Primary cell culture of adult zebrafish spinal neurons for electrophysiological studies.

ABSTRACT

BACKGROUND: Zebrafish (Danio rerio) are growing in popularity as a vertebrate model organism for the study of spinal neurocircuitry and locomotion. While many studies have used the zebrafish model system for electrophysiological analyses in embryonic and larval stages, there is a growing interest in studying spinal circuits and neurons from adult fish. NEW METHOD: To expand upon the existing toolset available to the zebrafish research community, we have developed the first primary cell culture system of adult zebrafish spinal neurons. The intact spinal cord is dissected, and neurons are isolated through enzymatic digestion and mechanical dissociation. Identifiable neurons are viable for electrophysiological analyses after two days in culture. RESULTS: Spinal neurons in culture were confirmed by immunofluorescence labeling and found to exhibit distinct morphologies from other cell types, allowing neurons to be identified based on morphology alone. Neurons were suitable for calcium imaging and whole cell patch clamp recordings, which revealed excitable cells with voltage-gated whole cell currents, including tetrodotoxin-sensitive sodium currents. COMPARISON WITH EXISTING METHODS: This primary cell culture system is the only methodology available to isolate neurons from the adult zebrafish spinal cord. Other methods rely on keeping the spinal cord intact or the utilization of embryonic or larval stage fish. This method provides a robust platform for use in neurophysiological and pharmacological studies. CONCLUSIONS: The novel primary cell culture system described here provides the first in vitro methodology available to isolate and culture neurons from the adult zebrafish spinal cord for use in electrophysiological analyses.