Lastly, there was no apparent change in the levels of GABAB2 receptor protein (Figure 7F), suggesting little METH-dependent degradation of receptor. Dephosphorylation of GABAB2-p-S783 has been previously shown to be regulated by protein phosphatase 2A (PP2A; Terunuma et al., 2010), raising the possibility that in vivo exposure to METH enhances the phosphatase activity in VTA GABA neurons. To address this, we examined the effect of acutely inhibiting PP1/PP2A phosphatases with okadaic

acid (OA; 100 nM). In saline-injected mice, check details there was no significant difference in the amplitude of IBaclofen with OA in the pipet, suggesting that basal activity of PP1/PP2A does not significantly regulate GABABR-GIRKs (Figures 7G–7J). In METH-injected mice, however, intracellular application of OA promoted recovery of the IBaclofen (Figures 7H and 7J). Note the slow time course of activation for IBaclofen in the presence of OA in METH-injected mice. This increase could reflect insertion of GABAB receptors and GIRK channels on the plasma membrane or restoration of

functional G protein coupling. For control, we examined the effect of PKC(19-36), a peptide inhibitor of PKC (Figure 7K). Unlike OA, the presence see more of PKC inhibitor in the pipet did not restore IBaclofen, similar to the effect of METH alone. Taken together, these findings suggest that in vivo exposure to METH triggers a phosphatase-dependent downregulation of GABABRs and GIRK channels from the plasma membrane of GABA neurons, which results in reduced GABABR-GIRK signaling and accumulation of GABAB receptor complexes in intracellular compartments. To investigate the functional consequence of reduced GABABR-GIRK currents these in GABA neurons of METH-injected mice, we examined the effect of baclofen on the induced firing rate of GABA neurons (Figure 8). We predicted that a loss of GABABR-GIRK signaling would attenuate GABABR-mediated

suppression of firing in GABA neurons. To test this, a series of current steps (20–100 pA) were injected to elicit a train of action potentials in GABA neurons (Figures 8A and 8B). In saline- and METH-injected mice, the input-output (I-O) plot shows a linear increase in firing rate with larger current injections (Figures 8B and 8D). As expected, baclofen (100 μM) significantly suppressed firing in GABA neurons of saline-injected mice, decreasing the slope of the I-O curve (Figures 8A and 8B). By contrast, a saturating dose of baclofen (100 μM) did not significantly change the I-O curve in METH-injected mice (Figures 8B and 8C). These results demonstrate that a loss of GABABR-GIRK currents in GABA neurons removes an important “brake” on GABA neuron firing in the VTA. Drug-evoked synaptic plasticity can cause persistent modifications of neural circuits that eventually lead to addiction.