Supported by the NIH grant SBC COBRE 1P20GM121310-01.

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 &#176;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
<ol>
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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...

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&#39;s retinotectal circuit that render it a powerful experimental model1,2,3. One major attribute, and the focus of this article, is the ability to carry out whole cell patch clamp recordings from tectal neurons, in vivo or using a whole brain preparation. With an electrophysiology rig outfitted with an amplifier that supports voltage- and current-clamp recording modes, whole cell patch clamp recordings allow a neuron&#39;s electrophysiology to be characterized at high resolution. As a result, whole cell patch clamp recordings from tectal neurons across the key stages of retinotectal circuit formation have provided a detailed and comprehensive understanding of the development and plasticity of intrinsic4,5,6,7 and synaptic8,9,10,11 properties. Combining whole cell patch clamp tectal neuron recordings, the ability to express genes or morpholinos of interest in these neurons12, and a method to assess visual guided behavior via an established visual avoidance test13 promotes the identification of links between molecules, circuit function, and behavior.
It is important to note that the type of high resolution data acquired from whole cell patch clamp recordings is not possible using newer imaging approaches such as the genetic calcium indicator GCaMP6, because although using calcium indicators permits the imaging of calcium dynamics across large populations of neurons simultaneously, there is no direct or obvious way that the specific electrical parameters can be obtained by measuring delta fluorescence in the somata, and there is no way to voltage clamp the neuron to measure current-voltage relationships. Clearly these two distinct approaches, electrophysiological recordings and calcium imaging, possess non-overlapping strengths and generate different types of data. Thus, the best approach depends on the specific experimental question being addressed.
Here, we describe our method for acquiring whole cell patch clamp recordings from neurons of the tadpole optic tectum using an in vivo preparation, whole brain preparation, and a newer modified whole brain preparation that was developed in our lab14. In the Representative Results section, we demonstrate the experimental advantages of each preparation and the different types of data that can be obtained. The limits and strengths of the different preparations, as well as tips for troubleshooting, are included in the Discussion section.

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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Stemi Stereo 508</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:148px;">495009-0006-000&#160;</td>
			<td style="width:299px;">Dissecting microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS-222 &#34;Tricane&#34;</td>
			<td>Finquel</td>
			<td>ARF5G</td>
			<td>Amphibian general anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-1KG</td>
			<td>Used to prepare Stienberg&#39;s solution and external solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium nitrate tetrahyrate (Ca(NO3)&#8226;4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>237124-500G</td>
			<td>Used to prepare Stienberg&#39;s solution&#160;&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Sulfate (MgSO4)</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare Steinberg&#39;s solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080-500G</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Magnesium Chloride (MgCl2)</td>
			<td>J.T. Baker</td>
			<td>2444-01</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose Anhydrous</td>
			<td>Mallinckrodt Chemicals</td>
			<td>6066-04</td>
			<td>Used to prepare external recording solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubocurarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379</td>
			<td>Nicotinic acetylcholine receptor antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>0.1mm diameter stainless steel pins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>761028</td>
			<td>Preweighed monomer and curing agent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Polystyrene Petri Dish - 60x15mm</td>
			<td>Fisher Scientific</td>
			<td>AS4052</td>
			<td>Small petri dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)</td>
			<td>BD</td>
			<td>305122</td>
			<td>Syringe needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL Slip Tip Tuberculin Syringe&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td>Disposable, sterile syringes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate pipette glass</td>
			<td>Sutter Instrument</td>
			<td>BF150-86-10HP</td>
			<td>Pulled to desired specifications using pipette pulling machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Fabricates micropipettes for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes Kimtech wipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>Delicate task lint-free wipers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axon Instruments MultiClamp 700B Headstage CV-7B</td>
			<td>Molecular Devices</td>
			<td>1-CV-7B</td>
			<td>Current clamp and voltage clamp headstage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MP-285 Motorized Manipulator with Tabletop Controller</td>
			<td>Sutter Instrument</td>
			<td>MP-285/T</td>
			<td>Control for headstage on electrophysiology rig</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber-Coupled LED (Green)</td>
			<td>Thorlabs</td>
			<td>M530F2</td>
			<td>Fiber optic cable paired with green LED</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cluster Bipolar Electrode (25&#181;m diameter)</td>
			<td>FHC</td>
			<td>30207</td>
			<td>Bipolar stimulating electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISO-Flex Stimulator</td>
			<td>A.M.P.I. (Israel)&#160;</td>
			<td>Contact manufacturer</td>
			<td>Flexible stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axon Instruments 700B Multipatch Amplifier</td>
			<td>Molecular Devices</td>
			<td>2500-0157</td>
			<td>Amplifier for voltage- and current-clamp recording&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1322A digitizer</td>
			<td>Molecular Devices</td>
			<td>2500-135</td>
			<td>Data acquisition system for electrophysiology recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axio Examiner.A1</td>
			<td>Zeiss</td>
			<td>491404-0001-000&#160;</td>
			<td>Microscope for electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-g Lab Table</td>
			<td>TMC</td>
			<td>63-533</td>
			<td>Air table for electrophysiology microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inspiron 620 Personal Desktop Computer with Windows 7 64-bit</td>
			<td>Dell</td>
			<td>D06D001</td>
			<td>Computer running electrophysiology software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">c2400 CCD camera</td>
			<td>Hamamatsu</td>
			<td>70826-5</td>
			<td>Charge-coupled device camera for electrophysiology imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7 O&#39;Clock Super Platinum Stainless Razorblades</td>
			<td>Gillette</td>
			<td>CMM01049</td>
			<td>Platinum-coated stainless razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td>Disposable Polyethylene transfer pipets</td>
		</tr>
	</tbody>
</table>

preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons

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.

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 (&#34;On&#34; response) and then turning off 15 s later to measure the &#34;Off response.&#34;&#160;Tectal neurons typically exhibit robust On and Off responses (show...

Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

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

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.

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

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

The overall goal of this procedure is to obtain whole cell electrophysiological recordings from neurons of the tadpole optic tectum in order to learn about their pattern of connectivity during development and ultimately to understand how neural circuits form and function. This method can help answer key questions in the field of neural development such as how do neurons change over the course of development and how do circuits function to give rise to behavior. The main advantage of these preparations is that they allow for the electrophysiological recording of optic tectum neurons to provide insight about the function of the developing neural circuits.Demonstrating the procedure will be Zhenyu Lui, a graduate student in my lab. Using a disposal transfer pipette, move the anesthetized tadpole to a dissection recording dish containing external recording solution. Secure the anesthetized tadpole to a submerged silicone block on the floor of the dissecting recording dish.For a clear view of the brain, remove the skin overlying the brain by making a superficial incision along the midline using a sterile 25 gauge needle. Filet the brain along the same midline axis by inserting the needle into the neural tube and by gently pulling upward such that the dorsal portion of the tube is cleanly cut while leaving the floor plate intact. To isolate the brain, first use a 25 gauge needle to sever the hindbrain.Then gently run the needle underneath the brain in a caudal to rostral direction to sever all lateral and ventral connective tissue and nerve fibers. Next, secure the brain to a block of silicone elastomer by placing one pin through one of the olfactory bulbs and another pin through the hindbrain. This is the optimal configuration for recording from tectal neurons.Then move the dish containing the pinned whole brain preparation from the dissecting scope to the electrophysiology rig. Remove the ventricular membrane using a broken glass pipette. To directly activate the RGC axons, gently lower a bipolar stimulating electrode onto the optic chiasm such that a small dent is formed in the tissue.The bipolar stimulating electrode should be rostral and almost adjacent to the large middle ventricle where the optic chiasm is located. The bipolar electrode is driven by a pulse stimulator which allows the strength of stimulation to be precisely controlled. To prepare the horizontal brain slice, start with the whole brain preparation.Then use a razor blade to excise the most lateral fourth of one side of one optic tectum. This cut is made parallel to the rostral caudal plane. Afterward, re-pin the brain to the side of the silicone elastomer with the sliced side facing upward so that the somata and neuropil can be directly accessed for recording and the ventricular surface of the brain facing away from the silicone elastomer block so that a bipolar electrode can be placed on the optic chiasm.Then perform the whole cell patch clamp or local field potential recording. Here is the schematic showing the configuration of a horizontal brain slice preparation. Note that although the stimulating electrode remains at the optic chiasm, the recording pipette is now positioned to access cells across all the tectal layers exposed by the horizontal brain slice preparation.This is an RGC-evoked response from a neuron residing in the superficial layer of the tectum. In this figure, the recorded field potentials across the neuropil and the converted current source densities are shown by image plot. Once mastered, this technique can be done within 10 minutes and will typically for up to three or four hours.After watching this video, you should have a good understanding of how to perform whole brain and horizontal brain slice preparation for whole cell recordings in order to quantify the electrical properties of the tectal neurons and their connectivity.

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Neuroscience

9.3K 조회수.  University of Wyoming. The overall goal of this procedure is to obtain whole cell electrophysiological recordings from neurons of the tadpole optic tectum in order to learn about their pattern of connectivity during development and ultimately to understand how neural circuits form and function. This method can help answer key questions in the field of neural development such as how do neurons change over the course of development and how do circuits function to give rise to behavior. The main advantage of these pre...