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

Støttet af NIH grant SBC COBRE 1P20GM121310-01.

Alle metoder beskrevet her er blevet godkendt af institutionelle Animal Care og brug udvalg (IACUC) af Wyoming Universitet. Alle procedurer, herunder elektrofysiologiske optagelser, udføres ved stuetemperatur, ca 23 ° C. Alle metoder beskrevet her er optimeret til optagelse af tectal neuroner fra haletudser mellem udviklingsstadiet 42 og 49 (iscenesat ifølge Neiuwkoop og Faber15).
1. in Vivo forberedelse
<ol><li>Bedøver haletudse.<ol>
<li>Placer haletudse...

<ol>
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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. 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Alle metoder, der beskrives i dette arbejde er optimeret til optagelse af tectal neuroner fra haletudser mellem udviklingsstadiet 42 og 49 (iscenesat ifølge Neiuwkoop og Faber15). Tidspunkt 42, haletudser er tilstrækkeligt stort og tilstrækkeligt udviklede så at insekt benene kan placeres på enten side af hjernen i vivo optagelser og udfører hele hjernen dissektion. På tidligere stadier, når haletudser er hovedsagelig todimensional (dvs.flade), metoderne beskrevet her er ...

Retinotectal kredsløb er det vigtigste element i padde visuelle system. Det består af RGCs i øjet, som projektet deres axoner til det optiske tectum, hvor de danner synaptiske forbindelser med postsynaptiske tectal neuroner. Xenopus haletudse retinotectal kredsløb er en populær udviklingsmæssige model til at studere neurale kredsløb dannelsen og funktionen. Der er mange attributter af denne haletudse retinotectal kredsløb, der gør det en kraftfuld eksperimentel model1,2,3. Én stor attribut, og fokus i denne artikel, er evnen til at udføre hele cellen patch klemme optagelser fra tectal neuroner, in vivo eller ved hjælp af en hele hjernen forberedelse. Med et Elektrofysiologi rig outfitted med en forstærker, der understøtter spænding - og aktuelle-klemme optagelsestilstande, tillade hele cellen patch klemme optagelser en neuron Elektrofysiologi til karakteriseres i høj opløsning. Som følge heraf har hele cellen patch klemme optagelser fra tectal neuroner på tværs af de vigtigste faser af retinotectal kredsløb dannelse givet en detaljeret og omfattende forståelse af udviklingen og plasticitet af iboende4,5 , 6 , 7 og synaptic8,9,10,11 egenskaber. Ved at kombinere hele cellen patch klemme tectal neuron optagelser, evnen til at udtrykke gener eller morpholinos af interesse i disse neuroner12, og en metode til at vurdere visuel guidede adfærd via en etableret visuelle undgåelse test13 fremmer den identifikation af forbindelserne mellem molekyler, kredsløb funktion og adfærd.
Det er vigtigt at bemærke, at typen af høj opløsning data erhvervet fra hele cellen patch klemme optagelser ikke er muligt ved hjælp af nyere billeddiagnostiske tilgange såsom indikatoren genetiske calcium GCaMP6, fordi selv ved hjælp af calcium indikatorer tillader imaging af calcium dynamics på tværs af store populationer af neuroner samtidigt, der er ikke direkte eller indlysende måde at de specifikke elektriske parametre kan fås ved at måle delta fluorescens i somata, og der er ingen måde at spænding klemme neuron til at måle Aktuel spænding relationer. Klart disse to forskellige tilgange, elektrofysiologiske optagelser og calcium imaging, besidder ikke-overlappende styrker og generere forskellige typer af data. Den bedste fremgangsmåde afhænger således af det konkrete eksperimentelle spørgsmål bliver behandlet.
Her beskriver vi vores metode for at erhverve hele cellen patch klemme optagelser fra neuroner i haletudse optiske tectum ved hjælp af en i vivo forberedelse, hele hjernen forberedelse, og en nyere ændret hele hjernen forberedelse, der blev udviklet i vores lab14 . I afsnittet repræsentant resultater vise vi eksperimentelle fordelene ved hvert præparat og de forskellige typer af data, der kan opnås. Grænser og styrker af forskellige præparater, samt tips til fejlfinding, er inkluderet i afsnittet diskussion.

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

Xenopus haletudse retinotectal banen, består af retinal ganglion celler (RGCs) i øjet som danner synapser direkte til neuroner i det optiske tectum, er en populær model til at studere hvordan neurale kredsløb selv samle. Evnen til at udføre hele celle patch klemme optagelser fra tectal neuroner og registrere RGC-fremkaldte svar, enten i vivo eller ved hjælp af en hele hjernen forberedelse, har genereret en stor krop af høj opløsning data om de underliggende normale mekanismer , og unormal, kredsløb dannelsen og funktionen. Vi beskrive her, hvordan man udfører i vivo forberedelse, den oprindelige hele hjernen forberedelse, og en mere nylig udviklet vandrette hjernen skive forberedelse for at opnå hele cellen patch klemme optagelser fra tectal neuroner. Hvert præparat har unikke eksperimentelle fordele. I vivo forberedelse giver mulighed for optagelse af tectal neuroner direkte reaktion på visuelle stimuli projiceret op på øjet. Hele hjernen forberedelse giver mulighed for RGC axoner skal aktiveres i et stærkt kontrolleret måde, og horisontale hjernen skive forberedelse giver mulighed for optagelse fra på tværs af alle lag af tectum.

For at registrere lys-fremkaldte svar er en hele feltet lysglimt projiceres nethinden mens de resulterende svar er optaget fra enkelte tectal neuroner (figur 4A). Denne særlige protokol er designet til at måle både svaret af neuron til lyset tænde ("om" reaktion) og derefter slukke 15 s senere til at måle den "Off svar." Tectal neuroner typisk udviser robust på og ned af svar (vist her optaget i spænding klemme tilstand, med neuron fa...

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

I dette papir diskuterer vi tre hjernen præparater bruges til optagelse af hele cellen patch klemme til at studere retinotectal kredsløb af Xenopus laevis haletudser. Hvert præparat, med sin egen specifikke fordele, bidrager til den eksperimentelle sporbarhed af Xenopus haletudse som en model til at studere neurale kredsløb funktion.

Præparater og protokoller for hele cellen Patch klemme optagelsen af Xenopus laevis Tectal neuroner

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

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JoVE Journal

Neuroscience

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

Neural Explant Cultures from Xenopus laevis

Neural Explant Cultures from Xenopus laevis

Neurale eksplantatkulturer fra<em&gt; Xenopus laevis</em

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

Hele Cell Patch Clamp for Undersøgelse af mekanismerne i Infrarød 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 ...

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

Helcelle-patch-clamp Optagelser fra Morphologically- og Neurochemically identificerede Hippocampale interneuroner

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

Optagelse Light-fremkaldte Postsynaptiske Responses i neuroner i Dark-tilpassede, Mouse Retinal Slice Forberedelser Brug Patch Clamp Techniques

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-celle Patch-clamp Optagelser i Brain Skiver

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

Whole-cell Patch-clamp optagelser til elektrofysiologisk bestemmelse af ion selektivitet i Channelrhodopsins

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

Auditive hjernestammen svar og ydre hår celle hele-celle Patch klemme optagelse i Postnatal rotter

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

Forberedelse af akut rygmarven skiver til hele-celle Patch-klemme optagelse i Substantia Gelatinosa neuroner

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

Evaluering af Synaptic mangfoldighed ved hjælp af hele-celle Patch-klemme Elektrofysiologi

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

Hypothalamus kisspeptin neuroner som et mål for helcelle patch-clamp optagelser

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