Serum Concentrations of Some Neuroactive Steroids in Women Suffering from Mixed Anxiety-Depressive Disorder

Concentrations of neurosteroids may be influenced by some physiological or pathological factors. We investigated neuroactive steroids in the serum of women suffering from anxiety-depressive disorder treated with fluoxetine and in a control group, in both the follicular and the luteal phases of the menstrual cycle. Two groups of neuroactive steroids were measured by radioimmunoassays: 1) the positive allosteric modulator of GABA_A receptors, allopregnanolone with its precursor progesterone and 2) pregnenolone sulfate and dehydroepiandrosterone sulfate (DHEAS) acting on GABA_A receptors by an opposite mechanism. Significantly higher levels of pregnenolone sulfate (p < 0.0001) were found in patients in both phases of the menstrual cycle. Significantly higher values were recorded in pregnenolone (p < 0.001) and 17-hydroxy-pregnenolone (p < 0.01) levels in the patients group in the follicular phase. Our results indicate that imbalance in neuroactive steroids may play a negative role in origin and course of psychiatric and neurological disorders.     

The effects of bicuculline and muscimol on glutamate-induced feeding behavior in broiler cockerels

This study was designed to examine the effects of intracerebroventricular (ICV) injection of bicuculline (GABA_A receptor antagonist) and muscimol (GABA_A receptor agonist) on glutamate-induced eating response in 24-h food-deprived (FD24) broiler cockerels. At first, guide cannula was surgically implanted in the right lateral ventricle of chickens. In experiment 1, birds were ICV injected with different doses of glutamate. In experiment 2, birds were administered with effective dose of glutamate after bicuculline. In experiment 3, chickens received muscimol prior to the injection of glutamate, and cumulative food intake was determined at 3-h postinjection. The results of this study showed that glutamate decreases food consumption in FD24 broiler cockerels (P ≤ 0.05), and this reduction occurs in a dose-dependent manner. Moreover, the inhibitory effect of glutamate on food intake was significantly increased with bicuculline pretreatment, and this effect was attenuated with muscimol (P ≤ 0.05). These results suggest that there is an interaction between glutamatergic and GABAergic systems (through GABA_A receptor) on food intake in broiler cockerels.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

GABAA agonists: Resolution and pharmacology of (+)- and (−)-isoguvacine oxide

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABA_A receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABA_A receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyloxycarbonyl)-3,4-epoxypiperidine-4-carboxylate ( 1 ) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee ≥ 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [³H]GABA to GABA_B receptor sites (IC₅₀ > 100 μM), (+)-isoguvacine oxide (IC₅₀ = 0.20 ± 0.03 μM) and (?)-isoguvacine oxide (IC₅₀ = 0.32 ± 0.05 μM) showed comparable potencies as inhibitors of the binding of [³H]GABA to GABA_A receptor sites. Furthermore, (+)-isoguvacine oxide (EC₅₀ = 6 μM; 33% relative efficacy) and (?)-isoguvacine oxide (EC₅₀ = 5 μM; 38% efficacy relative to 10 μM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [³H]diazepam to the GABA_A receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABA_A agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABA_A recognition site(s), derived from extensive structure—activity studies on GABA_A agonists. Thus, the model of the GABA_A recognition site(s) comprising a narrow cleft or pocket, in which the anionic moiety of the zwitterionic GABA_A agonists is assumed to be embedded during receptor activation, may have to be revised. © 1995 Wiley-Liss, Inc.     

Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures

Summary It is known that estrogen can protect neurons from excitotoxicity. Since isoflavones possess estrogen-like activity, it is of interest to determine whether isoflavones can also protect neurons from glutamate-induced neuronal injury. Morphological observation and lactate dehydrogenase (LDH) release assay were used to estimate the cellular damage. It is surprising that, contrary to estrogen, isoflavones, specifically genistein and daidzein, are toxic to primary neuronal culture at high concentration. Treatment of neurons with 50 μM genistein and daidzein for 24 h increased LDH release by 90% and 67%, respectively, indicating a significant cellular damage. Under the same conditions, estrogen such as 17β-estradiol did not show any effect on primary culture of brain cells. At 100 μM, both genistein and daidzein increased LDH release by 2.6- and 3-fold, respectively with a 30-min incubation. Furthermore, both genistein and daidzein at 50 μM increased the intracellular calcium level, [Ca²⁺]_i, significantly. To determine their mode of action, genistein and daidzein were tested on glutamate and GABA_A receptor binding. Both genistein and daidzein were found to have little effect on glutamate receptor binding, while the binding of [³H]muscimol to GABA_A receptors was markedly inhibited. However, 17β-estradiol did not affect GABA_A receptor binding suggesting that the toxic effect of genistein and daidzein could be due to their inhibition of the GABA_A receptor resulting in further enhancement of excitation by glutamate and leading to cellular damage. Ying Jin, Heng Wu contributed equally to this article.     

Sensitivity of hippocampal interneurons and pyramidal cells to GABA and some of its agonists:in vitro experiments

Effects of GABA and its agonists baclofen and muscimol on the background spike activity of single hippocampal neurons were studied in rat brain slices using an intracellular recording technique. Interneurons localized in thestratum alveus-oriens and pyramidal neurons of thestratum pyramidale showed high sensitivity to GABA (mean ID₅₀=65 µM and 40 µM, ranges 10–140 µM and 3–200 µM), baclofen (ID₅₀=2.6 µM and 3.5 µM, ranges 0.6–20.0 µM and 0.4–30.0 µM), and muscimol (ID₅₀=0.85 µM and 0.21 µM, ranges 0.11–4.0 µM and 0.05–0.45 µM, respectively). Responses of hippocampal neurons to application of GABA or either of its agonists were predominantly inhibitory. A part of interneurons (30%) differed from pyramidal neurons in their irresponsivity or low sensitivity to baclofen applications. GABA- or muscimol-induced inhibition of spike activity in many pyramidal cells was preceded by a short-lasting excitation. Our findings indicate that a part of hippocampal interneurons are very poorly supplied with GABAb receptors. Inhibition of pyramidal cells evoked by activation of GABAa receptors probably develops against the background of accompanying depolarization, which in some cases can result in a provisional excitation of these neurons. The excitatory effects of GABA on the pyramidal cells are mediated by GABAa receptors.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 36–44, January–February, 1995.     

Mechanism of Action of Nitrogen Pressure in Controlling Striatal Dopamine Level of Freely Moving Rats is Changed by Recurrent Exposures to Nitrogen Narcosis

In rats, a single exposure to 3 MPa nitrogen induces change in motor processes, a sedative action and a decrease in dopamine release in the striatum. These changes due to a narcotic effect of nitrogen have been attributed to a decrease in glutamatergic control and the facilitation of GABAergic neurotransmission involving NMDA and GABA_A receptors, respectively. After repeated exposure to nitrogen narcosis, a second exposure to 3 MPa increased dopamine levels suggesting a change in the control of the dopaminergic pathway. We investigated the role of the nigral NMDA and GABA_A receptors in changes in the striatal dopamine levels. Dopamine-sensitive electrodes were implanted into the striatum under general anesthesia, together with a guide-cannula for drug injections into the SNc. Dopamine level was monitored by in vivo voltammetry. The effects of NMDA/GABA_A receptor agonists (NMDA/muscimol) and antagonists (AP7/gabazine) on dopamine levels were investigated. Rats were exposed to 3 MPa nitrogen before and after five daily exposures to 1 MPa. After these exposures to nitrogen narcosis, gabazine, NMDA and AP7 had no effect on the nitrogen-induced increase in dopamine levels. By contrast, muscimol strongly enhanced the increase in dopamine level induced by nitrogen. Our findings suggest that repeated nitrogen exposure disrupted NMDA receptor function and decreased GABAergic input by modifying GABA_A receptor sensitivity. These findings demonstrated a change in the mechanism of action of nitrogen at pressure.     

GABAA receptors in nucleus accumbens shell mediate the hyperphagia and weight gain following haloperidol treatment in rats

AimsWeight gain is a common outcome of antipsychotics therapy in schizophrenic patients. However, the underlying neuronal mechanisms are unclear. The present study was undertaken to investigate the role of GABA_A receptors within the framework of nucleus accumbens shell (AcbSh) in haloperidol-induced hyperphagia and body weight gain in sated rats.Main methodsIn acute studies, GABA_A receptor agonists muscimol, diazepam or antagonist bicuculline were administered by AcbSh route, alone or in combination with haloperidol (intraperitoneal/ip). Immediately after these treatments, preweighed food was offered to the animals at commencement of dark phase. Cumulative food intake was measured at 2 and 6 h post-injection time-points. Furthermore, effects of subacute haloperidol treatment, alone or in combination with muscimol, diazepam or bicuculline, on food intake and body weight were investigated.Key findingsWhile acute treatment with haloperidol, muscimol or diazepam dose dependently stimulated the food intake, bicuculline suppressed the same. Prior administration of muscimol (20 ng/rat, intra-AcbSh) and diazepam (5 µg/rat, intra-AcbSh) significantly potentiated, whereas bicuculline (40 ng/rat, intra-AcbSh) negated the hyperphagic effect of acute haloperidol (0.005 or 0.01 mg/kg/rat, ip). Subacute administration of haloperidol (0.01 mg/kg/rat/day, ip) for 15 days produced increase in food intake and body weight. Although, concomitant administration of muscimol (20 ng/rat/day, intra-AcbSh) or diazepam (5 μg/rat/day, intra-AcbSh) markedly enhanced, bicuculline (40 ng/rat/day, intra-AcbSh) prevented the subacute haloperidol-induced hyperphagia and weight gain.SignificanceThe results of present study suggest that increased food intake and body weight following haloperidol treatment in rats, may be mediated via AcbSh GABA_A receptors.     

GABAA receptor-related chloride flux of rats receiving continuous intracerebroventricular infusion of pentobarbital

The in vitro receptor-stimulated synaptoneurosomal chloride uptake induced by pentobarbital or muscimol was studied in the brains of rats which had been rendered tolerant to or dependent on pentobarbital. In the chronic study, rats received continuous administration of sodium pentobarbital by intracerebroventricular (i.c.v.) infusion. In rats continuously infused with pentobarbital for 6 days or in rats which had the infusion terminated for 24 h, the basal synaptoneurosomal chloride uptake was not altered in either the cerebral cortex or the cerebellum. On the other hand, pentobarbital (500 µM)-stimulated chloride uptake was significantly decreased in both cerebral cortices and cerebella of rats which received pentobarbital for 6 days as compared with the saline control groups. Twenty-four hours after termination of pentobarbital infusion, this high concentration of pentobarbital-stimulated chloride uptake remained low in both cerebral cortices and cerebella of rats which had been infused with pentobarbital for 6 days. In addition, similar results were also obtained in muscimol (2.5 µM)-stimulated chloride uptake in the cerebral cortices of pentobarbital-infused animals. However, pentobarbital infusion failed to alter muscimol-stimulated chloride uptake in the cerebellum. These results suggest that the GABA_A receptor regulated chloride uptake is downregulated after chronic pentobarbital administration. The results also suggest that down-regulation of the GABA_A receptor chloride channel complex takes place at different recognition sites on the complex. It further substantiates the allosteric effects of GABA and barbiturate recognition sites.     

GABA Increases Electrical Excitability in a Subset of Human Unmyelinated Peripheral Axons

Background

A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established.

Methodology/Principal Findings

Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1–100 µM) increased electrical excitability in a subset (ca. 40%) of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABA_A receptors, being mimicked by bath application of the GABA_A agonist muscimol (0.1–30 µM) while the GABA_B agonist baclofen (10–30 µM) was without effect. Increases in excitability produced by GABA (10–30 µM) were blocked by the GABA_A antagonists gabazine (10–20 µM), bicuculline (10–20 µM) and picrotoxin (10–20 µM).

Conclusions/Significance

Functional GABA_A receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABA_A receptor modulation may therefore regulate segmental and peripheral components of nociception.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Pregnenolone Sulfate and Cortisol Induce Secretion of Acyl-CoA-binding Protein and Its Conversion into Endozepines from Astrocytes

Acyl-CoA-binding protein (ACBP) functions both intracellularly as part of fatty acid metabolism and extracellularly as diazepam binding inhibitor, the precursor of endozepine peptides. Two of these peptides, ODN and TTN, bind to the GABA_A receptor and modulate its sensitivity to γ-aminobutyric acid (GABA). We have found that depolarization of mouse primary astrocytes induces the rapid release and processing of ACBP to the active peptides. We previously showed that ODN can trigger the rapid sporulation of the social amoeba Dictyostelium. Using this bioassay, we now show that astrocytes release the endozepine peptides within 10 min of exposure to the steroids cortisol, pregnenolone, pregnenolone sulfate, or progesterone. ACBP lacks a signal sequence for secretion through the endoplasmic reticulum/Golgi pathway and its secretion is not affected by addition of brefeldin A, a well known inhibitor of the classical secretion pathway, suggesting that it follows an unconventional pathway for secretion. Moreover, induction of autophagy by addition of rapamycin also resulted in rapid release of ACBP indicating that this protein uses components of the autophagy pathway for secretion. Following secretion, ACBP is proteolytically cleaved to the active neuropeptides by protease activity on the surface of astrocytes. Neurosteroids, such as pregnenolone sulfate, were previously shown to modulate the excitatory/inhibitory balance in brain through increased release of glutamate and decreased release of GABA. These effects of steroids in neurons will be reinforced by the release of endozepines from astrocytes shown here, and suggest an orchestrated astrocyte-neuron cross-talk that can affect a broad spectrum of behavioral functions.     

Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation

A developmental “switch” in chloride transporters occurs in most neurons resulting in GABA_A mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABA_A receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABA_A receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABA_A receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABA_A activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABA_A mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABA_A receptor activation in NKCC1^-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1^-/- mice retained relatively normal responses to the GABA_A agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO₃ ^- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABA_A mediated transmission in GnRH neurons.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

GABAergic Modulation of Striatal Cholinergic Interneurons: An In Vivo Microdialysis Study

Abstract: Striatal cholinergic interneurons have been shown to receive input from Striatal γ-aminobutyric acid (GABA)-containing cell elements. GABA is known to act on two different types of receptors, the GABA_A and the GABA₆ receptor. Using in vivo microdialysis, we have studied the effect of intrastriatal application of the GABA_A-selective compounds muscimol and bicuculline and the GA- BA_B-selective compounds baclofen and 2-hydroxysaclofen, agonists and antagonists, respectively, at GABA receptors, on the output of Striatal acetylcholine (ACh). Intrastriatal infusion of 1 and 10 μmol/L concentrations of the GABA_A antagonist bicuculline resulted in a significant increase in Striatal ACh output, whereas infusion of 1 and 10 /μmol/L concentrations of the GABA_A agonist muscimol significantly decreased the output of Striatal ACh. Both compounds were ineffective in changing the output of Striatal ACh at lower concentrations. Infusion of concentrations up to 100 μmol/L of the GABA_B-selective antagonist 2-hydroxy-saclofen failed to affect Striatal ACh output, whereas infusion of 10 and 100 μmol/L baclofen, but not 0.1 and 1 μmol/L baclofen, significantly decreased the output of Striatal ACh. Thus, agonist-stimulation of GABAA and GABA_B receptors decreases the output of striatal ACh in a dose-dependent fashion, whereas the GABAergic system appears to inhibit tonically the output of striatal ACh via GABA_A receptors, but not via GABA_B receptors. We hypothesize that although GABA_A mediated regulation of striatal ACh occurs via GABA receptors on the cholinergic neuron, the GABA_B mediated effects may be explained by presynaptic inhibition of the glutamatergic input of the striatal cholinergic neuron.     

Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3α-substituted analogues such as the 3α-fluoro derivative. GABA_A receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [³H]flunitrazepam and [³H]muscimol. The 3α-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [³H]flunitrazepam. For the binding of [³H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC₅₀. The 3α-fluoro derivative was inactive in both assays.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

Effects of taurine on GABA release from synaptosomes of rat olfactory bulb

Summary Superfusion of synaptosomes prepared from rat olfactory bulb revealed constant basal release of endogenous taurine (Tau), aspartate (Asp), glutamate (Glu) and-aminobutyrate (GABA): their release rates were 110.4 ± 13.0, 30.3 ± 6.7, 93.7 ± 13.1, and 53.3 ± 8.8 pmol/min/mg protein, respectively. The depolarizing-stimulation with 30mM KCl evoked 1.17-, 2.18-, 2.55- and 1.53-fold increases, respectively. Tau release was calcium-independent. However, the perfusion of synaptosomes with Tau (10µM) inhibited the evoked increase in GABA release by 63% without changing basal release, although it did not affect release of Asp and Glu. Phaclofen (10µM, a GABA_B receptor antagonist), but not bicuculline (10µM, a GABA_A receptor antagonist), counteracted the Tau-induced reduction in GABA release. These data suggest that Tau may be abundantly released from nerve endings of rat olfactory bulb and that it may regulate GABA release through the activation of presynaptic GABA_B autoreceptors.     

Isoliquiritigenin, a chalcone compound, is a positive allosteric modulator of GABAA receptors and shows hypnotic effects

Isoliquiritigenin (ILTG) is a chalcone compound and has valuable pharmacological properties such as antioxidant, anti-inflammatory, anticancer, and antiallergic activities. Recently, the anxiolytic effect of ILTG has been reported; however, its action mechanism and hypnotic activity have not yet been demonstrated. Therefore, we investigated the hypnotic effect and action mechanism of ILTG. ILTG significantly potentiated the pentobarbital-induced sleep in mice at doses of 25 and 50 mg/kg. The hypnotic activity of ILTG was fully inhibited by flumazenil (FLU), a specific gamma-aminobutyric acid type A (GABA_A)–benzodiazepine (BZD) receptor antagonist. The binding affinity of ILTG was 0.453 μM and was found to be higher than that of the reference compound, diazepam (DZP, 0.012 μM). ILTG (10⁻⁵ M) potentiated GABA-evoked currents to 151% of the control level on isolated dorsal raphe neurons. ILTG has 65 times higher affinity for GABA_A–BZD receptors than DZP, and the dissociation constant for ILTG was 4.0 × 10⁻¹⁰ M. The effect of ILTG on GABA currents was blocked by 10⁻⁷ M FLU and ZK-93426. These results suggest that ILTG produces hypnotic effects by positive allosteric modulation of GABA_A–BZD receptors.