The information available regarding the effect of METH on

The information available regarding the effect of METH on neurogenesis describes that, in the dentate gyrus (DG), cell proliferation is decreased in gerbils (postnatal day 30) administered once with the drug at postnatal day 14–20 (50mg/kg) (Hildebrandt et al., 1999). On the other hand, a lower dose of METH (25mg/kg) transiently decreased cell proliferation in the same region (Teuchert-Noodt et al., 2000). Furthermore, a chronic METH administration (1mg/kg/day for 14days) had no effect on the number of proliferating cells in mice DG (Maeda et al., 2007). Interestingly, Wistar rats self-administered with METH (0.05mg/kg/infusion, 1h intermittent access, 2× a week during 28days) displayed an increase in DG cell proliferation as well as in neuronal differentiation, whereas both short (1h/day) and long (6h/day) access decreased proliferation and differentiation followed by a reduced number of DG granule cell neurons (Mandyam et al., 2008). Furthermore, self-administration of METH (1h/day access METH for 13days) increased the number of radial glia-like cells (type 1 cells), but decreased the proportion of preneuronal neuroblasts (type 2a cells) (Yuan et al., 2011) showing that at different maturation stages cells respond differently to an external stimuli (Tashiro et al., 2007). Also, daily access to METH (6h/day for 4 or 13days) decreased the number of proliferating cells in the DG without changing, however, the length of S-phase of the PRIMA-1MET Supplier (Yuan et al., 2011). In vitro studies also point that METH reduced proliferation of rat hippocampal neural progenitor cells (Tian et al., 2009; Venkatesan et al., 2011). Additionally, our group recently verified that a nontoxic concentration of METH (1nM for 7days) decreased the number of mature neurons in DG-derived neurosphere cultures (Baptista et al., 2012). Concerning the subventricular zone (SVZ), we have also shown that METH decreases cell proliferation, neuronal differentiation and maturation of stem/progenitor cells (Bento et al., 2011).
Material and methods
Results
Discussion The present work addresses the effect of the drug of abuse METH on DG stem cell properties. Indeed, our results show that METH at nontoxic concentrations impaired stem cell properties, specifically by decreasing self-renewal capacity, delaying cell cycle progression and directing cell fate division towards differentiation. Firstly, we verified that METH (100nM) induced cell death of Sox2+ DG stem/progenitor cells from free floating neurospheres, having no effect at a lower concentration (10nM). Interestingly, we have previously shown that exposure to 10nM METH for 24h induced cell death on plated DG-derived neurosphere cultures (Baptista et al., 2012). Thus, we may conclude that neurospheres plated on poly-d-lysine and in a medium devoid of growth factors initiate differentiation and are more sensitive to METH toxicity as compared to free floating Sox2+ neurospheres cultured in the presence of growth factors. This difference may rely on differential abilities to repair DNA damages (Fernando et al., 2011), to counteract oxidative stress (Le Belle et al., 2011) and/or METH-induced apoptosis mechanisms displayed by stem/progenitor cells versus committed progenitors. Herein, we also explored for the first time the impact of METH on the cell cycle of DG stem cells. It was possible to observe a delay in the transition from G0/G1 to the S phase, an increase of cell population in the quiescence state (phase G0), and a down-regulation of cyclin E protein, which forms a complex with cyclin-dependent kinase 2 (Cdk2) being involved in the progression through the G1 phase and initiation of DNA replication in the S phase (Mazumder et al., 2004). Accordingly, to confirm that METH delays cell cycle progression, the doubling time for cell division was assessed and we observed that DG cells undergo two cell divisions in 24h (data not shown). On the other hand, METH did not induce any alterations in both cyclin D1 and cyclin A protein levels, which lead us to conclude that METH specifically impairs G1-to-S transition. In fact, little information is available regarding the effect of METH in cell cycle. Nevertheless, Yuan et al. (2011) assumed that hippocampal proliferating cells may be arrested in the G1 phase by METH (self-administration of 0.05mg/kg/injection, 6h/daily for 13days) since they observed a decreased number not only of cells in the S phase, but also of cells that enter and exit the S phase without changing, however, its length. In fact, other psychostimulants like cocaine (10 or 100μM, nontoxic concentrations) also interfere with the transition from G1-to-S phase in AF5 progenitor cells (a rat mesencephalic cell line), confirmed by the down-regulation of cyclin A2 (Lee et al., 2008). The authors further demonstrated that this effect was triggered by oxidative endoplasmic reticulum stress (Lee et al., 2008). Additionally, Hu et al. (2006) observed that cocaine inhibited proliferation in human fetal neural progenitor cells with simultaneous increase of the cyclin-dependent kinase inhibitor, p21. Moreover, very recently Blanco-Calvo et al. (2014) showed that acute administration of cocaine (10mg/kg) decreases the number of proliferating cells in the subgranular zone, which was prevented by the inhibition of cannabinoid CB1 or CB2 receptors. Cannabinoid receptor blockade was also able to prevent hippocampal-dependent contextual memories induced by cocaine (Blanco-Calvo et al., 2014).