Loss of NMDA receptor function during development results in decreased KCC2 expression and increased neurons in the zebrafish forebrain

Abstract

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysfunction implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed and well-studied for their synaptic and activity-dependent function. Nevertheless, they play important roles during development in cell types and stages where there are no synapses or activity-dependence. Numerous mutations in their subunits are associated with neurodevelopmental disorders yet despite this interconnection, the contribution of NMDA receptors to neurogenesis is ambiguous and contradictory in the literature. To rigorously define the role of NMDA receptors in developmental neurogenesis, we capitalized on our mutant zebrafish line (grin1−/−) that lacks all NMDA receptors. Forebrain analysis shows thatgrin1−/−fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis but extended to all forebrain regions during postembryonic neurogenesis. Based on analysis of neural stem and progenitor cells and the ontogenetically-expressed chloride transporter, KCC2, we find that without NMDA receptors, immature neuroblasts acting as transit amplifying cells do not mature into postmitotic neurons in a timely manner. This prolongs their mitotic stage and leads to inappropriate amplification of forebrain neurons. Pharmacological blockade with MK-801 recapitulates this phenotype, indicating that NMDA receptor-mediated maturation of neuroblasts requires ionotropic signaling despite the absence of synapses in these cells. Our results demonstrate that NMDA receptors are required for maturational termination of mitosis in neuroblasts. Disruption of this process results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in neurodevelopmental disorder pathogenesis.SignificanceThe role of NMDA receptors in neurogenesis is ambiguous and complicated by contradictory studies. We show that NMDA receptors are required for the regulation of neurogenesis in neural progenitor cells during the postembryonic period: without NMDA receptors, these mitotically-active neuroblasts do not mature to the post-mitotic phase in a timely manner, causing supernumerary neurons in the forebrain. Dysregulated neurogenesis is a common phenotype in neurodevelopmental disorders and, notably, numerous mutations in NMDA receptor genes are also associated with these disorders. Thus, our work rigorously defines a specific role for NMDA receptors in neurogenesis and post-embryonic brain maturation and offers new perspective on the intersection of developmental neurogenesis, NMDA receptor dysfunction, and neurodevelopmental disorder etiology.