As METH impairs self renewal capacity we also explored the

As METH impairs self-renewal capacity, we also explored the influence of METH in DG stem cell-fate division, a suitable method to characterize the phenotype of cells that are derived from the division of one DG stem cell. Indeed, a stem cell can undergo symmetric cell division towards self-renewal, where the two resulting cells are both stem cells (Sox2+/Sox2+ pairs of daughter cells), and towards differentiation, resulting to two cells that are committed to differentiate (Sox2−/Sox2− pairs of daughter cells). Moreover, one stem cell can divide asymmetrically, originating to one pair of cells consisting of one Sox2+ and one Sox2− cell. Accordingly, we found that METH (10nM) decreased self-renewal symmetric cell division (Sox2+/Sox2+ pairs of daughter cells), directing division towards Sox2−/Sox2− pairs of daughter cells consistent with cell commitment. In fact, an animal study showed that Sox2-positive cells in the SGZ are able to undergo symmetric or asymmetric cell division, in which one Sox2-positive cell can give rise to one neuron and one astrocyte, or to one neural stem cell and one neuron, respectively (Suh et al., 2007). Also, we observed that these alterations could be, in part, due to the activation of NMDA receptor signaling because inhibition of these receptors prevented the shift of cell division towards differentiation. Moreover, we had previously demonstrated that METH induces an increase of glutamate release from DG neurospheres and the inhibition of NMDA receptors protects DG cells from METH toxicity (Baptista et al., 2012). In fact, several Wortmannin injuries, such as status epilepticus (Sugaya et al., 2010), cerebral ischemia (Choi et al., 2012) and traumatic brain injury (Zheng et al., 2013) can increase neurogenesis, and glutamate seems to play a central role upon activation of NMDA receptors (Arvidsson et al., 2001; Urbach et al., 2008). Indeed, Nacher et al. (2003) observed that a single injection of NMDA receptor antagonist (CGP-43487, 5mg/kg) prevents age-induced decrease of SGZ cell proliferation and neurogenesis in both middle-aged (10month-old) and aged (20month-old) Fisher F344 rats. Moreover, administration of NMDA (30mg/kg) to Sprague–Dawley rats decreased the population of proliferating cells in the DG, whereas administration of MK-801 or CGP37849 resulted in the opposite effect (Cameron et al., 1995). Also, MK-801 administration (3mg/kg) to NMDA-infused (2mg/ml) Wistar rats increases the phosphorylated levels of ERK in newly generated neurons (Okuyama et al., 2004), which strengths the involvement of NMDA receptors in decreasing cell proliferation through regulation of the MAPK pathway. Moreover, Deisseroth et al. (2004) demonstrated that NMDA receptors play an active role in neuronal differentiation, showing that excitatory stimuli induced by NMDA receptor activation enhances expression of NeuroD, a downstream regulator of neuronal differentiation, and consequently increases neurogenesis in neural progenitor cells culture. The present study also reveals that METH (10nM) enhanced differentiation into immature neurons in DG neurospheres, verified by the increase of DCX protein levels. This effect correlates with the fact that METH increases the pairs of cells that did not express Sox2 (Sox2−/Sox2− cell pairs), i.e., differentiating cells. Furthermore, cell cycle alterations induced by METH suggest a differentiation shift. In fact, the length of G1 phase may influence the decision for a neural stem cell to proliferate or differentiate as reviewed by Salomoni and Calegari (2010). Furthermore, METH did not induce any alterations in GFAP expression, indicating that immature neurons are preferentially generated rather than astrocytes. In accordance with our findings, it was described by Mandyam et al. (2008) that METH self-administration (0.05mg/kg/infusion, 1h/day, 2days/week for 49days) increases neuronal differentiation and maturation of hippocampal progenitor cells.