Name: single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons
Uploaded: 2020-10-06T13:30:00
Description: Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Dette arbejde blev støttet af National Institutes of Health Grants (R01NS085215 til K.F., T32 GM107000 og F30MH122146 til A.C.). Forfatterne takker Ms Naoe Watanabe for dygtig teknisk bistand.

Alle dyreprotokoller blev gennemgået og godkendt af Institutional Animal Care and Use Committee (IACUC) ved University of Massachusetts Medical School. Skivekulturforberedelse, plasmidpræparat og elektroporation er også beskrevet i vores tidligere offentliggjorte metoder og kan henvises til for yderligere information10.
1. Skær kultur forberedelse
<ol><li>Forbered mus organotypiske hippocampal skive kulturer som tidligere beskrevet11, ved h...

<ol>
	<li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Current Opinion in Neurobiology. 12 (5), 566-573 (2002).</li><li>Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+by+direct+microinjection+of+DNA+into+cultured+mammalian+cells.">High efficiency transformation by direct microinjection of DNA into cultured mammalian cells.</a> Cell. 22 (2), 479-488 (1980).</li><li>Rae, J. L., Levis, R. A. <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.">Single-cell electroporation.</a> Pflugers Archives: European Journal of Physiology. 443 (4), 664-670 (2002).</li><li>Teruel, M. N., Blanpied, T. A., Shen, K., Augustine, G. J., Meyer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+microporation+technique+for+the+transfection+of+cultured+CNS+neurons.">A versatile microporation technique for the transfection of cultured CNS neurons.</a> Journal of Neuroscience Methods. 93 (1), 37-48 (1999).</li><li>Rathenberg, J., Nevian, T., Witzemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+individual+neurons+using+modified+electrophysiology+techniques.">High-efficiency transfection of individual neurons using modified electrophysiology techniques.</a> Journal of Neuroscience Methods. 126 (1), 91-98 (2003).</li><li>Tanaka, M., Yanagawa, Y., Hirashima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+small+interfering+RNA+by+single-cell+electroporation+in+cerebellar+cell+cultures.">Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures.</a> Journal of Neuroscience Methods. 178 (1), 80-86 (2009).</li><li>Wiegert, J. S., Gee, C. E., Oertner, T. G. <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+of+Neurons.">Single-Cell Electroporation of Neurons.</a> Cold Spring Harbor Protocols. 2017 (2), 094904 (2017).</li><li>Li, L., 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=A+robot+for+high+yield+electrophysiology+and+morphology+of+single+neurons+in+vivo.">A robot for high yield electrophysiology and morphology of single neurons in vivo.</a> Nature Communication. 8, 15604 (2017).</li><li>Steinmeyer, J. D., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-cell+manipulation+in+brain+tissue.">High-throughput single-cell manipulation in brain tissue.</a> PLoS One. 7 (4), 35603 (2012).</li><li>Keener, D. G., Cheung, A., Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+method+for+single-cell+electroporation+in+mouse+organotypic+hippocampal+slice+culture.">A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.</a> Journal of Neuroscience Methods. 337, 108632 (2020).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Ibata, K., Sun, Q., Turrigiano, G. 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Vi beskriver her en elektroporationsmetode, der transfects både excitatoriske og hæmmende neuroner med høj effektivitet og præcision. Vores optimerede elektroporationsprotokol har tre innovative gennembrud for at opnå højeffektiv gentransfekt. Vores første ændring var at øge pipettestørrelsen sammenlignet med tidligere offentliggjorte protokoller3,5,6. Denne ændring gjorde det muligt for os at elektroporate mange neur...

I molekylærbiologi er en af de vigtigste overvejelser for en efterforsker, hvordan man leverer et gen af interesse til en celle eller population af celler for at belyse dens funktion. De forskellige leveringsmetoder kan kategoriseres som enten biologiske (f.eks. en viral vektor), kemisk (f.eks. calciumphosphat eller lipid) eller fysisk (f.eks. elektroporation, mikroinjection eller biolistics)1,2. Biologiske metoder er yderst effektive og kan være celletypespecifikke, men er begrænset af udviklingen af specifikke genetiske værktøjer. Kemiske tilgange er meget kraftige in vitro, men transfekt er generelt tilfældige; Desuden er disse tilgange for det meste forbeholdt primærceller. Af de fysiske tilgange er biolistik den enkleste og nemmeste fra et teknisk synspunkt, men producerer igen tilfældige transfektresultater med en relativt lav effektivitet. For applikationer, der kræver overførsel til specifikke celler uden behov for at udvikle genetiske værktøjer, ser vi mod enkeltcelleelektroporation3,4.
Mens elektroporation kun refererer til feltelektroporation, er der i løbet af de sidste tyve år udviklet flere in vitro- og in vivo-elektroporationsprotokoller med en enkelt celle for at forbedre specificitet og effektivitet5,6,7,hvilket viser, at elektroporation kan bruges til at overføre gener til individuelle celler og derfor kan være ekstremt præcis. Procedurerne er imidlertid teknisk krævende, tidskrævende og relativt ineffektive. Faktisk har nyere papirer undersøgt muligheden for mekaniserede elektroporationsplatforme8,9, som kan bidrage til at fjerne flere af disse barrierer for efterforskere, der er interesseret i at installere sådan robotteknologi. Men for dem, der leder efter enklere midler, er problemerne med elektroporation, nemlig celledød, transfekturesvigt og pipettestopning, fortsat et problem.
Vi har for nylig udviklet en elektroporation metode, der bruger større tippet glas pipetter, mildere elektrisk puls parametre, og et unikt tryk cykling skridt, som genererede en langt højere transfekt effektivitet i excitatoriske neuroner end tidligere metoder, og gjorde det muligt for os for første gang at transfekt gener i hæmmende interneurons, herunder somatostatin-udtrykke hæmmende internurons i hippocampal CA1 region af mus organotypic skive kultur10. Pålideligheden af denne elektroporationsmetode i forskellige hæmmende interneurontyper og neuronale udviklingsstadier er imidlertid ikke blevet behandlet. Her demonstrerede vi, at denne elektroporationsteknik er i stand til at transfektere gener til både excitatoriske neuroner og forskellige klasser af interneuroner. Det er vigtigt, at transfekteffektiviteten var høj, uanset hvilke dage in vitro (DIV) skivekulturalderen blev testet. Denne etablerede og brugervenlige teknik anbefales stærkt til enhver efterforsker, der er interesseret i at bruge enkeltcelleelektroporation til forskellige celletyper i forbindelse med in vitro-musehjernevæv.

<table>
	<tbody>
		<tr>
			<td>Plasmid preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Purification Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Organotypic slice culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>FST</td>
			<td>#5/45</td>
			<td>Angled dissection forceps for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Flask Filter Unit</td>
			<td>Millipore</td>
			<td>SCHVU02RE</td>
			<td>Filtration and storage of culture media</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>BD C150-UL</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>TED PELLA, INC.</td>
			<td>10180</td>
			<td>Tissue chopper for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm</td>
			<td>Millipore</td>
			<td>PIHP03050</td>
			<td>Organotypic slice culture inserts</td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Precision Systems</td>
			<td>OSMETTE II</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE coated spatulas</td>
			<td>Cole-Parmer</td>
			<td>SK-06369-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14958-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Filtration System</td>
			<td>Millipore</td>
			<td>SCGPT01RE</td>
			<td>Filtration and storage of aCSF</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glasses</td>
			<td>Warner Instruments</td>
			<td>640796</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetter Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder</td>
			<td>BD (E2)</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller Filament</td>
			<td>Sutter Instrument</td>
			<td>FB330B</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-cell electroporation and fluorescence imaging #1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 mm Falcon Petri Dishes</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtable</td>
			<td>TMC</td>
			<td>63-7512E</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Q Imaging</td>
			<td>Retiga-2000DC</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Electroporation System</td>
			<td>Molecular Devices</td>
			<td>Axoporator 800A</td>
			<td>Electroporator</td>
		</tr>
		<tr>
			<td>Fluorescence Illumination System</td>
			<td>Prior</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Sutter Instrument</td>
			<td>MPC-385</td>
			<td>Manipulator</td>
		</tr>
		<tr>
			<td>Metamorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Rainin</td>
			<td>Dynamax, RP-2</td>
			<td>Perfusion pump</td>
		</tr>
		<tr>
			<td>Shifting Table</td>
			<td>Luigs &#38; Neuman</td>
			<td>240 XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Unknown</td>
			<td></td>
			<td>Speakers connected to the electroporator</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ30</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Incubator</td>
			<td>Thermo Scientific</td>
			<td>MIDI 40</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging #2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons

Elektroporation har etableret sig som en kritisk metode til overførsel af specifikke gener til celler for at forstå deres funktion. Her beskriver vi en enkeltcellet elektroporationsteknik, der maksimerer effektiviteten (~ 80%) af in vitro-gentransfekt i excitatoriske og klassespecifikke hæmmende neuroner i musorganotypisk hippocampal skivekultur. Ved hjælp af store glaselektroder, tetrodotoxinholdige kunstige cerebrospinalvæske og milde elektriske impulser leverede vi et gen af interesse i dyrkede hippocampale CA1 pyramide neuroner og hæmmende internuroner. Desuden kunne elektroporation udføres i dyrkede hippocampale skiver op til 21 dage in vitro uden reduktion i transfekteffektivitet, hvilket giver mulighed for undersøgelse af forskellige skivekulturudviklingsstadier. Med stigende interesse for at undersøge geners molekylære funktioner på tværs af en bred vifte af celletyper demonstrerer vores metode en pålidelig og ligetil tilgang til in vitro-gentransfekt i musehjernevæv, der kan udføres med eksisterende elektrofysiologiudstyr og -teknikker.

Vores enkeltcellede elektroporation er i stand til præcist at levere gener til visuelt identificerede excitatoriske og hæmmende neuroner. Vi elektroporated tre forskellige neuronale celletyper på tre forskellige tidspunkter. Parvalbumin (Pv) eller vesikulær glutamat type 3 (VGT3), der udtrykker neuroner, blev visualiseret ved at krydse Pvcre-linjer (JAX #008069) eller VGT3cre-linjer (JAX #018147) med TdTomato (en variant af rødt fluorescerende protein) (Jax #007905), henholdsvis kaldet Pv/TdTom...

Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Præsenteret her er en protokol for enkelt-celle elektroporation, der kan levere gener i både excitatoriske og hæmmende neuroner på tværs af en række in vitro hippocampal skive kultur aldre. Vores tilgang giver præcis og effektiv ekspression af gener i individuelle celler, som kan bruges til at undersøge celle-autonome og intercellulære funktioner.

Single-Cell Electroporation på tværs af forskellige organotypiske Slice Kultur Af Mouse Hippocampal Excitatory og klasse-specifikke hæmmende neuroner

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

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The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Isolering og dyrkning af hippocampus neuroner fra Prænatal Mus

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Primary Culture of Mouse Dopaminergic Neurons

Primær Kultur Mouse dopaminerge neuroner

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

Organotypiske skivekulturer at studere oligodendrocyt Dynamics og myelinering

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Forbundne Helcelle optagelser i Organotypiske hippocampusskiver

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Analysen af ​​neurovaskulære Remodeling i Entorhino-hippocampus organotypisk skivekulturer

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic hippocampus skive kulturer som en Model til undersøgelse Neuroprotection og invasionsevne af tumorceller

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...