Nicotine Enhances Goal-Tracking in Ethanol and Food Pavlovian Conditioned Approach Paradigms

RationaleNicotine promotes alcohol intake through pharmacological and behavioral interactions. As an example of the latter, nicotine can facilitate approach toward food- and alcohol-associated stimuli (“sign-tracking”) in lever-Pavlovian conditioned approach (PavCA) paradigms. However, we recently reported that nicotine can also enhance approach toward locations of reward delivery (“goal-tracking”) triggered by ethanol-predictive stimuli when the location of ethanol delivery is non-static (i.e., a retractable sipper bottle).ObjectiveTo determine whether the non-static nature of the reward location could have biased the development of goal-tracking in our previous study (Loney et al., 2019); we assessed the effect of nicotine in a lever-PavCA paradigm wherein the location of ethanol delivery was static (i.e., a stationary liquid receptacle). Then, to determine whether nicotine’s enhancement of goal-tracking is unique to ethanol-predictive stimuli, we assessed the effect of systemic nicotine on approach triggered by food-predictive stimuli in a lever-PavCA paradigm.MethodsLong–Evans rats were used in two PavCA experiments wherein a lever predicted the receipt of ethanol (15% vol/vol; experiment 1) or food (experiment 2) into a stationary receptacle. Prior to testing, rats were administered nicotine (0.4 mg/kg subcutaneously) or saline systemically.ResultsIn both experiments, nicotine increased measures of goal-tracking, but not sign-tracking.ConclusionNicotine can facilitate approach to reward locations without facilitating approach to reward-predictive stimuli. As such, conceptualization of the mechanisms by which nicotine affects behavior must be expanded to explain an enhancement of goal-tracking by nicotine.

Neuroendocrine and behavioral measures of negative valence in male sign-tracker and goal-tracker rats

Cues, or stimuli in the environment, attain the ability to guide behavior via learned associations. As predictors, cues can elicit adaptive behavior and lead to valuable resources (e.g., food). For some individuals, however, cues are transformed into incentive stimuli and can elicit maladaptive behavior. The goal-tracker/sign-tracker animal model captures individual differences in cue-motivated behaviors, with reward-associated cues serving as predictors of reward for both goal-trackers and sign-trackers, but becoming incentive stimuli only for sign-trackers. While these distinct phenotypes are characterized based on Pavlovian conditioned approach behavior, they exhibit differences on a number of behaviors of relevance to psychopathology. To further characterize the neurobehavioral endophenotype associated with individual differences in cue-reward learning, we investigated neuroendocrine and behavioral profiles associated with negative valence in male goal-trackers, sign-trackers, and intermediate responders. We found that baseline corticosterone increases with Pavlovian learning, and that this increase is positively associated with the development of sign-tracking. We did not observe significant differences between goal-trackers and sign-trackers in behavior during an elevated plus maze or open field test, nor did we see differences in the corticosterone response to the open field test or physiological restraint. We did, however, find that sign-trackers have greater glucocorticoid receptor mRNA expression in the ventral hippocampus, with no phenotypic differences in the dorsal hippocampus. These findings suggest that goal-trackers and sign-trackers do not differ on indices of negative valence; rather, differences in neuroendocrine measures between these phenotypes can be attributed to distinct cue-reward learning styles.Significance StatementWhile the goal-tracker/ sign-tracker animal model derives from individual differences in Pavlovian conditioned approach behavior, other traits, including some of relevance to addiction and post-traumatic stress disorder, have been shown to co-exist with the propensity to sign-track. The extent to which this model encompasses differences in negative valence systems, however, remains largely unexplored. Here we show that behavioral and corticosterone response to paradigms associated with negative valence do not differ between goal-trackers and sign-trackers; but baseline corticosterone levels appear to be linked to the development of sign-tracking, as do differences in glucocorticoid receptor expression in the ventral hippocampus. These findings suggest that neuroendocrine measures typically associated with negative valence may, in fact, play an important role in positive valence systems.

The lateral hypothalamus and orexinergic transmission in the paraventricular thalamus promote the attribution of incentive salience to reward-associated cues

RationalePrior research suggests that inputs from the lateral hypothalamic area (LHA) to the paraventricular nucleus of the thalamus (PVT) contribute to the attribution of incentive salience to Pavlovian-conditioned reward cues. However, a causal role for the LHA in this phenomenon has not been demonstrated. In addition, it is unknown which hypothalamic neurotransmitter or peptide system(s) are involved in mediating incentive salience attribution.ObjectivesTo examine: 1) the role of the LHA in the propensity to attribute incentive salience to reward cues, and 2) the role of orexinergic signaling in the PVT on the expression of Pavlovian conditioned approach (PavCA) behavior, a reflection of incentive salience attribution.MethodsMale Sprague-Dawley rats received bilateral excitotoxic lesions of the LHA prior to the acquisition of Pavlovian conditioned approach (PavCA) behavior. A separate cohort of male rats acquired PavCA behavior and were characterized as sign-trackers (STs) or goal-trackers (GTs) based on their conditioned response. The orexin 1 receptor (OX1r) antagonist SB-334867, or the orexin 2 receptor (OX2r) antagonist TCS-OX2-29, were then administered directly into the PVT to assess the effects of these pharmacological agents on the expression of PavCA behavior and on the conditioned reinforcing properties of the Pavlovian reward cue.ResultsLesions of the LHA before training attenuated the development of lever-directed (sign-tracking) behaviors in the PavCA paradigm, without affecting magazine-directed (goal-tracking) behaviors. In STs, administration of the OX1r antagonist into the PVT reduced lever-directed behaviors and increased magazine-directed behaviors; while administration of the OX2r antagonist only reduced lever-directed behaviors. Further, OX2r, but not OX1r, antagonism was able to reduce the incentive motivational value of the conditioned stimulus on a conditioned reinforcement test in STs. The behavior of GTs was unaffected by orexinergic antagonism in the PVT.ConclusionsThe LHA is necessary for the attribution of incentive salience to reward cues and, thereby, the development of a sign-tracking conditioned response. Furthermore, blockade of orexin signaling in the PVT attenuates the incentive value of a Pavlovian reward cue. These data suggest that hypothalamic orexin inputs to the PVT are a key component of the circuitry that encodes the incentive motivational value of reward cues and promotes maladaptive cue-driven behaviors.

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 to goal-trackers in the prelimbic cortex, but there were no significant differences in CB1 or FAAH expression in the infralimbic cortex, dCA1, dCA3, dorsal 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 as well as drug relapse and craving in humans. The influence of peers and other social cues on drug-seeking has garnered more attention recently, but few studies have investigated the ability of social cues to gain incentive-motivational value. In the present study, a Pavlovian conditioned approach procedure was used to identify rats that are more (sign-trackers) or less (goal-trackers) prone to attribute incentive salience to food reward cues. A novel procedure then employed social ‘peers’ to compare the tendency of sign-trackers and goal-trackers to attribute incentive salience to social reward cues. Social behavior of sign-trackers and goal-trackers was also compared using social interaction and choice tests. Finally, basal levels of plasma oxytocin were measured in sign-trackers and goal-trackers, because oxytocin is known to modulate the mesolimbic reward system and social behavior. Compared to goal-trackers, sign-trackers attributed more incentive salience to social cues and exhibited more prosocial behaviors. No group differences were observed in baseline plasma oxytocin levels. Taken together, these experiments demonstrate a concordance of individual variation in social behavior, the attribution of incentive salience to social cues following peer interaction, and attribution of incentive salience to food cues. This general tendency to attribute motivational value to reward cues has important implications for the pathophysiology of addiction and other disorders of reward learning.

Sign-tracking behavior is difficult to extinguish and resistant to multiple cognitive enhancers

The attribution of incentive-motivational value to drug-related cues underlies relapse and craving in drug addiction. One method of addiction treatment, cue-exposure therapy, utilizes repeated presentations of drug-related cues in the absence of drug (i.e., extinction learning); however, its efficacy has been limited due to an incomplete understanding of extinction and relapse processes after cues have been imbued with incentive-motivational value. To investigate this, we used a Pavlovian conditioned approach procedure to screen for rats that attribute incentive-motivational value to reward-related cues (sign-trackers; STs) or those that do not (goal-trackers; GTs). In Experiment 1, rats underwent Pavlovian extinction followed by reinstatement and spontaneous recovery tests. For comparison, a separate group of rats underwent PCA training followed by operant conditioning, extinction, and tests of reinstatement and spontaneous recovery. In Experiment 2, three cognitive enhancers (sodium butyrate, D-cycloserine, and fibroblast growth factor 2) were administered following extinction training to facilitate extinction learning. STs but not GTs displayed enduring resistance to Pavlovian, but not operant, extinction and were more susceptible to spontaneous recovery. In addition, none of the cognitive enhancers tested affected extinction learning. These results expand our understanding of extinction learning by demonstrating that there is individual variation in extinction and relapse processes and highlight potential difficulties in applying extinction-based therapies to drug addiction treatment in the clinic.

Genetic characterization of outbred Sprague Dawley rats and utility for genome-wide association studies

Sprague Dawley (SD) rats are one of the most commonly used outbred rat strains. Despite this, the genetic characteristics of SD are poorly understood. We collected behavioral data from 4,625 SD rats acquired predominantly from two commercial vendors, Charles River Laboratories and Harlan Sprague Dawley Inc. Using double-digest genotyping-by-sequencing (ddGBS), we obtained dense, high-quality genotypes at 234,887 SNPs across 4,061 rats. This genetic data allowed us to characterize the variation present in Charles River vs. Harlan SD rats. We found that the two populations are highly diverged (FST > 0.4). We also used these data to perform a genome-wide association study (GWAS) of Pavlovian conditioned approach (PavCA), which assesses the propensity for rats to attribute motivational value to discrete, reward-associated cues. Due to the genetic divergence between rats from Charles River and Harlan, we performed two separate GWAS by fitting a linear mixed model that accounted for within vendor population structure and using meta-analysis to jointly analyze the two studies. We identified 18 independent loci that were significantly associated with one or more metrics used to describe PavCA; we also identified 3 loci that were body weight, which was only measured in a subset of the rats. The genetic characterization of SD rats is a valuable resource for the rat community that can be used to inform future study design.Author SummaryOutbred Sprague Dawley rats are among the most commonly used rats for neuroscience, physiology and pharmacological research. SD rats are sold by several commercial vendors, including Charles River Laboratories and Harlan Sprague Dawley Inc. (now Envigo). Despite their wide spread use, little is known about the genetic diversity of SD. We genotyped more than 4,000 SD rats, which we used to characterize genetic differences between SD rats from Charles River Laboratories and Harlan. Our analysis revealed that the two SD colonies are highly divergent. We also performed a genome-wide association study (GWAS) for Pavlovian conditioned approach (PavCA), which assesses the propensity for rats to attribute motivational value to discrete, reward-associated cues. Our results demonstrate that, despite sharing an identical name, SD rats are obtained from different vendors are genetically very different. We conclude that results obtained using SD rats should not be presented without also carefully noting the vendor.