cGMP MODULATES STEM CELLS DIFFERENTIATION TO NEURONS IN BRAIN IN VIVO U. GÓMEZ-PINEDO, a R. RODRIGO, b O. CAULI, b S. HERRAIZ, c J.-M. GARCIA-VERDUGO, d B. PELLICER, c A. PELLICER c AND V. FELIPO b * a Laboratorio de Morfologia Celular, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, Valencia, 46012, Spain b Laboratory of Neurobiology, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, Valencia, 46012, Spain c Fundacion IVI (Instituto Valenciano de Infertilidad), Instituto Univer- sitario IVI, Universidad de Valencia, C/Guadassuar, Valencia, 46015, Spain d Instituto Cavanilles, Universidad de Valencia, Polígono La Coma s/n, Paterna, Valencia 46071, Spain CIBERNED and Laboratorio de Mor- fologia Celular, Centro de Investigacion Principe Felipe, Avda. Auto- pista del Saler, Valencia, 46012, Spain Abstract—During brain development neural stem cells may differentiate to neurons or to other cell types. The aim of this work was to assess the role of cGMP (cyclic GMP) in the modulation of differentiation of neural stem cells to neurons or non-neuronal cells. cGMP in brain of fetuses was reduced to 46% of controls by treating pregnant rats with nitroargin- ine-methylester (L-NAME) and was restored by co-treatment with sildenafil.Reducing cGMP during brain development leads to reduced differentiation of stem cells to neurons and increased differentiation to non-neuronal cells. The number of neurons in the prefrontal cortex originated from stem cells proliferating on gestational day 14 was 71514/mm 2 in con- trol rats and was reduced to 44029/mm 2 (61% of control) in rats treated with L-NAME. In rats exposed to L-NAME plus sildenafil, differentiation to neurons was completely normal- ized, reaching 68311 neurons/mm 2 . In rats exposed to sil- denafil alone the number of cells labelled with bromode- oxyuridine (BrdU) and NeuN was 84116/mm 2 . In prefrontal cortex of control rats 48% of the neural stem cells prolif- erating in gestational day 14 differentiate to neurons, but only 24% in rats exposed to L-NAME. This was corrected by sildenafil, 40% of cells differentiate to neurons. Similar results were obtained for neurons proliferating during all developmental period. Treatment with L-NAME did not re- duce the total number of cells labelled with BrdU, further supporting that L-NAME reduces selectively the differenti- ation of stem cells to neurons. Similar results were ob- tained in hippocampus. Treatment with L-NAME reduced the differentiation of neural stem cells to neurons, al- though the effect was milder than in prefrontal cortex. These results support that cGMP modulates the fate of neural stem cells in brain in vivo and suggest that high cGMP levels promote its differentiation to neurons while reduced cGMP levels promote differentiation to non-neu- ronal cells. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: rat, nitric oxide, sildenafil. During brain development there is a strict control of the proliferation, migration and differentiation of neural stem cells to different cell types. Alterations in the control of these processes may result in altered balance in the for- mation of different cell types resulting in a long-lasting impairment of important cerebral processes. For example, exposure to alcohol during key stages of brain develop- ment accelerates glial cells formation, impairs neuron for- mation (Miller and Robertson, 1993) and results in im- paired cognitive function. In vitro studies have shown that ethanol exposure increases the differentiation of neural stem cells to glial cells and reduces neuronal differentiation (Tateno et al., 2005). The underlying mechanisms have not been clarified. The molecular mechanisms modulating differentiation of neural stem cells to neurons or non-neuronal cells are not well known. Nitric oxide (NO) plays a relevant role in this process. NO increases the activity of soluble guan- ylate cyclase about 200-fold and is a main modulator of cyclic GMP (cGMP) levels in brain (Russwurm and Koesling, 2004; Garthwaite, 2008; Domek-Lopacin ´ska and Strosznajder, 2005). It has been proposed that cGMP-mediated NO signal- ling may be involved in the early differentiation events of embryonic stem cells (Krumenacker and Murad, 2006; Madhusoodanan and Murad, 2007; Gibbs, 2003). If this is the case, pathological situations in which the production of cGMP is altered during brain development could lead to altered differentiation of stem cells to neurons or glial cells, resulting in cognitive impairment in the children. Moreover, normalizing cGMP levels in these situations could prevent the alterations in neural stem cells differentiation and cog- nitive impairment. The role of cGMP in stem cell differentiation has not been studied in vivo. The aims of this work were to assess in rats in vivo whether: (1) reduced cGMP levels during brain development alters the differentiation of stem cells to neurons or non-neuronal cells in vivo; and (2) restoration of cGMP levels prevents the alterations in neural stem cells differentiation. To assess the role of cGMP in neural stem cell differ- entiation we used a rat model in which brain cGMP levels are reduced during brain development by treating pregnant rats with nitroarginine-methylester (L-NAME), an inhibitor *Corresponding author. Tel: +34-963289680; fax: +34-963289701. E-mail address: vfelipo@cipf.es (V. Felipo). Abbreviations: BrdU, bromodeoxyuridine; cGMP, cyclic 3=-5=-guanosine monophosphate; FAD, flavin adenine nucleotide; L-NAME, nitroarginine- methylester; NADPH, nicotiamide adenine dinucleotide phosphate; NO, nitric oxide; PBS, phosphate buffer saline. Neuroscience 165 (2010) 1275–1283 0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.11.046 1275