Operant alcohol and saccharin self-administration

The rats were trained to self-administer 10% (v/v) alcohol solution during daily 30-min sessions in standard operant conditioning chambers (Med Associates) until stable responding was maintained (± 10% over the last three sessions). The rats were first subjected to an overnight session (12 h) in the operant chambers with access to one lever (right lever) that delivered water on a fixed-ratio 1 (FR1) schedule of reinforcement (i.e., each operant response was reinforced with 0.1 ml of water). After 1 d off, the rats were subjected to a 3-h session (FR1) with access to the right lever that delivered 0.1 ml of alcohol. In the next two sessions, the rats were subjected to 2-h and 1-h FR1 sessions, respectively, with the right lever delivering 0.1 ml of alcohol. After this training phase, all subsequent sessions lasted 30 min, with both levers available for water and alcohol (left lever for water and right lever for alcohol) until stable levels of intake were reached. For the saccharin self-administration study, the rats underwent daily 30 min of FR1 sessions. Responses on the right lever resulted in the delivery of 0.1 ml of saccharin (0.04%, w/v). Lever presses on the left lever delivered 0.1 ml of water. This training lasted two weeks until a stable baseline of intake was reached. Behavioral testing occurred three times per week during the dark phase (for the experimental design, see Figs. 1A, ​,66A).

Timeline of the experiment, escalation of alcohol self-administration, and Blood alcohol levels: BALs after exposure to alcohol vapor. A, Timeline of the experiment with systemic JNJ-63533054 administration in alcohol-dependent rats. B, Rats that were exposed to alcohol vapor escalated their alcohol intake after the 6th session of operant self-administration; *p < 0.05, versus pre-vapor baseline. C, BALs significantly increased after eight weeks of alcohol vapor exposure; ****p < 0.0001.

Intra-habenular but not intra-IPN JNJ-63533054 administration decreases alcohol intake and increases paw withdrawal thresholds in alcohol-dependent rats during withdrawal. A, Timeline of microinfusions of JNJ-63533054 in alcohol-dependent rats. B, Intra-habenular infusion of JNJ-63533054 (0.25 µg/0.5 µl) decreased alcohol self-administration in dependent rats (**p < 0.01), without affecting water self-administration (n = 6). C, Intra-habenular infusion of JNJ-63533054 increased paw withdrawal thresholds during alcohol withdrawal (**p < 0.01). D, Histology of accurate injection sites in the habenula (black circles) and misplaced injection sites (white circles); 5× magnification. E, In situ hybridization of GPR139 receptors in mouse habenula. Modified from Allen Mouse Brain Atlas (AllenMouseBrainAtlas, 2004). F, Intra-IPN infusion of JNJ-63533054 did not affect alcohol or water self-administration in alcohol-dependent rats (n = 7). G, Paw withdrawal thresholds during alcohol withdrawal were unaffected by intra-IPN infusion of JNJ-63533054. H, Histology of accurate injection sites in the IPN (black circles) and misplaced injection sites (white circles); 2.5× magnification. I, In situ hybridization of GPR139 receptors in the mouse IPN. Modified from Allen Mouse Brain Atlas (AllenMouseBrainAtlas, 2004). cp, cerebral peduncle; DG, dentate gyrus; D3V, dorsal third ventricle; LHb, lateral habenula; MHb, medial habenula; ml, medial lemniscus; SN, substantia nigra; VTA, ventral tegmental area.