Generation of stable brain cell cultures from embryonic zebrafish to interrogate phenotypes in zebrafish mutants of neurodevelopmental disorders.

Abstract

Background: Zebrafish are a popular model system to study the genetic and neural basis of perception and behavior. Cultured primary neurons provide a complementary tool for such studies, but existing protocols for culturing embryonic zebrafish 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 brain cell cultures from zebrafish that could be used to study the mechanistic contributions of genes to neuronal networks.

New method: This protocol improves the viability of embryonic zebrafish primary brain cell cultures. We successfully optimized several parameters to generate long lived mixed cell type or pure neuronal cultures derived from embryonic zebrafish.

Results: Our optimized protocol produces cultures that form stable networks of neurons expressing the structural hallmarks of mature synaptic connections. As proof of principle, we apply our protocol to explore the cellular consequences of scn1lab loss of function. We find that loss of scn1lab results in increased prevalence of non-neuronal cells consistent with transcriptional signatures from embryonic tissue, providing support for the utility of our protocol.

Comparison with existing method(s): Most existing embryonic zebrafish primary neuron culture protocols describe growing mixed cell types for short durations, with a reported maximum of 9 days in vitro. Here, we describe a protocol that produces cultures viable for over 100 days.

Conclusions: The protocol reported in this study raises embryonic zebrafish primary brain cell culture to similar standards observed by well-established methods using cell lines or mammalian tissue.