The identification and isolation of multipotent neural stem and progenitor cells in the brain, giving rise to neurons, astrocytes, and oligodendrocytes initiated many studies in order to understand basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system and to develop novel therapeutic strategies for cellular regeneration therapies in brain disease. A previous review (Trujillo et al., Cytometry A 2009;75:38-53) focused on the importance of extrinsic factors, especially neurotransmitters, for directing migration and neurogenesis in the developing and adult brain. Here, we extend our review discussing the effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms. Many of these mechanisms have been elucidated by in vitro studies for which neural stem cells were isolated, grown as neurospheres, induced to neural differentiation under desired experimental conditions, and analyzed for embryonic, progenitor, and neural marker expression by flow and imaging cytometry techniques. The better understanding of neural stem cells proliferation and differentiation is crucial for any therapeutic intervention aiming at neural stem cell transplantation and recruitment of endogenous repair mechanisms. ' 2012 International Society for Advancement of Cytometry

BackgroundNovel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.Principal FindingsP2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.ConclusionsIn embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.

Purinergic receptors belong to the most ancient neurotransmitter system. While their relevance in neurotransmission is well characterized, it has become clear that they have many other cellular functions. During development, they participate in regulation of proliferation and differentiation of stem cells. Here, we used rat embryonic telencephalon neurosphere cultures to detect purinergic P2 receptor subtype expression and possible synergistic actions of these receptors with NGF. Neurospheres proliferate in the presence of EGF and FGF-2; however, upon depletion of these growth factors, they migrate and differentiate into neurons and glial phenotypes. Expression patterns of P2X and P2Y receptors changed along neural differentiation. Gene expression of P2X2-7 and P2Y1,2,4,6,12,14 receptors was confirmed in undifferentiated and neural-differentiated neurospheres, with an up-regulation of P2X2 and P2X6 subtypes, together with a down-regulation of P2X4, P2X7 and P2Y subtypes upon induction to differentiation. BrdU-labeling and subsequent flow cytometry analysis was used to measure cell proliferation, which was increased by chronic exposure to NGF and increasing concentrations of ATP, in line with the expression levels of PCNA. Furthermore, a synergistic effect on proliferation was observed in conditions of co-incubation with ATP and NGF. While ATP and NGF independently promoted neural migration, no inter-relation between these factors was detected for this cellular process. As conclusion, an unknown synergism of ATP and NGF in proliferation is described. Future efforts may elucidate the underlying mechanisms of the interrelationship of ATP and NGF during neurogenesis.

Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyri-dostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 μM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 μM pyri-dostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.

Sleep deprivation is known to affect memory formation, but how it interacts with different memory systems is not completely understood. Adenosine, a homeostatic regulator of sleep that has an increased extracellular concentration during sleep deprivation, is one of the neuromodulators that may be involved in this interaction. The A adenosine receptor is involved in both sleep regulation and memory formation. Among other pathways, the A receptor decreases cAMP levels in the cytosol and thus also regulates protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) activity. To verify the role of the A receptor in the memory impairment caused by sleep deprivation, we tested the effect of 96 h of sleep deprivation (SD) and the administration of DPCPX, an A receptor antagonist on male Wistar rats prior to the training sessions for two memory tasks that relies on the hippocampal function: the multiple trial inhibitory avoidance (MTIA) task, which also requires the striatum, and the contextual fear conditioning (CFC) task, which does not. We also evaluated the effect of SD, DPCPX and the MTIA training session on the protein expression levels of the A1 receptor, PKA phosphorylation and EPAC activity in both the hippocampus and the striatum. Sleep deprivation impaired the performance in the test sessions of both tasks; DPCPX was able to prevent the impairment in the MTIA test but not in the CFC test. SD increased A receptor protein expression levels in the striatum but not in the hippocampus and also decreased PKA phosphorylation in both structures; DPCPX prevented this decrease in the striatum, but not in the hippocampus. Finally, SD had no effect on EPAC activity in either of the structures. These results indicate that the A adenosine receptors play a role in the memory impairment caused by sleep deprivation in tasks that involve the striatum through modulation of the cAMP/PKA pathway.