Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Summary

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Abstract

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Introduction

The development of optogenetic methods for promoting or inhibiting neuronal excitability with defined wavelengths of light has made it possible to study the function of distinct populations of neurons in neural circuits controlling behavior ^{1, 19, 21}. This technique is often used to activate groups of neurons, but it can also be used to activate individual neurons. Zebrafish larvae are particularly amenable to these methods since they are translucent, their nervous system develops quickly, and creating transgenic animals is fast and routine. However, significant technical hurdles must be overcome to reliably achieve single neuron activation.

Protocol

Prepare the following ahead of time.

1. Prepare Optic Cable

1. Create a storage unit for the optic cable by melting the tapered neck of a glass Pasteur pipette over a Bunsen burner to create a ~150 ° angle.
2. Using a wire cutter or a razor blade, carefully cut the optic cable into two pieces. Each piece should have one end with a FC/PC adaptor and one exposed end. Store one piece as a reserve cable.
3. Strip the optic cable down to the cladding by removing the fiber jacket and strengthening fibers (Figure 1A) from 5.0 cm of cut end of the cable.
4. Insert optic cable into prepared Pasteur ....

Results

Figure 1. Optic cable set up. (A) Layers of a fiber optic cable. (B) Stripped fiber optic cable in a Pasteur pipette. (C) Fiber optic cable in Pasteur pipette positioned using a micromanipulator.

Discussion

We have described an approach for optogenetic activation of single RB neurons in live zebrafish. Our method employs transient transgenesis to express a fluorescently tagged channelrhodopsin variant, ChEF-tdTomato¹³, in specific somatosensory neurons. This approach could easily be adapted for use in other larval zebrafish cell populations.

Using this approach we consistently elicited behavioral responses from 34-48 hpf larvae expressing ChEF-tdTomato. Using a 5 msec pulse of bl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Glass Pasteur pipette	Fisher	1367820B	or equivalent (10-15 mm diameter)
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
Adjustable Stripping Tool	ThorLabs	AFS900	or Three-Hole Stripping Tool (FTS4)
Diamond Wedge scribe	ThorLabs	S90W
Flaming/Brown Micropipette Puller	Sutter Instruments	P-97	or equivalent
Borosilicate glass tubing with filament	Sutter Instruments	BF-100-78-10
Injection mold	n/a	n/a	Figure 5
1.5 ml centrifuge tubes	Any	Any
Petri dish (100x15 mm)	Any	Any
Petri dish (60x15 mm)	Any	Any
Pressure injector	ASI	MPPI-3	or equivalent
Micromanipulator and metal stand	Narashige	M152	or equivalent
Disposable plastic pipettes	Fisherbrand	13-711-7	or equivalent
Poker (Pin holder and Insect pin)	Fine Science Tools, Inc.	26018-17 and 26000-70	or equivalent
Forceps	Fine Science Tools, Inc.	11255-20	or equivalent
Microloader pipette tips	Eppendorf	9300001007
28.5 °C incubator	any	any
42 °C heat block	Any	Any
Non-Sterile scalpel blades #11	Fine Scientific Tools, Inc.	10011-00	or equivalent
Dissecting scope	Zeiss	Stemi	or equivalent
Fluorescent dissecting scope with standard filter	Any	any	or equivalent
Confocal microscope	Zeiss	LSM 510 or 710	or equivalent with lasers for GFP and RFP, and 10x, 20x and 40x objectives
High speed camera	AOS Technologies, Inc.	X-PRI (130025-10)	or equivalent
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
FC/PC to FC/PC mating sleeve	ThorLabs	ADAFC1	May need for optic cable connection
Laser Safety Glasses	ThorLabs	LG10	or equivalent
24 culture plates	Genesee	25-102	or equivalent
Single depression slides	Fisher	S175201	Or equivalent
Reagent
Instant ocean	Aquatic Ecosystems	IS50
Methylene blue	Fisher	S71325
Phenol red	Sigma	P4758
Agarose	EMD	2125	or equivalent
Low Melt agarose	Sigma	A9045	or equivalent
PTU	Sigma	P7629
Tricaine	Sigma	A5040
blue/embryo water			10 L ddH2O 0.6 g Instant Ocean 6 drops methylene blue
phenol red			(5 mg/ml in 0.2 M KCl)
100x PTU			0.150 g PTU 50 ml ddH2O dissolve at 70 °C, shake often aliquot and store at -20 °C
1x PTU			1 ml 100x PTU 99 ml blue/fish water
Tricaine stock solution			400 mg tricaine 97.9 ddH2O
~2.1 ml 1M Tris, pH9.0			adjust pH to ~7.0 store in 4 °C or -20 °C for long term storage