To determine whether increased anxiety like behavior

To determine whether increased anxiety-like behavior emerges immediately upon amphetamine withdrawal or during protracted withdrawal, rats were allocated to a 24-h or 2-week withdrawal group (=10 per treatment group at each time period). The 24-h period represents the shortest withdrawal period that can be easily distinguished and is the time point that reflects expectation of a subsequent injection. Rats were tested on the elevated plus maze (EPM) at 24h or 2 weeks after the last amphetamine or saline injection, and testing took place 1h after the onset of the dark phase of the light Famprofazone in a dark room under red lighting. Each arm of the EPM was 50cm×11cm, and the maze was elevated 1 meter from the ground with a camera positioned overhead (Noldus Information Technology, Wageningen, The Netherlands). Rats were introduced to the center of the EPM facing an open arm and allowed to explore the maze for 5min. Latency to enter an open arm (s), time spent on open arms (s), and total distance traveled (cm) were measured by Ethovision 3.1 (Noldus Information Technologies). To determine whether antagonism of CRF receptors in the dRN could reverse increased anxiety-like behavior exhibited by amphetamine-treated rats in withdrawal, rats were tested for anxiety-like behavior 2 weeks following the last treatment. Seven days after last injection, saline and amphetamine-treated rats (=14 per treatment group) underwent aseptic stereotaxic surgery for implantation of guide cannula into the dRN as described by . Rats were anesthetized with a ketamine (80mg/kg, i.p.; Met-Vet, Libertyville, IL) xylazine (16mg/kg, i.p.; Met-Vet) mixture and placed into a small mammal stereotaxic frame (David Kopf Institute, CA, USA). A 22-gauge, 5mm long stainless steel guide cannula (Plastics One, Roanoke, VA) was stereotaxically implanted 1mm above the dRN (AP: −7.8mm from bregma; ML: −2.6 from midline ) at a 23° lateral to medial angle, so to avoid the cerebral aqueduct and to ensure a bilateral infusion . At the conclusion of surgery, the analgesic Ketoprofen (5mg/kg, i.m.; Met-Vet) was administered. All rats were allowed 3 days of recovery before being acclimated to the dRN infusion procedure. Following 3 days of acclimation to the infusion procedures as detailed by , rats were infused with either vehicle (2% ethanol) or the selective CRF receptor antagonist antisauvagine-30 (ASV-30; 2μg/0.5μl; Sigma ) using a 30-gauge stainless steel infusion cannula (1mm longer than the guide) inserted into the guide cannula. This vehicle has no effect on 5-HT release when infused into the dRN, and the dose of ASV-30 used is sufficient to completely block CRF receptor-mediated effects in the dRN , . Furthermore, this volume infused into the dRN is thought to provide a specific bilateral effect, with little effective diffusion outside the dRN , . Twenty minutes after dRN infusion, rats were introduced to the EPM and behaviors measured as described above.