Approval of Mycophenolate Mofetil for Prophylaxis of Organ Rejection in Pediatric Recipients of Heart or Liver Transplants: A Regulatory Perspective.

On June 6, 2022, the FDA expanded the indications for mycophenolate mofetil (MMF) to include the prophylaxis of organ rejection in combination with other immunosuppressants in pediatric recipients of allogeneic heart or liver transplants aged 3 months and older. The approved oral dosing regimen for these patients was a starting dose of 600 mg/m2 with titration up to a maximum of 900 mg/m2 twice daily. Data to support efficacy in pediatric patients were derived from established pharmacokinetic (PK) relationships across approved populations, a PK study in pediatric liver transplant recipients, and information from the Scientific Registry of Transplant Recipients database. Information supporting safety was based on comparing mycophenolic acid (MPA) exposure with that in pediatric kidney transplant recipients, the published literature, and post-marketing safety reports. Efficacy in pediatric patients was established based on extrapolation of efficacy from studies in adult liver, adult heart, and pediatric kidney transplant populations, and similarity in MPA exposure between pediatric and adult patients. Review of the data supported an oral dosing regimen for pediatric heart transplant and liver transplant recipients consisting of a starting dose of 600 mg/m2 up to a maximum of 900 mg/m2 b.i.d. A dosage range for MMF is recommended recognizing that the MMF dose may be modified in clinical practice for myriad factors. The dosage recommendations in the labeling for pediatric liver and pediatric heart transplant patients are intended to permit individualized dosing based on clinical assessment of these factors.

Augmenting glutamatergic, but not dopaminergic, activity in the nucleus accumbens shell disrupts responding to a discrete alcohol cue in an alcohol context.

Discrete alcohol cues and contexts are relapse triggers for people with alcohol use disorder exerting particularly powerful control over behaviour when they co-occur. Here, we investigated the neural substrates subserving the capacity for alcohol-associated contexts to elevate responding to an alcohol-predictive conditioned stimulus (CS). Specifically, rats were trained in a distinct 'alcohol context' to respond by entering a fluid port during a discrete auditory CS that predicted the delivery of alcohol and were familiarized with a 'neutral context' wherein alcohol was never available. When conditioned CS responding was tested by presenting the CS without alcohol, we found that augmenting glutamatergic activity in the nucleus accumbens (NAc) shell by microinfusing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) reduced responding to an alcohol CS in an alcohol, but not neutral, context. Further, AMPA microinfusion robustly affected behaviour, attenuating the number, duration and latency of CS responses selectively in the alcohol context. Although dopaminergic inputs to the NAc shell were previously shown to be necessary for CS responding in an alcohol context, here, chemogenetic excitation of ventral tegmental area (VTA) dopamine neurons and their inputs to the NAc shell did not affect CS responding. Critically, chemogenetic excitation of VTA dopamine neurons affected feeding behaviour and elevated c-fos immunoreactivity in the VTA and NAc shell, validating the chemogenetic approach. These findings enrich our understanding of the substrates underlying Pavlovian responding for alcohol and reveal that the capacity for contexts to modulate responding to discrete alcohol cues is delicately underpinned by the NAc shell.

Blocking µ-opioid receptors attenuates reinstatement of responding to an alcohol-predictive conditioned stimulus through actions in the ventral hippocampus.

The µ-opioid system is involved in the reinstatement of responding that is immediately evoked by alcohol-predictive cues. The extent of its involvement in reinstatement observed in a new model that evaluates the delayed effects of re-exposure to alcohol, however, is unclear. The current study investigated the role of µ-opioid receptors (MORs) in the delayed reinstatement of an extinguished, Pavlovian conditioned response that was evoked 24 h after alcohol re-exposure. Female and male Long-Evans rats received Pavlovian conditioning in which a conditioned stimulus (CS) was paired with the delivery of an appetitive unconditioned stimulus (US; Experiments 1, 2, 4: 15% v/v alcohol; Experiment 3: 10% w/v sucrose) that was delivered into a fluid port for oral intake. During subsequent extinction sessions, the CS was presented as before but without the US. Next, the US was delivered but without the CS. A reinstatement test was conducted 24 h later, during which the CS was presented in the absence of the US. Silencing MORs via systemic naltrexone (0.3 or 1.0 mg/kg) attenuated reinstatement of port entries elicited by an alcohol-CS, but not those elicited by a sucrose-CS. Finally, blocking MORs in the ventral hippocampus via bilateral microinfusion of D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 2.5 or 5.0 µg/hemisphere) prevented reinstatement of port alcohol-CS port entries. These data show that MORs are involved in the delayed reinstatement of a Pavlovian conditioned response in an alcohol-specific manner. Importantly, these data illustrate, for the first time, that MORs in the ventral hippocampus are necessary for responding to an alcohol-predictive cue.

Context-induced renewal of passive but not active coping behaviours in the shock-probe defensive burying task.

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.

Optogenetic stimulation of infralimbic cortex projections to the paraventricular thalamus attenuates context-induced renewal.

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.

Neural correlates of recall and extinction in a rat model of appetitive Pavlovian conditioning.

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.

Learning processes in relapse to alcohol use: lessons from animal models.

RATIONALE: Alcohol use is reliably preceded by discrete and contextual stimuli which, through diverse learning processes, acquire the capacity to promote alcohol use and relapse to alcohol use. OBJECTIVE: We review contemporary extinction, renewal, reinstatement, occasion setting, and sex differences research within a conditioning framework of relapse to alcohol use to inform the development of behavioural and pharmacological therapies. KEY FINDINGS: Diverse learning processes and corresponding neurobiological substrates contribute to relapse to alcohol use. Results from animal models indicate that cortical, thalamic, accumbal, hypothalamic, mesolimbic, glutamatergic, opioidergic, and dopaminergic circuitries contribute to alcohol relapse through separable learning processes. Behavioural therapies could be improved by increasing the endurance and generalizability of extinction learning and should incorporate whether discrete cues and contexts influence behaviour through direct excitatory conditioning or occasion setting mechanisms. The types of learning processes that most effectively influence responding for alcohol differ in female and male rats. CONCLUSION: Sophisticated conditioning experiments suggest that diverse learning processes are mediated by distinct neural circuits and contribute to relapse to alcohol use. These experiments also suggest that gender-specific behavioural and pharmacological interventions are a way towards efficacious therapies to prevent relapse to alcohol use.

Supplementary dataset of context-dependent conditioned responding to an alcohol-predictive cue in female and male rats.

This supplementary dataset is supportive of the research article entitled 'The role of context on responding to an alcohol-predictive cue in female and male rats' [1]. This article describes the raw data pertaining to the behaviour of male and female rats during intermittent to ethanol and Pavlovian conditioning training and testing procedures. Specifically, the dataset describes the alcohol consumption and ingested-dose of ethanol during home-cage ethanol exposure, as well as the conditioned responding during Pavlovian discrimination training, a test assessing the effect of context on responding to an alcohol-predict cue in the absence of alcohol, and a reinstatement test assessing the effect of context on conditioned responding to an extinguished alcohol-predictive cue.

The role of context conditioning in the reinstatement of responding to an alcohol-predictive conditioned stimulus.

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.

The role of context on responding to an alcohol-predictive cue in female and male rats.

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.

Corticostriatal Suppression 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 (IL) cortex to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behavior. 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 male Long Evans 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, however, does not appear to be 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 nonreinforcement. We investigated the role of the IL cortex inputs to the 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 used by future research aimed at understanding how corticostriatal circuits contribute to behavioral flexibility and mental disorders that involve the suppression of learned behaviors.

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

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

Dissociable mesolimbic dopamine circuits control responding triggered by alcohol-predictive discrete cues and contexts.

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.

Comparing ABA, AAB, and ABC Renewal of Appetitive Pavlovian Conditioned Responding in Alcohol- and Sucrose-Trained Male 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.

Considering Drug-Associated Contexts in Substance Use Disorders and Treatment Development.

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.

The medial prefrontal cortex is required for responding to alcohol-predictive cues but only in the absence of alcohol delivery.

BACKGROUND: 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. AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cue-alcohol associative learning in female rats.

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.

Context and topography determine the role of basolateral amygdala metabotropic glutamate receptor 5 in appetitive Pavlovian responding.

Preclinical data have shown that the excitatory metabotropic Gαq-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.

Alcohol-associated antecedent stimuli elicit alcohol seeking in non-dependent rats and may activate the insula.

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.

Modeling Relapse to Pavlovian Alcohol-Seeking in Rats Using Reinstatement and Spontaneous Recovery Paradigms.

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.