Effects of opioid receptor blockade on the renewal of alcohol seeking induced by context: relationship to c-fos mRNA expression Peter W. Marinelli, 1 Douglas Funk, 1 Walter Juzytsch, 1 Zhaoxia Li 1 and A. D. Le ˆ 1,2 1 Neurobiology of Alcohol Section, Department of Neuroscience, Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario, M5S 2S1, Canada 2 Departments of Pharmacology and Psychiatry, University of Toronto, Toronto, Ontario, Canada Keywords: alcohol, amygdala, in-situ hybridization, opioid receptors, rat hippocampus Abstract Contextual stimuli associated with alcohol availability can induce alcohol seeking during abstinence. Using a renewal procedure, we tested the effect of opioid receptor blockade on context-induced alcohol seeking, and its relationship to the activity of brain sites involved in learning and reward. Thirty-six male Wistar rats were trained to lever press for a 12% (w ⁄ v) alcohol solution before undergoing extinction sessions (no alcohol delivery). Half of the rats underwent training, extinction and testing in a single context with a distinct set of olfactory, visual, auditory and tactile properties [training, extinction and test in Context A (AAA)]. The other half were trained and extinguished in different contexts and returned to the training context on the test day [training, extinction and test in Contexts A, B and A, respectively (ABA)]. On the test day, the rats from each condition were pre-treated with either saline or 1 mg ⁄ kg naltrexone (s.c.) and tested for alcohol seeking. Immediately following the test session, rats were killed and their brains were analysed for c-fos mRNA expression using in-situ hybridization. Re-exposure to the alcohol-associated context (ABA) significantly increased operant behaviour on the previously active lever relative to the AAA groups and this increased responding was associated with increased c-fos mRNA expression in the basal and lateral amygdala and the CA3 subregion of the hippocampus. Naltrexone pre- treatment attenuated context-induced alcohol seeking and inhibited c-fos mRNA expression in the lateral amygdala and CA3. Our findings point to a critical role for the basolateral amygdala and dorsal hippocampus in mediating context-induced renewal of alcohol seeking and suggest that opioidergic mechanisms mediate this effect. Introduction Through pairing, environmental cues can become important predictors of events. This is particularly relevant to drug and alcohol relapse in humans where cues associated with the availability of the drug provoke craving (Ludwig, 1986; McCusker & Brown, 1990; O’Brien et al., 1998; Litt et al., 2000). This has also been established with an animal model of relapse, the reinstatement procedure. In the reinstatement model, the presentation of stimuli associated with the self-administration of drugs and alcohol reinstates their seeking following extinction of the operant response (de Wit & Stewart, 1981, 1983). Cues shown to reinstate alcohol seeking include discrete response- contingent cues (Liu & Weiss, 2002; Nie & Janak, 2003), discrim- inative cues (Katner et al., 1999; Dayas et al., 2007) and context, a multimodal cue complex (Burattini et al., 2006; Tsiang & Janak, 2006; Zironi et al., 2006). Context can elicit patterns of drug seeking that are different from those elicited by discrete or discriminative cues (Zhou et al., 2005). Context can also gate responding for alcohol induced by discrete (Tsiang & Janak, 2006) or discriminative (Burattini et al., 2006) cues. In addition, separate but overlapping brain substrates mediate drug seeking by each of these types of cues (Bossert et al., 2005; Fuchs et al., 2005). Growing evidence also suggests that the endogenous opioid system plays a significant role in mediating alcohol seeking induced by different types of cues. For example, the relatively non-selective opioid receptor antagonist naltrexone can attenuate or block alcohol seeking induced by discrete cues (Liu & Weiss, 2002), discriminative cues (Katner et al., 1999), compounds of these cues (Ciccocioppo et al., 2002, 2003) and context (Burattini et al., 2006). As naltrexone has no effect on stress-induced reinstatement, the effects of naltrexone appear to be specific to craving elicited by stimuli conditioned to alcohol (Le et al., 1999a; Liu & Weiss, 2002). The analysis of c-fos can be a useful tool to delineate neural pathways that underlie context-induced alcohol seeking. The c-fos immediate early gene is induced in the cell nucleus following its depolarization and is widely used as a marker of neuronal excitation (Cole et al., 1989). In one study, c-fos protein was examined in the rat brain following presentation of a water- or alcohol-associated discriminative odour in the presence or absence of naltrexone (Dayas et al., 2007). The alcohol-associated odour elevated c-fos in a variety of limbic sites and naltrexone reversed this response in the hippocampus. The effect of naltrexone on c-fos induced by re-exposure to a context predictive of alcohol availability remains unknown. Correspondence: Dr Peter Marinelli, as above. E-mail: Peter_marinelli@camh.net Received 22 June 2007, revised 7 September 2007, accepted 18 September 2007 European Journal of Neuroscience, Vol. 26, pp. 2815–2823, 2007 doi:10.1111/j.1460-9568.2007.05898.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd