Presentation Title

Primary Cell Culture of Adult Zebrafish Spinal Neurons for Electrophysiological Studies

Faculty Mentor

Dr. Joseph Schulz

Start Date

23-11-2019 10:45 AM

End Date

23-11-2019 11:30 AM

Location

114

Session

poster 4

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Zebrafish (Danio rerio) have increasingly become a popular organism in biological research fields, especially in neuroscience, as a paradigmatic vertebrate model. The mature adult zebrafish model can be used to study neural circuits and the physiological processes within the spinal cord. Building upon previous zebrafish research regarding the spinal cord, we used novel methods to successfully develop in vitro primary cell culture for the adult zebrafish spinal neurons. After dissecting and isolating neurons from an intact spinal cord, we cultured robust cells displaying distinct neuronal morphology and behavior with large cell somas, long axons and dendrites, and highly active growth cones. To characterize cells in culture as neurons, immunofluorescence labeling, calcium imaging, and patch clamping were performed. The staining of the cells with NeuN antibody, neuronal nuclear marker, and axonal marker acetyltubulin confirmed neuronal characteristics. Morphologic analysis was supported by calcium imaging which confirmed an excitable cell type. Lastly, analysis from the recordings of cell patch clamping revealed strong voltage-dependent currents, both inward and outward, as well as tetrodotoxin-blocked sodium conductance, both of which are consistent with neuronal behavior. Altogether, these tests successfully and effectively identified the spinal neurons of adult zebrafish from other surrounding cells in culture confirming successful isolation.

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Nov 23rd, 10:45 AM Nov 23rd, 11:30 AM

Primary Cell Culture of Adult Zebrafish Spinal Neurons for Electrophysiological Studies

114

Zebrafish (Danio rerio) have increasingly become a popular organism in biological research fields, especially in neuroscience, as a paradigmatic vertebrate model. The mature adult zebrafish model can be used to study neural circuits and the physiological processes within the spinal cord. Building upon previous zebrafish research regarding the spinal cord, we used novel methods to successfully develop in vitro primary cell culture for the adult zebrafish spinal neurons. After dissecting and isolating neurons from an intact spinal cord, we cultured robust cells displaying distinct neuronal morphology and behavior with large cell somas, long axons and dendrites, and highly active growth cones. To characterize cells in culture as neurons, immunofluorescence labeling, calcium imaging, and patch clamping were performed. The staining of the cells with NeuN antibody, neuronal nuclear marker, and axonal marker acetyltubulin confirmed neuronal characteristics. Morphologic analysis was supported by calcium imaging which confirmed an excitable cell type. Lastly, analysis from the recordings of cell patch clamping revealed strong voltage-dependent currents, both inward and outward, as well as tetrodotoxin-blocked sodium conductance, both of which are consistent with neuronal behavior. Altogether, these tests successfully and effectively identified the spinal neurons of adult zebrafish from other surrounding cells in culture confirming successful isolation.