Pavlovian Conditioned Approach Training Research Articles

Does vendor breeding colony influence sign- and goal-tracking in Pavlovian conditioned approach? A preregistered empirical replication

Vendor differences are thought to affect Pavlovian conditioning in rats. After observing possible differences in sign-tracking and goal-tracking behaviour with rats from different breeding colonies, we performed an empirical replication of the effect. 40 male Long-Evans rats from Charles River colonies ‘K72’ and ‘R06’ received 11 Pavlovian conditioned approach training sessions (or “autoshaping”), with a lever as the conditioned stimulus (CS) and 10% sucrose as the unconditioned stimulus (US). Each 58-min session consisted of 12 CS-US trials. Paired rats (n = 15/colony) received the US following lever retraction. Unpaired control rats (n = 5/colony) received sucrose during the inter-trial interval. Next, we evaluated the conditioned reinforcing properties of the CS, by determining whether rats would learn to nose-poke into a new, active (vs. inactive) port to receive CS presentations alone (no sucrose). Preregistered confirmatory analyses showed that during autoshaping sessions, Paired rats made significantly more CS-triggered entries into the sucrose port (i.e., goal-tracking) and lever activations (sign-tracking) than Unpaired rats did, demonstrating acquisition of the CS-US association. Confirmatory analyses showed no effects of breeding colony on autoshaping. During conditioned reinforcement testing, analysis of data from Paired rats alone showed significantly more active vs. inactive nosepokes, suggesting that in these rats, the lever CS acquired incentive motivational properties. Analysing Paired rats alone also showed that K72 rats had higher Pavlovian Conditioned Approach scores than R06 rats did. Thus, breeding colony can affect outcome in Pavlovian conditioned approach studies, and animal breeding source should be considered as a covariate in such work.

Effects of dopamine receptor antagonism and amphetamine-induced psychomotor sensitization on sign- and goal-tracking after extended training

The dopamine system is important for incentive salience attribution, where motivational value is assigned to conditioned cues that predict appetitive reinforcers. However, the role of dopamine in this process may change with extended training. We tested the effects of dopamine D1-like and D2-like receptor antagonism on the expression of sign-tracking and goal-tracking conditioned responses following extended Pavlovian conditioned approach (PCA) training. We also tested if amphetamine-induced psychomotor sensitization accelerates the enhanced acquisition of sign-tracking that is observed with extended training. In experiment 1, 24 male Long-Evans rats received 20 PCA sessions in which one lever (CS+, 10 s) predicted 0.2 ml sucrose (10 %, w/v) delivery and the other lever (CS–) did not. SCH-23390 (D1-like antagonist) or eticlopride (D2-like antagonist) were administered before non-reinforced behavioural tests at doses of 0, 0.01, and 0.1 mg/kg (s.c.). In experiment 2, rats received vehicle or 2 mg/kg amphetamine (i.p.) for 7 days (n = 12/group). Ten days later, they received 16 PCA training sessions. Both doses of SCH-23390 reduced sign- and goal-tracking, but also reduced locomotor behaviour. A low dose of eticlopride (0.01 mg/kg) selectively reduced goal-tracking, without affecting sign-tracking or locomotor behaviour. Amphetamine produced psychomotor sensitization, and this did not affect the acquisition of sign- or goal-tracking. Following extended PCA training, dopamine D2-like receptor activity is required for the expression of goal-tracking but not sign-tracking. Psychomotor sensitization to amphetamine did not impact incentive salience attribution; however, more selective manipulations of the dopamine system may be needed.

Ultrasonic vocalizations and individual differences in rats performing a Pavlovian conditioned approach task

Rats emit distinct types of ultrasonic vocalizations (USV), including high-frequency 50-kHz USV, which occur in appetitive situations. Such 50-kHz USV are thought to reflect positive affective states, for example in case of reward anticipation, and are linked to dopamine signaling. The present study was conducted to investigate whether rats emit 50-kHz USV during a Pavlovian conditioned approach task and whether trait-like differences in 50-kHz USV emission are associated with sign- versus goal-tracking. We hypothesize that individuals engaging more with a cue predicting a food reward will also elicit more 50-kHz USV. In order to test this, we investigated 34 female rats and gauged USV while they underwent a Pavlovian conditioned approach training and extinction paradigm. For one, we found a high subject-dependent variability in the emission of 50-kHz calls. These were not largely affected by state differences, since these 50-kHz USV were observed throughout the task. During task progress and in most subjects, there was a rather complete shift toward goal-tracking, but subjects engaging more with the cue predicting a reward also emitted higher numbers of appetitive 50-kHz calls. This supports the hypothesis that sign-tracking is positively associated with the emission of 50-kHz USV. The high subject-dependent variability in the emission of 50-kHz calls warrants special attention in future appetitive studies.

Effects of the cannabinoid receptor agonist CP-55,940 on incentive salience attribution.

Pavlovian conditioned approach paradigms are used to characterize the nature of motivational behaviors in response to stimuli as either directed toward the cue (i.e., sign-tracking) or the site of reward delivery (i.e., goal-tracking). Recent evidence has shown that activity of the endocannabinoid system increases dopaminergic activity in the mesocorticolimbic system, and other studies have shown that sign-tracking behaviors are dependent on dopamine. Therefore, we hypothesized that administration of a cannabinoid agonist would increase sign-tracking and decrease goal-tracking behaviors. Forty-seven adult male Sprague-Dawley rats were given a low, medium, or high dose of the cannabinoid agonist CP-55,940 (N = 12 per group) or saline (N = 11) before Pavlovian conditioned approach training. A separate group of rats (N = 32) were sacrificed after PCA training for measurement of cannabinoid receptor type 1 (CB1) and fatty acid amide hydrolase (FAAH) using in situ hybridization. Contrary to our initial hypothesis, CP-55,940 dose-dependently decreased sign-tracking and increased goal-tracking behavior. CB1 expression was higher in sign-trackers compared with that in goal-trackers in the prelimbic cortex, but there were no significant differences in CB1 or FAAH expression in the infralimbic cortex, dorsal or ventral CA1, dorsal or ventral CA3, dorsal or ventral dentate gyrus, or amygdala. These results demonstrate that cannabinoid signaling can specifically influence behavioral biases toward sign- or goal-tracking. Pre-existing differences in CB1 expression patterns, particularly in the prelimbic cortex, could contribute to individual differences in the tendency to attribute incentive salience to reward cues.

Individual variation in the attribution of incentive salience to social cues

Research on the attribution of incentive salience to drug cues has furthered our understanding of drug self-administration in animals and addiction in humans. The influence of social cues on drug-seeking behavior has garnered attention recently, but few studies have investigated how social cues gain incentive-motivational value. In the present study, a Pavlovian conditioned approach (PCA) procedure was used to identify rats that are more (sign-trackers; STs) or less (goal-trackers; GTs) prone to attribute incentive salience to food reward cues. In Experiment 1, a novel procedure employed social ‘peers’ to compare the tendency of STs and GTs to attribute incentive salience to social reward cues as well as form a social-conditioned place preference. In Experiment 2, social behavior of STs and GTs was compared using social interaction and choice tests. Finally, in Experiment 3, levels of plasma oxytocin were measured in STs and GTs seven days after the last PCA training session, because oxytocin is known to modulate the mesolimbic reward system and social behavior. Compared to GTs, STs attributed more incentive salience to social-related cues and exhibited prosocial behaviors (e.g., social-conditioned place preference, increased social interaction, and social novelty-seeking). No group differences were observed in plasma oxytocin levels. Taken together, these experiments demonstrate individual variation in the attribution of incentive salience to both food- and social-related cues, which has important implications for the pathophysiology of addiction.

Sex differences in nicotine-enhanced Pavlovian conditioned approach in rats

BackgroundNicotine exposure enhances Pavlovian conditioned approach (PCA), or the learned approach to reward-predictive cues. While females show elevated approach to conditioned stimuli compared to males, potentially indicating heightened addiction vulnerability, it is unknown how sex may interact with nicotine to influence approach behavior. Additionally, brain-derived neurotrophic factor (BDNF) levels can be altered significantly after repeated nicotine exposure, suggesting a potential mechanism contributing to nicotine-induced behavioral phenotypes. The present study investigated the role of sex on nicotine-induced changes to stimulus-response behavior and associated BDNF protein levels.MethodsMale and female rats were exposed to nicotine (0.4 mg/kg, subcutaneously) or saline 15 min prior to each PCA session. PCA training consisted of 29 sessions of 15 trials, in which a 30-s cue presentation ended concurrently with a sucrose reward (20% w/v in water, 100 μL), and a 120-s variable intertrial interval occurred between trials. Approach behavior to the cue and reward receptacle was recorded. Preference toward the reward receptacle indicated a goal-tracking phenotype, and preference toward the cue indicated a sign-tracking phenotype, demonstrating that the cue had gained incentive salience. Twenty-four hours after the last PCA session, brain tissue was collected and BDNF levels were measured in the basolateral amygdala, orbitofrontal cortex, and nucleus accumbens using Western blot analysis.ResultsNicotine exposure enhanced both sign- and goal-tracking conditioned approach, and females showed elevated sign-tracking compared to males. There were no sex-by-drug interactions on conditioned approach. Day-to-day variability in conditioned approach was similar between sexes. In contrast to prior studies, neither repeated exposure to nicotine nor sex significantly affected BDNF expression.ConclusionsDrug-naïve females exhibited heightened sign-tracking compared to males, and nicotine enhanced conditioned approach similarly in males and females. Further, non-significant changes to BDNF expression in brain regions highly associated with PCA indicate that BDNF is unlikely to drive nicotine-enhanced conditioned behavior.

Nicotine-enhanced Pavlovian conditioned approach is resistant to omission of expected outcome

Conditioned stimuli contribute to the resilience of nicotine addiction in that nicotine-associated cues can influence smokers and promote relapse. These stimuli are thought to acquire incentive motivational properties through a Pavlovian mechanism, and this phenomenon can be measured in animals by observing conditioned approach to the conditioned stimulus (sign-tracking) or to the location of unconditioned stimulus delivery (goal-tracking). Goal-tracking is thought to be more flexible than sign-tracking in response to changes in expected outcome. Nicotine exposure can increase the expression of conditioned responses, and we hypothesized that animals exposed to nicotine would also exhibit less flexible conditioned responses after a change in the expected unconditioned stimulus. Adult male rats were exposed to nicotine (0.4mg/kg, s.c.) or saline before Pavlovian conditioned approach training sessions. After training, animals underwent test sessions that reduced (water substitution) or withheld (omission) the unconditioned stimulus (US, 20% sucrose). As expected, nicotine enhanced sign- and goal-tracking. Water substitution moderately and nonspecifically reduced both sign- and goal-tracking in all rats. In contrast, US omission only reduced goal-tracking, with robust effects in saline-exposed rats and smaller effects in nicotine-exposed rats. These data support the hypothesis that both sign-tracking and nicotine exposure confer behavioral inflexibility under US omission.

Thalamic mast cell activity is associated with sign-tracking behavior in rats

Mast cells are resident immune cells in the thalamus that can degranulate and release hundreds of signaling molecules (i.e., monoamines, growth factors, and cytokines) both basally and in response to environmental stimuli. Interestingly, mast cell numbers in the brain show immense individual variation in both rodents and humans. We used a Pavlovian conditioned approach (PCA) procedure to examine whether mast cells are associated with individual variation in the attribution of incentive-motivational value to reward-related cues. During the PCA procedure, a lever response-independently predicts the delivery of a food pellet into a magazine, and over training sessions three conditioned responses (CRs) develop: sign-tracking (lever-directed CRs), goal-tracking (magazine-directed CRs), and an intermediate response (both CRs). In Experiment 1, we measured thalamic mast cell number/activation using toluidine blue and demonstrated that sign-trackers have increased degranulated (activated) but not granulated (inactive) mast cells. In Experiment 2, we infused the mast cell inhibitor, cromolyn (200µg/rat; i.c.v.), immediately before five daily PCA training sessions and demonstrated that mast cell inhibition selectively impairs the acquisition of sign-tracking behavior. Taken together, these results demonstrate that thalamic mast cells contribute to the attribution of incentive-motivational value to reward-related cues and suggest that mast cell inhibition may be a novel target for addiction treatment.

Unresponsive Choline Transporter as a Trait Neuromarker and a Causal Mediator of Bottom-Up Attentional Biases.

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.

Pavlovian Conditioned Approach Training in Rats.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, "wanted' stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Pavlovian Conditioned Approach Training in Rats

Pavlovian Conditioned Approach Training in Rats

Sign-trackers have elevated myo-inositol in thenucleus accumbens and ventral hippocampus following Pavlovian conditioned approach.

Pavlovian conditioned approach (PCA) is a behavioral procedure that can be used to assess individual differences in the addiction vulnerability of drug-naïve rats and identify addiction vulnerability factors. Using proton magnetic resonance spectroscopy (1 H-MRS) exvivo, we simultaneously analyzed concentrations of multiple neurochemicals throughout the mesocorticolimbic system 2weeks after PCA training in order to identify potential vulnerability factors to addiction in drug-naïve rats for future investigations. Levels of myo-inositol (Ins), a 1 H-MRS-detectable marker of glial activity/proliferation, were increased in the nucleus accumbens (NAc) and ventral hippocampus, but not dorsal hippocampus or medial prefrontal cortex, of sign-trackers compared to goal-trackers or intermediate responders. In addition, Ins levels positively correlated with PCA behavior in the NAc and ventral hippocampus. Because the sign-tracker phenotype is associated with increased drug-seeking behavior, these results observed in drug-naïve rats suggest that alterations in glial activity/proliferation within these regions may represent an addiction vulnerability factor. Sign-tracking rats preferentially approach reward cues during Pavlovian conditioning, while goal-trackers instead approach the location of impending reward. Sign-trackers are also more prone to cue-induced drug-seeking behavior. We used magnetic resonance spectroscopy to show that myo-inositol levels are higher in the ventral hippocampus and nucleus accumbens of sign-trackers relative to goal-trackers. Thus, elevated myo-inositol may be a vulnerability factor for addiction.

Amphetamine-induced incentive sensitization of sign-tracking behavior in adolescent and adult female rats.

Age-specific behavioral and neural characteristics may predispose adolescents to initiate and escalate use of alcohol and drugs. Adolescents may avidly seek novel experiences, including drugs of abuse, because of enhanced incentive motivation for drugs and natural rewards, perhaps especially when that incentive motivation is sensitized by prior drug exposure. Using a Pavlovian conditioned approach (PCA) procedure, sign-tracking (ST) and goal-tracking (GT) behavior was examined in amphetamine-sensitized and control adolescent and adult female Sprague-Dawley rats, with expression of elevated ST behavior used to index enhanced incentive motivation for reward-associated cues. Rats were first exposed to a sensitizing regimen of amphetamine injections (3.0 mg/kg/ml d-amphetamine per day) or given saline (0.9% wt/vol) once daily for 4 days. Expression of ST and GT was then examined over 8 days of PCA training consisting of 25 pairings of an 8-s presentation of an illuminated lever immediately followed by response-independent delivery of a banana-flavored food pellet. Results showed that adults clearly displayed more ST behavior than adolescents, reflected via both more contacts with, and shorter latencies to approach, the lever. Prior amphetamine sensitization increased ST (but not GT) behaviors regardless of age. Thus, when indexed via ST, incentive motivation was found to be greater in adults than adolescents, with a prior history of amphetamine exposure generally sensitizing incentive motivation for cues predicting a food reward regardless of age.

Post-training, but not post-reactivation, administration of amphetamine and anisomycin modulates Pavlovian conditioned approach

The psychostimulant, amphetamine (AMPH), and the protein synthesis inhibitor, anisomycin (ANI), have been shown to modulate the consolidation and reconsolidation of several types of learning. To determine whether Pavlovian conditioned approach (PCA) is modulated in a similar manner, we examined the effects of post-training and post-reactivation administration of both AMPH and ANI on memory for PCA. Male Long-Evans rats received PCA training sessions during which presentations of a CS+ were followed by sucrose delivery. AMPH (1 mg/kg, s.c.) injected immediately but not 6 h after the first training session enhanced PCA behavior. ANI (150 mg/kg, s.c.) injected immediately but not 3 h after the first training session impaired PCA behavior. This impairment was not due to the development of a conditioned taste aversion. To examine whether PCA can also be modulated by post-reactivation administration of AMPH and ANI, rats were given an injection of AMPH, ANI, or vehicle immediately after a memory reactivation session. Upon testing, the behavior of both the AMPH- and the ANI-treated rats was unaffected. This result remained consistent when the experiment was repeated with changes to various behavioral parameters (i.e., amount of training, length of memory reactivation). These findings indicate that AMPH and ANI act during the post-training but not the post-reactivation period to enhance and impair, respectively, the learning of PCA. This suggests that the consolidation of PCA can be modulated in a manner comparable to other types of learned associations, but once learned, the memory appears to be relatively robust and stable.

Medullary cystic disease and juvenile nephronophthisis in separate members of the same family.

<h3>Abstract</h3> Vendor differences are thought to affect Pavlovian conditioning in rats. After observing possible differences in sign-tracking and goal-tracking behaviour with rats from different breeding colonies, we performed an empirical replication of the effect. 40 male Long-Evans rats from Charles River colonies ‘K72’ and ‘R06’ received 11 Pavlovian conditioned approach training sessions (or “autoshaping”), with a lever as the conditioned stimulus (CS) and 10% sucrose as the unconditioned stimulus (US). Each 58-min session consisted of 12 CS-US trials. Paired rats (n = 15/colony) received the US following lever retraction. Unpaired control rats (n = 5/colony) received sucrose during the inter-trial interval. Next, we evaluated the conditioned reinforcing properties of the CS, by determining whether rats would learn to nose-poke into a new, active (vs. inactive) port to receive CS presentations alone (no sucrose). Preregistered confirmatory analyses showed that during autoshaping sessions, Paired rats made significantly more CS-triggered entries into the sucrose port (i.e., goal-tracking) and lever activations (sign-tracking) than Unpaired rats did, demonstrating acquisition of the CS-US association. Confirmatory analyses showed no effects of breeding colony on autoshaping. During conditioned reinforcement testing, analysis of data from Paired rats alone showed significantly more active vs. inactive nosepokes, suggesting that in these rats, the lever CS acquired incentive motivational properties. Analysing Paired rats alone also showed that K72 rats had higher Pavlovian Conditioned Approach scores than R06 rats did. Thus, breeding colony can affect outcome in Pavlovian conditioned approach studies, and animal breeding source should be considered as a covariate in such work.