Enhanced ultrasonic vocalization and Fos protein expression following ethanol withdrawal: effects of flumazenil

Rationale: Administration of flumazenil, a benzodiazepine (BZD) antagonist, has therapeutic efficacy against some anxiogenic effects of ethanol withdr awal. This observation has led to the suggestion that anxiety associated wi th ethanol withdrawal is related to release in brain of an endogenous BZD i nverse agonist. Objective: The present studies further tested this hypothes is by assessing the effect of flumazenil on withdrawal-induced changes in a behavioral task and on the expression of the neuronal protein, Fos. Method s: Male Sprague-Dawley rats were withdrawn from a chronic ethanol regimen a nd tested, with or without flumazenil pretreatment, for either ultrasonic v ocalization in response to air puff or for the induction of Fos protein-lik e immunoreactivity (Fos-LI) in brain. In addition, flumazenil effects on Fo s-LI were measured in a group of animals treated with the BZD inverse agoni st DMCM (0.75 and 1.0 mg/kg). Results: Flumazenil (5.0 mg/kg) significantly reduced the number of ultrasonic vocalizations observed following withdraw al from chronic ethanol. In contrast, flumazenil (5.0 mg/kg), given either 14 h before withdrawal from chronic ethanol, or during hours 3 and 5 follow ing withdrawal, did not attenuate the effects of withdrawal on Fos-LI. Subs equent testing with DMCM confirmed that a benzodiazepine inverse agonist ca n induce Fos-LI in most of the same brain regions as observed following eth anol withdrawal, and that this change in Fos protein can be attenuated by p retreatment with flumazenil (5.0 mg/kg). Conclusions: Overall, these result s demonstrate that specific behavioral indices of anxiety, but not measures of Fos-LI, support the contribution of an endogenous BZD inverse agonist i n the ethanol withdrawal syndrome.