Name: visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy
Uploaded: 2019-06-07T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

Vi takker fru Tess J. Lameyer, MS. Ruth Frank, og Dr. Michael A. Muniak for redigeringer og kommentarer, og Dr. Ryohei Yasuda på Max Planck Florida for 2pFLIM erhvervelse software. Dette arbejde blev støttet af to BRAIN Initiative Awards U01NS094247 (H.Z. og T.M.) og R01NS104944 (H.Z. og T.M.), en R01 Grant R01NS081071 (T.M.), og en R21 Grant R21NS097856 (H.Z.). Alle priser er fra National Institute of neurologiske lidelser og slagtilfælde, USA.

Alle metoder, der er beskrevet her, er blevet godkendt af Udvalget for institutionel dyrepasning og-anvendelse (IACUC) i Oregon Health and Science University.
1.2pFLIM mikroskop opsætning
<ol><li>Installer et foton timing tælle modul (PTCM, tabel over materialer) og Tilslut til computeren (figur 1) i henhold til producentens manual. Bemærk: PTCM er typisk en computer bord, der modtager en "Sync" input til laser puls timing...

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Denne protokol demonstrerer brugen af FRET-FLIM sensor tAKARα til at visualisere Neuromodulation-udløst PKA aktivitet i hoved-fast opfører mus. Denne applikation er baseret på omfattende tests og karakteriseringer af tAKARα in vitro og in vivo for at påvise, at det opnåede FLIM-signal er relevant for fysiologiske neuromodulatoriske hændelser17. Her bruges en in vivo-applikation, bevægelses induceret PKA-aktivitet i motorisk cortex, til at beskrive procedurerne for levering af sensoren til...

Neuromodulation, også kendt som langsom synaptisk transmission, pålægger stærk kontrol over hjernens funktion under forskellige adfærdsmæssige tilstande, såsom stress, ophidselse, opmærksomhed og bevægelse1,2,3, 4. på trods af sin betydning, studiet af Hvornår og hvor neuromodulatory begivenheder finder sted, er stadig i sin vorden. Neuromodulatorer, herunder acetylcholin, dopamin, noradrenalin, serotonin, og mange neuropeptider, aktivere G protein-koblede receptorer (GPCRs), som igen udløser intracellulære anden Messenger veje med et bredt vindue af tidshorisonter spænder fra sekunder til timer. Mens hver neuromodulator udløser et særskilt sæt signalerings hændelser, er Camp/protein kinase a (PKA)-vejen en fælles downstream-vej for mange neuromodulatorer1,5. Lejren/PKA-vejen regulerer neuronal ophidelse, synaptisk transmission og plasticitet6,7,8,9, og derfor melodier neuronal netværks dynamik. Fordi forskellige neuroner eller neuronal typer udtrykker forskellige typer eller niveauer af neuromodulator receptorer10, de intracellulære virkninger af samme ekstracellulære neuromodulator kan være heterogene på tværs af forskellige neuroner, og dermed, nødt til at være undersøgt med cellulær opløsning. Til dato, det er fortsat udfordrende at overvåge neuromodulatory begivenheder i individuelle neuroner in vivo under opførsel.
For at studere neuromodulations rumlige dynamik kræves en passende optagelses modalitet. Mikrodialyse og hurtig scanning cyklisk voltammetri bruges ofte til at studere frigivelse af neuromodulatorer, men de mangler den rumlige opløsning til at overvåge cellulære begivenheder11,12. Analogt med calcium dynamik anvendes som en proxy for neuronal elektrisk aktivitet i befolkningen Imaging13, pKa Imaging kan bruges til at læse ud neuromodulatory begivenheder på tværs af en neuronal befolkning ved cellulær opløsning. Denne protokol beskriver brugen af en forbedret A-kinase Activity reporter (AKAR) til at overvåge PKA aktiviteter in vivo under dyrs adfærd. Den beskrevne metode giver mulighed for simultan billeddannelse af neuronal populationer ved subcellulær opløsning med en tidsmæssig opløsning, der sporer fysiologiske neuromodulatoriske hændelser.
Akars er sammensat af en donor og en acceptor fluorescerende proteiner forbundet med en PKA fosforylering substrat peptid og en forkhead-associeret (FHA) domæne, der binder sig til fosforyleret Serin eller Threonin af substrat14,15. Ved aktivering af PKA-vejen er det substrat peptid af AKAR fosforyleret. Som følge heraf binder FHA-domænet sig til det fosforylerede substrat peptid, hvorved de to fluorophorer bringes i umiddelbar nærhed, benævnt den lukkede tilstand AKAR. Den lukkede tilstand af en fosforyleret AKAR resulterer i øget Förster Resonance energioverførsel (FRET) mellem donor og acceptor fluorophores. Da andelen af phosphorylerede akarer er relateret til niveauet af PKA-aktivitet16, kan mængden af Fret i en biologisk prøve anvendes til at kvantificere niveauet af PKA-aktivitet16,17,18, 19,20.
Tidlige versioner af AKARs var primært designet til to-farve ratiometrisk billeddannelse14. Når Imaging dybere ind i hjernevæv, den ratiometriske metode lider af signalforvrængning på grund af bølgelængde-afhængige lys spredning17,18,21. Som beskrevet nedenfor eliminerer fluorescens livstids billedbehandlings mikroskopi (Flim) dette problem, fordi Flim kun måler fotoner udsendt af donor fluoroforet18,21. Som følge heraf påvirkes FLIM-kvantificering af FRET ikke af vævs dybden17. Desuden kan en "mørk" (dvs. lav Quantum Yield [QY]) variant af acceptor fluoroforet anvendes. Dette frigør en farvekanal til at lette multiplexed måling af ortogonale neuronal egenskaber via simultan billeddannelse af en anden sensor eller en morfologiske markør17,19,20.
Flim Imaging kvantificerer den tid, en fluoroforet tilbringer i den ophidsede tilstand, dvs fluorescens levetid18. Tilbagelevering af en fluoroforet til jorden tilstand, således enden af den ophidsede tilstand, ofte samtidens med emission af en foton. Selv om emissionen af en foton for en individuel spændt molekyle er stokastisk, i en population den gennemsnitlige fluorescens levetid er en karakteristisk for denne særlige fluorophore. Når en ren population af fluorophorer er spændt samtidig, vil den resulterende fluorescens følge en enkelt eksponentiel forfald. Tids konstanten for dette eksponentielle forfald svarer til den gennemsnitlige fluorescens levetid, som typisk spænder fra et til fire nanosekunder for fluorescerende proteiner. Tilbagevenden af en ophidset donor fluoroforet til jorden tilstand kan også forekomme ved fret. Ved tilstedeværelse af Fret reduceres fluorescens levetid for donor fluoroforet. De unfosforylerede Akarer udviser en relativ længere donor fluorescens levetid. Ved fosforylering af PKA udviser sensoren en kortere levetid, fordi donoren og acceptoren fluorophores bringes i nærheden af hinanden, og FRET øges. Kvantificeringen af fluorescens levetid i en population af AKARs repræsenterer derfor niveauet for PKA-aktiviteten.
Tidlige versioner af AKARs er ikke blevet anvendt med succes til in vivo-billeddannelse ved en enkelt celle opløsning. Dette skyldes primært den lave signal amplitude af AKAR-sensorerne til fysiologiske aktiveringer17. For nylig, ved systematisk at sammenligne tilgængelige AKAR sensorer for to-photon fluorescens levetid Imaging mikroskopi (2pFLIM), en sensor kaldet FLIM-AKAR blev fundet at overgå alternative sensorer. Desuden blev der udviklet en række Flim-Akar varianter kaldet målrettede akars (takars) for at visualisere PKA aktivitet på specifikke subcellulære steder: microtubuler (takarα), cytosol (takarβ), actin (takarδ), filamentøse actin (takarε), membran (takarγ), og postsynaptisk tæthed (tAKARζ). Blandt Takarer øgede tAKARα signal amplitude fremkaldt af noradrenalin med 2,7 gange. Dette er i overensstemmelse med den viden, at størstedelen af PKA i neuroner er forankret til microtubuler i hviletilstand22,23. tAKARα var den bedste performer blandt eksisterende AKARs for 2pFLIM. Desuden opdagede tAKARα fysiologisk relevant PKA-aktivitet, som blev fremkaldt af flere neuromodulatorer, og ekspression af tAKARα ændrede ikke neuronal funktion17.
For nylig blev tAKARα anvendt med succes til at visualisere PKA-aktiviteter i hoved-Fixed opfører mus17. Det blev påvist, at tvungen bevægelse udløste PKA aktivitet i Soma af overfladiske lag neuroner (lag 1 til 3, op til en dybde på ~ 300 μm fra Pia) i motoren, tønde, og visuelle cortices. Den bevægelses udløste PKA-aktivitet var delvis afhængig af signalering via β-adrenerge receptorer og D1 dopamin receptorer, men blev ikke påvirket af en D2 dopamin receptor antagonist. Dette arbejde illustrerer evnen af takars til at spore Neuro hændelser in vivo ved hjælp af 2pflim.
I den nuværende protokol er hele metoden til PKA-aktivitetsbilleddannelse i hoved rettede vågne mus under et tvungen bevægelses paradigme beskrevet i seks trin. For det første tilføjelsen af 2pFLIM kapaciteter til en konventionel to-foton mikroskop (figur 1). For det andet opførelsen af et motoriseret løbebånd (figur 2). For det tredje ekspression af tAKARα-sensoren i muse barken ved in utero elektroporation (IUE) af DNA-plasmider eller stereotaxisk injektion af adeno-associeret virus (AAV). Fremragende protokoller for kirurgi for iue24,25 og stereotaxisk injektion af virale partikler26 er tidligere blevet offentliggjort. De vigtigste parametre, vi brugte, er beskrevet nedenfor. Frem, installation af et kranie vindue. Fremragende protokoller er tidligere blevet offentliggjort for kranie Window kirurgi27,28. Flere trin, der er blevet ændret fra standardprotokollerne, er beskrevet. Femte, udfører in vivo 2pFLIM. For det sjette analyserne af 2pFLIM billeder (figur 3 og figur 4). Denne tilgang bør være let anvendelig på mange andre hoved-faste adfærdsmæssige paradigmer og hjerneområder.

<table>
	<tbody>
		<tr>
			<td>0.2 &#956;m cellulose acetate syringe filter</td>
			<td>Nalgene</td>
			<td>190-2520</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>16x 0.8 NA water-immersion objective</td>
			<td>Nikon</td>
			<td>MRP07220</td>
			<td>Step 5.5.</td>
		</tr>
		<tr>
			<td>3-pin cable</td>
			<td>US digital</td>
			<td>CA-MIC3-SH-NC</td>
			<td>Step 2.5. To connect rotation sensor to the DAQ input of the microscope</td>
		</tr>
		<tr>
			<td>Aluminum bread board</td>
			<td>Thorlabs</td>
			<td>MB1012</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>AnimalTracker MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.5 and sections 5 - 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Band-pass barrier filter</td>
			<td>Chroma</td>
			<td>ET500-40m</td>
			<td>Step 1.4.</td>
		</tr>
		<tr>
			<td>Cage plate</td>
			<td>Thorlabs</td>
			<td>CP01</td>
			<td>Step 2.4. Used as mount for rotation sensor</td>
		</tr>
		<tr>
			<td>Carbon steel burrs for micro drill, 0.5 mm tip diameter</td>
			<td>FST</td>
			<td>19007-05</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Circular coverslip (5mm diameter)</td>
			<td>VWR</td>
			<td>101413-528</td>
			<td>Step 4.5.</td>
		</tr>
		<tr>
			<td>Custom-made injection needle holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 3.2.4. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>Yates Motloid</td>
			<td>44114</td>
			<td>Steps 4.3. and 4.5.</td>
		</tr>
		<tr>
			<td>Dental drill; Microtorque ii</td>
			<td>Ram products</td>
			<td>66699</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Dowsil transparent polymer</td>
			<td>The Dow Chemical Company</td>
			<td>3-4680</td>
			<td>Step 4.5. Artificial dura</td>
		</tr>
		<tr>
			<td>Electroporation electrode</td>
			<td>Bex</td>
			<td>LF650P5</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bex</td>
			<td>CUY21</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Fast green FCF</td>
			<td>Sigma-aldrich</td>
			<td>F7258-25G</td>
			<td>Step 3.1.1.</td>
		</tr>
		<tr>
			<td>FLIMimage MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida</td>
		</tr>
		<tr>
			<td>FLIMview MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sections 5. and 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Foam-compatible glue (Gorilla White Glue)</td>
			<td>Gorilla</td>
			<td>5201204</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 4.3. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket</td>
		</tr>
		<tr>
			<td>Hydraulic micromanipulator</td>
			<td>Narishige</td>
			<td>MO-10</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Krazy glue</td>
			<td>Krazy glue</td>
			<td>KG82648R</td>
			<td>Step 4.3. Cyanoacrylate-based glue</td>
		</tr>
		<tr>
			<td>Low-noise fast photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422PA-40 or H10769PA-40</td>
			<td>Step 1.3.</td>
		</tr>
		<tr>
			<td>MATLAB 2012b</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software</td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Zhengke</td>
			<td>ZGA37RG</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Motor speed controller</td>
			<td>Elenker</td>
			<td>EK-G00015A1-1</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>Motorized micromanipulator</td>
			<td>Sutter</td>
			<td>MP-285</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Mounting base</td>
			<td>Thorlabs</td>
			<td>BA1S</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2</td>
		</tr>
		<tr>
			<td>Mounting post</td>
			<td>Thorlabs</td>
			<td>P14</td>
			<td>Step 2.6. Used for headplate holder post in combination with PB2</td>
		</tr>
		<tr>
			<td>Mounting post base</td>
			<td>Thorlabs</td>
			<td>PB2</td>
			<td>Step 2.6. Used for headplate holder post in combination with P14</td>
		</tr>
		<tr>
			<td>Mounting post bracket</td>
			<td>Thorlabs</td>
			<td>C1515</td>
			<td>Step 2.6. Used in combination with right-angle bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Optical post</td>
			<td>Thorlabs</td>
			<td>TR2</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>&#925;/&#913;</td>
			<td>&#925;/&#913;</td>
			<td>Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>FDS010</td>
			<td>Step 1.2.</td>
		</tr>
		<tr>
			<td>Photon timing counting module</td>
			<td>Becker and Hickl</td>
			<td>SPC-150</td>
			<td>Step 1.1.</td>
		</tr>
		<tr>
			<td>Plasmid: tAKAR&#945; (CAG-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119913</td>
			<td>Step 3.1.3.</td>
		</tr>
		<tr>
			<td>Post holder</td>
			<td>Thorlabs</td>
			<td>PH4</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2</td>
		</tr>
		<tr>
			<td>Right-angle bracket</td>
			<td>Thorlabs</td>
			<td>AB90</td>
			<td>Step 2.6 Used in combination with mounting post bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Rotation sensor</td>
			<td>US digital</td>
			<td>MA3-A10-250-N</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Rubber mat</td>
			<td>Rubber-Cal</td>
			<td>B01DCR5LUG</td>
			<td>Step 2.1.</td>
		</tr>
		<tr>
			<td>Shaft coupling (1/4 inch x 1/4 inch)</td>
			<td>McMaster</td>
			<td>6208K433</td>
			<td>Steps 2.3. and 2.4.</td>
		</tr>
		<tr>
			<td>ScanImage 3.6</td>
			<td>Svoboda Lab/Vidrio Technology</td>
			<td>N/A</td>
			<td>Steps 5.9. and 6.1.</td>
		</tr>
		<tr>
			<td>Signal splitter</td>
			<td>Becker and Hickl</td>
			<td>HPM-CON-02</td>
			<td>Step 1.3.1.</td>
		</tr>
		<tr>
			<td>Stainless steel axle (diameter 1/4 inch, L = 12 inch)</td>
			<td>McMaster</td>
			<td>1327K66</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment systsem</td>
			<td>David kopf</td>
			<td>1900</td>
			<td>Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator</td>
		</tr>
		<tr>
			<td>Two-photon microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>14006</td>
			<td>Step 3.2.6.</td>
		</tr>
		<tr>
			<td>Virus: tAKAR&#945; (AAV2/1 hSyn-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119921</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>White PE foam roller (8 x 12 inch)</td>
			<td>Fabrication enterprises INC.</td>
			<td>30-2261</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>White polystyrene fom ball halves</td>
			<td>GrahamSweet</td>
			<td>200mm diameter 2 hollow halves</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>Zipkicker</td>
			<td>PACER</td>
			<td>PT29</td>
			<td>Step 4.3. Hardening accelerator</td>
		</tr>
	</tbody>
</table>

visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy

Neuromodulation udøver kraftfuld kontrol over hjernens funktion. Dysfunktion af neuromodulatoriske systemer resulterer i neurologiske og psykiske lidelser. På trods af deres betydning, teknologier til sporing af neuromodulatory begivenheder med cellulære opløsning er lige begyndt at dukke op. Neuromodulatorer, såsom dopamin, noradrenalin, acetylcholin, og serotonin, udløser intracellulære signalering begivenheder via deres respektive G protein-koblede receptorer til at modutere neuronal ophidelse, synaptisk kommunikation, og andre neuronal og dermed regulere informationsbehandlingen i neuronal netværket. De ovennævnte neuromodulatorer konvergerer på lejren/protein kinase A (PKA) pathway. Derfor, in vivo PKA Imaging med enkelt celle opløsning blev udviklet som en udlæser for neuromodulatoriske begivenheder på en måde analogt med calcium Imaging for neuronal elektriske aktiviteter. Heri præsenteres en metode til at visualisere PKA aktivitet på niveauet af individuelle neuroner i cortex af hoved-fast opfører mus. For at gøre dette, en forbedret A-kinase Activity reporter (AKAR), kaldet tAKARα, anvendes, som er baseret på Förster resonans energioverførsel (FRET). Denne genetisk kodede PKA-sensor introduceres i motorisk cortex via in utero elektroporation (iue) af DNA-plasmider eller stereotaxisk injektion af adeno-associeret virus (AAV). FRET ændringer er afbildet ved hjælp af to-foton fluorescens Lifetime Imaging mikroskopi (2pFLIM), som giver fordele i forhold til ratiometriske FRET-målinger til kvantificering af FRET-signalet i et let spredt hjernevæv. For at studere PKA aktiviteter under tvungen bevægelse, takarα er afbildet gennem en kronisk kranie vindue over cortex af vågen, hoved-faste mus, der kører eller hvile på en hastigheds kontrolleret motoriseret løbebånd. Denne billeddannelses tilgang vil være gældende for mange andre hjerneregioner for at studere tilsvarende adfærds inducerede PKA-aktiviteter og for andre FLIM-baserede sensorer til in vivo-billeddannelse.

FRET-FLIM sensorer giver mulighed for visualisering af mange forskellige signalering veje, herunder cAMP/PKA pathway involveret i Neuromodulation. Den nuværende protokol udnytter den nyudviklede tAKARα-sensor i kombination med 2pFLIM til at visualisere PKA-aktiviteter i hoved-fikseret opfører mus. De fleste eksisterende to-foton mikroskoper kan opgraderes med 2pFLIM kapaciteter ved at tilføje tre til fire komponenter, som illustreret i figur 1 (Se også p...

Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

En procedure er præsenteret for at visualisere protein kinase A aktiviteter i hovedet-fast, opfører mus. En forbedret A-kinase-aktivitets reporter, tAKARα, udtrykkes i kortikale neuroner og gøres tilgængelig for billeddannelse gennem et kranie vindue. To-foton fluorescens Lifetime Imaging mikroskopi bruges til at visualisere PKA aktiviteter in vivo under tvungen bevægelse.

Visualisering af proteinkinase en aktivitet i hoved-fast opfører mus ved hjælp in vivo to-foton fluorescens levetid Imaging mikroskopi

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Vores metode udnytter en ny protein kinase En sensor kaldet target En kinase aktivitet reporter alpha, eller tAKAR

Research

JoVE Journal

Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging af Mouse Spinal Cord ved hjælp af to-foton mikroskopi

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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 ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

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The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In Vivo to-foton mikroskopi af Single nerveender i hud

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

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Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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