mTORC1 includes regulatory-associated protein of mTOR (Raptor) and proline-rich AKT substrate 40 kDa and promotes protein synthesis and cell growth through phosphorylation of two main substrates, eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal S6 kinase 1 (p70S6K). This complex is sensitive to inhibition by rapamycin and is activated in response to several stimuli including nutrients and amino phosphatase inhibitor library acids. In contrast, mTORC2 specifically contains rapamycin-insensitive companion of mTOR (Rictor), mammalian stress-activated protein

kinase interacting protein, and protein observed with rictor-1, and phosphorylates the hydrophobic motif (HM) of multiple kinases including AKT,

protein kinase Cα (PKCα), and serum- and glucocorticoid-inducible kinase 1. mTORC2 activity was originally implicated in cytoskeletal remodeling (Sarbassov et al., 2004), and recent evidence suggests a role in cell survival and growth as well; however, the upstream activators are poorly understood (Pearce et al., 2010). Results of the present study show that the morphine-induced decrease in VTA DA soma size occurs concomitantly with an increase in the Nutlin 3a intrinsic excitability of these neurons, and that the net functional effect of chronic morphine is to decrease DA output to target regions. This net effect is consistent with morphine reward tolerance observed under these conditions. We go on to show that these adaptations induced by chronic morphine—including decreased soma size, increased excitability, and reward tolerance—are mediated via downregulation of IRS2-AKT and mTORC2 activity in this brain region. These results are surprising since our starting hypothesis was that chronic morphine might decrease mTORC1 activity, in concert with downregulation of IRS2-AKT, based on several reports that tie mTORC1 activity to regulation of neuronal growth and size (Kwon et al., 2003 and Zhou et al., 2009). Counter to this hypothesis, mTORC1 signaling was increased in VTA by chronic morphine, Digestive enzyme an effect

not related to the other actions of morphine on VTA DA neurons. Together, the findings reported here describe a fundamentally novel molecular pathway, involving decreased mTORC2 signaling, possibly as a result of decreased IRS2 signaling, through which chronic opiates alter the phenotype of VTA DA neurons to produce reward tolerance. We set out to characterize the effects of chronic morphine on several phenotypic characteristics of VTA DA neurons. We first determined whether morphine induces a morphological change in the mouse VTA similar to that seen in rats. We found an ∼25% decrease in the mean surface area of mouse VTA DA neurons in response to chronic morphine (Figure 1A), very similar to the magnitude of soma size decrease observed in rats (Russo et al., 2007 and Sklair-Tavron et al., 1996).