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Nicotine has been shown to activate stress-related brain nuclei, including the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CEA), through complex mechanisms involving direct and indirect pathways. To determine the neurochemical identities of rat brain neurons which are activated by a low dose (0.175 mg/kg) of nicotine given 30 minutes before sacrifice, we have used single- and double-label in situ hybridization. Neuronal activation was quantified by localization of (35)S-labeled probe for the immediate early gene, c-fos. Corticotrophin releasing factor (CRF), enkephalin (ENK), and dynorphin (DYN) mRNAs were colocalized using a colorimetric, digoxigenin-labeled probe. Film autoradiographic studies showed that nicotine significantly increased c-fos mRNA expression in both PVN and CEA. Pretreatment with the centrally acting nicotinic antagonist, mecamylamine (1 mg/kg), blocked nicotine's effects, whereas pretreatment with the peripherally acting antagonist, hexamethonium (5 mg/kg), did not, indicating that c-fos induction was mediated by a central nicotinic receptor. Double labeling studies showed that nicotine induced c-fos expression within CRF cells in the PVN, as well as in a small population of ENK cells, but not in PVN DYN cells. In contrast, there was no significant nicotine-induced increase in c-fos expression in CEA CRF or DYN cells, whereas nicotine treatment did increase c-fos expression within CEA ENK cells.
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