,

2004), however, we see no effect of rapamycin on morphine-induced downregulation of the AKT-mTORC2 Ulixertinib pathway. Clearly, delineating these complex signaling pathways underlying chronic morphine regulation of VTA DA neurons is very difficult using an in vivo paradigm, yet morphine regulation of VTA DA soma size is not observed in cultured neurons (unpublished observations), demonstrating the importance of focusing on in vivo systems. Indeed, we show here unique patterns of regulation of IRS2/AKT/mTOR signaling in VTA DA neurons in response to chronic morphine in vivo. Our evidence for a novel role of mTORC2 signaling in mediating morphine’s regulation of VTA DA neuron excitability and size raises fundamentally new approaches for the development of treatment agents that counteract these effects of chronic morphine and its important downstream functional consequences related to opiate addiction. For all experiments, animals were male, fed ad libitum, and

kept on a 12 hr light/dark cycle. Sprague-Dawley rats (250–275 g, Charles River) and 8–9 week c57BL/6 mice (Jackson Labs) were given PLX-4720 concentration s.c. morphine pellets (75 or 25 mg, respectively) as described previously (McClung et al., 2005 and Fischer et al., 2008). Homozygous floxed-Rictor mice and wild-type littermates were generated as described previously (Shiota et al., 2006 and Siuta et al., 2010). See Supplemental Experimental Procedures for further details. Human specimens were obtained from the Forensic Medicine Departments of Semmelweis University (Budapest, Hungary) and of Karolinska Institutet (Stockholm, Sweden) under approved local protocols (Horvath et al., 2007). Fresh-frozen brain sections (20 μm) were analyzed from control or heroin-overdose subjects. All samples had a postmortem interval (PMI) of <24 hr and were tested for common drugs of abuse and therapeutic drugs; demographic data are given in Table S1. For stereotaxic surgeries,

mice were anaesthetized with ketamine (100 mg/kg) and xylazine (100 mg/kg) and VTA was targeted using established coordinates. Bilateral 33 g syringes were used to infuse HSV at a flow rate of ∼0.1 μl/min. of HSV vectors encoding GFP, IRS2dn, AKTdn, AKTca, dnK, Kir2.1, GSK3β, and GSK3βdn have been previously used and validated (Krishnan et al., 2007 and Russo et al., 2007). Rictor cDNA (T1135A mutant) was provided by Dr. Brandon Manning (Harvard) and was cloned into the p1005 HSV vector. Rictor overexpression was verified by RT-PCR and western blot analysis (Figure S2B). AAV-GFP and AAV-Cre-GFP were used as described previously (Berton et al., 2006). Tissue was collected and processed as described previously (Krishnan et al., 2008 and Russo et al., 2007). Samples were processed in RIPA buffer, quantified, electrophoresed, transferred to PVDF, and blotted using standard procedures. Mice were perfused and sections (30 μm) containing VTA were selected for analysis.