Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

Abstract

Source: Palanca A. M. S., et al. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato. J. Vis. Exp. (2013).

This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

Protocol

1. Mount Larvae for Behavior Experiments

1. Make 1.5% low melt agarose in ddH2O and store in a 42 °C heat block to prevent it from solidifying.
2. Using a glass Pasteur pipette, transfer one of the pre-screened larvae into a tube of 1.5% low melt agarose with as little blue/embryo water as possible.
3. Transfer larvae in a drop of agarose onto a small Petri dish.
4. Under a dissecting microscope, position larva dorsal up.
5. When agarose has solidified, cut away the agarose with a thin razor blade (#11 scalpel), leaving a wedge of agar around the entire larva.
6. Make two diagonal cuts at either side of....

Results

Figure 1. Diagram of a mounted zebrafish larva and representative neurons involved in the larval touch response. Zebrafish larvae were partially mounted in 1.5% low melt agarose (represented by dashed lines surrounding the rostral portion of the larva). A trigeminal neuron .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Low Melt agarose 	Sigma	A9045	or equivalent
Petri dish (100x15 mm)	Any	Any
Non-Sterile scalpel blades	Fine Scientific Tools, Inc.	10011-00	or equivalent
blue/embryo water			10 L ddH2O 0.6 g Instant Ocean 6 drops methylene blue
High speed camera	AOS Technologies, Inc.	X-PRI (130025-10)	or equivalent
Glass Pasteur pipette	Fisher	1367820B	or equivalent (10-15 mm diameter)
473 nm portable laser	Crystal lasers	CL-473-050	or higher power, with TTL option
S48 Stimulator	Astro-Med, Inc. Grass Instrument division	S48K	or equivalent
Tricaine	Sigma	A5040
200 μm optic fiber	ThorLabs	AFS200/220Y-CUSTOM	Patch Cord, Length: 3 m, End A: FC/PC, End B: FC/PC, Jacket: FT030
50 μm optic fiber	ThorLabs	AFS50/125Y-CUSTOM	Patch Cord, Length: 3 m, End A: FC/PC, End B: FC/PC, Jacket: FT030
24 culture plates	Genesee	25-102	or equivalent