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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.

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.

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.

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.