FR5TO20 sec schedule. As soon as food responding criteria was reestablished, subjects were permitted to acquire intravenous nicotine selfadministration by autoshaping during 1 h each day sessions, 7 days per week. Nicotine was delivered through the tubing in to the intravenous catheter by a Razel syringe pump (Med Associates). Each and every nicotine selfadministration session was performed working with 2 retractable levers (1 active, 1 inactive) that extend 1 cm into the chamber. Completion in the response criteria around the active lever resulted in the delivery of an intravenous nicotine infusion (0.03 ml infusion volume for mice; 0.1 ml for rats). Responses around the inactive lever had been recorded but had no scheduled consequences. For doseresponse studies (fixed and progressive ratio schedules), animals have been presented with each dose of nicotine for at the least five days (mice) or 3 days (rats); the mean intake over the final 3 (mice) or 2 (rats) sessions for each dose was calculated and utilized for statistical analysis. Nicotine doses had been presented in line with a withinsubjects Latin square design. In between each dose, subjects were placed back around the instruction dose for at least 2 days or till their intake returned to baseline levels before getting tested on the next dose within the Latinsquare design. Surgical procedures for microinjections and ICSS electrode placement Animals were anesthetized as above and positioned in a stereotaxic frame (Kopf Instruments, Tujunga, CA). Unless otherwise noted, the incisor bar was set towards the `flatskull’ position. To test the efficacy in the reexpressing and knockdown viruses in vivo, bilateral injections have been made in to the hippocampus of mice or rats, respectively. This location was selected according to the constitutive expression of 5 nAChR subunit mRNA in wildtype animals. In mice, six bilateral injections (1 l each and every at a flow rate of 1 l per min) have been created in the following coordinates: anteriorposterior (AP): 1.7 mm from bregma; mediallateral (ML): .75 mm from midline; dorsalventral (DV): 2.05 mm, 1.80 mm and 1.3 5mmNature. Author manuscript; accessible in PMC 2011 September 30.Fowler et al.Pagefrom brain surface2. In rats, the six hippocampal injections (3 two l injections per side at a flow rate of 1 l per min) had been created at the following coordinates: AP: 3.three mm from bregma; ML: .1 mm from midline; DV: three.6 mm, 3.0 mm and 2.four mm from brain surface3. For habenular injections in mice, the needle was angled 20toward midline, and bilateral injections (0.375 l every single) have been administered at a price of 0.375 l per min. For habenular injections in rats, the lentivirus was injected bilaterally depending on previously published coordinates4.Methyl 2-chloro-3-methylisonicotinate custom synthesis The incisor bar was set to five mm above plane, plus the injector needle was at a 10angle toward midline (AP: 2.5371-70-0 Price 2 mm from bregma; ML: .PMID:23916866 five mm from midline; DV: four.9 mm from brain surface). The bilateral injections (1 l each) have been administered at a rate of 1 l per min. For all of the injections, the injector needle was remained in spot for any minimum of 2 min postinjection. For IPN and VTA microinjections in rats, guide cannula (Plastics One) have been implanted as follows: IPN (flat skull; 10angle toward midline; AP: 6.72 mm from bregma; ML: .six mm from midline; DV: six.5 mm from brain surface) or VTA (bilateral; flat skull; 6angle toward midline; AP: 5.four mm from bregma; ML: .three mm from midline; DV: 7.0 mm from skull)three. The MHb guide cannula coordinates had been the same as for the lentiviral injections, except with DV at two.9 mm from brain surface.