Down-Regulation of Benzodiazepine Binding to a5 Subunit- Containing g-Aminobutyric AcidA Receptors in Tolerant Rat Brain Indicates Particular Involvement of the Hippocampal CA1 Region

Chronic benzodiazepine treatment can produce tolerance and changes in g-aminobutyric acid (GABA)A receptors. To study the effect of treatment on a selected population of receptors, assays were performed using [H]RY-80, which is selective for GABAA receptors with an a5 subunit. Rats were given a flurazepam treatment known to produce tolerance and down-regulation of benzodiazepine binding, or a diazepam treatment shown to produce tolerance but not receptor down-regulation. Quantitative receptor autoradiography using sagittal brain sections bound with [H]RY-80 showed binding in areas known to express a5 mRNA. Brains from flurazepam-treated rats showed significantly decreased 1 nM [H]RY-80 binding in hippocampal formation (e.g., 32% decrease in CA1) and superior colliculus, but not other areas. Using 5 nM [H]RY-80 showed similar decreases in hippocampus. A corresponding 29% decrease in Bmax but no change in Kd was found with a filtration binding assay using hippocampal homogenates. Down-regulation of [H]RY-80 binding had returned to control by 2 days after withdrawing flurazepam treatment. The magnitude of downregulation of [H]RY-80 binding suggested that GABAA receptors with an a5 subunit may play a prominent role in the adaptive responses associated with benzodiazepine tolerance. Chronic diazepam treatment also resulted in decreased [H]RY80 binding. However, the regional selectivity was even more pronounced than in flurazepam-treated rats, and only the hippocampal CA1 region showed decreased binding (27%). This localized down-regulation persisted for several days after the end of diazepam treatment. These data indicate that synapses in the hippocampal CA1 region are particularly involved in the adaptive response to chronic benzodiazepine treatments. Benzodiazepines are psychoactive agents used for sleep disorders, sedation, anxiety, epilepsy, and other conditions. Tolerance to some benzodiazepine actions, particularly the antiepileptic action, can be pronounced. Several experimental correlates of tolerance have been reported (Hutchinson et al., 1996). Although many neurotransmitter systems may be affected during benzodiazepine treatment of intact animals, the g-aminobutyric acid (GABA)A receptor has been the focus of most studies of tolerance. The GABAA receptor is a ligand-gated Cl 2 channel with several modulatory sites, including the benzodiazepine site (Macdonald and Olsen, 1994). The receptor is a pentamer (Nayeem et al., 1994), and has a very complex stoichiometry with evidence of mammalian genes for at least 6a-, 3b-, and 3g-subunits, as well as gand e-subunits (Lüddens et al., 1995). The subunit composition of a GABAA receptor determines its benzodiazepine pharmacology. Only receptors incorporating a g-subunit form benzodiazepine binding sites (Lüddens et al., 1995). GABAA receptors with an a4 or a6 subunit do not recognize benzodiazepines such as diazepam, but bind agents such as Ro15-4513 (“diazepam-insensitive” binding). Diazepam-sensitive receptors can be categorized based on their affinity for zolpidem: those with an a1 subunit have high affinity for zolpidem, those with an a2 or a3 subunit have a much lower affinity, and receptors with an a5 subunit are essentially insensitive to zolpidem (Pritchett and Seeburg, 1990; Hadingham et al., 1993; Lüddens et al., 1995). Benzodiazepine agonists act as positive modulators, increasing the anion channel opening frequency produced by the binding of GABA to its own recognition site (Macdonald and