Polyclonal antibodies to agonist benzodiazepines.

Benzodiazepine-binding, immunoglobulin G class antibodies have been raised in three rabbits immunised with a conjugate of kenazepine coupled to keyhole limpet haemocyanin. The antibodies were assayed by [3H]flunitrazepam binding, followed by adsorption onto Staphylococcus aureus cells. Measurement of the rates of association and dissociation of [3H]flunitrazepam binding, together with saturation analysis of equilibrium binding, revealed varying degrees of heterogeneity in the affinity constants of the three rabbit antisera (equilibrium KD values 0.18 to 4.13 nM at 20-22 degrees). Specificity of the antibodies was investigated by testing a wide variety of compounds (at concentrations of up to 10-100 microM) for their ability to inhibit [3H]flunitrazepam binding. Only benzodiazepines known to act as agonists at their receptor sites in the central nervous system (CNS) caused an inhibition of binding. The rank orders of the IC50 values of these drugs for inhibition of [3H]flunitrazepam binding to IgG from two out of the three rabbits correlated significantly with that previously published for displacement of CNS receptor binding. The agonist beta-carboline derivative ZK 93423, the anxiolytic cyclopyrrolones suriclone and zopiclone and the purines inosine and hypoxanthine all failed to inhibit antibody binding, supporting previous suggestions that these drugs may bind at non-benzodiazepine recognition sites on the CNS receptor. The antibodies described are expected to provide useful reagents for raising anti-idiotypic antibodies directed against the CNS receptor and for the identification and purification of possible endogenous benzodiazepine receptor agonists in the CNS.

On the location of gamma-aminobutyrate and benzodiazepine receptors in the cerebellum of the normal C3H and Lurcher mutant mouse.

Binding of gamma-aminobutyrate and benzodiazepine receptor ligands has been studied in the cerebellum of adult normal (C3H) and Lurcher mutant mice. The adult mutant has lost all Purkinje cells and more than 90% of the granule cells in the cerebellar cortex. When compared with their normal littermates Lurcher mice displayed large decreases in the number of high-affinity binding sites for [3H]muscimol, a synaptic gamma-aminobutyrate receptor ligand, in washed cerebellar homogenates. This observation was consistent with the extensive loss of gamma-aminobutyrate receptive Purkinje and granule cells from the Lurcher cerebellum. However, specific binding of the benzodiazepine-receptor ligand [3H]flunitrazepam to Lurcher cerebellum remained unchanged. Indeed quantitative autoradiography, employing [3H]flunitrazepam as a photoaffinity label, showed no significant differences in the density of labelling between Lurcher and normal littermate mice in any region of the cerebellum. These benzodiazepine binding sites in washed homogenates or tissue sections displayed a gamma-aminobutyrate-induced enhancement of [3H]flunitrazepam binding which occurred to the same extent in both Lurcher and normal cerebellum, a facilitatory effect which could be blocked by the addition of bicuculline methobromide. Our results suggest that a large proportion of the high-affinity, specific benzodiazepine binding sites in mouse cerebellum are not coupled to the synaptic gamma-aminobutyrate receptors thought to be labelled by high affinity [3H]muscimol binding. Further, that benzodiazepine binding sites do not appear to be enriched on either the soma or dendrites of Purkinje cells, as has been suggested from previous studies. Investigations at the electron microscope level are now required to elucidate the cellular location of benzodiazepine binding sites in the cerebellar cortex and to examine whether or not they are likely to be exposed to gamma-aminobutyrate in vivo.

Autoradiography of benzodiazepine receptor binding in the central nervous system of the normal C57BL6J mouse.

[3H]flunitrazepam has been used as a photoaffinity label for the specific, clonazepam-displaceable 1,4-benzodiazepine binding sites in sections of normal C57BL6J mouse brain and spinal cord. Binding was visualized by light microscope autoradiography and quantified by a simple microdensitometric procedure. Specific flunitrazepam binding was seen to be highest in the colliculi, cerebral cortex, hippocampal formation, interpeduncular nucleus, mamillary body, hypothalamus, olfactory tubercle, and in the molecular layer and deep nuclei of the cerebellum. The distribution of specific flunitrazepam binding sites in mouse brain and spinal cord is discussed in terms of the known actions of the benzodiazepines.

Changes in benzodiazepine receptor binding as seen autoradiographically in the central nervous system of the spastic mouse.

Quantitative light-microscope autoradiography has been used to compare the specific, clonazepam-displaceable binding of [3H]flunitrazepam, a photoaffinity label for the 1,4-benzodiazepine receptor, in different regions of the brain and spinal cord of spastic mice and their unaffected littermates. Specific binding of [3H]flunitrazepam in the central nervous system of the spastic mouse showed significant increases in the anterior colliculus and pretectal area and in all laminae of the grey matter in the lumbar spinal cord. These results confirm homogenate binding assays suggesting an increased number of benzodiazepine receptors in the spinal cord of the spastic mouse. Possible sites are therefore provided at which disorders of function could arise, associated with changes seen at the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex in spinal cord homogenates from the mutant mouse spastic.

Histogenesis of the cochlear nucleus of the mouse.

In Nissl-stained preparations of the cochlear nucleus there are nine recognizable cell types. These cells are born during three periods of histogenesis prenatally. On gestation days 10.0, 10.5, and 11.0 the pyramidal, giant, and dark-staining cells are born. The spherical, globular, multipolar, and horizontal cells are formed on gestation days 12.0, 12.5, and 13.0 and small cells follow on gestation day 14.5. The onset of granule cell formation is gestation day 14.5 continues to birth on gestation day 19. At birth, and for at least the first 2 postnatal weeks, glial cells are born. There are no regional gradients in cell birth dates, cells from all birth dates being intermixed. Cell birth proceeds in an orderly sequence that is related only to cell size. Although there were no apparent spatiotemporal patterns, some clustering of labeled cells was evident. These observations do not support the hypothesis that Golgi Type I cells precede Golgi Type II cells in their order of birth since both large and small neurons project beyond the nucleus. There is, nonetheless, a sequential pattern in the onset of cell birth for the auditory system, with cochlear nucleus neurons preceding cochlear neurons.