Hypocretins regulate the anxiogenic-like effects of nicotine and induce reinstatement of nicotine-seeking behavior

Abstract

Emerging evidence suggests that the hypocretinergic system is involved in addictive behavior. In this study, we investigated the role of these hypothalamic neuropeptides in anxiety-like responses of nicotine and stress-induced reinstatement of nicotine-seeking behavior. Acute nicotine (0.8 mg/kg, s.c.) induced anxiogenic-like effects in the elevated plus-maze and activated the paraventricular nucleus of the hypothalamus (PVN) as revealed by c-Fos expression. Pretreatment with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 or preprohypocretin gene deletion blocked both nicotine effects. In the PVN, SB334867 also prevented the activation of corticotrophin releasing factor (CRF) and arginine-vasopressin (AVP) neurons, which expressed Hcrtr-1. In addition, an increase of the percentage of c-Fos-positive hypocretin cells in the perifornical and dorsomedial hypothalamic (PFA/DMH) areas was found after nicotine (0.8 mg/kg, s.c.) administration. Intracerebroventricular infusion of hypocretin-1 (Hcrt-1) (0.75 nmol/1 mul) or footshock stress reinstated a previously extinguished nicotine-seeking behavior. The effects of Hcrt-1 were blocked by SB334867, but not by the CRF1 receptor antagonist antalarmin. Moreover, SB334867 did not block CRF-dependent footshock-induced reinstatement of nicotine-seeking while antalarmin was effective in preventing this nicotine motivational response. Therefore, the Hcrt system interacts with CRF and AVP neurons in the PVN and modulates the anxiogenic-like effects of nicotine whereas Hcrt and CRF play a different role in the reinstatement of nicotine-seeking. Indeed, Hcrt-1 reinstates nicotine-seeking through a mechanism independent of CRF activation whereas CRF mediates the reinstatement induced by stress.

Figure 1.

Modulation of the anxiogenic-like effects of nicotine by Hcrts. A, B, Percentage of time spent in the open arms of the elevated plus-maze in response to acute nicotine (0.8 mg/kg, s.c.) in vehicle (□)- or SB334867 (5 mg/kg, i.p.) (■)-pretreated C57BL/6J mice (n = 16–18 mice per group) (A) and in wild-type (□) or preprohypocretin knock-out (■) mice (n = 8–9 mice per group) (B). SB334867 was administered 30 min before nicotine. C, Percentage of c-Fos-positive Hcrt-1-expressing neurons after saline or nicotine (0.8 mg/kg, s.c.) administration in the PFA/DMH (n = 3 mice per group) and the LH (n = 3 mice per group). Data are expressed as mean ± SEM, *p < 0.05, ** p < 0.01 compared with the control group; p < 0.05 compared with KO or SB334867-pretreated nicotine-treated mice (one-way ANOVA). D, Schematic anatomic representation of LH subdivisions adapted from Paxinos and Franklin's stereotaxic atlas (Paxinos and Franklin, 1997). Labeled areas delineate regions where c-Fos expression was examined. E, Representative images of sections of the PFA/DMH obtained via confocal microscopy after direct double labeling combining rabbit polyclonal antiserum to c-Fos with mouse monoclonal antibody to Hcrt-1. Arrowheads indicate c-Fos-positive Hcrt-1-expressing neurons. Scale bar, 100 μm.

Figure 2.

Nicotine-induced activation of the PVN is regulated by Hcrts. A, B, c-Fos expression in the PVN after nicotine (0.8 mg/kg, s.c.) administration in vehicle (□)- or SB334867 (5 mg/kg, i.p.) (■)-pretreated C57BL/6J mice (n = 5 mice per group) (A) and in wild-type (□) or preprohypocretin knock-out (■) mice (n = 3 mice per group) (B). SB334867 was administered 30 min before nicotine. Quantification of c-Fos-positive nuclei in the PVN was performed by ImageJ software. Data are expressed as mean ± SEM, **p < 0.01 compared with the control group; p < 0.01 compared with KO or SB334867-pretreated nicotine-treated mice (one-way ANOVA). C, D, Representative images of the PVN obtained by microscopy after direct-labeling with rabbit polyclonal antiserum to c-Fos. Scale bar, 100 μm.

Figure 3.

Nicotine-induced activation of CRF and AVP neurons of the PVN is dependent on Hcrtr-1. A, B, Percentage of c-Fos-positive CRF (A)- and AVP (B)-expressing neurons after nicotine (0.8 mg/kg, s.c.) administration in vehicle (□)- or SB334867 (5 mg/kg, i.p.) (■)-pretreated C57BL/6J mice (n = 3 mice per group). SB334867 was administered 30 min before nicotine. Data are expressed as mean ± SEM, **p < 0.01 compared with the control group; p < 0.05, p < 0.01 compared with SB334867-pretreated nicotine-treated mice (one-way ANOVA). Representative images of sections of the PVN are shown in supplemental Figure 5 (available at www.jneurosci.org as supplemental material). C, D, CRF- and AVP-expressing neurons of the PVN contain Hcrtr-1. Rabbit polyclonal antibodies to Hcrtr-1 were combined with guinea-pig polyclonal antibodies to CRF (C) and AVP (D). Scale bar for colocalization images, 25 μm.

Figure 4.

Acquisition and extinction of nicotine self-administration behavior. A, B, Mean number of nose-poking responses in the active (●) and inactive (○) holes during nicotine self-administration training and extinction. The complete group of animals used in the different experiments of reinstatement is shown (n = 50). A, Mice were trained daily in 1 h sessions to obtain nicotine (85.5 μg/kg per infusion) during 10 d under a fixed ratio 1 schedule of reinforcement. B, The first 15 d of the extinction phase and the last 3 d achieving the extinction criterion (E1, E2, E3) are shown. Sessions were conducted daily and lasted for 1 h. Animals were no longer reinforced after an active nose-poke. Data are expressed as mean ± SEM, **p < 0.01 comparison between holes (one-way ANOVA).

Figure 5.

Hcrtr-1, but not CRF1 receptor, regulates the reinstatement of nicotine-seeking behavior induced by Hcrt-1. Mice that achieved the extinction criterion received an intracerebroventricular saline infusion preceded by SB334867 (5 mg/kg, i.p.) (n = 4), antalarmin (30 mg/kg, s.c.) (n = 4), or vehicle (n = 9) 10 min before the reinstatement session. Next day, the same mice were infused with Hcrt-1 (0.75 nmol/1 μl, i.c.v.) preceded by the same SB334867 (5 mg/kg, i.p.) (n = 4), antalarmin (30 mg/kg, s.c.) (n = 4), or vehicle (n = 9) and tested for reinstatement 10 min later. SB334867 and antalarmin were administered 30 min and 1 h, respectively, before intracerebroventricular Hcrt-1/vehicle infusions. Mean number of nose-pokes in the active (■) and inactive (□) holes during the different experimental phases: acquisition of nicotine self-administration behavior (mean of 3 d acquisition criteria), extinction (mean of 3 d criterion), and reinstatement (intracerebroventricular infusion of vehicle/Hcrt-1). *p < 0.05, **p < 0.01, differences between experimental phases when considering the active hole; ⁺p < 0.05, ⁺⁺p < 0.01, differences between holes within the same experimental phase (Newman–Keuls test).

Figure 6.

CRF1 receptor, but not Hcrtr-1, regulates footshock stress-induced reinstatement of nicotine-seeking behavior. Mice that achieved the extinction criterion received intermittent footshock stimuli (0.22 mA, 5 min) immediately before the reinstatement session preceded by vehicle (n = 14), SB334867 (5 mg/kg, i.p.) (n = 6), SB334867 (10 mg/kg, i.p.) (n = 7), or antalarmin (30 mg/kg, s.c.) (n = 6). SB334867 and antalarmin were administered 30 min and 1 h, respectively, before electric footshock. Mean number of nose-pokes in the active (■) and inactive (□) holes during the different experimental phases: acquisition of nicotine self-administration behavior (mean of 3 d acquisition criteria), extinction (mean of 3 d criterion), and reinstatement (0.22 mA footshock stress). *p < 0.05, **p < 0.01, differences between experimental phases when considering the active hole; ⁺p < 0.05, ⁺⁺p < 0.01, significant differences between holes within the same experimental phase (Newman–Keuls test).