Ethanol withdrawal activates nitric oxide-producing neurons in anxiety-related brain areas.

The present study investigated whether nitric oxide (NO)-producing neurons localized in brain areas related to anxiety are also activated after ethanol withdrawal. Male Wistar rats were subjected to an oral ethanol self-administration procedure, in which they were offered 6-8% (vol/vol) ethanol solution for a period of 21 days followed by abrupt discontinuation of the treatment. Control animals received control dietary fluid for similar periods of time. Twenty-four or 48 h after ethanol discontinuation, the animals were exposed to the open field for 10 min. Two hours later, their brains were removed and processed for Fos immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry (which is used to detect NO-producing neurons). Decreased exploratory activity was observed in animals subjected to 24-h withdrawal, characterized by a shorter distance traveled in the open field. Additionally, increased Fos expression was detected in brain areas, such as the cingulate and piriform cortices, several hypothalamic nuclei, amygdaloid nuclei, most subdivisions of the periaqueductal gray matter, and dorsal raphe nucleus (DRN). Ethanol withdrawal activated NO-producing neurons in the paraventricular nucleus (PVN) of the hypothalamus, dorsolateral periaqueductal gray matter (DLPAG), and DRN. The results show that ethanol withdrawal activates NO-producing neurons in the PVN, DLPAG, and DRN, which are brain areas implicated in the modulation of emotional, autonomic, and motor expression of anxiety-like behaviors.