Waglerin-1 inhibits GABAA current of neurons in the nucleus accumbens of neonatal rats

The effect of Waglerin-1, a 22-amino acid peptide purified from the venom of Wagler's pit viper on the whole cell current response (I_GABA) to γ-aminobutyric acid (GABA) was examined for neurons freshly isolated from the nucleus accumbens of 3- to 7-day-old rats. Waglerin-1 depressed I_GABA induced by subsaturating concentrations of GABA; the IC₅₀ for I_GABA induced by 10 μM GABA was 2.5 μM Waglerin-1. This concentration of Waglerin-1 shifted the GABA concentration–response curve to the right in a parallel manner, increasing the GABA EC₅₀ from 12±3 to 27±5 μM. The depressant effect of Waglerin-1 was greater at negative holding potentials. Zn²⁺ also inhibited I_GABA with an IC₅₀ of 0.3 μM. Phosphorylation state appeared to modulate GABA_A receptor sensitivity to the inhibitory effect of Waglerin-1 since dialysis of neurons with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl (H-89), an inhibitor of protein kinase A, prevented inhibition. The data are discussed in terms of developmental influences on the subunit composition of GABA_A receptors in neurons of the nucleus accumbens.     

Differential modulation of γ-aminobutyric acid receptors by caprolactam derivatives with central nervous system depressant on convulsant activity

The effects of a series of caprolactam derivatives with central depressant, convulsant or muscle relaxant activity were investigated upon γ-aminobutyric acid (GABA) receptor-ionophore binding to rat brain membranes using [³H]GABA, [³H]muscimol and [³⁵S]-tert.-butylbicyclophophorothionate ([³⁵S]TBPS) as ligands, and GABA resonses in mouse spinal cord neurones in dissociated cell culture. Some caprolactams produced a picrotoxin-like chloride-dependent partial inhibition of muscimol binding and were potent inhibitors of TBPS binding. One compound that was further investigated (4,4,6,6,-tetramethylhexahydro-2H-azepin-2-one), inhibited GABA responses and increased the frequency of paroxysmal depolarizations in cultured neurones. Other caprolactams enhanced muscimol binding and were relatively weak inhibitors of TBPS binding, and one (3,3-diallyl-6,6-dimethylhexahydro-2H-azepin-2,4-dione) was shown to enhance GABA responses and produced quiescence of activity in cultured neurones. There was a direct correaltion between caprolactam effects on muscimol binding in the presence of chloride ions and their effects on TBPS binding suggesting a similar site of action for the caprolactams influencing the binding of these two ligands. For the two classes of caprolactams, with respect to inhibition or enhancement of muscimol binding, there appeared to be a relationship between in vitro effects and their convulsant or depressant activity in mice. Caprolactams may be useful low molecular weight probes for the study of GABA receptor-ionophore complexes.     

Effect of GABA‐ergic mechanisms on synaptosomal NO synthesis and the nitrergic component of NANC relaxation in rat ileum

Background  γ‐Aminobutyric acid (GABA) acts on specific neural receptors [A, B and C(Aρ)] to modulate gastrointestinal function. The precise role of GABA receptor activation in the regulation of presynaptic nitric oxide (NO) synthesis in nerve terminals is unknown. Methods  Rat ileal nerve terminals were isolated by differential centrifugation. Nitric oxide synthesis was analysed using a L‐[³H]arginine assay. In vitro studies were performed under non‐adrenergic non‐cholinergic (NANC) conditions on isolated ileal segments. Key Results  γ‐Aminobutyric acid inhibited NO synthesis significantly (n = 6, P < 0.05) [(fmol mg⁻¹ min⁻¹) control: 27.7 ± 1.5, 10⁻⁶ mol L⁻¹: 19.7 ± 1.3; 10⁻⁵ mol L⁻¹: 17.5 ± 3.0]. This effect was antagonized by the GABA A receptor antagonist bicuculline and the GABA C receptor antagonist (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA), but not by the GABA B receptor antagonist SCH 50911. The GABA A receptor agonist muscimol [(fmol mg⁻¹ min⁻¹) control: 27.6 ± 1.0, 10⁻⁶ mol L⁻¹: 19.1 ± 1.7, n = 5, P < 0.05] and the GABA C receptor agonist cis‐4‐aminocrotonic acid (CACA) [(fmol mg⁻¹ min⁻¹) control: 29.5 ± 3.2, 10⁻³ mol L⁻¹: 20.3 ± 2.5, n = 6, P < 0.05], mimicked the GABA‐effect, whereas the GABA B agonist baclofen was ineffective. Bicuculline reversed the inhibitory effect of muscimol, TPMPA antagonized the effect of CACA. In functional experiments the GABA A and C receptor agonists reduced the NANC relaxation induced by electrical field stimulation in rat ileum by about 40%. After NOS‐inhibition by Nε‐nitro‐l ‐arginine methyl ester (l ‐NAME) the GABA A receptor agonist had no effect, whereas the GABA C receptor agonist still showed a residual response. Conclusions & Inferences  γ‐Aminobutyric acid inhibits neural NO synthesis in rat ileum by GABA A and GABA C(Aρ) receptor‐mediated mechanisms.     

Lentivirus‐Mediated α‐Melanocyte‐Stimulating Hormone Overexpression in the Hypothalamus Decreases Diet Induced Obesity in Mice

Melanocyte stimulating hormone (MSH) derived from the pro‐hormone pro‐opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long‐term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long‐term anti‐obesity effects of hypothalamic α‐MSH overexpression in mice. An α‐MSH lentivirus (LVi‐α‐MSH‐EGFP) vector carrying the N‘‐terminal part of POMC and the α‐MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi‐α‐MSH‐EGFP or control LVi‐EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high‐fat diet. The effects of 6‐week‐treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14‐day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi‐α‐MSH‐EGFP treatment is dependent on decreased food intake. The 6‐week LVi‐α‐MSH‐EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi‐EGFP treated mice. Food intake was decreased during the first week in the LVi‐α‐MSH‐EGFP treated mice but subsequently increased to the level of LVi‐EGFP treated mice. The LVi‐EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi‐α‐MSH‐EGFP treated mice. We demonstrate that gene transfer of α‐MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α‐MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site‐specific effects of POMC peptides.     

Differential effects of single and repeated ketamine administration on dopamine, serotonin and GABA transmission in rat medial prefrontal cortex

Cognitive functions regulated by the prefrontal cortex are sensitive to changes in dopaminergic and serotoninergic transmission. The non-competitive N-methyl-

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-aspartate (NMDA) receptor antagonist ketamine influences dopaminergic transmission and induces psychotic symptoms in normal and schizophrenic individuals. This study examined the effect of single and repeated ketamine (25 mg/kg, i.p.) administration on extracellular levels of dopamine, GABA and the serotonin metabolite 5-hydroxyindoleacetic (5-HIAA) acid in the medial prefrontal cortex using in vivo microdialysis in conscious rat. In line with earlier studies, we observed a transient five-fold increase in dopamine release following single ketamine administration in drug naive animals. However, we also observed a two-fold increase in basal dopamine levels and an almost complete attenuation of the ketamine-induced increase in dopamine release in animals pre-treated with ketamine once daily for 7 days. Extracellular 5-HIAA levels were increased by ketamine in both drug naive and even more enhanced in ketamine-pre-treated animals but without a change in basal 5-HIAA levels. GABA levels were unaffected by either single or repeated ketamine administration. We demonstrate evidence for a differential effect of single and repeated ketamine administration on dopamine, serotonin and GABA transmission in the medial prefrontal cortex. We provide new evidence for a complex adaptation of neurotransmission following repeated NMDA receptor blockade whereby in the presence of increased basal dopamine levels the ketamine-induced increase in dopamine is attenuated and the increase in 5-HIAA is enhanced. It appears from our results that ketamine pre-treatment reduces the dynamics of dopaminergic transmission in the prefrontal cortex and may possibly alter the balance between dopamine and serotonin transmission.     

Effects of d-aspartate on excitatory amino acid-induced release of [H]GABA from goldfish retina

The rate of release of [³H]GABA from isolated intact goldfish retinas was studied. Release of [³H]GABA is markedly stimulated by the inclusion in the incubation medium of the photoreceptor neurotransmitter candidates l-glutamate (l-Glu) and l-aspartate (l-Asp), and the glutamate analogs, kainate and quisqualate. At micromolar concentrations, kainate and quisqualate are effective releasers of [³H]GABA, whereas millimolar concentrations of l-Glu and l-Asp are required to release comparable amounts of [³H]GABA. The d-isomers of aspartate (d-Asp) and glutamate (d-Glu) are able to release [³H]GABA, but only when applied at high concentrations (3–30 mM). In the presence of 5 mM d-Asp, the effect of l-Glu in releasing [³H]GABA was markedly potentiated. This dose-response curve of l-Glu was shifted to the left in the presence of d-Asp, although the maximal amount of release was unchanged. d-Asp at 5 mM only slightly increased the GABA release induced by quisqualate, and it did not increase the GABA release induced by kainate. Finally, low concentrations of l-Asp were potentiated by d-Asp, but higher concentrations of l-Asp (3–10 mM) were clearly inhibited by this agent. This biphasic effect of d-Asp on l-Asp-induced release of [³H]GABA is a possible explanation for previously conflicting reports of d-Asp's effect on l-Asp action^2,8,29. Our data suggest that d-Asp has both pre- and postsynaptic sites of action.     

Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii

Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero‐ oligomeric receptors. Extensive research on the molecular structure of the γ‐aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo‐oligomeric GABA‐activated Cl^? channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo‐oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA‐activated Cl^? channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo‐oligomeric complex. To investigate the structure–function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo‐oligomeric ionotropic GABAR that gates a bicuculline‐insensitive Cl^? current. The order of potency of the agonists was GABA > trans‐4‐amino‐crotonic acid = cis‐4‐aminocrotonic acid > muscimol. These data support the notion that X‐organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero‐oligomeric and homo‐oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X‐organ neurons from crayfish eyestalk express the isolated pcGABA_Aβ subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates. © 2015 Wiley Periodicals, Inc.     

Excitatory amino acid-evoked release of [H]GABA from hippocampal neurons in primary culture

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [³H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion butN-methyl-d-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [³H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.     

Endogenous GABA release from rat striatal slices: Effects of the GABAB receptor antagonist 2-hydroxy-saclofen

The reproducibility of endogenous GABA release evoked by multiple periods of electrical field stimulation was examined in rat striatal slices. In these experiments, NO-328 was used to block GABA uptake, and evoked GABA release (overflow) was completely Ca²⁺ dependent. A seemingly invariant observation in these experiments was that spontaneous GABA release (outflow) progressively decreased as a function of superfusion time and that GABA overflow decreased 25–30% in response to the second of two periods of stimulation (S2/S1 ratios = 0.70 to 0.75). The attenuation of GABA release was not explained by the amount of GABA lost to the superfusion buffer (fractional release), direct depletion of releasable pools of GABA, or slice viability. Furthermore, the decreases in GABA release were not dependent on stimulation frequency (5–15 Hz) or the absolute amount of GABA evoked by electrical stimulation. However, the GABA_B receptor antagonist 2-hydroxy-saclofen (2-OH-saclofen; 316 μM) not only enhanced GABA overflow, when superfused throughout both periods of stimulation, but also resulted in S2/S1 ratios of unity. When 2-OH-saclofen was superfused throughout the second stimulation period only, GABA overflow was almost two-fold greater than that evoked by the initial period of stimulation (2-OH-saclofen-free). In addition, these S2 responses were ～30% greater than S1 responses that were observed when 2-OH-saclofen was present throughout the entire superfusion period. These results indicate that activation of GABA_B receptors was involved in the progressive attenuation of GABA release and further emphasize that GABA_B receptors play an important role in modulating endogenous GABA release from striatal slices. © Wiley-Liss, Inc.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

α2‐Adrenergic drugs modulate the binding of [18F]fallypride to dopamine D2/3 receptors in striatum of living mouse

Aim. To test for α₂ adrenergic modulation of dopamine D_2/3 receptor availability in striatum of living mice using the high‐affinity ligand [¹⁸F]fallypride and microPET. Methods. Groups of anesthetized mice were pretreated with saline, the α₂‐agonist clonidine (1 mg/kg), and the α₂‐antagonists RX821002 (1 mg/kg) and yohimbine (1 mg/kg). Dynamic microPET recordings lasting 120 min were then initiated upon i.v. tracer injection of [¹⁸F]fallypride. Parametric maps of [¹⁸F]fallypride binding potential (BP_ND) were calculated using the Logan method, with cerebellum serving as the reference region. Results. Mean striatal [¹⁸F]fallypride BP_ND was 10.6 ± 1.7 in the saline control animals, 8.9 ± 1.7 (?16%; P < 0.05) in the RX821002 group, 8.3 ± 2.6 (?22%; P < 0.05) in the yohimbine group and 10.3 ± 2.2 (n.s.) in the clonidine group. Conclusions. These findings are consistent with a tonic inhibition of dopamine release by α₂ adrenergic receptors, such that α₂ blockade increased the competition from endogenous dopamine at D_2/3 receptors, thus reducing the [¹⁸F]fallypride BP_ND by about 20%. Absent effects of clonidine suggest a ceiling effect in the tonic inhibition of dopamine release. This in vivo PET evidence for α₂/dopaminergic interaction may be relevant to putative actions of atypical antipsychotic medications via adrenergic receptors. Synapse, 2010. © 2010 Wiley‐Liss, Inc.     

Kindling increases the K -evoked Ca -dependent release of endogenous GABA in area CA1 of rat hippocampus

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K⁺) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K⁺ was significantly increased (40–46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45–49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.     

Heterogeneity in EC50 and nH of GABAA receptors on dorsal root ganglion neurons freshly isolated from adult rats

GABA activates a Cl⁻ current through the GABA_A receptor/ionophore complex that influences excitability of neurons. Studies using expression of cloned cDNAs coding for different GABA_A receptor/ionophore subunits suggest that the EC₅₀ and Hill coefficient for GABA are influenced by subunit composition. However, no direct evidence for such heterogeneity has been reported for vertebrate neurons. I have investigated the heterogeneity of EC₅₀ and Hill coefficients (n_H) of isolated dorsal root ganglion neurons using the whole-cell patch clamp technique. The EC₅₀ for GABA varied from 26 to 107 μM among neurons, n_H calculated from the logistic equation varied from 1.18 to 2.0. A negative correlation was found between the EC₅₀ and n_H (r= −0.81). Both n_H and EC₅₀ differed between some cells. However, in some instances, n_H differed between cells while EC₅₀ values were similar, and in other cells, EC₅₀ values differed and n_H was similar. In addition, when cells were categorized according to action potential shape, the EC₅₀ and Hill coefficients differed among cell types in some instances and were similar in other instances. These findings demonstrate that different pharmacological profiles for GABA can be observed in adult mammalian neurons. Selective distribution of such pharmacological subtypes of GABA_A receptors may contribute to control of neuronal excitability.     

Involvement of protein kinase C in the Ca-dependent vesicular release of GABA from central and enteric neurons of the guinea pig

The involvement of protein kinase C (PKC) in the release of endogenous γ-aminobutyric acid (GABA) was studied using slices of deep cerebellar nucleus and strips of small intestine from the guinea pig.12-O-tetradecanoylphorbol 13-acetate (TPA), but not 4α-phorbol-12,13-dedecanoate (4α-PDD), potentiated the high K⁺-evoked release of GABA from both preparations in the presence of tetrodotoxin. Ouabain evoked the release of GABA from both preparations, and this release was not altered by TPA. Therefore, the activation of protein kinase C potentiates the Ca²⁺-dependent vesicular release of GABA from nerve terminals of the central and enteric GABAergic neurons of the guinea pig.     

Dopamine inhibits the release of immunoreactive β-endorphin from rat hypothalamus in vitro

Mediobasal hypothalamus tissue (MBH) from adult male rats was incubated in Krebs-Ringer bicarbonate medium (KRB). KRB was changed at 15 min intervals and the concentration of immunoreactive β-endorphin (β-ENDi) in the medium was measured by radioimmunoassay. Incubation of MBH tissue in normal KRB resulted in a constant release rate of β-ENDi of approximately 1% of the tissue content per h. KRB containing 45 mM K⁺ causes a two fold increase in the release rate of β-ENDi which was Ca²⁺ dependent. Dopamine (0.01–1.0 μM) inhibits both the spontaneous and the K⁺-stimulated release of β-ENDi in a dose related manner. The dopamine receptor blocking agent haloperidol prevents this inhibitory effect of dopamine. The selective D-1 receptor agonist SKF 38393 does not affect the release rate of β-ENDi; whereas the selective D-2 receptor agonist LY 141865 inhibits both the spontaneous and K⁺-stimulated release of β-ENDi. The effects of LY 141865 can be blocked by (−)-sulpiride, a selective D-2 receptor antagonist. Norepinephrine only weakly inhibits the K⁺-stimulated release of β-ENDi, and effect that can be blocked by haloperidol but not by the α-adrenoceptor blocker phentolamine. At concentrations tested (0.01–1.0 μM), isoproterenol, 5-hydroxytryptamine, carbachol and 8-Br-cAMP (1.0 μM) do not affect β-ENDi release. It is concluded that dopamine can inhibit the release of β-ENDi from hypothalamic neurons via a D-2 receptor mechanism.     

GABAA and GABAB receptor dysregulation in superior frontal cortex of subjects with schizophrenia and bipolar disorder

Schizophrenia and bipolar disorder are complex psychiatric disorders that affect millions of people worldwide. Evidence from gene association and postmortem studies has identified abnormalities of the gamma‐aminobutyric acid (GABA) signaling system in both disorders. Abnormal GABAergic signaling and transmission could contribute to the symptomatology of these disorders, potentially through impaired gamma oscillations which normally occur during cognitive processing. In the current study, we examined the protein expression of 14 GABA_A and two GABA_B receptor subunits in the superior frontal cortex of subjects with schizophrenia, bipolar disorder, and healthy controls. Analyses of Variance (ANOVAs) identified significant group effects for protein levels for the α1, α6, β1, β3, δ, ?, and π GABA_A receptor subunits and R1 and R2 GABA_B receptor subunits. Follow‐up t tests confirmed changes for these subunits in subjects with schizophrenia, subjects with bipolar disorder, or both groups. Alterations in stoichiometry of GABA receptor subunits could result in altered ligand binding, transmission, and pharmacology of GABA receptors in superior frontal cortex. Thus, impaired GABAergic transmission may negatively contribute to symptoms such as anxiety or panic as well as impaired learning and information processing, all of which are disrupted in schizophrenia and bipolar disorder. Taken together, these results provide additional evidence of GABAergic receptor abnormalities in these disorders.     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

The effect of phencyclidine on the basal and high potassium evoked extracellular GABA levels in the striatum of freely-moving rats: an in vivo microdialysis study

The effect of phencyclidine (PCP) on the γ-aminobutyric acid-ergic (GABAergic) transmission in the striatum of freely-moving rats was investigated using an in vivo microdialysis. The high potassium (100 mM) increased the extracellular GABA level to 4000% of the basal level. Although the basal GABA level in the striatal dialysate did not show either calcium dependency or tetrodotoxin (TTX) sensitivity, the high potassium evoked GABA level was reduced by 82% under calcium-free conditions (with 12.5 mM magnesium) and by 54% in the presence of 10 μM TTX. The systemic administration of PCP (7.5 mg/kg) or the local perfusion of PCP (100 μM and 1 mM) significantly inhibited the high potassium evoked GABA release in the rat striatum. The local perfusion of MK-801 (10 μM and 100 μM), a more potent and selective N-methyl- -aspartate (NMDA) receptor antagonist, also inhibited the high potassium evoked striatal GABA release. These drugs did not show any significant effect on the basal extracellular GABA level. NMDA (1 mM) either partly or completely blocked the effect of PCP (1 mM) or MK-801 (100 μM) on the high potassium evoked striatal GABA release. On the other hand, nomifensine (100 μM), a dopamine uptake blocker, did not show any effect on the high potassium evoked GABA release. These results suggest that PCP inhibited the striatal GABAergic neuronal transmission through its antagonism of the NMDA receptor.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

Regulation of α-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus by serotonin and the interaction of serotonin with the dopaminergic system inhibiting peptide release

Release of α-melanocyte-stimulating hormone (α-MSH) from frontal slices of rat hypothalamus superfused with oxygenated artificial cerebrospinal fluid (ACSF) was quantified by radioimmunoassay. Control depolarisations with 50 mM KCl-containing ACSFm produced significant increases in α-MSH release which were partially blocked by 10⁻⁶ M cinnaserin, a serotonin (5-HT) receptor antagonist. Superfusion of the tissues with varying concentrations of 5-HT *10⁻⁷ M to 10⁻⁴ M) resulted in an inverted U-shaped dose-response curve, maximum α-MSH release being obtained with 10⁻⁶ M 5-HT. Addition of 10⁻⁶ M cinanserin shifted the 5-HT dose-response curve to the right whilst the presence of 10⁻⁸ M flupenthixol, a dopamine receptor antagonist, resulted in a sigmoidal 5-HT dose-response curve. Superfusion with ACSF containing either 10⁻⁷ M fluoxetine, a 5-HT re-uptake inhibitor, or 10⁻⁷ M p-chloroamphetamine, an agent releasing 5-HT, induced significant increases in α-MSH release which were abolished in the presence of 10⁻⁶ M cinanserin. These data demonstrate the presence of an endogenous 5-HT system that exerts a biphasic effect on α-MSH release. A stimulatory effect caused by lower 5-HT concentrations appears to be a direct action whilst an inhibitory effect at higher concentrations is mediated through an inhibitory endogenous dopaminergic system. A significant proportion of K⁺-stimulated peptide release is 5-HT-mediated.