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

Summary

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.

Abstract

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 pin^1,2,3,4,5. 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 stages^6,7,8,9.

Introduction

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 wh....

Protocol

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

1. Raise and maintain adult zebrafish (Danio rerio) at 28.5 °C on a 10 h dark/14 h light cycle and with appropriate water treatment and exchange⁵².
2. Raise zebrafish embryos/larvae at 28.5 °C in embryo medium until they reach the desired stage (e.g., 2 dpf).

2. Preparation of Dissection Materials

1. Glue dispenser for zebrafish d....

Results

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.......

Discussion

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.

Materials

List of materials used in this article

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