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.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA_A and GABA_B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA_A receptor binding without significantly altering GABA_B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA_A, but not GABA_B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA_A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA_B receptors located on non-cholinergic neuronal elements in the striatum.     

Effect of embryonic knock-down of GABAA receptors on the levels of monoamines and their metabolites in the CNS of the mouse

In vitro evidence indicates that gamma-aminobutyric acid (GABA), acting at GABA(A) receptors, exerts a positive trophic effect on monoaminergic neurons during embryogenesis. To determine whether in vivo antagonism of GABA(A) receptors during embryogenesis interferes with the development of monoaminergic neurons, we used mice in which the number of GABA(A) receptors was decreased by 50% by targeted deletion of the beta(3) subunit gene of the GABA(A) receptor. Levels of serotonin, dopamine, norepinephrine, and the metabolites 3,4-deoxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in the brainstem, cortex, striatum and spinal cord of female adult homozygous null (beta3-/-) and wild-type (beta3+/+) mice, as well as progenitor C57BL/6J and Strain 129/SvJ mice. The level of norepinephrine in the spinal cord of beta3-/- mice was 44% less than that of beta3+/+ mice, and did not differ in the brainstem, cortex or striatum. This finding suggests that beta3 subunit-containing GABA(A) receptors mediate the trophic effects of GABA on a subpopulation of spinally-projecting noradrenergic neurons. In contrast, the levels of serotonin, dopamine or their metabolites were unaffected, suggesting that the development of serotonergic and dopaminergic neurons may require activation of only a small fraction of GABA(A) receptors or may not be dependent on beta3 subunit-containing GABA(A) receptors. Finally, Strain 129/SvJ and C57BL/6J mice differed with respect to the levels of dopamine and its metabolites in the brainstem, spinal cord and cortex. These differences may need to be considered when assessing the phenotype of gene-targeted mice for which these mice serve as progenitor strains.     

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 and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Cerebellar GABAA receptors and anxiolytic action of diazepam

Alcohol-sensitive ANT rats have a point mutation in the cerebellum-enriched GABA_A receptor α6 subunit, which makes this subunit and the ANT rats in vivo highly sensitive to benzodiazepine agonists. In the elevated plus maze test of anxiety, diazepam produced a greater anxiolytic response in the ANT rats than in the control, alcohol-insensitive AT rats. The ANT rats were less sensitive to the sedative effect of diazepam in the staircase test of exploration. The results thus suggest that the mutant cerebellar granule cell layer receptors can participate in GABA_A receptor-activation-induced anxiolysis.     

Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

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.     

Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site

An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA), SR 95103, has been shown to be a selective antagonist of GABA at the GABAA receptor site. Subsequent structure-activity studies showed that suppressing the methyl in the 4-position of the pyridazine ring, and substituting the phenyl ring at the para position with a chlorine (SR 42641) or a methoxy group (SR 95531) led to compounds which exhibited the highest affinities for the GABA receptor site in this series. In the present study we examined the biochemical interaction of these compounds with the GABA receptor as well as their biochemical selectivity for this receptor. SR 95531 and SR 42641 displaced [3H]GABA from rat brain membranes with apparent Ki values of 0.15 microM and 0.28 microM respectively and Hill numbers near 1.0. The two compounds antagonized the GABA-elicited enhancement of [3H]diazepam-binding in a concentration-dependent manner without affecting [3H]diazepam-binding per se. Scatchard and Lineweaver-Burk analysis of the interaction of the two compounds with the GABAA receptor sites, revealed that the compounds were competitive at the high affinity site, but non-competitive at the low affinity site. Neither compound interacted with other GABAergic processes or with a variety of central receptor sites. When administered intravenously, SR 95531 and SR 42641 elicited tonic-clonic seizures in mice. Based on these results, it is postulated that SR 95531 and SR 42641 are specific, potent and competitive GABAA antagonists.     

Dynamic regulation of GABAA receptors at synaptic sites

γ-Aminobutyric acid type A receptors (GABA_ARs) mediate fast synaptic inhibition in brain and spinal cord. They are ligand-gated ion channels composed of numerous distinct subunit combinations. For efficient synaptic transmission, GABA_ARs need to be localized to and anchored at postsynaptic sites in precise apposition to presynaptic nerve terminals that release the neurotransmitter GABA. Neurons therefore require distinct mechanisms to regulate intracellular vesicular protein traffic, plasma membrane insertion, synaptic clustering and turnover of GABA_ARs. The GABA_A receptor-associated protein GABARAP interacts with the γ2 subunit of GABA_ARs and displays high homology to proteins involved in membrane fusion underlying Golgi transport and autophagic processes. The binding of GABARAP with NSF, microtubules and gephyrin together with its localization at intracellular membranes suggests a role in GABA_AR targeting and/or degradation. Growth factor tyrosine kinase receptor activation is involved in the control of GABA_AR levels at the plasma membrane. In particular insulin recruits GABA_ARs to the cell surface. Furthermore, the regulation of GABA_AR surface half-life can also be the consequence of negative modulation at the proteasome level. Plic-1, a ubiquitin-like protein binds to both the proteasome and GABA_ARs and the Plic1-GABA_AR interaction is important for the maintenance of GABA-activated current amplitudes. At synaptic sites, GABA_ARs are clustered via gephyrin-dependent and gephyrin-independent mechanisms and may subsequently become internalized via clathrin-mediated endocytosis underlying receptor recycling or degradation processes. This article discusses these recent data in the field of GABA_AR dynamics.     

Influence of gender on chronic ethanol-induced alterations in GABAA receptors in rats

Ethanol dependence, arising from chronic ethanol exposure, is associated with neuroadaptations of GABA_A receptors, evidenced by alterations in various behaviors, receptor responsiveness and subunit gene expression. The present studies explored the effects of ethanol dependence in female rats for comparison with previous studies in our laboratory using male rats. We found that ethanol dependence resulted in differential effects on GABA_A receptor gene expression in female rat cerebral cortex compared to ethanol dependent male rats. Notably, chronic ethanol consumption did not change GABA_A receptor α1 subunit peptide levels in ethanol dependent female rat cortex, in contrast to previously observed decreases in α1 subunit expression in ethanol dependent male rat cortex. The effects of ethanol dependence on additional GABA_A receptor subunit peptide levels (α4, β2/3 and γ2) were similar, but not identical, between female and male rat cortex. When directly compared within the experiment, male and female rats had similar baseline bicuculline seizure thresholds and displayed a similar increase in seizure susceptibility during ethanol withdrawal. Ethanol withdrawn female rats were cross tolerant to the anticonvulsant effects of diazepam, similar to the findings in ethanol withdrawn male rats. Ethanol withdrawn female rats showed a dose-dependent enhancement of the anticonvulsant effect of the neuroactive steroid, THDOC (3α,21-dihydroxy-5α-pregnan-20-one) compared to control animals. This finding is similar to previous observations of increased sensitivity to the anticonvulsant effect of 3α,5α-THP (3α-hydroxy-5α-pregnan-20-one) in ethanol withdrawn male and female rats. In addition, low dose administration of THDOC elevated seizure thresholds in ethanol withdrawn female but not male rats, suggesting that ethanol withdrawn female rats were more responsive to the anticonvulsant effects of this neurosteroid than were ethanol withdrawn male rats. These findings show that gender impacts on adaptations in GABA_A receptors elicited by ethanol dependence. However, the physiological outcomes of the differential alterations are not clear. Taken together, these studies suggest that additional mechanisms, beyond effects on GABA_A receptor gene expression are involved in the mediation of ethanol dependence and withdrawal.     

The expression of GABAA receptor subunits in the substantia nigra is developmentally regulated and region-specific

The substantia nigra pars reticulata (SNR) controls the spread of seizures. GABA_Aergic drug (agonist or antagonist) infusions into the SNR have age-specific and site-specific effects on flurothyl-induced seizures. Developmental and cell-specific regulation of GABA_A receptor subunit expression may be responsible for these specific effects. To test this hypothesis,in situ hybridization was used to examine regional expression of α1 and γ2L GABA_A receptor subunit mRNAs in the SNR during development. Distinct temporal and spatial patterns of expression were observed. In rats at postnatal days (PN) 21–60, fewer neurons were labeled with probes directed to α1 and γ2L subunits in SNR_anterior compared with SNR_posterior. In addition, neurons in SNR_anterior contained higher amounts of hybridization grains than in SNR_posterior. In PN 15 rats, the labeling of neurons was relatively diffuse throughout the anterior and posterior SNR regions with moderate amounts of hybridization grains for both subunits. The finding of age-related differential distribution of α1 and γ2L subunit mRNAs in the SNR suggests that GABA_A receptor heterogeneity may play a role in the age-specific and site-specific effects of GABA_Aergic agents on seizures in the SNR.

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Sommario La sostanza nera pars reticulata (SNR) controlla la propagazione delle crisi. Infusioni di GABA agonisti o antagonisti nella SNR hanno effetti specifici in rapporto all’età e alla sede sulle crisi indotte con flurothyl, per la probabile influenza di fattori regolatori dell’espressione di subunità del receptore GABA_A specificamente legati allo sviluppo e al tipo cellulare. L’ipotesi è stata verificata ana-lizzando l’ espressione regionale del mRNA delle subunità recettoriali GABA_A α1 e γ2L durante lo sviluppo. Diverse modalità di espressione temporale e spaziale sono state osservate. Nel ratto ad una età postnatale (PN) di 21–60 giorni, il numero di neuroni marcati con sonde dirette alle subunità α1 e γ2L è risultato inferiore rispetto alla posteriore. Inoltre, i neuroni di SNR anteriore contenevano una quantità più elevata di granuli di ibridizzazione rispetto a quelli di SNR posteriore. In ratti PN 15, invece, la marcatura di neuroni è risultata relativamente diffusa nell’ambito delle regioni anteriori e posteriori di SNR con una quantità moderata di granuli di ibridizzazione per entrambe le subunità. Il rilievo di una distribuzione differenziale età dipendente dell’mRNA delle subunità α1 e γ2L nella SNR suggerisce che un’eterogeneità recettoriale possa giocare uno specifico ruolo nel determinare gli effetti età e sito-specifici sulle crisi di agenti GABAergici iniettati nella SNR.

     

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.     

Modulation of native and recombinant GABAA receptors by endogenous and synthetic neuroactive steroids

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABA_A receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABA_A receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABA_A receptor modulators and that their effects at ‘physiological’ concentrations (low nanomolar) may be influenced by the subunit composition of the GABA_A receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABA_A receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.     

Adrenalectomy prevents the stress-induced decrease in in vitro [H]Ro15-1788 binding to GABAA benzodiazepine receptors in the mouse

The effect of a single or repeated swim stress on in vivo benzodiazepine receptor binding to various brain regions in adrenalectomized and sham-operated (control) mice was assessed using the benzodiazepine receptor antagonist, [³H]Ro15-1788. In sham-operated mice the binding of [³H]Ro15-1788 to benzodiazepine receptors was reduced in the hippocampus and hypothalamus (single or repeated stress) and cerebral cortex (repeated swim stress) compared to non-stressed mice. In contrast, no alterations in [³H]Ro15-1788 binding were observed in any brain region in adrenalectomized mice after either single or repeated swim stress. These data suggest that an intact hypothalamic-pituitary-adrenal axis is required for the stress-induced decrease in benzodiazepine receptor occupancy measured using the in vivo binding method.     

Functional analysis of GABAA receptors in nucleus tractus solitarius neurons from neonatal rats

To gain insight into specific GABA_A receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1–14 day old rats using whole-cell patch clamp recordings. GABA_A− receptor-mediated currents were detected in all neurons tested, with an average EC₅₀ of 22.2 μM. GABA currents were consistently stimulated by diazepam (EC₅₀=63 nM), zolpidem (EC₅₀=85 nM), loreclezole (EC₅₀=10.1 μM) and the neurosteroid 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn²⁺ (IC₅₀=33.6 μM) picrotoxin (IC₅₀=2.4 μM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10–1000 μM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant α subunit isoform present in GABA_A receptors of the NTS appears to be the α1 and/or α2 subunit. β2 and/or β3 subunits are the major β isoform, while the predominant γ subunit is likely γ2. Our data suggest the contribution to NTS GABA currents by α3–α6, β1, γ1 and δ subunits, if present, is minor by comparison.     

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.     

Freeze-fracture immunocytochemical study of the expression of native and recombinant GABAA receptors

To assess the density and distribution of native and recombinant GABA_A receptors we used label-fracture and fracture-flip technologies combined with immunocytochemistry using monoclonal and polyclonal Abs directed against the extracellular domain of the GABA_A receptor protein located in the freeze-fracture replicas. In cortical neurons there is a high density of GABA_A receptors on both soma and dendrites with some areas were the density of receptors is higher, but there are no well defined clusters. In cerebellar granule cells most of the receptors are distributed in round clusters both in neurites and soma. In astroglial cells the receptor density is lower than in neurons and only occasionally they appear in clusters. In cells transfected with cDNAs encoding for various molecular forms of GABA_A receptor subunits, the receptor density is moderate when cDNAs for α, β and γ subnits are cotransfected; however, on cells cotransfected with cDNAs for β and γ subunits the receptor density is significantly lower. Recombinant receptors appear randomly distributed and occasionally they aggregate in small groups.     

Developmental changes of GABAA receptor-chloride channels in rat Meynert neurons

The developmental changes of GABA_A receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration–response relationships for GABA indicated the developmental alteration of the GABA_A receptor subunits and the channel density. The GABA-induced

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measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA_A response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA_A receptor·Cl⁻ channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.