Aging reduces the mRNA of alpha 1 GABAA receptor subunit in rat cerebral cortex.

The effect of aging on the binding of ligands to the GABA, benzodiazepine and picrotoxin binding sites as well as alpha subunit mRNA level of GABAA receptor was investigated in cerebral cortex of male Fischer F-344 rats. In aged (730- to 770-day-old) rats, the binding of [35S]t-butylbicyclophosphorothionate (TBPS) was significantly reduced. Also, alpha 1 mRNA level was markedly decreased (86% suppression). In contrast, alpha 1 mRNA remained unchanged in cerebellum. These findings indicate a selective age-related structural change in GABAA receptor in rat cerebral cortex.     

Gender-selective effects of ethanol dependence on NMDA receptor subunit expression in cerebral cortex, hippocampus and hypothalamus

Previous investigations have shown subunit-selective alterations in NMDA receptors in ethanol dependent male rats. In the present study, we found pronounced gender differences in the effects of ethanol dependence on NMDA receptor subunit expression in all brain regions investigated. Ethanol dependent female rats exhibited increased NR1 subunit levels in cerebral cortex and hypothalamus, whereas males displayed increased NR1 levels only in hippocampus. NR2A subunit levels were significantly increased only in hippocampus from ethanol dependent male rats, whereas NR2B subunit levels significantly increased in cerebral cortex of both female and male rats. These findings suggest that gender influences neuroadaptations elicited by ethanol dependence at the level of NMDA receptor subunit expression.     

Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat

Ethanol has been shown to enhance gamma-aminobutyric acid (GABA)ergic transmission. In this study an examination was made of the effect of chronic treatment with ethanol and its withdrawal at 24 h on the binding of [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate (TBPS) to brain regions in rat. Rats were rendered tolerant to, and dependent on, ethanol by an intragastric intubation method. The affinity (KD) or the binding capacity (Bmax) of [3H]flunitrazepam or [35S]TBPS was not altered by chronic treatment with ethanol or during withdrawal from ethanol. Neither the enhancing effect of GABA on the binding of [3H]flunitrazepam nor its inhibitory effect on the binding of [35S]TBPS were affected by chronic treatment with ethanol or its withdrawal at 24 h. These results suggest that the sensitivity of benzodiazepine and picrotoxin sites on the oligomeric GABA receptor complex is not affected during tolerance to, or withdrawal from ethanol. It is suggested that the effects of ethanol on GABAergic transmission may be produced at the level of coupled chloride ion channels.     

Physiological modulation of GABA(A) receptor plasticity by progesterone metabolites.

The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of gamma-aminobutyric acid type A (GABA(A)) receptors in the brain during pregnancy and after delivery was investigated in rats. The concentrations in the cerebral cortex and plasma of pregnenolone as well as of progesterone and its neuroactive derivatives allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) and allotetrahydrodeoxycorticosterone (5alpha-hydroxy-3alpha,21-diol-20-one) increased during pregnancy, peaking around day 19, before returning to control (estrus) values immediately before delivery (day 21). In the postpartum period, steroid concentrations in plasma and brain did not differ from control values. The densities of [3H]GABA, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorotionate (TBPS) binding sites in the cerebral cortex also increased during pregnancy, again peaking on day 19 and returning to control values on day 21; receptor density was decreased further 2 days after delivery and again returned to control values within 7 days. These changes were accompanied by a decrease in the apparent affinity of the binding sites for the corresponding ligand on day 19 of pregnancy. The amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, returned to control value around the time of delivery and did not change in the postpartum period. On the contrary, the amount of alpha4 subunit mRNA was not modified during pregnancy both in the cerebral cortex and hippocampus whereas significantly increased 7 days after delivery only in the hippocampus. No significant changes were apparent for alpha1, alpha2, alpha3, beta1, beta2, beta3 and gamma2S subunit mRNAs. Administration of finasteride, a specific 5alpha-reductase inhibitor, to pregnant rats from days 12 to 18 markedly reduced the increases in the plasma and brain concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone as well as prevented both the increase in the densities of [3H]flunitrazepam and [35S]TBPS binding sites and the decrease of gamma2L mRNA normally observed during pregnancy. The results demonstrate that the changes in the plasticity of GABA(A) receptors that occur in rat brain during pregnancy and after delivery are related to the physiological changes in plasma and brain concentrations of neurosteroids.     

Behavioral action of ethanol in Porsolt's forced swim test: modulation by 3 alpha-hydroxy-5 alpha-pregnan-20-one

Ethanol is known to increase cortical and plasma content of GABAergic neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) which is responsible for some of its behavioral and electrophysiological effects. We have previously demonstrated the antidepressant like effect of 3alpha,5alpha-THP in mice. This study investigated the role of 3alpha,5alpha-THP in acute, chronic and withdrawal effects of ethanol using mouse forced swim test (FST) paradigm. While acute systemic ethanol (2 or 2.5 g/kg) administration exhibited an antidepressant like effect, its prolonged consumption produced tolerance to this effect and its withdrawal, on the other hand, elicited enhanced behavioral despair (depression). The antidepressant like effect of ethanol was potentiated by GABA(A) receptor agonist, muscimol (0.5 mg/kg, i.p.), 3alpha,5alpha-THP (0.5, 1 or 2 microg/mouse, i.c.v.) and by neurosteroidogenic drugs viz. selective serotonin reuptake inhibitor (SSRI), fluoxetine (5 or 20 mg/kg, i.p.), agonist at mitochondrial diazepam binding inhibitor receptor, FGIN 1-27 (0.5 or 1 microg/mouse, i.c.v.), or 11beta-hydroxylase inhibitor, metyrapone (0.5 or 1 microg/mouse, i.c.v.) which are known to increase endogenous 3alpha,5alpha-THP content. Furthermore, inhibition of the endogenous neurosteroid biosynthesis by drugs like 5alpha-reductase inhibitor, finasteride (50 mg/kg, s.c.), 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg i.p.) or 3alpha-hydroxysteroid dehydrogenase inhibitor, indomethacin (5 mg/kg, i.p.) and GABA(A) receptor antagonist, bicuculline (1 mg/kg, i.p.) blocked the antidepressant like effect of ethanol. Withdrawal of ethanol from mice consuming it chronically displayed enhanced behavioral despair and elicited tolerance to antidepressant like action of acute ethanol (2.5, 3 or 3.5 g/kg). Moreover, sub-antidepressant doses (0.25 or 0.5 microg/mouse, i.c.v.) of 3alpha,5alpha-THP and fluoxetine (5 mg/kg, i.p.) but not imipramine (1 mg/kg, i.p.) reversed the depression associated with ethanol withdrawal indicating sensitization to their antidepressant action. Thus, 3alpha,5alpha-THP plays a pivotal role in the actions of ethanol and in the depression associated with ethanol withdrawal. These findings may be of potential ramification to contribute to the depression associated with alcoholism and its treatment using neurosteroids.     

Chronic intermittent ethanol (CIE) administration in rats decreases levels of neurosteroids in hippocampus, accompanied by altered behavioral responses to neurosteroids and memory function

The administration of ethanol on a chronic intermittent regimen (CIE) involving multiple withdrawal episodes is a model for ethanol dependence. After CIE, rats exhibited reduced seizure threshold, increased anxiety, tolerance to GABAergic sedative-hypnotic drugs, and changes in GABA(A) receptor function and subunit composition in hippocampus. Previous studies have shown that acute and chronic ethanol may induce changes in the levels of the neurosteroid 3alpha-hydroxysteroid-5alpha-pregnan-20-one (3alpha, 5alpha-THP) in the brain. Therefore, the current study analyses the correlation between chronic intermittent ethanol effects on the level of 3alpha, 5alpha-THP in hippocampus of CIE rats and the behavioral changes in sensitivity to neurosteroids. After CIE, the levels for 3alpha, 5alpha-THP were significantly reduced in hippocampus of rats. The mRNA levels for the enzymes 5alpha-reductase and 3alpha-HSD in hippocampus were also reduced. In vivo, (in contrast to a tolerance to the hypnotic effect of steroids), CIE rats showed increased sensitivity to the anticonvulsant and to the anxiolytic effect of the steroid alphaxalone. Perhaps, this is a response to lowered levels of endogenous neuroactive steroids. CIE rats also showed impairment of hippocampus-dependent memory function. These results suggest that changes in neurosteroids level and in vivo sensitivity to these compounds are involved in the development of ethanol dependence in the CIE model.     

Chronic ethanol administration increases the binding of the benzodiazepine inverse agonist and alcohol antagonist [3H]RO15-4513 in rat brain

Chronic ethanol treatment which produced intoxication and physical dependence in rats, produced an increase in the specific binding of ethanol antagonist [3H]RO15-4513 in rat brain cerebral cortex and cerebellum, but not in hippocampus and striatum. The increase in both the regions was due to an increase in the number (Bmax) of receptor sites. These results suggest that the RO15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of RO15-4513 as an ethanol antagonist.     

The effects of acute and chronic ethanol administration and its withdrawal on gamma-aminobutyric acid receptor binding in rat brain.

1 The effects of acute and chronic ethanol administration, and withdrawal on the binding of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), was investigated in rat brain. 2 Acute ethanol (2 to 4 g/kg i.p. 30 min before removal of brain) produced an increase in the binding capacity of the low affinity GABA receptor binding site. 3 Following chronic ethanol administration (1 to 21 days), the GABA receptor binding characteristics were not altered. These results suggest a possible adaptation of GABA receptors to the continuous presence of ethanol at the GABA synapse. 4 During ethanol withdrawal, the affinity of the low affinity GABA receptor binding site was significantly lower than pair-fed controls at 8 and 16 h withdrawal. 5 These results suggest that GABA receptor sensitivity may play a role in some of the neuropharmacological effects of ethanol and in its withdrawal symptoms.     

The effects of acute and chronic morphine administration on GABA receptor binding

The effect of acute and chronic morphine administration and naloxone-precipitated withdrawal on the binding of [3H]GABA to its receptor sites in rat brain membranes was investigated. Acute morphine (25 mg/kg) administration produced a decrease in the GABA binding in cerebellum, cortex and striatum. This decrease appears to be due to a selective decrease in the number of high-affinity GABA receptor binding sites. In contrast, rats chronically treated with morphine by pellet implantation did not exhibit any changes in GABA receptor binding, except for an increase in pons medulla. However, in rats which were physically dependent, as indicated by naloxone-precipitated withdrawal, GABA binding was decreased significantly in cerebellum and striatum, relative to chronic morphine treatment or placebo pellet controls. This decrease was due to a decrease in the number of low affinity GABA receptor binding sites. Both chronic morphine and naloxone-precipitated withdrawal treatments produced an increase in GABA binding in the pons medulla. These results suggest that morphine may produce some of its effects by modulating GABAergic systems and that high and low affinity GABA receptor sites may play a differential role during various morphine treatments.     

Effects of chronic ethanol administration and ethanol withdrawal on cyclic guanosine 3',5'-monophosphate (cGMP) levels in the rat brain

The main objective of the present study is to investigate the possible effects of chronic ethanol consumption and ethanol withdrawal on cyclic guanosine 3', 5'-monophosphate (cGMP) levels in cerebral cortex, striatum, hippocampus and hypothalamus of rat brain. Ethanol was given to female Wistar rats (225-270g) by a liquid diet for 21 days. cGMP levels were measured in respective brain regions using an EIA kit at 7th, 14th and 21st days of ethanol ingestion and at 6th and 24th h of ethanol withdrawal. cGMP levels in cortex, striatum and hippocampus but not hypothalamus were found significantly increased at 14th and 21st days of ethanol consumption. The most prominent increase was observed in striatal tissues (approximately 350%). cGMP levels of striatum and hippocampus were still remaining significantly high at 6th h of ethanol withdrawal. Blood ethanol levels were found as 115.60, 50.0 and 7.0mg/dl just before and after 6 and 24h of ethanol withdrawal, respectively and audiogenic seizures also occurred at 6th h of ethanol withdrawal with an incidence of 75% in individual parallel groups. Our results suggest that changes of cGMP levels in cerebral cortex, striatum and hippocampus might participate in the mechanism of ethanol dependence and withdrawal in rats.     

Progesterone withdrawal increases the alpha4 subunit of the GABA(A) receptor in male rats in association with anxiety and altered pharmacology - a comparison with female rats

Withdrawal from the neurosteroid 3alpha,5alpha-allopregnanolone after chronic administration of progesterone increases anxiety in female rats and up-regulates the alpha4 subunit of the GABA(A) receptor (GABA(A)-R) in the hippocampus. We investigated if these phenomena would also occur in male rats. Progesterone withdrawal (PWD) induced higher alpha4 subunit expression in the hippocampus of both male and female rats, in association with increased anxiety (assessed in the elevated plus maze) comparable to effects previously reported. Because alpha4-containing GABA(A)-R are insensitive to the benzodiazepine (BDZ) lorazepam (LZM), and are positively modulated by flumazenil (FLU, a BDZ antagonist), we therefore tested the effects of these compounds following PWD. Using whole-cell patch clamp techniques, LZM-potentiation of GABA ((EC20))-gated current was markedly reduced in CA1 pyramidal cells of male rats undergoing PWD compared to controls, whereas FLU had no effect on GABA-gated current in control animals but increased it in PWD animals. Behaviorally, both male and female rats were significantly less sensitive to the anxiolytic effects of LZM. In contrast, FLU demonstrated significant anxiolytic effects following PWD. These data suggest that neurosteroid regulation of the alpha4 GABA(A)-R subunit may be a relevant mechanism underlying anxiety disorders, and that this phenomenon is not sex-specific.     

Allosteric modulation of the GABA(A) receptor in rat hypothalamus by somatostatin is altered by stress

1. This autoradiographic study was conducted to investigate somatostatin modulation of GABA(A) receptor binding in hypothalamic structures of immobilization-stressed rats. 2. GABA(A) receptor binding was labelled with [35S]-t- butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel. 3. Several structures of the rat hypothalamus (i.e. the peri- and paraventricular nuclei) display an increase in [35S]-TBPS binding as well as an alteration of the modulatory effect of somatostatin on the GABA(A) receptor complex under stress. Furthermore, these results demonstrate for the first time that somatostatin is particularly effective in modifying [35S]-TBPS binding to the GABA(A) receptor in rat hypothalamus.     

Gamma-hydroxybutyric acid and diazepam antagonize a rapid increase in GABA(A) receptors alpha(4) subunit mRNA abundance induced by ethanol withdrawal in cerebellar granule cells

Both benzodiazepines and gamma-hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrome. The molecular basis for this therapeutic efficacy was investigated with primary cultures of rat cerebellar granule cells. Long-term exposure of these cells to ethanol (100 mM, 5 days) reduced the abundance of mRNAs encoding the gamma(2)L and gamma(2)S subunits of the GABA type A receptor (-32 and -23%, respectively) but failed to affect that of alpha(1), alpha(4), or alpha(6) subunit mRNAs. Subsequent ethanol withdrawal resulted in decreases in the amounts of alpha(1) (-29%), alpha(6) (-27%), gamma(2)L (-64%), and gamma(2)S (-76%),subunit mRNAs that were maximal after 6 to 12 h. In contrast, 3 h after ethanol withdrawal, the abundance of the alpha(4) subunit mRNA was increased by 46%. Ethanol withdrawal did not affect neuronal morphology but reduced cellular metabolic activity. The increase in alpha(4) subunit was confirmed by functional studies showing a positive action of flumazenil in patch clamp recordings of GABA-stimulated currents after ethanol withdrawal. Diazepam (10 microM) or GHB (100 mM) prevented the increase in the amount of the alpha(4) subunit mRNA, the metabolic impairment, and the positive action of flumazenil induced by ethanol withdrawal but failed to restore the expression of the alpha(1) and gamma(2) subunits. The antagonism by GHB seems not to be mediated by a direct action at GABA(A)R because GHB failed to potentiate the effects of GABA or diazepam on Cl(-) currents mediated by GABA type A receptor.     

Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [3H]flunitrazepam and the potency of gamma-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities.     

Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [35S]TBPS binding to 0-10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor (Mw = 55,000 daltons) is included in the 137,000-dalton [35S]-TBPS binding complex; the [35S]TBPS binding protein alone accounts for 137,000-55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000-500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) enhance and reduce [35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [35S]TBPS (145 pmol/g tissue) was only slightly higher than for [3H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.     

Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines

In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha-THDOC concentrations are implicated in the eliciting of the sedative/hypnotic action of GHB.     

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the “cage” convulsant [³⁵S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [³⁵S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [³⁵S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.     

Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABA(A) receptor subtypes.

1. [35S]t-butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA(A) receptors. Here we examined TBPS binding to the cloned receptors made of alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses. 2. In alpha beta subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the alpha isoform (alpha 3 beta 2 >> alpha 1 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (alpha 6 beta 2) to 1 microM (alpha 6 beta 2 gamma 2). 3. Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem

Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.