Diazepam-insensitive Receptors Research Articles

Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones

Thalamic ventrobasal (VB) relay neurones express multiple GABA(A) receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABA(A) receptors of developing VB neurones and investigate the role of the alpha(1) subunit during maturation of GABA-ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and alpha(1)(0/0) mice and mice engineered to express diazepam-insensitive receptors (alpha(1H101R), alpha(2H101R)). In immature brain, rapid (P8/9-P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8-27) formed by the alpha(4) and delta subunit occurred independently of the alpha(1) subunit. Subsequently (> or = P15), synaptic alpha(2) subunit/gephyrin clusters of WT VB neurones were replaced by those containing the alpha(1) subunit. Surprisingly, in alpha(1)(0/0) VB neurones the frequency of mIPSCs decreased between P12 and P27, because the alpha(2) subunit also disappeared from these cells. The loss of synaptic GABA(A) receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA-ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABA(A) receptors in VB follows a developmental programme independent of the alpha(1) subunit. Changes to synaptic GABA(A) receptor function and the increased expression of extrasynaptic GABA(A) receptors represent two distinct mechanisms for fine-tuning GABA-ergic control of thalamic relay neurone activity during development.

Benzodiazepine Binding to GABAA Receptors

This unit describes ligand binding assays for the characterization of the benzodiazepine (BZ) site of GABA(A) receptors in the central nervous system. The Basic Protocol provides a procedure for analyzing benzodiazepine binding to GABA(A) receptors from brain. The experimental design of the method is suitable for all commercially available radioligands that interact with the BZ site of central GABA(A) receptors. By using the appropriate radioligand in combination with particular displacer compounds, individual (or groups of) GABA(A) receptor subtypes can be identified. Preparation of membrane suspensions for this procedure is described in a Support Protocol, and five Alternate Protocols are provided for the identification of diazepam-sensitive GABA(A) receptors, the α1-subtype of diazepam-sensitive receptors, the α5 subtype of diazepam-sensitive receptors, both diazepam-sensitive and diazepam-insensitive receptors, and diazepam-insensitive receptors exclusively. Each protocol contains a brief description of the requirements for radioligand binding to these five types of BZ sites.

Selective labelling of diazepam‐insensitive GABAA receptors in vivo using [3H]Ro 15‐4513

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.

Selective GABAA ??5 Benzodiazepine Inverse Agonist Antagonizes the Neurobehavioral Actions of Alcohol

Previous research has implicated the alpha5-containing GABAA receptors of the hippocampus in the reinforcing properties of alcohol. In the present study, a selective GABAA alpha5 benzodiazepine inverse agonist (e.g., RY 023) was used in a series of in vivo and in vitro studies to determine the significance of the alpha5-receptor in the neurobehavioral actions of alcohol. In experiment one, systemic injections of RY 023 (1 to 10 mg/kg IP) dose-dependently reduced ethanol-maintained responding by 52% to 86% of controls, whereas bilateral hippocampal infusions (0.3 to 20 microg) reduced responding by 66% to 84% of controls. Saccharin responding was reduced only with the highest intraperitoneal (e.g., 10 mg) and microinjected (e.g., 20 microg) doses. In experiment two, RY 023 (3.0 to 15 mg/kg IP) reversed the motor-impairing effects of a moderate dose of alcohol (0.75 g/kg) on an oscillating bar task in the absence of intrinsic effects. In the open field, RY 023 (3.0 to 7.5 mg/kg) produced intrinsic effects alone but attenuated the suppression of the 1.25 g/kg ethanol dose. Because the diazepam-insensitive receptors (e.g., alpha4 and alpha6) have been suggested to play a role in alcohol motor impairing and sedative actions, experiment three compared the efficacy of RY 023 with Ro 15-4513 and two prototypical benzodiazepine antagonists (e.g., flumazenil and ZK 93426) across the alpha4beta3gamma2-, alpha5beta3gamma2-, and alpha6beta3gamma2-receptor subtypes in Xenopus oocytes. RY 023 produced classic inverse agonism at all receptor subtypes, whereas Ro15-4513 and the two antagonists displayed a neutral or agonistic profile at the diazepam-insensitive receptors. Overall, the results extend our previous findings by demonstrating that an alpha5-subtype ligand is capable of attenuating not only the rewarding action of alcohol but also its motor impairing and sedative effects. We propose that these actions are mediated in part by the alpha5-receptors of the hippocampus. The hippocampal alpha5-receptors could represent novel targets in understanding the neuromechanisms regulating the neurobehavioral actions of alcohol in humans.

Altered receptor subtypes in the forebrain of GABA A receptor δ subunit-deficient mice: recruitment of γ2 subunits

A GABA A receptor δ subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA A receptors. High-affinity [ 3H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of δ −/− animals. [ 3H]Ro 15-4513 binding to benzodiazepine sites was increased in δ −/− animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of γ2 subunits with α4 subunits. In the western blots of forebrain membranes of δ −/− animals, the level of γ2 subunit was increased and that of α4 decreased, while the level of α1 subunits remained unchanged. In the δ −/− forebrains, the remaining α4 subunits were associated more often with γ2 subunits, since there was an increase in the α4 subunit level immunoprecipitated by the γ2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[ 35S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of α4γ2 and α6γ2 subunit-containing receptors, respectively. The altered pharmacology of forebrain GABA A receptors and the decrease of the α4 subunit level in δ subunit-deficient mice suggest that the δ subunit preferentially assembles with the α4 subunit. The δ subunit seems to interfere with the co-assembly of α4 and γ2 subunits and, therefore, in its absence, the γ2 subunit is recruited into a larger population of α4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA A receptors.

Mechanism of α-subunit selectivity of benzodiazepine pharmacology at γ-aminobutyric acid type A receptors

Benzodiazepine pharmacology at the GABA A receptor is dependent on the α and γ subunit isoforms present. Ligands with higher affinity for certain isoforms—selective compounds—have been classified into benzodiazepine type I and II and into diazepam-sensitive and diazepam-insensitive receptors. A single amino acid position (α1G201/α3E225) has been identified which discriminates BZI and BZII receptors. The role of this residue has been explored by mutagenesis of α1 position 201 and the pharmacology of recombinant receptors examined using BZI receptor agonists. Ligand affinity is reduced by increasing side chain volume at α1G201 suggesting that steric inhibition underlies α-subunit selectivity. A second amino acid (α1H102/α6R100) determines diazepam sensitivity. The nature of the amino acid at this position was also examined by mutagenesis. Flumazenil and Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) binding affinity correlated weakly with the amino acid hydrophobicity suggesting a weak hydrophobic interaction between the ligand and α1H102.

Differential sensitivity to inverse agonists of GABA A/benzodiazepine receptors in rats with genetic absence-epilepsy

A strain of Wistar rats, genetic absence epilepsy rats from Strasbourg (GAERS), was selected and inbred over 40 generations for occurrence of spontaneous spike-wave discharges characteristic of absence seizures, simultaneously with a strain of non-epileptic rats (NER). GAERS demonstrate an excessive sensitivity to antagonists of the GABA A receptor. The sensitivity to convulsions induced by various inverse agonists of the GABA A/benzodiazepine receptor was compared in GAERS and NERs. The β-carbolines FG 7142 and DMCM, and the imidazobenzodiazepines RO 19-4603 and the alpha 5-selective RY 024 were several times more convulsant in GAERS than in NERs. The largest differences were found with the non-selective RO 19-4603- and FG 7142. The proconvulsant imidazobenzodiazepine RO 15-4513, binding also to diazepam-insensitive receptors, had low efficacy. The high affinity binding of GABA A/BZD receptors with (3H) RO 15-1788 in the brain of naive rats and after administration of FG 7142 did not differ in GAERS and NERs. The data indicate that the hypersensitivity of GAERS to various inverse agonists of the GABA A/benzodiazepine receptor involves cortical GABA A receptors and is not related to differential activity of a subunit-selective receptor.

Synthesis and evaluation of [ 123I]labelled analogues of the partial inverse agonist Ro 15-4513 for the study of diazepam-insensitive benzodiazepine receptors

The imidazobenzodiazepines ethyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4] benzodiazepine-3-carboxylate 1 and tert-butyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4] benzodiazepine-3-carboxylate 2 were prepared to study the diazepam-insensitive (DI) benzodiazepine receptor (BZR) subtype. The [ 123I] analogues were prepared via iododestannylation reactions in radiochemical yields of 70–80% and a specific activity >2,500 Ci/mmol. The tert-butyl analogue [ 123I]-2 exhibited nanomolar affinity for BZRs in homogenate membranes of rat cerebellum with K d values for the diazepam-sensitive (DS) and DI receptors of 3.18 ± 0.58 and 13.55 ± 2.72 nM, respectively. The B max for cerebellar DS and DI receptors were 1,276 ± 195 and 518 ± 26 fmol/mg protein, respectively.

P1C56 - Cytoprotective effect of anethole dithiole thione (sulfarlem®) as a free radical scavenger-evaluation by microplate cytofluorimetry

A GABAA receptor δ subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABAA receptors. High-affinity [3H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of δ−/− animals. [3H]Ro 15-4513 binding to benzodiazepine sites was increased in δ−/− animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of γ2 subunits with α4 subunits. In the western blots of forebrain membranes of δ−/− animals, the level of γ2 subunit was increased and that of α4 decreased, while the level of α1 subunits remained unchanged. In the δ−/− forebrains, the remaining α4 subunits were associated more often with γ2 subunits, since there was an increase in the α4 subunit level immunoprecipitated by the γ2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[35S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of α4γ2 and α6γ2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABAA receptors and the decrease of the α4 subunit level in δ subunit-deficient mice suggest that the δ subunit preferentially assembles with the α4 subunit. The δ subunit seems to interfere with the co-assembly of α4 and γ2 subunits and, therefore, in its absence, the γ2 subunit is recruited into a larger population of α4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABAA receptors.

The novel benzodiazepine inverse agonist RO19-4603 antagonizes ethanol motivated behaviors: neuropharmacological studies

The novel imidazothienodiazepine inverse agonist RO19-4603 has been reported to attenuate EtOH intake in home cage drinking tests for at least 24 h post-drug administration after systemic administration. In the present study, selectively bred alcohol-preferring (P) rats were trained under a concurrent (FR4–FR4) operant schedule to press one lever for EtOH (10% v/v) and another lever for saccharin (0.05% or 0.75% g/v), then dose-response and timecourse effects of RO19-4603 were evaluated. Systemic RO19-4603 injections (0.0045–0.3 mg/kg; i.p.) profoundly reduced EtOH responding by as much as 97% of vehicle control on day 1. No effects were seen on saccharin responding except with the highest dose level (0.3 mg/kg). In a second experiment, microinjections of RO19-4603 (2–100 ng) directly into the nucleus accumbens (NA) suppressed EtOH responding on day 1 by as much as 53% of control: Control injections dorsal to the NA or ventral tegmental area did not significantly alter EtOH or saccharin responding. On day 2, rats in both experiments received no RO19-4603 treatments; however, all 7 of the i.p. doses, and all 3 of the intra-NA infusions continued to significantly suppress EtOH responding by 43–85% of vehicle control levels. In addition, i.p. injections of RO19-4603 produced a dose-dependent decrease in the slope of the cumulative record for EtOH responding, while concomitantly producing a dose-dependent increase in the slope for saccharin responding. RO19-4603's actions appear to be mediated via recognition sites at GABA A-BDZ receptors which regulate EtOH reinforcement, and not via mechanisms regulating ingestive behaviors. Based on recent in situ hybridization studies in our laboratory, we hypothesize that occupation of α 4 containing GABA A diazepam insensitive (DI) receptors in the NA, may mediate in part, the RO19-4603 suppression of EtOH responding in EtOH-seeking P rats.

GABAA Receptors Containing the α4‐Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ

Recombinant GABA(A) receptors, expressed from alpha-, beta-, and gamma2-subunits, are diazepam-insensitive when the alpha-subunit is either alpha4 or alpha6. In situ, diazepam-insensitive receptors containing the alpha6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABA(A) receptors in various brain areas containing the alpha4-subunit. GABA(A) receptors immunoprecipitated with a newly developed alpha4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of alpha4beta2gamma2-recombinant receptors and diazepam-insensitive [3H]Ro 15-4513 binding sites in rat brain membranes. In addition, alpha4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of alpha4-subunit containing receptors revealed that the alpha4-subunit contributes to several receptor subtypes. Depending on the brain region, the alpha4-subunit can be coassembled with a second type of alpha-subunit variant being alpha1, alpha2, or alpha3. The data demonstrate that native receptors containing the alpha4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [3H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.

P-11-2 - Withdrawal of lorazepam long-term administered using six different plans

A strain of Wistar rats, genetic absence epilepsy rats from Strasbourg (GAERS), was selected and inbred over 40 generations for occurrence of spontaneous spike-wave discharges characteristic of absence seizures, simultaneously with a strain of non-epileptic rats (NER). GAERS demonstrate an excessive sensitivity to antagonists of the GABAA receptor. The sensitivity to convulsions induced by various inverse agonists of the GABAA/benzodiazepine receptor was compared in GAERS and NERs. The β-carbolines FG 7142 and DMCM, and the imidazobenzodiazepines RO 19-4603 and the alpha 5-selective RY 024 were several times more convulsant in GAERS than in NERs. The largest differences were found with the non-selective RO 19-4603- and FG 7142. The proconvulsant imidazobenzodiazepine RO 15-4513, binding also to diazepam-insensitive receptors, had low efficacy. The high affinity binding of GABAA/BZD receptors with (3H) RO 15-1788 in the brain of naive rats and after administration of FG 7142 did not differ in GAERS and NERs. The data indicate that the hypersensitivity of GAERS to various inverse agonists of the GABAA/benzodiazepine receptor involves cortical GABAA receptors and is not related to differential activity of a subunit-selective receptor.

P-11-1 - Gender differences in body temperature during lorazepam tolerance and withdrawal

A strain of Wistar rats, genetic absence epilepsy rats from Strasbourg (GAERS), was selected and inbred over 40 generations for occurrence of spontaneous spike-wave discharges characteristic of absence seizures, simultaneously with a strain of non-epileptic rats (NER). GAERS demonstrate an excessive sensitivity to antagonists of the GABAA receptor. The sensitivity to convulsions induced by various inverse agonists of the GABAA/benzodiazepine receptor was compared in GAERS and NERs. The β-carbolines FG 7142 and DMCM, and the imidazobenzodiazepines RO 19-4603 and the alpha 5-selective RY 024 were several times more convulsant in GAERS than in NERs. The largest differences were found with the non-selective RO 19-4603- and FG 7142. The proconvulsant imidazobenzodiazepine RO 15-4513, binding also to diazepam-insensitive receptors, had low efficacy. The high affinity binding of GABAA/BZD receptors with (3H) RO 15-1788 in the brain of naive rats and after administration of FG 7142 did not differ in GAERS and NERs. The data indicate that the hypersensitivity of GAERS to various inverse agonists of the GABAA/benzodiazepine receptor involves cortical GABAA receptors and is not related to differential activity of a subunit-selective receptor.

Mapping of GABA A receptor sites that are photoaffinity-labelled by [ 3H]flunitrazepam and [ 3H]Ro 15-4513

The GABA A receptor in brain membranes prepared from bovine cerebral cortex and cerebellum has been photoaffinity-labelled by the classical benzodiazepine agonist, [ 3H]flunitrazepam, or by the partial inverse agonist [ 3H]Ro 15-4513. Following solubilization and precipitation with trichloroacetic acid, the photoaffinity-labelled receptor preparations were subjected to specific chemical cleavage using hydroxylamine, a reagent which cleaves specifically at a relatively rare Asn-Gly bond. The resulting peptides were resolved by denaturing polyacrylamide gel electrophoresis and mapping of these peptides to the known amino acid sequences of the GABA A receptor subunits has localized the photoaffinity-labelling sites for these two ligands to distinct portions of the α subunits. It is shown that the site for [ 3H]flunitrazepam photoaffinity-labelling in the receptor populations of both the cerebral cortex and cerebellum occurs within residues 1–103 of the bovine α 1 subunit sequence (or within analogous segments of homologous α subunits). In contrast, the site of photoaffinity-labelling by [ 3H]Ro 15-4513 in the cerebral cortex and in the diazepam-sensitive GABA A receptor population of the cerebellum lies between residues 104 and the carboxy-terminus of the bovine α 1 or homologous α subunits. However, the [ 3H]Ro 15-4513 photoaffinity-labelling site for the diazepam-insensitive receptors of the cerebellum is shown to occur within residues 1–101 ( α 6 subunit numbering). These results demonstrate that the photoaffinity-labelling sites for [ 3H]flunitrazepam and [ 3H]Ro 15-4513 on the GABA A receptor are localized to distinct domains of the α 1 subunit and that [ 3H]Ro 15-4513 photoaffinity labels a site on the α 6 subunit that is unique from its site of labelling on the α 1 subunit.

Phenotypic and genotypic analysis of rats with cerebellar GABAA receptors composed from mutant and wild-type alpha 6 subunits.

The alcohol-sensitive (ANT) rat line, developed for high behavioral sensitivity to ethanol, also exhibits enhanced sensitivity to benzodiazepines, such as diazepam. The rat line carries a point mutation in the cerebellum-specific gamma-aminobutyric acid type A (GABAA) receptor subunit alpha 6, making their diazepam-insensitive (DIS) receptors sensitive to diazepam. We now report that phenotypes of individual ANT and alcohol-insensitive rats, classified on diazepam sensitivity of cerebellar [3H]Ro 15-4513 binding, correlated well with homozygous wild-type, homozygous mutant, and heterozygous genotypes, although some heterozygotes were biased toward the parental phenotypes. GABA down-modulated DIS [3H]Ro 15-4513 binding in mutant homozygotes but tended to up-modulate it in heterozygotes and wild-type homozygotes. Slopes for GABA inhibition of cerebellar t-butyl- bicyclophosphoro[35S]thionate binding were larger in mutant than in wild-type homozygotes, with heterozygotes being intermediate. Diazepam displacement of [3H]Ro 15-4513 binding in heterozygotes revealed three components, with their affinities indistinguishable from those in combined wild-type and mutant homozygotes. This lack of interaction in DIS binding between wild-type and mutant alpha 6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the alpha 6 subunit-containing GABAA receptors in the heterozygotes are formed from individual mutant and wild-type subunits with their relative expression differing from animal to animal.

Stereospecific transduction of behavioral effects via diazepam-insensitive GABA A receptors

Previous studies reported a positive correlation between ligand affinities at diazepam-insensitive GABA A receptors and substitution for the discriminative stimulus effects of the benzodiazepine receptor antagonist, flumazenil, in pigeons. In the present experiments, bretazenil and Ro 14-5974 (ethyl-( S)-11,12,13,13a-tetrahydro-9-oxo-9 H-imidazo[1,5-a]-pyrrolo-[2,1-c][1,4]benzodiazepine-1-carboxylate) partially substituted for, and blocked the discriminative stimulus effects of midazolam, congruent with their actions at diazepam-sensitive GABA A receptors in vitro. In addition, bretazenil and Ro 14-5974, but not their R-enantiomers, had high affinity for diazepam-insensitive receptors and fully substituted for the discriminative stimulus effects of flumazenil. The R-enantiomers of these compounds had low affinity ( K i > 1 μM) for diazepam-sensitive and diazepam-insensitive receptors, and did not share discriminative stimulus effects with flumazenil or midazolam. Ro 19-0528 (7-chloro-3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-4,5-dihydro-5-methyl-6 H-imidazo[1,5-a][1,4]benzodiazepin-6-one), a structurally related compound with full agonist actions at diazepam-sensitive GABA A receptors, had high diazepam-insensitive receptor affinity ( K i = 96 nM) and partially substituted for the discriminative stimulus effects of flumazenil. These results are consistent with stereospecific mediation of the discriminative stimulus effects of flumazenil through high affinity binding to diazepam-insensitive receptors in pigeons.

Different functional effect of Ro 15-4513 and Ro 19-4603 on synaptic responses of Purkinje cells in the rat cerebellar slice

Modulation of GABA-mediated neurotransmissions by Ro 15-4513 in cerebellar slices was assessed following stimulation of the parallel fibre input, which, in this preparation, preferentially activates the inhibitory interneurones innervating Purkinje cells. Peristimulus-time histogram analysis of inhibitory responses of spontaneously-active Purkinje cells showed only a decrease in the duration of inhibition induced by Ro 19-4603. This is consistent with inverse agonism on the BZ 1 receptors associated with postsynaptic GABA a receptors on Purkinje cells. 1 μM Ro 15-4513 induced a similar response but 100 nM Ro 15-4513 induced a biphasic response, with an increase in duration of inhibition preceding the decrease during continued perfusion of the compound. At lower concentrations of Ro 15-4513 the increase in inhibition predominated, the minimal effective concentration being 10 pM. 1 μM flumazenil blocked both components of this response to 100 nM Ro 15-4513, but at 100 nM flumazenil only blocked the decrease in inhibition. The ability of Ro 15-4513 but not Ro 19-4603 to enhance inhibition and its relative insensitivity to 100 nM flumazenil, parallel the affinities of these compounds for diazepam-insensitive (DI) binding sites in the cerebellum. These data suggest that the enhancement of inhibition induced by Ro 15-4513 results from its inverse agonist activity on DI receptors causing disinhibition of both granule cells and their parallel fibres and increased sensitivity to the electrical stimuli inducing activation of the inhibitory interneurones innervating Purkinje cells.

Labelling of diazepam-sensitive and -insensitive benzodiazepine receptors with [ 3H]tert-butyl-8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a][1,4]benzodiazepine 3-carboxylate (ZG-63)

A diazepam-insensitive subtype of benzodiazepine receptor has been identified in the cerebella of several species, including man. t-Butyl-8-chloro-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo[1,5- a][1,4]benzodiazepine 3-carboxylate (ZG-63) was recently described as a selective, high affinity ligand at diazepam-insensitive benzodiazepine receptors. This compound was tritiated, and its properties as a radioligand evaluated in rat brain membranes. Consistent with the high affinity and selectivity described for the non-radioactive form of this compound, saturation analyses of [ 3H]ZG-63 binding to cerebellar diazepam-insensitive and other, diazepam-sensitive benzodiazepine receptors revealed K d values of 2.6 ± 0.2 nM and 10.6 ± 1.4 nM, respectively. The density ( B max) of cerebellar diazepam-insensitive receptors labelled with [ 3H]ZG-63 was not significantly different from values obtained with the prototypical diazepam-insensitive receptor ligand [ 3H]Ro 15-4513, representing ∼30% of total cerebellar benzodiazepine receptors. [ 3H]ZG-63 also labelled cortical diazepam-sensitive benzodiazepine receptors, with B max values that were not significantly different from those obtained with [ 3H]flunitrazepam. Diazepam-insensitive benzodiazepine receptors in rat cerebral cortex could be detected with [ 3H]ZG-63, but the densities of these sites are a very minor component ( $ ̌ = 5% ) of total benzodiazepine receptors. In the presence of GABA, [ 3H]ZG-63 behaved as a ‘γ-aminobutyric acid (GABA) -positive’, ‘GABA-negative’, and ‘GABA-neutral’ ligand at cortical diazepam-sensitive receptors, cerebellar diazepam-sensitive receptors, and cerebellar diazepam-insensitive benzodiazepine receptors, respectively. This profile differs from the prototype diazepam-insensitive receptor ligand, [ 3H]Ro 15-4513. Competition studies demonstrated a very high correlation ( r 2 = 0.98; P < 0.002) between the potencies of a series of benzodiazepine receptor ligands to inhibit [ 3H]ZG-63 and [ 3H]Ro 15-4513 binding to cerebellar diazepam-insensitive receptors. The high affinity and selectivity of [ 3H]ZG-63 for diazepam-insensitive receptors (diazepam-insensitive/diazepam-sensitive ratio of ∼ 0.25) together with a GABA-shift profile which differs from Ro 15-4513 suggests that this compound may be useful in elucidating the function(s) of this benzodiazepine receptor subtype.

Natural mutation of GABA A receptor α6 subunit alters benzodiazepine affinity but not allosteric GABA effects

The binding of the imidazobenzodiazepine, [ 3H]Ro 15-4513, to cerebellar granule cell-specific GABA A/benzodiazepine receptors is typically insensitive to benzodiazepine receptor agonists such as diazepam. A mutation in the α6 subunit, causing replacement of the arginine at the 100 position by glutamine (Q100), has recently been found in an alcohol- and benzodiazepine-sensitive rat line. The mutant α6(Q100)β2γ2 recombinant receptors are sensitive to diazepam. The binding of [ 3H]Ro 15-4513 to cerebellar diazepam-insensitive receptors is enhanced by GABA, whereas binding to diazepam-sensitive receptors is inhibited. Recombinant receptors consisting of β2 and γ2 subunits together with the wildtype α6 or mutant α6(Q100) subunit showed positive modulation of [ 3H]Ro 15-4513 binding by GABA, whereas α1β2γ2 receptors showed negative modulation. The picrotoxin-sensitive binding of a convulsant, t-butylbicyclophosphoro[ 35S]thionate ([ 35S]TBPS),was inhibited in the α6β2γ2 and α6(Q100)β2γ2 receptors by GABA at concentrations less than one-tenth of those required in the α1β2γ2 receptors. GABA effects on [ 35S]TBPS binding were only slightly affected by diazepam in the α6(Q100)β2γ2 receptors, while profound effects were seen in the α1β2γ2 receptors in the presence of diazepam. The results with the mutant receptor suggest that the α1 and α6 subunits are responsible for differential allosteric actions by GABA on other binding sites, independently of the structures defining the benzodiazepine binding pharmacology.