Alcohol-associated antecedent stimuli elicit alcohol seeking in non-dependent rats and may activate the insula
Introduction
Classical conditioning allows antecedent sensory stimuli to become associated with alcohol availability, ingestion, and/or pharmacological effects. Conditioned behavioral and physiological reactivity to these alcohol-predictive cues contributes to the risk for relapse to problem drinking. We recently developed a model of alcohol cue reactivity in rats (Cofresí et al., 2017, Cofresí et al., 2018). Here, we verify whether reactivity to the alcohol cue in our model stems from associative learning and whether memory for that cue-alcohol association maps onto brain regions that are implicated in alcohol addiction.
In our paradigm, conditioning trials involve illumination of a houselight (the cue) and brief availability of a retractable sipper from which rats can lick out unsweetened alcohol. Houselight illumination appears to gain the ability to elicit anticipatory approach to the sipper. If this response reflects a learned association with alcohol availability, then it should only be acquired when illumination is explicitly paired with alcohol availability, in a classic “Paired” conditional-unconditional stimulus (CS-US) arrangement. However, if the same alcohol-seeking response is acquired when alcohol availability is explicitly “unpaired” with illumination by presenting the sipper only during the interval between illuminations (“Unpaired” CSUS arrangement), then it may stem from a non-associative learning process (e.g., sensitization). The associative basis of cue-alcohol associations has been shown previously in studies that used Paired and Unpaired groups (Srey, Maddux, & Chaudhri, 2015); however, in that task alcohol delivery occurred immediately after presentation of the CS, and the CS was a retractable lever.
Another reason for comparing a Paired and Unpaired group in our paradigm is that since alcohol delivery occurs via a sipper tube, the presentation of this sipper tube may itself be learned as a cue, and acquire the ability to initiate drinking bouts. However, sipper presentation is always experienced during houselight illumination. As such, what looks like a response to another distinct cue (sipper presentation) may actually represent facilitation of consummatory sipper licking behavior by the houselight cue-alcohol association. If so, then once the houselight cue-alcohol association is formed, rats in the “Paired” cue group should initiate drinking bouts faster than rats in the “Unpaired” cue group.
Cue-related behavioral reactivity in other rodent models of alcohol cue reactivity has been shown to reflect associative learning. However, we cannot assume that cue-related reactivity in our procedure also reflects associative learning, as our procedure differs substantially from others. In the conditioned place avoidance/preference procedure, which has taught us that alcohol-associated cues can acquire both appetitive and aversive properties in rodents, the cue predicts experimenter-administered alcohol (e.g., Bormann and Cunningham, 1998, Bozarth, 1990, Ciccocioppo et al., 1999, Cunningham, 1979, Cunningham, 1981, Fidler et al., 2004, Nentwig et al., 2017, Torres et al., 2014). In procedures where the rodent self-administers alcohol, cues typically acquire appetitive properties such as the ability to invigorate instrumental responding (Corbit and Janak, 2007, Glasner et al., 2005, Krank et al., 2008, Milton et al., 2012), the ability to elicit sign-tracking conditioned responses (e.g., Krank, 2003, Krank et al., 2008, Srey et al., 2015, Villaruel and Chaudhri, 2016), and the ability to act as conditioned positive reinforcers for the acquisition of new instrumental responses (e.g., Schramm et al., 2016, Srey et al., 2015). However, in some cases, even cues paired with self-administered alcohol can acquire aversive properties (e.g., Stewart & Grupp, 1986). In most of the self-administration models, the cue predicts delivery of a fixed amount of alcohol, which is available for ingestion even when the cue is not present (e.g., Krank et al., 2008, Remedios et al., 2014). In many studies, access to food and/or water in the homecage is restricted or alcohol is sweetened to motivate rodents to drink alcohol in the conditioning chamber (e.g., Krank, 2003, Millan et al., 2015, Tomie et al., 2002). In our procedure, rats have free access to food and water in the homecage and alcohol is never sweetened. Moreover, rats are free to drink as much alcohol as they want during conditioning trials. Importantly, our procedure also allows us to measure drinking behavior (consummatory sipper licking) directly for each conditioning trial.
Many therapies for alcohol use disorder (e.g., naltrexone, cue exposure, counterconditioning) are predicated on the idea that maladaptive alcohol-associative memories (implicit or not) drive relapse. Knowing whether alcohol-related behavior stems from associative or non-associative memory is important because different approaches (behavioral and pharmacological) are needed to target associative and non-associative aspects of behavior. Ideally, we would know the mechanisms how (associative vs. non-associative), as well as the sites where alcohol-related cue memories are encoded in the brain. Consequently, in the present study, using the same rats in which we characterized behavior and blood ethanol, we evaluated expression of the immediate-early gene product c-Fos as an index of cellular activity in brain regions that may be involved in maintaining and/or expressing alcohol-related cue memories, with a focus on those regions implicated in cue-induced relapse to alcohol-seeking (Koob & Volkow, 2010).
Section snippets
Subjects
Adult male Long-Evans rats were used (Envigo; Indianapolis, Indiana, United States). Upon arrival, rats weighed 250–275 g. All were singly housed in a temperature- and humidity-controlled room (22 ± 2 °C; 12-h light cycle). The homecage contained Sani-Chips® bedding and a Bio-Serv Gummy Bone (polyurethane; 5 cm long × 2.5 cm wide). Metal wire cage-tops were used. Standard chow pellets were loaded into a large cup inside the cage. Tap water was provided via gravity-fed sipper inserted at
Acquisition of alcohol drinking and behavioral reactivity
A total of 35 rats were obtained for this study. Of those screened, 29 ingested doses ≥1 g/kg/24 h on average over the last week of homecage drinking, and were retained for conditioning. Of those conditioned, 17 ingested doses ≥0.30 g/kg/session on average across the last three sessions (“Learners”), which is the dose threshold for conditioning effects of ingested ethanol that we observed in our previous study (Cofresí et al., 2018). The other 12 rats ingested doses that were consistently below
Discussion
In the present study, we characterized the behavioral reactions of rats to specific alcohol-related stimuli (houselight and sipper) in an oral alcohol conditioning task to determine whether reactivity was driven by associative or non-associative memory. We also sampled blood after a conditioning session to characterize alcohol exposure level in the same rats. Finally, we used c-Fos expression as an index of cellular activation to map which brain areas might contribute to alcohol-related
Conclusion
In the present study, we showed that behavioral reactivity to an antecedent visual stimulus signaling the opportunity to self-administer alcohol resulted from associative learning, rather than from non-specific effects of repeated exposure to the oral alcohol or visual stimulus. We also showed that memory for this conditioned alcohol cue reactivity may involve cells in the insular cortex. Our findings support continued investigation of the progression of brain and behavioral adaptations to
Acknowledgments
Funding was provided by NIH NIAAA R37AA11852 (RAG), NIH NIAAA T32AA007471 (RUC), NIHM R01MH091147 (MHM), CIHR MOP-137030 (NC), and a University of Texas undergraduate research fellowship (DJG). We thank Dr. Greg Loney for suggesting the idea to automate sipper site/faceplate contact measurement. We thank Jonathan Rosenzweig for his help with designing, prototyping, and fabricating the frame used to automate sipper site/faceplate contact measurement in conjunction with the Longhorn Maker Studio,
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2020, Pharmacology and TherapeuticsCitation Excerpt :Yet, many studies fail to obtain BEC measurements in the animal at any point during its conditioning. Studies in our laboratory (Cofresi et al., 2019; Cofresi, Lee, Monfils, Chaudhri, & Gonzales, 2018) and others (Fiorenza et al., 2018; LeCocq, Lahlou, Chahine, Padillo, & Chaudhri, 2018; Tomie et al., 2006; Tomie, Lewis, Curiotto, & Pohorecky, 2007; Tomie, Uveges, Burger, Patterson-Buckendahl, & Pohorecky, 2004) provide direct evidence for the role of ethanol's primary reinforcing properties in the acquisition of cue-elicited ethanol-related behavior by animals (see Section 3.1.1). Typically, BEC at the end of conditioning sessions in these studies is low (0.020–0.060 g/dL), but can be high (0.060–0.100 g/dL) under certain circumstances (e.g., Tomie et al., 2004; Tomie et al., 2007; Tomie, Hosszu, et al., 2006).
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2019, Neuroscience and Biobehavioral ReviewsCitation Excerpt :When neurons and astrocytes are activated, the transcription and translation of immediate early genes (e.g., arc, c-fos, erk) occurs, and this allows researchers to use the relative density of transcripts or translated protein product as an index of regional brain activity (typically, post-mortem) (Herdegen and Leah, 1998; Morgan and Curran, 1989; Sheng and Greenberg, 1990). Using these indices, alcohol cues conditioned by voluntary oral alcohol self-administration have been shown to activate cells in various IS circuit nodes in the rat brain, including the medial and orbital prefrontal cortices, insular cortex, basolateral nucleus of the amygdala, nucleus accumbens, and central nucleus of the amygdala (Barak et al., 2013; Cofresí et al., 2019a; Dayas et al., 2007; Jupp et al., 2011; Radwanska et al., 2008). These alcohol cues appear gain incentive salience as a function of time since acute withdrawal from alcohol.
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2019, AlcoholCitation Excerpt :This is strong behavioral evidence that cue-triggered alcohol-seeking behaviors arise from associative learning and are not merely due to repeated exposure to alcohol or the cue within the same context. Additionally, it confirms that associative learning about antecedent conditional stimuli for alcohol access in this (Cofresí et al., 2019) and similar paradigms (Srey et al., 2015) is not restricted to male rats. Despite equivalent alcohol exposure, rats in the Unpaired group did not develop cue-triggered alcohol-directed behavior.