, 2000). Alternatively, such differences in ERK1/2 phosphorylation may result from the submaximal defeat protocol used. We also observed induction of total levels of Ras in NAc of mice treated chronically with cocaine followed by repeated social defeat (Figure 6C). In contrast, total levels of other proteins in this cascade, including BDNF, TrkB, Raf, MEK1/2, ERK1/2, and CREB, were not altered. Also unaltered was Protein Tyrosine Kinase inhibitor Ras-GRF1, a guanine nucleotide exchange factor that activates Ras (Figure 6C, inset). These results suggest that induction of Ras expression per se may be responsible for activating downstream components of this signaling pathway

necessary for the cocaine enhancement of depressive-like behaviors (Figure 6C). Overexpression of G9a, which blocks cocaine-induced vulnerability to stress, significantly reduced cocaine- and stress-mediated increases in SCH 900776 order Ras expression in NAc, without affecting total

protein levels of any of the other members of this pathway examined (Figure 6C and Figures S5A–S5H). NAc-specific overexpression of G9a did, however, significantly reduce the phosphorylation state of all downstream-signaling molecules, including phospho-Raf, phospho-MEK1/2, phospho-ERK1/2, and phospho-CREB, indicating that transcriptional repression of Ras activity by G9a suppresses the activity of this entire signaling cascade. It is important to note that G9a overexpression had no effect on total levels or phosphorylation state of any of the proteins examined in this pathway under control conditions (i.e., HSV-G9a in cocaine- and stress-naive mice), suggesting that G9a acts in a stimulus-dependent manner to block BDNF-TrkB signaling. To further investigate the possibility that Cell press cocaine’s induction of Ras contributes to increased stress vulnerability, we examined expression of H-Ras1, the Ras transcript most predominately expressed in adult brain, in NAc of animals receiving either saline, acute, or repeated cocaine, either immediately after cocaine exposure (1 hr)

or 24 hr after the final drug dose. Although H-Ras1 mRNA expression was unaffected by a single dose of cocaine, it was significantly induced by repeated cocaine at 1 hr, an effect that persisted for 24 hr ( Figure 6D). A substantial literature indicates functional differences between NAc and dorsal striatum in the development of stress- and drug-induced behaviors ( Saka et al., 2004, Di Ciano et al., 2008 and Dias-Ferreira et al., 2009). We thus investigated Ras, ERK, and CREB in dorsal striatum in response to repeated cocaine or stress ( Figures S6A–S6G). Phospho-CREB was decreased in dorsal striatum by acute cocaine, and increased by repeated cocaine, similar to our observations in NAc ( Figure S6D). Cocaine had no effect on Ras or ERK in dorsal striatum. Ras protein levels were increased in dorsal striatum after chronic social stress, but only in unsusceptible mice ( Figure S6E). In contrast, Ras induction in NAc was observed exclusively in susceptible animals.