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PubMed1982

Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro.

When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.

Animals↗
PubMed1983

Autoradiographic differentiation of multiple benzodiazepine receptors by detergent solubilization and pharmacologic specificity.

We have used light microscopic autoradiography to localize differentially Type 1 and 2 benzodiazepine receptors using pharmacologic and detergent treatment techniques. The triazolopyridazine CL218872 has preferential affinity for Type 1 receptors from which it displaces [3H]flunitrazepam selectivity. In biochemical experiments [3H]flunitrazepam binding sites with pharmacologic properties of Type 2 receptors are preferentially solubilized from brain membranes by detergents. We have now treated rat brain slices either with CL218872 or 2% sodium cholate and evaluated the autoradiographic distribution of [3H]flunitrazepam-labeled receptors. Whether calculated from the drug or detergent treated preparations, the relative densities of apparent Type 1 and 2 receptors are the same. Thus differential sensitivity to determine solubilization distinguishes the same two populations of receptors as Type 1 and 2 specific drugs.

Animals↗
PubMed1989

Evidence for the existence of several different alpha- and beta-subunits of the GABA/benzodiazepine receptor complex from rat brain.

gamma-Aminobutyric acid (GABA)/benzodiazepine receptors purified from the brains of young and adult rats were photolabeled by [3H]flunitrazepam or [3H]muscimol. Gel electrophoresis revealed 3 different proteins with apparent molecular weight (Mr) 51,000, 53,000 and 59,000 which were specifically and irreversibly labeled by [3H]flunitrazepam and recognized by the alpha-subunit specific antibody bd-28. Similarly, 3 different proteins with Mrs 51,000, 53,000 and 56,000 were irreversibly labeled by [3H]muscimol and recognized by the beta-subunit-specific antibody bd-17. Comparison of the photolabeling and immunological staining pattern from young and adult rats seems to indicate that proteins labeled by [3H]flunitrazepam and bd-28 are different from those labeled by [3H]muscimol and bd-17.

Affinity Labels↗
PubMed1991

Characterization of recombinant GABAA receptors produced in transfected cells from murine alpha 1, beta 1, and gamma 2 subunit cDNAs.

In order to explore the structural basis of GABAA receptor function, we have expressed murine alpha 1, beta 1, and gamma 2 subunit cDNAs by transient transfection of human 293 cells. Expression of GABAA receptors was measured by ligand binding assay and by electrophysiological analysis. As in other species, expression of the alpha 1 and beta 1 subunits produced a receptor that was insensitive to modulation by benzodiazepines as measured by electrophysiological analysis; however, a small number of flunitrazepam binding sites were detectable. The coexpression of the gamma 2 subunit was found to be essential for this modulation, and also resulted in a dramatic (14-fold) increase in the number of binding sites for flunitrazepam. On the coexpression of all 3 subunit cDNAs, a receptor was produced that exhibited a similar number of binding sites for flunitrazepam and muscimol.

Animals↗
PubMed1991

Changes in benzodiazepine receptors of rat brain after long-term treatment with the Ca(2+)-antagonists nifedipine, verapamil, flunarizine and with the calmodulin antagonist trifluoperazine.

1. The binding of [3H]flunitrazepam to benzodiazepine receptors in the cerebral cortex and hippocampus (membrane fraction P2) was studied after 13-day oral treatment of male Wistar rats with the Ca(2+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin (CaM)-antagonist trifluoperazine (TFP) (3 mg/kg). 2. The changes in the binding characteristics of the benzodiazepine receptors in the frontal cortex were studied in vitro after the addition of nifedipine (10(-6) and 10(-5) M) and verapamil (10(-6) and 10(-5) M). 3. A significant decrease of the binding capacity (Bmax) of [3H]flunitrazepam was established after in vivo treatment with the three Ca(2+)-antagonists as well as after TFP, the decrease being much more pronounced in the hippocampus. 4. Changes in the affinity values (Kd) of [3H]flunitrazepam binding were found in neither of the groups. 5. No data for a direct interaction of nifedipine and verapamil with the brain benzodiazepine receptors were obtained in in vitro experiments.

Animals↗
PubMed1985

On the location of gamma-aminobutyrate and benzodiazepine receptors in the cerebellum of the normal C3H and Lurcher mutant mouse.

Binding of gamma-aminobutyrate and benzodiazepine receptor ligands has been studied in the cerebellum of adult normal (C3H) and Lurcher mutant mice. The adult mutant has lost all Purkinje cells and more than 90% of the granule cells in the cerebellar cortex. When compared with their normal littermates Lurcher mice displayed large decreases in the number of high-affinity binding sites for [3H]muscimol, a synaptic gamma-aminobutyrate receptor ligand, in washed cerebellar homogenates. This observation was consistent with the extensive loss of gamma-aminobutyrate receptive Purkinje and granule cells from the Lurcher cerebellum. However, specific binding of the benzodiazepine-receptor ligand [3H]flunitrazepam to Lurcher cerebellum remained unchanged. Indeed quantitative autoradiography, employing [3H]flunitrazepam as a photoaffinity label, showed no significant differences in the density of labelling between Lurcher and normal littermate mice in any region of the cerebellum. These benzodiazepine binding sites in washed homogenates or tissue sections displayed a gamma-aminobutyrate-induced enhancement of [3H]flunitrazepam binding which occurred to the same extent in both Lurcher and normal cerebellum, a facilitatory effect which could be blocked by the addition of bicuculline methobromide. Our results suggest that a large proportion of the high-affinity, specific benzodiazepine binding sites in mouse cerebellum are not coupled to the synaptic gamma-aminobutyrate receptors thought to be labelled by high affinity [3H]muscimol binding. Further, that benzodiazepine binding sites do not appear to be enriched on either the soma or dendrites of Purkinje cells, as has been suggested from previous studies. Investigations at the electron microscope level are now required to elucidate the cellular location of benzodiazepine binding sites in the cerebellar cortex and to examine whether or not they are likely to be exposed to gamma-aminobutyrate in vivo.

Animals↗
PubMed1988

Benzodiazepine receptors in the human hippocampal formation: a pharmacological and quantitative autoradiographic study.

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the human hippocampal formation were studied in seven cases aged 4-68 years. The pharmacology of the receptors was studied by computerized, non-linear least squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes and the anatomical localization of these receptors was demonstrated using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam. The pharmacological studies indicated that the human hippocampal formation contained equal numbers of benzodiazepine receptors with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate. The autoradiograms demonstrated that the benzodiazepine receptors were distributed in a heterogeneous fashion throughout the major regions of the human hippocampal formation; the highest concentrations of receptors were present in the dentate gyrus (molecular layer) and field CA1 of Ammon's horn (strata pyramidale, oriens, lacunosum), with moderate concentrations in field CA2 of Ammon's horn (stratum pyramidale) and in regions of the subicular complex and entorhinal cortex, and with considerably lower densities in fields CA3 and CA4. Quantitative analyses of the autoradiograms showed that the regions containing the highest densities of receptors (molecular layer of dentate gyrus and the strata oriens, pyramidale and lacunosum of CA1) were enriched with Type 1 receptors whereas other regions of lower receptor densities were enriched with either Type I or Type II receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Multiple benzodiazepine receptors in the human basal ganglia: a detailed pharmacological and anatomical study.

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the striatum (dorsal striatum, comprising the caudate nucleus and putamen, and ventral striatum) and globus pallidus (dorsal pallidum, comprising the external and internal segments, and ventral pallidum) of the human basal ganglia were examined in twelve cases aged 4-71 years. The pharmacology of the receptors was studied using computerized, non-linear least-squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes. The results showed that the dorsal striatum (caudate nucleus and putamen) contained higher concentrations of receptors than the dorsal pallidum (external and internal segments). The dorsal striatum contained equal numbers of sites with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate whereas the globus pallidus contained sites with only high affinity (Type I) for these ligands. The anatomical localization of the benzodiazepine receptor subtypes (Type I and II) was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam in the absence or presence of the discriminating ligand CL218,872. The autoradiograms showed that benzodiazepine receptors were distributed throughout all regions of the human striatum in a heterogeneous fashion, i.e. high-density patches of receptors were set against a background matrix of lower receptor densities. The highest densities of receptors were seen in the ventral striatum where the patches were particularly extensive and showed densities 56% higher than the receptor densities in the dorsal striatal patches. Quantitative analysis showed that the patches in all striatal regions contained mainly Type II receptors (83%-86%) whereas the matrix regions in the ventral and dorsal striatum contained different proportions of the receptor subtypes; Type I receptors predominated (60%) in the matrix of the ventral striatum and Type II receptors predominated (67%-71%) in the matrix of the dorsal striatum. By contrast, the autoradiograms showed that the globus pallidus contained considerably lower concentrations of receptors than the striatum. The highest density of receptors in the globus pallidus was present in the ventral pallidum with successively lower concentrations in the external (26% less) and internal (66% less) segments of the dorsal pallidum. In agreement with the membrane binding studies the receptors in the globus pallidus were mainly of the Type I variety.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1992

Changes in benzodiazepine-receptor activity modify morphine withdrawal syndrome in mice.

The effects of different benzodiazepine-receptor ligands on morphine withdrawal were studied: a benzodiazepine agonist, flunitrazepam; a benzodiazepine antagonist, flumazenil; a partial inverse benzodiazepine agonist, Ro 15-4513; and a partial benzodiazepine agonist, Ro 16-6028. Benzodiazepine-ligands were administered i.p. 30 min before naloxone-induced morphine withdrawal syndrome. Jumping behavior was significantly increased by Ro 15-4513 at 10 and 20 mg/kg. Flunitrazepam decreased jumps at all the doses used. Wet dog shakes were decreased by flumazenil and Ro 15-4513 and increased by Ro 16-6028 (only at the highest dose) and flunitrazepam. Our results show that the activation of the benzodiazepine receptor by agonists or high doses of partial agonists decreases jumping and increases wet dog shake behaviour, while the antagonists or the partial inverse agonists enhance jumping and decrease wet dog shakes. These modifications could be interpreted as an attenuation in the severity of the morphine withdrawal syndrome by benzodiazepine agonists.

Animals↗
PubMed1991

Quantitative autoradiographic study of the postnatal development of benzodiazepine binding sites and their coupling to GABA receptors in the rat brain.

The postnatal development of benzodiazepine binding sites in the rat brain was studied by quantitative receptor autoradiography using [3H]flunitrazepam. The coupling of these sites to GABA receptors was assessed in 43 cerebral structures by examining the effects of in vitro addition of GABA on flunitrazepam specific binding. Benzodiazepine-specific binding was relatively high at birth and exhibited an heterogeneous distribution pattern, anatomically different from the adult one. Data showed a sequential development of benzodiazepine receptors in relation to the time course of maturation of cerebral structures. A proliferation peak which paralleled rapid brain growth was noticed. High levels of benzodiazepine sites were transiently observed in some areas, e.g. thalamus and hypothalamus, and might be related to maturational events. In every brain structure examined, benzodiazepine binding sites were linked to GABA receptors. However, enhancement of flunitrazepam specific binding by exogenous GABA differed according to the structures studied and decreased during development, suggesting some changes in the control of GABA/benzodiazepine regulation during postnatal maturation.

Aging↗
PubMedDate not supplied

GABA/benzodiazepine receptor/chloride ionophore complex in brains of rat lines selectively bred for differences in ethanol-induced motor impairment.

In vitro ligand binding studies were used to compare GABA/benzodiazepine receptor/chloride ionophore complexes in various brain regions of ethanol-sensitive ANT and ethanol-insensitive AT rats. In naive rats, there were several, but fairly small line differences in the binding parameters of [3H]muscimol and [3H]flunitrazepam to cerebral cortical, cerebellar or hippocampal membranes washed with or without a detergent. GABA-stimulation of flunitrazepam binding in the cerebral cortex membranes was slightly greater in the AT than ANT rats. In detergent solubilized receptors, the GABA-stimulation of flunitrazepam binding emerged only in the presence of ethanol in most AT samples, whereas the GABA-stimulation was always observed in ANT samples and ethanol had no further effect. Pharmacological characteristics of [3H]t-butylbicycloorthobenzoate binding displaceable by picrotoxin were similar in both lines. Chronic ethanol administration tended to increase the number of these binding sites in the cerebral cortex of AT rats and to decrease them in the ANT rats. Although many differences between the lines were observed, our results indicate that the ethanol-sensitivity difference between the AT and ANT rat lines cannot be explained by enhanced function of the GABA/benzodiazepine receptor/chloride ionophore complex as far as this is revealed by in vitro binding studies. It remains to be studied whether these lines differ in presynaptic GABAergic mechanisms or in the actual function of the postsynaptic chloride channels before the role of GABA can be more accurately assessed in this genetic model for ethanol-induced motor impairment.

Animals↗
PubMed1994

Immunohistochemical and neurochemical evidence for GABAA receptor heterogeneity between the hypothalamus and cortex.

This study examined both the function of the GABAA receptor complex and the expression of its alpha 1, alpha 2 and alpha 3 subunits within the hypothalamus as compared to that of the cerebral cortex. A large number of different GABAA receptor subunit combinations potentially exist in various brain regions which, presumably, would intimate differing receptor structure and function. Here, we present evidence that the average functional characteristics of GABAA receptors within the rat hypothalamus are considerably different from those of the cerebral cortex. We assessed two neurochemical measures of GABAA receptor function: namely, chloride-facilitation of [3H]flunitrazepam binding and GABA-mediated 36chloride uptake. [3H]Flunitrazepam binding in the rat cortex was facilitated by increasing concentrations (12.5-500 mM) of chloride, and this facilitation was responsive to 15 min restraint. Yet, hypothalamic [3H]flunitrazepam binding was not responsive to increasing chloride-concentration in either the basal or restraint conditions. Also, maximal facilitation of GABA-mediated 36chloride uptake was significantly blunted in the hypothalamus relative to cortex (7.4 +/- 0.9 versus 35.8 +/- 1.5 nmoles/mg protein, respectively). While in vitro addition of 10 microM diazepam shifted GABA-mediated 36chloride uptake curves of the cortex to the left, diazepam addition appeared to be without effect in the hypothalamus. However, the blunted maximal facilitation of GABA on hypothalamic 36chloride uptake made accurate determination of the EC50 for the diazepam-potentiation difficult. In addition to these functional disparities between the regions, differences in subunit expression were also apparent. Distributions of alpha 1, alpha 2 and alpha 3 subunit immunoreactivities within cingulate, parietal and temporal cortices and 8 major hypothalamic regions were assessed. Staining of the alpha 1 subunit was prevalent throughout the hypothalamus and cortex, and dense in both regions. However, the alpha 2 and alpha 3 subunits, while of intermediate density in cortex, were of low density or absent (alpha 3) in the hypothalamus. The alpha 2-immunoreactivity was restricted to cell bodies of the arcuate nucleus, dorsomedial nucleus and overlying dorsal area and to neuropil staining of the median eminence. Thus, functional responsiveness of the GABAA receptor differs in the hypothalamus relative to the cortex and this would seem related to the presence of different receptor alpha subunits in homogenate preparations of the two regions.

Animals↗
PubMed1993

A system for testing the development and reversal of anticonvulsant tolerance to benzodiazepines in mice.

Tolerance to the anticonvulsant effects of benzodiazepines limits their use in epilepsy treatment. Animal models producing tolerance have been developed, but they require repetitive injections over several days or use silastic capsules which must be made for each drug and do not provide a constant infusion rate. Alzet 2001 osmotic pumps deliver at a constant rate (1 microliter/h) and dosage can be easily adjusted. Various solvents, PEG 400, propylene glycol, 2% Tween, 50% DMSO, saline, Molecusol, and 0.5% methyl cellulose, were tried and found unsuitable because benzodiazepines were not maintained in solution or proconvulsant activity was seen. Tetraglycol was chosen as it did not demonstrate these shortcomings. Anticonvulsant activity was evaluated by PTZ i.v. tail infusion using forelimb clonus as the endpoint. This study describes a simple method for testing the development of tolerance and its reversal with flumazenil or ZK 93426. At 72 h of pump infusion with diazepam or flunitrazepam, tolerance to anticonvulsant activity was evident. Acute treatment with flumazenil or ZK 93426 reversed this tolerance. When flumazenil or ZK 93426 was given to diazepam tolerant mice, this reversal was complete. In flunitrazepam tolerant mice reversal with flumazenil was partial, but significant. When flumazenil was chronically coinfused with diazepam or flunitrazepam, anticonvulsant activity was antagonized. Similarly, when ZK 93426 was coinfused with diazepam, anticonvulsant activity was antagonized. The method described is suitable for screening putative anticonvulsant drugs for development of tolerance and the reversal of tolerance by other compounds.

Animals↗
PubMed1993

Absence of modifications of the pharmacological properties of the GABAA receptor complex during aging, as assessed in 3- and 24-month-old rat cerebral cortex.

We have investigated the possible modifications of the pharmacological properties of Type I and Type II benzodiazepine binding sites of the gamma-aminobutyric acidA (GABAA) receptor complex in cortical membranes from 3- and 24-month-old Wistar or Fischer rats. No major changes were found in the binding parameters of [3H]zolpidem (a Type I-specific ligand) or [3H]flunitrazepam (a non-selective benzodiazepine). Neither the Kd values nor the Bmax for either ligand were modified during aging in cortical membranes from Wistar or Fischer rats. Consequently, the proportion of Type I binding sites was also unmodified in aged cortical membranes. The absence of modifications of Type I and Type II binding sites was also confirmed by Cl 218,872 displacement of [3H]flunitrazepam binding in aged cortical membranes from Wistar rats. Furthermore, the [3H]muscimol binding and the allosteric interactions between Type I or total benzodiazepine binding sites and GABA binding sites also remained unaltered with aging in cortical membranes from Wistar rats. Moreover, the pattern and proportion of the [3H]flunitrazepam photoaffinity labeled peptides were also unmodified by aging. These results demonstrate the absence of modifications in Type I or total benzodiazepine binding sites of the GABAA receptor complex from adult and aged cortical membranes in Fischer or Wistar rats.

Affinity Labels↗
PubMed1994

The neuroactive steroid 3 alpha-hydroxy-5 beta-pregnan-20-one is a two-component modulator of ligand binding to the GABAA receptor.

Neuroactive steroids allosterically inhibit [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and enhance [3H]flunitrazepam binding to the GABAA receptor complex. In the presence of 5 microM GABA, 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) inhibits [35S]TBPS binding with high- (IC50 21-32 nM) and low- (IC50 24-63 microM) affinity components in bovine cortical, cerebellar, and hippocampal membranes. The percentage of high-affinity sites ranges from 53% in cortex to 65% in cerebellum and hippocampus. However, 3 alpha, 5 beta-P is a single-site inhibitor in thalamus (IC50 43 nM). In the absence of GABA, similar affinities for the high- and low-affinity components were detected, although the percentages of high-affinity sites were reduced. Similarly, 3 alpha, 5 beta-P enhances [3H]flunitrazepam binding with high- (EC50 44-58 nM) and low- (EC50 2-13 microM) affinity components which account for 71-77% and 23-29% of the sites, respectively, in cortex, cerebellum and hippocampus. 3 alpha, 5 beta-P is a single-site enhancer in thalamus (EC50 80 nM). In contrast to 3 alpha,5 beta-P, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) is a single site modulator of [35S]TBPS and [3H]flunitrazepam binding in all regions examined. These data provide pharmacological evidence consistent with receptor heterogeneity for neuroactive steroids.

20-alpha-Dihydroprogesterone↗
PubMed2004

Characterization of a new synthetic isoflavonoid with inverse agonist activity at the central benzodiazepine receptor.

Research aimed at developing selective drugs acting on gamma-aminobutyric acid (GABA)A receptors introduced compounds from diverse chemical classes unrelated to the 1,4-benzodiazepines, including flavonoids. These studies also revealed the potential use of inverse agonists as cognition-enhancing agents. Here we report pharmacological properties of the novel synthetic isoflavonoid 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). PCALC36 displaced [3H]flunitrazepam binding to rat brain synaptosomes with an IC50 of 13.8 microM. Scatchard analysis of the effect of PCALC36 showed a concentration-dependent reduction of the Bmax of [3H]flunitrazepam, without a marked change in Kd. This effect could be reversed by diluting and washing the preparation. Addition of 20-microM GABA shifted to the right the inhibition curve of PCALC36 on [3H]flunitrazepam binding (IC50 ratio of 0.68), which is characteristic for inverse agonists. PCALC36 produced little change in the GABAergic tonic currents recorded by whole-cell patch clamp in cultured rat hippocampal neurones, but it caused a 20% reduction in miniature inhibitory postsynaptic current amplitude and completely antagonised the full (direct) agonist midazolam in a quickly reversible manner. The data suggest that the coumestan backbone can be useful for developing novel ligands at the GABAA receptor.

Analysis of Variance↗
PubMed2005

Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons.

Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABA(A) receptor-operated chloride channel activity and increased binding affinity of [(3)H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABA(A) receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 microM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC(50) = 12 microM and 135 microM, respectively). This direct effect was inhibited by competitive and non-competitive GABA(A) receptor antagonists. Thymol increased [(3)H]flunitrazepam binding (EC(50) = 131 microM) and showed a tendency to increase [(3)H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABA(A) receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [(3)H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABA(A) receptors. Thymol (0-1 mM) did not affect cellular viability.

Anesthetics, Intravenous↗
PubMed2000

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801.

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA(A) receptor binding and GABA(A) receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA(A) receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA(A) receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA(A) receptor bindings were analyzed by quantitative autoradiography using [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 microl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2 ML). The levels of [3H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [3H]flunitrazepam binding and [35S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of beta2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of beta3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of alpha6- and delta-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [3H]muscimol binding throughout the brain, increases regional [3H]flunitrazepam and [35S]TBPS binding, and alters GABA(A) receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA(A) receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.

Animals↗