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Contexts associated with prior reinforcement can renew extinguished conditioned responding. The prelimbic (PL) and infralimbic (IL) cortices are thought to mediate the expression and suppression of conditioned responding, respectively. Evidence suggests that PL inputs to the paraventricular nucleus of the thalamus (PVT) drive the expression of cue-induced reinstatement of drug seeking and that IL inputs to the PVT mediate fear extinction retrieval. However, the role of these projections in renewal of appetitive Pavlovian conditioned responding is unknown. We trained male and female Long-Evans rats to associate a conditioned stimulus (CS; 10 s white noise) with delivery of a 10% sucrose unconditioned stimulus (US; .2 ml/CS) to a fluid port in a distinct context (Context A). We then extinguished responding by presenting the CS without the US in a different context (Context B). At test, rats were returned to Context A, and optogenetic stimulation was delivered to either the IL-to-PVT or PL-to-PVT pathway during CS presentations. Optically stimulating the IL-to-PVT, but not the PL-to-PVT pathway, attenuated ABA renewal of CS port entries, and this effect was similar in males and females. Further, rats self-administered optical stimulation of the IL-to-PVT but not the PL-to-PVT pathway suggesting that activation of the IL-to-PVT pathway is reinforcing. The effectiveness of optical stimulation parameters to activate neurons in the IL, PL and PVT was confirmed using Fos immunohistochemistry. These findings provide evidence for novel neural mechanisms in renewal of responding to a sucrose-predictive CS, as well as more generally in contextual processing and appetitive associative learning.

In male rats, physical contexts that are associated with alcohol can amplify the response to a discrete, alcohol-predictive conditioned stimulus (CS), and amplify prime-induced reinstatement. Here, we examined these effects as a function of biological sex. Male and female Long-Evans rats were acclimated to drinking ethanol (15% v/v) in their home cages. Next, they were trained to associate an auditory conditioned stimulus (CS) (10 s; white noise or clicker; 15 trials per session) with ethanol 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 wherein they did not receive ethanol. At test, CS trials occurred in both contexts without ethanol 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 ethanol was presented at the start of the session and during the first CS trial, after which no ethanol was delivered for the remainder of the session. 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 the test in both the alcohol context and the neutral context. The reinstatement observed in female rats was also not affected by context. These findings suggest that the capacity of an alcohol-associated context to modulate responding to a discrete, alcohol-predictive cue is less pronounced in female than male rats.

Renewal is the return of extinguished responding after removal from the extinction context. Renewal has been extensively studied using classical aversive conditioning procedures that measure a passive freezing response to an aversive conditioned stimulus. However, coping responses to aversive stimuli are complex and can be reflected in passive and active behaviours. Using the shock-probe defensive burying task, we investigated whether different coping responses are susceptible to renewal. During conditioning, male, Long-Evans rats were placed into a specific context (context A) where an electrified shock-probe delivered a 3 mA shock upon contact. During extinction, the shock-probe was unarmed in either the same (context A) or a different context (context B). Renewal of conditioned responses was assessed in the conditioning context (ABA) or in a novel context (ABC or AAB). Renewal of passive coping responses, indicated by an increased latency and a decreased duration of shock-probe contacts, was observed in all groups. However, renewal of passive coping, measured by increased time spent on the side of the chamber opposite the shock-probe, was only found in the ABA group. Renewal of active coping responses linked to defensive burying was not observed in any group. The present findings highlight the presence of multiple psychological processes underlying even basic forms of aversive conditioning and demonstrate the importance of assessing a broader set of behaviours to tease apart these different underlying mechanisms. The current findings suggest that passive coping responses may be more reliable indicators for assessing renewal than active coping behaviours associated with defensive burying.

Extinction is a fundamental form of inhibitory learning that is important for adapting to changing environmental contingencies. While numerous studies have investigated the neural correlates of extinction using Pavlovian fear conditioning and appetitive operant reward-seeking procedures, less is known about the neural circuitry mediating the extinction of appetitive Pavlovian responding. Here, we aimed to generate an extensive brain activation map of extinction learning in a rat model of appetitive Pavlovian conditioning. Male Long-Evans rats were trained to associate a conditioned stimulus (CS; 20 s white noise) with the delivery of a 10% sucrose unconditioned stimulus (US; 0.3 ml/CS) to a fluid port. Control groups also received CS presentations, but sucrose was delivered either during the inter-trial interval or in the home-cage. After conditioning, 1 or 6 extinction sessions were conducted in which the CS was presented but sucrose was withheld. We performed Fos immunohistochemistry and network connectivity analyses on a set of cortical, striatal, thalamic, and amygdalar brain regions. Neural activity in the prelimbic cortex, ventral orbitofrontal cortex, nucleus accumbens core, and paraventricular nucleus of the thalamus was greater during recall relative to extinction. Conversely, prolonged extinction following 6 sessions induced increased neural activity in the infralimbic cortex, medial orbitofrontal cortex, and nucleus accumbens shell compared to home-cage controls. All these structures were similarly recruited during recall on the first extinction session. These findings provide novel evidence for the contribution of brain areas and neural networks that are differentially involved in the recall versus extinction of appetitive Pavlovian conditioned responding.

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 STATEMENTExtinction 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.

Extinction is a fundamental form of inhibitory learning that is important for adapting to changing environmental contingencies. While numerous studies have investigated the neural correlates of extinction using Pavlovian fear conditioning and appetitive operant reward-seeking procedures, less is known about the neural circuitry mediating the extinction of appetitive Pavlovian conditioned responding. Here, we aimed to generate an extensive brain activation map of extinction learning in a rat model of appetitive Pavlovian conditioning. Male Long-Evans rats were trained to associate a conditioned stimulus (CS; 20 s white noise) with the delivery of a 10% sucrose unconditioned stimulus (US; 0.3 ml/CS) to a fluid port. Control groups also received CS presentations, but sucrose was delivered either during the inter-trial interval or in the home-cage. After conditioning, 1 or 6 extinction sessions were conducted in which the CS was presented but sucrose was withheld. We performed Fos immunohistochemistry and network connectivity analyses on a set of cortical, striatal, thalamic, and amygdalar brain regions. Neural activity in the prelimbic cortex, ventral orbitofrontal cortex, nucleus accumbens core, and paraventricular nucleus of the thalamus was greater during recall relative to extinction. Conversely, prolonged extinction following 6 sessions induced increased neural activity in the infralimbic cortex, medial orbitofrontal cortex, and nucleus accumbens shell compared to home-cage controls. All these structures were similarly recruited during recall on the first extinction session. These findings provide novel evidence for the contribution of brain areas and neural networks that are differentially involved in the recall versus extinction of appetitive Pavlovian conditioned responding.

Environmental contexts that are reliably associated with the use of pharmacologically active substances are hypothesized to contribute to substance use disorders. In this review, we provide an updated summary of parallel preclinical and human studies that support this hypothesis. Research conducted in rats shows that environmental contexts that are reliably paired with drug use can renew extinguished drug-seeking behavior and amplify responding elicited by discrete, drug-predictive cues. Akin to drug-associated contexts, interoceptive drug stimuli produced by the psychopharmacological effects of drugs can also influence learning and memory processes that play a role in substance use disorders. Findings from human laboratory studies show that drug-associated contexts, including social stimuli, can have profound effects on cue reactivity, drug use, and drug-related cognitive expectancies. This translationally relevant research supports the idea that treatments for substance use disorders could be improved by considering drug-associated contexts as a factor in treatment interventions. We conclude this review with ideas for how to integrate drug-associated contexts into treatment-oriented research based on 4 approaches: pharmacology, brain stimulation, mindfulness-based relapse prevention, and cognitive behavioral group therapy. Throughout, we focus on alcohol- and tobacco-related research, which are two of the most prevalent and commonly misused drugs worldwide for which there are known treatments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00824-2) contains supplementary material, which is available to authorized users.

The ability of environmental cues to trigger alcohol-seeking behaviors is believed to facilitate problematic alcohol use. We previously showed that the development of this cue-evoked alcohol approach reflects cue-alcohol learning and memory in the adult male rat; however, we do not know whether the same is true for similarly aged female rats. Consequently, adult Long-Evans female rats were allowed to drink unsweetened alcohol in the home cage (Monday, Wednesday, Friday; 24-h two-bottle choice; 5 weeks) and were subsequently split into two experimental groups: Paired and Unpaired. Groups were matched for ingested doses and alcohol bottle preference across the pre-conditioning home cage period. Both groups were trained in conditioning chambers using a Pavlovian procedure. For the Paired group, the chamber houselight was illuminated to signal access to an alcohol sipper. Houselight onset was yoked for the Unpaired group, but access to the alcohol sipper was scheduled to occur only during the intervening periods (in the absence of light). We found that in the Paired, but not Unpaired group, an alcohol approach reaction was conditioned to houselight illumination, and the level of cue-conditioned reactivity predicted drinking behavior within trials. Groups experienced equivalently low but non-negligible blood alcohol concentrations over the course of conditioning sessions. We conclude that cue-triggered alcohol-seeking behavior in adult female rats reflects associative learning about the relationship between alcohol availability and houselight illumination.

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.

BackgroundIn 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.MethodsMale 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.ResultsAt 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.ConclusionsThese 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., Alcohol craving can be enhanced by alcohol-associated cues and by alcohol-associated contexts. Here the authors investigate the role of the ventral tegmental area (VTA)-to-nucleus accumbens (NAc) core and VTA-to-NAc shell circuits in mediating these distinct aspects of alcohol seeking behaviour in rats.

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.

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 Animal models are critical for studying causal explanations of relapse. Using a Pavlovian conditioning procedure with alcohol, we examined relapse after extinction triggered by either re-exposure to alcohol (reinstatement) or a delay between extinction and test (spontaneous recovery). Methods Male, Long-Evans rats were acclimated to 15% alcohol in the home-cage using an intermittent-access 2-bottle choice procedure. Next, they received Pavlovian conditioning sessions in which an auditory-conditioned stimulus (CS; 20 second white noise; 8 trials/session; variable time 240 seconds) was paired with 15% alcohol (0.3 ml/CS; 2.4 ml/session) that was delivered into a fluid port for oral ingestion. In subsequent extinction and test sessions, CS presentations occurred as before, but without alcohol. Results In experiment 1, exposure to either alcohol or water in the fluid port following extinction reinstated CS-elicited port entries at test 24 hours later. In a follow-up study using the same procedure (experiment 2), reinstatement was more robustly stimulated by alcohol, compared to a familiar lemon-flavored liquid. In experiment 3, systemic alcohol injections (0, 0.5, or 1.0 g/kg, intraperitoneal) administered either 24 hours or 15 minutes before test did not reinstate CS-elicited alcohol-seeking. Importantly, enzymatic assays in experiment 4 revealed detectable levels of alcohol in the blood following oral alcohol intake or intraperitoneal injection, suggesting that a pharmacological effect was likely with either route of administration. Last, in experiment 5, a 23-day delay between extinction and test resulted in a robust spontaneous recovery of CS-elicited alcohol-seeking. Conclusions The reinstatement and spontaneous recovery effects revealed herein provide evidence of viable new behavioral paradigms for testing interventions against relapse.

Implicit learning about antecedent stimuli and the unconditional stimulus (US) properties of alcohol may facilitate the progressive loss of control over drinking. To model this learning, Cofresi et al. (2017) developed a procedure in which a discrete, visual conditional stimulus (houselight illumination; CS) predicted the availability of a retractable sipper that rats could lick to receive unsweetened alcohol [Alcoholism: Clinical and Experimental Research, 41, 608–617]. Here we investigated the possibility that houselight illumination, sipper presentation, and oral alcohol receipt might each exert control over alcohol seeking and drinking. We also determined the relationship between ingested dose and blood alcohol concentration, in order to validate the idea that the US is a post-ingestive action of alcohol. Finally, we tested a major prediction from the conditioning account of problematic drinking [Tomie, A., & Sharma, N. (2013). Current Drug Abuse Reviews, 6, 201–219], which is that once learned, responses elicited by a CS will promote drinking. We found that despite having constrained opportunities to drink alcohol during the conditioning procedure, ingested doses produced discriminable blood concentrations that supported cue conditioning. Based on our analysis of the dynamics of cue reactivity in well-trained rats, we found that houselight illumination triggered conditioned approach, sipper presentation evoked licking behavior, and alcohol receipt promoted drinking. Reactivity to these cues, which varied in terms of their temporal proximity to the alcohol US, persisted despite progressive intoxication or satiety. Additionally, rats with the greatest conditioned reactivity to the most distal alcohol cue were also the fastest to initiate drinking and drank the most. Our findings indicate that the post-ingestive effects of alcohol may condition multiple cues simultaneously in adult rats, and these multiple cues help to trigger alcohol seeking and drinking. Moreover, identification and characterization of these cues should be helpful for designing interventions that attenuate the power of these cues over behavior.

The infralimbic medial prefrontal cortex (IL) is important for suppressing learned behavior after extinction, but whether this function extends to responses acquired through appetitive Pavlovian conditioning is unclear. We trained male, Long-Evans rats to associate a white-noise conditional stimulus (CS; 10 s; 14 presentations per session) with 10% liquid sucrose (0.2 mL per CS presentation), and recorded entries into the fluid port during the CS. The CS was presented without sucrose in subsequent extinction and test sessions. Increasing IL activity with pretest microinfusions of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 0, 0.3 nmol; 0.3 μl/side) reduced the reinstatement of CS-elicited port entries. The same result was obtained when IL neurons that expressed Channelrhodopsin-2 (ChR2) were optically stimulated during CS presentations at test (473 nm, 5 ms pulses at 20 Hz for 10.2 s, unilateral). Optical stimulation of ChR2-expressing IL neurons during CS presentations also reduced spontaneous recovery and context-induced renewal. Furthermore, optical stimulation (1) during intertrial intervals had no impact on renewal, (2) depolarized ChR2-expressing IL pyramidal neurons in vitro, and (3) preferentially increased Fos in ChR2-expressing neurons. These novel converging data highlight a critical role for the IL in suppressing the return of appetitive Pavlovian-conditioned responding following extinction.

Environmental stimuli that reliably accompany alcohol intake can become associated with the pharmacological effects of alcohol through classical (Pavlovian) conditioning. Of growing interest to addiction researchers is whether or not this process results in the attribution of incentive salience to alcohol-predictive cues, which could motivate alcohol-seeking behavior and relapse. To evaluate this question, we present a review of rodent behavioral studies that examined the capacity of alcohol-predictive cues to (i) support sign-tracking behavior, (ii) serve as conditioned reinforcers, and (iii) produce Pavlovian-to-instrumental transfer. A second, emerging area of research is focused on delineating the role of context in alcohol-seeking behavior and relapse. Here, we review studies showing that alcohol-associated contexts (i) support conditioned place preference, (ii) renew extinguished alcohol-seeking behavior, and (iii) modulate alcohol-seeking responses elicited by discrete alcohol-predictive cues. These behavioral effects may be mediated by unique psychological processes, and have important implications for cue-reactivity studies and neurobiological research.

Background Conditioned responses to alcohol-associated cues can hinder recovery from alcohol use disorder (AUD). Cue exposure (extinction) therapy (CET) can reduce reactivity to alcohol cues, but its efficacy is limited by phenomena such as spontaneous recovery and reinstatement that can cause a return of conditioned responding after extinction. Using a preclinical model of alcohol cue reactivity in rats, we evaluated whether the efficacy of alcohol CET could be improved by conducting CET during the memory reconsolidation window after retrieval of cue-alcohol associations. Methods Rats were provided with intermittent access to unsweetened alcohol. Rats were then trained to predict alcohol access based on a visual cue. Next, rats were treated with either standard extinction (n = 14) or postretrieval extinction (n = 13). Rats were then tested for long-term memory of extinction and susceptibility to spontaneous recovery and reinstatement. Results Despite equivalent extinction, rats treated with postretrieval extinction exhibited reduced spontaneous recovery and reinstatement relative to rats treated with standard extinction. Conclusions Postretrieval CET shows promise for persistently attenuating the risk to relapse posed by alcohol cues in individuals with AUD.

Context can influence the number of responses elicited by a discrete, appetitive conditioned stimulus (CS) but can context control when a CS elicits a response? To test this fundamental question, we gave male, Long-Evans rats Pavlovian conditioning sessions in which the same auditory conditioned stimulus (CS, 30 s, 15 trials/session) was presented in 2 different physical contexts on alternating days, according to a within-subjects design. In one context, called the early context, alcohol (15 % ethanol, 0.2 ml/trial) was delivered from the onset of the 5th second until the termination of the 10th second of the 30 s CS. In the second late context, alcohol was delivered from the onset of the 25th second until the termination of the 30th second of the same CS. In a comparison of the last session of training, the probability of making a conditioned response during the first four seconds of the CS was significantly higher in the early context than in the late context. This result shows that context can signal when an unconditioned stimulus occurs in relation to a CS and highlights a role for context in controlling precisely timed alcohol-seeking responses.

The prelimbic medial prefrontal cortex is implicated in promoting drug-seeking in relapse tests. However, drug-seeking behaviour is typically extinguished before a test and tests normally occur without drug delivery.We investigated the involvement of the prelimbic and the infralimbic cortex in responding elicited by a non-extinguished cue for alcohol that was presented without alcohol in an alcohol-associated context or a neutral context, and in responding to the same cue when it was paired with alcohol.Male, Long-Evans rats (220-240 g on arrival) were acclimated to 15% ethanol (v/v; 'alcohol') and then trained to associate a conditioned stimulus (10 s white noise; 15 trials/session) with alcohol delivery into a fluid port (0.2 mL/conditioned stimulus, 3 mL per session) for oral intake. Conditioning sessions occurred in a specific 'alcohol context' and were alternated daily with exposure to a second 'neutral' context that contained neither the conditioned stimulus nor alcohol.At test, functional prelimbic cortex inactivation using baclofen/muscimol reduced fluid port entries elicited by a non-extinguished conditioned stimulus that was presented without alcohol, but had no subsequent impact on port entries when the conditioned stimulus was paired with alcohol. Similar results were obtained following infralimbic cortex inactivation; however, infralimbic cortex inactivation also non-specifically reduced port entries in the absence of alcohol.These data indicate that the prelimbic and infralimbic cortex are involved in responding to cues for alcohol when alcohol delivery is omitted, but suggest that other brain regions are engaged in responding to such cues in the presence of alcohol.

Preclinical data have shown that the excitatory metabotropic Gα(q)-coupled glutamate receptor, mGluR5, has a role in substance abuse and relapse. However, little is known about the contribution of mGluR5 to the expression of conditioned responding elicited by appetitive Pavlovian cues. We investigated this question in rats that were trained to associate a discrete, auditory conditioned stimulus (CS) with a fructose-glucose solution (5.5% fructose/4.5% glucose; “sugar”). In subsequent tests for the expression of conditioned responding without sugar delivery, CS-elicited fluid port entries were elevated in a context associated with sugar, relative to an equally familiar, neutral context. Inhibiting mGluR5 via systemic injections of a negative allosteric modulator (MTEP; 5 mg/kg) reduced CS port entries in both the sugar context and neutral context. Targeting MTEP microinjections (3 µg/side; 0.3 µl/min) to the nucleus accumbens (Acb) core had no effect on CS port entries at test, whereas the same manipulation in the basolateral amygdala (BLA) produced effects that were topographically dependent. Specifically, microinjecting MTEP in the posterior BLA had no effect on behavior, whereas inhibiting mGluR5 in the anterior BLA enhanced the contextual discrimination of CS port entries. These data are the first to show a role of mGluR5 in the context-dependent expression of appetitive Pavlovian conditioned responding, with a topographically defined arrangement of mGluR5 in the BLA being particularly important for context-based responding to a discrete, appetitive cue.

Discrete and contextual cues that predict alcohol trigger alcohol-seeking. However, the extent to which context influences alcohol-seeking triggered by discrete cues, and the neural mechanisms underlying these responses, are not well known. We show that, relative to a neutral context, a context associated with alcohol persistently elevated alcohol-seeking triggered by a discrete cue, and supported higher levels of priming-induced reinstatement. Alcohol-seeking triggered by a discrete cue in a neutral context was reduced by designer receptor-mediated inhibition of ventral tegmental area (VTA) dopamine neurons in TH::Cre rats. Inhibiting terminals of VTA dopamine neurons in the nucleus accumbens (NAc) core reduced alcohol-seeking triggered by a discrete cue, irrespective of context, whereas inhibiting VTA dopamine terminals in the NAc shell selectively reduced the elevation of alcohol-seeking triggered by a discrete cue in an alcohol context. This dissociation highlights unique roles for divergent mesolimbic dopamine circuits in alcohol-seeking driven by discrete and contextual environmental cues.

An appetitive Pavlovian conditioned stimulus (CS) can predict an unconditioned stimulus (US) and acquire incentive salience. We tested the hypothesis that US intensity and motivational state of the subject would influence Pavlovian learning and impact the attribution of incentive salience to an appetitive Pavlovian CS. To this end, we examined the effects of sucrose concentration and water deprivation on the acquisition of Pavlovian conditioning and responding for a conditioned reinforcer. Male Long-Evans rats (Harlan; 220-240 g) receiving 3% (3S) or 20% (20S) sucrose were either non-water deprived or given water for 1 hr per day. During Pavlovian conditioning sessions, half the rats in each concentration and deprivation condition received a 10-s CS paired with 0.2 ml of sucrose (16 trials/session; 3.2 ml/session). The remainder received unpaired CS and US presentations. Entries into a port where sucrose was delivered were recorded. Next, responding for conditioned reinforcement was tested, wherein pressing an active lever produced the CS and pressing an inactive lever had no consequences. CS-elicited port entries increased, and latency to the first CS-elicited port entry decreased across sessions in paired groups. Water deprivation augmented these effects, whereas sucrose concentration had no significant impact on behavior. Responding for conditioned reinforcement was observed in the 20S water-deprived, paired group. Thus, water deprivation can facilitate the acquisition of Pavlovian conditioning, potentially by enhancing motivational state, and a high-intensity US and a high motivational state can interact to heighten the attribution of incentive salience to an appetitive Pavlovian CS. (PsycINFO Database Record

The environmental context in which a discrete Pavlovian conditioned stimulus (CS) is experienced can profoundly impact conditioned responding elicited by the CS. We hypothesized that alcohol-seeking behavior elicited by a discrete CS that predicted alcohol would be influenced by context and require glutamate signaling in the basolateral amygdala (BLA). Male, Long-Evans rats were allowed to drink 15% ethanol (v/v) until consumption stabilized. Next, rats received Pavlovian conditioning sessions in which a 10 s CS (15 trials/session) was paired with ethanol (0.2 ml/CS). Entries into a port where ethanol was delivered were measured. Pavlovian conditioning occurred in a specific context (alcohol context) and was alternated with sessions in a different context (non-alcohol context) where neither the CS nor ethanol was presented. At test, the CS was presented without ethanol in the alcohol context or the non-alcohol context, following a bilateral microinfusion (0.3 μl/hemisphere) of saline or the AMPA glutamate receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt) in the BLA (0, 0.3, or 1.0 μg/0.3 μl). The effect of NBQX (0, 0.3 μg/0.3 μl) in the caudate putamen (CPu) on CS responding in the non-alcohol context was also tested. The discrete alcohol CS triggered more alcohol-seeking behavior in the alcohol context than the non-alcohol context. NBQX in the BLA reduced CS responding in both contexts but had no effect in the CPu. These data indicate that AMPA glutamate receptors in the BLA are critical for alcohol-seeking elicited by a discrete CS and that behavior triggered by the CS is strongly invigorated by an alcohol context.

Alcohol self-administration produces brain and behavior adaptations that facilitate a progressive loss of control over drinking and contribute to relapse. One possible adaptation is the ability of antecedent environmental stimuli that are consistently paired with alcohol to trigger alcohol-seeking behaviors. We previously modeled this adaptation in rats using a Pavlovian conditioning procedure in which illumination of a houselight preceded the presentation of a sipper tube that produced unsweetened alcohol when licked. However, in our previous work we did not demonstrate whether this adaptation represented a consequence of repeated exposure to alcohol or the houselight, or whether it was the consequence of associative learning and memory. Thus, in the present study, we tested the associative basis of alcohol seeking in response to houselight illumination in our task using adult male rats that were not food- or water-deprived and were not dependent on alcohol. Separate groups of rats received houselight illumination that was explicitly paired or unpaired with presentation of the retractable sipper that provided access to unsweetened alcohol. Our primary dependent variable was appetitive alcohol-directed behavior: the frequency of movement toward and interaction with the hole in the wall of the chamber through which the sipper was presented during the period of houselight illumination trial before each sipper presentation. However, we also analyzed consummatory sipper licking behavior and blood ethanol concentration in the same rats. Finally, we explored the brain basis of cue-elicited alcohol seeking using c-Fos immunohistochemistry. Our findings confirmed the associative basis of cue-elicited alcohol seeking in our paradigm and mapped these onto the insular cortex, suggesting a role for this brain region in early stages of brain and behavior adaptation to regular alcohol use.

Graphical abstract, In vivo optogenetic experiments commonly employ long lengths of optical fiber to connect the light source (commonly a laser) to the optical fiber implants in the brain. Commercially available patch cords are expensive and break easily. Researchers have developed methods to build these cables in house for invivo experiments with rodents [1], [2], [3], [4]. However, the half-life of those patch cords is greatly reduced when they are used with behaving rats, which are strong enough to break the delicate cable tip and to bite through the optical fiber and furcation tubing. Based on [3] we have strengthened the patch-cord tip that connects to the optical implant, and we have incorporated multiple layers of shielding to produce more robust and resistant cladding. Here, we illustrate how to build these patch cords with FC or M3 connectors. However, the design can be adapted for use with other common optical-fiber connectors. We have saved time and money by using this design in our optical self-stimulation experiments with rats, which are commonly several months long and last four to eleven hours per session. The main advantages are: • Long half-life. • Resistant to moderate rodent bites. • Suitable for long in vivo optogenetic experiments with large rodents.

Introduction Drug craving can be independently stimulated by cues that are directly associated with drug intake (discrete drug cues), as well as by environmental contexts in which drug use occurs (contextual drug cues). We tested the hypothesis that the context in which a discrete alcohol-predictive cue is experienced can influence how robustly that cue stimulates alcohol-seeking behavior. Methods Male, Long-Evans rats received Pavlovian discrimination training (PDT) sessions in which one conditioned stimulus (CS+; 16 trials/session) was paired with ethanol (0.2 mL/CS+) and a second stimulus (CS−; 16 trials/session) was not. PDT occurred in a specific context, and entries into a fluid port where ethanol was delivered were measured during each CS. Next, rats were acclimated to an alternate (nonalcohol) context where cues and ethanol were withheld. Responses to the nonextinguished CS+ and CS− were then tested without ethanol in the alcohol-associated PDT context, the nonalcohol context or a third, novel context. Results Across PDT the CS+ elicited more port entries than the CS−, indicative of Pavlovian discrimination learning. At test, the CS+ elicited more port entries than the CS− in all three contexts: however, alcohol seeking driven by the CS+ was more robust in the alcohol-associated context. In a separate experiment, extinguishing the context-alcohol association did not influence subsequent CS+ responding but reduced alcohol seeking during non-CS+ intervals during a spontaneous recovery test. Conclusion These results indicate that alcohol-seeking behavior driven by a discrete Pavlovian alcohol cue is strongly invigorated by an alcohol context, and suggest that contexts may function as excitatory Pavlovian conditioned stimuli that directly trigger alcohol-seeking behavior.

Environmental contexts associated with drug use promote craving in humans and drug-seeking in animals. We hypothesized that the basolateral amygdala (BLA) itself, as well as serial connectivity between the basolateral amygdala (BLA) and nucleus accumbens core (NAC core), were required for context-induced renewal of Pavlovian-conditioned alcohol-seeking. Male, Long-Evans rats were trained to discriminate between two conditioned stimuli (CS) - a CS+ that was paired with ethanol (EtOH, 20%, v/v) delivery into a fluid port (0.2 ml/CS+, 3.2 ml/session) and a CS− that was not. Entries into the port during each CS were measured. Next, rats received extinction in a different context where both cues were presented without EtOH. At test, responding to the CS+ and CS− without EtOH was evaluated in the prior training context. Control subjects showed a selective increase in CS+ responding relative to extinction, indicative of renewal. This effect was blocked by pre-test, bilateral inactivation of the BLA using a solution of gamma-amino-butyric-acid receptor agonists (0.1 mM muscimol and 1.0 mM baclofen; M/B; 0.3 µl/side). Renewal was also attenuated following unilateral injections of M/B into the BLA, combined with either M/B, the dopamine D1 receptor antagonist SCH 23390 (0.6 µg/side), or saline infusion in the contralateral NAC core. Hence, unilateral BLA inactivation was sufficient to disrupt renewal, highlighting a critical role for functional activity in the BLA in enabling the reinstatement of alcohol-seeking driven by an alcohol context.

Individual differences exist in the attribution of incentive salience to conditioned stimuli associated with food. Here, we investigated whether individual differences also manifested with a Pavlovian alcohol conditioned stimulus (CS). We compiled data from five experiments that used a Pavlovian autoshaping paradigm and tests of conditioned reinforcement. In all experiments, male, Long-Evans rats with unrestricted access to food and water were acclimated to 15% ethanol. Next, rats received Pavlovian autoshaping training, in which a 10 s presentation of a retractable lever served as the CS and 0.2 mL of 15% ethanol served as the unconditioned stimulus (US). Finally, rats underwent conditioned reinforcement tests in which nose-pokes to an active aperture led to brief presentations of the lever-CS, but nose-pokes to an inactive aperture had no consequence. Rats were categorized as sign-trackers, goal-trackers, and intermediates based on a response bias score that reflected their tendencies to sign-track or goal-track at different times during training. We found that distinct groups of rats either consistently interacted with the lever-CS (‘sign-trackers’) or routinely approached the port during the lever-CS (‘goal-trackers’) across a majority of the training sessions. However, some individuals (‘shifted sign-trackers’) with an early tendency to goal-track later shifted to comparable asymptotic levels of sign tracking as the group identified as sign-trackers. The lever-CS functioned as a conditioned reinforcer for sign-trackers and shifted sign-trackers, but not for goal-trackers. These results provide evidence of robust individual differences in the extent to which a Pavlovian alcohol cue gains incentive salience and functions as a conditioned reinforcer.

Varenicline, a pharmacotherapy for tobacco addiction, reduces alcohol consumption in humans and rodents. The therapeutic potential of varenicline would escalate if it also diminished conditioned responses elicited by alcohol-predictive cues, which can precipitate relapse in abstinent individuals. We investigated this application, along with the underlying neural substrates, using a robust preclinical assay in which relapse to alcohol-seeking was triggered by re-exposure to an alcohol-associated environmental context. Male, Long-Evans rats received Pavlovian conditioning sessions in which one auditory conditioned stimulus (CS+) was paired with 15% ethanol and a second conditioned stimulus (CS-) was not. Ethanol was delivered into a port for oral consumption and port entries triggered by each CS were recorded. Extinction was then conducted in a different context where the CS+ and CS- were presented without ethanol. To stimulate relapse, both cues were subsequently presented without ethanol in the prior conditioning context. Systemic varenicline (0, 0.5 or 2.5 mg/kg; intraperitoneal) blocked context-induced relapse to alcohol-seeking without affecting the ability to make a port entry. It also reduced context-induced relapse to sucrose-seeking, but only at the 2.5 mg/kg dose. Neuropharmacological studies showed that context-induced relapse to alcohol-seeking was attenuated by bilateral microinfusion of varenicline (0.3 μl/side) into the nucleus accumbens (NAc; 0 or 3.5 μg), but not the ventral tegmental area (0, 2 or 4 μg). These data show for the first time that varenicline reduces relapse triggered by contexts that predict alcohol, and suggest that nicotinic acetylcholine receptors in the NAc are critical for this effect.

Nicotine enhances responding elicited by Pavlovian cues that predict positive outcomes. We tested the hypothesis that nicotine acting at nicotinic acetylcholine receptors (nAChRs) would augment Pavlovian alcohol-seeking. Male, Long-Evans rats with unrestricted access to food and water were acclimated to drinking 15% ethanol in their home cages and then given Pavlovian conditioning sessions in which each trial of a 15-s conditioned stimulus (CS, 12 trials/session) was paired with 0.2 ml of ethanol (unconditioned stimulus, US, 2.4 ml/session). Entries into a port where ethanol was delivered were used to assess conditioning. Control groups received explicitly unpaired trials of the CS and US. In experiment 1, systemic injections of saline (1 ml/kg) or nicotine (0.4 mg/kg, freebase) were administered before each session. In experiments 2 and 3, an identical regimen of saline or nicotine injections was administered before the start of Pavlovian conditioning sessions. All paired groups acquired conditioned port-entry responding to the CS, indicative of Pavlovian alcohol-seeking, whereas unpaired control group did not. Pre-session nicotine injections increased CS port-entries relative to saline, only in the paired group. This nicotine-induced enhancement of Pavlovian alcohol-seeking was blocked by pre-treatment with the nAChR antagonist mecamylamine. Prior exposure to nicotine did not influence the subsequent acquisition of Pavlovian alcohol-seeking. These findings highlight for the first time that nicotine acting at nAChRs augments Pavlovian alcohol-seeking, specifically in non-restricted rats. Individuals who smoke and drink may thus be particularly susceptible to alcohol cues that could trigger further drinking.

Experimenter-administered nicotine produces reliable increases in blood pressure and changes in heart rate. However, an extensive literature demonstrates that the effects of psychoactive drugs are dependent on whether administration is contingent on behavior. The present study assessed the cardiovascular effects of nicotine and whether those effects vary as a function of whether nicotine was self-administered or response-independent. Rats were divided into three groups according to a yoked design. The pattern of infusions for each triad was determined by the animal self-administering nicotine; the other two animals received either yoked nicotine or saline. Heart rate and blood pressure were measured during eighteen daily, 1 hour drug sessions by radiotelemetry. Each session was preceded and followed by a 20 minute period during which cardiovascular function was monitored in the operant chambers, but drug was not available. Acute exposure to yoked nicotine produced a rapid rise in blood pressure that was larger than the increase observed with self-administered nicotine. Additional infusions during the first session resulted in a similar sustained elevation in blood pressure in the nicotine groups. Over subsequent sessions, self-administered nicotine produced a larger effect on systolic blood pressure particularly early in each session, although for both self-administered and yoked nicotine the hypertensive effects waned partially with repeated test sessions. This decrease was fully accounted for by a pre-session decrease in pressure; relative to pre-session levels the strong hypertensive effects of nicotine persisted. Initial exposure to nicotine produced a short-lived bradycardia that in subsequent sessions was replaced with a longer-lasting nicotine-induced tachycardia; neither effect was related to the behavioral contingency of nicotine delivery. Together, these data provide a rich picture of the cardiovascular effects of nicotine. Effects of behavioral contingency were observed, but differences were limited. Other non-pharmacological factors such as baseline shifts potentially related to nicotine-associated cues deserve further attention.

The reinstatement of ethanol (EtOH)-seeking induced by an EtOH-predictive light-tone stimulus is enhanced in an environment associated with prior EtOH self-administration (SA) compared with a context associated with EtOH unavailability (Tsiang & Janak, 2006). Here we hypothesized that EtOH-seeking would be elicited by the conditioned sensory stimulus properties of EtOH and that this reinstatement would be similarly modulated by context. We also determined whether pharmacologically inactivating the nucleus accumbens (NAc), a key structure in relapse circuitry, would attenuate reinstated EtOH-seeking. Rats lever-pressed for oral EtOH (10% v/v) in operant conditioning chambers distinguished by specific visual, olfactory and tactile stimuli. Responding was then extinguished by withholding EtOH in a different context. EtOH-seeking, expressed as elevated responding without EtOH delivery, was subsequently tested by presenting an oral EtOH prime (two aliquots of 0.1 mL EtOH) in either the extinction or the prior EtOH-SA context. Rats received a microinfusion (0.3 μL/hemisphere) of saline or GABA agonists (muscimol/baclofen) into the NAc core or shell immediately before the reinstatement test. Robust EtOH-seeking was observed in the prior EtOH-SA but not the extinction context in saline-pretreated rats. This effect was significantly attenuated by inactivating the NAc core but not shell. Conversely, NAc shell inactivation significantly elevated lever-pressing in the extinction context. These data suggest that the sensory stimulus properties of oral EtOH can reinstate EtOH-seeking when experienced in the appropriate context and that functional activity in the NAc core is required for this effect. In contrast, the shell may normally inhibit incorrect behavioral responses.

Background The environmental context in which drug-conditioned cues are encountered could modulate the capacity of such cues to trigger relapse in abstinent addicts. We explored this hypothesis using a behavioral animal model. Methods Rats were trained to discriminate between two auditory stimuli; the first (CS+) was paired with 10% ethanol and the second (CS−) was presented without ethanol. Training occurred in operant conditioning chambers equipped with distinct contextual stimuli, and entries into the ethanol delivery port during the stimuli were measured. Behavior was then extinguished by presenting both stimuli without ethanol in a second, different context. Context-dependent renewal of port entries was tested by presenting the CS+ and CS− without ethanol in the original training context. Results At test, port entries during the CS+ increased compared with extinction levels, while responding during the CS− remained unchanged ( n = 11). This effect was attenuated after multiple extinction sessions in three distinct contexts ( n = 18), compared with an equivalent number of extinction sessions in a single unique context ( n = 16). Context-dependent renewal of port entries was also observed to a CS+ paired with 14% sucrose ( n = 7) but not to a CS+ paired with 2% sucrose ( n = 8). Conclusions Environmental contexts can trigger the relapse of behavioral responding to ethanol- and sucrose-predictive cues in rats. For ethanol, this effect can be reduced by extinguishing responses to the ethanol cue in multiple distinct contexts, a manipulation that could increase the efficacy of cue-reactivity treatments for addiction.

Addiction is characterized by compulsive alcohol or drug taking and seeking, and the dorsal striatum has been implicated in such maladaptive persistent habits. The NMDA receptor (NMDAR), which is a major target of alcohol, is implicated in striatal-based habit learning. We found that, in the dorsal striatum, alcohol (ethanol) exposure produced an increase in the phosphorylation of the NR2B subunit of the NMDAR, and a corresponding increase in the activity of Fyn kinase, which phosphorylates NR2B. We further observed an ethanol-mediated long-term facilitation (LTF) of the activity of NR2B-containing NMDARs (NR2B-NMDARs) in the dorsal striatum. This LTF is Fyn kinase dependent, because it was observed in Fyn wild-type but not in Fyn knock-out mice. Importantly, none of these biochemical and physiological changes was observed in the ventral striatum. Finally, dorsal but not ventral striatum infusion of a Fyn or NR2B-NMDAR inhibitor reduced rat operant self-administration of ethanol. Our results suggest that the Fyn-mediated phosphorylation and LTF of NR2B-NMDAR activity in the dorsal striatum after exposure to ethanol may underlie aberrant plasticity that contributes to mechanisms underlying alcohol drinking behavior.

Neurobiological mechanisms that influence behavior in the presence of alcohol-associated stimuli involve processes that organize behavior during the presence of these cues, and separately, regulation of behavior in their absence. However, little is known about anatomical structures that might mediate this regulation. Here we examined nucleus accumbens shell (AcbSh) as a possible neural substrate mediating behavior modulation triggered by the presence and absence of alcohol-associated environmental cues and contexts. We also examined subregions of basal amygdala nuclei— rostral basolateral (BLA) and basal posterior (BAP)— as they provide a major source of glutamatergic input to the AcbSh. Animals were trained to associate an auditory conditioning stimulus with alcohol in a discriminative context and then subsequently tested for conditioned port-entries across contexts either previously associated or not associated with alcohol. We found that, on test to the alcohol cue alone, AcbSh inactivation prevented conditioned port-entries in contexts that either were associated with alcohol or were novel, while also increasing unconditioned port-entries during the intertrial intervals. When tested to alcohol-reinforced cues, AcbSh inactivation produced more cue-trial omissions and elevated unconditioned port-entries. Interestingly, BLA and BAP inactivation produced dissociable effects. BAP but not BLA increased unconditioned port-entries, while both manipulations prevented conditioned port-entries during the alcohol-cue. We conclude that AcbSh is necessary for modulating control over behavior otherwise guided by the presence of alcohol-predictive environmental stimuli and contexts. Moreover, this role may involve integration of functionally segregated inputs from rostral and posterior portions of basal amygdala nuclei.

Environmental stimuli that are reliably paired with alcohol may acquire incentive salience, a property that can operate in the use and abuse of alcohol. Here we investigated the incentive salience of Pavlovian alcohol cues using a preclinical animal model. Male, Long-Evans rats (Harlan) with unrestricted access to food and water were acclimated to drinking 15% ethanol (v/v) in their home-cages. Rats then received Pavlovian autoshaping training in which the 10 second presentation of a retractable lever served as the conditioned stimulus (CS) and 15% ethanol served as the unconditioned stimulus (US) (0.2 ml/CS; 12 CS presentations/session; 27 sessions). Next, in an operant test of conditioned reinforcement, nose pokes into an active aperture delivered presentations of the lever-CS, whereas nose pokes into an inactive aperture had no consequences. Across initial autoshaping sessions, goal-tracking behavior, as measured by entries into the fluid port where ethanol was delivered, developed rapidly. However, with extended training goal-tracking diminished, and sign-tracking responses, as measured by lever-CS activations, emerged. Control rats that received explicitly unpaired CS and US presentations did not show goal-tracking or sign-tracking responses. In the test for conditioned reinforcement, rats with CS-US pairings during autoshaping training made more active relative to inactive nose pokes, whereas rats in the unpaired control group did not. Moreover, active nose pokes were positively correlated with sign-tracking behavior during autoshaping. Extended training may produce a shift in the learned properties of Pavlovian alcohol cues, such that after initially predicting alcohol availability they acquire robust incentive salience.

Nicotine self-administration in rats is modest when response-contingent nicotine infusions are delivered alone (primary reinforcement) but robust when nicotine infusions are combined with a mildly reinforcing non-pharmacological stimulus. Furthermore, response-independent (non-contingent) nicotine administration also elevates responding for that same non-pharmacological stimulus, suggesting that in addition to primary reinforcement, nicotine can enhance the incentive value of other reinforcers. In this study, we tested the hypothesis that the reinforcement-enhancing effects of non-contingent nicotine are more dependent on the reinforcing strength of the non-pharmacological stimulus than are the effects of contingent nicotine. A weakly reinforcing light-tone stimulus was established as a conditioned reinforcer by repeated pairings with sucrose for some rats, or by delivery in an explicitly unpaired design with sucrose to other rats. Subsequently, both groups lever pressed for the stimulus with contingent nicotine, non-contingent nicotine (0.06 mg kg−1 per infusion, freebase), or non-contingent saline, according to fixed ratio and progressive ratio reinforcement schedules. Compared to sucrose-unpaired training, repeated association with sucrose established the light-tone stimulus as a robust conditioned reinforcer. Contingent and non-contingent nicotine equally elevated responding for this conditioned stimulus. Conversely, for the less reinforcing (sucrose-unpaired) stimulus contingent nicotine more effectively elevated behavior compared to non-contingent nicotine. The reinforcement-enhancing effect of nicotine increases behavior controlled by both conditioned and unconditioned reinforcers; however, for less salient stimuli associative processes derived from the primary reinforcing effects of contingent nicotine may also be important. These data suggest that nicotine present in tobacco may differentially modulate stimulus-driven behavior in smokers.

Invasive cervical carcinomas almost invariably carry extra copies of chromosome arm 3q, resulting in a gain of the human telomerase gene (TERC). This provided the rationale for the development of a multicolor fluorescence in situ hybridization (FISH) probe set as a diagnostic tool for the direct detection of TERC gains in Pap smears. We previously used this probe set to show that cervical intraepithelial neoplasia (CIN) 2 and CIN3 lesions could be distinguished from normal samples, atypical squamous cell of undetermined significance (ASCUS) and CIN1, with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In the current study, we explored whether gain of 3q and amplification of TERC could predict progression from CIN1/CIN2 to CIN3 and invasive carcinoma. We applied our probe set to a series of 59 previously stained Pap smears for which repeat Pap smears and clinical follow-up were available. The samples included CIN1/CIN2 lesions that progressed to CIN3 (progressors), CIN1/CIN2 lesions that regressed spontaneously (regressors), and normal Pap smears from women who subsequently developed CIN3 or cervical cancer. Here, we show that progressors displayed a gain of 3q whereas none of the regressors showed this genetic aberration. These data suggest that 3q gain is required for the transition from CIN1/CIN2 to CIN3 and that it predicts progression. Of note, 3q gain was found in 33% of cytologically normal Pap smears from women who were diagnosed with CIN3 or invasive cervical carcinoma after a short latency. The sensitivity of our test for predicting progression from CIN1/CIN2 to CIN3 was 100% and the specificity, ie, the prediction of regression, was 70%. We conclude that the detection of 3q gain and amplification of TERC in routinely collected Pap smears can assist in identifying low-grade lesions with a high progression risk and in decreasing false-negative cytological screenings.

Sex differences have been reported for the impact of nicotine and nonpharmacological cues on smoking. While nonpharmacological environmental stimuli have also been shown to influence nicotine self-administration in rats, there have been no attempts to examine the impact of sex differences in the contributions of nicotine and nondrug stimuli to this behavior.This experiment investigated sex differences in operant responding for nicotine in rats when drug infusions were delivered either in the absence of, or in combination with, a nonpharmacological stimulus.Initially, male and female rats acquired self-administration for nicotine alone across a range of doses (0.03, 0.06, and 0.15 mg kg(-1) inf(-1), freebase). After stable acquisition, nicotine infusions were combined with a weakly reinforcing, compound visual stimulus.While there was no overall effect of dose on active lever responding for nicotine in the absence of the visual stimulus, female rats responded more on the reinforced lever than males at 0.06 and 0.15 mg kg(-1) inf(-1) on an FR5 schedule. However, they also showed increased responding on the nonreinforced lever compared to males at the same doses. Combining nicotine infusions with the visual stimulus doubled responding compared to nicotine alone at 0.03 and 0.06, but not at 0.15 mg kg(-1) inf(-1): this effect was significantly greater for female rats.These data highlight the prominent contribution of nonpharmacological stimuli to nicotine-reinforced behavior across a range of doses in both male and female rats. They also reveal sex differences in operant responding for nicotine under conditions where a nonpharmacological stimulus is either absent, or combined with drug delivery.

Current conceptualizations of drug reinforcement assume that drug-taking behavior is a consequence of the contingent, temporal relationship between the behavior and drug reward. However, stimulant drugs also potentiate the rewarding effects of other reinforcers when administered noncontingently. These studies were designed to determine whether noncontingent nicotine enhances the reinforcing properties of a nonpharmacological reinforcer and whether this direct effect facilitates operant behavior within the context of a nicotine self-administration procedure. Rats self-administered nicotine or food, or received noncontingent nicotine, saline, or food either with or without a response-contingent, unconditioned reinforcing visual stimulus (VS). Noncontingent nicotine, whether delivered as discrete injections based on a pattern of self-administered nicotine or as a continuous infusion, increased response rates maintained by the VS. There were no significant differences in responding by animals that received contingent compared with noncontingent nicotine when a VS was available. This increase was not observed in the absence of the VS or as a consequence of noncontingent food delivery. Operant behavior was equally attenuated and reinstated by the removal and subsequent replacement of contingent and noncontingent nicotine. Nicotine supported self-administration in the absence of response-contingent, nicotine-paired stimuli; however, response rates were drastically reduced compared with nicotine self-administration with the VS. Nicotine influences operant behavior in two ways: by acting as a primary reinforcer when it is contingent upon behavior, and by directly potentiating the reinforcing properties of other stimuli through a nonassociative mechanism. Nicotine self-administration and smoking may be largely dependent upon this later action.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

All recipients of non-genetically identical solid organ transplants are at risk for rejection initiated by alloantigens and must be pharmacologically immunosuppressed to prevent rejection mediated by T cell recognition of these nonself HLA molecules or peptides as foreign. However, a potential transplant recipient may also be at risk for rejection initiated by alloantibodies if they were exposed pretransplant to foreign antigens that are also present on the allograft. When these preexisting alloantibodies are directed against ABO group antigens, HLA class I antigens, endothelial-monocyte antigens, and perhaps HLA class II antigens that are also present on the allograft, they can initiate immediate (hyperacute) or delayed humoral immune responses against the graft (N Engl J Med 280:735–9, 1969; Clinical Transplants 1985, Los Angeles. p. 139). Patients who possess such alloantibodies prior to transplantation are considered to be “sensitized.” Highly sensitized patients, especially those who have high levels of circulating anti-HLA class I antibodies, face significant difficulties in finding a compatible donor and encounter a worse prognosis for the organ after transplantation (Engl J Med 348:1033–44, 2003; Clin Transpl 1–18, 2000). According to the Organ Procurement and Transplant Network (UNOS data), graft survival rates for patients transplanted between 1997 and 2004 in the United States were 72.2 % at 5 years for those with a PRA of 0–9 % versus 65.7 % for those with a PRA of 10–80 % (http://optn.transplant.hrsa.gov). As such, these patients present difficult challenges to transplant physicians and to organ allocation systems. Until recent years, these patients faced hopelessly long waits for a crossmatch-negative kidney. However, in the past several years, much progress has been made in managing highly sensitized patients by more optimally pairing the recipient with a compatible organ and, more recently, by suppressing the powerful humoral immune response elicited by the alloantibodies.