An autoradiographic study of [³H]flunitrazepam binding sites in the brain of rat made tolerant to and dependent on pentobarbital

The effects of continuous administration of pentobarbital on the benzodiazepine receptor labeled by [³H]flunitrazepam were investigated. Animals were made tolerant to pentobarbital by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 μg/10 μl/h) for 6 days through pre-implanted cannulae connected to osmotic mini-pumps. The dependent rats were assessed 24 h after cessation of pentobarbital infusion. Changes in [³H]flunitrazepam binding were examined in 37 brain regions at a concentration of [³H]flunitrazepam of 1 nM. In subsequent saturation studies, the binding parameters B(max) and K(D) were also investigated in 17 brain regions, most of which showed significant changes in [³H]flunitrazepam binding in experiments using a fixed concentration of radioligand. The pentobarbital-tolerant rats showed a significant increase in B(max) with an increase in K(D) for [³H]flunitrazepam in the ventroposterior nucleus of thalamus. In the dependent rats, a significant increase in B(max) for [³H]flunitrazepam binding, without a change in K(D), was observed in all layers of the frontal cortex, the caudate-putamen, olfactory tubercle, and some nuclei in thalamus, compared to those in the control. Increased [³H]flunitrazepam binding in the molecular layer of the olfactory bulb, the ventral pallidum, and the cerebellum of the pentobarbital dependent rats at a fixed concentration of [³H]flunitrazepam was also observed. There was no significant change in [³H]flunitrazepam binding in the hippocampus and several nuclei of the brain stem. These findings suggest that benzodiazepine receptors are closely involved in the development of tolerance to and dependence on pentobarbital. Further studies on changes in γ-aminobutyric acid (GABA)A receptor subunit mRNA or the effects of pentobarbital on GABA(A) receptor phosphorylation would be necessary for an explanation of the precise mechanisms underlying the development of tolerance to and dependence on pentobarbital.