We did not observe a significant decrease in phospho-p70S6K, but this may be due to an increase in total p70S6K levels induced by this treatment. This effect of rapamycin was specific for mTORC1, since there was no evidence of altered mTORC2 activity, based on normal levels of phospho-AKT (Ser-473)

and phospho-PKCα, in VTA. Rapamycin treatment of morphine-naive mice had no effect on VTA DA cell surface area, demonstrating that decreasing mTORC1 activity per se is not sufficient to alter the size of these neurons (Figure 5H). Further, when mice were pretreated with rapamycin and then treated chronically with morphine, we still observed the expected morphine-induced decrease in DA soma size. These findings show that preventing the morphine-induced increase in mTORC1 signaling in VTA does not block the morphine-induced decrease in soma size. Since there is no selective small selleck compound molecule inhibitor of mTORC2, we used a conditional neuronal knockout strategy, with recently developed floxed-Rictor mice (Siuta et al., 2010) to directly study the contribution of mTORC2 in morphine action. Knocking out Rictor enables a selective reduction in mTORC2 activity, without any discernable effect on mTORC1. To achieve a local knockout of Rictor from VTA, we

BTK inhibitor concentration injected AAV-Cre into VTA of floxed-Rictor mice or into wild-type littermates as a control (Figure 6A). Knockout was validated by RT-PCR and western blot analysis, where we observed a significant decrease in Rictor mRNA in VTA and decreased phosphorylation of the mTORC2 substrates AKT (Ser-473) and PKCα (Figure S2A). Local Rictor knockout also decreased DA cell surface area by ∼20% (Figure 6B). We next developed an HSV to overexpress Rictor-T1135A. This Rictor mutant increases mTORC2 activity, and lacks the p70S6K Montelukast Sodium phosphorylation site, eliminating

the possibility of mTORC1 negative feedback regulation of mTORC2 (see Discussion). This vector increased Rictor expression and mTORC2 signaling in VTA (Figure S2B), and blocked the morphine-induced decrease in DA neuron soma size (Figure 6B). These results demonstrate that downregulation of mTORC2 signaling in VTA is both necessary and sufficient for mediating the morphine-induced decrease in DA soma size. In addition to the mTOR pathway, another downstream target of AKT that has been observed to affect neuronal size and structure in other systems is GSK3β (van Diepen et al., 2009). Since we observe changes consistent with increased GSK3β activity (decreased phospho-GSK3β, Figure S1A) in VTA after chronic morphine, we studied the possible influence of GSK3β in regulating VTA DA soma size. Overexpression of wild-type GSK3β in VTA, which mimics morphine regulation of the protein, did not alter soma size (Figure S3).