gamma-Aminobutyric acid-stimulated chloride permeability in crayfish muscle.

gamma-Aminobutyric acid selectively increased Cl- permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in Van Harrevald's solution at room temperature took up 36Cl- to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-S incubations, the control 36Cl- uptake space was 131 +/- 4 ml/kg (n = 60) and this was significantly increased by gamma-aminobutyric acid at 200 muM or higher concentrations to 177 +/- 4 ml/kg (n = 48, P less than 0.05). This effect was specific for chloride since gamma-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. gamma-Aminobutyric acid stimulation of 36Cl- uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The gamma-aminobutyric acid stimulation of Cl- permeability is dose dependent with 50% of the maximal effect at 40 muM gamma-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The gamma-aminobutyric acid agonist muscimol causes the same maximal effect on Cl- uptake as gamma-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist gamma-amino, beta-hydroxybutyric acid produced little or no stimulation of 36Cl- flux. The response to gamma-aminobutyric acid was blocked by 2 mM beta-guanidinopropionate or gamma-guanidinobutyrate, 0.5 mM bicuculline, and 10 muM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 muM. Antagonists did not affect control 36Cl- uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter gamma-aminobutyric acid involves a rapid increase in membrane permeability to Cl-.

Benzodiazepine-GABA receptor-ionophore complex. Current concepts.

The benzodiazepine--gamma-aminobutyric acid (GABA) receptor--ionophore system is an oligomeric complex, composed of at least three interacting components. These three components have been well characterized in vitro by radioreceptor binding assays. A variety of centrally acting anxiolytic, depressant, anticonvulsant and convulsant drugs, which affect GABAergic transmission, bind to one of the sites and modulate the binding of ligands at the other sites. Thus, depressant barbiturates, nonbarbiturate hypnotics (like etomidate) and pyrazolopyridines (like etazolate), while inhibiting the binding of alpha-dihydropicrotoxinin (DHP), enhance the binding of GABA and benzodiazepines. These enhancing effects are blocked by convulsant drugs that inhibit the binding of dihydropicrotoxinin and also by bicuculline. These interactions involving barbiturates and other modulatory drugs, exhibit stereoselectivity, anion dependence and brain regional selectivity. Several classes of drugs which facilitate GABAergic transmission appear to interact with the sites for GABA and benzodiazepines allosterically via the dihydropicrotoxinin site of the oligomeric complex. The GABA system has also been implicated in a variety of pathological conditions, including anxiety, seizure activity, movement disorders, cardiovascular control, pain and in drug dependence. Since most of the GABA agonists do not pass the blood-brain barrier, future trends in the pharmacology of GABA may be the development of drugs that will activate the GABA receptor system via picrotoxinin or benzodiazepine sites.

Baclofen induces catatonia in rats.

Baclofen (10 and 20 mg/kg, i.p.) induced catatonia in rats within 10 min of its administration and the effect lasted for 3 hr. Muscimol (100 ng i.c.v. or 1 mg/kg, i.p.) as well as GABA (5 micrograms i.c.v.) potentiated the effect without producing any effect per se. Bicuculline, bromocriptine and scopolamine failed to modify the catatonia induced by baclofen, thereby ruling out the involvement of GABAA receptors, dopaminergic and cholinergic mechanisms. However, GABAB receptor antagonists, such as homotaurine and delta-amino-n-valeric acid, reversed the catatonia induced by baclofen in rats. Since baclofen is known to bind to a subpopulation of GABA receptors (bicuculline-insensitive) and baclofen-induced catatonia was susceptible to reversal by homotaurine and delta-amino-n-valeric acid, it is suggested that this effect could be mediated through GABAB receptors.

Unsulfated and sulfated neurosteroids differentially modulate the binding characteristics of various radioligands of GABA(A) receptors following chronic ethanol administration.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) inhibited the binding of [(3)H]flunitrazepam (2 nM), [(3)H]muscimol (5 nM) and 4 nM [(35)S]t-butylbicyclophosphorothionate [(35)S]TBPS in the rat cerebellum as well as cerebral cortex. DHEAS-induced inhibition of binding of these radioligands (62% to 100%) was more pronounced as compared to that in the case of DHEA (5% to 31%). DHEAS, unlike DHEA, inhibited [(3)H]flunitrazepam binding significantly to a lesser extent in the cerebellum of ethanol-dependent rats as compared to the control group (I(max):82+/-1vs.92+/-2%, p<0.005). However, DHEA, unlike DHEAS, inhibited [(35)S]TBPS binding to a greater extent in the ethanol-dependent rat cerebellum as compared to the control group (I(max):31+/-2vs.19+/-2%, p<0.005). Furthermore, DHEA was more potent in inhibiting [(35)S]TBPS binding in the cerebellum (IC(50):55+/-5 vs. 74+/-7 microM, p<0.05) and cerebral cortex (IC(50):26+/-4vs.64+/-9 microM, p<0.05) of ethanol-dependent rats as compared to the control group. These observations indicate that unsulfated and sulfated androstane-steroids modulate the GABA(A) receptors in the control as well as the ethanol-dependent rats differentially, and also suggest that the androstane-steroid binding sites associated with the GABA(A) receptors play an important role during ethanol dependence.

Anticonvulsant profile of drugs which facilitate GABAergic transmission on convulsions mediated by a GABAergic mechanism.

The effects of selected modulators of GABAergic transmission, either alone or in combination, were tested for their potency on the seizure pattern and mortality induced by convulsant drugs in rat. Pentobarbital and diazepam were effective against both tonic and clonic seizure components induced by bicuculline and picrotoxin. The anticonvulsant profile of ethanol closely resembled that of pentobarbital. Pentobarbital, diazepam and ethanol did not modify seizures induced by strychnine. In contrast, progabide, a central gamma-aminobutyric acid (GABA) receptor agonist, caused significant protection only against convulsions induced by strychnine and its lethality, but did not protect against seizures induced by bicuculline or picrotoxin. Data on interaction of drugs with subprotective doses of these agents clearly demonstrated potentiation of the anticonvulsant actions of these modulators. Thus, seizures induced by bicuculline were more sensitive to the inhibition by pentobarbital in combination with diazepam or ethanol, while pentobarbital with progabide was equally effective against convulsions induced by GABA antagonists. Diazepam, in combination with progabide, blocked only convulsions induced by picrotoxin. Ethanol, in combination with either pentobarbital or with diazepam, was effective against all the three convulsant drugs. These results are consistent with the concept that drugs which facilitate GABAergic transmission are effective against seizures related to an impairment of GABA transmission. Further, the present data indicate that tonic seizures are more susceptible to the actions of drugs than the clonic component. Smaller doses of these drugs, alone or in combination, modified the seizure patterns and mortality, whereas at larger doses, the possible involvement of a nonspecific depressant action cannot be ruled out.

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.

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.

Chronic ethanol treatment upregulates the GABA receptor beta subunit expression.

The molecular mechanisms associated with ethanol-induced tolerance and physical dependence have yet to be elucidated. In previous studies we have demonstrated that chronic ethanol administration produced a decrease in the GABAA receptor mRNA level of alpha 1, alpha 2, alpha 5 subunits, and a decrease in the polypeptide (alpha 1, alpha 2, and alpha 3) expression in the rat cerebral cortex. In this study we examined the effect of chronic ethanol treatment on the mRNA levels and the expressions of the beta-subunits of the GABAA receptors in rat cerebral cortex. The results indicate that chronic ethanol administration produced an upregulation of the beta 1 subunit mRNA (12 kb) by 29 +/- 10%, beta 2 mRNA (8 kb) by 55 +/- 6% and the beta 3-subunit (6 kb) mRNA by 72 +/- 9% in cerebral cortex. The levels of the beta 2 and beta 3 subunit mRNAs remains elevated at 24 hr withdrawal. We also investigated the effect of chronic ethanol administration on the beta-subunit polypeptide expression using monoclonal antibody BD17, which recognizes the beta 2 (P56) and beta 3 (P58) polypeptides. Chronic ethanol treatment increased the levels of both of these polypeptides in cerebral cortex. Taken together, chronic ethanol administration produced an upregulation of the beta-subunit mRNA and the polypeptide expression of these subunits in rat cerebral cortex. In contrast, chronic ethanol treatment decreased the expression of various alpha-subunits in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons.

Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [3H]flunitrazepam binding was rapid and saturable, with an apparent Kd of 4.2 +/- 1.5 nM and Bmax of 776 +/- 54 fmol/mg protein. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC50 = 9 +/- 2 microM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [3H]flunitrazepam, [3H]GABA, [3H]Ro15-1788, [3H]Ro15-4513 and [35S]TBPS binding, a reduced enhancement of [3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABAA-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABAA receptor-mediated slow process.

Chronic ethanol treatment upregulates the NMDA receptor function and binding in mammalian cortical neurons.

In the present study, we investigated the effects of chronic ethanol exposure on NMDA-mediated increase in intracellular calcium concentration ([Ca2+]i) by means of fluorescent measurement of [Ca2+]i with Fura-2AM in mammalian cortical cultured neurons, and the radioligand [3H]MK-801 binding to cortical neuronal membranes. Chronic exposure of the cortical neurons to ethanol (50 mM, 5 days) did not produce any change in the cell protein, morphological appearance, and the resting [Ca2+]i; however, it significantly enhanced the NMDA-mediated increase in [Ca2+]i. The EC50 value of NMDA was not significantly altered following chronic ethanol exposure, however, its Emax value was increased by approximately 45%. Furthermore, chronic ethanol exposure increased the specific [3H]MK-801 binding in cortical neuronal membrane preparation by approximately 30%. The enhancement of the NMDA-mediated increase in [Ca2+]i and the increase in [3H]MK-801 specific binding were reversed following 48 h ethanol withdrawal. Additionally, this enhanced NMDA response and the increased [3H]MK-801 specific binding were susceptible to blockade by the concomitant chronic exposure of the cortical neurons to the NMDA receptor competitive (20 microM CPP), and non-competitive (1 microM MK-801) antagonists, but not by the non-NMDA receptor antagonist, CNQX (10 microM), and the L-type calcium channel blocker, nitrendipine (10 microM). Taken together, these results suggest that chronic ethanol exposure upregulated the NMDA receptor function and binding in cortical cultured neurons, and this increased NMDA receptor function is a NMDA receptor-mediated process. This altered NMDA receptor function may be responsible for the chronic ethanol-induced behavioral consequences and withdrawal syndrome associated with chronic ethanol exposure.

Chronic ethanol treatment differentially regulates NMDA receptor subunit mRNA expression in rat brain.

Previous studies have implicated the N-methyl-D-aspartate receptor (NMDAR) complex in the physical dependence and withdrawal effects of chronic ethanol administration. In this study, we examined the effect of chronic ethanol administration and ethanol withdrawal on the NMDAR subunit R1, R2A, R2B, and R2C mRNA levels in rat hippocampus, cerebral cortex, and cerebellum. Using the RNase protection assay, we compared the levels of the NMDAR subunits mRNAs in ethanol-treated and control rats. Our results indicate that chronic ethanol administration and ethanol withdrawal do not change the NMDAR R1 subunit mRNA levels in cerebral cortex, hippocampus, or cerebellum at any time point. In contrast, 9 h after the last ethanol administration the R2A and R2B mRNA subunits were elevated by approximately 40% in cerebral cortex, and approximately 30% in hippocampus with respect to the levels in control animals. At 48 h the mRNA levels returned to the control levels. The chronic ethanol treatment did not alter R1, R2A, and R2C subunit mRNA levels in cerebellum. Our results demonstrate that chronic ethanol administration produces a differential regulation of the genes encoding the various subunits of the NMDAR.

Aging related alterations in GABAA receptor subunit mRNA levels in Fischer rats.

The influence of aging on the binding of ligands to picrotoxin binding sites as well as steady state levels of mRNA for various alpha subunits of gamma-aminobutyric acid (GABA) receptor complex was investigated in male Fischer F-344 rats. In aged rats, the binding of [35S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced. This decrease in TBPS binding derived from a reduced density of binding sites, rather than from affinity changes, in both cerebral cortex and cerebellum. In aged rats, alpha 1 mRNA level decreased approximately 70% between age 6 months and 24 months in the cerebral cortex (P less than 0.005). In contrast, alpha 1 mRNA remained unchanged in the cerebellum of old rats. The association of a decrease in picrotoxin binding sites in the cerebral cortex with a decline in alpha 1 mRNA level in the cerebral cortex and in alpha 2 mRNA level in the cerebellum is indicated. alpha 6 mRNA level increased with age in the cerebellum. These findings indicate a selective age related modulation in the stoichiometry of GABAA receptor in aging.

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons.

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.

Effect of ethanol on phosphorylation of the NMDAR2B subunit in mouse cortical neurons.

There is evidence that phosphorylation plays a crucial role in the regulation of the NMDA receptors in the brain. In this study we examined the effect of acute and chronic ethanol treatment on the phosphorylation of the R2B subunit of the NMDA receptors in fetal cortical neurons. Additionally, the effect of acute ethanol treatment on the phosphorylation of the R2B subunit in adult cerebral cortex and hippocampus was also examined. The results show that acute or chronic ethanol treatments did not affect the total phosphorylation of the R2B subunit in cortical neurons. In adult mice, we observed that acute ethanol treatment increased the tyrosine phosphorylation of the R2B subunit in hippocampus but not in cerebral cortex. We also observed that acute or chronic ethanol treatments did not alter the Fyn or Csk levels in cortical neurons. Although Fyn, but not Csk, was present in adult cerebral cortex, ethanol did not phosphorylate the R2B subunit in this region. Like ethanol, MK-801 (NMDA antagonist) did not affect the phosphorylation of the R2B subunit in cortical neurons. Taken together, these results suggest that acute and chronic ethanol and MK-801 treatments do not affect the phosphorylation of the R2B subunit in fetal cortical neurons and adult cerebral cortex. Based on these observations, we speculate that the R2B subunit of the NMDA receptors is regulated by multiple cascades and in a brain region specific manner.

Prevalence of the GABAA receptor assemblies containing alpha1-subunit in the rat cerebellum and cerebral cortex as determined by immunoprecipitation: lack of modulation by chronic ethanol administration.

The anti-alpha1 antibody elicited higher immunoprecipitation (%) values of the [3H]flunitrazepam and [3H]muscimol binding activity in the rat cerebellum vs. cerebral cortex, whereas immunoprecipitation values for [3H]Ro 15-4513 and [3H]zolpidem were comparable in these brain regions. Chronic ethanol administration neither changed the radioligand binding to the immunoprecipitated pellet nor the percentage immunoprecip-itation values, thereby indicating that chronic ethanol did not result in down-regulation of the GABAA receptor assemblies containing alpha1-subunit.

Potential involvement of tyrosine kinase pathway in the antagonist induced upregulation of the NMDA receptor NR2B subunit in cortical neurons.

The aim of this study was to study the potential mechanism(s) involved in the antagonist induced upregulation of the N-methyl-d-aspartate receptor (NMDA) NR2B subunit. The results show that chronic treatment of cortical neurons with tyrosine kinase inhibitor (genistein) resulted in downregulation of the NR2B subunit polypeptide levels, while daidzein, an inactive analog of genistein, did not alter the levels of NR2B subunit, implying that tyrosine kinases may be involved in the regulation of the NMDA NR2B subunit content. Chronic treatment of cortical neurons with the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cycloheptane-5,10-iminemaleate (MK-801) enhanced the membrane associated tyrosine kinase activity and upregulated the NR2B receptor subunit. These results suggest that MK-801 induced upregulation of NMDA (NR2B) receptor subunit might be mediated by tyrosine kinases.

Caloric restriction retards the aging associated changes in gamma-aminobutyric acidA receptor gene expression in rat cerebellum.

It is widely accepted that calorie restriction is an effective way of delaying the aging process. Also, there is an indication that the beneficial effects exerted by dietary manipulation may be due to a direct effect at the molecular level like gene expression. The studies were conducted to determine whether calorie restriction prevents any age-related changes in the structural and molecular aspects of the GABAA-BZ receptor. In aged (24-month old diet ad libitum) rats, the binding of [35S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced in the cerebellum. In contrast, [35S]TBPS binding remained unchanged in the cerebellum of calorie restricted old rats. In order to evaluate the molecular basis of these changes, the alpha sub-unit mRNA levels were measured. The GABAA receptor alpha1 sub-unit mRNA level remained unchanged in both the old groups of rats. The alpha2 subunit mRNA level was significantly decreased in the cerebellum of aged rats (24-month old ad libitum), whereas it remained unchanged in the cerebellum of calorie restricted old animals. These findings indicate a selective age and diet related modulation in the stoichiometry of the GABAA receptor in aging.

Postnatal phencyclidine treatment differentially regulates N-methyl-D-aspartate receptor subunit mRNA expression in developing rat cerebral cortex.

The present study was designed to determine the effects of chronic neonatal exposure to the NMDA receptor antagonist phencyclidine (PCP) on [3H]MK-801 binding and on gene expression of NMDA receptor subunits in juvenile male rats. Rat pups were injected daily with PCP from day 5 to 15 and killed on day 21. [3H]MK-801 binding was measured by quantitative autoradiography. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of NR1 subunit (comprising all different splice variants) and three NR2 subunits (NR2A-NR2C). The relative distribution profile of NMDA receptor subunits in the cerebral cortex was NR2B > NR1 > NR2A > NR2C and in the cerebellum NR2C = NR1 > NR2A = NR2B. Chronic PCP administration in postnatal rats produced significant reduction in both [3H]MK-801 binding and mRNA level of the NR2B subunit in the cerebral cortex. Expression of the other NMDA receptor subunits in the cerebral cortex did not change following the drug treatment. In the cerebellum, neither [3H]MK-801 binding nor any of the NMDA receptor subunit expression levels showed any alteration. Together, these data provide a molecular correlate for chronic postnatal PCP-induced down-regulation of [3H]MK-801 binding in rat cerebral cortex and suggest that the NR2B subunit plays an important role in developmental plasticity.

Chronic ethanol treatment produces a selective upregulation of the NMDA receptor subunit gene expression in mammalian cultured cortical neurons.

Our previous work has shown that chronic ethanol treatment upregulated NMDA receptor function and binding in mammalian cortical neurons. However, the potential molecular mechanisms involved in these phenomenon have yet to be elucidated. In the present study, using RNase protection assay, we investigated the effect of chronic ethanol treatment on the NMDA receptor subunits R1, R2A, and R2B mRNA levels in cultured cortical neurons. We found that chronic ethanol (50 mM, 5 days) exposure did not change the NMDA receptor R1 and R2A subunits mRNA levels. In contrast, the NMDA receptor R2B subunit mRNA level was increased by approximately 40% with respect to the control values. The levels of the R2B subunit mRNA returned to the control values following the removal of ethanol for 72 h. In order to determine the involvement of the NMDA receptors in the action of chronic ethanol exposure, we further investigated the effect of the NMDA receptor antagonists on the upregulation induced by chronic ethanol exposure. The results indicate that the increased R2B subunit level was reversed by concomitant chronic exposure of the cortical neurons to the NMDA receptor competitive (10 microM; CPP), and non-competitive (1 microM; MK-801) antagonists, but not by the non-NMDA receptor antagonist, CNQX (10 microM), or the L-type calcium channel blocker, nitrendipine (10 microM). Taken together, these results suggested that chronic ethanol exposure selectively upregulated the NMDA receptor subunit R2B mRNA level in cortical neurons, and this increased NMDA receptor gene expression appears to be a NMDA receptor mediated process. The altered NMDA receptor gene expression may be responsible for the observed upregulation of the NMDA receptor binding and function in the cortical neurons following chronic ethanol exposure.

Down-regulation of the GABA receptor subunits mRNA levels in mammalian cultured cortical neurons following chronic neurosteroid treatment.

We have recently shown that chronic neurosteroid, 5 alpha 3 alpha, treatment produced down-regulation of the GABA receptor binding and function, and heterologous uncoupling on the GABAA receptor complex in cultured mammalian cortical neurons. In order to explore the underlying mechanism of these observed down-regulation and heterologous uncoupling phenomenon, we investigated the effect of chronic 5 alpha 3 alpha (1 microM; 5 days) treatment on the GABAA receptor subunits mRNA levels, using RNase protection assay. We found that chronic neurosteroid, 5 alpha 3 alpha, treatment decreased the beta- and alpha-subunits mRNA levels while not altering the gamma 2S-subunit mRNA levels in the cortical neurons. The decrease in the beta-subunits mRNA levels suggests a decrease in the presence of the beta-subunits in the composition of GABAA receptors. This phenomenon may explain the down-regulation of the GABAA receptor binding and function. A decrease in the alpha 3-subunit mRNA level suggests a corresponding decrease in the alpha 3-subunit in the composition of GABAA receptor isoforms, relative to other isoforms. This observation may be responsible for the chronic neurosteroid-induced uncoupling and decreased efficacy. In summary, chronic 5 alpha 3 alpha treatment produced down-regulation of the GABAA receptor beta- and alpha-subunit mRNA levels, and these changes may be associated with the down-regulation, heterologous uncoupling, and decreased efficacy of GABAA receptor complex in the cultured mammalian cortical neurons.