, 2007). For colocalization studies, sections were check details incubated with antibodies to TH and phospho-S6,

confocal scans were obtained, and the number of TH+, p-S6+, and dual-labeled cells were counted by a blind observer. Mice were implanted with sham or morphine pellets, perfused ∼48 hr later with cold artificial CSF (aCSF), and 250 μm slices containing VTA were cut and transferred into a recording chamber containing aCSF, 5 μM morphine, or the opioid receptor antagonist naloxone (1 μM). The firing rates of VTA DA neurons from sham- or morphine-pelleted mice were determined using extracellular single unit recording and for Kir2.1 channel studies, single unit recordings were obtained click here from DA neurons in VTA slice cultures generated ∼48 hr after the

last pellet, as described previously (Krishnan et al., 2007). For HSV-Rictor-T1135A studies, mice were pelleted with sham or morphine ∼48 hr after HSV injection and perfused ∼48 hr later, and VTA slices were made. VTA DA firing rate of GFP+ and − neurons was determined by cell-attached recording configuration as described previously (Cao et al., 2010). Three to four days following viral surgery, rats were anesthetized with urethane (300 μg/kg), placed in a stereotactic frame, and prepared for recordings of electrically evoked DA transmission using fast-scan cyclic voltammetry (Cheer et al., 2004). For morphine studies, rats were pelleted Oxygenase as described above, then anesthetized 3–7 days later, a time range previously shown to exhibit decreased DA soma size (Russo et al., 2007). A bipolar stainless-steel stimulating electrode was advanced to VTA, a glass-encased cylindrical carbon fiber microelectrode targeted the medial shell of the NAc and a reference

electrode (Ag/AgCl) was inserted into the right hemisphere posterior to Bregma. Electrical stimulation of VTA was delivered through the stimulating electrode and DA release was evoked using trains of 60 bipolar pulses of 300 μA amplitude at 60 Hz. DA was detected using fast-scan cyclic voltammetry at the carbon fiber microelectrode. The two hemispheres were recorded from successively and the order of recording was counterbalanced across rats. Experiments were only included in analysis when a full set of dorsoventral recordings from both hemispheres was obtained. Punches from rat or mouse VTA were homogenized in Trizol and processed according to the manufacturer’s protocol. RNA was then purified using RNAesy Micro columns (QIAGEN) and quality was assessed by spectroscopy. RNA was then reverse transcribed (iScript, BioRad) and quantified by quantitative PCR using SYBR green. Glyceraldehyde-3-phosphate dehydrogenase was utilized as a normalization control and all samples were run in triplicate and analyzed using the ΔΔCt method as described previously (Tsankova et al., 2006).