Name: implantation of a cranial window for repeated in vivo imaging in awake mice
Uploaded: 2021-06-22T15:00:00
Description: Watch this Scientific Journal Video about Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice at JoVE.com

Alle dyreforsøg blev udført i overensstemmelse med retningslinjer godkendt af IACUC ved University of Nebraska Medical Center.
1. Før operationen
<ol><li>Forbered pipetter til virale injektioner. Træk borosilikatglaskapillærer ved hjælp af en pipettetrækker, og begle pipetten i en 20 ° vinkel. Steriliser pipetterne natten over.</li>
	<li>Forbered friske stykker sterilt gelskum ved at skære dem i små firkanter. Nedsænk gelskummet i et sterilt mikrofugerør indeholdende 0,5 ml saltv...

<ol>
	<li>Cichon, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branch-specific+dendritic+Ca2++spikes+cause+persistent+synaptic+plasticity.">Branch-specific dendritic Ca2+ spikes cause persistent synaptic plasticity.</a> Nature. 520 (7546), 180-185 (2015).</li><li>Goncalves, J. 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T., 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=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420 (6917), 788-794 (2002).</li><li>Agarwal, A., 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=Transient+opening+of+the+mitochondrial+permeability+transition+pore+induces+microdomain+calcium+transients+in+astrocyte+processes.">Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes.</a> Neuron. 93 (3), 587-605 (2017).</li><li>Bindocci, E., 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=Three-dimensional+Ca2++imaging+advances+understanding+of+astrocyte+biology.">Three-dimensional Ca2+ imaging advances understanding of astrocyte biology.</a> Science. 356 (6339), (2017).</li><li>Dana, H., 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=High-performance+calcium+sensors+for+imaging+activity+in+neuronal+populations+and+microcompartments.">High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.</a> Nature Methods. 16 (7), 649-657 (2019).</li><li>Han, S., Yang, W., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+volumetric+imaging+of+neuronal+activity+of+cortical+columns.">Two-color volumetric imaging of neuronal activity of cortical columns.</a> Cell Reports. 27 (7), 2229-2240 (2019).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308 (5726), 1314-1318 (2005).</li><li>Stowell, R. D., 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=Noradrenergic+signaling+in+the+wakeful+state+inhibits+microglial+surveillance+and+synaptic+plasticity+in+the+mouse+visual+cortex.">Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.</a> Nature Neuroscience. 22 (11), 1782-1792 (2019).</li><li>Madisen, 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+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neuroscience. 13 (1), 133-140 (2010).</li><li>Chen, S. X., 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=Subtype-specific+plasticity+of+inhibitory+circuits+in+motor+cortex+during+motor+learning.">Subtype-specific plasticity of inhibitory circuits in motor cortex during motor learning.</a> Nature Neuroscience. 18 (8), 1109-1115 (2015).</li><li>Stobart, J. 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=Cortical+circuit+activity+evokes+rapid+astrocyte+calcium+signals+on+a+similar+timescale+to+neurons.">Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons.</a> Neuron. 98 (4), 726-735 (2018).</li><li>Matsui, A., 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=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> Journal of Visualized Experiments: JoVE. (54), e3024 (2011).</li><li>Roth, R. H., 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=Cortical+synaptic+AMPA+receptor+plasticity+during+motor+learning.">Cortical synaptic AMPA receptor plasticity during motor learning.</a> Neuron. 105 (5), 895-908 (2020).</li><li>Stogsdill, J. A., 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=Astrocytic+neuroligins+control+astrocyte+morphogenesis+and+synaptogenesis.">Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis.</a> Nature. 551 (7679), 192-197 (2017).</li><li>Suresh, A., Dunaevsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+synaptic+AMPAR+and+spine+dynamics:+impairments+in+the+FXS+mouse.">Relationship between synaptic AMPAR and spine dynamics: impairments in the FXS mouse.</a> Cerebral Cortex. 27 (8), 4244-4256 (2017).</li><li>Yang, G., 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=Transcranial+two-photon+imaging+of+synaptic+structures+in+the+cortex+of+awake+head-restrained+mice.">Transcranial two-photon imaging of synaptic structures in the cortex of awake head-restrained mice.</a> Methods in Molecular Biology. 1010, 35-43 (2013).</li><li>Dombeck, D. A., 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=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56 (1), 43-57 (2007).</li><li>Kislin, M., 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=Flat-floored+air-lifted+platform:+a+new+method+for+combining+behavior+with+microscopy+or+electrophysiology+on+awake+freely+moving+rodents.">Flat-floored air-lifted platform: a new method for combining behavior with microscopy or electrophysiology on awake freely moving rodents.</a> Journal of Visualized Experiments: JoVE. (88), e51869 (2014).</li><li>Holtmaat, A., 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=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li><li>Hauglund, N. 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=Meningeal+lymphangiogenesis+and+enhanced+glymphatic+activity+in+mice+with+chronically+implanted+EEG+electrodes.">Meningeal lymphangiogenesis and enhanced glymphatic activity in mice with chronically implanted EEG electrodes.</a> Journal of Neuroscience. 40 (11), 2371-2380 (2020).</li><li>De Paola, V., 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=Cell+type-specific+structural+plasticity+of+axonal+branches+and+boutons+in+the+adult+neocortex.">Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex.</a> Neuron. 49 (6), 861-875 (2006).</li><li>Cheng, A., 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=Simultaneous+two-photon+calcium+imaging+at+different+depths+with+spatiotemporal+multiplexing.">Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing.</a> Nature Methods. 8 (2), 139-142 (2011).</li><li>Yang, G., 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=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nature Protocols. 5 (2), 201-208 (2010).</li><li>Shih, A. Y., 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+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> Journal of Visualized Experiments: JoVE. (61), e3742 (2012).</li><li>Helm, P. J., Ottersen, O. P., Nase, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+optical+properties+of+the+mouse+cranium--implications+for+in+vivo+multi+photon+laser+scanning+microscopy.">Analysis of optical properties of the mouse cranium--implications for in vivo multi photon laser scanning microscopy.</a> Journal of Neuroscience Methods. 178 (2), 316-322 (2009).</li><li>Stobart, J. 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Her har vi præsenteret en protokol for implantation af kroniske kranievinduer til in vivo-billeddannelse af kortikale astrocytter og neuroner i vågne, hovedbeherskede mus på et luftløftet hjemmebur. Der er givet specifikke eksempler på kranievinduesapplikationen til billeddannelse af astrocytter, der udtrykker GECIS'er og neuronale synaptiske strukturer. Ved brug af multifotonmikroskopi kan astrocytisk calciumsignaldynamik og strukturel synaptisk dynamik registreres gentagne gange over dage.
<p class="j...

Evnen til at udføre in vivo multifotonfluorescensmikroskopi i musebarken er altafgørende for undersøgelsen af cellulær signalering og struktur 1,2,3,4,5,6,7,8,9, sygdomspatologi 10,11 og cellulær udvikling12,13 . Med implantation af kroniske kranievinduer er langsgående billeddannelse mulig, hvilket muliggør gentagen billeddannelse af kortikale områder i dage, uger eller måneder13,14 hos levende dyr. Multifotonmikroskopi er ideel til in vivo, gentagen billeddannelse på grund af forbedret dybdesondering og reduceret fotoskade forbundet med den anvendte infrarøde laser. Dette gør det muligt at studere molekylær og cellulær dynamik af specifikke celler i forskellige kortikale regioner.
Multifotonmikroskopi er blevet anvendt til in vivo-billeddannelse af neuronale og glialceller i mus 15,16,17,18,19,20. Forskellige strategier kan implementeres for at mærke bestemte celletyper og interesseområder. En almindelig tilgang er at drive ekspressionen af genetisk kodede fluorescerende proteiner på en cellespecifik måde ved hjælp af Cre-Lox-rekombinationssystemet. Dette kan udføres med genetisk modificerede mus, f.eks. krydsning af tdTomato "floxed" mus (Ai14) med en mus, der udtrykker Cre-recombinase under en promotor af interesse21. Alternativt kan celle- og stedspecifik mærkning opnås med virale injektioner. Her injiceres en virus, der koder for Cre-rekombinase under en cellespecifik promotor, og en virus, der koder for et floxet gen af interesse, i en defineret region. Passende celletyper, der modtager begge virale vektorer, vil derefter udtrykke det eller de ønskede gener. Disse gener kan være strukturelle markører, såsom tdTomato, for at se ændringer i cellulær morfologi22 eller genetisk kodede calciumindikatorer (GECIS'er), såsom GCaMP og / eller RCaMP, for at undersøge calciumdynamik23. Metoder til genetisk rekombination kan anvendes individuelt eller i kombination til mærkning af en eller flere celletyper. En tredje tilgang, der ikke kræver transgene mus eller virale konstruktioner (som har begrænset emballagekapacitet), er in utero elektroporation af DNA-konstruktioner24. Afhængigt af tidspunktet for elektroporationen kan forskellige celletyper målrettes 25,26,27.
Når du udfører multifotonbilleddannelse, kan mus afbildes, mens de er vågne eller anæstetiserede. Billeddannelse af vågne mus kan udføres ved at fastgøre musen via en vedhæftet hovedplade28. Denne fremgangsmåde gøres mindre stressende ved at tillade relativt fri bevægelighed for dyret ved hjælp af metoder, såsom fritsvævende, luftunderstøttede styrofoamkugler29, fritsvævende løbebånd1 eller et luftløftet hjemmebursystem, hvor mus fastgøres med en fastgjort hovedplade og får lov til at bevæge sig i et åbent kammer30. For hver af disse billeddannelsesbetingelser vil det først være nødvendigt at vænne musene til billeddannelsesopsætningen. Dette papir beskriver tilvænnings- og billeddannelsesproceduren ved hjælp af et luftløftet hjemmebursystem.
Denne protokol beskriver implantationen af et kronisk kranievindue til langsgående in vivo-billeddannelse i cortex. Her vil vi bruge mus, der betinget udtrykker GCaMP6f i astrocytter til at overvåge calciumsignaldynamikken. Endvidere beskriver dette papir proceduren for virale injektioner ved hjælp af tdTomato som en etiket for neuroner. Dette gør det muligt at bestemme ændringer i neuronal synaptisk struktur og / eller tilgængeligheden som en strukturel markør, der muliggør gentagen billeddannelse af den samme astrocyt. I hele protokollen vil afgørende trin blive fremhævet for at sikre den bedst mulige kvalitet af billeder opnået ved multifotonmikroskopi.

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		<tr>
			<td>15o Pointed Blade</td>
			<td>Surgistar</td>
			<td>6500</td>
			<td>Surgery Tools</td>
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		<tr>
			<td>19 G Needles</td>
			<td>BD</td>
			<td>305186</td>
			<td>Surgery Supply</td>
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		<tr>
			<td>AAV1-CAG-FLEX-tdTomato</td>
			<td>Addgene</td>
			<td>28306-AAV1</td>
			<td>Viral Vector</td>
		</tr>
		<tr>
			<td>AAV1-CaMKII-0-4-Cre</td>
			<td>Addgene</td>
			<td>105558-AAV1</td>
			<td>Viral Vector</td>
		</tr>
		<tr>
			<td>Acteone</td>
			<td>Fisher Scientific</td>
			<td>A16P4</td>
			<td>Reagent</td>
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			<td>Alcohol Prep Pads</td>
			<td>Fisher Scientific</td>
			<td>Covidien 5750</td>
			<td>Surgery Supply</td>
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		<tr>
			<td>Beveler</td>
			<td>Narishige</td>
			<td></td>
			<td>Equipment</td>
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		<tr>
			<td>Borosilicate Glass</td>
			<td>World Precision Instruments</td>
			<td>TW100F-4</td>
			<td>Surgery Supply</td>
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			<td>Carbide Burs</td>
			<td>SS White Dental</td>
			<td>14717</td>
			<td>Surgery Tools</td>
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			<td>Carprofen (Rimadyl), 50 mg/mL</td>
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			<td>Cover Glass, No. 1 thickness, 3 mm/5 mm</td>
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			<td>Aseptico</td>
			<td></td>
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			<td>Dexamethasone, 4 mg/mL</td>
			<td>Mylan Institutional, LLC.</td>
			<td></td>
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			<td>Dissecting Microscope</td>
			<td>Nikon</td>
			<td></td>
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		<tr>
			<td>Duralay Liquid&#160; (dental cement liquid)</td>
			<td>Patterson Dental</td>
			<td>602-8518</td>
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			<td>Duralay Powder&#160; (dental cement powder)</td>
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			<td>Enrofloxacin, 2.27%</td>
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			<td>Eye Ointment</td>
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			<td>Fiber Lite High Intensity Illuminator</td>
			<td>Dolan-Jenner Industries</td>
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		<tr>
			<td>Forceps (Large)</td>
			<td>World Precision Instruments</td>
			<td>14099</td>
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		<tr>
			<td>Forceps (Small)</td>
			<td>World Precision Instruments</td>
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		<tr>
			<td>GCaMP6f B6; 129S-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 024105</td>
			<td>Mouse line</td>
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		<tr>
			<td>Germinator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Equipment</td>
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		<tr>
			<td>GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 012586</td>
			<td>Mouse Line</td>
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		<tr>
			<td>Headplate</td>
			<td>Neurotar</td>
			<td>Model 1, Model 3</td>
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		<tr>
			<td>Hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501705</td>
			<td>Surgery Tools</td>
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		<tr>
			<td>Holder for 15o Pointed Blade</td>
			<td>World Precision Instruments</td>
			<td>501247</td>
			<td>Surgery Tools</td>
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		<tr>
			<td>Holder for Scalpel Blades</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
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		<tr>
			<td>Iodine Prep Pads</td>
			<td>Avantor</td>
			<td>15648-926</td>
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		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td></td>
			<td>Surgery Supply</td>
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		<tr>
			<td>Isoflurane table top system with Induction Box</td>
			<td>Harvard Apparatus</td>
			<td></td>
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		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>SurgiVet</td>
			<td></td>
			<td>Equipment</td>
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		<tr>
			<td>Krazy Glue</td>
			<td>Office Depot</td>
			<td>KG517</td>
			<td>Reagent</td>
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		<tr>
			<td>Loctite 401</td>
			<td>Henkel</td>
			<td>40140</td>
			<td>fast-curing instant adhesive</td>
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		<tr>
			<td>Loctite 454</td>
			<td>Fisher Scientific</td>
			<td>NC9194415</td>
			<td>cyanoacrylate adhesive gel</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Multiphoton Microscope</td>
			<td></td>
			<td></td>
			<td>Equipment</td>
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		<tr>
			<td>Nitrogen</td>
			<td>Matheson</td>
			<td>NI M200</td>
			<td>Gas</td>
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		<tr>
			<td>Oxygen</td>
			<td>Matheson</td>
			<td>OX M250</td>
			<td>Gas</td>
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		<tr>
			<td>Picospritzer</td>
			<td>Parker</td>
			<td></td>
			<td>intracellular microinjection dispense system</td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Rainin</td>
			<td>17014340</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Rodent Hair Trimmer</td>
			<td>Wahl</td>
			<td></td>
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		<tr>
			<td>Saline (0.9% Sodium Chloride)</td>
			<td>Med Vet International</td>
			<td>RX0.9NACL-30BAC</td>
			<td>Surgery Supply</td>
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		<tr>
			<td>Scalpel Blades, Size 11</td>
			<td>Integra</td>
			<td>4-111</td>
			<td>Surgery Tools</td>
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		<tr>
			<td>Scissors</td>
			<td>World Precision Instruments</td>
			<td>503667</td>
			<td>Surgery Tools</td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Sugi Sponge Strips (sponge strips)</td>
			<td>Kettenbach Dental</td>
			<td>31002</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>SURGIFOAM (gel foam)</td>
			<td>Ethicon</td>
			<td>1972</td>
			<td>Surgery Supply</td>
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		<tr>
			<td>Syringe with 26 G Needle</td>
			<td>BD</td>
			<td>309625</td>
			<td>Surgery Supply</td>
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		<tr>
			<td>Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>T5648-1G</td>
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			<td>Ti:Sapphire Laser</td>
			<td>Coherent</td>
			<td></td>
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		<tr>
			<td>Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>Surgery Supply</td>
		</tr>
		<tr>
			<td>Water Blanket</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL</td>
			<td>Fresenius Kabi USA</td>
			<td></td>
			<td>Drug</td>
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implantation of a cranial window for repeated in vivo imaging in awake mice

For fuldt ud at forstå den cellulære fysiologi af neuroner og glia i at opføre dyr, er det nødvendigt at visualisere deres morfologi og registrere deres aktivitet in vivo i at opføre mus. Dette papir beskriver en metode til implantation af et kronisk kranievindue for at muliggøre langsgående billeddannelse af hjerneceller i vågne, hovedbeherskede mus. I kombination med genetiske strategier og virale injektioner er det muligt at mærke specifikke celler og regioner af interesse med strukturelle eller fysiologiske markører. Denne protokol viser, hvordan man kombinerer virale injektioner for at mærke neuroner i nærheden af GCaMP6-ekspressiv astrocytter i cortex til samtidig billeddannelse af begge celler gennem et kranievindue. Multifotonbilleddannelse af de samme celler kan udføres i dage, uger eller måneder i vågen, opførende dyr. Denne tilgang giver forskere en metode til at se cellulær dynamik i realtid og kan anvendes til at besvare en række spørgsmål inden for neurovidenskab.

Kvaliteten af kranievinduet kan vurderes ud fra, hvor skarpe neuronale strukturer ser ud. I et godt vindue er dendritiske rygsøjler tydeligt synlige (figur 1). Med de strukturelle og positionelle data lagret kan det samme dyr afbildes gentagne gange i dage, uger eller måneder for at undersøge de samme celler (figur 1). Billederne i figur 1 blev opnået fra forbenet i den primære motoriske cortex (i et 5 mm vindue). En række par...

Watch this Scientific Journal Video about Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice at JoVE.com

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Præsenteret her er en protokol til implantation af et kronisk kranievindue til langsgående billeddannelse af hjerneceller i vågne, hovedbeherskede mus.

Implantation af et kranievindue til gentagen In Vivo-billeddannelse i vågne mus

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Protokollen beskriver, hvordan man udfører virale injektioner og implanterer et kranievindue til billeddannelse af hjerneceller i vågne, hovedbeherskede mus. Fordelen ved denne metode er, at struktur og aktivitet af neuroner og astrocytter kan afbildes gentagne gange over flere sessioner. Denne tilgang kan bruges til at bestemme, hvordan neurale celler ændres i modeller af neuroudviklingsmæssige eller neurodegenerative lidelser, eller om oplevelsesafhængig plasticitet er svækket.Begynd med at fastgøre en anæstesimus til en stereotaksisk ramme ved hjælp af snudklemmen og ørestængerne. Brug sterile kirurgiske værktøjer til at skære og fjerne huden fra de frontale suturer forrest til bregma til posteriorly til lambda. Brug et sterilt kirurgisk blad i kulstofstål i størrelse 11 til at skrabe forsigtigt alt bindevævet fra kraniet.Ved hjælp af en tandbor skal du gradvist bore kraniet langs omridset af åbningen i koncentriske cirkler for at lette jævn udtynding af knoglen. Efter hvert koncentrisk pas skal du tilføje en dråbe eller to saltvand til det borede område og lade saltvandet sidde i mindst 10 sekunder, før boringen genoptages. Skub forsigtigt på det centrale område af kraniet med fine tang for at kontrollere, at kraniet er tilstrækkeligt tyndt og bevæger sig.Sørg for, at den underliggende vaskulatur, hvor boringen fandt sted, er intakt, og at knoglen ikke er revnet. Indsæt forsigtigt et miniature 15-graders spidst blad i den tynde knogle og skær og brug gelskummet gennemblødt i saltvand for at stoppe eventuelle blødninger, der kan opstå. Brug tang til forsigtigt at løfte og fjerne knoglen, og pas på ikke at beskadige dura mater.Til injektionen fyldes en steril skrå glaspipette med en 20 mikrometer spids med virusblandingen. Sænk derefter pipetten for at røre ved hjernens overflade, og fortsæt med at sænke i yderligere 200 til 300 mikrometer til lag 2/3 injektioner. Ved hjælp af et intracellulært mikroinjektionsdispenseringssystem injiceres systemet 12 til 15 gange i løbet af to minutter.Til kranievinduesimplantationen skal du placere dækglasset over åbningen i kraniet. Brug tang til at sikre, at glasset skyller over åbningen. For at forsegle glasvinduets kanter til kraniet skal du anvende cyanoacrylatklæbende gel omkring glasoverfladens omkreds.Påfør et lag lim over klæbegelen. Tilsæt derefter et lag tandcementvæske. Når tandcement hærder, påføres et tyndt lag lim omkring den centrale åbning af helikoptertypens hovedplade og anbring helikoptertypens hovedplade af passende størrelse over dækglasset.Lad limen tørre. Tilsæt dental cementpulver i et 1,5 ml mikrofugerør op til 0,1 milliliter mærket. Bland syv til otte dråber hurtighærdende øjeblikkeligt klæbemiddel i pulveret, og træk den resulterende blanding i en sprøjte på en milliliter med en 19 gauge nål, der er skåret for at skabe en større åbning.Indsprøjt pulverblandingen gennem helikopterstangens sidehuller, indtil den siver fra begge sider. Påfør tandcementklæbemiddelblandingen på resten af det udsatte kranium for at fastgøre hovedpladen til kraniet. Pak musen ind i klud, og fastgør den via hovedpladen til hovedfikseringsarmen på det luftløftede hjemmebur, og lad derefter hjemmeburet være udsat for lys.Efter en tilvænningstid på 15 minutter skal du fjerne musen fra hjemmeburet. Brug mikroskopets brede felttilstand til at vælge to til tre positioner med let identificerbar vaskulatur. Gem billederne af blodkarrene, og optag x- og y-koordinaterne, der vises på motorstyringen.Forestil dig neuronernes synaptiske strukturer og GCaMP6f-aktivitet i astrocytter. I den repræsentative analyse blev kvaliteten af kranievinduet evalueret med gentagen billeddannelse af dendritter og GCaMP6f-ekspressiv astrocytter over dage. I et godt vindue syntes neuronale strukturer skarpe med tydeligt synlige dendritiske rygsøjler.To nye rygsøjler dukkede op på dag to af billeddannelsen. Kun en rygsøjle fortsatte og var synlig på dag fem. Calciumaktivitet blev undersøgt i astrocytter, der udtrykte GCaMP6f på forskellige tidspunkter.En global begivenhed, der omfattede hele cellen, blev vidne til under en bevægelseskamp. De to kritiske trin i denne protokol er, at knoglen skal tyndes tilstrækkeligt inden fjernelse af knoglen og korrekt placering af dækglasset over åbningen. Billeddannelse kan kombineres med adfærdsanalyse, enten under billeddannelse eller efter adfærd, såsom at lære en ny motorisk færdighed, for at bestemme, hvordan neuronal og astrocytstruktur og funktion moduleres ved læring.

Research

JoVE Journal

Neuroscience

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Multimodal Imaging af stamcelle implantation i det centrale nervesystem mus

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funktionel Neuroimaging Med Ultrasonic blod-hjerne barrieren forstyrrelser og mangan-forstærket MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Tyndet-skull Cortical Window Teknik for<em&gt; In Vivo</em&gt; Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Lateral Kronisk Kranie Window Forberedelse Aktiverer<em&gt; In vivo</em&gt; Observation Efter Distal mellem-cerebral arterieokklusion i mus

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Piezo høj nøjagtighed kirurgisk Osteal fjernelse (PHASOR): En teknik til forbedret kraniel vindue kirurgi i mus

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

Laminektomi og rygmarv vindue implantation i musen

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo To-foton billeddannelse af kortikale neuroner i Neonatal mus

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging i Mus Ringere Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Konstruktion og implementering af kulfibermikroelektrodesystemer til kroniske og akutte In Vivo-optagelser

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...