These data suggest that GSK3β activity in VTA is not involved in morphine-induced changes in the morphology of VTA DA neurons. To investigate whether mTORC2 downregulation might also contribute to the morphine-induced increase in DA neuron firing rate, we injected HSV-Rictor-T1135A into mouse VTA, treated the mice with sham or morphine pellets, and recorded DA neuron firing rates in acute VTA slices. Similarly to Figure 1C, chronic morphine increased DA neuron firing rate in both GFP-positive and GFP-negative cells compared with sham-treated mice (Figure 6C).

However, GSK2656157 cell line in cells that overexpressed Rictor, the morphine-induced increase in firing rate was

completely abolished (Figure 6C). Further, it was the overexpression of Rictor in the DA neurons themselves driving the effect, as GFP-negative DA neurons from the Rictor-morphine mice still showed the morphine-induced increase in firing rate. These data support a cell-autonomous link between decreased mTORC2 activity and increased VTA DA neuron excitability induced by chronic morphine. Given that Rictor overexpression prevented morphine-induced changes in VTA neuron morphology and excitability, we next assessed whether altered mTORC2 activity might also affect morphine reward as measured by place conditioning. We found that Rictor overexpression caused a significant place preference to a low dose of morphine (5 mg/kg) that Selleckchem VX 809 does not induce preference in GFP-injected mice (Figure 6D). Rictor overexpression also increased morphine-induced locomotor activity (Figure 6D). Conversely, local knockout

of Rictor in VTA decreased morphine place preference (15 mg/kg) without affecting locomotor activity (Figure 6E). These data are consistent with our previous findings that treatments that decrease VTA DA soma size—chronic morphine or decreased AKT signaling—decrease morphine reward. Results of the present study establish that chronic morphine induces a pattern of phenotypic changes in VTA DA neurons characterized by decreased soma size, increased Casein kinase 1 cell excitability, and decreased DA output to NAc. The dramatic decrease in DA output is consistent with the profound reward tolerance observed previously (Russo et al., 2007). Such reward tolerance would be expected to lead to an escalation of drug intake to overcome this cellular break, as seen clinically (O’Brien, 2001). These morphine-induced changes in VTA could thus be viewed as homeostatic adaptations to counter the effects of sustained morphine exposure. We provide several lines of evidence that these adaptations to chronic morphine are mediated via downregulation of AKT-mTORC2 signaling in VTA DA neurons.