Induction of Fos-like proteins and ultrasonic vocalizations during ethanol withdrawal: further evidence for withdrawal-induced anxiety.

The ethanol withdrawal syndrome includes anxiety as a prominent symptom. Because the extent that specific regions of brain are critical to the generation of this emotional state is unknown, Fos-like immunoreactivity (Fos-LI) was used to associate specific regions of the rat brain with the anxiety component of the ethanol withdrawal syndrome exacerbated by an air puff challenge in rats. Chronic ethanol liquid diet was administered intragastrically for 4 days or by having the rats consume the diet for 14 days. During withdrawal from either treatment protocol, Fos-LI was induced most prominently in forebrain areas, although the midbrain and hindbrain were also represented. Included in these Fos-LI positive regions were many cortical regions, septum, accumbens, claustrum, amygdala, paraventricular nucleus of the thalamus and hypothalamus, hippocampus, locus coeruleus, and central gray. Fos-LI expression differed mostly in intensity between the two treatment and withdrawal protocols, with the gastric protocol producing the greatest Fos-LI induction in most brain regions. The threshold for air puff-induced ultrasonic vocalizations was decreased, and the number of vocalizations was increased and the period of vocalization was extended. These behavioral data indicate that aversively motivated responding in rats during ethanol withdrawal can be readily quantified with the ultrasonic vocalizations test without precipitating convulsive activity. Furthermore, a comparison of the effects of the air puff challenge versus withdrawal on Fos-LI indicated that the behavioral state induced in these two situations share functional neuroanatomical features. Some regions--such as the accumbens core, medial septum, subregions of the amygdala, hippocampus, substantia nigra, and cerebellum--exhibited little Fos-LI during withdrawal and also did not exhibit strong increases after the addition of the air puff challenge. However, other regions-such as the cerebral cortex (medial prefrontal, frontal, cingulate and ventrolateral orbital, claustrum, and tenia tecta), hypothalamus, and locus ceoruleus- exhibited Fos-LI at levels higher than that seen after either the ethanol withdrawal or puff challenge alone. These overlapping patterns of Fos-LI in specific regions of the brain, activated by both ethanol withdrawal and an anxiety provoking behavioral challenge, suggest that specific neuroanatomical sites in brain are associated with the symptom of anxiety observed during the "ethanol withdrawal syndrome."