, 2008). Another possibility is that K+ channels may be modulated, since many conditions associated with altered neuronal excitability involve changes in K+ channel expression (Lüscher and Slesinger,

2010). To test this possibility, we examined VTA from mice that received sham or morphine pellets and analyzed by PCR the expression of K+ channels whose regulation has been implicated in other systems. We observed a significant decrease in expression levels of two K+ channel subunits, KCNAB2 and GIRK3, with a trend seen for several others ( Figure 3A). To examine whether decreased K+ channel expression is regulated at the transcriptional level, we performed chromatin immunoprecipitation (ChIP) on VTA dissected from sham- and morphine-treated rats. Overall,

we observed epigenetic changes consistent with K+ channel downregulation learn more (Figures 3B and 3C). Modifications consistent with increased transcription—acetylation of histones H3 and H4, trimethylation of Lys-4 of H3, and binding of RNA polymerase II (POL2)—were significantly decreased at the KCNAB2 and KCNF1 promoters ( Figure 3B) and decreased trimethylation of Lys-4 of H3 and binding of RNA polymerase II (POL2) was observed at the GIRK3 promoter. These changes suggest that, in addition to morphine potentially reducing GABAA responses in VTA DA neurons, expression of specific K+ channel subunits is reduced via transcriptional mechanisms to further mediate enhanced excitability of these neurons. see more Since both chronic morphine and decreased AKT signaling increase VTA DA neuron firing rate, we next examined whether decreasing AKT activity also Thymidine kinase decreases K+ channel

expression similarly to chronic morphine. To test this possibility, we analyzed VTA from mice that had received intra-VTA injections of either HSV-GFP or HSV-IRS2dn. We overexpressed IRS2dn because this is the most direct way of reducing AKT activity without affecting total levels of the enzyme, as seen with chronic morphine. We observed a significant decrease in levels of expression of three K+ channel subunits, KCNF1, KCNJ2, and GIRK3 ( Figure 4A). These data suggest that, in addition to altering DA neuronal activity via GABAA channel regulation, altering AKT signaling can also modulate K+ channel expression. Finally, since both chronic morphine and decreased IRS2/AKT signaling control VTA DA neuronal morphology and excitability, we determined whether decreased AKT signaling also affects DA output to NAc. As found with chronic morphine, HSV-IRS2dn in VTA decreased electrically evoked DA output in rat NAc (Figure 4B). Based on our prior research in rats showing that IRS2 downregulation mediates the chronic morphine-induced decrease in VTA DA soma size (Russo et al., 2007), we assumed that this morphological change was likewise dependent on AKT downregulation.