Immunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABAA receptor complex

The distribution of the γ-aminobutyric acid (GABA)_A receptor/benzodizepine receptor/CI⁻ channel complex in the rat brain was examined immunohistochemically using the specific antibody against purified GABA_A receptor complex. The immunization of white albino rabbit with purified GABA_A receptor complex resulted in the formation of specific antibody as indicated by the immunoprecipitation test. Immunohistochemical examinations using the antiserum on rat brain slices by the peroxidase-antiperoxidase method revealed the presence of the following immunoreactive sites which coincided with a previous report using antibody againstl-glutamic acid decar☐ylase; ventromedial nucleus of hypothalamus, red nucleus, globus pallidus, zona compacta and zona reticulata of substantia nigra, layers of Purkinje cells and granular cells of cerebellum, layers III–V of cerebral cortex and stratum radiatum of hippocampus. These results strongly suggest that immunohistochemical application of the antibody against the purified GABA_A receptor complex is a useful tool for identifying GABAergic neurons having GABA_A receptor complex-mediated synapses.     

Expression of GABAA receptor polypeptides in clonal rat cell lines

The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [³H-]muscimol binding; none bound [³⁵S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed α subunits. Northern blot and polymerase chain reaction analysis that showed that out of α₁, α₄, β₁ and γ₂ subunits, only α₁ subunit mRNA was present. Thus, GABA_A-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.     

Tonic inhibition of striatal dopamine transmission: effects of benzodiazepine and GABAA receptor antagonists on extracellular dopamine levels

At present, it is unclear whether ligands which bind at the benzodiazepine/GABA receptor complex play a tonic modulatory role with regard to striatal dopamine (DA) transmission. The present study was designed to examine the effects of Ro15-1788, a benzodiazepine (BZ) receptor antagonist, and SR 95531, a GABA_A receptor antagonist, on striatal extracellular DA (DA_[e]) concentrations in anesthetized and awake rats using the technique of in vivo microdialysis. Local administration of Ro15-1788 resulted in a dose-dependent increase in DA_[e] in both anesthetized and awake animals. The Ro15-1788-induced increase in DA_[e] was blocked by coadministration of the BZ agonist diazepam, as well as GABA. Local administration of SR 95531 also resulted in a dose-dependent alteration in striatal DA levels in both anesthetized and awake animals. The SR 95531-induced increase in DA was blocked by coadministration of GABA. The results suggest that GABA may play a tonic inhibitory role with regard to striatal DA transmission.     

Temperature-dependent effect of zolpidem on the GABAA receptor-mediated response at recombinant human GABAA receptor subtypes

The effects of zolpidem on the two forms of recombinant human GABA_A receptors (α1β2γ2s and α3β2γ2s) at different temperatures were functionally investigated, using the whole-cell patch recording configuration. In both forms, zolpidem potentiated the response to GABA in a concentration-dependent manner. At 16°C, the apparent dissociation constant (K_D) values for the α1β2γ2s and α3β2γ2s forms were 3.7×10⁻⁸ and 5.6×10⁻⁷ M, respectively. When the temperature was increased to 36°C, the K_D values for the α1β2γ2s and α3β2γ2s forms were 2.1×10⁻⁷ and 1.5×10⁻⁶ M, respectively. Although the affinity ratio was reduced from 15.1 to 7.1-fold the selectivity of zolpidem for the α1β2γ2s still remained at 36°C.     

GABAA/benzodiazepine receptor localization in the circadian timing system

gamma-Aminobutyric acid (GABA) and exogenous benzodiazepines are thought to play a role in the neural regulation of circadian rhythms. Because binding sites for the benzodiazepines and GABAA ligands are functionally coupled as part of the GABAA/benzodiazepine receptor complex (GABAA/BZR), we analyzed the localization of GABA neurons and GABAA/BZR within 3 nuclei involved in circadian rhythm regulation using autoradiographic and immunohistochemical techniques. Glutamic acid decarboxylase-immunoreactive axons are present in the suprachiasmatic nuclei (SCN), intergeniculate leaflet (IGL), and dorsal raphe nucleus (DR). Immunoreactivity for the GABAA/BZ receptor complex is absent from the SCN and the IGL whereas the DR shows a dense, uniform immunoreactivity. Semiquantitative analysis of autoradiograms for [3H]diazepam and [3H]flunitrazepam binding reveals a moderate level of binding in the SCN, a low level of binding in the IGL, and the highest level of the DR. Based on both the pattern of benzodiazepine binding and of receptor immunoreactivity the DR would appear to be a likely target site for GABAA and benzodiazepine action. The SCN would also appear to be a possible target site. The results suggest the IGL is not a site for direct GABAA and benzodiazepine action, but do not exclude a role for the IGL in the neural circuitry mediating GABA and benzodiazepine interactions with the circadian system.     

Cocaine and lidocaine have additive inhibitory effects on the GABAA current of acutely dissociated hippocampal pyramidal neurons

Inhibition mediated by γ-aminobutyric acid (GABA) is a major target for the central actions of cocaine and lidocaine, which can result in seizures, especially when these drugs are abused in combination. In the present study, we investigated how cocaine and lidocaine interact to depress GABA current (I_GABA), recorded by the whole-cell technique in freshly isolated rat hippocampal neurons. Cocaine depressed I_GABA in a concentration dependent manner, such that cocaine was more potent against lower than higher GABA concentrations: the cocaine IC₅₀ was 0.13, 0.62 and 1.2 mM for GABA at 2, 10 and 100 μM, respectively. Cocaine depressed I_GABA to the same extent in the absence and presence of 1 μM tetrodotoxin, indicating that cocaine inhibition of I_GABA is distinct from its Na⁺ channel blocking action. Lidocaine reversibly depressed I_GABA evoked by 10 μM GABA, with an IC₅₀ of 9.8 mM. In the presence of 3 mM lidocaine, 0.3 mM cocaine depressed I_GABA (10 μM GABA) to 30±7%. The significantly greater depression by the combined agents (p<0.05) indicates additive effects on the GABA receptor/channel complex, which are likely to contribute to the additive convulsant effects noted when these drugs are abused in combination.     

Benzodiazepine- and barbiturate-interactions with GABAA receptor responses on lactotrophes

Modulation of the biphasic effect of muscimol on prolactin secretion by benzodiazepines and secobarbital was investigated, using an in vitro superfusion system. The stimulatory effect of low concentrations of muscimol was potentiated by both classes of drugs, and the effect of benzodiazepines appeared to be mediated by central-type benzodiazepine receptors. Neither benzodiazepines nor secobarbital affected the inhibitory response to muscimol. Clonazepam reduced the potency of bicuculline methiodide as an antagonist of the stimulatory effect, but did not alter the potency of picrotoxinin. These results demonstrate a selective potentiation of one component of the GABAA receptor effect on lactotrophs by benzodiazepines and barbiturates and provide evidence for a functional effect of these drugs at a site without the CNS.     

Regionally specific alterations in the low-affinity GABAA receptor following perinatal exposure to diazepam

Alterations in a low affinity form of the GABA_A receptor were examined with [³H]bicuculline methylchloride in the adult rat following perinatal exposure to diazepam. Perinatal exposure resulted in a significant reduction in [³H]bicuculline binding in the cingulate cortex. A significant decrease in the ability of GABA to displace bound [³H]bicuculline was observed only in the hypothalamus. The results suggest that the effects of perinatal exposure to diazepam are regionally specific and that benzodiazepine receptors and low affinity GABA_A receptors are functionally linked during the perinatal period.     

Localization of GABAA and GABAB receptor subtypes on serotonergic neurons

The effect of selective destruction of serotonin (5-HT)-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT) on [3H] muscimol and (-)-[3H]baclofen binding was investigated in various rat brain regions. Ten days after intracerebroventricular 5,7-DHT, serotonin levels and [3H]imipramine binding were markedly decreased. 5,7-DHT reduced [3H]muscimol binding only in the mesencephalon, and (-)-[3H]baclofen binding was unmodified in all the areas considered. These results suggest that except in the mesencephalon GABA receptors may not be localized on serotonergic nerve terminals.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

Calcineurin-mediated GABAA receptor dephosphorylation in rats after kainic acid-induced status epilepticus

Calcineurin (CaN) is a neuronally enriched, calcium-dependent phosphatase, which plays an important role in a number of neuronal processes including development of learning and memory, and modulation of receptor's function and neuronal excitability as well as induction of apoptosis. It has been established in kindling model that the status epilepticus (SE)-induced increase in CaN activity is involved in the development of seizures through down-regulation of γ-aminobutyric acid A receptor (GABA_AR) activation. However, the mechanism by which CaN mediates GABA_A receptor dephosphorylation in SE is not fully understood. Here, using a model of kainic acid (KA)-induced SE and CaN inhibitor FK506, we observed the behaviors induced by KA and levels of CaN activity and CaN expression in hippocampus by immunobloting. The results showed that the SE-induced CaN activity was time-dependent, with a peak at 2 h and a return to basal level at 24 h, whereas a significant increase in CaN expression was seen at 24 h after SE. It is proposed that the rapid elevation in CaN activity after KA-induced SE is not likely due to an increase in CaN expression but rather an increase in CaN activation state or kinetics. In addition, we also demonstrated that pre-treatment with FK506 remarkably suppressed the SE-induced CaN activity and its expression, and reversed the SE-induced dephosphorylation of GABA_AR 2/3 subunits. Taken together, our data suggest that down-regulation in inhibition of GABA_AR 2/3 by CaN activity contributes to an elevation in neuronal excitability of hippocampus, which may be involved in development of chronic processes of seizures.     

Effects of repeated amphetamine treatment on regional GABAA receptor binding

The present study examined the effects of repeated exposure to amphetamine on GABA_A receptor binding in cortical and subcortical areas. The goal of the study was to determine whether changes in specific binding were related to behavioral sensitization. Animals were exposed to either saline (0.3 ml, s.c.; n=12) or -amphetamine (2.5 mg/kg, s.c.; n=12) for 6 consecutive days and sacrificed after a 14-day withdrawal period. Differences in GABA_A receptor binding in these two groups of animals were assessed using the GABA_A receptor antagonist [³H]SR 95531. To verify that the preceding treatment regimen led to the development of behavioral sensitization, a separate set of animals (n=8/group) was exposed to the same regimen and challenged with -amphetamine (2.5 mg/kg, s.c.) after the 14-day withdrawal period. As expected, preexposure to amphetamine led to the development of amphetamine sensitization. There were no differences in GABA_A receptor binding in animals preexposed to saline and amphetamine in the prefrontal cortex, caudate-putamen, hypothalamus, or cerebellum. These findings do not provide support for the idea that changes in GABA_A receptor binding in the medial prefrontal cortex or various subcortical areas are related to the development of behavioral sensitization.     

The GABAA/benzodiazepine receptor complex in rat brain neuronal cultures. Characterization by immunoprecipitation

The expression of the GABA_A/benzodiazepine receptor (GABAR/BZDR) complex in primary neuronal cultures from rat brain embryos has been investigated. The GABAR/BZDR complex was photoaffinity labeled with [³H]flunitrazepam [³H]FNZ and immunoprecipitated with subunit specific antibodies. These were the mAb 62-3G1 which is specific for the 57-kDa GABA binding subunit, and the rabbit antiserum A which recognizes the 51-kDa [³H]FNZ binding subunit. The results indicate that the cultured neurons express 5 different peptides of 51, 53, 54, 57 and 59 kDa that can be photoaffinity labeled with [³H]FNZ and that all of them are physically coupled to the GABA_A receptor. Most of the [³H]FNZ photolabeled peptides have similar mobilities to those found in the brain of the newborn rat. Nevertheless, some of the quantitative changes in the photolabeled peptides observed during the normal development of the rat brain were not observed or occurred at much slower pace in the cultured neurons.     

Propofol increases agonist efficacy at the GABAA receptor

Using the whole-cell patch-clamp technique, we have determined that propofol, but not midazolam, increases the efficacy of piperidine-4-sulphonic acid (P4S), a partial agonist at α₁β₁γ_2s GABA_A receptors expressed in HEK 293 cells. These findings are consistent with the idea that propofol facilitates receptor gating, while midazolam increases receptor occupancy by the agonist.     

The maturation of GABAA receptor-mediated control of luteinizing hormone secretion in immature male rats

In male rats, a GABA_A receptor antagonist, bicuculline methiodide or an agonist, muscimol, was infused intravenously for 30 min into the male rat before (postnatal days 16–17 and days 30–31) and after (over day 45) the sexual maturation. Neither bicuculline nor muscimol infusion significantly altered serum LH concentrations at days 16–17. At the days 30–31 and after day 45, bicuculline infusion significantly increased and muscimol infusion significantly decreased serum LH. In conclusion, in male rats, the functional GABA_A receptor system to inhibit the release of LH seems to be established before the sexual maturation, between 18 and 29 days of age.     

Changes in neuroactive steroid content during social isolation stress modulate GABAA receptor plasticity and function

Rats deprived of social contact with other rats at a young age experience a form of prolonged stress that leads to long-lasting alteration in their behavior profile. This chronic stress paradigm is thus thought to be anxiogenic for these normally gregarious animals and their abnormal reactivity to environmental stimuli, when reared under this condition, is thought to be a product of prolonged stress. Neurochemical, molecular, and electrophysiological evidences demonstrate that social isolation is associated with alteration in the structure and function of GABA_A receptors and suggest that endogenous content of the progesterone metabolite 3α,5α-TH PROG may be an important determinant in regulating brain excitability and sensitivity to stimuli and point out its possible role in psychiatric and neurological disorder.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Positive and negative modulation of the GABAA receptor and outcome after traumatic brain injury in rats

Glutamate-mediated excitotoxicity has been shown to contribute to cellular dysfunction following traumatic brain injury (TBI). Increasing inhibitory function through stimulation of γ-aminobutyric acid (GABA_A) receptors may attenuate excitotoxic effects and improve outcome. The present experiment examined the effects of diazepam, a positive modulator at the GABA_A receptor, on survival and cognitive performance in traumatically brain-injured animals. In experiment 1, 15 min prior to central fluid percussion brain injury, rats (n=8 per group) were injected (i.p.) with saline or diazepam (5 mg/kg or 10 mg/kg). Additional rats (n=8) were surgically prepared but not injured (sham-injury). Rats pre-treated with the 5 mg/kg dose of diazepam had significantly lower mortality (0%) than injured, saline-treated rats (53%). Also, diazepam-treated (5 mg/kg) rats had significantly shorter latencies to reach the goal platform in the Morris water maze test performed 11–15 days post-injury. In experiment 2, at 15 min post-injury, rats were given either saline (n=5) or 5 mg/kg diazepam (n=6). Rats treated with diazepam did not differ in mortality from injured rats treated with vehicle. However, rats treated with diazepam at 15 min post-injury had significantly shorter latencies to reach the goal platform in the Morris water maze than injured, vehicle-treated rats. In experiment 3, the post-injury administration of bicuculline (1.5 mg/kg, n=8), a GABA_A antagonist, increased Morris water maze goal latencies compared to injured animals treated with saline (n=8). These results suggest that enhancing inhibitory function during the acute post-injury period produces beneficial effects on both survival and outcome following experimental TBI.     

Evidence for the involvement of nigral GABAA receptors in seizures of adult rats

Several studies have implicated the substantia nigra GABAergic system in the mediation of seizures in adult rats. The present study examines whether the different GABA receptors (GABA_A and GABA_B), are preferentially involved in this GABAergic seizure suppression mechanism. Adult rats were intranigrally infused with muscimol (GABA_A receptor agonist), bicuculline (GABA_A receptor antagonist) or baclofen (GABA₁ receptor agonist) and were exposed to flurothyl seizures Results indicated that while infusions of muscimol had an anticonvulsant effect, infusions of bicuculline had a proconvulsant effect. Baclofen infusions were found to have no effect on seizures. These findings suggest an involvement of the nigral GABA_A receptors in the mediation of seizures in adult rats.     

Subunit dependent modulation of GABAA receptor function by neuroactive steroids

Neurosteroids are potent, endogenous modulators of GABA_A receptor function in the central nervous system. The endogenous progesterone metabolite allopregnanolone (ALP) and the synthetic steroid compound alphaxalone (AFX) have been shown to both directly activate and potentiate GABA_A receptor-activated membrane current (I_GABA). The role of different α and γ subunit subtypes in modulation of I_GABA by ALP and AFX was investigated using recombinant GABA_A receptor isoforms expressed in Xenopus oocytes. Changing or removal of the α subunit subtype altered the efficacy of both ALP and AFX (α2β1γ2L>α1β1γ2Lβ1γ2L) to potentiate I_GABA, but did not alter the potency of the neuroactive steroids at these receptor isoforms. The efficacy of ALP to enhance I_GABA was also dependent on the γ subunit subtype (α1β1γ3>α1β1γ2L=α1β1γ1). AFX also had higher efficacy in the α1β1γ3 receptor isoform compared to α1β1γ1. In contrast to ALP, the potency of AFX was greater in the α1β1γ3 and α1β1γ1 receptor isoforms compared to α1β1γ2L. This study provides evidence that the α subunit subtype determines the efficacy, but not the potency, of these neuroactive steroids to potentiate I_GABA. The γ3 subunit subtype increases the maximal efficacy of neuroactive steroids compared to other γ subunit subtypes. These results suggest that the heteromeric assembly of different GABA_A receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA_A receptors that in turn produce distinctly different modulatory interactions between neuroactive steroids acting at the GABA_A receptor.