Chronic benzodiazepine antagonist treatment and its withdrawal upregulates components of GABA-benzodiazepine receptor ionophore complex in cerebral cortex of rat

Effect of chronic administration of benzodiazepine (BZ) receptor antagonist Ro 15-1788 (flumazenil) (4 mg/kg once daily for 14 days) treatment and its withdrawal on locomotor activity, body temperature, and the binding pattern of receptor ligands that bind to GABA-BZ receptor ionophore complex in different regions of the brain of the rat was studied. Ro 15-1788 (x 14 d) increased the specific binding of [3H]ethyl-8-fluoro-5-6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5 alpha][1,4]benzodiazepine-3-carboxylate [( 3H]Ro 15-1788), [3H]ethyl-8-azido-5-6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5 alpha][1,4]benzodiazepine-3-carboxylate [( 3H]Ro 15-4513), [3H]flunitrazepam, and [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) in cerebral cortex, and this increase in binding remained upregulated during the drug withdrawal at 24 h. The binding of [3H]Ro 15-1788 was also found significantly increased in the hippocampus, but not in cerebellum and striatum. The chronic Ro 15-1788 treatment did not alter the specific binding of [3H]GABA. Rosenthal analysis of the saturation isotherms indicated that the observed upregulation in the binding pattern of [3H]Ro 15-1788 and [3H]Ro 15-4513 in the cerebral cortex was due to an increase in the binding capacity (Bmax). The receptor affinity (Kd) was not changed. The withdrawal of Ro 15-1788 following its chronic administration also enhanced locomotor activity. However, no apparent change in body temperature was observed either due to chronic treatment or withdrawal. These data indicate that chronic Ro 15-1788 treatment and its withdrawal may produce an upregulation of subunits which bind the positive (benzodiazepines), negative (inverse agonist), and neutral (antagonist) ligands of benzodiazepine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rats with different thresholds for DMCM-induced clonic convulsions differ in the sleep-time of diazepam and [(3)H]-Ro 15-4513 binding

The current study investigated the possible inherent relationship between convulsions and sleep involving the GABA(A)/benzodiazepine site complex. The aim of this study was to determine if rats with high (HTR) and low (LTR) thresholds for clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ in the following aspects: (1) sensitivity to the hypnotic effects of the GABA(A) positive allosteric modulators diazepam, pentobarbital and ethanol and (2) in the binding of [(3)H]-flunitrazepam, a benzodiazepine agonist, measured by autoradiography, and [(3)H]-Ro 15-4513, a benzodiazepine partial inverse agonist, to membranes from discrete brain regions. The LTR subgroup presented a shorter diazepam-induced sleeping time compared to that of the HTR subgroup. Biochemical assays revealed that the LTR subgroup did not differ in [(3)H]-flunitrazepam binding compared to the HTR subgroup. With respect to the binding of [(3)H]-Ro 15-4513, the LTR subgroup had higher binding in the brainstem and lower binding in the striatum compared to the HTR subgroup. These results suggest that differences in the benzodiazepine site on the GABA(A) receptor may underlie the susceptibility to DMCM-induced convulsions and sensitivity to the hypnotic effect of diazepam.     

Imaging of [C]-labelled RO 15-1788 binding to benzodiazepine receptors in the human brain by positron emission tomography

The benzodiazepine antagonist Ro 15-1788 was labelled with [¹¹C] and examined for possible use as ligand for PET scan studies on benzodiazepine receptors in the brain of cynomolgus monkeys and human subjects. [¹¹C] Ro 15-1788 allowed the in vivo visualization of benzodiazepine receptor binding in cerebral and cerebellar cortical areas as well as in basal brain nuclei in PET scan images. [¹¹C] Ro 15-1788 exhibited a high ratio of specific benzodiazepine receptor binding (cerebral cortex) to non-specific binding (pons) and the kinetics of binding should be satisfactory for quantitative clinical PET scan studies using [¹¹C]. The in vivo binding of [¹¹C] Ro 15-1788 in the cerebral cortex of cynomolgus monkeys and healthy human subjects was reduced by approximately 90% within 10 min after the intravenous injection of a high dose of unlabelled Ro 15-1788 (0.5 mg/kg i.v.). Different areas of the healthy human brain showed an approximately 10-fold variation in maximal [¹¹C] Ro 15-1788 binding that corresponded to the previously known distribution of benzodiazepine receptors in these regions. The highest degree of binding was obtained in the medial occipital cerebral cortex followed by frontal cortex, cerebellum, thalamus, striatum and pons. Two psychiatric patients with anxiety syndromes who had been treated for a long time with high doses of benzodiazepines had roughly the same degree of maximal [¹¹C] Ro 15-1788 binding in brain regions as the healthy subjects but the rate of decline of [¹¹C] Ro 15-1788 in the brain was higher. This indicates that there is measurable competition between [¹¹C] Ro 15-1788 binding and clinical benzodiazepine concentrations in the body fluids of psychiatric patients. The results demonstrate that [¹¹C] Ro 15-1788 should be a valuable tool for quantitative analyses of benzodiazepine receptor characteristics and receptor occupancy in the brain of patients with neuropsychiatric disorders.     

Involvement of benzodiazepine recognition sites in the foot shock-induced decrease of low affinity GABA receptors in the rat cerebral cortex

The cerebral cortices of rats habituated to the handling manipulation that precedes sacrifice by guillotine (unstressed rats) have a higher number of low affinity GABA receptors than naive rats (stressed rats). Foot shock stress delivered to handling-habituated rats 5 min before sacrifice decreased the number of low affinity GABA receptors to the level found in naive animals, while leaving almost unchanged the [3H]GABA binding in the latter group. Since benzodiazepine (BZ) recognition sites are the target through which benzodiazepines modulate the emotional states of the animals, we investigated whether these receptors were involved in the action of foot shock stress on GABA binding. The in vitro addition of diazepam (5 X 10(-6) M) to cortical membranes from foot-shocked handling-habituated rats brought back the number of low affinity GABA receptors to the level found in cortical membranes from handling habituated rats. Moreover, the effect of foot shock on low affinity GABA receptors was completely antagonized in vivo by pretreatment with the specific benzodiazepine antagonist Ro15-1788 (30 mg/kg per os). Since the effect of foot shock on [3H]GABA binding is mimicked by the in vitro addition of beta-carbolines to cortical membranes from handling habituated rats, our working hypothesis is that an endogenous ligand for BZ recognition sites, possessing beta-carboline-like properties, is released during foot shock stress.     

Characterization of benzodiazepine receptors in the cerebellum

1. The goals of the work reported here were to further characterize benzodiazepine/GABA(A) (BDZR) receptor heterogeneity in the cerebellum and to measure the affinities and selectivities of structurally diverse benzodiazepines at each site identified. 2. Five chemical families were included in these studies. These were 1,4-benzodiazepines (flunitrazepam), imidazobenzodiazepines (RO15-1788 and RO15-4513 and RO16-6028), beta-carbolines (Abecarnil) and pyrazoloquinolines (CGS 8216, CGS 9895 and CGS 9896). 3. Saturation and competition binding assays were combined with powerful data analysis software developed in our laboratory. Among the capabilities of this software is the identification of multiple binding sites for a cold ligand using a non-selective labeled ligand that binds with equal, but high, affinity to all the binding sites 4. Saturation binding assays using either [3H]-RO15-1788 or [3H]-RO15-4513 revealed only one apparent binding site, with a higher affinity for RO15-4513 than for RO15-1788. However, using [3H]-RO15-4513 for the competition binding studies in the cerebellum, together with our data analysis software, led to the identification of two distinct binding sites with equal densities for the diverse benzodiazepines studied. 5. In rat cerebellum one of the sites identified corresponds to GABA(A) receptors exhibiting alpha1 subunit pharmacology and the other to GABA(A) receptors exhibiting alpha6 subunit pharmacology. In general, the diverse families of BDZR ligands studied had much lower affinities for the alpha6 containing receptors.     

The shark GABA-benzodiazepine receptor: further evidence for a not so late phylogenetic appearance of the benzodiazepine receptor

Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark gamma-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [3H]muscimol binding sites. The immunoreaction obtained with the beta-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.     

Benzodiazepine receptors in normal human brain, in Parkinson''s disease and in progressive supranuclear palsy

Benzodiazepine receptors have been characterized in human brain. They have been localized mainly in the cerebral cortex and a synaptosomal enrichment was observed after brain fractionation by differential centrifugation. Benzodiazepine receptors were studied in Parkinson's disease and in progressive supranuclear palsy (PSP). In both diseases, the [3H]flunitrazepam specific binding was unchanged when compared to control groups (Bmax and KD values) except in the caudate nucleus of parkinsonian patients where an increase of the specific binding was observed. The subcellular distribution profile of benzodiazepine receptors in Parkinson's disease was similar to that of controls. gamma-Aminobutyric acid (GABA) still enhanced the [3H]flunitrazepam-specific binding (increase of binding affinity), indicating that the functional link between GABA and benzodiazepine receptors remained intact in Parkinson's disease. The present results suggest that benzodiazepine receptors in human striatum are localized on neuronal elements which do not degenerate in Parkinson's disease and PSP.     

Adrenalectomy prevents the stress-induced decrease in in vitro [H]Ro15-1788 binding to GABAA benzodiazepine receptors in the mouse

The effect of a single or repeated swim stress on in vivo benzodiazepine receptor binding to various brain regions in adrenalectomized and sham-operated (control) mice was assessed using the benzodiazepine receptor antagonist, [³H]Ro15-1788. In sham-operated mice the binding of [³H]Ro15-1788 to benzodiazepine receptors was reduced in the hippocampus and hypothalamus (single or repeated stress) and cerebral cortex (repeated swim stress) compared to non-stressed mice. In contrast, no alterations in [³H]Ro15-1788 binding were observed in any brain region in adrenalectomized mice after either single or repeated swim stress. These data suggest that an intact hypothalamic-pituitary-adrenal axis is required for the stress-induced decrease in benzodiazepine receptor occupancy measured using the in vivo binding method.     

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Benzodiazepine ([3H])-flunitrazepam) binding sites in cerebellar and cerebral cortical slices of mouse brain

We have characterized and quantified the specific binding of [3H]-flunitrazepam ( FNZ ) to thick (230 micron) slices of mouse brain. The binding site has the characteristics of a benzodiazepine receptor, i.e., binding of FNZ is reversible, stereospecific, saturable and of high affinity. Clonazepam, but not R05 -4864, readily displaces the label. In contrast to results from homogenate assays, neither GABA nor bicuculline has any effect on [3H]- FNZ binding. However, as previously reported, the slice assay confirms the lower number of benzodiazepine receptors in "emotional" mouse brain. In addition, we have confirmed that the neurotoxin DSP4 can modify [3H]- FNZ binding though in our hands this compound elevates rather than reduces binding. The speed, simplicity and minimal tissue preparation involved suggests that this slice assay could be a valuable addition to neurochemical studies of neurotransmitter receptors.     

Modulation of the GABA receptor complex by a steroid anaesthetic

The interactions of a steroid anaesthetic, alphaxalone, with the GABA receptor-ionophore complex were investigated by two different experimental approaches. In the rat cuneate nucleus slice, alphaxalone (0.1-10 microM) potentiated depolarizing responses to superfused GABA and muscimol, but not those to glycine. The potentiating effect of alphaxalone was unaltered by the benzodiazepine antagonist Ro 15-1788. Alphaxalone (0.1-30 microM) also enhanced [3H]muscimol binding to rat brain membranes in the presence of Cl-ions; the enhancing effect on [3H]muscimol binding was abolished by Triton X-100. Analysis of binding curves for [3H]muscimol indicated that the steroid anaesthetic increases the affinity for [3H]muscimol of low affinity binding sites; this property is shared by pentobarbitone. The physiologically inactive beta-hydroxy isomer of the steroid was without activity in either of the experimental situations at 30 microM. It is suggested that alphaxalone and pentobarbitone share a common mode of action on the GABA system, which may be relevant to the mechanisms by which these drugs produce anaesthesia.     

Changes in GABAergic transmission induced by stress, anxiogenic and anxiolytic beta-carbolines

The cerebral cortex of unstressed (handling-habituated) rats has a higher number of low affinity GABA receptors than stressed (naive) rats. Foot shock stress delivered to unstressed rats decreases the density of cortical low affinity GABA receptors to the level found in the naive animals. The effect of stress on GABA receptors is mimicked by anxiogenic beta-carbolines, both after in vitro addition (10(-6) M) to cortical membrane preparations or after the in vivo administration (20 mg/kg IP) to unstressed rats. Vice versa, benzodiazepines or anxiolytic beta-carbolines (ZK 93423, 10(-5) M) added to membranes from naive rats increase GABA binding to the level of unstressed rats and remove the decrease in the density of GABA receptors elicited by anxiogenic beta-carbolines. Rats chronically treated with the anxiogenic beta-carboline, FG 7142 (15 mg/kg IP twice a day for 10 consecutive days) have an enhanced sensitivity to punishment at 5 and 15 days after the last treatment. The behavioural effect is paralleled by a marked decrease in the total number of cortical low affinity GABA receptors. Both biochemical and behavioural effects elicited by chronic FG 7142 are prevented by the concurrent administration of the benzodiazepine antagonist Ro15-1788. These results suggest that (a) anxiolytic beta-carbolines, like benzodiazepines, increase the GABAergic transmission, (b) acute and chronic anxiogenic beta-carboline administration, like stress, decreases GABAergic transmission. Since all these effects are antagonized by the benzodiazepine receptor blocker Ro15-1788, it is tempting to speculate that stress releases an endogenous ligand for benzodiazepine recognition sites.     

Laminar distribution of receptors in monkey (Macaca fascicularis) geniculostriate system

We have examined the laminar distributions of eight types of receptor in the primary visual cortex (area 17) and the lateral geniculate nucleus (LGN) of the macaque monkey. The receptor populations and subpopulations examined included those selective for gamma-aminobutyric acid (GABA) (using [3H]-muscimol as ligand), L-glutamate-related receptors (using [3H]-L-glutamate and [3H]-AMPA), muscarinic acetylcholine (using [3H]-quinuclidinyl benzilate--QNB and [3H]-N-methyl scopolamine--NMS), cholecystokinin (CCK) (using [3H] pentagastrin), benzodiazepine (using [3H]-flunitrazepam), and adenosine (using [3H]-cyclohexyladenosine--CHA). Each of the receptors examined exhibited characteristic and differing laminar patterns of binding in the striate cortex. Perhaps reflecting the high density of cell bodies and synapses in layer 4C, most receptors, except those labelled by [3H]-L-glutamate or [3H]-AMPA, showed dense concentrations in this layer. Layers 4B and 5, which contain relatively few cell bodies and heavy myelin concentrations, were in general lightly labelled. Layer 6 showed relatively heavy labelling when [3H]-AMPA (quisqualate) or [3H]-pentagastrin (CCK) were used as ligands. The superficial layers of the cortex were zones of relative concentration of GABA, benzodiazepine, acetylcholine, glutamate-related, and adenosine receptors. In general, the binding patterns resembled those previously described for cat visual cortex, but there were also some clear differences. The distributions of all of these receptors likely reflect the differential input substances to different laminae of the visual cortex. Of the receptors examined, only those for GABA, benzodiazepine, and acetylcholine were found in substantial concentration in the LGN. Of these, GABA and benzodiazepine receptors showed especially dense binding in the magnocellular layers of the LGN compared to the parvicellular layers.     

Cerebellar and frontal cortical benzodiazepine receptors in human alcoholics and chronically alcohol-drinking rats.

Postmortem cerebellar and frontal cortical membrane homogenates from human alcoholics, control subjects without neurological or psychiatric illnesses, and rats that chronically drank alcohol were studied to determine the binding characteristics of an imidazobenzodiazepine, [3H]Ro 15-4513. This ligand binds to classical gamma-aminobutyric acidA (GABAA)/benzodiazepine receptors, as well as to a "diazepam-insensitive" site associated with the GABAA receptor complex in the cerebellar granule cell layer. There were no differences in the density of the binding sites between alcoholics and their controls, between alcohol-drinking AA rats that had a choice between 10% alcohol or water for about 10 weeks and their controls, or between Wistar rats that had been given 20% alcohol as their only fluid for 4 months and their controls, which were pair-fed isocalorically with sucrose. The affinity for the cerebellar binding of [3H]Ro 15-4513 was higher in the alcoholics than the controls. No differences were observed in the frontocortical binding. No affinity differences were observed in the rat models. There were no differences between the groups in the characteristics of [3H]Ro 15-4513 binding to human cerebellum in the presence of micromolar diazepam, thus revealing the diazepam-insensitive binding. When this component was subtracted from the total cerebellar binding, to reveal the diazepam sensitive binding, both the KD and Bmax were lower in the alcoholic than the control group. The binding of [3H]muscimol, a GABAA agonist, tended to be higher in the frontal cortices of alcoholics; a similar trend for greater effects was observed in the alcoholics for the GABA inhibition of [3H]Ro 15-4513 binding. These results suggest that no drastic changes occur through chronic alcohol abuse in the numbers of cerebellar and frontocortical benzodiazepine receptors in humans and rodent models; however, the data indicate that the alcoholics have either acquired or innate differences in classical benzodiazepine recognition sites of the cerebellum and in the coupling of these sites to GABAA sites in the frontal cortex, without any differences in cerebellar granule cell-specific diazepam-insensitive [3H]Ro 15-4513 binding sites.     

Relationships between benzodiazepine receptors, impairment of GABAergic transmission and convulsant activity of beta-CCM: a PET study in the baboon Papio papio.

Central type benzodiazepine receptors were studied in vivo by positron emission tomography in brain areas of 2 different groups of the baboon Papio papio: non-photosensitive (group 1) and those with an allylglycine-induced decrease in GABA-mediated inhibition (group 2). Further, a naturally photosensitive Papio papio (+3 level of photosensitive response) was compared to both groups. Regional brain binding of the specific benzodiazepine receptor ligand, [11C]Ro 15-1788, was not significantly different between groups 1 and 2. In addition, the data from the naturally photosensitive Papio papio did not seem to differ markedly from groups 1 and 2 either. Pharmacological effects of increasing doses of beta-CCM (0.05-3 mg/kg i.v.) and regional benzodiazepine receptor occupancy by the drug were simultaneously studied using electroencephalographic activity recording and positron emission tomography. A positive correlation was observed between the degree of photosensitivity of the baboon and sensitivity to the action of beta-CCM, with increasing convulsant efficacy of beta-CCM in going from group 1 to the naturally photosensitive baboon, then to group 2. Dose-related displacement curves of [11C]Ro 15-1788 binding by beta-CCM revealed that reduction in brain GABA concentration did not modify the inhibitory potency of beta-CCM on [11C]Ro 15-1788 binding in cerebral cortex. This suggests a lack of detectable in vivo allosteric effects of GABA on beta-CCM binding during beta-CCM-induced seizures. Thus, a given dose of beta-CCM displayed increasing pharmacological potency in going from baboons with the lowest photosensitivity to those with the highest, whereas benzodiazepine receptor occupancy by beta-CCM was similar in the cerebral cortex of the different baboons. Conversely, a given level of convulsant activity of beta-CCM was related to a different benzodiazepine receptor occupancy by the drug, depending on the photosensitivity of Papio papio. A given dose of a drug may, thus, have a different pharmacological potency when occupying the same number of receptors, depending on the physiopathological state of the subject.     

Benzodiazepine ([H])-flunitrazepam) binding sites in cerebellar and cerebral cortical slices of mouse brain

We have characterized and quantified the specific binding of [³H]-flunitrazepam (FNZ) to thick (230 μm) slices of mouse brain. The binding site has the characteristics of a benzodiazepine receptor, i.e., binding of FNZ is reversible, stereospecific, saturable and of high affinity. Clonazepam, but not R05-4864, readily displaces the label. In contrast to results from homogenate assays, neither GABA nor bicuculline has any effect on [³H]-FNZ binding. However, as previously reported, the slice assay confirms the lower number of benzodiazepine receptors in “emotional” mouse brain. In addition, we have confirmed that the neurotoxin DSP4 can modify [³H]-FNZ binding though in our hands this compound elevates rather than reduces binding. The speed, simplicity and minimal tissue preparation involved suggests that this slice assay could be a valuable addition to neurochemical studies of neurotransmitter receptors.     

Transient increase in [3H]Ro 15-4513 specific binding in the superficial gray layer of the rat superior colliculus induced by visual deafferentation.

The imidazodiazepine compound [3H]Ro 15-4513, a partial inverse agonist of benzodiazepine receptors of the central type, binds with high affinity (order of 10(-8) M) to a single population of benzodiazepine binding sites in the mammalian central nervous system. A quantitative autoradiographic study was carried out to determine the effects of one eye removal on [3H]Ro 15-4513 specific binding to rat brain sections in the superficial gray layer or stratum griseum superficiale (SGS) of the superior colliculus. Retinal afferent degeneration due to right eye removal, performed 3 and 7 days before sacrifice, led to a significant and symmetrical increase in the [3H]Ro 15-4513 specific binding in both right and left SGS by enhancing the binding affinity of the radioligand. This transient phenomenon disappeared when a longer survival period of 45 days was allowed to elapse. Conversely, unilateral lesion of the primary visual areas had no apparent effects on the specific binding of the radioligand. The absence of any loss of binding sites after either type of lesion suggests that the benzodiazepine receptors are probably not situated on the optic nerve axon terminals, nor on the cortical axon terminals originating from primary visual areas. In the SGS, as in other rat brain structures, benzodiazepine receptors of the central type are functionally coupled with GABAA receptors and form 'GABAA receptors/benzodiazepine receptors/chloride channel' complexes. The involvement of the local GABAergic system in the postlesion plasticity of benzodiazepine receptors was studied by testing the effects of exogenously applied GABA on [3H]Ro 15-4513 specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of benzodiazepine receptors causes altered agonist-antagonist interactions

Previous studies have shown that [3H]flunitrazepam forms irreversible cross-links with brain tissue when exposed to ultraviolet irradiation. Comparison of the amount of [3H]flunitrazepam irreversibly incorporated and the number of benzodiazepine binding sites blocked after photolabeling has indicated that several binding sites are inactivated for each molecule of [3H]flunitrazepam incorporated. To learn the cause of this discrepancy, binding to the benzodiazepine binding sites has been examined using several radiolabeled benzodiazepine antagonists. Binding of a beta-carboline ester, CGS-8216, and Ro 15-1788 was not altered by photolabeling; however, displacement studies revealed that photolabeling converted a homogeneous set of benzodiazepine binding sites into two subsets: one of high affinity (unaltered sites) and one of low affinity. The low affinity sites could be detected by displacement studies of antagonist binding by benzodiazepines, and conversion to a low affinity form accounts for the discrepancy observed after photolabeling using [3H]flunitrazepam as ligand.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

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

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