To determine the cell autonomous and environmental factors that control the differentiation of neurons, astrocytes, and oligodendrocytes, we have used neurospheres made of primary neural progenitor cells. These organoids are amenable to the live cell imaging of several parameters which are central to the proper control of neuron and glial cell differentiation, as well as to the function of the resulting fully differentiated neural cells. Here we report on the methods to study in living cells the connexin-dependent cell-to-cell coupling, the oscillations in intracellular Ca2+ , and specifically the intercellular synchronization of such events, and the ATP release by either exocytosis of vesicles or through specialized membrane channels. The methods rely on the combination of a variety of state-of-the art microscopy and biophysical methods.