Vol. 291, Issue 3, 932-942, December 1999

Antagonist-Induced Reversal of Functional and Structural Measures of Hippocampal Benzodiazepine Tolerance¹

Elizabeth I. Tietz, Xu J. Zeng, Shanyi Chen, Scott M. Lilly, Howard C. Rosenberg and Peter Kometiani

Department of Pharmacology, Medical College of Ohio, Toledo, Ohio

One week oral flurazepam (FZP) administration in rats results in anticonvulsant tolerance in vivo, tolerance measured in vitro in hippocampal CA1 pyramidal cells, and regulation of hippocampal -aminobutyric acid_A-receptor subunit protein expression. A single injection (4 or 20 mg/kg i.p) of the benzodiazepine antagonist flumazenil (FLM) was given 1 day after FZP treatment, and tolerance and subunit protein expression were evaluated 1 day later. In vivo tolerance was measured by a reduced ability of the ₁-subunit-selective agonist zolpidem to suppress pentylenetetrazole-induced seizures. This tolerance was reversed by 20 but not 4 mg/kg FLM. In in vitro hippocampal slices, there was tolerance to the effect of zolpidem to prolong the decay of pyramidal cell miniature inhibitory postsynaptic currents, which was reversed by FLM (4 mg/kg) pretreatment. A reduction in miniature inhibitory postsynaptic current amplitude (~50%) was also restored by FLM injection. [³H]Zolpidem binding measured 0, 2, and 7 days after FZP treatment was significantly decreased in the hippocampus and cortex at 0 days but not thereafter. Changes in ₁- and ₃-subunit protein expression were examined via quantitative immunohistochemical techniques. ₁-Subunit protein levels were down-regulated in the CA1 stratum oriens and  subunit levels were up-regulated in the stratum oriens and stratum radiatum of the CA3 region. Chronic FZP effects on ₁- and ₃-subunit protein levels were also reversed by prior FLM injection. FLM's effect on both functional and structural correlates of benzodiazepine tolerance suggests that each of these measures plays an interdependent role in mediating benzodiazepine tolerance.