Ringraziamo la signora Tess J. Lameyer, la signora Ruth Frank, e il dr. Michael A. Muniak per le modifiche e i commenti, e il dottor Ryohei Yasuda a Max Planck Florida per il software di acquisizione 2pFLIM. Questo lavoro è stato sostenuto da due premi BRAIN Initiative U01NS094247 (H.e T.M.) e R01NS104944 (H. e T.M.), una sovvenzione R01 R01 R01NS081071 (T.M.) e una concessione R21 R21 R21NS097856 (H. ) . Tutti i premi sono dal National Institute of Neurological Disorders and Stroke, Stati Uniti.

<ol>
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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=Liberated+PKA+Catalytic+Subunits+Associate+with+the+Membrane+via+Myristoylation+to+Preferentially+Phosphorylate+Membrane+Substrates.">Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates.</a> Cell Reports. 19 (3), 617-629 (2017).</li><li>Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+spatiotemporal+dynamics+of+neuronal+signaling+using+fluorescence+resonance+energy+transfer+and+fluorescence+lifetime+imaging+microscopy.">Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy.</a> Current Opinion in Neurobiology. 16 (5), 551-561 (2006).</li><li>Theurkauf, W. 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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=Intracellular+dynamics+of+hippocampal+place+cells+during+virtual+navigation.">Intracellular dynamics of hippocampal place cells during virtual navigation.</a> Nature. 461 (7266), 941-946 (2009).</li><li>Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+signaling+using+two-photon+fluorescent+lifetime+imaging+microscopy.">Imaging intracellular signaling using two-photon fluorescent lifetime imaging microscopy.</a> Cold Spring Harbor Protocols. 7 (11), 1121-1128 (2012).</li><li>Mehta, S., 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=Single-fluorophore+biosensors+for+sensitive+and+multiplexed+detection+of+signalling+activities.">Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities.</a> Nature Cell Biology. 20 (10), 1215-1225 (2018).</li></ol>

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Questo protocollo dimostra l'uso del sensore FRET-FLIM tAKAR per visualizzare l'attività PKA innescata dalla neuromodulazione nei topi che si comportano con la testa fissa. Questa applicazione si basa su test approfonditi e caratterizzazioni di tAKAR , in vitro e in vivo per dimostrare che il segnale FLIM ottenuto è rilevante per gli eventi neuromodulatori fisiologici17. Qui, un'applicazione in vivo, l'attività PKA indotta dalla locomozione nella corteccia motoria, viene utilizzata per descrive...

La neuromodulazione, nota anche come trasmissione sinaptica lenta, impone un forte controllo sulla funzione cerebrale durante i diversi stati comportamentali, come lo stress, l'eccitazione, l'attenzione e la locomozione1,2,3, 4. Nonostante la sua importanza, lo studio di quando e dove si svolgono eventi di neuromodulatori è ancora agli inizi. Neuromodulatori, tra cui acetilcolina, dopamina, noradrenalina, serotonina, e molti neuropeptidi, attivare G recettori accoppiati a proteina (GPCR), che a sua volta innescano percorsi di messaggeri intracellulari secondo con una vasta finestra di scale temporali che vanno da secondi a ore. Mentre ogni neuromodulatore innesca una serie distinta di eventi di segnalazione, il percorso cAMP/proteina kinase A (PKA) è un percorso a valle comune per molti neuromodulatori1,5. Il percorso cAMP/PKA regola l'eccitabilità neuronale, la trasmissione sinaptica e la plasticità6,7,8,9e quindi sintonizza le dinamiche della rete neuronale. Poiché neuroni o tipi neuronali diversi esprimono diversi tipi o livelli di recettori neuromodulatori10, gli effetti intracellulari dello stesso neuromodulatore extracellulare possono essere eterogenei tra neuroni diversi e, quindi, devono essere studiato con risoluzione cellulare. Ad oggi, rimane difficile monitorare gli eventi neuromodulatori nei singoli neuroni in vivo durante il comportamento.
Per studiare la dinamica spatiotemporale della neuromodulazione, è necessaria una modalità di registrazione adeguata. La microdialisi e la cimmimetria a scansione rapida sono spesso utilizzate per studiare il rilascio di neuromodulatori, ma mancano della risoluzione spaziale per monitorare gli eventi cellulari11,12. Analoga alla dinamica del calcio utilizzata come proxy per l'attività elettrica neuronale nell'imaging della popolazione13,l'imaging PKA può essere utilizzato per leggere eventi neuromodulatori in una popolazione neuronale a risoluzione cellulare. Il presente protocollo descrive l'uso di un activity reporter A-kinase (AKAR) migliorato per monitorare le attività PKA in vivo durante il comportamento animale. Il metodo qui descritto consente l'imaging simultaneo di popolazioni neuronali a risoluzione subcellulare con una risoluzione temporale che tiene traccia degli eventi neuromodulatori fisiologici.
Gli AKAR sono composti da un donatore e da proteine fluorescenti accettatore collegate da un peptide del substrato pkA phosphorylation e da un dominio associato alla forchetta (FHA) che si lega al serino fosforolato o alla treonina del substrato14,15. Al momento dell'attivazione della via PKA, il peptide substrato di AKAR viene fosforato. Di conseguenza, il dominio FHA si lega al peptide del substrato fosforilo, portando così i due fluorofori in stretta vicinanza, indicato come lo stato chiuso dell'AKAR. Lo stato chiuso di un AKAR fosforolato si traduce in un aumento del trasferimento di energia di risonanza del fàrster (FRET) tra il donatore e l'accettatore fluorofori. Poiché la percentuale di ACR al fosforo è correlata al livello di attività PKA16, la quantità di FRET in un campione biologico può essere utilizzata per quantificare il livello di attività PKA16,17,18, 19,20.
Le prime versioni degli AKAR sono state progettate principalmente per l'imaging ratiometrico a due colori14. Quando si immagina più in profondità nel tessuto cerebrale, il metodo ratiometrico soffre di distorsione del segnale a causa della dispersione della luce dipendente dalla lunghezza d'onda17,18,21. Come discusso di seguito, la microscopia per l'imaging a vita di fluorescenza (FLIM) elimina questo problema perché Il FLIM misura solo i fotoni emessi dal donatore fluoroforo18,21. Di conseguenza, la quantificazione FLIM di FRET non è influenzata dalla profondità tissutale17. Inoltre, può essere utilizzata una variante "scura" (cioè bassa resa quantistica [QY]) del fluoroforo dell'accettatore. Questo libera un canale di colore per facilitare la misurazione multiplexata delle proprietà neuronali ortogonali tramite l'imaging simultaneo di un secondo sensore o di un marcatore morfologico17,19,20.
L'imaging FLIM quantifica il tempo che un fluoroforo trascorre nello stato eccitato, cioè la durata della fluorescenza18. Il ritorno di un fluoroforo allo stato di terra, così la fine dello stato eccitato, spesso si contempla con l'emissione di un fotone. Anche se l'emissione di un fotone per una singola molecola eccitata è stocastica, in una popolazione la durata media della fluorescenza è una caratteristica di quel particolare fluoroforo. Quando una popolazione pura di fluorofori è eccitata contemporaneamente, la fluorescenza risultante seguirà un singolo decadimento esponenziale. La costante temporale di questo decadimento esponenziale corrisponde alla durata media della fluorescenza, che in genere varia da uno a quattro nanosecondi per le proteine fluorescenti. Il ritorno di un fluoroforo donatore eccitato allo stato di terra può avvenire anche da PARTE di FRET. In presenza di FRET, la durata della fluorescenza del fluoroforo del donatore è ridotta. Gli AKAsphorylated unphosphorylated mostrano una durata di fluorescenza del donatore relativamente più lunga. Al fosforo della PKA, il sensore presenta una vita più breve perché i fluorofori donatore e accettatore sono portati l'uno vicino all'altro e FRET è aumentato. La quantificazione della durata della fluorescenza in una popolazione di AKAR rappresenta quindi il livello di attività PKA.
Le prime versioni degli AKA non sono state utilizzate con successo per l'imaging in vivo a risoluzione a cella singola. Ciò è dovuto principalmente alla bassa ampiezza del segnale dei sensori AKAR alle attivazioni fisiologiche17. Recentemente, confrontando sistematicamente i sensori AKAR disponibili per la microscopia di imaging a durata della fluorescenza a due fotoni (2pFLIM), è stato scoperto che un sensore chiamato FLIM-AKAR ha sovraperformato i sensori alternativi. Inoltre, una serie di varianti FLIM-AKAR chiamate AKAR mirati (tAKAR) sono state sviluppate per visualizzare l'attività PKA in posizioni subcellulari specifiche: microtubuli (tAKAR), citosol (tAKAR), actina (tAKAR) , actina mentoluta (tAKAR) , membrana (tAKAR) densità postsinaptica (tAKAR). Tra i tAKARR, tAKAR ha aumentato l'ampiezza del segnale suscitata dalla noradrenalina di 2,7 volte. Questo è coerente con la conoscenza che la maggior parte della PKA nei neuroni sono ancorati ai microtubuli allo stato di riposo22,23. il tAKAR è stato il migliore tra gli AKAR esistenti per 2pFLIM. Inoltre, tAKAR ha rilevato un'attività PKA fisiologicamente rilevante suscitata da più neuromodulatori, e l'espressione di tAKAR non ha alterato le funzioni neuronali17.
Di recente, tAKAR è stato utilizzato con successo per visualizzare le attività PKA nei mouse che si comportano alla testa17. È stato dimostrato che la locomozione forzata ha innescato l'attività PKA nel soma dei neuroni a strati superficiali (strato da 1 a 3, fino a una profondità di 300 m da pia) nel motore, nella canna e nelle cortici visive. L'attività PKA innescata dalla locomozione dipendeva in parte dalla segnalazione tramite recettori adrenergici e recettori della dopamina D1, ma non era influenzata da un antagonista del recettore della dopamina D2. Questo lavoro illustra la capacità dei tAKAR di tenere traccia degli eventi di neuromodulazione in vivo utilizzando 2pFLIM.
Nel protocollo corrente, l'intero metodo per l'imaging dell'attività PKA nei topi svegli fissati alla testa durante un paradigma di locomozione forzata è descritto in sei passaggi. In primo luogo, l'aggiunta di funzionalità 2pFLIM a un microscopio convenzionale a due fotoni (Figura 1). In secondo luogo, la costruzione di un tapis roulant motorizzato (Figura 2). In terzo luogo, l'espressione del sensore tAKAR nella corteccia del topo per elettroporazione inutero (IUE) di plasmidi di DNA, o iniezione stereotassica di virus adeno-associato (AAV). Protocolli eccellenti per interventi chirurgici per IUE24,25 e iniezione stereotassica di particelle virali26 sono stati precedentemente pubblicati. I parametri chiave che abbiamo usato sono descritti di seguito. Forth, l'installazione di una finestra cranica. Protocolli eccellenti sono stati precedentemente pubblicati per la chirurgia della finestra cranica27,28. Vengono descritti diversi passaggi che sono stati modificati dai protocolli standard. Quinto, esecuzione in vivo 2pFLIM. Sesto, le analisi delle immagini 2pFLIM (Figura 3 e Figura 4). Questo approccio dovrebbe essere facilmente applicabile a molti altri paradigmi comportamentali fissati alla testa e aree cerebrali.

<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

Neuromodulazione esercita un potente controllo sulla funzione del cervello. La disfunzione dei sistemi neuromodulatori si traduce in disturbi neurologici e psichiatrici. Nonostante la loro importanza, le tecnologie per monitorare gli eventi neuromodulatori con risoluzione cellulare stanno appena cominciando ad emergere. I neuromodulatori, come dopamina, noradrenalina, acetilcolina e serotonina, innescano eventi di segnalazione intracellulare tramite i rispettivi recettori accoppiati alle proteine G per modulare l'eccitabilità neuronale, le comunicazioni sinaptiche e altri neuronali funzioni, regolando così l'elaborazione delle informazioni nella rete neuronale. I neuromodulatori di cui sopra convergono sul percorso cAMP/protein a chinasi A (PKA). Pertanto, l'imaging PKA in vivo con risoluzione a singola cellula è stato sviluppato come lettura per gli eventi neuromodulatori in modo analogo all'imaging del calcio per le attività elettriche neuronali. Qui, viene presentato un metodo per visualizzare l'attività PKA a livello di singoli neuroni nella corteccia di topi che si comportano alla testa.Here, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mic. A tale scopo, viene utilizzato un giornalista di attività A-kinasi (AKAR) migliorato, chiamato tAKAR, che si basa sul trasferimento di energia di risonanza del Fàrster (FRET). Questo sensore PKA geneticamente codificato viene introdotto nella corteccia motoria tramite elettroporazione in utero (IUE) di plasmidi di DNA, o iniezione stereotassica di virus adeno-associato (AAV). I cambiamenti freT sono immagini utilizzando la microscopia di imaging a vita a durata a due fotoni (2pFLIM), che offre vantaggi rispetto alle misurazioni geometriche FRET per quantificare il segnale FRET nel tessuto cerebrale che disperde la luce. Per studiare le attività della PKA durante la locomozione forzata, tAKAR è immagine attraverso una finestra cranica cronica sopra la corteccia di topi svegli, fissati per la testa, che corrono o poggiano su un tapis roulant motorizzato a velocità controllata. Questo approccio di imaging sarà applicabile a molte altre regioni del cervello per studiare le corrispondenti attività PKA indotte dal comportamento e ad altri sensori basati su FLIM per l'imaging in vivo.

I sensori FRET-FLIM consentono la visualizzazione di molti percorsi di segnalazione diversi, tra cui il percorso cAMP/PKA coinvolto nella neuromodulazione. L'attuale protocollo utilizza il sensore tAKAR, sviluppato di recente, in combinazione con 2pFLIM per visualizzare le attività PKA nei mouse che si comportano con la testa fissa. La maggior parte dei microscopi a due fotoni esistenti può essere aggiornata con capacità 2pFLIM aggiungendo da tre a quattro componenti, come illustrato n...

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

Viene presentata una procedura per visualizzare le attività della chinasi proteica A nei topi con problemi alla testa e che si comportano. Un giornalista di attività A-kinase migliorato, tAKAR, è espresso in neuroni corticali e reso accessibile per l'imaging attraverso una finestra cranica. La microscopia di imaging a durata della fluorescenza a due fotoni viene utilizzata per visualizzare le attività PKA in vivo durante la locomozione applicata.

Visualizzazione della proteina Kinase un'attività nel mouse behaving fissato per la testa utilizzando la microscopia fluorescenza a due fotoinina in vivo

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

Il nostro metodo utilizza una nuova protein chinasi Un sensore chiamato bersaglio A chinasi attività reporter alfa, o tAKAR

visualizing-protein-kinase-activity-head-fixed-behaving-mice-using

Research

JoVE Journal

Neuroscience

8.3K Views.  Oregon Health & Science University. Il nostro metodo utilizza una nuova protein chinasi Un sensore chiamato bersaglio A chinasi attività reporter alfa, o tAKAR

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

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

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 significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

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 ...

Ricerca

Neuroscienze

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced 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 Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

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

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

In Vivo microscopia a due fotoni di Endings singolo nervo in pelle

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon<em&gt; In vivo</em&gt; Imaging di spine dendritiche nella corteccia mouse Uso un teschio Diluito-Preparazione

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Due-fotone Imaging dei neuroni corticali in topi neonatali

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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillare Due fotoni In vivo Imaging della retina del topo

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Valutazione e manipolazione dell'attività neurale mediante microscopia olografica a due fotoni

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In vivo Imaging del calcio a campo largo e a due fotoni da un topo utilizzando una grande finestra cranica

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Monitoraggio del comportamento e modello di attività neurale nel proencefalo del pesce zebra

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Autore in primo piano: Progressi nella ricerca sul cervello del pesce zebra adulto 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Imaging del calcio a due fotoni dell'attività del prosencefalo nel comportamento del pesce zebra adulto

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...