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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Psychopharmacology (Berl). 2013 May 29;230(1):137–147. doi: 10.1007/s00213-013-3144-3

The effects of amphetamine sensitization on conditioned inhibition during a Pavlovian-instrumental transfer task in rats

Michael W Shiflett 1, Meaghan Riccie 1, RoseMarie DiMatteo 1
PMCID: PMC3797263  NIHMSID: NIHMS486273  PMID: 23715640

Abstract

Rationale

Psychostimulant sensitization heightens behavioral and motivational responses to reward-associated stimuli; however, its effects on stimuli associated with reward absence are less understood.

Objectives

We examined whether amphetamine sensitization alters performance during Pavlovian-instrumental transfer (PIT) to conditioned excitors and inhibitors. We further sought to characterize the effects of amphetamine sensitization on learning versus performance by exposing rats to amphetamine prior to Pavlovian training or between training and test.

Methods

Adult male Long Evans rats were given conditioned inhibition (A+/AX−) and Pavlovian (B+) training, followed by variable-interval instrumental conditioning. Rats were sensitized to d-amphetamine (2 mg/kg daily injections for seven days), or served as non-exposed controls. Rats were given a PIT test, in which they were presented with stimulus B alone or in compound with the conditioned inhibitor (BX).

Results

During the PIT test, control rats significantly reduced instrumental responding on BX trials (to approximately 50% of responding to B). Amphetamine sensitization prior to Pavlovian conditioning increased lever-pressing on BX trials and reduced lever-pressing on B trials compared to controls. Amphetamine sensitization between training and test increased lever-pressing on B and BX trials compared to controls. No effects of sensitization were observed on conditioned food-cup approach.

Conclusions

Amphetamine sensitization increases instrumental responding during PIT to a conditioned inhibitor, by enhancing excitation of conditioned stimuli and reducing inhibition of conditioned inhibitors.

Keywords: psychostimulant, instrumental, Pavlovian, incentive, reward, learning

Introduction

Stimuli present in the environment have a strong influence on reward-seeking behavior, by affecting the choice and vigor with which animals pursue resources. In an experimental setting, a Pavlovian-instrumental transfer (PIT) paradigm clearly demonstrates the motivational effects that stimuli wield on an animal’s reward-seeking behavior. PIT measures the effects of conditioned stimulus (CS) presentations on ongoing instrumental action. During tests of PIT in humans and laboratory animals, CS’s associated with rewarding outcomes invigorate reward-seeking instrumental responses (Bray et al. 2008; Colwill and Rescorla 1988; Corbit et al. 2007; Edgar et al. 1981; Holland 2004; Lovibond 1981; Nadler et al. 2011; Talmi et al. 2008). Response invigoration during PIT provides a measure of incentive motivation, or the stimuli’s ability to engender “wanting” for a particular outcome (Berridge 2009; Berridge et al. 2009; Holmes et al. 2010). Furthermore, PIT illustrates how drugs of abuse influence incentive motivation: repeated exposure to psychostimulants elevates PIT (i.e., greater instrumental responding during CS presentation) (Saddoris et al. 2011; Shiflett 2012; Smith et al. 2011; Tindell et al. 2005; Wyvell and Berridge 2001).

Understanding the behavioral and neural basis for PIT is important for devising ways to control detrimental responses to stimuli, such as craving and relapse caused by drug-associated cues (Childress et al. 2002; Epstein et al. 2010; O’Brien et al. 1998; Robinson and Berridge 1993). One way of reducing responses to conditioned stimuli comes from training in a conditioned inhibition paradigm. During a conditioned inhibition paradigm, a stimulus A is consistently associated with reward delivery, whereas when A is presented with a second stimulus (X), the compound (AX) is unrewarded. As a result of such training, the stimulus X becomes a conditioned inhibitor (Rescorla 1969). Evidence of its inhibitory properties is shown in a summation test, during which X is paired with a novel conditioned excitor (B), and this pairing (BX) dampens responses to B relative to presentations of B alone (Rescorla 1969; Rescorla and Holland 1977).

The effects of psychostimulant sensitization on conditioned inhibition remain to be fully understood. Incentive-sensitization models predict that sensitized animals should be overwhelmed by “wanting” elicited by conditioned excitors, and would therefore be less susceptible to the effects of conditioned inhibitors. However, psychostimulant sensitization tends to increase the effectiveness of conditioned inhibitors on approach behavior (Harmer and Phillips 1999). One explanation for this finding is based on the different effects on behavior of psychostimulants depending on the timing of drug exposure. Psychostimulants administered prior to or immediately following Pavlovian conditioning sessions are known to enhance the acquisition of conditioned approach and disrupt expression of PIT (Blaiss and Janak 2007; Hall and Gulley 2010; Simon et al. 2009; Taylor and Jentsch 2001) whereas amphetamine administered between training and test elevates PIT (Saddoris et al. 2011; Shiflett 2012; Smith et al. 2011; Tindell et al. 2005; Wyvell and Berridge 2001).

The purpose of this study was to examine the effects of amphetamine sensitization on conditioned inhibition using PIT as a summation test. We further sought to characterize the effects of amphetamine sensitization on learning versus performance by exposing rats to amphetamine prior to Pavlovian training or between training and test. To this end, we trained rats in a conditioned inhibition (A+/AX−) paradigm. As a control, we trained a second group of rats in a differential conditioning (A+/X−) paradigm. We exposed some rats to a sensitizing regimen of amphetamine, either prior to Pavlovian training (Experiment 1) or between training and test (Experiment 2). We then administered a PIT test, in which we presented rats with a second conditioned excitor (B) alone or in compound with the conditioned inhibitor (BX). Following the PIT test we assayed rats for locomotor sensitization to amphetamine.

Experiment 1: the effects of pre-training amphetamine exposure on conditioned inhibition during PIT

Materials and methods

Subjects and Apparatus

Subjects were 48 adult male Long Evans Blue Spruce rats from Harlan Laboratories (Indianapolis IN, USA). Rats arrived weighing approximately 225–250 g, and were 58–64 d old. Rats were housed in pairs in 47.6 cm × 20.3 cm × 26 cm (w/h/d) polycarbonate containers with Alpha Chip bedding material (Northeastern Products Corp, Warrensburg NY) and had free access to water. One week after arrival, rats were placed on a restricted food diet of approximately 20 g of standard rat pellets per day (Purina, St. Louis MO, USA). Rats were fed after their daily behavioral training session. Food restriction continued for the duration of the experiment. All procedures were approved by the Rutgers University Institutional Animal Care and Use Committee.

Behavioral training and testing took place in twelve identical rat operant conditioning chambers (Med Associates, St. Albans VT, USA). Each operant conditioning chamber measured 30.5 cm × 24.1 cm × 21 cm (w/h/d) and was constructed of stainless steel and clear plastic walls and a stainless steel grid floor. A food cup with infrared detectors was centered on one wall of the operant conditioning chamber. Retractable levers were situated to the left and right of the food cup. Responses on one lever delivered a single 45-mg grain-based food pellet (Bio-serv, Frenchtown NJ USA) from a dispenser mounted outside the operant conditioning chamber. Three audio generators were located in the operant conditioning chambers: a Sonalert™, a clicker module and a white-noise generator. Each generator produced sounds at approximately 80 db. A 28 V light was located on the opposite wall from the food cup and illuminated the operant conditioning chamber during behavioral procedures. Each operant conditioning chamber was housed in a sound-attenuating shell and equipped with a ventilation fan that was activated during behavioral procedures. Control over the operant conditioning chambers was enabled by a personal computer operating through an interface. Operant conditioning chamber operation and data collection were carried out with Med Associates proprietary software (Med-PC).

Experiment 1 timeline

A timeline of behavioral procedures is depicted in Table 1. The reinforcement frequency during instrumental and Pavlovian conditioning, and the number and spacing of trials during training were designed to maximize the likelihood of observing PIT. Behavioral procedures commenced after one week of food restriction. We provided rats with two 30-min sessions to habituate to the operant conditioning chamber, after which they began instrumental training sessions. Following 8 days of instrumental training, rats were given daily injections of amphetamine, or saline for control subjects. Rats began Pavlovian training six days following the final amphetamine injection. Half the rats were trained using a conditioned inhibition procedure. The remaining rats were trained using a differential conditioning procedure. Once this training was complete, rats performed reminder instrumental sessions and a PIT test. The stimuli used as conditioned excitors were counterbalanced across drug and training groups. The clicker module was always used as stimulus X.

Table 1. Experiment 1 timeline of events.

Timeline of behavioral procedures in Experiment 1. During instrumental training rats pressed a lever to obtain a food outcome (R-O). Following amphetamine exposure, rats underwent Pavlovian conditioning with a conditioned excitor (A+), followed by conditioned inhibition training (A+/AX−) or differential conditioning (A+/X−). During the PIT test, instrumental responses (R) were measured during presentation of a second excitor (B) alone or paired with the conditioned inhibitor (BX).

Treatment Inst. cond. Drug Treatment Pav. cond. phase I Conditioned inhibition/differential conditioning Pav. cond. phase II Reminder Inst. session PIT test
Conditioned inhibition + amph VI 7.5–15 Amph A+ A+/AX− B+ VI-15 B, BX:R
Differential conditioning + amph VI 7.5–15 Amph A+ A+/X− B+ VI-15 B, BX:R
Conditioned inhibition + saline VI 7.5–15 Saline A+ A+/AX− B+ VI-15 B, BX:R
Differential conditioning + saline VI 7.5–15 Saline A+ A+/X− B+ VI-15 B, BX:R
Day 1–8 9–19 20–21 22–27 28–29 30 31
Trials per day/duration 20 outcomes/day or 25 min once/day for 7 days 6 trials/day; 30 min 12 trials/day; 60 min 6 trials/day; 30 min 20 outcomes or 25 min
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Instrumental conditioning

A single lever was inserted into the operant conditioning chamber and responses on the lever delivered a food pellet. Rats received a total of 8 daily training sessions. For the first two sessions each response the rat made resulted in pellet delivery. For the remaining sessions outcomes were delivered according to a variable-interval (VI) schedule, in which outcomes were delivered on the first response rats made after an interval had elapsed since the previous outcome delivery. The interval used for session 3 averaged 7.5 sec, after which rats were shifted to a VI-15 sec schedule for the remaining 5 sessions.

Amphetamine sensitization

Rats were divided into two groups of 24 animals. Rats assigned to the amphetamine group received an intraperitoneal injection of d-amphetamine (Sigma, St. Louis MO, USA) daily for 7 days at a dose of 2 mg/kg body weight, diluted in 0.8% saline to 2 mg/ml. Control rats received the same volume of saline injections. Rats were returned to their home cage after each injection.

Pavlovian conditioning phase I

Rats were trained to associate a 120-sec auditory stimulus (stimulus A Table 1) with delivery of grain pellets. During the training session, the stimulus was presented six times per session with an inter-stimulus interval that averaged 3 min (range 2–4 min). During stimulus presentation, food pellets were dispensed according to the following probability: for each second during presentation there was a probability of p = 0.06 of pellet delivery, which on average resulted in 7.2 pellets delivered per stimulus presentation. Rats received one training session per day for 2 d.

For stimuli paired with food delivery (A+, B+) we recorded head entries during a probe trial, which was identical to the other training trials involving these stimuli except that no food pellets were delivered during the first 30 sec of the trial. One probe trial occurred for each training session.

Conditioned inhibition training

During conditioned inhibition training, rats were presented with a compound stimulus consisting of stimulus A and a second auditory stimulus (the conditioned inhibitor; stimulus X Table 1). Both stimuli were presented in tandem (AX) for 120 s, during which no pellets were delivered. In each training session, rats received 8 trials with the compound stimulus and 4 trials with stimulus A. Trials involving A alone were coupled with pellet delivery using the same reward probabilities as during Pavlovian training. Rats received a total of 6 conditioned inhibition training sessions.

Differential conditioning

Rats in the differential conditioning group were presented separately with stimulus A and stimulus X. In each training session, rats received 8 trials with presentation of stimulus X and 4 trials with stimulus A. Trials involving stimulus A were coupled with pellet delivery using the same reward probabilities as during Pavlovian training. Presentation of stimulus X was never paired with food delivery. Rats received a total of 6 differential conditioning sessions.

Pavlovian conditioning phase II

Rats were trained to associate a second auditory stimulus (stimulus B Table 1) with grain pellet delivery. As with initial Pavlovian conditioning, rats received 6 pairings of stimulus B with grain pellet delivery. Rats received 2 training sessions.

Pavlovian-instrumental transfer (PIT) test

The PIT test was conducted in extinction, such that lever pressing and stimulus presentation produced no outcomes. During the test, the trained lever was inserted into the operant conditioning chamber. Stimulus B was presented, as were compound presentations of B and X (BX). Rats experienced a total of 6 trials, 3 with B and 3 with BX in pseudorandom order. A 3-min interval separated each trial. Prior to the first trial, rats underwent 10 min of instrumental extinction. Responses on the lever were recorded during each trial and during the 2 min preceding each trial (baseline).

Results

Instrumental training

During instrumental training, which occurred prior to drug exposure, all rats acquired an instrumental response: during the final day of training rats made 12.06 ± 5.06 (SD) responses per min. There was no difference in response rates between groups prior to receiving amphetamine or saline (P = 0.37).

Pavlovian conditioning phase I

Rats acquired a conditioned approach response. In the final training session, rats made significantly more head insertions during the CS probe trial compared to an equivalent interval preceding stimulus onset (ANOVA: F 1, 46 = 5.91, P < 0.05). No effect of drug on approach behavior was observed (P = 0.65).

Conditioned inhibition/Differential conditioning

Rats received 6 sessions of conditioned inhibition (A+/AX−) or differential conditioning (A+/X−). For the purposes of statistical analysis these sessions were grouped into three 2-session blocks. In both forms of conditioning, rats made significantly more head insertions to A alone compared to AX, X or the inter-stimulus interval (ANOVA: F 2, 88 = 80.43, P < 0.01) (Figure 1A–B). Importantly, there was no significant effect of amphetamine exposure on the rate of head insertions made during training, nor was there any significant interaction involving drug as a factor (all P’s > 0.25).

Fig. 1. Experiment 1: Pavlovian and instrumental conditioning.

Fig. 1

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(A) Approach during conditioned inhibition training across 3 trial blocks. A+ indicates approach during trials in which stimulus A was presented alone; AX− indicates approach during trials in which stimulus A was presented in tandem with stimulus X. (B) Approach during differential conditioning. (C) Approach during Pavlovian training for B+. (D) Instrumental response rates during the reminder instrumental session. * = significantly different at P < 0.01.

Pavlovian conditioning phase II

During B+ training, rats made significantly more head insertions during B compared to baseline (ANOVA: F 1, 46 = 109.25, P < 0.01) (Figure 1C). We observed no effect of prior amphetamine exposure on approach to B, or any interaction involving this factor (all P’s > 0.4).

Instrumental reminder

A comparison of mean response rates during the reminder session following drug treatment shows that rats that received amphetamine responded at a significantly lower rate than their saline counterparts (independent samples t-test, t 46 = 2.92, P < 0.01) (Figure 1D).

Instrumental extinction

Extinction of instrumental responding prior to the first stimulus onset during the PIT test was analyzed and is depicted in Figure 2A. Rats significantly reduced their response rate over the course of the ten-minute extinction period (ANOVA F 9, 414 = 20.57, P < 0.01). Importantly, amphetamine-exposed rats made significantly fewer responses during extinction compared to their non-exposed counterparts (ANOVA: F 1, 46 = 9.13, P < 0.01). However, this effect did not persist during the inter-stimulus interval, with no difference observed between amphetamine-exposed and control rats in baseline response rates (independent-samples t-test: t 46 = 1.34, P = 0.18) (Figure 2A).

Fig. 2. Experiment 1: Instrumental responses during PIT.

Fig. 2

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(A) Lever presses during the 10-min extinction period prior to the first stimulus presentation during PIT. Right bars indicate average baseline (inter-stimulus interval) response rates during the PIT test. (B) Lever presses during the PIT test minus baseline in rats trained under conditioned inhibition (left column) or differential conditioning (right column). * = significantly different at P < 0.05.

Pavlovian-instrumental transfer

Amphetamine sensitization altered performance during the PIT test. Instrumental responses during stimulus B and BX were subtracted from responses during the inter-stimulus interval. These data were analyzed with a three-factor ANOVA, with stimulus as a within-subjects factor and drug (amphetamine or saline) and training group (differential conditioning or conditioned inhibition) as between-subjects factors. This analysis revealed a significant stimulus by drug interaction (ANOVA: F 1, 44 = 9.15, P < 0.01) and a three-way interaction that approached significance (ANOVA: F 1, 44 = 3.15, P = 0.08) (Figure 2B). A comparison of means revealed significantly greater instrumental responses to B compared to BX among saline-treated animals trained under conditioned inhibition (paired samples t-test, t 11 = 2.3, P < 0.05). In contrast, rats treated with amphetamine made significantly more instrumental responses to BX compared to B (paired samples t-test, t 11 = 3.6, P < 0.01). No differences in instrumental response rates were observed between BX and B in rats trained under differential conditioning (all P’s > 0.5)Approach behavior during the PIT test was analyzed and is depicted in Figure 3. An ANOVA was carried out on approach responses during each stimulus minus responses during the inter-stimulus interval. Stimulus (B, BX), training group (conditioned inhibition or differential conditioning) and drug (amphetamine or saline) were included as factors. There was a significant effect of stimulus (ANOVA: F 1, 44 = 73.72, P < 0.01) and a significant stimulus by training group interaction (ANOVA: F 1, 44 = 6.87, P = 0.01). No effect of amphetamine was observed nor any interaction involving this factor (all P’s > 0.25). Post-hoc comparison of group means show that all groups made fewer head insertions during BX compared to B (paired samples t-test, P’s < 0.05, Bonferroni correction for multiple comparisons).

Figure 3. Experiment 1: Approach during PIT.

Figure 3

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Food cup head insertions during the PIT test. * = significantly different at P < 0.05.

Experiment 2: the effects of post-training amphetamine exposure on conditioned inhibition during PIT

Materials and Methods

Subjects and Apparatus

Subjects were 24 adult male Long Evans Blue Spruce rats from Harlan Laboratories. Rats arrived weighing approximately 225–250 g, and were 58–64 d old. Housing and diet conditions were identical to Experiment 1. Rats training was conducted in the same operant conditioning chambers as were used in Experiment 1.

Experiment 2 timeline

A timeline of behavioral procedures is depicted in Table 2. Pavlovian and instrumental conditioning procedures were identical to Experiment 1; however, we did not include a differential conditioning group in Experiment 2. Unlike Experiment 1, we did not include a probe trial during Pavlovian conditioning in Experiment 2. The number of head entries that rats made was measured during the entire stimulus presentation. For food-paired stimuli (A+, B+) head insertions were not counted for 5 sec after each pellet delivery to minimize the effects of food pellet retrieval on head insertion counts.

Table 2. Experiment 2 timeline of events.

Timeline of behavioral procedures in Experiment 2. See Table 1 caption for details.

Treatment Pav. cond. phase I Conditioned inhibition Inst. cond. Pav. cond. phase II Drug treatment Reminder sessions PIT test
Conditioned inhibition + amph A+ A+/AX− VI 7.5–15 B+ Amph B+; VI-15 B, BX:R
Conditioned inhibition + saline A+ A+/AX− VI 7.5–15 B+ Saline B+; VI-15 B, BX:R
Day 1–2 3–8 9–16 17–18 19–39 40–41 42
Trials per day/time 6 trials/day; 30 min 12 trials/day; 60 min 20 outcomes/day or 25 min 6 trials/day; 30 min once/day for 7 days 6/30 min; 20 outcomes or 25 min
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Amphetamine administration

Amphetamine source, dose and method of administration were identical to Experiment 1. The timing of drug exposure relative to behavioral procedures differed slightly from Experiment 1. In Experiment 1, behavioral procedures commenced 4 days after the final amphetamine injection. In Experiment 2, behavioral procedures commenced 14 days after the final amphetamine injection. This discrepancy was meant to keep the timing of the PIT task the same relative to drug exposure for the two experiments. In Experiment 1, the PIT test occurred 16 days after the final amphetamine injection; In Experiment 2, the PIT test occurred 17 days after the final amphetamine injection.

Results

Two rats were excluded from the study because of adverse reaction to injections (prolonged distress and irritability). A total of 22 rats were included in the study and their data are presented below.

Pavlovian conditioning phase I

Rats acquired a conditioned approach response. In the final training session, rats made significantly more head insertions during presentations of stimulus A compared to an equivalent interval preceding the stimulus (paired t-test: t21 = 9.40, P < 0.01).

Conditioned inhibition

Rats made significantly more head insertions during A compared to AX and baseline. A main effect of stimulus was observed (F2, 84 = 86.44 P < 0.01), as were significant effects comparing A to AX approach on the third training block (paired t-test t 21 = 9.42, P < 0.01) (Figure 4A). However, head insertions during AX were greater than baseline head insertions on the third training block as well (paired t-test: t 21 = 4.2, P < 0.01). These data must be interpreted with caution, because the measure of approach in this experiment differs from Experiment 1. Namely, approach scores were collected during the entire stimulus duration, except for 5-sec intervals following pellet delivery. Therefore some approach behavior may be attributable to the presence of the food pellet and are therefore not fully reflective of conditioned approach.

Figure 4. Experiment 2: Pavlovian and instrumental conditioning.

Figure 4

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(A) Food cup approach during 3 conditioned inhibition training blocks prior to drug exposure. (B) Food cup approach to stimulus B prior to and following amphetamine sensitization. (C) Lever presses during the instrumental reminder session following amphetamine sensitization. * = significantly different at P < 0.01.

Instrumental conditioning

Rats acquired an instrumental response. The mean instrumental response rate on the final day of training prior to drug treatment was 17.46 ± 5.6 (SD) responses per min.

Pavlovian conditioning phase II

Rats acquired an approach response to stimulus B. On the final training session, rats approached the food cup significantly more often during stimulus B compared to baseline (ANOVA: F 1, 21 = 42,81, P < 0.01).

Pavlovian conditioning reminder session

Amphetamine sensitization significantly altered Pavlovian approach during the reminder training session. Approach behavior was analyzed using a three-factor ANOVA, with sensitization drug (amphetamine or saline) as a between-subjects factor, and stimulus (baseline or stimulus B) and exposure (pre-exposure or post-exposure) as within-subject factors (Figure 4B). Significant main effects of stimulus (F1, 20 = 78.42, P < 0.01) and of exposure (F 1, 20 = 28.02 P < 0.01) were observed, as well as a significant interaction involving these factors (F 1, 20 = 14.31, P < 0.01). No main effect or two-way interactions involving drug were observed. A significant three-way interaction involving drug, stimulus and exposure was observed (F1, 20 = 4.96, P < 0.05). During the post-treatment reminder session, amphetamine-exposed rats trended toward greater approach during presentations of stimulus B compared to non-exposed rats, after subtracting baseline (independent samples t-test, t 20 = 1.94, P = 0.067). No such tendency was observed in the pre-treatment training session (P = 0.95).

Instrumental reminder session

The effect of amphetamine sensitization on instrumental response rates was assessed by examining response rates during a reminder training session following drug treatment. Amphetamine sensitization had no effect on instrumental performance. An independent samples t-test revealed no effect of drug on response rates during the reminder session (t 20 = 0.37, P = 0.71) (Figure 4C).

Instrumental extinction

Extinction of instrumental responding prior the first stimulus onset during the PIT test was analyzed and is depicted in Figure 5A. Rats significantly reduced their response rate over the course of the ten-minute extinction period (ANOVA F 9, 180 = 27.10, P < 0.01); however, no main effect of amphetamine on response rates or interaction with the time- course of extinction was observed (ANOVA: all P’s > 0.3).

Fig. 5. Experiment 2: Instrumental responses during PIT.

Fig. 5

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(A) Lever presses during the 10-min extinction period prior to the first stimulus presentation during PIT; (B) Instrumental responses minus baseline during the PIT test in amphetamine-sensitized rats (left columns) and control rats (right columns); (C) Instrumental response rates during BX trials as a proportion of responding during B trials. * = significantly different at P < 0.05.

Pavlovian-instrumental transfer

Amphetamine sensitization altered performance during the PIT test. Instrumental response rates for B and BX trials minus baseline were analyzed using a two-factor ANOVA with stimulus (B and BX) as a within-subjects factor and drug (amphetamine or saline) as a between-subjects factor. This analysis revealed significant main effects of stimulus (F 1, 20 = 13.61, P < 0.01) and drug (F 1, 20 = 5.86, P < 0.05) but no significant interaction (Figure 5B). Comparisons of group means found that control rats made significantly fewer instrumental responses during BX presentations compared to B alone (paired t-test: t10 = 3.77, P < 0.05). The difference between B and BX response rates among amphetamine-sensitized rats was maginally significant (P = 0.11). A between-groups comparison revealed that responses during BX trials were significantly greater in amphetamine-sensitized rats compared to control rats (independent-samples t-test: t 20 = 2.84, P = 0.01), whereas the difference between groups on responses during B trials was only marginally significant (P = 0.12). Overall, these data suggest that amphetamine sensitization increased instrumental responses on B and BX trials during PIT test. To further investigate the effects of the conditioned inhibitor during the PIT test, instrumental response rates during BX presentations were expressed as a percent of responding during B alone. This provides a measure of the extent to which X reduced responding to B. This analysis shows that X had a greater attenuating effect on responses in control animals compared to amphetamine-exposed animals (independent samples t-test, t20 = 2.10, P < 0.05) (Figure 5C).

Approach behavior during the PIT test was analyzed and is depicted in Figure 6. An ANOVA was carried out on responses during each stimulus minus responses during the inter-stimulus interval. Stimulus (B, BX), and drug (amphetamine or saline) were included as factors. There was a significant effect of stimulus (ANOVA: F 1, 20 = 4.68, P < 0.05). No effect of amphetamine was observed nor an interaction involving this factor (P’s > 0.6).

Fig 6. Experiment 2: Approach during PIT.

Fig 6

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Food cup head insertions during the PIT test.

Locomotor responses to acute amphetamine

The locomotor effects of acute amphetamine were assessed in both sensitized and control rats (Figure 7). Locomotor activity was recorded in 3 10-min blocks immediately after rats were injected with amphetamine or saline. Data were analyzed with a two factor ANOVA with acute drug injection (amphetamine or saline) and block (1–3) as within-subjects factors and sensitization (amphetamine-sensitized or control) as a between group factor. Acute amphetamine exerted a strong locomotor effect, as shown by a significant effect of drug injection on locomotor behavior (F1, 40 = 98.92, P < 0.01). A significant effect of sensitization on locomotor behavior was also observed (F1, 20 = 4.82, P <0.05). Most importantly, a significant acute drug × block × sensitization group interaction was observed (F2, 40 = 4.47, P < 0.05). These data indicate that the locomotor effects of acute amphetamine were elevated in rats that had previous amphetamine exposure.

Fig. 7. Experiment 2: Locomotor activity following amphetamine challenge.

Fig. 7

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The effects of amphetamine challenge on locomotor behavior. Locomotor activity is presented in three 10-min blocks following amphetamine injection (solid lines) or saline injection (hatched lines) in previously amphetamine-exposed rats (black lines) or control rats (gray lines).

Discussion

We found that a Pavlovian conditioned inhibitor reduces instrumental responding during PIT in unaltered rats. This is consistent with previous findings showing that stimuli associated with the absence of reward or with an aversive outcome inhibit instrumental behavior (Delamater et al. 2003; Estes and Skinner 1941; Kearns et al. 2002; Lombas et al. 2008; McDannald and Galarce 2011). In contrast, we found amphetamine sensitization decreases conditioned inhibition during PIT: in two experiments, rats continued to lever press in the presence of the conditioned inhibitor. Overall, the pattern of results we observed in this study reflects the different effects of amphetamine on PIT depending on the timing of its administration. In experiment 1, when amphetamine was administered prior to Pavlovian training, the excitatory stimulus B by itself did not elevate instrumental responses during PIT, yet rats responded quite vigorously during compound presentations of B and conditioned inhibitor X. This was the reverse of the pattern of behavior we observed in non-exposed rats. In experiment 2, when amphetamine was administered following Pavlovian training but before the PIT test, we observed enhanced instrumental responding to B and BX during the PIT test compared to control animals. Responses during BX in experiment 2 therefore appear to be partly a function of enhanced responding to B.

In contrast to its effects on instrumental responses, amphetamine sensitization appears to have spared conditioned inhibition of approach responses during PIT. Amphetamine-sensitized and control rats made fewer conditioned approach responses during BX trials compared to B trials during the PIT test. These findings are understandable within the context of dopamine’s known function in “goal-tracking” and “sign-tracking” behavior, which have been shown to be differentially susceptible to dopamine manipulations (Flagel et al. 2011; Robinson and Flagel 2009; Saunders and Robinson 2012). Behavior during our PIT task approximates goal-tracking and sign tracking behavior. Approach to the food cup during the CS during PIT is a form of goal-tracking. Instrumental responding during PIT to a non-discrete CS, such as a tone, is similar to sign tracking in that both behaviors reflect the incentive properties acquired by the conditioned stimulus. The fact that we found no effect of amphetamine sensitization on approach but found effects on instrumental responding lends further support to the notion that lends further support to the notion that amphetamine sensitization selectively alters the incentive properties of reward-associated stimuli.

In Experiment 1 we observed no elevation of instrumental responding during the PIT test for B trials in amphetamine-sensitized animals trained under conditioned inhibition training, whereas all other groups showed transfer to stimulus B. There is some evidence that a history of amphetamine exposure inhibits PIT- amphetamine given immediately after Pavlovian training sessions interferes with subsequent instrumental responding during the PIT test (Hall and Gulley 2010). In that study, amphetamine energized a conditioned approach response during the PIT test, which interfered with instrumental responding. Although we did not observe an enhancement of food-cup approach during PIT in amphetamine-treated animals, one possibility is that amphetamine sensitization in Experiment 1 enhances a distinct overt conditioned response (orienting to the food cup or conditioned stimulus, rearing, etc), which nevertheless interferes with instrumental responding during PIT. Conditioned inhibition training may be particularly suited to eliciting an orienting response: conditioned inhibition training requires increased attention to discriminate overlapping auditory stimuli. If amphetamine sensitization selectively facilitated an orienting response during the PIT test, this would interfere with PIT in animals that underwent conditioned inhibition training but not the other groups.

Amphetamine sensitization has been shown to facilitate acquisition of conditioned inhibition (Harmer and Phillips 1999). Although we did not observe any effects of sensitization on acquisition of conditioned inhibition, a number of differences between our training paradigm and that used by Harmer and Phillips (1999) may account for this discrepancy. For instance, the length of the CS used in our study was 120 sec, and 10 sec in the study by Harmer and Phillips; longer stimuli, as we used, tend to be less effective in controlling an approach response and may account for the lack of an effect of amphetamine sensitization on approach (Delamater and Holland 2008).

In Experiment 2, we found that amphetamine sensitization enhanced PIT by increasing instrumental responses during B and BX trials. One explanation for our results is that amphetamine sensitization enhances the excitatory properties of conditioned stimuli, which overwhelm the inhibitory effects of the conditioned inhibitor during PIT. It is well established that psychostimulant sensitization enhances behavioral and neural responses to excitatory stimuli (Ehrman, Robbins et al. 1992; Jentsch and Taylor 1999; Taylor and Jentsch 2001; Wyvell and Berridge 2001; Schiltz, Kelley et al. 2005; Hollander and Carelli 2007; Wan and Peoples 2008) If we assume that the excitatory properties of the CS sum with the inhibitory properties of the conditioned inhibitor to determine output during PIT (Weiss, Thomas et al. 1996), then enhancing the excitatory properties of the CS by amphetamine sensitization would increase instrumental responses during BX presentations. However, an enhancement of B alone does not entirely explain the increased response rate during BX trials in amphetamine-sensitized rats. We observed greater numerical reduction in responding on BX trials in control animals compared to amphetamine-treated animals, and significantly greater proportional reduction in responding (Figure 5B). The reduced effectiveness of X at inhibiting instrumental responses during PIT therefore contributes to elevated responses during BX trials in amphetamine-sensitized rats.

The effects of amphetamine sensitization on conditioned inhibition during PIT are likely a consequence of changes in dopamine-dependent neural mechanisms (Everitt and Wolf 2002; Vanderschuren and Kalivas 2000). Theoretical accounts of conditioned inhibition posit the acquisition of negative associative strength by the conditioned inhibitor (Rescorla 1969; Tobler et al. 2003). Presentation of a conditioned inhibitor has been shown to reduce phasic dopamine neuron activity (Tobler et al. 2003). The negative associative strength the conditioned inhibitor accrues may therefore be substantiated in terms of a reduction in dopamine neuron activity below baseline. Computationally, phasic dopamine signaling accomplishes some aspects of assigning incentive salience to stimuli to initiate actions (Berridge 2012; McClure et al. 2003). A reduction in dopamine signaling caused by the conditioned inhibitor could therefore be responsible for reducing responses during the PIT test, as is the case with dopamine receptor antagonism (Dickinson et al. 2000; Lex and Hauber 2008; Ostlund and Maidment 2012; Wassum et al. 2011). Consequently, excessive or dysregulated dopamine resulting from amphetamine sensitization could impair conditioned inhibition during PIT.

In conclusion, our results add to the growing body of literature documenting changes in PIT as a consequence of repeated drug exposure. The way in which amphetamine sensitization influenced PIT depended on the timing of amphetamine sensitization relative to Pavlovian training. Most significantly, we found that amphetamine sensitization impairs conditioned inhibition of instrumental responding during PIT. These results further illustrate the vulnerabilities of individuals repeatedly exposed to abused drugs, particularly with respect to behavior controlled by reward-associated cues.

Acknowledgments

This research was supported by grant 026559 from the National Institute on Drug Abuse. I would like to thank Joan Morrell and Julia Basso for assistance with collecting locomotor activity data. All experiments comply with current laws in the USA.

Footnotes

Conflict of Interest: None

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