Name: two-photon calcium imaging in mice navigating a virtual reality environment
Uploaded: 2014-02-20T22:00:00
Description: Watch this Scientific Journal Video about Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment at JoVE.com

Dette arbejde blev støttet af Friedrich Miescher Institut for biomedicinsk forskning, Max Planck Society, og Human Frontier Science Program.

Alle animalske procedurer blev godkendt og gennemført i overensstemmelse med retningslinjerne i Veterinary Department of Canton Basel-Stadt. 1.. Hardware og software Opsætning <ol><li> To-foton scanning mikroskop opsætning: <ol><li> Brug en pulseret infrarød laser som en belysningskilde (impulsbredde &lt;120 fsec). </li><li> Brug en scanning hoved bestående af en 8 eller 12 kHz resonant scanner og en standard galvanometer Bemærk:. Dette muliggør frame rates på 40 e...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+Calcium+Indicator+for+Neural+Activity+Imaging.">Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging.</a> J. Neurosci. 32 (40), 13819-13840 (2012).</li><li>Zhao, Y., Araki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca2++indicators.">An expanded palette of genetically encoded Ca2+ indicators.</a> Science. 333 (6051), 1888-1891 (2011).</li><li>Mittmann, W., Wallace, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+of+evoked+activity+from+L5+somatosensory+neurons+in+vivo.">Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo.</a> Nat. Neurosci. 14 (8), 1089-1893 (2011).</li><li>Katona, G., Szalay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+two-photon+in+vivo+imaging+with+three-dimensional+random-access+scanning+in+large+tissue+volumes.">Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes.</a> Nat. Methods. 9 (2), 201-208 (2012).</li><li>Mank, M., Santos, A. 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F., Plett, J., Mank, M., Griesbeck, O., Borst, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+Reality+for+Mice,+mousevr.blogspot.com.">Virtual Reality for Mice, mousevr.blogspot.com.</a> Nat. Neurosci. 13, 973-978 (2010).</li><li>Sakatani, T., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+spontaneous+saccade-like+rapid+eye+movements+in+C57BL/6+mice.">Quantitative analysis of spontaneous saccade-like rapid eye movements in C57BL/6 mice.</a> Neurosci. 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Nøglen til succes for adfærdsmæssige to-foton imaging er stabiliteten af ​​præparatet på to måder: <ol><li> I løbet af dage efter vindue implantation kan inflammatoriske reaktioner i vævet føre til en forbedring af dannelsen af ​​granulationsvæv og brusk, som vil hæmme eller endog forhindre billeddannelse. </li><li> Under eksperimentet har hjernen at være stabil nok til at forhindre bevægelsesartefakter fra korrumperende neurale aktivitet relateret fluorescens signal. </li></ol><p class="jove_co...

To-foton billeddannelse af calcium indikatorer (genetisk kodet ligesom GCaMP5 7 eller R-GECO 8 eller syntetiske farvestoffer som OGB eller Fluo4) er dukket op som en kraftfuld metode til måling af neuronal aktivitet i opfører mus 1-6. Det muliggør samtidig måling af aktiviteten af hundredvis af celler ved nær-enkelt virkningspotentiale opløsning, op til ca 800 um under hjernen overfladen 9,10. Desuden ved hjælp af genetisk indkodede calcium indikatorer (GECIs) kan måles, neuronal aktivitet kronisk 5,11,12, og i genetisk definerede celletyper 13.. Tilsammen udgør disse metoder giver en vis tidsmæssig og rumlig opløsning, der åbner op for en lang række nye muligheder i studiet af neuronal beregning in vivo. Kirurgisk indgreb er nødvendigt for at afsløre og mærke musehjernen til billeddannelse. Celler er typisk transficeret under anvendelse af en rekombinant adeno-associeret vir<html> (AAV) system til GECI levering og et kranie vindue os er implanteret over injektionsstedet for at få optisk adgang til hjernen. Et hoved bar derefter fastgjort til kraniet til hoved fiksering under to-foton mikroskop. Udformningen og gennemførelsen af ​​disse trin er kritisk, da de fleste af problemerne med vågen billedbehandling opstår ustabilitet i præparatet. Ideelt den her beskrevne fremgangsmåde skal give mulighed for kronisk billeddannelse af op til flere måneder efter operationen. For at aktivere visuelt guidet opførsel under to-foton billedbehandling, lederen fast musen sidder på en luft understøttet sfærisk løbebånd, som den kan bruge til at navigere en virtual reality miljø. Locomotion af musen på løbebåndet er koblet til bevægelse i det virtuelle miljø, der vises på en ringkerne skærm omgiver mus 14,15. Adfærdsmæssige variabler såsom bevægelse, visuel stimulus, og elev position kan optages 6. t &quot;&gt; Vi beskriver involveret i kronisk to-foton billeddannelse i mus udforske et virtual reality miljø procedurer De vigtigste punkter behandles, er:. reduktion af bevægelsesartefakter, reduktion af lys lækage, maksimering af antallet af samtidigt optagede celler, og minimering af foto skader. Vi leverer også oplysninger om opsætning af luft-støttede løbebånd, elev tracking, og virtual reality miljø. De her beskrevne procedurer kan bruges til billeddannelse fluorescensmærkede cellepopulationer i hoved faste mus i en potentielt lang række adfærdsmæssige paradigmer .

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			<td height="20" style="height:20px;width:216px;">cover slips (d = 3-5 mm)</td>
			<td style="width:151px;">Menzel</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">window implant</td>
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		<tr height="20">
			<td height="20" style="height:20px;">InSight DeepSee laser</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">12kHz resonance scanner</td>
			<td>Cambridge Technology</td>
			<td>G1-003-30026</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Galvometer</td>
			<td>Cambridge Technology</td>
			<td>G6215H</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>National Instruments</td>
			<td>NI 5772</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">FPGA</td>
			<td>National Instruments</td>
			<td>PXIe 7965R</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCIe 6363</td>
			<td>microscope</td>
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			<td height="20" style="height:20px;">Emission filter 525/50</td>
			<td>Semrock</td>
			<td>FF03-525/50-25</td>
			<td>microscope</td>
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			<td height="20" style="height:20px;">Piezo-electric z-drive</td>
			<td>Physikinstrumente</td>
			<td>P-726.1CD</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Controller for piezo-electric drive</td>
			<td>Physikinstrumente</td>
			<td>E665 LVPZT</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Objective 16x, 0.8NA</td>
			<td>Nikon</td>
			<td>CFI75</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Current amplifier</td>
			<td style="width:151px;">Femto</td>
			<td style="width:119px;">DHPCA-100</td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Photomultiplier tube</td>
			<td style="width:151px;">Hamamatsu</td>
			<td style="width:119px;"></td>
			<td>microscope</td>
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		<tr height="20">
			<td height="20" style="height:20px;">USB Camera without IR filter</td>
			<td>ImagingSource</td>
			<td>DMK22BUC03&#160;</td>
			<td>pupil tracking</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Objective 50 mm</td>
			<td>ImagingSource</td>
			<td>M5018-MP&#160;</td>
			<td>pupil tracking</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Macro adapter rings</td>
			<td>ImagingSource</td>
			<td>LAexSet</td>
			<td>pupil tracking</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Optical computer mouse</td>
			<td>Logitech</td>
			<td>G500</td>
			<td>motion tracking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Styrofoam ball 20 cm</td>
			<td>e.g. idee-shop.de</td>
			<td>08797.00.15</td>
			<td>virtual environment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LED projector</td>
			<td>Samsung</td>
			<td>SP-F10M&#160;</td>
			<td>virtual environment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acquisition card</td>
			<td>National Instruments</td>
			<td style="width:119px;">NI 6009</td>
			<td>virtual environment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Panda3D game engine</td>
			<td></td>
			<td><a href="http://www.panda3d.org/">www.panda3d.org</a></td>
			<td>virtual environment</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Numpy library for Python</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"><a href="http://www.scipy.org/">www.scipy.org</a></td>
			<td>virtual environment</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Scipy library for Python</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"><a href="http://www.scipy.org/">www.scipy.org</a></td>
			<td>virtual environment</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NI-DAQmx driver</td>
			<td style="width:151px;">National Instruments</td>
			<td style="width:119px;"><a href="http://www.ni.com/">www.ni.com</a></td>
			<td>virtual environment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ultrasound gel</td>
			<td style="width:151px;">Dahlhausen</td>
			<td style="width:119px;">5701.0342.10</td>
			<td>imaging</td>
		</tr>
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two-photon calcium imaging in mice navigating a virtual reality environment

I de senere år har to-foton billeddannelse blevet et uvurderligt værktøj i neurovidenskab, da det giver mulighed for kronisk måling af aktiviteten af genetisk identificerede celler under adfærd 1-6. Her beskriver vi metoder til at udføre to-foton billeddannelse i mus cortex mens dyret navigerer et virtual reality miljø. Vi fokuserer på aspekter af de eksperimentelle procedurer, der er nøglen til billeddannelse i en opfører dyr i et klart oplyst virtuelt miljø. De vigtigste problemer, der opstår i denne forsøgsopstilling, at vi her adresse er: minimering hjernen motion relaterede artefakter, minimering lys lækage fra virtual reality projektion system og minimere laser vævsbeskadigelse. Vi leverer også prøve software til at styre virtual reality miljø og gøre elev tracking. Med disse procedurer og ressourcer bør det være muligt at konvertere en konventionel to-foton mikroskop til anvendelse i opfører mus.

Billedkvaliteten i to-foton calcium imaging af cellepopulationer mærket med en GECI i høj grad afhænger af kvaliteten af ​​det kranie vindue implantat. To uger efter virus indsprøjtning kranie vindue bør inspiceres for klarhed. Der bør ikke være granulationsvæv eller fornyet knoglevækst synlig (figur 1A). Desuden bør mønsteret af overfladiske blodkar forbliver uændret, og grænserne for vaskulaturen bør skarpt defineret. Samtidig kan GECI udtryk også kontrolleres. Boli mærkede celler ...

Watch this Scientific Journal Video about Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment at JoVE.com

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

Her beskriver vi de eksperimentelle procedurer, der er involveret i to-foton-billeddannelse af muse cortex under opførsel i et virtual reality miljø.

To-foton Calcium Imaging i Mus navigerer en Virtual Reality Miljø

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

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