Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs

The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000-650,000 by gel filtration, the sedimentation coefficients being 11.0-11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for flunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in th hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.     

Solubilization of the central benzodiazepine receptor and study of its interaction with nicotinamide

It was shown that nicotinamide and NAD inhibit the specific binding of [3H]flunitrazepam to benzdiazepine receptors without causing a direct influence of gamma-aminobutyric acid (GABA) receptors. The GABA-benzdiazepine complex was separated by solubilization with 0.5% lubrol PX. The solubilized preparations contain the binding sites for [3H]flunitrazepam alone (Kd = 5.9 nm). The Kd value for the membrane-bound benzdiazepine receptor is 2.9 nM. NAD inhibited the specific binding of [3H]flunitrazepam to the solubilized membrane preparation when used at concentrations by several orders of magnitude lower than that of nicotinamide. Using gel filtration on Sepharose 6B-CL, the molecular mass of the soluble benzdiazepine receptor protein was determined.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

Increased specific binding of [3H]diazepam in rat brain following chronic diazepam administration.

Male rats (175 g) were given 30 mg of diazepam in their food daily for 35 days. The animals became drowsy and ataxic from this high dose of drug. After the 35-day dosing, the rats were killed daily, and specific binding of [3H]diazepam and [3H]flunitrazepam was determined in synaptosomal preparations from these and corresponding control rats. On days 3, 4, 6, and 7 after the treatment period the specific binding and specific binding of [3H]diazepam was double that of the control binding and specific binding of [3H]flunitrazepam was 1.67 times that of control. The data indicate that very high doses of diazepam, given for long periods, cause increased specific binding of radiolabeled ligand to brain subfractions. The possible mechanisms and implications are discussed. When lower doses or shorter dosage regimens are used, increased binding is not observed.     

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5-4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala-kindled and "yoked" control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of gamma-aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.     

[3H]Propyl β-Carboline-3-Carboxylate Binds Specifically to Brain Benzodiazepine Receptors

Ethyl beta-carboline-3-carboxylate has recently been isolated from human urine and it was proposed that derivatives of this compound might be related to an endogenous ligand for benzodiazepine receptors. In the present study we investigated high-affinity binding of [3H]propyl beta-carboline-3-carboxylate ([3H]PrCC) to rat brain membranes. [3H]PrCC binds specifically and with high affinity (half-maximal binding at ca. 1nM) to rat brain membranes. The regional and subcellular distributions of specific [3H]PrCC binding are similar, but not identical, to the distributions of [3H]flunitrazepam or [3H]-diazepam binding. The total numbers of binding sites labelled by [3H]PrCC and [3H]flunitrazepam in rat cerebellum are closely similar, and both ligands bind to cerebellar membranes in a mutually exclusive way. The pharmacological selectivity of [3H]PrCC and [3H]diazepam binding is almost identical. Binding of [3H]PrCC like binding of [3H]diazepam, can be increased in vitro by muscimol, GABA and SQ 20.009. Although subtle differences in binding characteristics were observed, these results indicate that [3H]PrCC and benzodiazepines bind to a common recognition site on benzodiazepine receptors.     

The gamma-aminobutyric-acid/benzodiazepine-receptor protein from rat brain. Large-scale purification and preparation of antibodies

The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.     

gamma-Aminobutyric acid/benzodiazepine receptor protein carries binding sites for both ligands on both two major peptide subunits

Affinity column-purified GABA-benzodiazepine receptor proteins from human, cow, and rat brain were photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol and examined by gel electrophoresis in sodium dodecyl sulfate. Using high receptor protein concentrations (1 microM), the benzodiazepine ligand [3H]flunitrazepam was incorporated covalently primarily into the expected 52 kiloDalton major subunit but also significantly into a second 57 kiloDalton peptide. Likewise the GABA ligand [3H]muscimol photolabeled primarily the 57 kiloDalton peptide but also to some extent the 52 kiloDalton peptide. This cross-labeling suggests strongly that both major subunits carry binding sites for both GABA and benzodiazepine.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.     

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [³H] flunitrazepam and [³H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [³H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [³H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [³H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [³H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [³H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

Characterization of Neurotransmitter Receptors in the Rat Hippocampal Formation

The hippocampal formation has been extensively research in terms of its putative neurotransmitters, anatomical connections, and behavioral relevance. An aspect of importance is the assessment of apparent neurotransmitter receptors by using receptor binding assays. In the present study, such assays were done in vitro to investigate alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic, muscarinic cholinergic, benzodiazepine, and opiate receptors in the rat hippocampal formation. The corresponding radioligands for these receptors were [3H]prazosin, [3H]p-aminoclonidine, [3H]dihydroalprenolol, [3H]quinuclidinyl benzilate, [3H]flunitrazepam, and [3H]naloxone. An analysis of the binding parameters for the ligands indicated saturable binding of a high affinity and the following rank order of maximal binding capacities: [3H]flunitrazepam greater than [3H]quinuclidinyl benzilate greater than [3H]naloxone greater than [3H]p-aminoclonidine greater than [3H]prazosin greater than [3H]dihydroalprenolol. Competition experiments with pharmacologic agonists and antagonists confirmed the specificity of each ligand. The results are integrated with information on other types of receptors and with neurotransmitter concentrations, and discussed in terms of hippocampal function.     

Benzodiazepine Receptors on Primary Cultures of Mouse Astrocytes

Benzodiazepines bind to glial membranes on a single type of site, with a high affinity (KD = 5 x 10(-9) M) on about 100 fmol of sites per mg protein. The number of binding sites is increased when the membranes are treated with Triton X-100. Antiepileptic drugs such as clonazepam and phenobarbital and hypnotic drugs such as Ro-11-3128 and Ro-11-6896 are able in pharmacological concentrations to displace [3H]flunitrazepam from its glial binding sites.     

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.