Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca²⁺) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca²⁺ channels that triggered Ca²⁺ oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca²⁺ activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation. Significance Synchronized activity among groups of interconnected cells is essential for diverse functions in the brain. Most studies on neuronal networks have been performed in the mature brain when chemical synapses have been established. However, less is known about networking during embryonic development. We have studied neural progenitors and found that they form gap junction-mediated small-world networks, which, via electrical depolarization, drive spontaneous calcium activity to stimulate cell proliferation. Our data underscore the critical role of intricate cell signaling during embryonic development and show that complex networks of immature cells exist in the brain before birth.

Progress in stem cell biology research is enhancing our ability to generate specific neuron types for basic and applied studies and to design new treatments for neurodegenerative diseases. In the case of Parkinson's disease (PD), alternative human dopaminergic (DAergic) neurons other than primary fetal tissue do not yet exist. One possible source could be human neural stem cells (hNSCs), although the yield in DAergic neurons and their survival are very limited.In this study, we found that Bcl-X_Lenhances (one-to-two orders of magnitude) the capacity for spontaneous dopaminergic differentiation of hNSCs, which then exceeds that of cultured human ventral mesencephalic tissue. Bcl-X_Lalso enhanced total neuron generation by hNSCs, but to a lower extent. Neuronal phenotypes other than DA were not affected by Bcl-X_L, indicating an exquisitely specific effect on DAergic neurons.In vivo, grafts of Bcl-X_L-overexpressing hNSCs do generate surviving human TH⁺neurons in the adult rat 6-OH-dopamine lesioned striatum, something never seen when naive hNSCs were transplanted. Most of the data obtained here in terms of the effects of Bcl-X_Lare consistent with an enhanced survival type of mechanism and not supportive of induction, specification, or proliferation of DAergic precursors.From thisin vitroandin vivoevidence, we conclude that enhancing Bcl-X_Lexpression is important to obtain human DAergic neurons from hNSCs. These findings may facilitate the development of drug-screening and cell-replacement activities to discover new therapeutic strategies for PD.