Role for hypocretin in mediating stress-induced reinstatement of cocaine-seeking behavior

Abstract

Hypocretin-1 and -2 (Hcrt-1 and Hcrt-2), also referred to as orexin-A and -B, are neuropeptides synthesized by a few thousand neurons in the lateral hypothalamus. Hypocretin-containing neurons project throughout the brain, with a prominent input to basal forebrain structures involved in motivation, reward, and stress. However, the role of hypocretins in addiction-related behaviors remains largely unexplored. Here we show that intracerebroventricular infusions of Hcrt-1 lead to a dose-related reinstatement of cocaine seeking without altering cocaine intake in rats. Hcrt-1 also dramatically elevates intracranial self-stimulation thresholds, indicating that, unlike treatments with reinforcing properties such as cocaine, Hcrt-1 negatively regulates the activity of brain reward circuitries. Hypocretin-induced reinstatement of cocaine seeking was prevented by blockade of noradrenergic and corticotropin-releasing factor systems, suggesting that Hcrt-1 reinstated drug seeking through induction of a stress-like state. Consistent with this interpretation, the selective Hcrt-1 receptor antagonist SB-334867 blocked footshock-induced reinstatement of previously extinguished cocaine-seeking behavior. These findings reveal a previously unidentified role for hypocretins in driving drug seeking through activation of stress pathways in the brain.

Fig. 1.

Icv infusions of Hcrt-1 reinstate previously extinguished rewarding behaviors. (A-C) Hcrt-1 dose-dependently reinstates cocaine-seeking behavior. Data are expressed as mean (±SEM) number of active or inactive lever responses during different phases of the experiment (n = 7 in each group), during extinction and after i.c.v. Hcrt-1 infusion (reinstatement). The dose-response effect of Hcrt-1 on relapse to cocaine seeking was determined by using a linear regression (P < 0.05). Hcrt-1 infusion into the lateral ventricle also reinstated previously extinguished food-seeking behavior. (D) Mean (±SEM) responses on the active lever in food-trained rats (n = 6) during different phases of the experiment, after a saline infusion before the last extinction session (extinction), and after i.c.v. infusion of Hcrt-1 (1.5 nmol) (reinstatement). (E) Mean (±SEM) responses on the active/inactive levers in food-trained rats (n = 4) for which pressing the active lever was previously paired with the delivery of food pellets. (F) Mean (±SEM) responses on both levers in non-food-trained rats for which pressing the active lever was not previously paired with the delivery of food pellets (n = 5). #, Significant difference (P < 0.05) between Hcrt-1 doses (0.3 vs. 1.5 nmol); $, significant difference (P < 0.05) between food-paired and non-food-paired lever; asterisks, significant differences (*, P < 0.05; **, P < 0.01) between experimental phases (extinction and reinstatement). See Statistical Analyses.

Fig. 2.

Elevated ICSS reward thresholds in rats (n = 9) measured up to 48 h after Hcrt-1 administration into the lateral ventricle. *, statistically significant differences (P < 0.05) compared with Pre thresholds (baseline); #, statistically significant differences (P < 0.05) compared with thresholds in saline-treated rats (n = 5) at the same time points, as determined by planned comparisons among means after statistically significant main effect in the ANOVA. See Statistical Analyses.

Fig. 3.

Noradrenergic and CRF systems regulate Hcrt-1-induced reinstatement of cocaine seeking. Data are expressed as mean (±SEM) number of active or inactive lever responses during different phases of the experiment, during extinction (Ext), and after i.c.v. Hcrt-1 infusion (reinstatement, Rein). (A) In absence of pretreatment, Hcrt-1 (n = 8; 1.5 nmol, i.c.v.) induced a robust reinstatement of lever pressing (P < 0.05). Pretreatment with the α2 agonist clonidine (Clon; n = 8; 20 μg/kg, i.p.) (B) or the CRF₁/CRF₂ antagonist d-Phe-CRF_12-41 (n = 10; 1.3 nmol, i.c.v.) (C) attenuated the effect of Hcrt-1 (1.5 nmol, i.c.v.) on reinstatement (P < 0.05), but responding on the previously cocaine-paired active lever remained significantly increased compared with extinction responses (P < 0.05). (D) Coadministration of clonidine (20 μg/kg, i.p.) and d-Phe-CRF_12-41(1.3 nmol, i.c.v.) completely blocked the Hcrt-induced reinstatement of cocaine seeking (n = 7, P < 0.05). #, significant difference (P < 0.05) compared with the Hcrt-1; *, statistically significant differences (P < 0.05) between experimental phases (extinction and reinstatement). See Statistical Analyses.

Fig. 4.

Involvement of the Hcrt system in stress-induced relapse of cocaine seeking. Footshock induced a significant increase in the number of responses on the previously cocaine-paired active lever in vehicle-treated rats (n = 14) (A), whereas pretreatment with the Hcrt-1 receptor antagonist SB-334867 (15 mg/kg i.p., n = 8; 30 mg/kg i.p., n = 5) did not induce a significant increase in responding on the active lever (B and C). Data are expressed as mean (±SEM) number of active or inactive lever responses during extinction and footshock-induced reinstatement for cocaine seeking. *, Significant difference (P < 0.05) between experimental phases (extinction and footshock); #, significant difference (P < 0.05) compared with vehicle-treated rats. See Statistical Analyses.