This work was supported by the World Class Institute (WCI) program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology of Korea Grant WCI 2009-003 and Korea Institute of Science and Technology Institutional Program Project 2E24210. Sungmoo Lee was supported by Global Ph.D. Fellowship program (NRF-2013H1A2A1033344) of the National Research Foundation (NRF) under the Ministry of Education (MOE, Korea).

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Il sistema nervoso utilizza tensione in diversi modi, inibizione provoca un leggero iperpolarizzazione, ingresso sinaptica provoca un leggero depolarizzazione e un&#39;azione potenziali risultati in un cambiamento relativamente grande tensione. La possibilità di misurare le variazioni del potenziale di membrana dalla Gevis offre il potenziale promettente di analizzare diversi componenti di circuiti neuronali contemporaneamente. In questo rapporto si dimostra un metodo fondamentale per le modifiche di imaging del potenziale di membrana utilizzando Gevis. Una chiave importante per modifiche di imaging in tensione è l&#39;espressione efficiente del GEVI nella membrana plasmatica. espressione intracellulare crea una fluorescenza non reattivo che riduce il SNR della sonda. Ottimizzare le condizioni di trasfezione migliora notevolmente la coerenza delle misure ottiche. Infatti, quando si verifica un romanzo GEVI è consigliabile testare anche una sonda nota per garantire che le differenze nelle attività ottica è dovuto unicamente alla neSonda w e non le condizioni delle celle. La Figura 6 mostra l&#39;effetto della fluorescenza interna diminuire la sensibilità dell&#39;imaging tensione, valore Af / F. La Figura 6B mostra Af valori una cellula HEK esprime il GEVI base singola FP, Bongwoori. Traccia 2 proviene dalla regione 2 (colore blu), dove relativamente brillante fluorescenza interna si vede in Figura 6A. Questa traccia ha analogo livello di valore Af come traccia 1. Tuttavia, nella Figura 6C dove le tracce della figura 6B sono stati divisi da valori RLI per i valori Af / F, la traccia 2 gocce in modo significativo a causa della fluorescenza interna luminoso che diminuisce Af / F valori. Fluorescenza trace 3 ha una piccolissima Af ma ha anche un valore molto piccolo RLI (F). Il risultato è un segnale / Af fuorviante F che ha un sostanziale aumento nel rumore della registrazione. Questo esempio è stato incluso per dimostrare la Af è ancheimportante. Un grande cambiamento di Af / F non è utile se F è molto basso per cominciare. L&#39;uso di un divisore immagini consente la misura contemporanea di due lunghezze d&#39;onda su una singola telecamera CCD. Questo aiuta notevolmente la misurazione di FRET variazioni fluorescenti dipendente per lo sviluppo sonda e riduce il costo dell&#39;installazione. Tuttavia, a causa di aberrazione cromatica, queste due immagini saranno leggermente fuori fuoco implicano la necessità di un obiettivo apocromatico. La luce più sono e meglio SNR, in modo da scegliere gli obiettivi con la massima apertura numerica migliora anche imaging di fluorescenza. Inoltre, si possono utilizzare sorgenti luminose forti quali diodi emettitori di luce (LED) o laser per aumentare SNR. I LED non richiedono un otturatore meccanico. In questo rapporto ci mostra i segnali ottici da un unico FP GEVI e un GEVI FRET-based. Il vantaggio principale di una GEVI FRET-based è che l&#39;imaging raziometrico consente la riduzione del rumore correlata due per la respirazione e il flusso di sangue in vivo. La polarità opposta dei segnali fluorescenti conferma un cambiamento reale nel potenziale di membrana. Tuttavia, poiché le intensità di fluorescenza dei due cromofori spesso variano notevolmente, il SNR sarà anche variare. Questo confonde l&#39;analisi raziometrico e limita la sua utilità 28. Non ci può essere anche un segnale FRET indipendente 29. È perciò talvolta preferibile utilizzare solo il cromoforo luminosa fluorescente utilizzando FRET analizzare variazioni del potenziale di membrana. Imaging tensione è più impegnativo di calcium imaging a causa della velocità e la variabilità delle variazioni di potenziale di membrana rispetto a calcium imaging che misura il flusso di calcio. I potenziali di membrana variazioni scale di tempo molto veloci rispetto al calcio ione flusso, ed eventi sottosoglia in neuroni non possono essere misurati con calcium imaging 30. Inoltre, la sonda di tensione deve essere nella membrana plasmaticaper essere efficace, che riduce la quantità di sonda disponibile ai cambiamenti relazione otticamente del potenziale di membrana. Di conseguenza, il numero di fotoni che può effettivamente arrivare al chip CCD è relativamente pochi. Ciò richiede la necessità di una alta velocità e fotocamera a basso rumore di lettura con alta efficienza quantica e una sonda che provoca un elevato SNR dalle variazioni del potenziale di membrana. Con l&#39;imaging cellule in vitro, i ricercatori potranno comprendere meglio i potenziali benefici e le insidie ​​di Gevis prima di tentare misure in vivo.

Il principale obiettivo di questo lavoro è quello di dimostrare l&#39;imaging ottico delle variazioni di potenziali di membrana in vitro utilizzando proteine ​​fluorescenti geneticamente codificate. cambiamenti di imaging del potenziale di membrana offre l&#39;eccitante possibilità di studiare l&#39;attività dei circuiti neuronali. Quando i cambiamenti nella membrana risultato potenziale in un cambiamento intensità di fluorescenza, ogni pixel della telecamera diventa un elettrodo surrogata per misure non intrusivi di attività neuronale. Da oltre quarant&#39;anni, coloranti organici voltaggio-sensibile sono stati utili per osservare i cambiamenti nel potenziale di membrana 1-4. Tuttavia, questi coloranti mancano di specificità cellulare. Inoltre, alcuni tipi di cellule sono difficili da macchiare. indicatori di tensione geneticamente codificati (GeViS) superare queste limitazioni avendo le cellule da studiare specificamente esprimono la sonda fluorescente tensione-sensibili. Ci sono tre classi di Gevis. La prima classe di GEVI utilizza il vodominio della fosfatasi tensione-sensing ensione-sensing con una singola proteina fluorescente (FP) 5-9 o un paio 10-12 un Förster trasferimento di energia di risonanza (FRET). La seconda classe di sensori utilizza microbica rodopsina come indicatore fluorescente direttamente 13-15 o tramite FRET elettrocromico 16,17. La terza classe utilizza due componenti, il componente genetica essendo un FP membrana ancorata ed un secondo componente essendo una membrana vincolato tempra colorante 18-20. Mentre il secondo e terzo classi sono utili per esperimenti in vitro e fetta 19,20, solo la prima classe di sensori sono attualmente utile per analisi in vivo 6. In questo rapporto ci sarà dimostrare l&#39;imaging del potenziale di membrana utilizzando la prima classe di Gevis (figura 1) in vitro. Questa prima classe di sensori di tensione è il più facile da passare a imaging in vivo. Dal momento che Gevis utilizing un dominio di rilevamento della tensione fuso ad una FP sono circa 50 volte più luminoso classe rodopsina dei sensori, possono essere ripreso con illuminazione lampada ad arco anziché richiedere un laser estremamente potente. Un&#39;altra conseguenza della disparità di luminosità è che la prima classe di GeViS può facilmente superare l&#39;auto-fluorescenza del cervello. Le sonde rodopsina-based non può. La terza classe di sensore è altrettanto chiaro come la prima classe, ma richiede l&#39;aggiunta di un quencher chimico che è difficile da gestire in vivo. Noi, quindi, dimostrare l&#39;acquisizione di una sonda con un singolo FP (Bongwoori) 8 e una sonda costituita da una coppia FRET (Nabi 2) 12. Il FRET costruisce in questo rapporto sono versioni farfalla di VSFP-CR (proteine ​​fluorescenti voltaggio-sensibile - Clover-mRuby2) 11 costituito da un donatore fluorescente verde, trifoglio, e un accettore fluorescente rossa, mRuby2, chiamato Nabi 2.242 e 2.244 Nabi <sup&gt; 12. L&#39;introduzione di questi tipi di registrazioni dovrebbe dare ai ricercatori una migliore comprensione del tipo di Gevis di informazione in grado di fornire. <img alt="Figura 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> Figura 1. due tipi di indicatori geneticamente codificati tensione (Gevis) Divisori stampati in questo rapporto (A) Un FP mono basato GEVI avere un dominio di tensione-sensing trans-membrana e una proteina fluorescente. (B) Un GEVI basato FRET composto da un dominio di tensione-sensing trans-membrana, un donatore tasto e accettore. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">IX71</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60X objective lens (numerical aperture = 1.35)</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">UPLSAPO 60XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">&#160;FF02-472/30</td>
			<td rowspan="3" style="width:187px;">For voltage imaging of super ecliptic pHluorin in Bongwoori</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-497/LP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">75W Xenon arc lamp</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">OptoSource Illuminator</td>
			<td style="width:187px;">LEDs and lasers are also effective light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Slow speed CCD camera</td>
			<td style="width:165px;">Hitachi</td>
			<td style="width:203px;">KP-D20BU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dual port camera adaptor</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">U-DPCAD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High speed CCD camera</td>
			<td style="width:165px;">RedShirtImaging, LLC</td>
			<td style="width:203px;">NeuroCCD-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image splitter</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">Optosplit 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-475/23-25</td>
			<td rowspan="5" style="width:187px;">For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET520/40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF560-FDi01-25X36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET645/75</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Vibration isolation system</td>
			<td style="width:165px;">Kinetic systems</td>
			<td style="width:203px;">250BM-IC, 5702E-3036-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patching chamber</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">RC-26G, 64-0235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">#0 Micro Coverglass (22x40mm)</td>
			<td style="width:165px;">Electron Microscopy Sciences</td>
			<td style="width:203px;">72198-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Temperature controller</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">#0 (0.08~0.13mm) - 10mm diameter glass coverslip</td>
			<td style="width:165px;">Ted Pella</td>
			<td style="width:203px;">260366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lipofection agent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">11668-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcium phosphate reagent</td>
			<td style="width:165px;">Clontech - Takara</td>
			<td style="width:203px;">631312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patch clamp amplifier</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">EPC 10 USB amplifier</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Multi-channel data acquisition software</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">Patchmaster</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image acquisition and analysis software</td>
			<td style="width:165px;">RedShirtImaging</td>
			<td style="width:203px;">Neuroplex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Spreadsheet application software</td>
			<td style="width:165px;">Microsoft</td>
			<td style="width:203px;">Microsoft Excel 2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Data analysis software</td>
			<td style="width:165px;">OriginLab</td>
			<td style="width:203px;">OriginPro 8.6.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Demagnifier</td>
			<td style="width:165px;">Qioptiq LINOS</td>
			<td style="width:203px;">Optem standard camera coupler 0.38x SC38 J clamp</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Confocal microscope</td>
			<td style="width:165px;">Nikon</td>
			<td style="width:203px;">Nikon A1R confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Anti-fade reagent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">P36930</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging membrane potential with two types of genetically encoded fluorescent voltage sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

Transitoriamente cellule transfettate possono presentare una significativa variazione di intensità di fluorescenza e il grado di espressione membrana plasmatica. Anche sullo stesso vetrino alcune cellule avranno diversi livelli di fluorescenza interna. Questo è probabilmente dovuto alla quantità di agente trasfezione assorbita dalla cella. Di tanto in tanto, troppo espressione fa sì che la cellula di sperimentare la risposta proteina spiegato con conseguente apoptosi 27 (luminoso, le cellule arrotondate, con elevata fluorescenza interna). Lo sperimentatore è quindi avvertito per testare sia cellule luminose e cellule dim con un minimo di fluorescenza interna possibile, in quanto espressione interno crea una fluorescenza non reattivo che abbassa il rapporto segnale rumore (SNR). Un&#39;altra considerazione è il tasso di maturazione dei cromofori. Figura 3 mostra disparità nella fluorescenza della chromoph accettoreore (mRuby2) che ha un tempo di maturazione più lungo del cromoforo donatore (trifoglio). <img alt="Figura 3" src="/files/ftp_upload/53566/53566fig3.jpg" /> Figura 3. confocale immagini di cellule HEK 293 trasfettate con un FRET Based GEVI, Nabi2.242. (A) immagini confocale di una cella che mostrano HEK membrana fluorescenza localizzata da FRET donatore e accettore, (B) immagini confocale che mostrano un potenziale conseguenza di lenta maturazione di mRuby2. Tutte e quattro le cellule presentano Clover fluorescenza mentre solo due mostre mRuby2 fluorescenza. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Le immagini sono state acquisite da un microscopio confocale. Per il donatore FRET, trifoglio, 488 nm laser è stato utilizzato per l&#39;eccitazione e l&#39;emissione è stata captrato con 525/50 nm banda passante del filtro. L&#39;accettore FRET, mRuby2, è stata illuminata con 561 nm laser per l&#39;eccitazione e 595/50 nm banda passante del filtro è stato utilizzato per l&#39;emissione. I campioni per l&#39;imaging confocale sono state fissate con paraformaldeide al 4% / soluzione di saccarosio in tampone fosfato regolata a pH 7,4 e poi montati con un reagente anti-sbiadimento. Una volta che le condizioni di trasfezione sono ottimizzate, la prossima fonte di variazione proviene dalla determinazione della regione di interesse da analizzare. Uso telaio sottrazione per identificare le regioni della cella con la massima variazione di fluorescenza è spesso usato per massimizzare il valore Af / F. Un&#39;alternativa è quella di selezionare tutti i pixel che ricevono luce dalla cella. Questo aumenta il numero di pixel con conseguente riduzione del rumore, ma diminuisce la dimensione del segnale dal non-responsive fluorescenza interna è incluso. Entrambi i metodi sono bene fintanto sperimentatore rimane costante. <pclass = &quot;jove_content&quot; fo: keep-together.within-page = &quot;1&quot;&gt; Figura 4A mostra la variazione di fluorescenza di una cellula HEK che esprimono un GEVI base single-FP, Bongwoori. Questa è una tipica variazione di fluorescenza in risposta ad impulsi di tensione a gradini. Da questi dati si può tracciare sensibilità tensione della GEVI e determinare l&#39;on e off costanti T si a tensioni diverse montando a singola o doppia funzione di decadimento esponenziale. 16 studi sono tipicamente mediati quando l&#39;imaging cellule HEK per migliorare il SNR di piccoli passi tensione e per rilevare qualsiasi potenziale sbiancamento durante la registrazione. Quelle sonde che danno un segnale forte a 100 mV possono essere testati nei neuroni dell&#39;ippocampo dissociati (Figura 4C). La traccia di fluorescenza dal neurone dell&#39;ippocampo è un singolo trial. <img alt="Figura 4" src="/files/ftp_upload/53566/53566fig4.jpg" /> Figura 4. Imaging di tensione con un FP Based GEVI unico espressain cellule HEK 293 e neuroni dell&#39;ippocampo primario. (A) Af / F traccia di una singola base di FP GEVI, Bongwoori, mostrando le risposte a impulsi di tensione a gradini registrati a 1 kHz con una telecamera CCD ad alta velocità. (B) Un HEK 293 cellule ripreso con la fotocamera CCD ad alta velocità; (A sinistra) Intensità Riposo Luce (RLI) di una cellula che esprime l&#39;immagine di sottrazione (a destra) cornice che indica i pixel in cui è stato osservato il cambiamento di fluorescenza Bongwoori &amp;. (C) la registrazione ottica dei potenziali d&#39;azione indotta da topo neuroni primari dell&#39;ippocampo che esprimono Bongwoori. I potenziali d&#39;azione sono stati evocati in modalità morsetto corrente cellula intera. La traccia Af / F è stato selezionato tra i pixel correlati alla soma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Un rappresentante fluorescence lettura dei cromofori donatore e accettore è mostrato in Figura 5A. La polarità del cambiamento di fluorescenza è opposto per il donatore e accettore consentendo analisi raziometrica per ridurre sorgenti di rumore correlate, ad es., Il movimento. Ad esempio, il movimento correlata rumore esporrà un cambiamento della fluorescenza nella stessa direzione per donatore e accettore di fluorescenza. Mentre la sonda basata FRET è capace di immagini raziometrico, spesso è meglio analizzare solo il cromoforo luminoso. Questo perché il cromoforo dimmer può aumentare sostanzialmente il rumore della registrazione. Figura 5B mostra questo effetto. La sottrazione struttura in Figura 5B dal brillante trifoglio mostra chiaramente dove il segnale ottico nella cella. Al contrario, il telaio sottrazione di mRuby2 fluorescenza mostra gradi più elevati del rumore in tutta la cella. Pertanto, solo il segnale donatore trifoglio è mostrato in un neurone dell&#39;ippocampo in Fifigura 5C. <img alt="Figura 5" src="/files/ftp_upload/53566/53566fig5.jpg" /> Figura 5. immagini di tensione con un FRET basata GEVI espresso in HEK 293 cellule e neuroni primari dell&#39;ippocampo. (A) Af / F traccia di un GEVI FRET basata, Nabi2.242, che mostra le risposte a due lunghezze d&#39;onda a impulsi di tensione a gradini registrati a 1 kHz con una telecamera CCD ad alta velocità. (B) superiore; il donatore (Clover-RLI, Clover-frame sottrazione) e accettore (mRuby2-RLI, mRuby2-frame sottrazione) immagini elaborate con due soglie diverse per ogni FP, in basso; la stessa soglia è stata utilizzata sia per il donatore (Clover-RLI, Clover-frame sottrazione) e accettore (mRuby2-RLI, mRuby2-frame sottrazione) immagini per illustrare la penombra relativo di mRuby2. Tutte le immagini sono state scattate dalla stessa cellula HEK 293 che esprimono Nabi2.242. (C) La 520 nm di lunghezza d&#39;onda trasso di potenziali d&#39;azione indotta da un topo neuroni dell&#39;ippocampo primaria che esprimono sensore Nabi2.244. La traccia Af / F è stato selezionato tra i pixel correlati alla soma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 6" src="/files/ftp_upload/53566/53566fig6.jpg" /> Figura 6. Conseguenze di diversi livelli di luce per Af e Af / F valori. (A) L&#39;immagine di uno HEK 293 cellule che esprimono un unico basato FP GEVI, Bongwoori, mostrati in Intensità Riposo Luce (RLI), (B) le tracce di fluorescenza che mostrano kernel media Af (F x -F 0) valori da tre diverse regioni, la regione 1: regione membrana con segnale di fluorescenza ben localizzato, regione 2: una regione con una brillante fluorescenza interna, unD regione 3: regione lontana dal segnale ottico, (C) le tracce di fluorescenza mostrano kernel in media valori Af / F dalle stesse regioni (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig6large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> concetti </td><td> equazioni </td><td> Osservazione </td></tr><tr><td> Frazionale cambiamento di fluorescenza (Af / F) </td><td><img alt="Equazione 2" src="/files/ftp_upload/53566/53566eq2.jpg" /></td><td> F 1 = intensità della luce misurata in un punto di tempo, F 0 = intensità della luce misurata a potenziale tenendo </td></tr><tr><td> funzione di Boltzmann </td><td><img alt="Equazione 3" src="/files/ftp_upload/53566/53566eq3.jpg" /></td><td> y = Af / F, V = potenziale di membrana in mV, V 1/2 è il potenziale di membrana in mV a metà-massimale Af / F, A <sub&gt; 1 = il valore minimo, A 2 = valore massimo, dx = pendenza </td></tr><tr><td> Doppia funzione decadimento esponenziale </td><td> y = y 0 + 1 A e - (t - t 0) / τ 1 + A 2 e - (t - t 0) / τ 2 </td><td> T si 1, τ 2 = costanti di tempo, a 1, a 2 = ampiezze, t, t 0 = due punti di tempo, compensati y 0 = </td></tr></tbody></table> Tabella 2. Equazioni

Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Imaging potenziale di membrana con due tipi di geneticamente codificati fluorescenti di sensori di tensione

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

imaging-membrane-potential-with-two-types-genetically-encoded

Universidad San Sebastian

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JoVE Journal

Neuroscience

10.6K Views.  Seoul National University. A method for imaging changes in membrane potential using genetically encoded voltage indicators is described. 

Video: Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Imaging ottico di neuroni nel ganglio Granchio Stomatogastric con tensione sensibili Coloranti

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Ricerca

Neuroscienze

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Determinazione della mitocondriale Oxygen Species potenziale di membrana e reattiva in neuroni corticali di ratto dal vivo

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Le risposte di imaging neuronale in preparazione Fetta di organo vomeronasale Esprimere un sensore di calcio geneticamente codificato

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Cella di misura singola di rilascio di dopamina con simultanea di tensione-clamp e Amperometria

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potenziale di membrana Dye Imaging di ventromediale dell&#39;ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging intracellulare Ca<sup&gt; 2+</sup&gt; Segnali di striatale astrociti da topi adulti utilizzando indicatori di calcio geneticamente codificate

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controllo ottico di una proteina neuronale Utilizzando un acido Innaturale Amino geneticamente codificata in neuroni

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Imaging potenziale di membrana con due tipi di geneticamente codificati fluorescenti di sensori di tensione

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