Role of acetylcholine transmission in nucleus accumbens and ventral tegmental area in heroin-seeking induced by conditioned cues

Abstract

The involvement of cholinergic transmission in heroin self-administration and the reinstatement of heroin-seeking was examined in rats trained to nose-poke for i.v. heroin. Systemic treatment with physostigmine, an inhibitor of acetylcholinesterase, modestly reduced the acquisition and rate of heroin self-administration, and this suppression of heroin intake was reversed by pretreatment with scopolamine but not by mecamylamine. Following 10-14 days of self-administration, rats were left in the home environment for 14 days. Subsequently, rats were evaluated for extinction of nose-pokes during the first hour after being returned to the self-administration apparatus. One hour later a conditioned stimulus (house light, light in the nose-poke hole, sound of the infusion pump) was presented to initiate cue-induced reinstatement. Physostigmine produced a dose-dependent inhibition of cue-induced reinstatement, but only the dose of 0.5 mg/kg significantly decreased nose-poke responding in the extinction test. Chronic treatment with physostigmine (0.1 mg/kg) did not impair performance during acquisition of heroin self-administration. However, during a subsequent reinstatement test conducted in the absence of physostigmine pretreatment, heroin seeking was significantly below that of rats chronically pretreated with saline. To evaluate brain regions mediating the effects of systemic drug treatment on reinstatement, physostigmine was microinjected into the nucleus accumbens (NAc) or ventral tegmental area (VTA). Microinjection of physostigmine into the NAc prior to presenting conditioned cues inhibited the reinstatement of heroin-seeking, without affecting extinction responding. In contrast, microinjection of physostigmine into the VTA augmented the reinstatement induced by conditioned cues and extinction responding. Inactivation of either NAc or VTA by microinjecting tetrodotoxin blocked both extinction responding and cue-induced reinstatement. These data demonstrate that cholinergic transmission influences heroin self-administration and reinstatement. Moreover, cue-induced reinstatement was inhibited by physostigmine in the NAc and potentiated by cholinergic stimulation in the VTA.

Figure 1

Systemic injection of physostigmine inhibits the maintenance of the heroin self-administration. (A) Physostigmine was injected (i.p.) 10 min prior to beginning access to heroin on day 11 of training. Data are shown as mean ± s.e.m. nose-pokes in the hole associated with heroin (active) and the hole not associated with heroin (inactive). * p<0.05, compared with the vehicle control. (B) Infusions earned during the heroin self-administration. * p<0.05, compared with the vehicle control. (C) Scopolamine (0.5 mg/kg) or mecamylamine(0.5 mg/kg) was injected 10 min before injection of physostigmine (0.5 mg/kg ) on day 11 of training. Data are shown as mean ± s.e.m. nose-pokes. * p<0.05, compared to the physostigmine treated group. (D) Infusions earned during the heroin self-administration. * p<0.05, compared to the physostigmine treated group. (E) Representative individual response records of heroin self-administration on day 11 after pretreatment with physostigmine or saline control. Each vertical tick mark indicates an active response. Note the lower response during the first 30 min of heroin training in the rat pretreated with 0.5 mg/kg physostigmine, and the reduction in responding throughout the session after 2.5 mg/kg physostigmine.

Figure 2

Effects of daily pretreatment with physostigmine on the acquisition of heroin self-administration and reinstatement of drug-seeking after withdrawal in Experiment 2. (A) Mean ± s.e.m. active and inactive responses (nose-pokes) during heroin self-administration for 10 consecutive days in rats pretreated 10 min prior to each training session with physostigmine or saline control. (B) Heroin injections earned during the training sessions. (C) When tested after 14 days withdrawal, drug-seeking elicited by the conditioned cues decreased in rats previously treated with physostigmine during heroin self-administration, while extinction responding was not different between the two groups. * p<0.05, compared to the vehicle control.

Figure 3

Systemic injection of physostigmine inhibits heroin seeking induced by conditioned cues. (A) Mean ± s.e.m. extinction responses over 60 min when rats were placed in the heroin self-administration context after 14 days of withdrawal. (B) Mean ± s.e.m. nose-pokes over 60 min induced by conditioned cues begun immediately after collecting data in panel A. (C) Time course of the active nose-poke responses. * p<0.05, compared to the vehicle control.

Figure 4

Effect of physostigmine treatment on horizontal locomotor activity after 14 days withdrawal. The rats were allowed to habituate to their novel environment. One hour later, rats were treated with physostigmine (0.02, 0.1, 0.5 mg/kg) or saline and immediately placed back in the AccuSan chamber and observed for two hours. (A) Each time point represents the mean ± s.e.m. photocell beam breaks per 5 min period. (B) Histograms represent the total horizontal locomotor activity during the one hr period following physostigmine treatment. * p<0.05, compared with the vehicle control.

Figure 5

Effect of intra-NAc or intra-VTA treatment with physostigmine (1.5 μg/side), physostigmine (5 μg/side) or TTX (0.3 ng/side) on reinstatement of drug seeking induced by conditioned cues. (A) Bilateral injection of physostigmine into the NAc inhibited the drug-seeking induced by the conditioned cues, but did not decrease extinction responding. The extinction responding and drug-seeking induced by conditioned cues were decreased by TTX infused into the NAc. (B) Bilateral injection of physostigmine (1.5 μg/side) into the VTA increased the drug-seeking induced by conditioned cues, physostigmine (5 μg/side) increased both extinction responding and drug-seeking induced by conditioned cues, while bilateral injection of TTX inhibited the extinction responding and drug seeking induced by conditioned cues. * p<0.05, compared to the vehicle control.