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• Consumption of benzodiazepines in Morocco between the years 2004 and 2017. • Use of benzodiazepines in terms of defined daily doses (DDDs)/1,000 inhabitants/day. • The Bromazepam was the molecule the most consumed during 2004–2016. • In 2017, Alprazolam was the most consumed. Although information exists regarding the rate of benzodiazepines (BZDs) use in different countries, little information is available concerning the BZDs consumption in Morocco. To describe prescription rate in Morocco, a retrospective descriptive analysis of BZDs and their agonists use with the instituteIQIVIA database was performed during the period 2004–2017. The obtained data provide a dynamic approach to total BZDs consumption using an annual collection of sales data in Morocco and were expressed in terms of daily defined doses/ 1000 inhabitants / day. Data analysis showed that the major BZDs sold in Morocco were Alprazolam, Bromazepam, Nordazepam, Lorazepam, Parazepam, Diazepam and two benzodiazepine agonists, Zolpidem and Zopiclone. The Bromazepam was the molecule the most consumed during 2004–2016. In 2017, Alprazolam was the most consumed followed by Bromazepam, Nordazepam, Zolpidem, Lorazepam, Parazepam, Diazepam, Dipotassium clorazepate, Dipotassium Clorazypate and Zopiclone with 0.94, 0.91, 0.6, 0.55, 0.45, 0.32, 0.18, 0.18, 0.07 and 0.05 daily defined doses/ 1000 inhabitants / day respectively. The total amount consumed each year for all BZDs and their agonists in Morocco was 2.69, 2.77, 3, 3.17, 3.32, 3.54, 3.61, 3.81, 4.06, 4.30, 4.06, 3.94, 3.78 and 3.66 daily defined doses/ 1000 inhabitants / day, respectively during 2004–2017. To our knowledge, this is the first study that describes the consumption of BZDs and their agonists (Zolpidem, Zopiclone) in Morocco. This data may help the analytical toxicology laboratory and health organizations operating in the field of analytical biochemistry to develop specific BZDs quantification and detection methods needed for the Moroccan population. [ABSTRACT FROM AUTHOR]

The extracellular α (+) /γ 2 (-) interface in the α 1,2,3,5 βγ 2 GABA A receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α 1 -preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α 3 -over-α 1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α 1,3 β 2 γ 2S receptors expressed in tsA201 cells and Xenopus oocytes by [ 3 H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly 201 in α 1 with the corresponding Glu in α 3 completely eliminated the α 1 -over-α 3 preference exhibited by zolpidem. In contrast, the reverse α 3 -E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α 3 β 2 γ 2S , and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α 1 -preference. Interestingly, the α 1 -Gly 201 /α 3 -Glu 225 residue was also a key determinant of the efficacy-based α 3 -over-α 1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α 1 -preferring modulator indiplon and the α 3 -over-α 1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α 1,2,3,5 βγ 2 GABA A receptor seem more complex than previously appreciated, and the importance of the α 1 -Gly 201 /α 3 -Glu 225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: High-dose benzodiazepine dependence constitutes a major clinical concern. Although withdrawal treatment is recommended, it is unsuccessful for a significant proportion of affected patients. More recently, a benzodiazepine maintenance approach has been suggested as an alternative for patients' failing discontinuation treatment. While there is some data supporting its effectiveness, patients' perceptions of such an intervention have not been investigated., Methods: An exploratory qualitative study was conducted among a sample of 41 high-dose benzodiazepine (BZD)-dependent patients, with long-term use defined as doses equivalent to more than 40 mg diazepam per day and/or otherwise problematic use, such as mixing substances, dose escalation, recreational use, or obtainment by illegal means. A qualitative content analysis approach was used to evaluate findings., Results: Participants generally favored a treatment discontinuation approach with abstinence from BZD as its ultimate aim, despite repeated failed attempts at withdrawal. A maintenance treatment approach with continued prescription of a slow-onset, long-acting agonist was viewed ambivalently, with responses ranging from positive and welcoming to rejection. Three overlapping themes of maintenance treatment were identified: "Only if I can try to discontinue…and please don't call it that," "More stability and less criminal activity…and that is why I would try it," and "No cure, no brain and no flash…and thus, just for everybody else!", Conclusions: Some patients experienced slow-onset, long-acting BZDs as having stabilized their symptoms and viewed these BZDs as having helped avoid uncontrolled withdrawal and abstain from criminal activity. We therefore encourage clinicians to consider treatment alternatives if discontinuation strategies fail.

Rationale: Basal forebrain cholinergic neurons modulate the activation of cortical neurons by several stimuli such as fear and anxiety. However, the role of the muscarinic receptor in the medial prefrontal cortex (MPFC) in the modulation of the conditioned emotional response (CER) evoked in the model contextual conditioned fear remains unclear., Objectives: The objective of this study is to test the hypothesis that inhibition of the muscarinic receptor in ventral MPFC modulates CER observed during animal's re-exposure to the aversive context., Methods: Rats implanted with cannulae aimed at the prelimbic (PL) or the infralimbic (IL) were submitted to a high-intensity contextual fear conditioning protocol. Before the test session, they received microinjections of the hemicholinium (choline reuptake blocker), atropine (muscarinic antagonist), J104129 fumarate (M1-M3 muscarinic antagonists), pirenzepine (M1 muscarinic antagonist), neostigmine (inhibitor acetylcholinesterase enzyme), or the systemic administration of the FG7142 (inverse benzodiazepine agonist). Additional independent groups received the neostigmine or FG7142 before the ineffective doses of J104129 fumarate in the low-intensity protocol of contextual fear conditioning., Results: In the high-intensity protocol, the administration of hemicholinium (1 nmol), atropine (0.06-6 nmol), J104129 fumarate (6 nmol), or pirenzepine (6 nmol) attenuated the expression of CER in rats. However, in the low-intensity protocol, only J10129 fumarate (0.06 nmol) reduced the expression of the CER. Finally, neostigmine (0.1-1 nmol) or FG7142 (8 mg/Kg) increased CER expression, an effect inhibited by the low dose of the J10129 fumarate., Conclusions: These results indicated that the blockade of M3 muscarinic receptor in the vMPFC attenuates the CER expression.

The aim of the present study was to examine the effects of nitric oxide synthase (NOS) inhibitors on responses, elicited by benzodiazepines (BZs) in a modified elevated plus-maze task in mice. It was shown that acute doses of diazepam (DZ; 1 and 2 mg/kg) and flunitrazepam (FNZ; 0.05, 0.1 and 0.2 mg/kg) significantly increased the time of transfer latency (TL2) in a retention trial, thus confirming memory impairing effects of BZs. l-NAME (N(G)-nitro-l-arginine methyl ester; 200 mg/kg), a non-selective inhibitor of NOS, and 7-NI (7-nitroindazole; 40 mg/kg), a selective inhibitor of NOS, further intensified DZ-induced memory impairment. On the other hand, L-NAME (50, 100 and 200 mg/kg) and 7-NI (10, 20 and 40 mg/kg) prevented FNZ-induced memory compromising process. The results of this study indicated that suppressed NO synthesis enhanced DZ-induced but prevented FNZ-induced memory impairment. Taken together, these findings could suggest NO involvement in BZs-induced impairment of memory processes. The precise mechanism of these controversial effects, however, remains elusive., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Combination with a low dose of pineal hormone melatonin increased the anxiolytic effect of tofisopam (subthreshold dose) in open-field and cross-maze tests, as well as its chronotropic activity in time-course of forced swimming test in rats.

The gabra5 gene is associated with pharmacological properties (myorelaxant, amnesic, anxiolytic) of benzodiazepines. It is tightly located (0.5 cM) close to the pink-eyed dilution (p) locus which encodes for fur color on mouse chromosome 7. We tested the putative role of the gabra5 gene in pharmacological properties of the full non specific agonist chlordiazepoxide (CDP), using behavioral and molecular approaches in mutated p/p mice and wild type F2 from crosses between two multiple markers inbred strain ABP/Le and C57BL/6By strain. From our results, using rotarod, light-dark box, elevated maze and radial arm maze tests, we demonstrate that p/p mice are more sensitive than WT to the sensory motor, anxiolytic and amnesic effect of CDP. This is associated with the presence of a haplotypic block on the murine chromosome 7 and with an up regulation of gabra5 mRNAs in hippocampi of p/p F2 mice., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Rationale: The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor positive allosteric modulators (AMPA-PAMs), Org 24448 and Org 26576, and the glycine transporter-1 (GlyT-1) inhibitor Org 25935 are developed for treatment of schizophrenia., Objectives: Here we examined experimentally the ability of co-administration of these AMPA-PAMs or the GlyT-1 inhibitor to augment the antipsychotic activity and effect on cortical N-methyl-D: -aspartate (NMDA) receptor-mediated transmission of risperidone, olanzapine, or haloperidol., Methods: We examined antipsychotic efficacy using the conditioned avoidance response (CAR) test, extrapyramidal side effect liability using a catalepsy test, and cortical NMDA receptor-mediated glutamatergic transmission using intracellular electrophysiological recording technique in vitro., Results: Both AMPA-PAMs enhanced the suppression of CAR induced by risperidone or olanzapine, and Org 24448 also enhanced the effect of haloperidol. In contrast, the GlyT-1 inhibitor did not cause any behaviorally significant effect in the CAR test. However, the GlyT-1 inhibitor, but not the AMPA-PAMs, produced a large facilitation of NMDA-induced currents. All three drugs potentiated the effect of risperidone but not haloperidol on these currents. The GlyT-1 inhibitor also facilitated the effect of olanzapine. All drugs potentiated the effect of risperidone on electrically stimulated excitatory postsynaptic potentials (EPSP) in cortical pyramidal cells, whereas only the GlyT inhibitor facilitated the effect of olanzapine., Conclusions: Our results suggest that the AMPA-PAMs, when compared to the GlyT-1 inhibitor, show differential effects in terms of augmentation of antipsychotic efficacy, particularly when combined with risperidone or olanzapine. Both AMPA-PAMs and the GlyT-1 inhibitor may also improve negative symptoms and cognitive impairments in schizophrenia, in particular when combined with risperidone.

Stress is a well-known risk factor in relapse to drug abuse. Several forms of stress in animals have been used with varied degrees of success to elicit reinstatement of drug-seeking after chronic drug self-administration. Here, we tested the ability of the benzodiazepine (BZ) inverse agonist, FG 7142, to elicit anxiety-like behavior and potentiate stress responses in rats as measured by standard behavioral and hormonal indices and for its ability to affect reinstatement of cocaine-seeking in rats with a prior history of cocaine self-administration. FG 7142 elicited anxiety-like behavior on the elevated plus maze (EPM) in cocaine-naïve rats, and cocaine-naïve rats injected with FG 7142 exhibited increased plasma corticosterone levels following EPM exposure. However, in animals with a history of cocaine self-administration, FG 7142 failed to affect elevated plus maze performance and did not affect plasma corticosterone response to the EPM. Furthermore, FG 7142 failed to reinstate cocaine-seeking, nor did it alter conditioned cue-induced reinstatement. These data indicate that the anxiety-related and stress potentiating qualities of BZ inverse agonism are attenuated in cocaine-experienced animals and do not lead to reinstatement of cocaine-seeking., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Key developments in GABA pharmacology over the last 30 years are reviewed with special reference to the advances pioneered by Erminio Costa. His passion for innovative science, and his quest for novel therapies for psychiatric disorders are particularly apparent in his fundamental contributions to the field of GABA research, with a focus on anxiety disorders and schizophrenia. He was a cofounder of the GABAergic mechanism of action of benzodiazepines. He envisaged partial agonists as novel anxiolytics. He identified DBI (diazepam binding inhibitor) as endogenous agonist of neurosteroidogenesis with multiple CNS effects and he pointed to the developmental origin of GABAergic dysfunctions in schizophrenia through his discovery of a reelin deficit, all this in collaboration with Sandro Guidotti. Today, the GABA pharmacology comprises selective hypnotics, non-sedative anxiolytics, memory enhancers and powerful analgesics. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have found repeatedly that medial septal (MS) infusions of glucose impair memory when co-infused with the gamma-amino butyric acid (GABA) agonist muscimol. The present experiments sought to determine whether the memory-impairing effects of this concentration of glucose would generalize to another GABA(A) receptor agonist and to an agonist from another neurotransmitter system that is known to impair memory. Specifically, we determined whether the dose of glucose that produces memory deficits when combined with muscimol in the MS would also impair memory when co-infused with the GABA(A) receptor modulator chlordiazepoxide (CDP) or the opiate morphine. Male Sprague-Dawley rats were given MS co-infusions and then 15 min later tested for spontaneous alternation or given shock avoidance training (retention tested 48 h later). The results showed that MS infusions of the higher dose of glucose with morphine did not produce memory deficits, whereas, the performance of rats given MS co-infusions of CDP with glucose was impaired. These findings suggest that the memory-impairing effects of brain glucose administration may involve an interaction with the GABA(A) receptor., ((c) 2009 Elsevier Inc. All rights reserved.)

Initial effects of drugs of abuse seem to converge on the mesolimbic dopamine pathway originating from the ventral tegmental area (VTA). Even after a single dose, many drugs of abuse are able to modulate the glutamatergic transmission activating the VTA dopamine neurons, which may represent a critical early stage in the development of addiction. Ligands acting on the benzodiazepine site of the inhibitory gamma-aminobutyric acid type A (GABA(A)) receptors are known to be rewarding in animal models and have abuse liability in humans, but notably little evidence exists on the involvement of the mesolimbic dopamine system in their effects. Here we report that single in vivo doses of benzodiazepine-site agonists, similar to morphine and ethanol, induce a modulation in the glutamatergic transmission of VTA dopamine neurons. This is seen 24 h later as an increase in the ratio between alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated excitatory currents using whole-cell patch-clamp configuration in mouse VTA slices. The effect was due to increased frequency of spontaneous miniature AMPA receptor-mediated currents. It lasted at least 3 days after the injection of diazepam, and was prevented by coadministration of the benzodiazepine-site antagonist flumazenil or the NMDA receptor antagonist dizocilpine. A single injection of the GABA(A) receptor alpha1 subunit-preferring benzodiazepine-site ligand zolpidem also produced an increase in the AMPA/NMDA ratio in VTA dopamine neurons. These findings suggest a role for the mesolimbic dopamine system in the initial actions of and on neuronal adaptation to benzodiazepines.

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (alpha/gamma interface) and at aligned positions in the alpha/beta interface. alpha(1)His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of alpha(1)H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of alpha(1)H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of alpha(1)H101C at the alpha/beta interface was significantly slower than modification of alpha(1)H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the alpha/gamma and alpha/beta interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near alpha(1)H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.

The function of gamma-aminobutyric acid type A receptors (GABA(A) receptors) is enhanced by various clinically important drugs including benzodiazepines that act on an allosteric site formed at the interface between the alpha and gamma subunits. In contrast to classical benzodiazepines, the novel pyrazolopyrimidine indiplon (N-methyl-N-{3-[7-(thiophene-2-carbonyl)-1,5,9-triazabicyclo[4.3.0]nona-2,4,6,8-tetraen-2-yl]phenyl}acetamide; N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide) demonstrates relative binding selectivity for the alpha1 subunit containing receptor subtypes, which are the most frequently expressed in the mammalian central nervous system. To investigate the pharmacological properties at GABA(A) receptors and to promote the development of alpha1 subunit selective radiotracers for positron emission tomography imaging, we have started with the evaluation of various fluorinated indiplon derivatives. Binding affinities were determined in homogenates from newborn and adult rats suggesting an alpha1 preference of the reference compounds indiplon, zaleplon as well as for all newly synthesized indiplon derivatives. In homogenated cerebellar tissue obtained from adult rat brain, known to primarily express alpha1 containing GABA(A) receptors, the high affinity of the basic indiplon structure was only slightly affected by an elongation of the alkyl substituent of the amide N from methyl (indiplon; K(i) 3.1 nM) via ethyl (2a, N-(2-fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 5.4 nM) to propyl (2b, N-(3-fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 2.4 nM). Whole cell patch-clamp recordings at neuronal and recombinant GABA(A) receptors indicated that the fluorinated derivatives 2a and 2b have a high potency at alpha1beta3gamma2L isoforms comparable to indiplon (EC(50): 105, 158, and 81 nM, respectively), with 2b displaying the most pronounced efficacy at alpha3beta3gamma2L subtypes. In conclusion, the affinity profiles and functional properties of the newly synthesised fluorinated indiplon derivatives make compounds 2a and 2b suitable for the development of [(18)F]-labelled ligands at GABA(A) receptors containing the alpha1 subunit.

Alcohol produces many subjective and objective effects in man including pleasure, sedation, anxiolysis, plus impaired eye movements and memory. In human volunteers we have used a newly available GABA-A/benzodiazepine receptor inverse agonist that is selective for the alpha5 subtype (a5IA) to evaluate the role of this subtype in mediating these effects of alcohol on the brain. After pre-treatment with a5IA, we found almost complete blockade of the marked impairment caused by alcohol (mean breath concentration 150mg/100ml) of word list learning and partial but non-significant reversal of subjective sedation without effects on other measures such as intoxication, liking, and slowing of eye movements. This action was not due to alterations in alcohol kinetics and so provides the first proof of concept that selectively decreasing GABA-A receptor function at a specific receptor subtype can offset some actions of alcohol in humans. It also supports growing evidence for a key role of the alpha5 subtype in memory. Inverse agonists at other GABA-A receptor subtypes may prove able to reverse other actions of alcohol, and so offer a new approach to understanding the actions of alcohol in the human brain and in the treatment of alcohol related disorders in humans.

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.

In epileptic patients, there is a high incidence of psychiatric comorbidities, such as anxiety. Gamma-aminobutyric acid (GABA) ionotropic receptor GABA(A)/benzodiazepine allosteric site is involved in both epilepsy and anxiety. This involvement is based on the fact that benzodiazepine allosteric site agonists are anticonvulsant and anxiolytic drugs; on the other hand, benzodiazepine inverse agonists are potent convulsant and anxiogenic drugs. The aim of this work was to determine if subgroups of rats selected according to their susceptibility to clonic convulsions induced by a convulsant dose 50% (CD50) of DMCM, a benzodiazepine inverse agonist, would differ in behavioral tests commonly used to measure anxiety (elevated plus-maze, open field) and depression (forced swimming test). In the first experiment, subgroups of adult male Wistar rats were selected after a single dose of DMCM and in the second experiment they were selected after two injections of DMCM given after an interval of 1 week. Those rats presenting full clonic convulsions were termed Low Threshold rats to DMCM-induced clonic convulsions (LTR) and those not having clonic convulsions High Threshold rats to DMCM-induced clonic convulsions (HTR). In both experiments, only those rats presenting full clonic convulsions induced by DMCM and those not showing any signs of motor disturbances were used in the behavioral tests. The results showed that the LTR subgroup selected after two injections of a CD50 of DMCM spent a significantly lower time in the open arms of the elevated plus-maze and in the off the walls area of the open field; moreover, this group also presented a higher number of rearings in the open field. There were no significant differences between HTR and LTR subgroups in the forced swimming test. LTR and HTR subgroups selected after only one injection of DMCM did not differ in the three behavioral tests. To verify if the behavioral differences between HTR and LTR subgroups of rats selected after two injections of DMCM were due to the clonic convulsion, another experiment was carried out in which subgroups of rats susceptible and nonsusceptible to clonic convulsions induced by a CD50 of picrotoxin, a GABA(A) receptor channel blocker, were selected and submitted to the elevated plus-maze and open field tests. The results obtained did not show any significant differences between these two subgroups in the elevated plus-maze and open field tests. In another approach to determine the relation between fear/anxiety and susceptibility to clonic convulsions, subgroups of rats were selected in the elevated plus-maze as more or less fearful/anxious. The CD50 for clonic convulsions induced by DMCM was determined for each of these two subgroups. The results showed a significantly lower CD50 for the more fearful/anxious subgroup, which means a higher susceptibility to clonic convulsions induced by DMCM. The present findings show a relation between susceptibility to clonic convulsions and fear/anxiety and vice versa which may be due to differences in the assembly of GABA(A)/allosteric benzodiazepine site receptors in regions of the brain.

Classical benzodiazepines (BZs) are the most widely prescribed drugs acting on the central nervous system (CNS). They exert their therapeutic effects via binding to the BZ-site of GABAA receptors, and allosterically modulating the chloride flux through the ion channel complex. Given the multiple actions of classical BZs, the serious limitations to their usefulness have directed much research into development of novel ligands for the BZ-site with retained therapeutic effectiveness and minimal side effects. From the studies of CNS-active chemical constituents of medicinal herbs, some members of the family of flavonoids were demonstrated to have moderate binding affinities for the BZ-site. In vivo studies revealed that these compounds were mostly partial agonists of GABAA receptors, and only a few flavonoids were shown to possess antagonistic activities. At effective anxiolytic doses, the actions of partial agonistic flavonoids were often not accompanied by sedative and myorelaxant side effects. Based on structure-activity relationship (SAR) studies, incorporation of electronegative groups to the C6 and C3' on the flavone backbone was found to yield significant increases in the binding affinities for the BZ-site. It was also shown that 2'-hydroxyl was a critical moiety on flavonoids with regard to BZ-site binding. These have guided the identification of several synthetic flavonoids with high BZ-site binding affinity and in vivo activity, and further quantitative SAR studies resulted in the development of several pharmacophore models. This review attempts to summarize these findings, which has led to the establishment of flavonoids as potential therapeutics for GABAA receptor-mediated disorders.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Within the diseased brain, glial cells and in particular, microglia, express a multimeric protein complex termed "peripheral benzodiazepine binding sites (PBBS)" or "peripheral benzodiazepine receptor (PBR)". The expression of the PBBS is dependent on the functional state of the cell and in glial cells is triggered by a wide range of activating stimuli. In the healthy brain, the PBBS are nearly absent with the notable exception of the choroid plexus, ependymal layer, perivascular cells, central canal, possibly certain nuclei in the brainstem and layers in the cerebellum where a constitutive presence of the PBBS is found. Likewise, areas that due to the absence of the blood-brain barrier contain more active glial cells, such as the pituitary gland, or the area postrema at floor of the 4th ventricle show a degree of constitutive expression. The tight correlation of the parenchymal de novo expression of the PBBS with the presence of activated glial cells, that in turn are usually only found in tissue affected by progressive disease, establishes the PBBS as a generic marker for the detection and measurement of active disease processes in the brain. Specific radioligands of the PBBS for use in positron emission tomography (PET) may thus provide a sensitive in vivo index of neuropathological activity. Whilst prototype ligands for the PBBS are available, future research needs to focus on the development of new ligands with improved pharmacodynamic properties and the ability to discriminate between the different, still insufficiently understood functional states of the peripheral benzodiazepine receptor complex.

Background and Objective: Little representative data of the epidemiology of attempted suicide exists in Germany. In this study the frequency of parasuicidal drug intoxication, the distribution of age and gender, as well as the kind and origin of used drugs were evaluated. Furthermore the knowledge about used drugs and possible adverse effects of a previously given medication were analysed., Patients and Methods: Over a period of 2 years (January 1998-December 1999) 155 patients (41 males, 114 females, average age 40.5 years) with drug intoxication by attempted suicide were recruited at the University Hospital of Dresden, Germany, for further retrospective analysis., Results: 74 % of these patients were women. Sedatives and hypnotics were most frequently used for parasuicide (44 %), followed by analgesics (18 %) and antidepressants (12 %). Benzodiazepines and benzodiazepine-agonists were the most commonly used drugs (32 %). Moreover, 80 % of all drugs used had been prescribed by physicians. Approximately half of the patients were well-informed about drugs taken. In 43 (47 %) of 92 patients with long-term medication an adverse effect was considered as a possible cause of the attempted suicide., Conclusion: Our data underline the importance of attempted suicide in view of the frequency of their use, the need of hospitalization, the required intensive care and possible relapses. Because the majority of drugs used were prescribed by physicians, before giving any medication to their possible suicidal use should be considered.

Sleep complaints are common among patients and may be associated with mild to severe clinical symptoms. The use of hypnotic agents to improve the onset and quality of sleep has a history dating back centuries. Currently, several benzodiazepines and nonbenzodiazepines are widely used as sedative hypnotic drugs. The pharmacokinetic and pharmacodynamic profiles of these agents vary substantially, allowing them to be chosen for different clinical situations and tailored to specific sleep complaints. The short-acting benzodiazepines and benzodiazepine agonists have become the most widely prescribed sedative hypnotics because of their short to ultrashort half-life, and low tendency to impair cognitive and psychomotor performance on the day following nighttime use. In the current review, we summarize the pharmacokinetic and drug interaction profiles of the Food and Drug Administration-approved benzodiazepine sedative hypnotics: estazolam, flurazepam, quazepam, temazepam, and triazolam. In addition, alprazolam is included because of its popularity for this use, and 2 benzodiazepine receptor agonists, zolpidem and zaleplon, are reviewed. The presented data will provide clinicians with insight into the pharmacokinetic profiles of several popular sedative hypnotic agents, and provide guidelines to help them recognize or avoid drug interactions.

A GABA(A) receptor delta 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 [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma(2)Y72-gamma(2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma(2)T73-gamma(2)T81 adopts a beta-strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma(2)A79 and gamma(2)T81, that line the BZD binding site. Sulfhydryl modification of gamma(2)A79C or gamma(2)T81C allosterically shifts the GABA EC(50) of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

This study identifies by microautoradiography activated microglia/macrophages as the main cell type expressing the peripheral benzodiazepine binding site (PBBS) at sites of active CNS pathology. Quantitative measurements of PBBS expression in vivo obtained by PET and [(11)C](R)-PK11195 are shown to correspond to animal experimental and human post-mortem data on the distribution pattern of activated microglia in inflammatory brain disease. Film autoradiography with [(3)H](R)-PK11195, a specific ligand for the PBBS, showed minimal binding in normal control CNS, whereas maximal binding to mononuclear cells was found in multiple sclerosis plaques. However, there was also significantly increased [(3)H](R)-PK11195 binding on activated microglia outside the histopathologically defined borders of multiple sclerosis plaques and in areas, such as the cerebral central grey matter, that are not normally reported as sites of pathology in multiple sclerosis. A similar pattern of [(3)H](R)-PK11195 binding in areas containing activated microglia was seen in the CNS of animals with experimental allergic encephalomyelitis (EAE). In areas without identifiable focal pathology, immunocytochemical staining combined with high-resolution emulsion autoradiography demonstrated that the cellular source of [(3)H](R)-PK11195 binding is activated microglia, which frequently retains a ramified morphology. Furthermore, in vitro radioligand binding studies confirmed that microglial activation leads to a rise in the number of PBBS and not a change in binding affinity. Quantitative [(11)C](R)-PK11195 PET in multiple sclerosis patients demonstrated increased PBBS expression in areas of focal pathology identified by T(1)- and T(2)-weighted MRI and, importantly, also in normal-appearing anatomical structures, including cerebral central grey matter. The additional binding frequently delineated neuronal projection areas, such as the lateral geniculate bodies in patients with a history of optic neuritis. In summary, [(11)C](R)-PK11195 PET provides a cellular marker of disease activity in vivo in the human brain.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABA(A) receptors and allosterically modulate the effect of GABA on chloride ion flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, and hyperphagic effects. In the work presented here, a computational procedure developed in our laboratory has been used to obtain a 3D pharmacophore for ligand recognition of the GABA(A)/BDZRs initiating the hyperphagic response. To accomplish this goal, 17 structurally diverse compounds, previously assessed in our laboratory for activity at the hyperphagic endpoint, were used. The result is a four-component 3D pharmacophore. It consists of two proton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobic moiety in a common geometric arrangement in all compounds with activity at this endpoint. This 3D pharmacophore was then assessed and successfully validated using three different tests. First, two BDZR ligands, which were included as negative controls in the set of seventeen compounds used for the pharmacophore development, did not fit the pharmacophore. Second, some benzodiazepine ligands known to have activity at the hyperphagia endpoint, but not included in the pharmacophore development, were used as positive controls and were found to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the present work to search 3D databases, over 50 classical benzodiazepines were found. Among them, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. In addition, the novel compounds also found in this search are promising therapeutic agents that could beneficially affect feeding behavior.

Drugs that bind to benzodiazepine recognition sites of gamma-aminobutyric acid type A receptor complexes may function as agonists in some behavioral assays and as antagonists in other behavioral assays. The present studies compared the effects of the benzodiazepines midazolam, flumazenil, bretazenil, Ro 41-7812, and Ro 42-8773 and the beta-carboline, beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) under two different types of schedule-controlled responding in squirrel monkeys. One group of monkeys responded under a fixed-ratio schedule of stimulus-shock termination, and a second group of monkeys responded under a multiple fixed-ratio schedule of food presentation involving suppressed and nonsuppressed behavior. Under the schedule of stimulus-shock termination, midazolam produced dose-related decreases in response rate, and these effects were surmountably antagonized by flumazenil, bretazenil, Ro 41-7812, Ro 42-8773, and beta-CCt. Schild plot analysis of these data revealed the following mean pA(2) values: flumazenil, 7.18; bretazenil, 7.62; Ro 41-7812, 7. 06; Ro 42-8773, 6.95. Apparent pA(2) values were not calculated for beta-CCt because the CL of the slope of the Schild plot included positive values. Under the multiple schedule, midazolam, bretazenil, and Ro 42-8773 dose-dependently increased rates of suppressed responding, whereas flumazenil, Ro 41-7812, and beta-CCt had no significant rate-altering effects. Flumazenil antagonized the antisuppressant effects of midazolam and bretazenil; however, individual variability in these effects prohibited the determination of apparent pA(2) values. These results indicate that in vivo pA(2) values may be determined for benzodiazepine-site ligands. These results further demonstrate that some benzodiazepine-site ligands, e. g., bretazenil and Ro 42-8773, may function as both agonists and as competitive antagonists in vivo.

The effect of benzodiazepine agonists of varying efficacy on gamma-aminobutyric acidA receptor alpha1 subunit protein expression was determined in primary cultured cerebellar granule cells. After 48 h exposure to 1 microM drug concentrations, flunitrazepam, diazepam, and the partial agonists Ro 19-8022 and bretazenil, but not the partial agonists Ro 42-8773, Ro 41-7812 or imidazenil, decreased alpha1 subunit protein expression. The grading of effect of the benzodiazepine partial agonists on alpha1 subunit protein expression is consistent with their agonist efficacies. This model, therefore, appears to act as a sensitive indicator of benzodiazepine agonist efficacy with the ability to differentiate between partial agonists.

The present study examined the relationship between the intrinsic efficacy of benzodiazepines and their ability to reproduce the discriminative stimulus effects of pentobarbital. Rhesus monkeys (n=5) were trained to discriminate pentobarbital (10mg/kg) from saline using a discrete trials shock avoidance procedure (fixed-ratio 5). Drug-appropriate responding and response rate were assessed after intragastric administration of pentobarbital, the high efficacy benzodiazepines triazolam and clonazepam, and the low efficacy benzodiazepines imidazenil and bretazenil. Pentobarbital increased drug-appropriate responding to 100% and decreased the response rate. Triazolam and clonazepam occasioned 80% or greater drug-appropriate responding in four out of four monkeys, and decreased response rate in three out of four monkeys. The antagonist flumazenil decreased drug-appropriate responding and increased response rate after triazolam and clonazepam, consistent with antagonism of the discriminative stimulus and the rate-altering effects of these ligands. Imidazenil occasioned 80% or greater drug-appropriate responding in three out of four monkeys and suppressed the response rate in two out of four monkeys, whereas bretazenil occasioned 80% or greater drug-appropriate responding in two out of four monkeys and decreased the response rate slightly. Bretazenil, when co-administered with a dose of triazolam that produced 90-100% drug-appropriate responding, decreased responding to 0-60% but did not alter the rate-suppressing effects of this ligand. The rank order of potency (based on pmol/kg ED, values) was: triazolam > clonazepam > or = imidazenil > bretazenil. These results demonstrate differences in the pentobarbital-like discriminative stimulus and response rate-decreasing effects among benzodiazepine agonists. Specifically, full agonists engendered pentobarbital-like responding in all subjects with decreased response rates in most subjects, whereas partial agonists engendered pentobarbital-like responding and decreased response rates in subsets of subjects.

The response to i.v. administration of bicuculline and its interaction with the benzodiazepine agonist diazepam and antagonist flumazenil were studied in male and female handling stressed and swim stressed rats. Both handling stressed and swim stressed male rats needed less bicuculline to produce myoclonic twitch and running/bouncing (RB) clonus than females. Besides, a lower dose of bicuculline produced tonic hindlimb extensor convulsion (THE) in male than in female swim stressed rats. Flumazenil failed to affect seizure thresholds for bicuculline either in handling stressed or in swim stressed animals. Sex differences remained present after diazepam pre-treatment as well. While diazepam enhanced doses of bicuculline producing all three convulsive signs similarly in both handling and swim stressed rats (141-162%), swim stress had the lowest anticonvulsive effect for the onset of myoclonic twitch (110% in males and 117% in females) and the highest for THE (148% in males and 188% in females). The anticonvulsive effect of diazepam was not sex-dependent, while the anticonvulsive effect of swim stress was greater in female than in male rats. The results suggest that greater sensitivity of male rats to bicuculline and the anticonvulsive effect of swim stress do not result from the release of endogenous modulators of benzodiazepine binding sites.

This study investigated the ability of the benzodiazepine inverse agonist, Ro 15-4513, to alter the expression of physical dependence on pentobarbital. Male Sprague-Dawley rats were made physically dependent on pentobarbital by continuous. IP, infusion of escalating doses of pentobarbital for 12 days. In Experiment 1, pentobarbital dependent rats received either vehicle or Ro 15-4513, in doses of 5, 10, or 15 mg/kg, IP, periodically during the pentobarbital abstinence period. As expected, Ro 15-4513 produced a significant, dose-dependent, exacerbation of withdrawal signs in the pentobarbital dependent rats. In Experiment 2, either vehicle or Ro 15-4513, at a dose of 15 mg/ kg, was administered, IP, once daily during the 12 days of continuous pentobarbital infusion. During the subsequent pentobarbital abstinence period it was noted that the withdrawal signs were significantly reduced in the rats receiving the daily administration of Ro 15-4513. It is hypothesized that the benzodiazepine inverse agonist, Ro 15-4513, may inhibit the development of physical dependence on pentobarbital through an opposing action on the GABA-A receptor.

The present study investigated dose dependence and time course effects of the benzodiazepine (BDZ) partial inverse agonist, RO19-4603 (0.005-0.30 mg/kg) alone, and in combination with the BDZ receptor antagonists flumazenil, ZK 93426, and CGS 8216 (20 mg/kg) in selectively bred alcohol-preferring (P) rats provided a two-bottle choice test between ethanol (EtOH) (10% v/v), and a palatable saccharin (0.0125% g/v) solution. A single dose of RO19-4603 as low as 0.009 mg/kg selectively reduced EtOH drinking during the initial 15 min of a 4 h access to 19-0% of control levels on day 1. The 0.08, 0.15 and 0.30 mg/kg doses of RO19-4603 significantly reduced total EtOH intake in the 4 h access period to 57-45% of controls on day 1. On day 2, no RO19-4603 injections were given; however, six of the seven doses of RO19-4603 (0.009, 0.02, 0.04, 0.08, 0.15, and 0.30 mg/kg) continued to reduce EtOH intake to 42-3% of control levels at the initial 15 min interval, while the 0.005, 0.009, 0.08, and 0.30 mg/kg doses reduced total 4 h EtOH intake to 60-42% of controls. Saccharin intake was either not altered by RO19-4603 or showed increases during the initial 15 min intervals and the total 4 h sessions on days 1 and 2. Food intake was also unaffected by RO19-4603. The CGS 8216, but neither flumazenil nor ZK 93426, reliably reversed the RO19-4603-induced suppression of EtOH intake on days 1 and 2. That certain BDZ inverse agonists can attenuate motivated behavior for EtOH reinforcement over a prolonged time course may provide a possible therapeutic approach to reducing EtOH consumption associated with alcoholism.

The time course of the novel benzodiazepine inverse agonist, RO19--4603 (0.075 or 0.150 mg/kg) in antagonizing the depressant effects of ethanol (EtOH) (0.50, 1.0 and 1.5 g/kg) and the development of tolerance on locomotor behaviors (e.g., ambulatory count, total distance and stereotypy count) were investigated in Sprague-Dawley rats given EtOH injections spaced at 24-hr intervals. A single dose of RO19--4603 prevented the development of tolerance to the 0.50- and 1.0-g/kg EtOH doses 24-hr post-RO19--4603 administration on most locomotor behaviors. On Day 1, the 0.150-mg/kg RO19--4603 dose prevented the reduction of motor behaviors after the 1.0- and 1.5-g/kg EtOH doses, whereas the 0.075-mg/kg RO19--4603 dose prevented the reduction of motor behaviors only after the 1.5-g/kg EtOH dose. The 0.075- and 0.150-mg/kg RO19--4603 doses also prevented the EtOH-induced reduction of motor behaviors after the 1.5-g/kg EtOH dose 24-hr post-RO19--4603 administration. RO19--4603 was without effect on activity when given alone. These data suggest that the motor impairing effects of EtOH and the development of tolerance to them may involve gamma-aminobutyric acidA-benzodiazepine receptor mechanisms that when occupied, even briefly by certain benzodiazepine inverse agonists, produce long-lasting effects on locomotion and tolerance.

Anti-anxiety drugs are widely used for patients with neurosis or psychosomatic diseases due to the stress of contemporary society. The high efficacy of benzodiazepine (BZD) or its analog, which contains diazepam, is well-known. While these compounds have strong anti-anxiety effects, it has recently been pointed out that they have some side effects when used as daytime tranquilizers and induce drug dependence. The cause of these side effects is thought to be that typical BZD is a full agonist of BZD receptors. For this reason, partial agonists, inverse agonists and antagonists of BZD receptors are being developed. Furthermore, some non-BZD anti-anxiety drugs are also being developed to avoid the side effects of BZD. One of these is a 5-HT1A agonist, which has a high affinity for 5-HT receptors, because it is reported that 5-HT is related to anxiety in the septum-hippocampus system. In addition, the anti-anxiety effect of 5-HT3 agonist is being investigated, while the anti-anxiety effect of cholecystokinin agonists is also attracting attention. Because these new anti-anxiety drugs are more potent and have fewer side effects than BZD, they may achieve widespread clinical use.

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (< 1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)-induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole-cell patch-clamp technique. This effect is specific for CCM and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA-induced currents because glutamate-induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagonist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist-like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA-induced current in GABA-responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAA receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist-like activity, glia cells could possibly modulate GABAA receptor-mediated responses.

The effect of benzodiazepines (BZP) on melatonin release was investigated in the pineal gland of the rainbow trout, Oncorhynchus mykiss, maintained under in vitro perifusion culture conditions. Melatonin and the methoxyindoles 5-methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptamine (5-MT) were determined directly in samples of the superfusion medium by HPLC with electrochemical detection. Melatonin release was significantly increased by addition of diazepam and clonazepam in a dose-related and reversible manner. The effects of benzodiazepines were more pronounced in light-adapted pineal organs, when melatonin secretion is low, than under scotopic conditions. When the perifusion medium was replaced by a medium containing low calcium, high magnesium concentrations, melatonin release was considerably decreased by 70% in light-adapted and 20% in dark-adapted pineal organs. Addition of diazepam to low Ca2+, high Mg(2+)-medium reversed the decrease of melatonin release and produced a clear rise in its secretion rate. Addition of the BZP antagonist flumazenil to the perifusion medium slightly decreased melatonin release in the light- and dark-adapted state, whereas the peripheral receptor antagonist PK 11195 did not alter melatonin release. The effect of diazepam is reduced by simultaneous addition of flumazenil to the superfusion medium, suggesting that the effects of diazepam are receptor-mediated. The methoxyindoles 5-MTOL, 5-MIAA, and 5-MT showed no significant changes of their release pattern after diazepam application in light- and dark-adapted pineal organs. These results suggest that BZP can influence melatonin production and release by an intrapineal action possibly on the melatonin synthesizing photoreceptor cell.