Name: patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina
Uploaded: 2016-10-13T14:00:00
Description: Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

Etisk godkjenning - prosedyrer som involverer dyr fag ble gjennomført i samsvar med regler og forskrifter av National Institutes of Health retningslinjer for forsøksdyr, som er godkjent av den institusjonelle dyr omsorg og bruk komité av Baylor College of Medicine. 1. Ekstern og intern løsning <ol><li> Bruk pattedyr Ringers oppløsning i løpet av retina disseksjon og som den eksterne løsningen i påfølgende elektrofysiologisk opptak. Forbered pattedyr Ringer løsning fra 10x stamløsning (uten kalsium) ...

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+phosphorylation+of+Cx36+gap+junctions+in+the+AII+amacrine+cells+of+RD+retina.">Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina.</a> Front Cell Neurosci. 9, 390 (2015).</li><li>Enoki, R., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+horizontally+sliced+preparation+of+the+retina.">A method of horizontally sliced preparation of the retina.</a> Methods Mol Biol. 935, 201-205 (2013).</li><li>Akrouh, A., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+function+of+three+VIP-expressing+amacrine+cell+types+in+the+mouse+retina.">Morphology and function of three VIP-expressing amacrine cell types in the mouse retina.</a> J Neurophysiol. 114 (4), 2431-2438 (2015).</li><li>Wei, W., Elstrott, J., Feller, M. 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Mange laboratorier har registrert fra GCL nevroner i flat-mount forberedelse 15-18, men våre prosedyrer tillater opptak fra INL nevroner. Vi vektlegger herved flere trinn som er avgjørende for vellykkede rutine opptak. Friskhet og planhet av netthinnen er viktig for å trenge det med et opptak pipette. I denne forbindelse, fast feste av netthinnen til stemplet nitrocellulosemembranen er viktig, og blir oppnådd ved forbigående absorpsjon av løsningen, etterfulgt av tidsriktig ...

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

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		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
			<td style="width:155px;">Olympus</td>
			<td style="width:219px;">BX51-WI</td>
			<td style="width:419px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micromanipulators</td>
			<td style="width:155px;">Sutter</td>
			<td style="width:219px;">MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
			<td style="width:155px;">A-M System</td>
			<td style="width:219px;">AM2400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">AD converter</td>
			<td style="width:155px;">National Instrument</td>
			<td style="width:219px;">NI-USB-6221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Heater controller</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">TC-324B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Inline heater</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">SC-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Peristaltic pump</td>
			<td style="width:155px;">Rainin</td>
			<td style="width:219px;">Dynamax</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pipette puller</td>
			<td style="width:155px;">Sutter Instrument</td>
			<td style="width:219px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
			<td style="width:155px;">King Precision Glass</td>
			<td style="width:219px;">Customized</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Stimulator</td>
			<td style="width:155px;">A.M.P.I.</td>
			<td style="width:219px;">Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Biocytin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">B4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5405</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:219px;">BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Na2HPO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaH2PO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgSO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">K-gluconate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">ATP-Mg</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KOH</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Cs-methanesulfonate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CsOH</td>
			<td style="width:155px;">Sigma</td>
			<td align="right" style="width:219px;">232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Syringer filter</td>
			<td style="width:155px;">Nalgene</td>
			<td align="right" style="width:219px;">171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1 ml syring</td>
			<td style="width:155px;">Rainin</td>
			<td align="right" style="width:219px;">17013002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10 ul pipette tip</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:219px;">24-130RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
			<td style="width:155px;">ThermoFisher</td>
			<td style="width:219px;">S-11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

Pattedyrnetthinnen er en lagdelt vev består av flere neuronale typer. For å forstå hvordan visuelle signaler behandles innen sine intrikate synaptiske nettverk, er elektrofysiologiske opptak ofte brukt til å studere forbindelser mellom enkeltnerveceller. Vi har optimalisert en flat-mount forberedelse for patch clamp opptak av genetisk merket nevroner i både GCL (ganglion celle laget) og INL (indre kjernefysiske lag) muse netthinne. Opptak INL nevroner i flat-mounts er favoriserte over skiver fordi både vertikale og laterale tilkoblinger er bevart i den tidligere konfigurasjon, slik at retinal kretser med store side komponenter som skal studeres. Vi har brukt denne fremgangsmåten for å sammenligne svarene fra speil samarbeid nevroner i netthinne slik som kolinerge starburst amacrine celler (sekker).

Representative opptak av on-og off-type sekker fra et deafferentated mus hinnen er vist i figur 1. Kolinerge celler i både GCL og INL kan bli pålitelig identifiseres av tdTomato fluorescens og målrettet for hel-celle patch clamp opptak i henhold til DIC (figur 1A) å avsløre svinging av sine membranpotensialer (øverst spor) og synaptic strømninger som driver det (nederst spor, Figur 1b). Hemmende og eksitatoriske synaptiske strømm...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Denne protokollen viser hvordan du utfører hel-celle patch clamp opptak på netthinnens nerveceller fra en flat-mount forberedelse.

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The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

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Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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Dyrking og Elektrofysiologi celler på NRCC Patch-Clamp Chips

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F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPase Vesikkel Forberedelse og teknikk for utøvende Patch Clamp Innspillinger av Submitochondrial vesikkelmikrosomer Membraner

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Patch Clamp Recordings fra embryonale Sebrafisk Mauthner Cells

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

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

Innspilling Lys-fremkalt postsynaptiske Responses i nevroner i Dark-tilpasset, Mouse Netthinne Slice Forberedelser Bruke Patch Clamp Teknikker

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

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Perforert Patch-clamp Opptak av Mouse olfactory sensoriske nevroner i Intakt neuroepithelium: Funksjonell analyse av nerveceller uttrykker en Identifiserte odoranten Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Hel-celle Patch-clamp Recordings i hjernen 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 ...

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

Auditory hjernestammen respons og ytre hår cellen hele celle Patch klemme opptak 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 ...

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

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

Forberedelser og protokoller for hele cellen Patch klemme opptak i Xenopus laevis Tectal nerveceller

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

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

Utarbeidelse av akutt ryggmargen skiver for hele celle Patch-klemme opptak i Substantia Gelatinosa nerveceller

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