Name: zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons
Uploaded: 2017-04-18T14:00:00
Description: Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

This work was supported by grants from the NIH (F32 NS059120 to RLM and R01NS25217 and P30NS048154 to ABR).

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC; Office of Laboratory Animal Resources, University of Colorado Anschutz Medical Campus).
1. Zebrafish Husbandry
<ol>
	<li>Raise and maintain adult zebrafish (Danio rerio) at 28.5 &#176;C on a 10 h dark/14 h light cycle and with appropriate water treatment and exchange52.</li>
	<li>Raise zebrafish embryos/larvae at 28.5 &#176;C in embryo medium until they reach the de...

The methods described here allow for the electrical and morphological characterization of sensory and motor neurons of zebrafish embryos after minimal dissection of the spinal cord. Neurons remain healthy for at least 1 h, the time limit imposed on these recordings. Neurons have been recorded using the standard whole-cell configuration, as well as from nucleated patches; the latter method minimizes space-clamp issues that can preclude a detailed biophysical study of ion currents9.
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George Streisinger pioneered the use of Danio rerio, commonly known as zebrafish, as a model system for the genetic analysis of vertebrate development10. The model offers several advantages including: (1) relatively simple and inexpensive animal husbandry; (2) external fertilization, allowing easy access to embryos from the earliest developmental stages; and (3) a transparent embryo, permitting direct and repeated observations of cells, tissues, and organs as they form.
Over the ensuing decades, several advances further increased the power of the zebrafish model. In particular, forward genetic screens and whole-genome sequencing efforts played key roles in the identification of mutations and genes critical to many developmental processes11,12,13,14,15,16. Gateway cloning methods have allowed the routine application of transgenic approaches17,18. Recent advances in genome editing, exemplified by transcription activator-like (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases, allow for the targeted introduction of mutations, as well as knock-out and knock-in approaches19,20,21,22. Combined, these methods make zebrafish a powerful model for the study of the genetic mechanisms underlying specific behaviors and several human diseases23,24,25,26,27.
This work focuses on developmental regulation and the role of electrical activity in neuronal development. The focus is on the spinal cord, for which the zebrafish model provides several advantages. First, it is relatively easy to access zebrafish at embryonic and larval stages; therefore, one can study spinal cord function during developmental stages that have fewer neurons and simpler circuitry28,29. Moreover, the zebrafish spinal cord has a diverse set of neurons, similar to other vertebrates, as demonstrated by characteristic and distinguishing patterns of transcription factors30,31,32,33,34,35.
The majority of studies in zebrafish that aim to uncover the mechanisms that underlie the function of spinal cord circuits, especially ones that support locomotion, are understandably focused on larval stages36,37,38,39,40,41,42,43. However, many of the neurons that form the spinal locomotive networks initiate their differentiation at early embryonic stages, ~9-10 h post-fertilization (hpf)44,45,46,47,48,49,50,51. In view of this, understanding how the morphological and electrical properties of spinal neurons arise and change between the embryonic and larval stages is important for an overall understanding of locomotor circuit formation and function.
The dissection methods described here allow patch clamp recordings from spinal neurons and have been successfully applied at embryonic stages (~17-48 hpf) and larval stages (~3-7 days post fertilization [dpf]). This approach limits the amount of dissection required to provide access to the neurons of interest. The protocol differs from the majority of the other published methods for recording from zebrafish spinal neurons in that veterinarian suture glue is used, rather than a fine tungsten pin, to attach the embryo or larva to the recording chamber. The availability of two different approaches (i.e., suture glue versus the tungsten pin) for mounting the zebrafish embryos or larvae for electrophysiological analysis provides researchers with alternative options to achieve their specific experimental goals.
First, procedures for accessing and recording from a population of primary sensory neurons, Rohon-Beard cells, are described. The cell bodies of these neurons lie within the dorsal spinal cord. Rohon-Beard cells exist in numerous vertebrate species, differentiate early in development, and underlie the embryonic touch response6,44,47,48.
Second, procedures for accessing and recording from spinal motor neurons are detailed. Zebrafish spinal motor neurons arise during two waves of neurogenesis. The earlier-born primary motor neurons arise at the end of gastrulation (~9-16 hpf), with only 3-4 primary motor neurons present per hemisegment45,46,49. In contrast, the later-born population of secondary motor neurons is more numerous and arises during a prolonged period, starting at ~14 hpf45,50. Secondary motor neuron genesis in mid-trunk segments is mostly completed by 51 hpf50. Secondary motor neurons are considered to be the counterpart of motor neurons in amniotes46. Interestingly, supraspinal neurons, via dopamine, regulate locomotion in the larva and secondary motor neuron genesis in the embryo and young larva50,51. Primary and secondary motor neurons each comprise several different subtypes. Each primary motor neuron subtype projects a peripheral axon that innervates a characteristic muscle group, resulting in a stereotypical, identifying axonal trajectory. Generally, secondary motor neurons follow the axonal pathways previously established by primary motor neurons. Thus, with respect to axonal trajectories, primary and secondary motor neurons are similar, with the exception that axonal thickness and somata size are greater for primary motor neurons45.
Third, methods for recording from a few types of interneurons are discussed. However, in these cases, a limited amount of removal of other spinal cord cells is required, and thus the spinal cord is less intact than for recordings from Rohon-Beard cells or motor neurons.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vacuum filter/Storage bottle, 0.22 mm pore</td>
			<td>Corning</td>
			<td>431096</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.2 mm</td>
			<td>Whatman</td>
			<td>6780-2502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma</td>
			<td>A-5040</td>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt&#160;</td>
		</tr>
		<tr>
			<td>a-bugarotoxin</td>
			<td>Tocris</td>
			<td>11032-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>4368-28-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-549 hydrazine salt</td>
			<td>Molecular Probes</td>
			<td>A-10438</td>
			<td>fluorescent dye</td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>Corning</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Fisher</td>
			<td>12-550C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>silicone elastomer</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>30-0038</td>
			<td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Drummond Scientific</td>
			<td>1-000-1000-100</td>
			<td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Miniature barbed polypropylene fitting&#160;</td>
			<td>Cole-Palmer</td>
			<td>6365-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive&#160;</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter Instruments</td>
			<td>&#160;Model P-97&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized micromanipulator&#160;&#160;</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher</td>
			<td>14-169-1B</td>
			<td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Molecular Devices</td>
			<td>1-HC-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmaseal Three-Way Stopcocks&#160;</td>
			<td>Baxter</td>
			<td>K75</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Axon Instruments</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td>Zeiss</td>
			<td>Axioskop2 FS plus</td>
			<td></td>
		</tr>
		<tr>
			<td>40x/0.80w Achroplan objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition and analysis software&#160;</td>
			<td>Axon Instruments</td>
			<td colspan="2">PClamp 10 - Clampex and Clampfit&#160;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="2">Dissection and Recording Solutions(in mM)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">All solutions, except the intracellular, are stable for ~ 2 - 3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td>
		</tr>
		<tr>
			<td colspan="4">The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20&#176;C) in small aliquots that are individually thawed on the day of use.&#160;</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="1">Dissection/Ringer&#8217;s solution</td>
			<td></td>
			<td colspan="2">145 NaCl, 3 KCl, 1.8 CaCl2.2H2O, 10 HEPES; pH 7.4 (with NaOH)</td>
		</tr>
		<tr>
			<td colspan="2">Pipette (intracellular) recording solution</td>
			<td></td>
			<td colspan="2">135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td>
		</tr>
		<tr>
			<td colspan="2">Bath (extracellular) recording solution/voltage and current-clamp</td>
			<td></td>
			<td colspan="2">125 NaCl, 2 KCl, 10 CaCl2.2H2O, 5 HEPES; pH 7.4 (with NaOH).</td>
		</tr>
		<tr>
			<td colspan="2">Alexa-594 hydrazine salt stock solution.&#160;</td>
			<td></td>
			<td colspan="2">Prepare a 13.2 mM stock in ddH2O, aliquot (~ 100 &#181;l) and store at -20&#176;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&#160; with a centrifuge tube filter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immobilizing agents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">0.4 % ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td></td>
			<td colspan="2">Prepare a 0.4% stock solution in 0.2M Tris, pH9 (0.4 g Tricaine/100 ml 0.2 M Tris</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">Adjust pH to 7 with NaOH and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute the stock solution ~ 25 fold in embryo media</td>
		</tr>
		<tr>
			<td>250 &#956;M &#945;-bungarotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 250 &#956;M stock in ddH2O (1 mg/500 &#956;l), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td>
		</tr>
		<tr>
			<td>1 mM Tetrodotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 1 mM stock in ddH2O (1 mg/3 ml), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td>
		</tr>
	</tbody>
</table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

We have successfully recorded from Rohon-Beard neurons in 17 hpf embryos through 7 dpf larvae (Figure 5A and 5B). When Rohon-Beard cells were recorded, the preparation was mounted dorsal-side up. Such mounting allows for the unambiguous identification of Rohon-Beard cells based on their superficial dorsal positions and large soma sizes. The identification is additionally confirmed by the stereotypical hyperpolarized resting membrane potential of these neu...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

The overall goal of this preparation is to allow the electrophysiological study of zebrafish spinal neurons in situ at the embryonic and larval stages. The electrophysiological analyses compliment the powerful genetic and imaging approaches that are also possible with the zebrafish model. While the method was originally developed to study rohon beard neurons, we have easily adapted the approach over time and now we can study various types of spinal neurons.Demonstrating the procedure will be Dr.Rosa Moreno, a research associate, with whom I've had the pleasure of collaborating for many years. To begin this procedure remove the black bulb from one glass bore, and replace it with a white rubber adaptor from another glass bore. Next, cut a piece of flexible tubing to a length of about 38 centimeters.Then, attach the flexible tubing via a small, straight polypropylene fitting. To prepare the silicone elastomer add the base and the curing agent to a plastic conical tube at a ratio of four to one. Afterward, mix the elastomer base and curing agent thoroughly.Pour the mixture into two 100 millimeter Petri dishes up to about one millimeter deep in one Petri dish and about 2.5 millimeters deep in the other. Allow the silicone elastomer to cure by exposure to air for four to five days. If cured elastomer is needed sooner, incubate it at 60 degrees Celsius.Afterward, cut out a 3.8 centimeter by 6.3 centimeter rectangle from the one millimeter thick cured elastomer to serve as the bottom of the chamber. From the 2.5 millimeter thick cured silicone cut a rectangle of 3.8 centimeters by 6.3 centimeters. Then from that, cut out an internal rectangle of 2.5 centimeters by five centimeters to generate a frame for the top of the chamber.To make the dissection recording chamber place the thin silicone rectangle directly on top of the glass slide, making sure to remove any air bubbles between the silicone and the glass. Then place the silicone rectangle frame on top of the thin bottom layer of silicone. In this procedure place an embryo in a dissection chamber containing two to three milliliters of ringer's solution.Immobilize the embryo by adding 100 microliters of 0.4%tricaine solution to the chamber. Then pull some injection micro pipettes with long thin tips from the thin wall glass with a micro pipette puller. Attach a pulled glass micro pipette to the glass floor of the glue dispenser.Under the dissecting microscope use tweezers to break the tip of the glass micro pipette resulting in a tip of about 75 micrometers in diameter. Now, load the glass micro pipette with three to five microliters of glue by applying suction through the mouth piece. Bring the glue filled tip to the dissection chamber.Then bring the tip of the glue loaded micro pipette near the head of the embryo while maintaining slight positive pressure to the micro pipette via the mouth tube. Once the tip is near the head of the embryo, apply sufficient positive pressure to expel a small drop of glue onto the bottom of the chamber. Use a dissecting pin to move the embryo towards the drop of glue so that the head makes contact with the glue.Orient the embryo dorsal side up, and press on the head to ensure good contact with the glue. As the glue slowly hardens use the dissecting pin to reposition the embryo so that it lies ventral side down, dorsal side up. Dip the dissecting pin into the drop of glue and drag a few threads of the glue over and across the head of the embryo to further secure its position.Once the head is securely attached to the silicone and the glue has solidified sacrifice the embryo by transection at the hind brain level with another glass micro pipette. Use a fresh glass micro pipette to superficially cut the skin several times at a position caudal the hind brain. Pierce the skin superficially by moving the pipette perpendicular to rostro caudal axis on each side of the trunk for dorsally mounted specimens, or on the exposed side of the trunk for laterally mounted specimens.To create a flap of skin for the tweezers to grab, scrape the skin several times with the micro pipette, at the level of and perpendicularly to the initial cut. Using tweezers, gradually lift the skin flap, and pull the skin caudally. After that, deliver a small drop of glue near the tail of the embryo.Use this glue to attach the tail to the bottom of the dissection chamber. As the glue hardens, adjust the position of the trunk with the dissecting pin to ensure that the trunk remains dorsally oriented, and that it is firmly attached to the chamber. Following this initial dissection, rinse the preparation extensively with ringer's solution to remove the tricaine and debris, and allow the preparation to rest for five minutes.Next, replace the dissection solution with extra cellular recording solution. If needed, add an immobilizing agent to the preparation. Next, transfer the dissection chamber with the mounted embryo to the stage of an upright compound microscope, equipped with a 40 X water immersion long working distance objective.Then mount an empty bore of silicate thick wall glass micro pipette onto the electrode holder of the head stage. Attach the tubing at one end to the air outlet of the electrode holder and at the other end to a three way stop cock. After that, place a mouth piece at one end of another piece of tubing, and attach it to the three way stop cock at the other end.Subsequently, bring the micro pipette tip to the most dorsal portion of the spinal cord, and gently pierce the meninges. Follow with swift, short, sideways movements to loosen the meninges. After the micro pipette tip has traversed the meninges advance and raise the micro pipette to pull the meninges away from the spinal cord.Subsequently move the micro pipette rostorally advancing over one to two hemi segments to expose the rohon beard cells. In our experience, rohon beard neurons are more fragile than other spinal neurons. To maintain them healthy we expose one or two rohon beards at a time.Voltage clamp recordings of outward and inward currents were obtained from the rohon beard neurons in one, two, and seven days post fertilization embryos and larvae. The holding potential was 80 millivolts, and the currents were elicited by a depolarizing step to positive 20 millivolts. In current clamp mode, 0.35 nano amp current injection for one millisecond elicits a single action potential from the rohon beard neurons in both embryos and larvae.In the absence of electrical stimulation, rohon beard neurons do not show spontaneous spiking. The resting potential of the rohon beard neurons becomes increasingly more negative as the development progresses. Once this method is mastered, embryos may be mounted and dissected to expose rohon beards within 10 minutes.As mentioned in the protocol, it is important to understand the factors that determine how quickly the glue hardens. This video highlights the critical steps for recording from spinal neurons in zebrafish.

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