Low-dose ethanol excites lateral habenula neurons projecting to VTA, RMTg, and raphe.

It is unclear how social drinking can contribute to the development of addiction in susceptible individuals. However, alcohol's aversive properties are a well-known factor contributing to its abuse. The lateral habenula (LHb) is a key brain structure responding to various aversive stimuli, including those related to alcohol. We recently reported that ethanol at 10 mM or less that can be achieved by social drinking activates many LHb neurons and drives aversive conditioning. The current study sought to identify LHb circuits that are activated by a low-dose of ethanol using immunohistochemistry and anatomic tracing techniques on adult Sprague-Dawley rats. We showed here that an intraperitoneal injection of ethanol (0.25 g/kg), resulting in a blood ethanol concentration of 5.6 mM, significantly increased the number of cFos immunoreactive (IR) neurons in the LHb. Most of the ethanol-activated cFos-IR LHb neurons expressed vGluT2 (vesicular glutamate transporters 2, a marker of a glutamatergic phenotype). These LHb neurons projected to the ventral tegmental area (VTA), rostromedial tegmental nucleus (RMTg), and dorsal raphe. Moreover, injections of the anterograde tracer AAV-CaMKIIa-eGFP into the lateral hypothalamus produced a significant amount of labeled fibers with vGluT2 positive terminals on the ethanol-activated LHb cells. These results indicate that the LHb neurons stimulated by a low-dose of ethanol project to the VTA, RMTg, and dorsal raphe, and receive excitatory projections from the lateral hypothalamus. These neurocircuits may play a crucial role in mediating the initial aversive effects produced by a low-dose of ethanol.