Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Summary

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Abstract

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Introduction

The retinotectal circuit is the major component of the amphibian visual system. It is comprised of the RGCs in the eye, which project their axons to the optic tectum where they form synaptic connections with postsynaptic tectal neurons. The Xenopus tadpole retinotectal circuit is a popular developmental model to study neural circuit formation and function. There are many attributes of this tadpole's retinotectal circuit that render it a powerful experimental model^1,2,3. One major attribute, and the focus of this article, is the ability to carry out whole cell pat....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wyoming. All procedures, including electrophysiological recordings, are carried out at room temperature, approximately 23 °C. All methods described here are optimized for recording tectal neurons from tadpoles between developmental stage 42 and 49 (staged according to Neiuwkoop and Faber¹⁵).

1. In Vivo Preparation

1. Anesthetize the tadpole.
   1. Place the tadpole in a small Petri dish containing Steinberg's solution with 0.01% MS-222 for approximately 5 min.<....

Results

To record light-evoked responses a whole field flash of light is projected onto the retina while the resulting response is recorded from individual tectal neurons (Figure 4A). This particular protocol is designed to measure both the response of the neuron to the light turning on ("On" response) and then turning off 15 s later to measure the "Off response." Tectal neurons typically exhibit robust On and Off responses (shown here recorded in vo.......

Discussion

All methods described in this work are optimized for recording tectal neurons from tadpoles between developmental stage 42 and 49 (staged according to Neiuwkoop and Faber¹⁵). By stage 42, the tadpoles are sufficiently large and sufficiently developed so that the insect pins can be placed on either side of brain for in vivo recordings and for carrying out the whole brain dissection. At earlier stages, when the tadpoles are essentially two-dimensional (i.e., flat), the approaches d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stemi Stereo 508	Zeiss	495009-0006-000	Dissecting microscope
MS-222 "Tricane"	Finquel	ARF5G	Amphibian general anesthetic
Sodium Chloride (NaCl)	Fisher Scientific	S271-3	Used to prepare Stienberg's solution and external solution
Potassium Chloride (KCl)	Fisher Scientific	P217-500	Used to prepare Stienberg's solution and external solution
HEPES	Sigma-Aldrich	H3375-1KG	Used to prepare Stienberg's solution and external solution
Calcium nitrate tetrahyrate (Ca(NO3)•4H2O)	Sigma-Aldrich	237124-500G	Used to prepare Stienberg's solution
Magnesium Sulfate (MgSO4)	Mallinckrodt Chemicals	6066-04	Used to prepare Steinberg's solution
Calcium Chloride (CaCl2)	Sigma-Aldrich	C5080-500G	Used to prepare external recording solution
Magnesium Chloride (MgCl2)	J.T. Baker	2444-01	Used to prepare external recording solution
D-glucose Anhydrous	Mallinckrodt Chemicals	6066-04	Used to prepare external recording solution
Tubocurarine hydrochloride pentahydrate	Sigma	T2379	Nicotinic acetylcholine receptor antagonist
Insect Pins	Fine Science Tools	26002-10	0.1mm diameter stainless steel pins
Sylgard 184 Silicone Elastomer Kit	Dow Corning	761028	Preweighed monomer and curing agent kit
Sterile Polystyrene Petri Dish - 60x15mm	Fisher Scientific	AS4052	Small petri dishes
PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)	BD	305122	Syringe needles
1mL Slip Tip Tuberculin Syringe	BD	309659	Disposable, sterile syringes
Borosilicate pipette glass	Sutter Instrument	BF150-86-10HP	Pulled to desired specifications using pipette pulling machine
Flaming/Brown Micropipette Puller	Sutter Instruments	P-97	Fabricates micropipettes for electrophysiology recording
Kimwipes Kimtech wipes	Kimberly-Clark	34120	Delicate task lint-free wipers
Axon Instruments MultiClamp 700B Headstage CV-7B	Molecular Devices	1-CV-7B	Current clamp and voltage clamp headstage
MP-285 Motorized Manipulator with Tabletop Controller	Sutter Instrument	MP-285/T	Control for headstage on electrophysiology rig
Fiber-Coupled LED (Green)	Thorlabs	M530F2	Fiber optic cable paired with green LED
Cluster Bipolar Electrode (25µm diameter)	FHC	30207	Bipolar stimulating electrode
ISO-Flex Stimulator	A.M.P.I. (Israel)	Contact manufacturer	Flexible stimulus isolator
Axon Instruments 700B Multipatch Amplifier	Molecular Devices	2500-0157	Amplifier for voltage- and current-clamp recording
Digidata 1322A digitizer	Molecular Devices	2500-135	Data acquisition system for electrophysiology recording
Axio Examiner.A1	Zeiss	491404-0001-000	Microscope for electrophysiology
Micro-g Lab Table	TMC	63-533	Air table for electrophysiology microscope
Inspiron 620 Personal Desktop Computer with Windows 7 64-bit	Dell	D06D001	Computer running electrophysiology software
c2400 CCD camera	Hamamatsu	70826-5	Charge-coupled device camera for electrophysiology imaging
7 O'Clock Super Platinum Stainless Razorblades	Gillette	CMM01049	Platinum-coated stainless razor blades
Transfer Pipets	Fisher Scientific	13-711-7M	Disposable Polyethylene transfer pipets