A novel anti-α4 antibody has been used for the purification and characterization of the α4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the α4 subunit peptide in rat brain and retina. The anti-α4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10–28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-α4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-α6 antibody but not by the anti-α4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-α4 revealed molecular colocalization of α4 and γ2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the α4 and γ2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of α4, γ2 and β2 subunits. Immunocytochemistry has revealed the abundance of α4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The α4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, α4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer. Copyright © 1996 Elsevier Science Ltd.

Aging-related alterations in both protein and mRNA expression of γ2S and γ2L subunits of the GABAA receptors have been observed in several brain areas of Sprague-Dawley and Fischer 344 rats. Subunit-specific antibodies to γ2S and γ2L as well as a riboprobe to the large intracellular loop of γ2, which recognizes both γ2S and γ2L mRNAs, in conjunction with computerized image analysis were used for quantitative immunocytochemistry and in situ hybridization. In addition, specific oligonucleotide probes to γ2S or γ2L mRNA were used for quantitative dot blot hybridization. A large increase in the number of heavily immunostained neurons with the anti-γ2L antibody was detected in the cerebral cortex (115%) of old rats. However, only a small (but significant) aging-related increase in the density of γ2L immunostaining (7%) was observed throughout the cerebral cortex whereas no significant aging-related change in γ2L mRNA was detected in this brain region. Contrary to γ2L, the γ2S immunostaining did not show aging-related increased number of heavily immunostained neurons in cerebral cortex. Moreover, the density of γ2S immunostaining and the expression of γ2S mRNA were significantly decreased in the cerebral cortex (9–24%). Important aging-related changes were also found in the cerebellum of old rats where the expression of both γ2S and γ2L peptides was significantly decreased (24% and 23% respectively). This decrease in γ2 protein expression was accompanied by decreased expression of γ2S (16–38%) and γ2L (24%) mRNAs. Nevertheless, the most important decrease of γ2S (48%) and γ2L protein (20%) was revealed in the molecular layer of the cerebellum. In addition, the expression of γ2S protein was increased (14%) whereas the expression of γ2L was decreased (13%) in the granule cell layer. Therefore, the relative expression of γ2S protein in both layers was reversed in old animals. The observed aging-related changes in the expression of GABAA receptor subunits might lead to altered GABAA receptor/benzodiazepine receptor subunit composition.

Aging-related changes in the subunit expression of some hippocampal GABAA receptors have been found. Quantitative in situ hybridization has revealed that α1 subunit messenger RNA expression was significantly increased in the hippocampus (34%) of old rats. The largest increases were observed in the dentate gyrus (76%) and in the CA1 field (30%). Quantitative immunocytochemistry also showed increased protein expression of the α1 subunit in the dentate gyrus (19%) and CA1 (14%) of old rats. The increased α1 messenger RNA and protein expression led to increased proportions of assembled GABAA receptors that contained α1 subunits, as revealed by quantitative immunoprecipitation of [3H]flunitrazepam and [3H]muscimol binding. In contrast, there were no significant changes in the expression of β2, β3 and total γ2 (γ2S + γ2L) subunits, although a slightly increased expression of γ2L peptide was detected in the hippocampus proper (7%), but not in the dentate gyrus. The results are consistent with the notion that in the rat hippocampus there is an aging-related change in the subunit composition of some GABAA receptors. Copyright © 1996 IBRO. Published by Elsevier Science Ltd.

Polymerase chain reaction was used to amplify the cDNA region that codes for the large intracellular loop of the β3 subunit of the γ-aminobutyric acidA/benzodiazepine receptors (GABAAR/BZDR) from rat brain. The amplified cDNA was inserted into the prokaryotic expression vector pGEX-3X and a fusion protein containing glut athione-S-transferase and β3 intracellular loop moieties was expressed in bacteria. The fusion protein was affinity-purified and it was used to raise a rabbit anti-β3 antiserum. The anti-β3 antiserum immunoprecipitated the γ-aminobutyric acidA receptor from rat and bovine brain. Immunoblots of the affinity-purified GABAAR/BZDR from bovine brain revealed that the anti-β3 antiserum reacted with a 57 kDa peptide, whereas the monoclonal antibody 62-3G1 that recognized both β2 and β3 reacted with 55 and 57 kDa peptides. The anti-β3 antiserum showed specificity for the β3 subunit vs β2 and β1.

Immunocytochemical techniques were used to investigate the distribution of gamma-aminobutyric acidA (GABAA) receptors in the rat primary somatosensory cortex (SI). Monoclonal antibody 62-3G1 (de Blas et al., 1988; Victorica et al., 1988), which recognizes an epitope common to the beta 2 and beta 3 subunits of the GABAA receptor, produces staining of small punctate structures throughout the neuropil, and around somata and linear processes in all laminae of SI. Receptor immunostaining is relatively intense in upper lamina I and in lamina IV, where patches of intense receptor staining are interleaved with narrow zones of moderate immunoreactivity. Staining is lightest in lamina Vb, where stained puncta appear to be aligned with radially oriented processes, and moderate in the remaining laminae. Tangential sections through lamina IV reveal that each large cortical barrel encompasses several patches of intense receptor staining that are aligned with the corners or edges of individual barrels; interbarrel septa are moderately of intense cytochrome oxidase (CO) histochemical staining. A similar correspondence is apparent between a complex lattice of dense receptor clustering and a plexus of dark CO staining in the cortical trunk representation. Six to eight weeks of tactile deprivation produced by simple whisker trimming have no visible effect on GABAA receptor distribution. This is the case for rats whose whiskers were trimmed only during adulthood and for rats deprived from the day of birth until examination 6-8 weeks later. However, electrocautery ablation of whisker follicles leads to a marked decline in GABAA receptor immunoreactivity in cortical barrels associated with the ablated follicles. Our findings indicate that there is reasonable, though not perfect, correspondence between the distribution of GABAA receptors and the distribution of GABA-containing neurons and terminals in rat SI. These elements are associated with regions of intense oxidative metabolic activity revealed by CO staining. The density of GABAA receptors is reduced in lamina IV following complete loss of peripheral afferent input. However, less severe tactile deprivation, which is known to affect cortical neuron responsiveness, produces little or no change in receptor distribution.

The large intracellular loop (IL) of the gamma 2 subunit of the cloned human gamma-aminobutyric acidA (GABAA) receptor (gamma 2 IL) was expressed in bacteria as glutathione-S-transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the gamma 2 IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABAA/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000-49,000-M(r) peptide band in immunoblots of affinity-purified GABAA receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABAA receptors in various brain regions.

An antiserum to the beta 2 subunit of the rat gamma-aminobutyric acid (GABAA) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the beta 2 subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABAA receptors. The anti-beta 2 antibodies were affinity purified on immobilized beta 2 intracellular loop peptide. The antibodies recognized a 55-57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABAA receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the beta 2 subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-beta 2 antibody and the monoclonal antibody 62-3G1 (which recognizes both beta 2 and beta 3 subunits) is presented. The procedure described for generating and preparing specific anti-beta 2 subunit antibodies that are valuable for immunocytochemistry could be extended to other GABAA receptor subunits.

The distribution of the short (gamma 2S) and long (gamma 2L) subunits of the GABAA receptors in the rat brain has been revealed by light microscopy immunocytochemistry with novel subunit-specific antibodies (anti-gamma 2S and anti-gamma 2L). We have also used other subunit-specific antibodies including anti-gamma 2IL2 (which recognizes both gamma 2S and gamma 2L), anti-alpha 1COOH, and the monoclonal antibody 62-3G1 to beta 2/3 for comparing the regional and cellular distribution of the most abundant GABAA receptor subunits in the rat brain. The distributions of gamma 2S and gamma 2L immunoreactivities are similar throughout the brain although the relative intensity of both signals varies depending on the brain area and neuronal type. In the hippocampus, cerebral cortex, and olfactory bulb (particularly mitral, periglomerular, and tufted neurons), gamma 2S was more abundant than gamma 2L. In contrast, the inferior colliculus, medulla, and the cerebellar Purkinje cells displayed more gamma 2L than gamma 2S immunolabeling. An important difference in the distribution of the various subunits was found in cerebellum: gamma 2S and gamma 2L were predominantly localized in the molecular layer, whereas alpha 1 and beta 2/3 were more abundant in the granular layer. In the thalamus, gamma 2L and gamma 2S were less abundant than either alpha 1 or beta 2/3 subunits. The results showed that there is colocalization of gamma 2S and gamma 2L subunits in some brain areas and neuronal types, as well as areas of mismatch. Colocalization and mismatches were also found among alpha 1, beta 2/3, and gamma 2, probably resulting from the heterogeneity in the subunit composition of the GABAA receptors through the brain.

A selective age-related decrease in both the protein and mRNA levels of the most abundant GABAA receptor subunits has been revealed in the rat inferior colliculus. The number (not affinity) of the native and fully assembled GABAA receptors assayed by 3H-muscimol binding was also decreased (35-49%). The decrease in GABA receptors was accompanied by a decrease in the protein and mRNA of the GABA-synthesizing enzyme glutamic acid decarboxylase. No other region of the rat brain showed such large age-related changes in these GABAergic synaptic molecules. Specific antibodies and riboprobes in conjunction with a computerized image analysis system were used to quantify immunocytochemistry and in situ hybridization. In old Sprague-Dawley rats, the combination of beta 2 and beta 3 peptide subunits was reduced 55%, while the beta 2 and beta 3 mRNAs were decreased 31% and 22%, respectively. The gamma 2S and gamma 2L subunit proteins decreased 43% and 21%, respectively, while the gamma 2 mRNA, including both short and long forms, was reduced 61%. The alpha 1 subunit protein was decreased 28%, whereas the alpha 1 mRNA decreased 40%. The glutamic acid decarboxylase protein was reduced 62% while GAD65 mRNA decreased 42%. Similar age-related changes were also observed in the inferior colliculus of Fischer-344 rats. In contrast, no changes were observed in the level of expression of some glial and/or neuronal proteins such as S-100, glial fibrillary acidic protein, and 160 KDa neurofilament protein in the inferior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)

The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA(A) receptors that show diazepam (or clonazepam)-insensitive [H-3]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have alpha(6), alpha(1), gamma(2S), gamma(2L), and beta(2) or beta(3) subunits colocalizing in the same receptor complex.

A gamma-aminobutyric acid, (GABA(A)) receptor (GABA(A)R) gamma, subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage lambdagt11. The 26 1 -bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors lambdagt11 and pGEX-3X. Both beta-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the gamma2IL were purified, and a rabbit antibody to the LacZ-gamma2IL was made. The antibody reacted with the gamma2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA(A)R/benzodiazepine receptor (GABA(A)R/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA(A)R/BZDR immunoblots with a wide peptide band of 44,000-49,000 M(r). Immunoprecipitation studies with the anti-gamma2IL antibody suggest that in the cerebral cortex, 87% of the GABA(A)Rs with high affinity for benzodiazepines and 70% of the GABA(A)Rs with high affinity for muscimol contain at least a gamma subunit, probably a gamma2. These results indicate that there are [H-3]muscimol binding GABA(A)Rs that do not bind [H-3]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA(A)R/BZDR antibodies indicate that the most abundant combination of GABA(A)R subunits in the cerebral cortex involves alpha1, gamma2 (or other gamma), and beta2 and/or beta3 Subunits. These subunits coexist in >60% of the GABA(A)R/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20-25%) of the cerebellar GABA(A)R/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain alpha6, also contain gamma2 (or other gamma) subunit(s). The alpha1 and beta2 or beta3 subunits are also frequently associated with gamma2 (or other gamma) and alpha6 in these cerebellar receptors.

Recent studies have identified several subunits (α, β, γ and δ) of the γ-aminobutyric acidA/benzodiazepine receptor; each consists of several variants. The γ subunit appears to mediate the interaction of the α and β subunits making the receptor capable of modulation by benzodiazepines. In the present studies, the expression of mRNA encoding the γ2 subunit was examined in the cerebellum during development and in adult Purkinje cell degeneration, lurcher and reeler mutant mice. In the normal adult cerebellum, in situ hybridization with [35S]cRNA probes revealed a strong signal over the Purkinje cell layer and deep cerebellar nuclei, and a weaker signal over basket, stellate and granule cells. Labeling over Purkinje cells was detectable at birth, gradually becoming stronger and more punctate during postnatal weeks 1 and 2, as Purkinje cells formed a monolayer between the molecular and granule cell layers. Adult levels of grain density were reached by P20. The external germinal layer, which contained proliferating granule cells, was unlabeled throughout development; however, weak labeling was detected over the internal granular layer at the end of postnatal week 1, as granule cells began their migration across the molecular layer. During the second postnatal week, punctate labeling became visible over the molecular layer in a distribution indicative of basket and stellate cells. In adult Purkinje cell degeneration and lurcher mutants, in which Purkinje cells have degenerated, no punctate labeling characteristic of mature Purkinje cells was detected. In adult and developing reeler mutants, where all classes of cells are malpositioned throughout the cerebellum, the punctate hybridization signal was present and clearly associated with Purkinje cells in all cortical regions. Our results suggest that developing Purkinje cells express the γ2 gene at a time prior to receiving GABAergic inhibitory input, and that the continued expression in the adult is not affected by the absence of afferents.

Some aspects of the GABA and cholinergic systems have been investigated in the cortex and thalamus of GAERS Wistar rats, a model of petit-mal epilepsy, and in a non-epileptic control strain. GABA and its synthetic enzyme, glutamic acid decarboxylase (GAD), were located by immunocytochemistry; the GABAA receptors were evaluated by autoradiography of GABA-enhanced 3H-flunitrazepam binding and by immunocytochemistry using specific antibodies against the beta 2-beta 3 subunits of GABAA receptor protein. GABA and GAD immunocytochemistry did not show up any difference in density or distribution of immunoreactive elements (fibers, terminals and neurons) between epileptic and control animals, but autoradiographic and immunocytochemical studies showed a decreased enhancement of 3H-flunitrazepam binding and of beta 2-beta 3 subunits of GABAA receptor in the sensorimotor cortex and anterior thalamic areas of the epileptic strain. No differences were found in benzodiazepine receptors in the two strains. GABAB receptors were measured as 3H-baclofen binding in a crude synaptic membrane preparation and there was no difference between epileptic and control animals. Choline acetyltransferase, the synthetic enzyme for acetylcholine, and muscarinic receptor subtypes (M1 and M2), visualized respectively by an immunocytochemical procedure and binding autoradiography, did not differ in epileptic and normal rats. The data suggest an impairment of the 'GABAA system' in restricted brain regions of epileptic rats, due to a reduction of receptor beta 2-beta 3 subunits and coupling to benzodiazepine receptors despite the normal synthesis and location of the neurotransmitter.

Abstract: The postnatal development of the γ‐Aminobutyric acidA/benzodiazepine receptor (GABAR/BZDR) complex of the rat brain has been investigated using the monoclonal antibody 62‐3G1 and the polyclonal rabbit antiserum A, specific for the 57,000 and 51,000 Mr, receptor subunits, respectively. Both GABAR and BZDR binding activities co‐precipitated during all postnatal ages. Adult rats showed a main 51,000 Mr [3H]flunitrazepam photoaffinity‐labeled peptide, whereas newborn rats showed several photolabeled peptides of higher Mr. All the photolabeled peptides could be immunoprecipitated with each antibody regardless of the age of the rats. These results suggest that the physical coupling between the GABAR and the BZDR is already present in newborn animals and it is maintained afterwards during development. Glycosidase and peptidase treatments of the immunoprecipitated GABAR/BZDR complex indicated that all the [3H]flunitrazepam‐photolabeled subunits are different peptides, although they seem to conserve a high degree of homology. In addition to the age‐dependent heterogeneity, the results also suggest that for each age, there is heterogeneity in the subunit composition of the GABAR/BZDR complex.

The expression of the GABAA/benzodiazepine receptor (GABAR/BZDR) complex in primary neuronal cultures from rat brain embryos has been investigated. The GABAR/BZDR complex was photoaffinity labeled with [3H]flunitrazepam [3H]FNZ and immunoprecipitated with subunit specific antibodies. These were the mAb 62-3G1 which is specific for the 57-kDa GABA binding subunit, and the rabbit antiserum A which recognizes the 51-kDa [3H]FNZ binding subunit. The results indicate that the cultured neurons express 5 different peptides of 51, 53, 54, 57 and 59 kDa that can be photoaffinity labeled with [3H]FNZ and that all of them are physically coupled to the GABAA receptor. Most of the [3H]FNZ photolabeled peptides have similar mobilities to those found in the brain of the newborn rat. Nevertheless, some of the quantitative changes in the photolabeled peptides observed during the normal development of the rat brain were not observed or occurred at much slower pace in the cultured neurons.

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62-3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus. Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cell's plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5-7 microns in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13-20 microns in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells. Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The mAb 62–3G1 to the GABAA receptor/benzodiazepine receptor/Cl- channel complex was used with light-microscopy immunocytochemistry for studying the localization of the GABAA receptors (GABAR) in the rat brain. The results have shown a receptor distribution identical to the one obtained by others using 3H-muscimol binding in combination with autoradiographic techniques. The external plexiform layer of the olfactory bulb, cerebral cortex, granule cell layer of the cerebellum, hippocampus, dentate gyrus, substantia nigra, dorsolateral and medium geniculate nuclei, and the lateral posterior thalamic nucleus, among other areas, were rich in GABAA receptor immunoreactivity. In the cerebellum the granule cell layer had more immunoreactivity than did the molecular layer. In the hippocampus the receptor was most abundant in the stratum oriens and in the molecular layer of the dentate gyrus. The immunocytochemical techniques have also allowed us to study the distribution of the GABAA receptor with high-resolution light microscopy. These studies have shown that the GABAA receptors are localized in neuronal membranes and concentrated in structures rich in GABAergic synapses, such as the cerebellar and olfactory glomeruli and the external plexiform layer of the olfactory bulb, the deep cerebellar nuclei, and the substantia nigra. The mAb 62–3G1 was generated by immunizing mice with the affinity-purified GABAA receptor/benzodiazepine receptor (BZDR) complex. This mAb bound to the 57,000 Mr peptide but not to the benzodiazepine binding 51,000 Mr peptide. The distribution of the GABAR immunoreactivity in the rat brain colocalized better with 3H-muscimol than with 3H-benzodiazepine binding. Therefore, it is suggested that (1) the 57,000 Mr peptide that is recognized by the mAb 62–3G1 is the muscimol (GABAA receptor agonist) binding subunit of the receptor complex, (2) there is an important population of brain GABAA receptors that is not functionally coupled to the benzodiazepine receptors, and (3) both the BZDR-coupled and uncoupled forms of the GABAA receptor are immunologically similar, if not identical.

The anti-benzodiazepine (BZD) monoclonal antibody 21-7F9 was used with light and electron microscopy immunocytochemistry techniques for studying the distribution of BZD-like molecules in the rat brain. With light microscopy, BZD-like immunoreactivity was found throughout the brain, mainly in neurons and occasionally in some glial cells (in periventricular areas, as well as in some perivascular astrocytes). Despite the fact that in the cerebellum the GABAergic neurons exhibit BZD-like immunoreactivity, co-localization of these two molecules is not exact, since there are also BZD-like positive neurons that are non-GABAergic (e.g., cerebellar granule cells, some neocortical and hippocampal pyramidal cells). Ultrastructural study of the cerebellar cortex disclosed that all neuronal categories were immunoreactive, as were some astrocytes within the granular layer. The reaction product was concentrated in neuronal perikarya and dendritic processes. Axons and axon terminals remained mostly unlabeled. The absence of immunoprecipitate within cytoplasmic organelles (Golgi apparatus, mitochondria, lumen of endoplasmic reticulum) and its presence at the cytoplasmic face of the cell membranes strongly suggests that endogenous BZD-like molecules are present in both the soluble cytoplasm (hyatoplasm), and also in association with both external and internal cell membranes. The results suggest that the brain BZD-like molecules might be functionally involved in either the modulation of GABA neurotransmission and/or the biotransformation, accumulation and elimination of benzodiazepines and benzodiazepine-like molecules in the brain.