Differential effects on natural reward processing in rats ... - Springer Link

Psychopharmacology (2013) 229:125–132 DOI 10.1007/s00213-013-3087-8

ORIGINAL INVESTIGATION

Differential effects on natural reward processing in rats after repeated heroin Ewa Galaj & Ivonne Cruz & Jordan Schachar & Matthew Koziolek & Robert Ranaldi

Received: 28 November 2012 / Accepted: 25 March 2013 / Published online: 23 April 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Rationale Heroin users report reward deficits as well as reward enhancements (to drug stimuli). To better understand the causal relation between chronic heroin and alterations in natural reward processing, we used experimental techniques in animal models. Methods Separate groups of rats were trained in several food reward paradigms: conditioned place preference (CPP), food-reinforced lever pressing under a progressive ratio schedule of reinforcement, free feeding, and lever pressing with conditioned reinforcement. After training, the rats were subjected to 10 daily heroin (2 mg/kg) or saline vehicle injections and tested at 3, 15, and 30 days post-treatment. Results Repeated heroin treatment abolished the CPP and significantly reduced break points for food reward at 3, 15, and 30 days post-treatment. Repeated heroin did not affect free feeding. Finally, repeated heroin significantly enhanced responding for a food-based conditioned reinforcer. Conclusions Repeated heroin decreases the attractiveness of food-associated cues and reduces motivation to work for natural reward. However, it appears to enhance natural conditioned reward processes that involve the acquisition of novel responding. Thus, repeated heroin appears to produce differential effects on natural reward processing depending on the nature of the reward-directed behavior. Keywords Behavioral sensitization . Heroin . Anhedonia . Motivation . Natural reward . Conditioned reinforcement E. Galaj : R. Ranaldi The Graduate Center, City University of New York, New York, NY, USA I. Cruz : J. Schachar : M. Koziolek : R. Ranaldi (*) Department of Psychology, Queens College, 65-30 Kissena Blvd, Flushing, NY 11367, USA e-mail: [email protected]

Research on the effects of heroin use reveals differential responses to reward stimuli. Some studies demonstrate reward enhancements in the form of greater neural and behavioral responsiveness to drug-related stimuli (Martin-Soelch et al. 2001; Sell et al. 1999; Zijlstra et al. 2009). Other studies indicate reduced responsiveness to pleasant, nondrug-related stimuli (i.e., anhedonia) (Lubman et al. 2009) as well as reduced activity in reward circuits (Sell et al. 1999; Zijlstra et al. 2009). This differential response pattern is not well understood and important questions remain. First, because in human studies heroin use is a subject variable, it is not clear if the reward deficits are a cause or consequence of repeated drug use. Second, whether or not the reduced hedonic processing experienced by people who have used heroin is derived from, or associated with, loss of motivation for natural reward has not been addressed. Third, given that the mesolimbic dopamine (DA) system is thought to be common to both drug and natural rewards (Wise 2004; Wise and Rompre 1989), it is not clear if sensitization of this system may also enhance appetitive processing of natural rewards. The present studies used animal models to address these questions. Some animal research indicates that repeated opiate injections enhance natural rewards such as social and sexual incentive stimuli (Nocjar and Panksepp 2007). Furthermore, repeated opiate injections enhance operant responding reinforced with food-associated stimuli (Morrison et al. 2011; Ranaldi et al. 2009) as well as with food reward itself (Babbini et al. 1976; Cooper et al. 2010; Ford and Balster 1976; Parker et al. 1973) with enhanced responding starting 1 day after treatment (Cooper et al. 2010) or only after 7 days after treatment (Parker et al. 1973). On the other hand, chronic opiates can also result in reward deficits during acute and protracted withdrawal. For example, rats exposed to repeated morphine injections showed decreased saccharine preference (up to 6 days post-treatment) (Lieblich et al. 1991; Parker et al. 1973) and reduced

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responding for saccharine (up to 10 days after termination of treatment) (Zhang et al. 2007). They also demonstrate elevated brain stimulation reward thresholds (Kenny et al. 2006; Liu and Schulteis 2004), suggesting reduced responsiveness of the brain’s reward system (Wise 1996). Comparison of the effects of chronic opiates on reward across studies is difficult because different procedures (e.g., conditioned place preference (CPP), self-administration), training (e.g., doses, routes of administration), and testing parameters (e.g., different lengths of withdrawal) were used. Thus, it is important to test the effects of repeated heroin on different reward-related tasks using similar training and testing parameters. In the present studies, we tested the hypothesis that repeated heroin intake reduces hedonic processing of food reward, as expressed by a reduction in the expression of a food-conditioned place preference. Others have investigated the effect of chronic morphine on food CPP and found a reduction in preference in morphine-treated animals (Harris and Aston-Jones 2003, 2007). However, in those studies, the morphine treatment occurred before conditioning, so it is not clear if it affected the animals’ capacity to experience the food-paired side as rewarding or if it reduced appetite, food reward, or associative learning during conditioning. This is a critical distinction. Of particular interest is whether or not repeated opiates reduce the capacity to experience reward in already established rewards. In the present set of experiments, we addressed this question by administering the intermittent heroin treatment after conditioning. Although some studies indicate repeated opiate intake produces reward deficits, it is not clear if these deficits derive from, or are associated with, reduced motivation. Here, we investigated the effects of repeated heroin on responding for food reward under a progressive ratio (PR) schedule of reinforcement, a procedure that measures motivation. We hypothesized that repeated heroin would significantly reduce motivation to respond for food reward at different post-treatment periods, at 3, 15, and 30 days post-treatment. Moreover, to better understand the effects of repeated heroin on food motivation, we also investigated its effects on free feeding. Finally, we were also interested in the effects of repeated heroin on incentive motivation. Thus, we tested the effects of repeated heroin on learning to press a lever reinforced by a food-associated stimulus. The results of this experiment serve as an important comparison to those of the CPP study. In both procedures, a neutral stimulus was converted to a conditioned stimulus (CS) through classical conditioning with food, but the two paradigms emphasize different types of behavioral control by these CSs: in the CPP paradigm, the ability of a CS (food-paired environment) to elicit approach, and in conditioned reinforcement, its ability to reinforce behavior. Furthermore, in the CPP paradigm, we investigate

Psychopharmacology (2013) 229:125–132

the expression of an already learned association (food and place) while in the conditioned reinforcement paradigm, we investigate the acquisition of a new response, pressing a lever reinforced by a food-paired stimulus. In the present study, we investigated the effects of repeated heroin injections that lead to behavioral sensitization on natural reward processing using different reward tasks. The goal was to better understand the effects of repeated heroin injections on conditioned and unconditioned stimuli and responding. These studies were not an attempt to characterize the effects of chronic heroin in an animal model of heroin addiction or in rats that are physically dependent on heroin but instead to better understand the changes in natural reward processing in animals that are behaviorally sensitized to heroin.

Methods All protocols followed the NIH Guide for Care and Use of Laboratory Animals and were approved by the Queens College Institutional Animal Care and Use Committee. Subjects Subjects were male Long Evans rats taken from our inhouse colony. They were housed individually on a reversed 12-h light:12-h dark cycle (lights on at 6 pm). Except for the CPP study, all rats had restricted access to food to maintain their weights at 85 % of their free feeding values and unlimited access to water. The CPP rats had access to food and water at all times except on conditioning and test days, when food was not available for 4 h prior to placement in conditioning chambers. All procedures were conducted during the dark cycle. Apparatus Operant conditioning chambers Details of these chambers can be found in Morrison et al. (2011) with the addition of a 1.5-kHz tone generator directly above the food trough. For free feeding sessions, the levers were removed and a mesh floor was added. Conditioned place preference chambers These were twocompartment chambers placed in sound-attenuating ventilated boxes. Each chamber measuring 43×43×30 cm was equipped with 16 photo-emitters positioned evenly along the length of the chamber 6 cm above the floor, each paired directly opposite a photocell. These photocells detected the position of the rats in one compartment or the other. Each compartment had a distinct combination of walls (striped or nonstriped) and floor (grid or rods), and their common wall


had an opening that could be closed by the placement of a guillotine door. Locomotor activity chambers Locomotor activity was measured in the same chambers used in the CPP study but without the two-compartment inserts. Locomotor activity counts were registered by photocells when adjacent beams were broken consecutively. Drug Heroin (a gift from the National Institute on Drug Abuse, Bethesda, MD) was dissolved in 0.9 % saline to achieve a dose of 2 mg/kg and injected in volumes of 1 ml/kg. Foods The foods used contained the following carbohydrate, protein, and fat percentages, respectively: rat chow, (LabDiet) 57.9, 28.5, and 13.5; BioServ food pellets, 59.1, 18.7, and 5.6; and Lucky Charms cereal, 84.8, 7.5, and 7.7. Procedure Experiment 1: conditioned place preference All rats were acclimated to Lucky Charms cereal by placing a bowl of it in their home cages on each of three consecutive days before the experiment. The CPP procedure consisted of one 15-min pre-exposure session, eight 30-min conditioning sessions, and one 15-min test session, each held on consecutive days. Prior to the pre-exposure session, animals were placed directly in the open doorway of the apparatus and allowed to freely explore the two compartments. The time spent in each compartment was measured. During the conditioning sessions, the doorway was closed; for four sessions, each rat was placed in one of these compartments with a bowl of food reward, and for the other four sessions, the rats were placed in the opposite compartment without food reward. For half the rats, the reward sessions were scheduled on conditioning days 1, 3, 5, and 7, for the other half, in the opposite manner. Also, for half the rats, the reward was paired with the preferred and for the other half with the nonpreferred compartment. Starting the day after conditioning, all rats were exposed to the treatment regimen. Assignment to the saline or heroin group was based on ranked magnitude of side preference determined during pre-exposure. On each of 10 consecutive days, the rats received intraperitoneal (IP) heroin (2 mg/kg) or saline injections in their home cages. Three days following the last injection, animals were placed directly in the open doorway and allowed to freely explore the compartments for 15 min. The time spent in each compartment was measured.

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Experiment 2: food-reinforced lever pressing This experiment consisted of three phases: training, treatment, and testing during three post-treatment periods: 3–5, 15–17, and 30–32 days post-injection. Training phase: All initial sessions were 10 min long and held daily. Pressing one lever delivered one food pellet (45 mg, Bioserv) and illuminated the light above that lever for 3 s. Pressing the other lever had no programmed consequences. After five consecutive sessions where the total number of rewards earned per session was greater than 100, the schedule was changed to a progressive ratio (PR) schedule of reinforcement where the number of active lever presses required for subsequent rewards increased as follows: 1, 2, 4, 6, 9, 12, 15, 20, and so on. All PR sessions were 90 min long. Break point (BP) was operationally defined as the total number of rewards earned prior to a 20-min period during which no rewards were obtained. This continued until the rats demonstrated stable BPs, operationally defined as three consecutive BPs that did not differ by more than 2 ratio steps and did not show ascending or descending trends. Treatment phase: After animals met the criteria for stable BP, they were injected with either saline or heroin (2 mg/kg) IP for 10 consecutive days. Testing phase: At 3, 15, and 30 days from the last injection, all animals were tested under the PR schedule of reinforcement for three sessions, each held on consecutive days. Experiment 3: free feeding Animals were placed in chambers loaded with 25–30 g of rat chow on a mesh grid floor for 60 min on three consecutive days. After each session, the rat chow remaining on the mesh grid floor and in a collection tray beneath it was weighed and subtracted from the original weight to determine the amount consumed. After the third session, rats were assigned to either saline or heroin treatment conditions based on ranked average chow consumption. Animals were injected with either saline or heroin (2 mg/kg, IP) for 10 consecutive days. Then they were tested for food consumption same as previously during three post-treatment test periods (3, 15, and 30 days post-injection). Each test period consisted of three test sessions, each held on consecutive days. Experiment 4: conditioned reinforcement This procedure consisted of four phases (see Morrison et al. 2011; Ranaldi et al. 2009 for details). Briefly, levers, one producing a light and the other a tone (both 3 s), were present only during the pre-exposure and test phases, and during conditioning, the light stimulus was paired with food. The treatment regimen (saline or heroin) was administered between the conditioning and test phases. Experiment 5: locomotor activity effects of repeated heroin injections All rats were administered an injection of saline or heroin and placed immediately into locomotor activity

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chambers for 30 min on each of 10 consecutive days. For the following 29 days, all rats did not receive injections or placement in activity monitors. On the 30th day following the previous treatment injection, all rats received a challenge injection of heroin (2 mg/kg) and were placed in activity monitors for 30 min. Data analysis For the CPP experiment, the number of seconds spent in the food-paired compartment during the test session was compared to the number of seconds spent in that compartment during the pre-exposure session for each rat. For the foodreinforced lever pressing experiments, BPs during each test session were expressed as a percentage of the mean of the last three baseline BPs, and these values were analyzed. For the free feeding experiments, the amount of food consumed during each test day was expressed as a percentage of the mean of the food eaten during the three baseline sessions, and these values were analyzed. For the conditioned reinforcement experiments, the number of responses made on each lever during the pre-exposure sessions was averaged, and the number of responses on each lever during the test sessions was averaged for each rat. These average values were analyzed. For the locomotor activity study, the number of locomotor activity counts per 5 min during each session was analyzed. All statistical analyses consisted of analyses of variance (ANOVAs): for the CPP experiment, a two-way ANOVA (treatment × phase); for the food-reinforced lever pressing and free feeding experiments, separate three-way ANOVAs (treatment × post-treatment period × session); for the conditioned reinforcement study, a three-way ANOVA (treatment × phase × lever); for the locomotor activity study, a three-way ANOVA comparing data from days 1 to 10 and a three-way ANOVA comparing data from day 1 and the challenge session (both treatments × day × time). Significant interactions were followed by tests of simple effects.

Results Experiment 1: conditioned place preference The saline group spent significantly more time in the food-paired compartment during the test session than during the preexposure session, but the heroin group spent the same amount of time in the food-paired compartment during both sessions (see Fig. 1). A two-way ANOVA revealed a significant phase by group interaction [F1, 14 =7.7, p