Effect of Chronic Ethanol on Enkephalin in the Hypothalamus and Extra‐Hypothalamic Areas

Abstract

Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague-Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real-time quantitative polymerase chain reaction (qRT-PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin-labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. With qRT-PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK-expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra-hypothalamic as well as hypothalamic areas, possibly to carry out specific area-related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response-reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC.