Name: preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons
Uploaded: 2018-03-15T15:00:00
Description: Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

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>
	<li>Anesth...

<ol>
	<li>Pratt, K. G., Khakhalin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+neurodevelopmental+disorders+in+the+Xenopus+tadpole:+from+mechanisms+to+therapeutic+targets.">Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets.</a> Dis. Model Mech. 6, 1057-1065 (2013).</li><li>Pratt, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+Order+in+Human+Neurological+Disorder+Using+a+Tadpole.">Finding Order in Human Neurological Disorder Using a Tadpole.</a> Curr. Pathobio. Rep. 3 (2), 129-136 (2015).</li><li>Liu, Z., Hamodi, A. S., Pratt, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+and+function+of+the+Xenopus+tadpole+retinotectal+circuit.">Early development and function of the Xenopus tadpole retinotectal circuit.</a> Curr. Opin. Neurobiol. 41, 17-23 (2016).</li><li>Hamodi, A. S., Pratt, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+regulation+of+voltage-gated+intrinsic+currents+in+the+developing+optic+tectum+of+the+Xenopus+tadpole.">Region-specific regulation of voltage-gated intrinsic currents in the developing optic tectum of the Xenopus tadpole.</a> J. Neurophysiol. 112 (7), 1644-1655 (2014).</li><li>Pratt, K. G., Aizenman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+regulation+of+intrinsic+excitability+and+synaptic+transmission+in+a+developing+visual+circuit.">Homeostatic regulation of intrinsic excitability and synaptic transmission in a developing visual circuit.</a> J. Neurosci. 27 (31), 8268-8277 (2007).</li><li>Cialeglio, C. M., Khakhalin, A. S., Wang, A. F., Constantino, A. C., Yip, S. P., Aizenman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+analysis+of+electrophysiological+diversity+of+Xenopus+visual+neurons+during+development+and+plasticity.">Multivariate analysis of electrophysiological diversity of Xenopus visual neurons during development and plasticity.</a> Elife. 4, 11351 (2015).</li><li>Aizenman, C. D., Akerman, C. J., Jensen, K. R., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+driven+regulation+of+intrinsic+neuronal+excitability+improves+stimulus+detection+in+vivo.">Visually driven regulation of intrinsic neuronal excitability improves stimulus detection in vivo.</a> Neuron. 39 (5), 831-842 (2003).</li><li>Wu, G., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cline+H.T.+of+a+central+glutamatergic+synapse.">Cline H.T. of a central glutamatergic synapse.</a> Science. , 972-976 (1996).</li><li>Van Rheed, J. J., Richards, B. A., Akerman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory-evoked+spiking+behavior+emerges+via+an+experience-dependent+plasticity+mechanism.">Sensory-evoked spiking behavior emerges via an experience-dependent plasticity mechanism.</a> Neuron. 87 (5), 1050-1060 (2015).</li><li>Schwartz, N., Schohl, A., Ruthazer, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+transcription+of+BDNF+enhances+visual+acuity+during+development.">Activity-dependent transcription of BDNF enhances visual acuity during development.</a> Neuron. 70 (3), 455-467 (2011).</li><li>Zhang, L. I., Tao, H. W., Holt, C. E., Harris, W. A., Poo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+window+for+cooperation+and+competition+among+developing+retinotectal+synapses.">A critical window for cooperation and competition among developing retinotectal synapses.</a> Nature. 395 (6697), 37-44 (1998).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation+in+vivo+within+the+intact+developing+brain.">Single cell electroporation in vivo within the intact developing brain.</a> J. Vis. Exp. (17), e705 (2008).</li><li>Dong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+avoidance+in+Xenopus+tadpoles+is+correlated+with+the+maturation+of+visual+responses+in+the+optic+tectum.">Visual avoidance in Xenopus tadpoles is correlated with the maturation of visual responses in the optic tectum.</a> J. Neurophysiol. 101 (2), 803-815 (2009).</li><li>Hamodi, A. S., Pratt, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+horizontal+brain+slice+preparation:+a+novel+approach+for+visualizing+and+recording+from+all+layers+of+the+tadpole+tectum.">The horizontal brain slice preparation: a novel approach for visualizing and recording from all layers of the tadpole tectum.</a> J. Neurophysiol. 113 (1), 400-407 (2015).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li><li>Segev, A., Garcia-Oscos, F., Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> J. Vis. Exp. (112), e54024 (2016).</li><li>Muldal, A. M., Lillicrap, T. P., Richards, B. A., Akerman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Relationships+Impact+Neuronal+Tuning+within+a+Phylogenetically+Ancient+Vertebrate+Brain+Structure.">Clonal Relationships Impact Neuronal Tuning within a Phylogenetically Ancient Vertebrate Brain Structure.</a> Curr. Biol. 24 (16), 1929-1933 (2014).</li><li>Khakhalin, A. S., Koren, D., Gu, J., Xu, H., Aizenman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation+and+inhibition+in+recurrent+networks+mediate+collision+avoidance+in+Xenopus+tadpoles.">Excitation and inhibition in recurrent networks mediate collision avoidance in Xenopus tadpoles.</a> Eur. J. Neurosci. 40 (6), 2948-2962 (2014).</li><li>Ruthazer, E. S., Aizenmann, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+to+see:+patterned+visual+activity+and+the+development+of+visual+function.">Learning to see: patterned visual activity and the development of visual function.</a> Trends Neurosci. 44 (4), 183-192 (2010).</li><li>Pratt, K. G., Aizenman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisensory+integration+in+mesencephalic+trigeminal+neurons+in+Xenopus+tadpoles.">Multisensory integration in mesencephalic trigeminal neurons in Xenopus tadpoles.</a> J. Neurophysiol. 102 (1), 399-412 (2009).</li></ol>

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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			<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>
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			<td height="21" style="height:21px;">MS-222 &#34;Tricane&#34;</td>
			<td>Finquel</td>
			<td>ARF5G</td>
			<td>Amphibian general anesthetic</td>
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			<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">Tubocurarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379</td>
			<td>Nicotinic acetylcholine receptor antagonist</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;">Sterile Polystyrene Petri Dish - 60x15mm</td>
			<td>Fisher Scientific</td>
			<td>AS4052</td>
			<td>Small petri dishes</td>
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			<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;">Kimwipes Kimtech wipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>Delicate task lint-free wipers</td>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;">Axio Examiner.A1</td>
			<td>Zeiss</td>
			<td>491404-0001-000&#160;</td>
			<td>Microscope for electrophysiology</td>
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			<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>
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			<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>
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		<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>
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			<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>
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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

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...