Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Summary

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Abstract

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

Introduction

In this article, a protocol is described for obtaining in vivo recordings of neural activity in adult zebrafish. Extracellular recording methods are used, providing voltage measurements of electrical activity within a small region of neural tissue. This method of investigation involves monitoring a large number of cells in a behaving animal¹. Previously, slice recordings have been performed in both adults and larvae, as have eye cup preparations and electroretinogram recordings. These experiments have largely been performed to detail physiological responses of various sensory systems^2-5. Until recently, intact brain preparations have onl....

Protocol

All experimental procedures were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed protocol #A2011 09-003, which was reviewed, approved, and overseen by the University of Georgia Institutional Animal Care and Use Committee.

1. Equipment Setup

1. Perfusion system for craniotomy
   Immobilization of the adult necessitates an intubation system to deliver dissolved oxygen to the fish.  A variety of systems can be utilized, but a simple gravity system consisting of a 60 ml syringe is used. Elevate the pressure head to a height that consi....

Results

This protocol has been used to measure the neural activity of adult zebrafish in vivo. These electrophysiological recordings are consistently and reproducibly obtained. Figure 5 shows a representative example of native and induced alterations of the neural activity of an adult zebrafish when pentylenetetrazol (PTZ), a common chemoconvulsant^6,7,25,26, is introduced in the intubation setup.

The native neurological activity of the adult zebrafish was monitored.......

Discussion

This protocol has been used to measure the neural activity of adult zebrafish in vivo. With practice, neural activity can be observed consistently, though the characteristics (amplitude and shape of events) of the recorded activity can vary between individual fish. Utilization of the extracellular recording technique can explain this observation. The method provides simultaneous monitoring of a large number of cells within a region¹, so variations in the positioning of the primary electrode can play a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70% Ethanol	Decon Laboratories	2750HC	Dilute 100% to 70% with DI water
2 M Potassium Chloride	J.T. Baker
2 M Sodium Chloride	J.T. Baker	3624-05
0.4% Tris-Buffered Tricaine	Sigma-Aldrich	E10521	pH 7.2-7.4; stored at -20 oC
Pancuronium Bromide	Sigma-Aldrich	P1918	Diluted to 1 μg/μl in 1x phosphate buffered saline
Habitat water			pH 7.0-7.4, conductivity of 400-450 μS; maintained by Instant Ocean and Sodium Bicarbonate
Pentylenetetrazol	Sigma-Aldrich	P6500	Diluted to 300 mM in 1x phosphate buffered saline
Nanofil syringe	World Precision Instruments, Inc.	06A
34 G Beveled needle	World Precision Instruments, Inc.	NF34BV
Sponge			Small pore and chemical-free
Foam-backed fine sand paper			5 x 5 cm2 is large enough
9 V Battery
Wires with alligator clips			Need 2
37 cm x 42 cm Kimwipe	Kimberly-Clark Professional	TW31KEM
11 cm x 21 cm Kimwipe	Kimberly-Clark Professional	TW31KWP
1/8 in diameter tube
1 cm diameter tube
1 mm diameter tube
Reducing valve with female Luer lock cap and silicone ferrule	Qosina	51505
Microscope (Leica MZ APO)			Another microscope can be used
Vanna scissors	Roboz Surgical Instruments Co., Inc.	15018-10
60 ml Luer lock syringe tubes	Becton, Dickinson and Company	309653
3-way Stopcocks with Luer connections
1-way Stopcock with Luer connection
Fisherbrand 100 mm x 15 mm Petri dish	Fisher Scientific	NC9299146
Fisherbrand 60 mm x 15 mm Petri dish	Fisher Scientific	S67961
4 in Borosilicate capillary tube	World Precision Instruments	TW100F-4	Can contain a filament to aid in filling with solution
P-97 Flaming/Brown Micropipette Puller	Sutter Instrument Co.
Digidata 1440	Molecular Devices
Axon Aloclamp 900A	Molecular Devices
Axoclamp software	Molecular Devices
HS-9Ax 1U headstage	Molecular Devices
0.010 in Silver wire	A-M Systems, Inc.
Q-series electrode holder	Warner Instruments	QSW-A10P
10 ml Luer lock syringe
1 mm x 15 in Tubing			Connect Luer lock syringe to Q-series electrode holder
Micromanipulator	Warner Instruments		Need 2
Microsoft-based PC	Dell
Faraday Cage
Air Table
Dissecting Microscope