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Benzodiazepine (BDZ) hypnotics bind at a specific receptor located on postsynaptic neurons. Some data support specificity of binding for several hypnotics to receptor subtypes. We evaluated BDZ receptor binding in cerebral cortical membranes using agonist, antagonist, and subtype-specific ligands for commonly used hypnotics and their metabolites. All hypnotics competed similarly at BDZ 1 and BDZ 2 receptor subtypes except quazepam and its metabolite 2-oxo-quazepam and to a lesser extent hydroxyethyl flurazepam (EtOH) flurazepam. These compounds had relative specificity for the BDZ 1 site. Triazolam, estazolam, and flurazepam bound equally to sites labeled by agonists but kesalkylflurazepam, EtOH flurazepam, temazepam, quazepam, and 2-oxo-quazepam did not; in addition, these four compounds did not bind to the “peripheral” BDZ site labeled by Ro 5-4864. BDZ hypnotics differ in their receptor subtype and ligand binding characteristics.

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the γ-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10−4M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 μM, or the agonist lorazepam, 10 μM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10−4M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ. Administration of EEDQ to senescent mice (20 months) led to a similar decrease in benzodiazepine sites compared to young mice, but receptor density returned more slowly to control levels (calculated receptor t1/2: young, 25.3 h; senescent, 75.1 h). EEDQ modifies all three major sites on the GABAA receptor, with the greatest effect at the benzodiazepine site. This compound may be useful in assessing benzodiazepine receptor half-life in vivo.

1. Chronic benzodiazepine administration has been associated with tolerance and with downregulation of gamma-aminobutyric acidA (GABAA)-receptor binding and function. However, effects of individual benzodiazepines on brain regions have varied. 2. To compare the effects of chronic lorazepam and alprazolam, we have administered these drugs to mice for 1 and 7 days (2 mg kg-1 day-1) and determined benzodiazepine receptor binding in vivo with and without administration of CL 218,872, 25 mg kg-1 i.p., and GABA-dependent chloride uptake in 3 brain regions at these time points. 3. Benzodiazepine binding was decreased in the cortex and hippocampus at day 7 compared to day 1 of lorazepam, with an increase in CL 218,872-resistant (Type 2) sites in both regions. Maximal GABA-dependent chloride uptake was also decreased in the cortex and hippocampus at day 7. 4. Binding was decreased only in the cortex after 7 days of alprazolam, with no significant change in Type 2 binding. Maximal GABA-dependent chloride uptake was also decreased only in the cortex. 5. These data suggest that the effects of chronic benzodiazepine administration on the GABAA-receptor may be both region-specific and receptor subtype-specific.

The relationship between the specific neuronal loss observed in Huntington's disease and the mutation in the IT15 gene responsible for this disease remains obscure. Using an antipeptide antibody against amino acids 3114-3141 of the human huntington protein, we demonstrate that striatal injection of quinolinic acid in mice induces increased immunoreactivity for huntington in some remaining neurons but not in glial cells. This increase is apparent in both neuronal cell bodies and in cell processes in the white matter six hours after excitotoxic challenge. This finding suggests that huntington may be involved in the response to excitotoxic stress in these neurons.