of PKD2L1 expressing taste cells, but anterior taste fields also contain a significant population of GAD67-only expressing cells. These differences in expression patterns may underlie the observed functional differences between anterior and posterior taste fields. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Alteration of putative amino acid levels and morphological findings in neural tissues of methylmercury-intoxicated mice

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Hirayama, K.; Inouye, M.; Fujisaki, T.

1985-04-01

Methylmercury chloride was administered PO to male Kud:ddY mice at a dose of 5 mg/kg/day for 20 days. The contents of taurine, aspartate, glutamate, glycine, and ..gamma..-aminobutyric acid were determined in tissue and crude synaptosomal (P/sub 2/) fraction of cerebellum, cerebral cortex, and spinal cord of methylmercury-treated mice with or without ataxia. In the cerebellum of ataxic mice, increased levels of taurine and glycine were found in the tissue and P/sub 2/ fraction, and increased levels of glutamate were found in the P/sub 2/ fraction. In the cerebral cortex, the levels of ..gamma..-aminobutylic acid decreased in the tissue and in the P/sub 2/ fraction of ataxic mice, but increased levels were found in the tissue of non-ataxic mice. A decreased asparate level in the cerebral cortex of ataxic mice and an increased taurine level in the cerebral cortex of non-ataxic mice were also found. In the spinal cord of ataxic mice, taurine increased in the tissue and in the P/sub 2/ fraction. Glutamate level decreased in the spinal cord of ataxic mice, but increased in the P/sub 2/ fraction of non-ataxic mice. Increased glycine levels in the P/sub 2/ fraction of the spinal cord were also found in non-axtaxic mice. Histologically, some degenerative changes were demonstrated in the cerebral and cerebellar cortices of ataxic mice. Such changes were also present to a mild degree in non-ataxic mice. In conclusion, methylmercury treatment altered the levels of putative neurotransmitter amino acids in neutral tissue of mice. These alterations might be caused by specific neural cell dysfunction and could be related to the appearance of ataxia.

Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress.

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Kaster, Manuella P; Machado, Nuno J; Silva, Henrique B; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E; Rodrigues, Ana Lúcia S; Porciúncula, Lisiane O; Chen, Jiang Fan; Tomé, Ângelo R; Agostinho, Paula; Canas, Paula M; Cunha, Rodrigo A

2015-06-23

The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function.

Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

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Hochheiser, Julia; Haase, Tobias; Busker, Mareike; Sömmer, Anne; Kreienkamp, Hans-Jürgen; Behrends, Sönke

2016-12-15

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α 1 and α 2 ) give rise to two heterodimeric enzymes α 1 /β 1 and α 2 /β 1 . Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α 2 /β 1 form is generally much lower and approaches levels similar to the α 1 /β 1 form in the brain only. In the present paper, we show that the α 2 /β 1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α 2 /β 1 form, but not the α 1 /β 1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α 2 subunit was sufficient for cell-cell contact localization. For the full length α 2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β 1 subunit, but not as isolated α 2 subunit. We conclude that the PDZ binding motif of the α 2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α 2 /β 1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective. Copyright © 2016 Elsevier Inc. All rights reserved.

The myth of nitric oxide in central cardiovascular control by the nucleus tractus solitarii

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Talman W.T.

1997-01-01

Full Text Available Considerable evidence suggests that nitroxidergic mechanisms in the nucleus tractus solitarii (NTS participate in cardiovascular reflex control. Much of that evidence, being based on responses to nitric oxide precursors or inhibitors of nitric oxide synthesis, has been indirect and circumstantial. We sought to directly determine cardiovascular responses to nitric oxide donors microinjected into the NTS and to determine if traditional receptor mechanisms might account for responses to certain of these donors in the central nervous system. Anesthetized adult Sprague Dawley rats that were instrumented for recording arterial pressure and heart rate were used in the physiological studies. Microinjection of nitric oxide itself into the NTS did not produce any cardiovascular responses and injection of sodium nitroprusside elicited minimal depressor responses. The S-nitrosothiols, S-nitrosoglutathione (GSNO, S-nitrosoacetylpenicillamine (SNAP, and S-nitroso-D-cysteine (D-SNC produced no significant cardiovascular responses while injection of S-nitroso-L-cysteine (L-SNC elicited brisk, dose-dependent depressor and bradycardic responses. In contrast, injection of glyceryl trinitrate elicited minimal pressor responses without associated changes in heart rate. It is unlikely that the responses to L-SNC were dependent on release of nitric oxide in that 1 the responses were not affected by injection of oxyhemoglobin or an inhibitor of nitric oxide synthesis prior to injection of L-SNC and 2 L- and D-SNC released identical amounts of nitric oxide when exposed to brain tissue homogenates. Although GSNO did not independently affect blood pressure, its injection attenuated responses to subsequent injection of L-SNC. Furthermore, radioligand binding studies suggested that in rat brain synaptosomes there is a saturable binding site for GSNO that is displaced from that site by L-SNC. The studies suggest that S-nitrosocysteine, not nitric oxide, may be an

Characterization of a novel and potentially lethal designer drug (±)-cis-para-methyl-4-methylaminorex (4,4'-DMAR, or 'Serotoni').

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Brandt, Simon D; Baumann, Michael H; Partilla, John S; Kavanagh, Pierce V; Power, John D; Talbot, Brian; Twamley, Brendan; Mahony, Olivia; O'Brien, John; Elliott, Simon P; Archer, Roland P; Patrick, Julian; Singh, Kuldip; Dempster, Nicola M; Cosbey, Simon H

2014-01-01

During the second half of 2013, a total of 26 deaths involving para-methyl-4-methylaminorex (4,4'-DMAR) were reported to the European Monitoring Centre for Drugs and Drug Addiction. While aminorex and 4-methylaminorex (4-MAR) are known psychostimulants, nothing is known about the comparatively new para-methyl analog. Analytical characterization of two independent samples obtained from online vendors confirmed the presence of the (±)-cis isomer that also appeared to be associated with at least 18 of the 26 deaths. Extensive characterizations included crystal structure analysis, single, tandem, and high-resolution mass spectrometry, liquid and gas chromatography, and nuclear magnetic resonance spectroscopy. For the work described here, both the (±)-cis and (±)-trans racemates were also synthesized, confirming that the differentiation between these two forms was straight-forward. Monoamine transporter activity was studied using rat brain synaptosomes which included the comparison with d-amphetamine, aminorex and (±)-cis-4-MAR. (±)-cis-4,4'-DMAR was a potent, efficacious substrate-type releaser at transporters for dopamine, norepinephrine and serotonin with EC50 values of 8.6 ± 1.1 nM (DAT), 26.9 ± 5.9 nM (NET) and 18.5 ± 2.8 nM (SERT), respectively. The potency of (±)-cis-4,4'-DMAR at DAT and NET rivalled that of other psychomotor stimulant drugs like d-amphetamine and aminorex. However, (±)-cis-4,4'-DMAR had much more potent actions at SERT and activity at SERT varied more than 100-fold across the four drugs. The potent releasing activity of (±)-cis-4,4'-DMAR at all three monoamine transporters predicts a potential for serious side-effects such as psychotic symptoms, agitation, hyperthermia and cardiovascular stimulation, especially after high-dose exposure or following combination with other psychostimulants. Copyright © 2014 John Wiley & Sons, Ltd.

Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

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Jennifer A Watson

2015-06-01

Full Text Available Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs, a component of the endosomal sorting complex required for transport (ESCRT. The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ, including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

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Roz, Netta; Rehavi, Moshe

2003-06-13

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

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Hong, Weimin Conrad; Yano, Hideaki; Hiranita, Takato; Chin, Frederick T; McCurdy, Christopher R; Su, Tsung-Ping; Amara, Susan G; Katz, Jonathan L

2017-07-07

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ 1 R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ 1 R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di- o -tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the B max values of [ 3 H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ 1 R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ 1 R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ 1 R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ 1 R antagonist CM304. Moreover, σ 1 R ligands had distinct effects on σ 1 R multimerization. CM304 increased the proportion of multimeric σ 1 Rs, whereas (+)-pentazocine increased monomeric σ 1 Rs. Together these results support the hypothesis that σ 1 R agonists promote dissociation of σ 1 R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ 1 R agonists in animal models. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Studies on the mechanism of the epileptiform activity induced by U18666A. II. concentration, half-life and distribution of radiolabeled U18666A in the brain

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Cenedella, R.J.; Sarkar, C.P.; Towns, L.

1982-06-01

The concentration, half-life, and distribution in brain of U18666A, a drug that can drastically alter cerebral lipids and induce a chronic epileptiform state, was determined following both acute and chronic drug administration. U18666A specifically labeled with tritium was prepared by custom synthesis. Brain levels of 1 x 10(-6)M and higher were reached soon after giving an acute 10-mg/kg dose (i.p. or s.c.) of U18666A containing 7-/sup 3/H-U18666A of known specific activity. A steady state concentration of 1 to 2 x 10(-6)M was reached with chronic injection of 10 mg/kg every 4th day, a treatment schedule that results in altered brain lipids and induction of epilepsy if begun soon after birth. The disappearance of U18666A from both brain and serum was described by two similar biexponential processes, a brief rapid clearance (t1/2 . 10 h) and a sustained and much slower one (t1/2 . 65 h). Brain levels of the drug were about 10 times higher than serum at all times examined. Few differences were seen in the regional distribution of radiolabeled drug in brain as determined by both direct analysis and by autoradiographic examination; but the drug did concentrate in lipid-rich subcellular fractions. For example, the synaptosome and myelin fractions each contained about 25-35% of both the total /sup 3/H-labeled drug and total lipid in whole brain. The lipid composition of these fractions was drastically altered in treated animals. In conclusion, the chronic epileptiform state induced by U18666A does not appear to involve localization of the drug in a specific brain region or particular cell type. Rather, the condition could involve localization of the drug in lipid-rich membranes and marked changes in the composition of these membranes.

Effects of chronic social isolation on Wistar rat behavior and brain plasticity markers.

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Djordjevic, Jelena; Djordjevic, Ana; Adzic, Miroslav; Radojcic, Marija B

2012-01-01

Chronic stress is a contributing risk factor in the development of psychiatric illnesses, including depressive disorders. The mechanisms of their psychopathology are multifaceted and include, besides others, alterations in the brain plasticity. Previously, we investigated the effects of chronic social stress in the limbic brain structures of Wistar rats (hippocampus, HIPPO, and prefrontal cortex, PFC) and found multiple characteristics that resembled alterations described in some clinical studies of depression. We extended our investigations and followed the behavior of stressed animals by the open field test (OFT) and forced swimming test (FST), and the expression and polysialylation of synaptic plasticity markers, neural cell adhesion molecule (NCAM) and L1, in the HIPPO and PFC. We also determined the adrenal gland mass and plasma corticosterone (CORT) as a terminal part of the hypothalamic-pituitary-adrenal axis activity. Our data indicated that stressed animals avoided the central zone in the OFT and displayed decreased swimming, but prolonged immobility in the FST. The animals exhibited marked hypertrophy of the adrenal gland cortex, in spite of decreased serum CORT. Simultaneously, the stressed animals exhibited an increase in NCAM mRNA expression in the HIPPO, but not in the PFC. The synaptosomal NCAM of the HIPPO was markedly polysialylated, while cortical PSA-NCAM was significantly decreased. The results showed that chronic social isolation of Wistar rats causes both anxiety-like and depression-like behavior. These alterations are parallel with molecular changes in the limbic brain, including diminished NCAM sialylation in the PFC. Together with our previous results, the current observations suggest that a chronic social isolation model may potentially be used to study molecular mechanisms that underlie depressive symptomatology. Copyright © 2012 S. Karger AG, Basel.

Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats

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Anibal Pacheco

2017-08-01

Full Text Available Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA and N-methyl-D-aspartate (NMDA receptor subunits in dorsal and ventral hippocampus (VH. Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH. By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.

Prion protein accumulation in lipid rafts of mouse aging brain.

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Federica Agostini

Full Text Available The cellular form of the prion protein (PrP(C is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C. In old mice, this change favors PrP(C accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C translocation into detergent-resistant membranes (DRMs, we looked at PrP(C compartmentalization in hippocampi from acid sphingomyelinase (ASM knockout (KO mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.

Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons.

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Varaschin, Rafael Koerich; Osterstock, Guillaume; Ducrot, Charles; Leino, Sakari; Bourque, Marie-Josée; Prado, Marco A M; Prado, Vania Ferreira; Salminen, Outi; Rannanpää Née Nuutinen, Saara; Trudeau, Louis-Eric

2018-04-15

Histamine H 3 receptors are widely distributed G i -coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H 3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H 3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H 3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H 3 -modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H 3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H 3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H 3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons. Copyright © 2018 IBRO

Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

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Kyle C Wilcox

Full Text Available Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs. AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs. This method gives a soluble membrane protein library (SMPL--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can

Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

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Vigelsø, Andreas; Gram, Martin; Wiuff, Caroline; Hansen, Christina Neigaard; Prats, Clara; Dela, Flemming; Helge, Jørn Wulff

2016-03-01

Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

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Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

2016-11-01

Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.

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Lebrun, B; Romi-Lebrun, R; Martin-Eauclaire, M F; Yasuda, A; Ishiguro, M; Oyama, Y; Pongs, O; Nakajima, T

1997-11-15

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

Decreased hippocampal homoarginine and increased nitric oxide and nitric oxide synthase levels in rats parallel training in a radial arm maze.

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Sase, Ajinkya; Nawaratna, Gayan; Hu, Shengdi; Wu, Guoyao; Lubec, Gert

2016-09-01

L-homoarginine (hArg) is derived from enzymatic guanidination of lysine. It was demonstrated that hArg is a substrate for nitric oxide (NO) synthesis, blocks lysine transport and inhibits the uptake of arginine into synaptosomes and modulates GABA responses ex vivo. As there is limited information on its physiological roles in the brain, the aim of the study was to show whether hippocampal or frontal lobe (FL) hArg is paralleling training in the radial arm maze (RAM) or NO formation. Hippocampi and FL of male Sprague-Dawley rats were taken from trained or yoked in a RAM. Then hArg and metabolites, NO and NO synthase (NOS) were determined by standard methods. The animals learned the task in the RAM showing significant reduction of working memory errors. hArg showed decreased levels in both brain regions of trained animals as compared to yoked animals. Nitrate plus nitrite (NOx) concentrations and NOS activity were significantly increased in hippocampi, F(1,36) = 170.5; P ≤ 0.0001 and FL, F(1,36) = 74.67; P ≤ 0.0001 of trained animals as compared to yoked animals. Levels of hArg were negatively correlated with NOx in hippocampus (r = -0.6355; P = 0.0483) but not in FL and with lysine in the FL (r = -0.6650; P = 0.0358). NOx levels were positively correlated with NOS in both the hippocampus (r = 0.7474; P = 0.0129) and FL (r = 0.9563; P ≤  0.0001). These novel findings indicate that hArg is linked to NO formation in hippocampus but not in FL and is paralleling spatial memory in the RAM.

Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease.

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Calon, Frédéric; Lim, Giselle P; Morihara, Takashi; Yang, Fusheng; Ubeda, Oliver; Salem, Norman; Frautschy, Sally A; Cole, Greg M

2005-08-01

Epidemiological data indicate that low n-3 polyunsaturated fatty acids (PFA) intake is a readily manipulated dietary risk factor for Alzheimer's disease (AD). Studies in animals confirm the deleterious effect of n-3 PFA depletion on cognition and on dendritic scaffold proteins. Here, we show that in transgenic mice overexpressing the human AD gene APPswe (Tg2576), safflower oil-induced n-3 PFA deficiency caused a decrease in N-methyl-D-aspartate (NMDA) receptor subunits, NR2A and NR2B, in the cortex and hippocampus with no loss of the presynaptic markers, synaptophysin and synaptosomal-associated protein 25 (SNAP-25). n-3 PFA depletion also decreased the NR1 subunit in the hippocampus and Ca2+/calmodulin-dependent protein kinase (CaMKII) in the cortex of Tg2576 mice. These effects of dietary n-3 PFA deficiency were greatly amplified in Tg2576 mice compared to nontransgenic mice. Loss of the NR2B receptor subunit was not explained by changes in mRNA expression, but correlated with p85alpha phosphatidylinositol 3-kinase levels. Most interestingly, n-3 PFA deficiency dramatically increased levels of protein fragments, corresponding to caspase/calpain-cleaved fodrin and gelsolin in Tg2576 mice. This effect was minimal in nontransgenic mice suggesting that n-3 PFA depletion potentiated caspase activation in the Tg2576 mouse model of AD. Dietary supplementation with docosahexaenoic acid (DHA; 22 : 6n-3) partly protected from NMDA receptor subunit loss and accumulation of fodrin and gelsolin fragments but fully prevented CaMKII decrease. The marked effect of dietary n-3 PFA on NMDA receptors and caspase/calpain activation in the cortex of an animal model of AD provide new insights into how dietary essential fatty acids may influence cognition and AD risk.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

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Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1999-01-01

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary

Psychostimulant Effect of the Synthetic Cannabinoid JWH-018 and AKB48: Behavioral, Neurochemical, and Dopamine Transporter Scan Imaging Studies in Mice.

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Ossato, Andrea; Uccelli, Licia; Bilel, Sabrine; Canazza, Isabella; Di Domenico, Giovanni; Pasquali, Micol; Pupillo, Gaia; De Luca, Maria Antonietta; Boschi, Alessandra; Vincenzi, Fabrizio; Rimondo, Claudia; Beggiato, Sarah; Ferraro, Luca; Varani, Katia; Borea, Pier Andrea; Serpelloni, Giovanni; De-Giorgio, Fabio; Marti, Matteo

2017-01-01

JWH-018 and AKB48 are two synthetic cannabinoids (SCBs) belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. In vivo studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB 1 receptor blockade and dopamine (DA) D 1/5 and D 2/3 receptors inhibition. SPECT-CT studies on dopamine transporter (DAT) revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [ 123 I]-FP-CIT binding in the mouse striatum. Conversely, in vitro competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc) shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [ 3 H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.

Psychostimulant Effect of the Synthetic Cannabinoid JWH-018 and AKB48: Behavioral, Neurochemical, and Dopamine Transporter Scan Imaging Studies in Mice

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Andrea Ossato

2017-08-01

Full Text Available JWH-018 and AKB48 are two synthetic cannabinoids (SCBs belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. In vivo studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB1 receptor blockade and dopamine (DA D1/5 and D2/3 receptors inhibition. SPECT-CT studies on dopamine transporter (DAT revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [123I]-FP-CIT binding in the mouse striatum. Conversely, in vitro competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [3H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.

The new psychoactive substances 5-(2-aminopropyl)indole (5-IT) and 6-(2-aminopropyl)indole (6-IT) interact with monoamine transporters in brain tissue.

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Marusich, Julie A; Antonazzo, Kateland R; Blough, Bruce E; Brandt, Simon D; Kavanagh, Pierce V; Partilla, John S; Baumann, Michael H

2016-02-01

In recent years, use of psychoactive synthetic stimulants has grown rapidly. 5-(2-Aminopropyl)indole (5-IT) is a synthetic drug associated with a number of fatalities, that appears to be one of the newest 3,4-methylenedioxymethamphetamine (MDMA) replacements. Here, the monoamine-releasing properties of 5-IT, its structural isomer 6-(2-aminopropyl)indole (6-IT), and MDMA were compared using in vitro release assays at transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) in rat brain synaptosomes. In vivo pharmacology was assessed by locomotor activity and a functional observational battery (FOB) in mice. 5-IT and 6-IT were potent substrates at DAT, NET, and SERT. In contrast with the non-selective releasing properties of MDMA, 5-IT displayed greater potency for release at DAT over SERT, while 6-IT displayed greater potency for release at SERT over DAT. 5-IT produced locomotor stimulation and typical stimulant effects in the FOB similar to those produced by MDMA. Conversely, 6-IT increased behaviors associated with 5-HT toxicity. 5-IT likely has high abuse potential, which may be somewhat diminished by its slow onset of in vivo effects, whereas 6-IT may have low abuse liability, but enhanced risk for adverse effects. Results indicate that subtle differences in the chemical structure of transporter ligands can have profound effects on biological activity. The potent monoamine-releasing actions of 5-IT, coupled with its known inhibition of MAO A, could underlie its dangerous effects when administered alone, and in combination with other monoaminergic drugs or medications. Consequently, 5-IT and related compounds may pose substantial risk for abuse and serious adverse effects in human users. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pharmacological evaluation of an [(123)I] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors.

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Mattner, Filomena; Mardon, Karine; Loc'h, Christian; Katsifis, Andrew

2006-06-13

In vitro binding of the iodinated imidazopyridine, N',N'-dimethyl-6-methyl-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [(123)I]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [(123)I]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K(d)=30 nM). The density of binding sites was 22+/-6 and 1.2+/-0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [(123)I]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [(123)I]IZOL by 30% (p<0.05) in olfactory bulbs and by 51-86% (p<0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p<0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p<0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [(123)I]IZOL in peripheral organs and in the brain. [(123)I]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites.

Lateral Fluid Percussion Injury Impairs Hippocampal Synaptic Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Complex Formation

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Shaun W. Carlson

2017-10-01

Full Text Available Traumatic brain injury (TBI and the activation of secondary injury mechanisms have been linked to impaired cognitive function, which, as observed in TBI patients and animal models, can persist for months and years following the initial injury. Impairments in neurotransmission have been well documented in experimental models of TBI, but the mechanisms underlying this dysfunction are poorly understood. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE complex facilitates vesicular docking and neurotransmitter release in the synaptic cleft. Published studies highlight a direct link between reduced SNARE complex formation and impairments in neurotransmitter release. While alterations in the SNARE complex have been described following severe focal TBI, it is not known if deficits in SNARE complex formation manifest in a model with reduced severity. We hypothesized that lateral fluid percussion injury (lFPI reduces the abundance of SNARE proteins, impairs SNARE complex formation, and contributes to impaired neurobehavioral function. To this end, rats were subjected to lFPI or sham injury and tested for acute motor performance and cognitive function at 3 weeks post-injury. lFPI resulted in motor impairment between 1 and 5 days post-injury. Spatial acquisition and spatial memory, as assessed by the Morris water maze, were significantly impaired at 3 weeks after lFPI. To examine the effect of lFPI on synaptic SNARE complex formation in the injured hippocampus, a separate cohort of rats was generated and brains processed to evaluate hippocampal synaptosomal-enriched lysates at 1 week post-injury. lFPI resulted in a significant reduction in multiple monomeric SNARE proteins, including VAMP2, and α-synuclein, and SNARE complex abundance. The findings in this study are consistent with our previously published observations suggesting that impairments in hippocampal SNARE complex formation may contribute to

Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

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Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

2015-01-01

Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

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Wilcox, Kyle C; Marunde, Matthew R; Das, Aditi; Velasco, Pauline T; Kuhns, Benjamin D; Marty, Michael T; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G; Klein, William L

2015-01-01

Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery

Caffeine triggers behavioral and neurochemical alterations in adolescent rats.

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Ardais, A P; Borges, M F; Rocha, A S; Sallaberry, C; Cunha, R A; Porciúncula, L O

2014-06-13

Caffeine is the psychostimulant most consumed worldwide but concerns arise about the growing intake of caffeine-containing drinks by adolescents since the effects of caffeine on cognitive functions and neurochemical aspects of late brain maturation during adolescence are poorly known. We now studied the behavioral impact in adolescent male rats of regular caffeine intake at low (0.1mg/mL), moderate (0.3mg/mL) and moderate/high (1.0mg/mL) doses only during their active period (from 7:00 P.M. to 7:00 A.M.). All tested doses of caffeine were devoid of effects on locomotor activity, but triggered anxiogenic effects. Caffeine (0.3 and 1mg/mL) improved the performance in the object recognition task, but the higher dose of caffeine (1.0mg/mL) decreased the habituation to an open-field arena, suggesting impaired non-associative memory. All tested doses of caffeine decreased the density of glial fibrillary acidic protein and synaptosomal-associated protein-25, but failed to modify neuron-specific nuclear protein immunoreactivity in the hippocampus and cerebral cortex. Caffeine (0.3-1mg/mL) increased the density of brain-derived neurotrophic factor (BDNF) and proBDNF density as well as adenosine A1 receptor density in the hippocampus, whereas the higher dose of caffeine (1mg/mL) increased the density of proBDNF and BDNF and decreased A1 receptor density in the cerebral cortex. These findings document an impact of caffeine consumption in adolescent rats with a dual impact on anxiety and recognition memory, associated with changes in BDNF levels and decreases of astrocytic and nerve terminal markers without overt neuronal damage in hippocampal and cortical regions. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

The non-neuronal and nonmuscular effects of botulinum toxin: an opportunity for a deadly molecule to treat disease in the skin and beyond.

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Grando, S A; Zachary, C B

2018-05-01

There is growing evidence that botulinum neurotoxins (BoNTs) exhibit biological effects on various human cell types with a host of associated clinical implications. This review aims to provide an update on the non-neuronal and nonmuscular effects of botulinum toxin. We critically analysed recent reports on the structure and function of cellular signalling systems subserving biological effects of BoNTs. The BoNT receptors and intracellular targets are not unique for neurotransmission. They have been found in both neuronal and non-neuronal cells, but there are differences in how BoNT binds to, and acts on, neuronal vs. non-neuronal cells. The non-neuronal cells that express one or more BoNT/A-binding proteins, and/or cleavage target synaptosomal-associated protein 25, include: epidermal keratinocytes; mesenchymal stem cells from subcutaneous adipose; nasal mucosal cells; urothelial cells; intestinal, prostate and alveolar epithelial cells; breast cell lines; neutrophils; and macrophages. Serotype BoNT/A can also elicit specific biological effects in dermal fibroblasts, sebocytes and vascular endothelial cells. Nontraditional applications of BoNT have been reported for the treatment of the following dermatological conditions: hyperhidrosis, Hailey-Hailey disease, Darier disease, inversed psoriasis, aquagenic palmoplantar keratoderma, pachyonychia congenita, multiple eccrine hydrocystomas, eccrine angiomatous hamartoma, eccrine sweat gland naevi, congenital eccrine naevus, Raynaud phenomenon and cutaneous leiomyomas. Experimental studies have demonstrated the ability of BoNT/A to protect skin flaps, facilitate wound healing, decrease thickness of hypertrophic scars, produce an anti-ageing effect, improve a mouse model of psoriasiform dermatitis, and have also revealed extracutaneous effects of BoNT arising from its anti-inflammatory and anticancer properties. BoNTs have a much wider range of applications than originally understood, and the individual cellular responses

Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain composition of rat gastrocnemius muscles.

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Hong, Bin; Chen, Min; Hu, Xing-yue

2013-11-01

Botulinum toxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of a synaptosomal-associated protein of 25 kDa (SNAP-25). It has gained widespread use as a treatment for cerebral palsy and skeletal muscle hypertrophy. In China, Chinese botulinum toxin type A (CBTX-A), a type of BoNT/A, is in widespread clinical use. However, the changes in the morphological and biochemical properties of treated muscles and in remote muscles from the CBTX-A injection site are relatively unknown. Therefore, we investigated the changes in histomorphology and myosin heavy chain (MyHC) isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A. The weakness of the injected muscles was assessed periodically to identify their functional deficiency. Muscle slices were stained with hematoxylin-eosin (HE) and adenosine triphosphatase (ATPase). MyHC isoform composition was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to uncover changes in morphological and biochemical properties. Our findings demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles, shifts in MyHC isoform composition emerged on the third day after injection and peaked in the fourth week. The composition remained distinctly different from that of the control group after the twelfth week. More specifically, there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx, type IIa, and type I isoforms. Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that the MyHC composition remained far from normal six months after a single injection. However, no noticeable remote muscle weakness was induced.

Exogenous hydrogen sulfide eliminates spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress via promoting glutamate uptake.

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He, Jin; Guo, Ruixian; Qiu, Pengxin; Su, Xingwen; Yan, Guangmei; Feng, Jianqiang

2017-05-14

Acute stress impairs the hippocampus-dependent spatial memory retrieval, and its synaptic mechanisms are associated with hippocampal CA1 long-term depression (LTD) enhancement in the adult rats. Endogenous hydrogen sulfide (H 2 S) is recognized as a novel gasotransmitter and has the neural protective roles. However, very little attention has been paid to understanding the effects of H 2 S on spatial memory retrieval impairment. We observed the protective effects of NaHS (a donor of H 2 S) against spatial memory retrieval impairment caused by acute stress and its synaptic mechanisms. Our results showed that NaHS abolished spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress, but not by glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic (tPDC), indicating that the activation of glutamate transporters is necessary for exogenous H 2 S to exert its roles. Moreover, NaHS restored the decreased glutamate uptake in the hippocampal CA1 synaptosomal fraction caused by acute stress. Dithiothreitol (DTT, a disulfide reducing agent) abolished a decrease in the glutamate uptake caused by acute stress, and NaHS eradicated the decreased glutamate uptake caused by 5,5'-dithio-bis(2-nitrobenzoic)acid (DTNB, a thiol oxidizing agent), collectively, revealing that exogenous H 2 S increases glutamate uptake by reducing disulfide bonds of the glutamate transporters. Additionally, NaHS inhibited the increased expression level of phosphorylated c-Jun-N-terminal kinase (JNK) in the hippocampal CA1 region caused by acute stress. The JNK inhibitor SP600125 eliminated spatial memory retrieval impairment, hippocampal CA1 LTD enhancement and the decreased glutamate uptake caused by acute stress, indicating that exogenous H 2 S exerts these roles by inhibiting the activation of JNK signaling pathway. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

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Zhang, Qiuxia; Na, Quan; Song, Weiwei

2017-10-01

Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

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Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

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Hyunjeong Yang

Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

[In vitro release of [5Met]- and [5Leu]-enkephalins from the rat brain crude synaptosomal (P2) fraction: Ca2+-dependency of K+-stimulation and effects of various drugs].

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Koida, M; Takahashi, M; Takenaga, K

1983-01-01

The rat brain P2 fraction was suspended in Krebs Ringer bicarbonate buffer containing 20 microM bacitracin and incubated at 37 degrees C for 10 min under an atmosphere of 95% O2-5% CO2. Incubation was terminated by centrifugation at 4 degrees C and 10,000 X g for 10 min. The supernatant was designated as the S1 fraction, and from the pellet, the S2 to S4 fractions were collected by repeated suspension, incubation, and centrifugation. The radioimmunoassays of each S fraction revealed the spontaneous release of [5Met]- and [5Leu]-enkephalins at the ratio of 2 to 1. The peptide contents gradually decreased from S1 to S4, but the release tended to become constant in S3 and S4. Thus, the effects of some ions and drugs on the release were compared at the stage of obtaining the S3 fraction. The release of both peptides were significantly stimulated in 50 mM KCl buffer, and the stimulatory effect appears to be dependent on Ca2+ concentration. Veratrine and A23187 were also effective stimulants of the release. On the other hand, neither spontaneous nor K+-stimulated release of enkephalins was affected by morphine (1 microM), naloxone (1 microM), kyotorphin (1 or 10 microM), and Li+ (50 mM). Similar results were obtained with the release of 3H-noradrenaline taken up in vitro by the P2 fraction. The usability of the P2 fraction as an in vitro model for the study of stimulus-coupled release of enkephalins was discussed with some limitations found herein.

A case of paroxysmal kinesigenic dyskinesia which exhibited the phenotype of anxiety disorder

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Kunii Y

2017-08-01

levels, and the recent advances in the molecular understanding of PKD suggested slight, but widespread alterations to the neurotransmitter systems in the brain. Keywords: paroxysmal kinesigenic dyskinesia, anxiety disorder, proline-rich transmembrane protein 2, synaptosomal-associated protein 25

A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

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Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D. (UIUC); (Scripps); (UCSF)

2010-08-13

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

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Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

2014-01-01

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Role of sodium-calcium exchange in regulation of intracellular calcium in nerve terminals

International Nuclear Information System (INIS)

Sanchez-Armass, S.; Blaustein, M.P.

1987-01-01

Ca efflux from rat brain presynaptic nerve terminals (synaptosomes) was examined after loading the terminals with 45 Ca during a brief depolarization, usually in media containing 20 μM Ca labeled with 45 Ca, to assure a small (physiological) load. Efflux of 45 Ca was very slow in the absence of external Na and Ca and was greatly accelerated by Na and/or Ca. The dependence of 45 Ca efflux on external Na was sigmoid, with a Hill coefficient of ∼ 4.5; this implies that more than two external Na ions are required to activate the efflux of one Ca ion. The external Na (Na 0 )-dependent Ca efflux was inhibited by 1 mM external La, by low temperature, and by raising external K. With small Ca loads, the mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), had negligible effect on either Ca uptake or efflux; with large loads, however, FCCP reduced the depolarization-stimulated Ca uptake and increased the Na 0 -dependent Ca efflux. These effects may be attributed to reduction of mitochondrial Ca sequestration. Mitochondria do not appear to sequester much Ca when the loads are smaller. Estimations of Ca efflux indicate that ∼ 20% of a small 45 Ca load may be extruded via Na + -Ca 2+ exchange within 1 s; this corresponds to a net Ca efflux of ∼ 110 pmol Ca x mg protein -1 x s -1 . This rate of extrusion is equivalent to the net Ca gain when the terminals fire at a frequency of ∼ 18/s. The data on the Ca efflux into Na- and Ca-free media indicate that the ATP-fueled Ca pump can only extrude ∼ 10-20 pmol Ca x mg protein -1 x s -1 . Thus the results imply that Na + -Ca 2+ exchange plays an important role in helping to extrude the Ca that enters during activity

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies

Energy Technology Data Exchange (ETDEWEB)

Zeng Zhizhen [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)], E-mail: zhizhen_zeng@merck.com; O' Brien, Julie A. [Sleep and Psychiatric Disorders, Merck Research Laboratories, West Point, PA 19486 (United States); Lemaire, Wei [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); O' Malley, Stacey S.; Miller, Patricia J. [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States); Zhao Zhijian [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); Wallace, Michael A. [Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065 (United States); Raab, Conrad [Drug Metabolism, Merck Research Laboratories, West Point, PA 19486 (United States); Lindsley, Craig W. [Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486 (United States); Departments of Pharmacology and Chemistry, Vanderbilt University, Nashville, TN 37232 (United States); Sur, Cyrille; Williams, David L. [Imaging, Merck Research Laboratories, West Point, PA 19486 (United States)

2008-04-15

Introduction: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [{sup 35}S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl) ethyl)benzamide ([{sup 35}S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl) benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. Methods: Functional potencies of unlabeled compounds were characterized by [{sup 14}C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. Results: ACPPB is a potent (K{sub d}=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [{sup 35}S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [{sup 35}S]ACPPB in rat brain tissues following iv administration of this radioligand. Conclusions: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop

Atomoxetine affects transcription/translation of the NMDA receptor and the norepinephrine transporter in the rat brain – an in vivo study

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Udvardi, Patrick T; Föhr, Karl J; Henes, Carolin; Liebau, Stefan; Dreyhaupt, Jens; Boeckers, Tobias M; Ludolph, Andrea G

2013-01-01

Attention-deficit/hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET) inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR) antagonist. The compound’s effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B) are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip]) or saline (0.9%, ip) for 21 consecutive days on postnatal days (PND) 21–41. In humans, atomoxetine’s earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR), mesencephalon (MES), and hippocampus (HC) was analyzed either directly after treatment (PND 42) or 2 months after termination of treatment (PND 101) to assess the compound’s long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine’s effects beyond NET inhibition. Taken together, these data reveal that atomoxetine seems to decrease glutamatergic transmission in a brain region-specific manner. Long-term data show that the compound’s impact is not due to an acute pharmacological effect but lasts or even amplifies after a drug-free period of 2 months, leading to altered development of

Role of nitric oxide in long-term potentiation of the rat medial vestibular nuclei.

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Grassi, S; Pettorossi, V E

2000-01-01

In rat brainstem slices, we investigated the role of nitric oxide in long-term potentiation induced in the ventral portion of the medial vestibular nuclei by high-frequency stimulation of the primary vestibular afferents. The nitric oxide scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide ] and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester were administered before and after induction of potentiation. Both drugs completely prevented long-term potentiation, whereas they did not impede the potentiation build-up, or affect the already established potentiation. These results demonstrate that the induction, but not the maintenance of vestibular long-term potentiation, depends on the synthesis and release into the extracellular medium of nitric oxide. In addition, we analysed the effect of the nitric oxide donor sodium nitroprusside on vestibular responses. Sodium nitroprusside induced long-term potentiation, as evidenced through the field potential enhancement and unit peak latency decrease. This potentiation was impeded by D, L-2-amino-5-phosphonopentanoic acid, and was reduced under blockade of synaptosomal platelet-activating factor receptors by ginkgolide B and group I metabotropic glutamate receptors by (R,S)-1-aminoindan-1, 5-dicarboxylic acid. When reduced, potentiation fully developed following the washout of antagonist, demonstrating an involvement of platelet-activating factor and group I metabotropic glutamate receptors in its full development. Potentiation induced by sodium nitroprusside was also associated with a decrease in the paired-pulse facilitation ratio, which persisted under ginkgolide B, indicating that nitric oxide increases glutamate release independently of platelet-activating factor-mediated presynaptic events. We suggest that nitric oxide, released after the activation of N-methyl-D-aspartate receptors, acts as a retrograde messenger leading to an enhancement of glutamate release to a

Spermidine decreases Na⁺,K⁺-ATPase activity through NMDA receptor and protein kinase G activation in the hippocampus of rats.

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Carvalho, Fabiano B; Mello, Carlos F; Marisco, Patricia C; Tonello, Raquel; Girardi, Bruna A; Ferreira, Juliano; Oliveira, Mauro S; Rubin, Maribel A

2012-06-05

Spermidine is an endogenous polyamine with a polycationic structure present in the central nervous system of mammals. Spermidine regulates biological processes, such as Ca(2+) influx by glutamatergic N-methyl-d-aspartate receptor (NMDA receptor), which has been associated with nitric oxide synthase (NOS) and cGMP/PKG pathway activation and a decrease of Na(+),K(+)-ATPase activity in rats' cerebral cortex synaptosomes. Na(+),K(+)-ATPase establishes Na(+) and K(+) gradients across membranes of excitable cells and by this means maintains membrane potential and controls intracellular pH and volume. However, it has not been defined whether spermidine modulates Na(+),K(+)-ATPase activity in the hippocampus. In this study we investigated whether spermidine alters Na(+),K(+)-ATPase activity in slices of hippocampus from rats, and possible underlying mechanisms. Hippocampal slices and homogenates were incubated with spermidine (0.05-10 μM) for 30 min. Spermidine (0.5 and 1 μM) decreased Na(+),K(+)-ATPase activity in slices, but not in homogenates. MK-801 (100 and 10 μM), a non-competitive antagonist of NMDA receptor, arcaine (0.5μM), an antagonist of the polyamine binding site at the NMDA receptor, and L-NAME (100μM), a NOS inhibitor, prevented the inhibitory effect of spermidine (0.5 μM). ODQ (10 μM), a guanylate cyclase inhibitor, and KT5823 (2 μM), a protein kinase G inhibitor, also prevented the inhibitory effect of spermidine on Na(+),K(+)-ATPase activity. Spermidine (0.5 and 1.0 μM) increased NO(2) plus NO(3) (NOx) levels in slices, and MK-801 (100 μM) and arcaine (0.5 μM) prevented the effect of spermidine (0.5 μM) on the NOx content. These results suggest that spermidine-induced decrease of Na(+),K(+)-ATPase activity involves NMDA receptor/NOS/cGMP/PKG pathway. Copyright © 2012 Elsevier B.V. All rights reserved.

Recruitment of GABA(A) receptors and fearfulness in chicks: modulation by systemic insulin and/or epinephrine.

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Cid, Mariana Paula; Toledo, Carolina Maribel; Salvatierra, Nancy Alicia

2013-02-01

One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation

The antidepressant-like effect of 7-fluoro-1,3-diphenylisoquinoline-1-amine in the mouse forced swimming test is mediated by serotonergic and dopaminergic systems.

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Pesarico, Ana Paula; Sampaio, Tuane Bazanella; Stangherlin, Eluza Curte; Mantovani, Anderson C; Zeni, Gilson; Nogueira, Cristina Wayne

2014-10-03

The aim of the present study was to investigate the role of monoaminergic system in the antidepressant-like action of 7-fluoro-1,3-diphenylisoquinoline-1-amine (FDPI), a derivative of isoquinoline class, in Swiss mice. The antidepressant-like effect of FDPI was characterized in the modified forced swimming test (FST) and the possible mechanism of action was investigated by using serotonergic, dopaminergic and noradrenergic antagonists. Monoamine oxidase (MAO) activity and [(3)H]serotonin (5-HT) uptake were determined in prefrontal cortices of mice. The results showed that FDPI (1, 10 and 20mg/kg, i.g.) reduced the immobility time and increased the swimming time but did not alter climbing time in the modified FST. These effects were similar to those of paroxetine (8mg/kg, i.p.), a positive control. Pretreatments with p-chlorophenylalanine (100mg/kg, i.p., an inhibitor of 5-HT synthesis), WAY100635 (0.1mg/kg, s.c., 5-HT1A antagonist), ondansetron (1mg/kg, i.p., a 5-HT3 receptor antagonist), haloperidol (0.2mg/kg, i.p., a non-selective D2 receptor antagonist) and SCH23390 (0.05mg/kg, s.c., a D1 receptor antagonist) were effective to block the antidepressant-like effect of FDPI at a dose of 1mg/kg in the FST. Ritanserin (1mg/kg, i.p., a 5-HT2A/2C receptor antagonist), sulpiride (50mg/kg, i.p., a D2 and D3 receptor antagonist), prazosin (1mg/kg, i.p., an α1 receptor antagonist), yohimbine (1mg/kg, i.p., an α2 receptor antagonist) and propranolol (2mg/kg, i.p., a β receptor antagonist) did not modify the effect of FDPI in the FST. FDPI did not change synaptosomal [(3)H]5-HT uptake. At doses of 10 and 20mg/kg FDPI inhibited MAO-A and MAO-B activities. These results suggest that antidepressant-like effect of FDPI is mediated mostly by serotonergic and dopaminergic systems. Copyright © 2014 Elsevier Inc. All rights reserved.

Presynaptic Dopamine D2 Receptors Modulate [3H]GABA Release at StriatoPallidal Terminals via Activation of PLC → IP3 → Calcineurin and Inhibition of AC → cAMP → PKA Signaling Cascades.

Science.gov (United States)

Jijón-Lorenzo, Rafael; Caballero-Florán, Isaac Hiram; Recillas-Morales, Sergio; Cortés, Hernán; Avalos-Fuentes, José Arturo; Paz-Bermúdez, Francisco Javier; Erlij, David; Florán, Benjamín

2018-02-21

Striatal dopamine D2 receptors activate the PLC → IP3 → Calcineurin-signaling pathway to modulate the neural excitability of En+ Medium-sized Spiny GABAergic neurons (MSN) through the regulation of L-type Ca 2+ channels. Presynaptic dopaminergic D2 receptors modulate GABA release at striatopallidal terminals through L-type Ca 2+ channels as well, but their signaling pathway is still undetermined. Since D2 receptors are Gi/o-coupled and negatively modulate adenylyl cyclase (AC), we investigated whether presynaptic D2 receptors modulate GABA release through the same signaling cascade that controls excitability in the striatum or by the inhibition of AC and decreased PKA activity. Activation of D2 receptors stimulated formation of [ 3 H]IP 1 and decreased Forskolin-stimulated [ 3 H]cAMP accumulation in synaptosomes from rat Globus Pallidus. D2 receptor activation with Quinpirole in the presence of L 745,870 decreased, in a dose-dependent manner, K + -induced [ 3 H]GABA release in pallidal slices. The effect was prevented by the pharmacological blockade of Gi/o βγ subunit effects with Gallein, PLC with U 73122, IP3 receptor activation with 4-APB, Calcineurin with FK506. In addition, when release was stimulated with Forskolin to activate AC, D2 receptors also decreased K + -induced [ 3 H]GABA release, an effect occluded with the effect of the blockade of PKA with H89 or stimulation of release with the cAMP analog 8-Br-cAMP. These data indicate that D2 receptors modulate [ 3 H]GABA release at striatopallidal terminals by activating the PLC → IP3 → Calcineurin-signaling cascade, the same one that modulates excitability in soma. Additionally, D2 receptors inhibit release when AC is active. Both mechanisms appear to converge to regulate the activity of presynaptic L-type Ca 2+ channels. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

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Sandeep eKumar

2012-04-01

Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

Oxidative stress and toxicity induced by the nucleoside reverse transcriptase inhibitor (NRTI)--2',3'-dideoxycytidine (ddC): relevance to HIV-dementia.

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Opii, Wycliffe O; Sultana, Rukhsana; Abdul, Hafiz Mohmmad; Ansari, Mubeen Ahmad; Nath, Avindra; Butterfield, D Allan

2007-03-01

Human immunodeficiency virus dementia (HIVD) is the most common form of dementia occurring among young adults. In HIVD, neuronal cell loss occurs in the absence of neuronal infection. With the advent of highly active anti-retroviral therapy (HAART), the incidence of HIVD has drastically reduced, though prevalence of milder forms of HIVD continues to rise. Though these agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. Here we examine the possible role of NRTIs, in particular 2',3'-dideoxycytidine (ddC), in the neuropathology of HIVD. Synaptosomes and isolated mitochondria treated and incubated for 6 h with CSF-achievable concentrations of ddC, i.e., 6-11 ng/ml, were found to show a significant increase in oxidative stress with 40 nM ddC as measured by protein carbonyls and 3-nitrotyrosine (3NT), effects that were not observed in the more tolerable NRTI, 3TC. Protection against protein oxidation induced by ddC was observed when brain mitochondria were isolated from gerbils 1 h after injection i.p. with the brain accessible antioxidant and glutathione mimetic, tricyclodecan-9-yl-xanthogenate (D609). In addition, there is a significant reduction in the levels of anti-apoptotic protein Bcl-2 and a significant increase in cytochrome c release and also a significant increase in the expression of pro-apoptotic protein caspase-3 after mitochondria were treated with 40 nM ddC. The results reported here show that ddC at 40 nM can induce oxidative stress, cause the release of cytochrome c, and in addition, reduce the levels of anti-apoptotic proteins, increase the levels of pro-apoptotic proteins, thereby increasing the possibility for induction of apoptosis. These findings are consistent with the notion of a possible role of the NRTIs, and in particular, ddC, in the mechanisms involved in HIVD.

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies

International Nuclear Information System (INIS)

Zeng Zhizhen; O'Brien, Julie A.; Lemaire, Wei; O'Malley, Stacey S.; Miller, Patricia J.; Zhao Zhijian; Wallace, Michael A.; Raab, Conrad; Lindsley, Craig W.; Sur, Cyrille; Williams, David L.

2008-01-01

Introduction: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [ 35 S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl) ethyl)benzamide ([ 35 S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl) benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. Methods: Functional potencies of unlabeled compounds were characterized by [ 14 C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. Results: ACPPB is a potent (K d =1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [ 35 S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [ 35 S]ACPPB in rat brain tissues following iv administration of this radioligand. Conclusions: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission

Long-term hippocampal glutamate synapse and astrocyte dysfunctions underlying the altered phenotype induced by adolescent THC treatment in male rats.

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Zamberletti, Erica; Gabaglio, Marina; Grilli, Massimo; Prini, Pamela; Catanese, Alberto; Pittaluga, Anna; Marchi, Mario; Rubino, Tiziana; Parolaro, Daniela

2016-09-01

Cannabis use has been frequently associated with sex-dependent effects on brain and behavior. We previously demonstrated that adult female rats exposed to delta-9-tetrahydrocannabinol (THC) during adolescence develop long-term alterations in cognitive performances and emotional reactivity, whereas preliminary evidence suggests the presence of a different phenotype in male rats. To thoroughly depict the behavioral phenotype induced by adolescent THC exposure in male rats, we treated adolescent animals with increasing doses of THC twice a day (PND 35-45) and, at adulthood, we performed a battery of behavioral tests to measure affective- and psychotic-like symptoms as well as cognition. Poorer memory performance and psychotic-like behaviors were present after adolescent THC treatment in male rats, without alterations in the emotional component. At cellular level, the expression of the NMDA receptor subunit, GluN2B, as well as the levels of the AMPA subunits, GluA1 and GluA2, were significantly increased in hippocampal post-synaptic fractions from THC-exposed rats compared to controls. Furthermore, increases in the levels of the pre-synaptic marker, synaptophysin, and the post-synaptic marker, PSD95, were also present. Interestingly, KCl-induced [(3)H]D-ASP release from hippocampal synaptosomes, but not gliosomes, was significantly enhanced in THC-treated rats compared to controls. Moreover, in the same brain region, adolescent THC treatment also resulted in a persistent neuroinflammatory state, characterized by increased expression of the astrocyte marker, GFAP, increased levels of the pro-inflammatory markers, TNF-α, iNOS and COX-2, as well as a concomitant reduction of the anti-inflammatory cytokine, IL-10. Notably, none of these alterations was observed in the prefrontal cortex (PFC). Together with our previous findings in females, these data suggest that the sex-dependent detrimental effects induced by adolescent THC exposure on adult behavior may rely on its

Synaptic Tau Seeding Precedes Tau Pathology in Human Alzheimer's Disease Brain

Directory of Open Access Journals (Sweden)

Sarah L. DeVos

2018-04-01

Full Text Available Alzheimer's disease (AD is defined by the presence of intraneuronal neurofibrillary tangles (NFTs composed of hyperphosphorylated tau aggregates as well as extracellular amyloid-beta plaques. The presence and spread of tau pathology through the brain is classified by Braak stages and thought to correlate with the progression of AD. Several in vitro and in vivo studies have examined the ability of tau pathology to move from one neuron to the next, suggesting a “prion-like” spread of tau aggregates may be an underlying cause of Braak tau staging in AD. Using the HEK293 TauRD-P301S-CFP/YFP expressing biosensor cells as a highly sensitive and specific tool to identify the presence of seed competent aggregated tau in brain lysate—i.e., tau aggregates that are capable of recruiting and misfolding monomeric tau—, we detected substantial tau seeding levels in the entorhinal cortex from human cases with only very rare NFTs, suggesting that soluble tau aggregates can exist prior to the development of overt tau pathology. We next looked at tau seeding levels in human brains of varying Braak stages along six regions of the Braak Tau Pathway. Tau seeding levels were detected not only in the brain regions impacted by pathology, but also in the subsequent non-pathology containing region along the Braak pathway. These data imply that pathogenic tau aggregates precede overt tau pathology in a manner that is consistent with transneuronal spread of tau aggregates. We then detected tau seeding in frontal white matter tracts and the optic nerve, two brain regions comprised of axons that contain little to no neuronal cell bodies, implying that tau aggregates can indeed traverse along axons. Finally, we isolated cytosolic and synaptosome fractions along the Braak Tau Pathway from brains of varying Braak stages. Phosphorylated and seed competent tau was significantly enriched in the synaptic fraction of brain regions that did not have extensive cellular tau

A Preliminary Report on Brain-Derived Extracellular Vesicle as Novel Blood Biomarkers for Sport-Related Concussions

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Keisuke Kawata

2018-04-01

Full Text Available The purpose of the study was to test the utility of unique panel of blood biomarkers as a means to reflect one’s recovery process after sport-related neurotrauma. We established a panel of biomarkers that reacted positive with CD81 (extracellular vesicle marker and various neuron- and glia-specific antigens [e.g., neurofilament light polypeptide (NF-L, tau, synaptosome-associated protein 25 (SNAP25, glial fibrillary acidic protein, and myelin basic protein]. We first evaluated test–retest reliabilities of brain-derived exosome markers, followed by an application of these markers in eight professional ice hockey players to detect cumulative neuronal burden from a single ice hockey season. During the season, two players were diagnosed with concussions by team physician based on an exhibition of symptoms as well as abnormality in balance and ocular motor testing. One player reached symptom-free status 7 days after the concussion, while the other player required 36 days for symptoms to completely resolve. Blood samples and clinical assessments including balance error scoring system and near point of convergence throughout recovery process were obtained. Biomarkers indicative of axonal damage, neuronal inflammation, and glial activation showed excellent test–retest reliabilities (intraclass correlation coefficient: 0.713–0.998, p’s < 0.01. There was a statistically significant increase in the NF-L marker at post-season follow-up compared to pre-season baseline (Z = −2.100, P = 0.036; however the statistical significance did not withstand Bonferroni correction for multiple comparisons. In concussion cases, neuronal and microglia markers notably increased after concussions, with the unique expression patterns being similar to that of concussion recovery process. These longitudinal data coupled with excellent test–retest reliabilities of novel array of blood biomarkers potentially reflect the damage in neural cell

Defective lysosomal proteolysis and axonal transport are early pathogenic events that worsen with age leading to increased APP metabolism and synaptic Abeta in transgenic APP/PS1 hippocampus.

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Torres, Manuel; Jimenez, Sebastian; Sanchez-Varo, Raquel; Navarro, Victoria; Trujillo-Estrada, Laura; Sanchez-Mejias, Elisabeth; Carmona, Irene; Davila, Jose Carlos; Vizuete, Marisa; Gutierrez, Antonia; Vitorica, Javier

2012-11-22

Axonal pathology might constitute one of the earliest manifestations of Alzheimer disease. Axonal dystrophies were observed in Alzheimer's patients and transgenic models at early ages. These axonal dystrophies could reflect the disruption of axonal transport and the accumulation of multiple vesicles at local points. It has been also proposed that dystrophies might interfere with normal intracellular proteolysis. In this work, we have investigated the progression of the hippocampal pathology and the possible implication in Abeta production in young (6 months) and aged (18 months) PS1(M146L)/APP(751sl) transgenic mice. Our data demonstrated the existence of a progressive, age-dependent, formation of axonal dystrophies, mainly located in contact with congophilic Abeta deposition, which exhibited tau and neurofilament hyperphosphorylation. This progressive pathology was paralleled with decreased expression of the motor proteins kinesin and dynein. Furthermore, we also observed an early decrease in the activity of cathepsins B and D, progressing to a deep inhibition of these lysosomal proteases at late ages. This lysosomal impairment could be responsible for the accumulation of LC3-II and ubiquitinated proteins within axonal dystrophies. We have also investigated the repercussion of these deficiencies on the APP metabolism. Our data demonstrated the existence of an increase in the amyloidogenic pathway, which was reflected by the accumulation of hAPPfl, C99 fragment, intracellular Abeta in parallel with an increase in BACE and gamma-secretase activities. In vitro experiments, using APPswe transfected N2a cells, demonstrated that any imbalance on the proteolytic systems reproduced the in vivo alterations in APP metabolism. Finally, our data also demonstrated that Abeta peptides were preferentially accumulated in isolated synaptosomes. A progressive age-dependent cytoskeletal pathology along with a reduction of lysosomal and, in minor extent, proteasomal activity could be

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications

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Kiss David S

2009-04-01

Full Text Available Abstract Background Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3 may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR hypothalamic structures in the rat brain, here we investigated: 1. The cellular and subcellular localization of NTPDase3; 2. The effects of 17β-estradiol on the expression level of hypothalamic NTPDase3; and 3. The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. Methods Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. Results Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD indicated that γ-amino-butyric-acid- (GABA ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of

Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons.

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Gaspar, J M; Castilho, Á; Baptista, F I; Liberal, J; Ambrósio, A F

2010-12-29

A few studies have reported the existence of depletion of synaptic vesicles, and changes in neurotransmitter release and in the content of exocytotic proteins in the hippocampus of diabetic rats. Recently, we found that diabetes alters the levels of synaptic proteins in hippocampal nerve terminals. Hyperglycemia is considered the main trigger of diabetic complications, although other factors, such as low insulin levels, also contribute to diabetes-induced changes. Thus, the aim of this work was to evaluate whether long-term elevated glucose per se, which mimics prolonged hyperglycemia, induces significant changes in the content and localization of synaptic proteins involved in exocytosis in hippocampal neurons. Hippocampal cell cultures were cultured for 14 days and were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose), for 7 days. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. The protein levels of vesicle-associated membrane protein-2 (VAMP-2), synaptosomal-associated protein-25 (SNAP-25), syntaxin-1, synapsin-1, synaptophysin, synaptotagmin-1, rabphilin 3a, and also of vesicular glutamate and GABA transporters (VGluT-1 and VGAT), were evaluated by immunoblotting, and its localization was analyzed by immunocytochemistry. The majority of the proteins were not affected. However, elevated glucose decreased the content of SNAP-25 and increased the content of synaptotagmin-1 and VGluT-1. Moreover, there was an accumulation of syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell body of some hippocampal neurons exposed to high glucose. No changes were detected in mannitol-treated cells. In conclusion, elevated glucose per se did not induce significant changes in the content of the majority of the synaptic proteins studied in hippocampal cultures, with the exception of SNAP-25, synaptotagmin-1 and VGluT-1. However, there was an accumulation of some proteins in cell bodies of hippocampal

Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

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Impagnatiello Francesco

2010-11-01

Full Text Available Abstract Background Current evidence suggests a role of neuroinflammation in the pathogenesis of Parkinson's disease (PD and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP model of basal ganglia injury. Reportedly, nonsteroidal anti-inflammatory drugs (NSAIDs mitigate DAergic neurotoxicity in rodent models of PD. Consistent with these findings, epidemiological analysis indicated that certain NSAIDs may prevent or delay the progression of PD. However, a serious impediment of chronic NSAID therapy, particularly in the elderly, is gastric, renal and cardiac toxicity. Nitric oxide (NO-donating NSAIDs, have a safer profile while maintaining anti-inflammatory activity of parent compounds. We have investigated the oral activity of the NO-donating derivative of flurbiprofen, [2-fluoro-α-methyl (1,1'-biphenyl-4-acetic-4-(nitrooxybutyl ester], HCT1026 (30 mg kg-1 daily in rodent chow in mice exposed to the parkinsonian neurotoxin MPTP. Methods Ageing mice were fed with a control, flurbiprofen, or HCT1026 diet starting ten days before MPTP administration and continuing for all the experimental period. Striatal high affinity synaptosomial dopamine up-take, motor coordination assessed with the rotarod, tyrosine hydroxylase (TH- and dopamine transporter (DAT fiber staining, stereological cell counts, immunoblotting and gene expression analyses were used to assess MPTP-induced nigrostriatal DAergic toxicity and glial activation 1-40 days post-MPTP. Results HCT1026 was well tolerated and did not cause any measurable toxic effect, whereas flurbiprofen fed mice showed severe gastrointestinal side-effects. HCT1026 efficiently counteracted motor impairment and reversed MPTP-induced decreased synaptosomal [3H]dopamine uptake, TH- and DAT-stained fibers in striatum and TH+ neuron loss in subtantia nigra pars compacta (SNpc, as opposed to age-matched mice fed with a control diet. These effects were associated to a significant decrease in reactive

A Multilevel Functional Study of a SNAP25 At-Risk Variant for Bipolar Disorder and Schizophrenia.

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Houenou, Josselin; Boisgontier, Jennifer; Henrion, Annabelle; d'Albis, Marc-Antoine; Dumaine, Anne; Linke, Julia; Wessa, Michèle; Daban, Claire; Hamdani, Nora; Delavest, Marine; Llorca, Pierre-Michel; Lançon, Christophe; Schürhoff, Franck; Szöke, Andrei; Le Corvoisier, Philippe; Barau, Caroline; Poupon, Cyril; Etain, Bruno; Leboyer, Marion; Jamain, Stéphane

2017-10-25

The synaptosomal-associated protein SNAP25 is a key player in synaptic vesicle docking and fusion and has been associated with multiple psychiatric conditions, including schizophrenia, bipolar disorder, and attention-deficit/hyperactivity disorder. We recently identified a promoter variant in SNAP25 , rs6039769 , that is associated with early-onset bipolar disorder and a higher gene expression level in human prefrontal cortex. In the current study, we showed that this variant was associated both in males and females with schizophrenia in two independent cohorts. We then combined in vitro and in vivo approaches in humans to understand the functional impact of the at-risk allele. Thus, we showed in vitro that the rs6039769 C allele was sufficient to increase the SNAP25 transcription level. In a postmortem expression analysis of 33 individuals affected with schizophrenia and 30 unaffected control subjects, we showed that the SNAP25b / SNAP25a ratio was increased in schizophrenic patients carrying the rs6039769 at-risk allele. Last, using genetics imaging in a cohort of 71 subjects, we showed that male risk carriers had an increased amygdala-ventromedial prefrontal cortex functional connectivity and a larger amygdala than non-risk carriers. The latter association has been replicated in an independent cohort of 121 independent subjects. Altogether, results from these multilevel functional studies are bringing strong evidence for the functional consequences of this allelic variation of SNAP25 on modulating the development and plasticity of the prefrontal-limbic network, which therefore may increase the vulnerability to both early-onset bipolar disorder and schizophrenia. SIGNIFICANCE STATEMENT Functional characterization of disease-associated variants is a key challenge in understanding neuropsychiatric disorders and will open an avenue in the development of personalized treatments. Recent studies have accumulated evidence that the SNARE complex, and more specifically

Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

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Sonntag William E

2011-10-01

Full Text Available Abstract Background Age-related cognitive dysfunction, including impairment of hippocampus-dependent spatial learning and memory, affects approximately half of the aged population. Induction of a variety of neuroinflammatory measures has been reported with brain aging but the relationship between neuroinflammation and cognitive decline with non-neurodegenerative, normative aging remains largely unexplored. This study sought to comprehensively investigate expression of the MHC II immune response pathway and glial activation in the hippocampus in the context of both aging and age-related cognitive decline. Methods Three independent cohorts of adult (12-13 months and aged (26-28 months F344xBN rats were behaviorally characterized by Morris water maze testing. Expression of MHC II pathway-associated genes identified by transcriptomic analysis as upregulated with advanced aging was quantified by qPCR in synaptosomal fractions derived from whole hippocampus and in hippocampal subregion dissections (CA1, CA3, and DG. Activation of astrocytes and microglia was assessed by GFAP and Iba1 protein expression, and by immunohistochemical visualization of GFAP and both CD74 (Ox6 and Iba1. Results We report a marked age-related induction of neuroinflammatory signaling transcripts (i.e., MHC II components, toll-like receptors, complement, and downstream signaling factors throughout the hippocampus in all aged rats regardless of cognitive status. Astrocyte and microglial activation was evident in CA1, CA3 and DG of intact and impaired aged rat groups, in the absence of differences in total numbers of GFAP+ astrocytes or Iba1+ microglia. Both mild and moderate microglial activation was significantly increased in all three hippocampal subregions in aged cognitively intact and cognitively impaired rats compared to adults. Neither induction of MHCII pathway gene expression nor glial activation correlated to cognitive performance. Conclusions These data demonstrate a

Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa.

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Aird, Steven D; da Silva, Nelson Jorge; Qiu, Lijun; Villar-Briones, Alejandro; Saddi, Vera Aparecida; Pires de Campos Telles, Mariana; Grau, Miguel L; Mikheyev, Alexander S

2017-06-08

Venom gland transcriptomes and proteomes of six Micrurus taxa ( M. corallinus , M. lemniscatus carvalhoi , M. lemniscatus lemniscatus , M. paraensis , M. spixii spixii , and M. surinamensis ) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2-6 toxin classes that account for 91-99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A₂ (PLA₂s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA₂s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1-2.0%) are found in all venoms except that of M. s. spixii . Other toxin families are present in all six venoms at trace levels (venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6-9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three

Atomoxetine affects transcription/translation of the NMDA receptor and the norepinephrine transporter in the rat brain – an in vivo study

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Udvardi PT

2013-12-01

Full Text Available Patrick T Udvardi,1,2 Karl J Föhr,3 Carolin Henes,1,2 Stefan Liebau,2 Jens Dreyhaupt,4 Tobias M Boeckers,2 Andrea G Ludolph11Department of Child and Adolescent Psychiatry and Psychotherapy, 2Institute of Anatomy and Cell Biology, 3Department of Anaesthesiology, 4Institute of Epidemiology and Medical Biometry, University of Ulm, Ulm, GermanyAbstract: Attention-deficit/hyperactivity disorder (ADHD is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR antagonist. The compound's effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip] or saline (0.9%, ip for 21 consecutive days on postnatal days (PND 21–41. In humans, atomoxetine's earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR, mesencephalon (MES, and hippocampus (HC was analyzed either directly after treatment (PND 42 or 2 months after termination of treatment (PND 101 to assess the compound's long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine's effects

Journal of Gravitational Physiology, Volume 12, Number 1

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Fuller, Charles A. (Editor); Cogoli, Augusto (Editor); Hargens, Alan R. (Editor); Smith, Arthur H. (Editor)

2005-01-01

The following topics were covered: System Specificity in Responsiveness to Intermittent -Gx Gravitation during Simulated Microgravity in Rats; A Brief Overview of Animal Hypergravity Studies; Neurovestibular Adaptation to Short Radius Centrifugation; Effect of Artificial Gravity with Exercise Load by Using Short-Arm Centrifuge with Bicycle Ergometer as a Countermeasure Against Disused Osteoporosis; Perception of Body Vertical in Microgravity during Parabolic Flight; Virtual Environment a Behavioral and Countermeasure Tool for Assisted Gesture in Weightlessness: Experiments during Parabolic Flight; Artificial Gravity: Physiological Perspectives for Long-Term Space Exploration; Comparison of the Effects of DL-threo-Beta-Benzyloxyaspartate on the Glutamate Release from Synaptosomes before and after Exposure of Rats to Artificial Gravity; Do Perception and Postrotatory Vestibulo-Ocular Reflex Share the Same Gravity Reference?; Vestibular Adaptation to Changing Gravity Levels and the Orientation of Listing's Plane; Compound Mechanism Hypothesis on +Gz - Induced Brain Injury and Dysfunction of Learning and Memory; Environmental Challenge Impairs Prefrontal Brain Functions; Effect of 6-Days of Support Withdrawal on Characteristics of Balance Function; Hypergravity-Induced Changes of Neuronal Activities in CA1 Region of Rat Hippocampus; Audiological Findings in Antiorthostatic Position Modelling Microgravitation; Investigating Human Cognitive Performance during Spaceflight; The Relevance of the Minimization of Torque Exchange with the Environment in Weightlessness is Confirmed by Asimulation Study; Characteristics of the Eyes Pursuit Function during Readaptation to Terrestrial Gravity after Prolonged Flights Aboard the International Space Station; Comparison of Cognitive Performance Tests for Promethazine Pharmacodynamics in Human Subjects; Structural Reappraisal of Dendritic Tree of Cerebellar Purkinje Cell for Novel Functional Modeling of Elementary Sensorimotor Adaptive

Coralsnake Venomics: Analyses of Venom Gland Transcriptomes and Proteomes of Six Brazilian Taxa

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Steven D. Aird

2017-06-01

Full Text Available Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2–6 toxin classes that account for 91–99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs and phospholipases A2 (PLA2s comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA2s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1–2.0% are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%. Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6–9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen

Effects of testosterone and its metabolites on aromatase-immunoreactive cells in the quail brain: relationship with the activation of male reproductive behavior.

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Balthazart, J; Foidart, A; Absil, P; Harada, N

1996-01-01

locally formed estrogens cannot control the behavior and the aromatase synthesis in an autocrine fashion in the cells where they were formed. Multi-neuronal networks need therefore to be considered. The behavioral activation could result from the action of estrogens in ER-positive cells located in the vicinity of the ARO-ir cells where they were produced (paracrine action). Alternatively, actions that do not involve the nuclear ER could be important. Immunocytochemical studies at the electron microscope level and biochemical assays of AA in purified synaptosomes indicate the presence of aromatase in presynaptic boutons. Estrogens formed at this level could directly affect the pre-and post-synaptic membrane or could directly modulate neurotransmission namely through their metabolization into catecholestrogens (CE) which are known to be powerful inhibitors of the catechol- omicron - methyl transferase (COMT). The inhibition of COMT should increase the catecholaminergic transmission. It is significant to note, in this respect, that high levels of 2-hydroxylase activity, the enzyme that catalyzes the transformation of estrogens in CE, are found in all brain areas that contain aromatase. On the other hand, the synthesis of aromatase should also be controlled by estrogens in an indirect, transynaptic manner very reminiscent of the way in which steroids indirectly control the production of LHRH. Fibers that are immunoreactive for tyrosine hydroxylase (synthesis of dopamine), dopamine beta-hydroxylase (synthesis of norepinephrine) or vasotocine have been identified in the close vicinity of ARO-ir cells in the POM and retrograde tracing has identified the origin of the dopaminergic and noradrenergic innervation of these areas. A few preliminary physiological experiments suggest that these catecholaminergic inputs regulate AA and presumably synthesis.

Poster Session B

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2014-01-01

Biol 8, 599. (2012) B.4 Extracellular Phosphorylation in the Murine Synaptosome Jonathan C Trinidad1, Ralf Schoepfer2, Alma L Burlingame3, Katalin F Medzihradszky3* 1Department of Chemistry, Indiana University, Bloomington, IN, USA; 2Department of Pharmacology, University College London, England, UK; 3Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, CA, USA Post-translational modifications (PTMs) play key regulatory roles in cellular localization and/or biological function of proteins. The site of modification within a protein; their fixed or transient nature; the stoichiometry and potential crosstalk between modifications have been the focus of numerous large-scale studies. Most of this research is focused on PTMs involved in intracellular processes, such as phosphorylation, methylation, GlcNAcylation, acetylation, and ubiquitination. Of these, phosphorylation is the most studied due to the biological importance of this modification as well as the availability of tools for such studies. While the majority of protein phosphorylation occurs on intracellular proteins, phosphorylation of secreted proteins is well established. Well-characterized examples include the secreted milk protein, beta-casein, and the serum protein fetuin. Our current understanding of the biological role of extracellular phosphorylation, as well as knowledge regarding the process by which they are modified, is incomplete. A kinase known as FAM20 has been identified and is localized to the Golgi as well as secreted. It has been shown to phosphorylate extracellular proteins with a SXE motif. We recently conducted extensive studies on the interplay between intracellular phosphorylation and GlcNAcylation on proteins isolated from murine synaptosomes. This data also allowed us to identify specific glycan structures on more than 500 secreted or transmembrane proteins. We have subsequently examined the extent to which phosphorylation was present in our