Relapse to drug seeking was studied using a rodent model of reinstatement induced by exposure to drug-related cues. Here, we used intravenous drug self-administration procedures in rats to further investigate the beneficial effects of electroacupuncture (EA) on heroin-seeking behavior in a reinstatement model of relapse. We trained Sprague-Dawley rats to nose-poke for i.v. heroin either daily for 4 h or 25 infusions for 14 consecutive days. Then the rats were abstinent from heroin for two weeks. 2 Hz EA stimulation was conducted once daily for 14 days during heroin abstinence. We tested these animals for contextual and discrete cue-induced reinstatement of active responses. We also applied immunohistochemistry to detect Fos-positive nuclei in the nucleus accumbens (NACc) core and shell after reinstatement test. We found that active responses elicited by both contextual cues and discrete cues were high in the rats trained with heroin than in saline controls. EA treatment significantly reduced active responses elicited by discrete cues. EA stimulation attenuated Fos expression in the core but not the shell of the NACc. Altogether, these results highlight the therapeutic benefit of EA in preventing relapse to drug addiction.
Drug addiction is characterized by relapse to drug-seeking behavior during periods of abstinence [
One factor that contributes to drug-seeking behavior is the presence of cues and contexts that it previously has associated with past drug use. The motivational effect of cue presentation is illustrated most dramatically by the reports of drug craving and relapse to drug-seeking behavior by addicts while in the presence of drug-associated cues and contexts [
The neural circuitry of cue-elicited drug seeking involves the nucleus accumbens (NACc) [
Male Sprague-Dawley rats (250–300 g) from the Zhejiang Center of Experimental Animals were used. They were randomly assigned and housed collectively (four per cage) under controlled environmental conditions (22°C, 12-h light/dark cycle) with free access to food and water. All animal treatments were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments were conducted during the light cycle.
The animals were implanted with chronically indwelling intravenous catheters under sodium pentobarbital (50 mg/kg, ip) anesthesia. A silicon catheter (3.5 cm length, 0.5 mm inner diameter, 0.94 mm outer diameter) was inserted into the right external jugular vein and secured with thread so that the tip reached the right atrium. The other end of the catheter (10 cm length, PE20) exited from an incision on the back of the body. The catheters were flushed daily with 0.2 mL saline containing sterile benzylpenicillin sodium (60,000 units) and heparin (5 units), to prevent bacterial infection and maintain catheter patency and capped daily. All the animals were allowed to recover for at least 7 days. The rats were housed individually in stainless-steel mesh home cages (size 25 × 30 × 30 cm) after surgical procedure.
Training and testing were conducted in stainless-steel operant chambers (size 30 × 30 × 30 cm) placed in a sound-attenuated, temperature-controlled room; the light of the room was turned off during training. The apparatus consisted of 24 chambers equipped with two nose-pokes (ENV-114 M, Med Associates, Lafayette, IN) in the back wall. There were three LED lights (green, red, and yellow) inside each nose-poke hole. A cue-light (28 V, 0.1 mA, ENV-215 M, Med Associates) was situated on the wall above the nose-pokes. Drug solution was delivered through Tygon tubing, protected by a leash assembly (PHM-120, Med Associates), and suspended through the ceiling of the chamber from a plastic fluid swivel (PHM-115, Med Associates). The leash assembly was modified to fit a custom-made fluid connector fixed with an animal jacket. The Tygon tubing was attached to a syringe pump (PHM-100, Med Associates) that delivered fluid at a speed of 1.08 mL/min using a 10-mL syringe. The experimental events were controlled by an IBM-compatible PC using a MED Associates interface, running self-programmed software (OBSM v4.0, operant behavioral schedule manager) written in Borland Delphi 6.0. Heroin was obtained from the Institute of Forensic Science, Ministry of Public Security of the People's Republic of China and dissolved in physiological saline.
All the rats underwent an identical sequence of behavioral training. Each rat was trained with one daily 4 h session for 14 consecutive days with either saline or heroin self-administration. The animals were transferred into the operant chambers before each training session and were put back in their individual home cages after the session where food was available. Water was always available both in the test cages and home cages. The rats were not placed in food restriction schedule. Enough food was provided to maintain natural weight gain.
The reinforcement schedule was a modified progressive ratio schedule that involved incrementing response requirements in a relatively gradual manner. The response requirement increased in a linear pattern as calculated according to the following equation: response requirement = truncate (0.2*(step-1) +1), where the results were truncated to integer value. The step number is the number of ratios completed. So in each daily session, the response requirements were 1 for the first five heroin infusions, 2 for the second five infusions, 3 for the third five infusions, 4 for the fourth five infusions, and 5 for the last five infusions. Based on our preliminary experiment, this schedule supported reliable heroin self-administration across a range of heroin doses, and the response training was as easy as a FR1 schedule but maintained a relatively high rate of responding, so the schedule could be kept constant throughout the sessions.
Each trial began with illumination of a green light inside the active nose-poke hole. Responding in the active hole resulted in an infusion of heroin (0.05 mg/kg) delivered by an infusion pump (PHM-100, Med Associates, Lafayette, IN). The green nose-poke light was turned off during heroin infusions. A 30 s intertrial interval (time out) followed and then another trial began. Responding in the inactive hole had no consequences. The response requirements started with one and increased one after each five heroin infusion. Each earned heroin infusion was also paired with a 5 s cue-light (situated on the wall above the nose-pokes) that served as the discrete cue stimulus. The session ended after 25 infusions were earned or 4 hours had passed, whichever came first.
After 14 days of self-administration training, the rats were made to abstain from heroin for another 14 days during which they were confined to their individual home cages. The choice of abstinence duration was based on our previous work [
The reinstatement testing lasted for 2 hours, and each animal was tested only once. During testing, the animals still wore their jackets, but the leash assemblies were not connected. The testing consisted of two consecutive 1-h phases. During the first phase, the rats were allowed to respond to the nose-pokes with all the conditioned stimulus lights kept off. The responses were recorded. This phase was used to measure contextual cue- (chamber environment) induced heroin seeking and was generally regarded as an extinction phase.
Immediately after the first phase, the second phase began. This was signaled by one 5 s presentation of the discrete cue stimulus (the nose-poke light, the cue light, and the pump noise), which was previously paired with each heroin infusion. During this phase, the green light inside the active nose-poke hole was turned on, and each active response resulted in another 5 s presentation of the discrete cue stimulus and the turning off of the green nose-poke light. After turning off the discrete cue stimulus, another trial began. This phase was used to measure discrete cue-induced heroin seeking.
Rats were kept in special holders with their hind legs and tails exposed. Two stainless steel needles of 0.3 mm diameter were inserted into each hind leg in the acupoints ST36 (5 mm lateral to the anterior tubercle of the tibia) and SP6 (3 mm proximal to the superior border of the medial malleolus, at the posterior border of the tibia). Constant current squarewave electric stimulation produced by an electroacupuncture apparatus (Model G-6805-2, Shanghai Medical Electronic Apparatus, China) was administered via the two needles. The frequency of stimulation used was 2 Hz. The intensity of the stimulation was increased stepwise from 0.5 to 1.0 mA, with each step lasting for 15 min.
Four rats from each group were randomly selected for c-Fos immunohistochemistry. Immediately after behavioral testing, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, ip) and killed by transcardial perfusion of 200 mL ice saline followed by 200 mL 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB). Brains were dissected and postfixed in the same fixative and then stored in 30% sucrose at 4°C for 3–5 days. Coronal sections (30
The NACc is an integral part of the basal ganglia located within the ventral striatum. It is composed of two regions: core and shell (see Figure
Forty rats were trained with heroin self-administration. Training sessions were conducted daily for 14 consecutive days. Then the rats were abstinent from heroin for two weeks, during which they lived in their individual home cages. The heroin-trained rats were divided randomly into four groups: the contextual-cue-induced reinstatement (No EA CONT,
Experimental data were expressed as mean ± SEM. The differences in total active responses and heroin infusions during heroin self-administration were analyzed using two-way analysis of variance (ANOVA) with session as a repeated within-subject factor and group as a between-subject factor. Cue-induced active responding during reinstatement testing was also analyzed using two-factor repeated ANOVA with time block (15 min) as a within subject factor and group as a between-subject factor. Significant effects were followed by post hoc Tukey tests. Fos protein expression was analyzed using one-way ANOVA. When significance was found using ANOVA procedures, post hoc analyses were conducted using Fisher LSD test.
As shown in Figure
Acquisition of intravenous heroin self-administration. Data were expressed with mean ± S.E.M. of total infusions (a) and total active nose-poke responses (b) during each daily session.
After 14 days of abstinence from heroin self administration, the rats were returned to the operant chambers for testing cue-evoked heroin-seeking behavior. Two-factor ANOVA revealed significant main effects of block (
Effect of EA treatment on cue-induced heroin-seeking after two weeks of abstinence from heroin self-administration. Data were expressed with mean ± S.E.M. Number of active response rates in 15 min blocks (a) or total responses (b) in contextual cue phase and discrete cue phase. *
When the total amount of active responses were analyzed, one-way ANOVA revealed significant effects of group for both contextual cues (
In the NACc core, One-way ANOVA revealed significant effects of group for Fos protein expression (
Quantitative analysis of Fos-positive nuclei in the NACc core and shell. Data are expressed as mean ± SEM. *
Representative coronal sections showing Fos immunoreactivity in the NACc core. No EA CONT/DIS group (a), restraint CONT/DIS group (b), EA CONT/DIS group (c), No EA CONT group (d), and No EA SAL group (e). Schematic coronal section analyzed. (f) Number at the top right of Figure
In the NACc shell, enhanced Fos protein expression was observed in No EA CONT/DIS, EA CONT/DIS, restraint CONT/DIS, and No EA CONT group relative to No EA SAL group. There were no differences in Fos expression among No EA CONT/DIS, EA CONT/DIS, restraint CONT/DIS, and No EA CONT rats, as determined by Fisher LSD post hoc test (
Exposure to environmental stimuli previously associated with drug intake can provoke drug relapse in humans. Environmental cues repeatedly associated with the subjective effects of heroin can elicit drug craving and, possibly, automatic behavioral responses that may lead to relapse in recovering heroin addicts [
In the present study, we found that 2 Hz EA attenuated discrete but not contextual cue-induced reinstatement of heroin seeking after heroin abstinence. These results demonstrate dissociable roles of EA in discrete versus contextual cue-induced reinstatement of heroin seeking. Discrete cues are different from contextual cues. In discrete cue appetitive Pavlovian conditioning, discrete cues with a defined onset and offset that typically activate one sensory modality are provided, accompanied by heroin delivery [
The NACc core and shell subregions are differentially involved in the reinstatement of cocaine seeking, depending on the type of trigger that elicits this behavior. Our present results that discrete cue evoked an increase in Fos-positive nuclei in the NACc core are consistent with data from several studies using cocaine-trained rats [
Despite knowledge of neuronal activation in the NACc core underlying EA’s effectiveness in discrete cue-elicited drug seeking, little is known about neurobiological mechanisms by which EA stimulation exerts a positive influence on drug-seeking behavior. It is possible that acupuncture reduces discrete cue-induced drug-seeking behavior by modulating dopamine release or activation of postsynaptic dopamine receptors in the NACc core. Kim et al. [
With regard to Experimental protocols in the present study, there are some variable factors that need to be taken into account. Firstly, it is still controversial as to how to set suitable control group for acupuncture or EA. In order to exclude interference from restraint stress, we run restraint group as a control. However, the perfect control, of course, is one in which needles are inserted into acupuncture points but without electric stimulation. Secondly, in our experimental protocol, the animals tested contextual cue-induce reinstatement firstly, and then tested discrete cue-induced reinstatement. This raises the possibility that contextual cue itself might influence discrete cue-induced heroin seeking. In fact, during contextual cue phase, the rats were allowed to respond to the nose-pokes with all the conditioned stimulus lights kept off. The responses were recorded, but no consequences were produced. This phase was used to measure contextual cue- (chamber environment) induced heroin-seeking and was generally regarded as an extinction phase [
EA treatment significantly reduced discrete cue-induced heroin seeking behavior in reinstatement of self-administration procedures. Fos protein expression consistent with conditioned enhancement by discrete cue stimuli was observed in the NACc core. 2 Hz EA stimulation attenuated Fos expression in the core but not the shell of the NACc. Altogether, these results support the hypothesis that EA can reduce drug-seeking and highlight the therapeutic benefit of EA in preventing relapse to drug addiction.
The authors do not have any conflicts of interest or any circumstances that could be perceived as a potential conflict of interest.
This work was supported by National Basic Research Program of China (2009CB522008), Nature Science Foundation of China (30870824; 81071077), Zhejiang TCM Technology R&D Program (2008CA102), and Ningbo Natural Science Foundation (2010A610042).