Subjects. Thirty-five adult male Lister Hooded rats (Charles River, Kent, UK),

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1 Supplemental Material: Subjects. Thirty-five adult male Lister Hooded rats (Charles River, Kent, UK), weighing between g at the beginning of the experiment, were housed singly in holding rooms maintained at 21 C on a reversed-light cycle (12 hours light: 12 hours dark; lights on at 19:00h). Throughout behavioral testing, all rats were food restricted to 20 g/day (Purina rat chow). All experiments were undertaken in accordance with the UK 1986 Animals (Scientific Procedures) Act (Project License PPL 80/1767). Surgery and Histology. Prior to behavioral training, all rats were implanted with a single indwelling intravenous catheter and also with bilateral chronic indwelling guide cannulas targeting the BLA. The coordinates for cannula implantation were (mm relative to bregma): AP 2.6; ML ± 4.5; DV 5.6 (from dura). The procedures for catheter implantation and stereotaxic surgical procedures are described elsewhere (Di Ciano and Everitt, 2001). Behavioral training and testing began a minimum of 7 days postoperatively. Drug. Heroin (diamorphine hydrocholoride; Macfarlan Smith Ltd., Edinburgh UK) was dissolved in sterile 0.9 % saline. All doses of heroin were calculated as the salt and intravenous infusions were delivered in a 0.1 ml solution over 7.3 s. Naloxone hydrochloride (Sigma-Aldrich, St Louis USA) was dissolved in sterile 0.9 % saline and administered s.c. Infusions. All infusions were carried out in a room separate from behavioral testing and delivered using a syringe pump connected to injectors (28 gauge, projecting 2 mm beyond the guide cannulae) by polyethylene tubing. Immediately following insertion of the injectors, animals were infused with either Zif268 antisense oligodeoxynucleotides

2 (ASO; 1.0 µl/side; µl/min) or missense sequences (MSO; 1.0 µl/side; µl/min). Injectors were withdrawn after 1 min to allow for diffusion away from the injector tips. Oligodeoxynucleotides were PAGE-purified phosphorothioate end-capped 18-mer sequences, resuspended in sterile PBS to a concentration of 2 nmol/µl as used previously (Lee et al., 2005). Rats were habituated to the infusion procedure using the PBS vehicle (1.0 µl/side; µl/min) on 2 occasions during self-administration training but prior to conditioning sessions. Behavioral Apparatus and Procedures. Training and testing took place in twelve operant chambers as previously described (Hellemans et al., 2006). Self-Administration Training: Rats were connected to the IV line with a tether connected to the syringe pump containing heroin. Each session began with the houselight turning on and insertion of the drug-taking lever. Each lever press resulted in the delivery of 0.06 mg/kg of heroin infused over 7.3 s. At the same time, the stimulus light above the lever was illuminated, and the house light was switched off. After 5 days, rats were then introduced to the multiple (heterogeneous chain (tandem FR 1 (RI 120-s) FR 1) time-out schedule, which for brevity we refer to as a seeking-taking chain schedule. Each cycle of the chain schedule began with the insertion of the seeking lever into the chamber, and the first press on this lever initiated a random interval (RI) schedule. The first seeking response meeting the RI contingency terminated the first link of the chain and resulted in the retraction of the seeking lever and the insertion of the taking lever. A single press on the taking lever produced an infusion of heroin (0.12 mg/kg over 7.3 s) followed by a time-out period of 24 min, after which the seeking lever was reinserted and the house light illuminated, signaling the next cycle of the chain. The parameter of the RI link was

3 increased from 2 to 120 s across successive sessions, each of which terminated after 10 infusions or 4 h, whichever occurred first. Dependence Induction. Following stable baseline responding, all rats were implanted subcutaneously with osmotic minipumps (Charles River Ltd, UK) that infused heroin at a continuous rate (3mg/kg/day) for up to 4 weeks. For a detailed description of the implantation procedure, see Hellemans et al., After a 48-hr recovery period, rats were allowed to re-establish baseline responding for heroin under the seeking-taking chain schedule. Once seeking rates were comparable to pre-implantation baseline levels, rats were then subjected to the withdrawal-conditioning phase. Histological and Western Blotting procedures The rats were decapitated and the brain was rapidly removed, frozen in dry ice and stored at 80 o C. Following mild thawing, a 1 mm section through the amygdala was cut, cannula placements verified by eye, and tissue from the BLA and CeN sampled bilaterally using a 1 mm diameter brain punch set (Stoelting, Wood Dale, Illinois). Tissue lysates and Western blotting were performed as previously described (Lee et al., 2004; Lee et al., 2005). Proteins were separated on 7.5% Tris-HCl gels at a constant voltage of 80 V and then transferred to nitrocellulose membranes at a constant current of 100 ma for 1 hr. Blots were blocked in 5% non-fat milk in 0.01 M Tris-buffered saline solution containing 0.05% Tween 20 (TBST), and this TBST solution was used for all subsequent incubations and washes. Primary and secondary antibodies were used at the following concentrations: Zif268 (kind gift from G.I. Evan, University of California, San Francisco), 1:10 000; β-actin (loading control), 1:20 000; goat anti-rabbit and goat antimouse IgG (whole-molecule)-peroxidase conjugates, 1: Blots were developed

4 using enhanced chemilluminescence and opposed to autoradiographic film. Autoradigraphic images were captured using a Photometrics CoolSnap CCD digital camera and quantified using Openlab software. Autoradiographs of each Western blot were developed to be linear in the range used for densitometry for both Zif268 and β- actin. For analysis, optical density (OD) values and the band areas were obtained for each microdissected BLA and CeN sample and an OD X Area calculation was made to derive an amount figure for both Zif268 and the β-actin loading control. Loading variation was corrected on each Western blot by deriving a normalization factor ([average amount of β- actin]/[sample amount of β-actin]) for each sample. This factor was multiplied to the raw sample amount of Zif268 to produce the corrected figure, which was itself normalized against the average corrected level of Zif268 in the control group (non-reactivated or MSO). Statistics. Data were analyzed using SPSS software, v. 12 for Windows (SPSS, Chicago, IL) against a Type I error rate of Suppression ratios were calculated for all animals using the formula a/(a + b), where a and b indicate the total number of heroinseeking responses made in the presence and absence of the CS, respectively. Data were compared between Reactivation (Pre-reactivation, Post-reactivation, Non-reactivated) and Infusion (ASO or MSO) conditions using a 2-way ANOVA. Planned comparisons were performed between pre- and post-reactivation, as well as post-reactivation and nonreactivated, groups, and Tukey s post-hoc tests were performed on any significant findings. Data for the Western blotting were checked for normality of distribution and homogeneity of variance, using the Shapiro-Wilk test for the former, and both Levene s

5 test and the Brown-Forsythe test for the latter. As these assumptions were not violated, the untransformed data were analysed using a one-way ANOVA. References Di Ciano P, Everitt BJ (2001) Dissociable effects of antagonism of NMDA and AMPA/KA receptors in the nucleus accumbens core and shell on cocaine-seeking behavior. Neuropsychopharmacology 25: Hellemans KGC, Dickinson A, Everitt BJ (2006) Motivational control of heroin seeking by conditioned stimuli associated with withdrawal and heroin taking by rats. Behav Neurosci 120: Lee JLC, Everitt BJ, Thomas KL (2004) Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science 304: Lee JLC, Di Ciano P, Thomas KL, Everitt BJ (2005) Disrupting reconsolidation of drug memories reduces cocaine-seeking behavior. Neuron 47: