Stable primary brain cell cultures from zebrafish reveal hyperproliferation of non-neuronal cells from scn1lab mutants

Zebrafish are a popular model system for studying the genetic and neural underpinnings of perception and behavior, both in wild-type animals and in the context of disease modelling. Cultured primary neurons provide a key complementary tool for such studies, but existing protocols for culturing embryonic zebrafish primary neurons are limited by short cell survival and low neuronal purity. In this study, we set out to establish a protocol to produce long lived, pure neuronal cultures from zebrafish that could be used to study the mechanistic contributions of genes to neuronal networks. We then used these primary cultures to characterize cell proliferation and differentiation in primary neurons derived from scn1lab mutant embryos, which lack a sodium channel relevant to Dravet syndrome and autism. Using our optimized protocol, we generated cultures that proliferate, diversify, and form stable networks of neurons surviving for months. These stable cultures allowed us to perform genetic experiments, in this case revealing dramatic differences in the cellular composition of cultures derived from scn1lab mutant embryos versus their wild type siblings. Specifically, we find that loss of scn1lab promotes hyperproliferation of non-neuronal cells in mixed cultures of brain cells. In pure neuronal cultures, we find alterations in neurotransmitter subtypes consistent with known effects of scn1lab loss of function. Validating the utility of this approach, we then identify a corresponding hyperproliferation phenotype in live scn1lab mutant embryos, shedding light on potential mechanisms that may be relevant for Dravet syndrome. Significance statementMost existing embryonic zebrafish primary neuron culture protocols describe growing mixed cell types for restricted durations. Here, we report generation of zebrafish mixed type or pure neuronal cultures that are viable for over 100 days. We apply these cultures to gain new insight into scn1lab, a zebrafish orthologue of the Dravet Syndrome-associated sodium channel gene SCN1A. We report that loss of scn1lab results in hyperproliferation of non-neuronal cells, revealing an underappreciated mechanism by which mutations in SCN1A impact the structure and function of neuronal networks. Our in vitro cultures thus faithfully recapitulate in vivo neurobiology and provide a powerful platform to interrogate brain function in health and disease.