Retrograde Labeling of Zebrafish Spinal Interneurons for Calcium Imaging Studies

Abstract

Teleost fish such as zebrafish utilize a rapid escape response, or C-start, in response to aversive stimuli. This response occurs when a stimulus triggers a ?C?-shaped posture through the contraction of the musculature on one side of the body and involves activation of circumferential descending (CiD) interneurons. The neuroexcitatory peptide c4a, a major component in the injected venom of the fish-hunting cone snail, Conus catus , elicits a qualitatively similar response. C4a induces repetitive firing of motor neurons in the snail?s prey, resulting in sustained muscle contractions that effectively paralyze the fish in the C-start posture. Knowing that CiD interneurons are involved in the rapid escape response, the goal of this study is to determine whether CiD interneurons are activated in the presence of c4a as well and convey the signal for rapid paralysis. Danio rerio (zebrafish) was used as a model system for in vivo calcium imaging of spinal neurons. Motor neurons and CiD interneurons were retrograde labeled with the calcium sensitive dye Calcium Green Dextran in 72-hour larvae. Calcium imaging of fluorescently labeled motor neurons in the presence of c4a showed an increase in intracellular calcium coinciding with muscle contractions, indicating the firing of these neurons. We are examining whether retrograde labeled CiD interneurons, like motor neurons, are also activated in the presence of c4a.