Name: optogenetic activation of zebrafish somatosensory neurons using chef-tdtomato
Uploaded: 2013-01-31T13:00:00
Description: Watch this Scientific Journal Video about Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato at JoVE.com

We thank Fumi Kubo, Tod Thiele and HerwigBaier (UCSF/Max Planck Institute) for advice on behavior experiments and DPSS laser set up; Heesoo Kim and Chiara Cerri from the MBL Neurobiology Course for assisting in ChEF-tdTomato experiments; PetronellaKettunen (University of Gothenburg)for initial collaboration on optogenetic experiments; BaljitKhakh, Eric Hudson, Mike Baca and John Milligan (UCLA) for technical advice; and Roger Tsien (UCSD) for the ChEF-tdTomato construct. This work was supported by an NRSA (5F31NS064817) award to AMSP and a grant from the NSF (RIG:0819010) to AS.

Prepare the following ahead of time.
1. Prepare Optic Cable
<ol>
 <li>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 &deg; angle.</li>
 <li>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.</li>
 <li>Strip the optic cable down to the cladding by removing the fiber...

<ol>
	<li>Arrenberg, A. B., Del Bene, F., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+zebrafish+behavior+with+halorhodopsin.">Optical control of zebrafish behavior with halorhodopsin.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (42), 17968-17973 (2009).</li><li>Burgess, H. A., Johnson, S. L., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unidirectional+startle+responses+and+disrupted+left-right+co-ordination+of+motor+behaviors+in+robo3+mutant+zebrafish.">Unidirectional startle responses and disrupted left-right co-ordination of motor behaviors in robo3 mutant zebrafish.</a> Genes Brain Behav. 8 (5), 500-511 (2009).</li><li>Douglass, A. D., Kraves, S., Deisseroth, K., Schier, A. F., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+behavior+elicited+by+single,+channelrhodopsin-2-evoked+spikes+in+zebrafish+somatosensory+neurons.">Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons.</a> Curr. Biol. 18 (15), 1133-1137 (2008).</li><li>Downes, G. B., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supraspinal+input+is+dispensable+to+generate+glycine-mediated+locomotive+behaviors+in+the+zebrafish+embryo.">Supraspinal input is dispensable to generate glycine-mediated locomotive behaviors in the zebrafish embryo.</a> J. Neurobiol. 66 (5), 437-451 (2006).</li><li>Drapeau, P., Saint-Amant, L., Buss, R. R., Chong, M., McDearmid, J. 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(29), e1394 (2009).</li><li>Kohashi, T., Oda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+Mauthner-+or+non-Mauthner-mediated+fast+escape+evoked+by+different+modes+of+sensory+input.">Initiation of Mauthner- or non-Mauthner-mediated fast escape evoked by different modes of sensory input.</a> J. Neurosci. 28 (42), 10641-10653 (2008).</li><li>Lai, S. L., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+with+dual+binary+transcriptional+systems+in+Drosophila.">Genetic mosaic with dual binary transcriptional systems in Drosophila.</a> Nat. Neurosci. 9 (5), 703-709 (2006).</li><li>Liao, J. 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K., Trauner, D., Baier, H., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+dissection+of+a+behavioural+module+in+the+vertebrate+spinal+cord.">Optogenetic dissection of a behavioural module in the vertebrate spinal cord.</a> Nature. 461 (7262), 407-410 (2009).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li></ol>

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...

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.
To optimize a procedure for optogenetic activation of single zebrafish neurons, we focused on somatosensory neurons. Zebrafish larvae detect a variety of somatosensory stimuli using two populations of neurons: trigeminal neurons, which innervate the head, and Rohon-Beard (RB) neurons, which innervate the rest of the body. Each trigeminal and RB neuron projects a peripheral axon that branches extensively in the skin to detect stimuli and a central axon that connects to downstream neural circuits. Animals respond to touch as early as 21 hr post-fertilization (hpf), indicating that coherent somatosensory circuits have formed 5, 18. During larval development at least some trigeminal and RB neurons synapse onto the Mauthner cell to activate classic escape responses, but accumulating evidence suggests that there are multiple classes of somatosensory neurons with different patterns of connectivity that may elicit variations on the escape behavior 2, 4, 10, 12, 14, 15, 16, 17. Our motivation for developing this method was to characterize the behavioral function of different classes of somatosensory neurons, but this approach could in principle be used to study the function of almost any neuron or population of neurons in larval zebrafish.
Douglass et al. previously described a method for activating Channelrhodopsin-2-expressing somatosensory neurons with blue light, eliciting escape behavior 3. Their approach used an enhancer element from the isl1 gene to drive expression of ChR2-EYFP in somatosensory neurons. This transgene, however, was reported to display relatively weak fluorescence, requiring co-injection of a second reporter, UAS::GFP, to allow visualization of cells expressing ChR2-EYFP. This approach was used to elicit behavior responses between 24-48 hpf, but could never elicit a response past 72 hpf. Thus, while this method works for studying neural circuitry at very early larval stages (24-48 hpf), it is inadequate for characterizing neural circuits and behavioral responses in older larvae, when more diverse behavioral responses are apparent and neural circuits are more mature.
We sought to improve the sensitivity of this technique in order to characterize the function of subpopulations of larval RB neurons. To improve expression we used a somatosensory-specific enhancer (CREST3) 20 to drive expression of LexA-VP16 and a stretch of LexA operator sequences (4xLexAop) 11 to amplify the expression of a fluorescently tagged light-activated channel. This configuration amplified expression of the channel, eliminating the need for co-expressing a second reporter and allowing us to directly determine the relative abundance of the channel in each neuron. Using the LexA/LexAop sequence had the additional advantage of allowing us to introduce the transgene into zebrafish reporter lines that use the Gal4/UAS system. Transient expression of this transgene resulted in varying levels of expression, but was usually robust enough to visualize both the cell body and axonal projections of individual neurons over several days. To optimize sensitivity to light we used the light activated channel ChEF, a channelrhodopsin variant consisting of a chimera of channelopsin-1 (Chop1) and channelopsin-2 (Chop2) with a crossover site at helix loop E-F 13. This channel is activated at the same wavelength as ChR2, but requires lower light intensity for activation, making it more sensitive than other commonly used channels, including ChR2. The ChEF protein was fused to the red fluorescent protein, tdTomato, enabling us to screen for protein expression without activating the channel. As a light source, we used a diode pumped solid-state (DPSS) laser coupled to a fiber optic cable to deliver a precise, high-powered pulse of blue light to a specific region of the larvae. This allowed us to focus laser light on individual neurons, eliminating the need for finding rare transgenic animals expressing the channel in a single neuron. Using this approach, we were able to activate single RB neurons, record behavioral responses with a high-speed video camera, and image the activated neurons at high resolution with confocal microscopy.

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Glass 			Pasteur pipette</td>
 <td>Fisher</td>
 <td>1367820B</td>
 <td>or 			equivalent (10-15 mm diameter)</td>
 </tr>
 <tr>
 <td>200 			&mu;m 			optic fiber</td>
 <td>ThorLabs</td>
 <td>AFS200/220Y-CUSTOM</td>
 <td>Patch Cord, Length: 3 m, End A: FC/PC, End B: FC/PC, Jacket: FT030</td>
 </tr>
 <tr>
 <td>50 			&mu;m 			optic fiber</td>
 <td>ThorLabs</td>
 <td>AFS50/125Y-CUSTOM</td>
 <td>Patch Cord, Length: 3 m, End A: FC/PC, End B: FC/PC, Jacket: FT030</td>
 </tr>
 <tr>
 <td>Adjustable 			Stripping Tool</td>
 <td>ThorLabs</td>
 <td>AFS900</td>
 <td>or 			Three-Hole Stripping Tool (FTS4)</td>
 </tr>
 <tr>
 <td>Diamond 			Wedge scribe</td>
 <td>ThorLabs</td>
 <td>S90W</td>
 <td></td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette 			Puller</td>
 <td>Sutter 			Instruments</td>
 <td>P-97</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Borosilicate 			glass tubing with filament</td>
 <td>Sutter 			Instruments</td>
 <td>BF-100-78-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Injection 			mold</td>
 <td>n/a</td>
 <td>n/a</td>
 <td>Figure 			5</td>
 </tr>
 <tr>
 <td>1.5 			ml centrifuge tubes</td>
 <td>Any</td>
 <td>Any</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri 			dish (100x15 mm)</td>
 <td>Any</td>
 <td>Any</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri 			dish (60x15 mm)</td>
 <td>Any</td>
 <td>Any</td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure 			injector</td>
 <td>ASI</td>
 <td>MPPI-3</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Micromanipulator 			and metal stand</td>
 <td>Narashige</td>
 <td>M152</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Disposable 			plastic pipettes</td>
 <td>Fisherbrand</td>
 <td>13-711-7</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Poker 			(Pin holder and Insect pin)</td>
 <td>Fine 			Science Tools, Inc.</td>
 <td>26018-17 			and 26000-70</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>Fine 			Science Tools, Inc.</td>
 <td>11255-20</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Microloader 			pipette tips</td>
 <td>Eppendorf</td>
 <td>9300001007</td>
 <td></td>
 </tr>
 <tr>
 <td>28.5 &deg;C 			incubator</td>
 <td>any</td>
 <td>any</td>
 <td></td>
 </tr>
 <tr>
 <td>42 &deg;C 			heat block</td>
 <td>Any</td>
 <td>Any</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-Sterile 			scalpel blades #11</td>
 <td>Fine 			Scientific Tools, Inc.</td>
 <td>10011-00</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Dissecting 			scope</td>
 <td>Zeiss</td>
 <td>Stemi</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Fluorescent 			dissecting scope with standard filter</td>
 <td>Any</td>
 <td>any</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Confocal 			microscope </td>
 <td>Zeiss</td>
 <td>LSM 			510 or 710</td>
 <td>or 			equivalent with lasers for GFP and RFP, and 10x, 20x and 40x 			objectives </td>
 </tr>
 <tr>
 <td>High 			speed camera</td>
 <td>AOS 			Technologies, Inc.</td>
 <td>X-PRI 			(130025-10)</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>473 			nm portable laser</td>
 <td>Crystal 			lasers</td>
 <td>CL-473-050</td>
 <td>or 			higher power, with TTL option</td>
 </tr>
 <tr>
 <td>S48 			Stimulator</td>
 <td>Astro-Med, 			Inc. Grass Instrument division</td>
 <td>S48K</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>FC/PC 			to FC/PC mating sleeve</td>
 <td>ThorLabs</td>
 <td>ADAFC1</td>
 <td>May 			need for optic cable connection</td>
 </tr>
 <tr>
 <td>Laser 			Safety Glasses</td>
 <td>ThorLabs</td>
 <td>LG10</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>24 			culture 			plates</td>
 <td>Genesee</td>
 <td>25-102</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Single 			depression slides</td>
 <td>Fisher</td>
 <td>S175201</td>
 <td>Or 			equivalent</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Instant 			ocean</td>
 <td>Aquatic 			Ecosystems</td>
 <td>IS50</td>
 <td></td>
 </tr>
 <tr>
 <td>Methylene 			blue</td>
 <td>Fisher</td>
 <td>S71325</td>
 <td></td>
 </tr>
 <tr>
 <td>Phenol 			red</td>
 <td>Sigma</td>
 <td>P4758</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>EMD</td>
 <td>2125</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>Low 			Melt agarose</td>
 <td>Sigma</td>
 <td>A9045</td>
 <td>or 			equivalent</td>
 </tr>
 <tr>
 <td>PTU</td>
 <td>Sigma</td>
 <td>P7629</td>
 <td></td>
 </tr>
 <tr>
 <td>Tricaine</td>
 <td>Sigma</td>
 <td>A5040</td>
 <td></td>
 </tr>
 <tr>
			<td>blue/embryo water</td>
			<td></td>
			<td></td>
			<td>10 L ddH2O 
			0.6 g Instant Ocean 
			6 drops methylene blue</td>
		</tr>
		<tr>
			<td>phenol red </td>
			<td></td>
			<td></td>
			<td>(5 mg/ml in 0.2 M KCl)</td>
		</tr>
		<tr>
			<td>100x PTU</td>
			<td></td>
			<td></td>
			<td>0.150 g PTU 
			50 ml ddH2O 
			dissolve at 70 &deg;C, shake often 
			aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>1x PTU</td>
			<td></td>
			<td></td>
			<td>1 ml 100x PTU 
			99 ml blue/fish water</td>
		</tr>
		<tr>
			<td>Tricaine stock solution</td>
			<td></td>
			<td></td>
			<td>400 mg tricaine 
			97.9 ddH2O</td>
		</tr>
		<tr>
			<td>~2.1 ml 1M Tris, pH9.0</td>
			<td></td>
			<td></td>
			<td>adjust pH to ~7.0 
			store in 4 &deg;C or -20 &deg;C for long term storage</td>
		</tr>
 </tbody>
</table>

optogenetic activation of zebrafish somatosensory neurons using chef-tdtomato

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.

<img src="/files/ftp_upload/50184/50184fig1.jpg" alt="Figure 1"/> 
 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.
<img src="/files/ftp_upload/50184/50184fig2.jpg" alt="Figure 2"/> 
 Figure 2. Injection mold template.
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Watch this Scientific Journal Video about Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato at JoVE.com

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

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.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

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Neuroscience

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...