Psychopharmacology (2001) 156:98–107 DOI 10.1007/s002130100748 Abstract Rationale and objectives: Footshock stress reliably reinstates heroin seeking in rats, but the time course of the development of this effect following drug withdrawal is not known. Here we studied the effect of intermittent footshock stress on reinstatement of heroin seeking following different withdrawal periods (1–66 days). We also studied whether changes in cortico- tropin-releasing factor (CRF) mRNA in the central nu- cleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) are correlated with this rein- statement after 1 day and 6 days of heroin withdrawal. Methods: Rats were trained to self-administer heroin (9 h/day; 0.1 mg/kg per infusion) for 10 days. Tests for extinction behavior and footshock-induced reinstatement of heroin seeking were then conducted after 1, 6, 12, 25, or 66 days of heroin withdrawal. On the test day, rats were given five to ten 60-min extinction sessions until they reached the extinction criterion of less than 15 re- sponses per 60 min on the lever previously associated with heroin. Rats were then exposed to intermittent foot- shock (0.8 mA; 10 min), and lever-pressing behavior was recorded for 120 min. Results: Reinstatement of le- ver-pressing behavior by footshock followed an inverted U-shaped curve with maximal responding after 6 days and 12 days of heroin withdrawal. Surprisingly, foot- shock did not reinstate lever-pressing behavior on day 1 of withdrawal. Lever pressing during extinction, prior to exposure to footshock, also followed an inverted U-shaped curve, with higher responding after 6, 12, and 25 days of heroin withdrawal. Finally, compared with control groups not exposed to shock, CRF mRNA levels in response to footshock were increased in the CeA (day 1 of withdrawal) and the dorsal BNST (day 1 and day 6), but not in the ventral BNST. Conclusions: The duration of the heroin withdrawal period is an important factor in the manifestation of (1) footshock stress- induced reinstatement of heroin seeking and (2) extinc- tion of the heroin-reinforced behavior. Finally, the time- dependent changes in footshock stress-induced reinstate- ment following withdrawal from heroin were not corre- lated with alterations in CRF mRNA in the CeA and BNST. Keywords Corticotropin-releasing factor · In situ hybridization · Heroin · Relapse · Stress Introduction High rates of relapse to drug use after prolonged with- drawal periods characterize the behavior of experienced drug users (Jaffe 1990). One factor thought to contribute to drug relapse and craving in humans, even after prolonged abstinence periods, is exposure to stress (Shiffman and Wills 1985; Kreek and Koob 1998; Sinha et al. 1999). Using a reinstatement procedure, we and others reported that exposure to footshock stress reliably reinstates heroin (Shaham and Stewart 1995; Ahmed et al. 2000), cocaine (Erb et al. 1996; Mantsch and Goeders 1999), alcohol (Lê et al. 1998; Martin-Fardon et al. 2000), and nicotine (Buczek et al. 1999) seeking in drug-free rats. Subsequent studies have shown that the effect of footshock stress on reinstatement is mediated by central noradrenaline (NA) and corticotropin-releas- ing factor (CRF) systems (Shaham et al. 2000a). One important issue that has not been systematically addressed in previous research is whether the response to stress following withdrawal from drugs changes over time. In two studies, it was reported that footshock rein- states drug seeking after 1–2 weeks or 4–6 weeks of her- oin or cocaine withdrawal (Shaham and Stewart 1995; Erb et al. 1996). In these studies, however, the same groups of rats were tested during the two withdrawal pe- riods, and the effect of footshock was not determined during early periods of withdrawal. Thus, it cannot be U. Shalev · B. Hope · J. Yap · Y. Shaham ( ✉ ) Behavioral Neuroscience Branch, IRP/NIDA/NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA e-mail: yshaham@intra.nida.nih.gov Tel.: +1-410-5501746, Fax: +1-410-5501612 M. Morales Cellular Neurophysiology, IRP/NIDA, Baltimore, Maryland, USA ORIGINAL INVESTIGATION Uri Shalev · Marisela Morales · Bruce Hope Jasmine Yap · Yavin Shaham Time-dependent changes in extinction behavior and stress-induced reinstatement of drug seeking following withdrawal from heroin in rats Received: 5 December 2000 / Accepted: 21 February 2001 / Published online: 28 April 2001 © Springer-Verlag 2001