Nadia Chaudhri

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.

The capacity to suppress learned responses is essential for animals to adapt in dynamic environments. Extinction is a process by which animals learn to suppress conditioned responding when an expected outcome is omitted. The infralimbic cortex (IL) to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behaviour. However, the role of the IL-to-NAcS neural circuit in the extinction of responding to appetitive Pavlovian cues is unknown and the psychological mechanisms involved in response suppression following extinction are unclear. We trained rats to associate a 10 s auditory conditioned stimulus (CS; 14 trials per session) with a sucrose unconditioned stimulus (US; 0.2 mL per CS) in a specific context and then, following extinction in a different context, precipitated a renewal of CS responding by presenting the CS alone in the original Pavlovian conditioning context. Unilateral, optogenetic stimulation of the IL-to-NAcS circuit selectively during CS trials suppressed renewal. In a separate experiment, IL-to-NAcS stimulation suppressed CS responding regardless of prior extinction and impaired extinction retrieval. Finally, IL-to-NAcS stimulation during the CS did not suppress the acquisition of Pavlovian conditioning but was required for the subsequent expression of CS responding. These results are consistent with multiple studies showing that the IL-to-NAcS neural circuit is involved in the suppression of operant cocaine-seeking, extending these findings to appetitive Pavlovian cues. The suppression of appetitive Pavlovian responding following IL-to-NAcS circuit stimulation does not, however, appear to require an extinction-dependent process. SIGNIFICANCE STATEMENT: Extinction is a form of inhibitory learning through which animals learn to suppress conditioned responding in the face of non-reinforcement. We investigated the role of infralimbic (IL) cortex inputs to the nucleus accumbens shell (NAcS) in the extinction of responding to appetitive Pavlovian cues and the psychological mechanisms involved in response suppression following extinction. Using in vivo optogenetics, we found that stimulating the IL-to-NAcS neural circuit suppressed context-induced renewal of conditioned responding after extinction. In a separate experiment, stimulating the IL-to-NAcS circuit suppressed conditioned responding in an extinction-independent manner. These findings can be leveraged by future research aimed at understanding how corticostriatal circuits contribute to behavioural flexibility and mental disorders that involve the suppression of learned behaviours.

Re-exposure to an unconditioned stimulus (US) can reinstate extinguished conditioned responding elicited by a conditioned stimulus (CS). We tested the hypothesis that the reinstatement of responding to an appetitive CS is driven by an excitatory association formed between the US and the context that the US was ingested in during US re-exposure. Male, Long-Evans rats were acclimated to drinking alcohol (15%, v / v ) in the home-cage, then trained to associate an auditory CS with an alcohol-US that was delivered into a fluid port for oral intake. During subsequent extinction sessions, the CS was presented as before, but without alcohol. After extinction, rats were re-exposed to alcohol as in training, but without the CS (alcohol re-exposure). 24 h later at test, the CS was presented as in training, but without alcohol. First, we tested the effect of extinguishing the context-alcohol association, formed during alcohol re-exposure, on reinstatement. Conducting four context extinction sessions across four days (spaced extinction) after the alcohol re-exposure session did not impact reinstatement. However, four context extinction sessions conducted across two days (massed extinction) prevented reinstatement. Next, we conducted alcohol re-exposure in a context that either differed from, or was the same as, the test context. One alcohol re-exposure session in a different context did not affect reinstatement, however, three alcohol re-exposure sessions in a different context significantly reduced reinstatement during the first CS trial. These results partially support the view that a context-US association formed during US re-exposure drives the reinstatement of responding to an appetitive, alcohol-predictive CS.

Background In male rats, physical contexts that are associated with alcohol can invigorate responding to a discrete, alcohol-predictive conditioned stimulus (CS), and amplify priming-induced reinstatement. Here, we examined these effects as a function of biological sex. Methods Male and female Long-Evans rats were acclimated to drinking ethanol (EtOH, 15% v/v) in their home cages. Next, they were trained to associate an auditory CS (10 s; white noise; 15 trials per session) with EtOH delivery (0.2 ml per CS; 3.0 ml per session) into a fluid port for oral intake. Training occurred in a distinctive context containing specific visual, olfactory, and tactile stimuli. During alternating sessions rats were exposed to a second context where they did not receive EtOH. At test, CS presentations occurred in both contexts without EtOH delivery. Rats then underwent extinction using repeated unreinforced presentations of the CS in both contexts. An alcohol-primed reinstatement test was then conducted, in which 0.2 ml of EtOH was presented both at the start of the session and during the first CS presentation, after which no EtOH was delivered for the remainder of the session. Results At both test and reinstatement, male rats made significantly more CS port-entries in the context associated with alcohol delivery than in the context in which alcohol was never experienced. Unlike males, female rats made a similar number of CS port-entries at test in both the alcohol context and the neutral context. The reinstatement observed in female rats was not affected by context. Conclusions These findings identify novel sex differences in the capacity of an alcohol-associated context to modulate responding to a discrete, alcohol-predictive cue.

Context can influence reactions to environmental cues and this elemental process has implications for substance use disorder. Using an animal model, we show that an alcohol-associated context elevates entry into a fluid port triggered by a conditioned stimulus (CS) that predicted alcohol (CS-triggered alcohol-seeking). This effect persists across multiple sessions and, after it diminishes in extinction, the alcohol context retains the capacity to augment reinstatement. Systemically administered eticlopride and chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons reduce CS-triggered alcohol-seeking. Chemogenetically silencing VTA dopamine terminals in the nucleus accumbens (NAc) core reduces CS-triggered alcohol-seeking, irrespective of context, whereas silencing VTA dopamine terminals in the NAc shell selectively reduces the elevation of CS-triggered alcohol-seeking in an alcohol context. This dissociation reveals new roles for divergent mesolimbic dopamine circuits in the control of responding to a discrete cue for alcohol and in the amplification of this behaviour in an alcohol context.

Conditioned responding can be renewed by re-exposure to the conditioning context following extinction in a different context (ABA renewal) or by removal from the extinction context (AAB or ABC renewal). ABA renewal is robust in Pavlovian and operant conditioning paradigms. However, fewer studies have investigated AAB and ABC renewal of appetitive conditioning, and those that did predominantly used operant conditioning tasks. Renewal has theoretical relevance for extinction and for exposure-based treatments for substance use disorders that aim to extinguish reactivity to drug-predictive cues. We therefore investigated ABA, AAB, and ABC renewal of Pavlovian conditioned responding to cues that predicted either alcohol or sucrose. Male, Long-Evans rats (Charles River) were exposed to either 15% ethanol (Study 1: “alcohol”) or 10% sucrose (Study 2: “sucrose”) in their home cages. Next, they were trained to discriminate between two auditory stimuli (white noise and clicker; 10 s) in conditioning chambers equipped with distinct olfactory, visual, and tactile contextual stimuli (context A). One conditioned stimulus (CS+) was paired with fluid delivery (0.2 ml/CS+; 3.2 ml/session; alcohol or sucrose in separate experiments), and the second CS (CS−) was not. In all sessions (conditioning, extinction, and test), each CS was presented 16 times/session on a variable-time 67-s schedule, and entries into the fluid port were recorded. CS+ port entries were then extinguished by withholding fluid delivery either in context A or in a second, different context (context B). Next, we assessed ABA, AAB, and ABC renewal in the absence of fluid delivery. During extinction, CS+ port entries were initially elevated in context A relative to context B. ABA renewal of CS+ port entries occurred in both alcohol- and sucrose-trained rats. ABC renewal approached statistical significance when data from both experiments were combined. No AAB renewal was observed, and, in fact, alcohol-trained rats showed AAB suppression. These results corroborate the reliability of ABA renewal and suggest that ABC renewal is a modest effect that may require greater statistical power to detect. From a treatment perspective, the lack of AAB renewal suggests that exposure-based treatments for substance use disorders might benefit from implementation in real-world, drug-use contexts.

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.