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S5-03 LIVE IMAGING AND SINGLE CELL TRACKING REVEALS CELL DYNAMICS FROM EARLY CEREBELLAR NEURAL STEM CELLS. ROLE OF VNUT IN THEIR LINEAGE PROGRESSION. Felipe Ortega 1,2,3 , Aida Menendez-Mendez 1,2,3 , Lucía Paniagua-Herranz 1,2,3 ,Rosa Gomez-Villafuertes 1,2,3 , Raquel Pérez-Sen 1,2,3 , Esmerilda G Delicado 1,2,3 , María Teresa Miras-Portugal 1,2,3 . 1 Biochemistry and Molecular Biology Department, Faculty of Veterinary Medicine, Complutense University,Madrid, Spain; 2 Institute of Neurochemistry (IUIN), Madrid, Spain; 3 Health Research Institute of the Hospital Clínico San Carlos (IdISSC), Spain . The cerebellum, among the structures within the central nervous system, is the one whose development has been more studied. Cerebellar morphogenesis starts as early as embryonic day 7.5 but holds the particular feature of finalizing at postnatal stage (day 15). Afterbirth, cerebellar neural progenitors reside in three specific proliferative niches; the external granular layer (EGL) that produces the granule cells of the internal granular layer, the Purkinje cell layer (PCL) and the prospective white matter (PWM), which constitutes the main source of cerebellar interneurons. However, despite active research on cerebellar development, major questions of cerebellar neural stem cell (NSC) biology remain unanswered. For example, are cerebellar NSCs dependent or independent of their local niche to modulate their lineage progression? In addition we don’t know which are the cell dynamics exhibited by the NSCs located in these proliferative areas. We refer here to cell dynamics as the cell cycle length, mode of division (symmetric or asymmetric), multi/bi/unipotency of NSCs, self-renewal capacity, heterogeneity of NSCs populations etc. Most of the studies that tried to disentangle these questions were classically performed with cerebellar NSCs cultivated in the presence of high doses of growth factors that (i) do not ensure isolation from the niche and (ii) may modify the NSCs cell biology. In order to unravel these questions, we have adapted a previously developed preparation of the adult subependymal zone to the early (P0) postnatal cerebellum. In this preparation, cells are cultured in adherent conditions and in absence of growth factors or feeding layer, in isolation from their niche. Combining this preparation with continuous live imaging by timelapse videomicroscopy we were able to monitor the lineage progression of neural progenitors studying the cell-intrinsic processes that drive their transitions from neural progenitors to neurons (either glutamatergic or GABAergic). Moreover, the isolation from the niche provides a unique tool to study the effect of single factors on NSCs lineage progression. Among the different extracellular signaling pathways present in the cerebellar niche, purinergic signaling modulates crucial aspects of cerebellar granule neurons and astrocytes maturation and survival. Purinergic-mediated cell to cell communication begins with the loading of its fundamental molecule, the ATP, into secretory vesicles which is mediated by the recently cloned vesicular nucleotide transporter (VNUT). In this study we have assessed the role of VNUT in the modulation of cerebellar NSCs behavior.