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).

<ol>
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Yazarlar ifşa hiçbir şey yok.

sinir sistemi çeşitli yollarla voltaj inhibisyon hafif hiperpolarizasyon, sinaptik girişi nispeten büyük bir gerilim değişikliği hafif bir depolarizasyonu ve hareket potansiyeli sonuçlar neden olur kullanır. GeViS tarafından membran potansiyeli değişiklikleri ölçmek yeteneği aynı anda nöronal devrelerin çeşitli bileşenleri analiz umut verici bir potansiyel sunmaktadır. Bu yazıda GeViS kullanarak membran potansiyelindeki görüntüleme değişiklikler için temel bir yöntem ortaya koymaktadır. Gerilim görüntüleme değişiklikler için önemli bir anahtar plazma membranında GeVi etkin ifadesidir. Hücre içi ifade prob SNR azaltan duyarlı olmayan floresan oluşturur. transfeksiyon koşulları optimize çok optik ölçümlerin tutarlılığını artırır. yeni bir GeVi test Gerçekte, optik aktivite farklılıklar KD tamamen bağlı olduğundan emin olmak için, aynı zamanda, bilinen bir sonda test edilmesi önerilirw prob ve hücrelerin değil koşullar. Şekil 6, gerilim görüntüleme duyarlılığını azaltmak iç floresan etkisini göstermektedir, mesafe kadar taşınmış / F değeri. Şekil 6B, mesafe kadar taşınmış tek AP göre GeVi, Bongwoori ifade eden HEK hücre değerlerini gösterir. İz 2 nispeten parlak iç floresan Şekil 6A görülen bölge 2 (mavi renk) geliyor. Bu iz yüzünden kadar taşınmış / F azalır parlak iç floresan 2 damla anlamlı Şekil 6B&#39;de izleri kadar taşınmış / F değerleri için RLI değerlerine göre ayrıldı Şekil 6C, iz de, ancak iz 1 olarak mesafe kadar taşınmış değeri benzer düzeyine sahip değerleri. Floresan iz 3 çok küçük bir mesafe kadar taşınmış vardır ama aynı zamanda çok küçük RLI değeri (F) sahiptir. Sonuç kayıt gürültü önemli bir artış vardır yanıltıcı bir mesafe kadar taşınmış / F sinyaldir. Ayrıca, Bu örnekte kadar taşınmış göstermek için dahil edilmiştirönemli. F ile başlamak çok düşükse kadar taşınmış / F büyük bir değişim yararlı değildir. Bir görüntü ayırıcı kullanılması tek bir CCD kamera üzerinde iki dalga boyunda eş zamanlı ölçüm sağlar. Bu büyük ölçüde prob gelişimi için FRET bağımlı floresan değişikliklerin ölçümü yardımcı olur ve kurulum maliyetini düşürür. Ancak, renk sapmaları nedeniyle, bu iki görüntü bir apochromatic amaç için ihtiyaç gerektiren odak dışında hafifçe olacak. bu yüzden en yüksek sayısal diyafram ile hedefleri seçerek daha fazla ışık daha iyi SNR, aynı zamanda floresan imajını geliştirir. Buna ek olarak, böyle bir ışık yayan diyotlar (LED) ya da SNR artırmak için lazer gibi kuvvetli ışık kaynakları kullanabilirsiniz. LED&#39;ler mekanik bir deklanşör gerektirmez. Bu yazıda bir tek FP GeVi ve FRET tabanlı GeVi optik sinyaller. FRET tabanlı GeVi ana avantajı rasyometrik görüntüleme ilişkili ses d azaltılmasını mümkün kılmasıdırin vivo solunum ve kan akışına ue. Floresan sinyallerin ters polarite membran potansiyeli gerçek bir değişim doğruluyor. İki kromomorfların floresan yoğunlukları genellikle önemli ölçüde değişebilir çünkü Ancak, SNR de değişecektir. Bu oranlı metrik analiz boşa ve yararlılığını 28 sınırlar. Bir FRET bağımsız sinyal 29 olabilir. Bu nedenle bazen daha iyi zar potansiyeli değişiklikleri analiz etmek için FRET kullanırken sadece parlak floresan kromofor kullanmaktır. Gerilim görüntüleme nedeniyle hız ve kalsiyum akı ölçen kalsiyum görüntüleme ile karşılaştırıldığında zar potansiyeli değişim değişkenlik kalsiyum görüntüleme daha zordur. Nöronlarda membran kalsiyum iyonu akı ile karşılaştırıldığında çok hızlı zaman ölçekleri potansiyel değişiklikler ve eşik altı olaylar kalsiyum görüntüleme 30 ile ölçülen olamaz. Bundan başka, voltaj prob plazma membranında olmalıdırMembran potansiyeli optik raporu değişikliklere uygun prob miktar olan etkili olduğu bulunmuştur. Sonuç olarak, aslında CCD çipi varabilir fotonların sayısı nispeten azdır. Bu yüksek kuantum verimliliği ve membran potansiyelindeki değişiklikler üzerine yüksek SNR ortaya çıkarır bir prob ile yüksek hız ve düşük okuma-gürültü kamera ihtiyacını gerektirir. In vitro hücre görüntüleme, araştırmacılar daha iyi in vivo ölçümlerinde çalışmadan önce GeViS potansiyel yararları ve tuzaklar anlayacaksınız.

Bu çalışmanın temel odak noktası genetik olarak kodlanmış floresan proteinleri kullanılarak in vitro membran potansiyelleri değişikliklerin optik görüntüleme göstermektir. membran potansiyeli Görüntüleme değişiklikleri nöronal devrelerin aktivitesini okuyan heyecan verici imkanı sunar. Bir floresan yoğunluğu değişikliği membran potansiyeli sonuç değişiklikler, kameranın her bir piksel nöronal aktivitenin nonintrusive ölçümlerini sağlayan bir vekil elektrot olduğunda. Kırk yıl boyunca, organik voltaja duyarlı boyalar 1-4 zardaki potansiyel değişimlerinin gözlemlemek için yararlı olmuştur. Bununla birlikte, bu boyalar, selüler özgüllük yoksundur. Buna ek olarak, bazı hücre türleri leke zordur. Genetik olarak kodlanmış gerilim göstergeleri (GeViS) hücreleri, özellikle çalışılan floresan voltaja duyarlı prob ifade edilecek olan bu sınırlamaları aşmak. GeViS üç sınıf vardır. GeVi birinci sınıf vo kullanırTek bir floresan proteini (FP) 5-9 veya Förster rezonans enerji transferi (FRET) çifti 10-12 biriyle voltaj algılama fosfataz alan adını ltage algılama. Sensörlerin ikinci sınıf bir floresan göstergesi doğrudan 13-15 veya elektrokromik FRET 16,17 aracılığıyla mikrobiyal rodopsin kullanır. Üçüncü sınıf iki bileşeni, bir zara bağlı AP ve zara bağlı söndürme boyasının 18-20 olan bir ikinci bileşen olan genetik bileşen kullanır. İkinci ve üçüncü sınıf nitro ve kesiti deneyleri 19,20 olarak yararlı olsa da, sensör, sadece birinci sınıf an in vivo 6 analizleri için de yararlıdır. Bu yazıda in vitro GeViS ilk sınıfını (Şekil 1) kullanılarak membran potansiyelinin görüntüleme gösterecektir. Gerilim sensörlerinin Bu birinci sınıf in vivo görüntüleme geçiş için en kolay yoldur. GeViS u yanayaklaşık sensörlerin rodopsin sınıfından daha parlak 50 kat bir FP kaynaşmış bir gerilim algılayan bölge olan tilizing, onlar ark lambası aydınlatma kullanılarak yerine son derece güçlü bir lazer gerektiren görüntülü olabilir. parlaklık eşitsizlik diğer sonucu GeViS birinci sınıf kolayca beynin otomatik floresan aşabilir olmasıdır. rodopsin tabanlı sondalar olamaz. Sensörün üçüncü sınıf birinci sınıf kadar parlak fakat in vivo olarak uygulanması zor bir kimyasal söndürücü eklenmesini gerektirir. Bu nedenle biz, tek bir FP (Bongwoori) 8 bir FRET çiftinin oluşan bir prob (Nabi 2) 12, bir probun satın gösterecektir. Nabi 2.242 ve Nabi 2,244 adlandırılan yeşil fluoresan verici, yonca oluşan 11 ve kırmızı flüoresan alıcıya, mRuby2, - VSFP-CR (yonca mRuby2 voltaja duyarlı flüoresan protein) kelebek versiyonları FRET bu raporda yapılarıdır <s&gt; 12 kadar. Kayıtların bu tür tanıtım araştırmacılara sağlayabilir bilgi GeViS türü daha iyi fikir verecektir. <img alt="figür 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> Şekil 1. Bu Rapor (A) görüntülenmistir Genetik kodlanmış Gerilim Göstergeleri (GeViS) bir trans-membran voltaj algılama alanını ve bir floresan proteini olan GeVi dayalı bir mono FP İki Türleri. (B) trans-membran voltaj algılama etki, bir FRET verici ve alıcı oluşan bir FRET tabanlı GeVi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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.

Geçici olarak transfekte hücrelerde anlamlı floresan değişimi ve plazma membranı ifade derecesi sergileyebilirler. Hatta aynı lamel bazı hücreler iç floresan çeşitli düzeylerde olacaktır. Bu durum, hücre tarafından emilen transfeksiyon ajanı miktarı muhtemeldir. Bazen çok fazla ifade apoptosis 27 sonuçlanan gelişeceğini protein yanıt yaşamaya hücreyi neden (parlak, yüksek iç floresan ile, hücreler yuvarlak). deneyci, bu nedenle iç ifade gürültü oranı (SNR) sinyali düşürür duyarlı olmayan bir floresan oluşturur beri mümkün olduğunca az iç floresan ile parlak hücreleri ve loş hücreler hem test etmek için uyarılır. Diğer bir faktör Şekil 3. Kromomorfların olgunlaşma oranı alıcı chromoph bir floresan uyumsuzluğunu gösterirDonör kromofor (Clover) daha uzun bir olgunlaşma süresi vardır cevheri (mRuby2). <img alt="Şekil 3," src="/files/ftp_upload/53566/53566fig3.jpg" /> HEK 293 hücreleri Şekil 3. Konfokal Çıkan Geçici FRET Tabanlı GeVi ile transfekte Nabi2.242. (A) (B), potansiyel bir sonucu gösteren Konfokal görüntü FRET verici ve alıcı zar lokalize floresan gösteren bir HEK hücre Konfokal görüntüler mRuby2 yavaş olgunlaştırma. Sadece iki sergi mRuby2 floresan. Iken Dört hücreleri Clover floresan sergileyen <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig3large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> görüntüleri konfokal mikroskop tarafından satın alındı. FRET donör için, Clover, 488 nm lazer uyarma kullanıldı ve emisyon Kaptan edildiedili- ile 525/50 nm bant geçiş filtresi. FRET kabul edici, mRuby2, 561 nm uyarım için lazer ve 595/50 nm bant-geçiş filtresi ile aydınlatılmıştır emisyonu için kullanıldı. Konfokal görüntüleme için örnekler, pH 7.4 ayarlanır, fosfat tamponlu tuzlu su içinde% 4 paraformaldehid / sukroz çözeltisi ile işlem görmüş ve daha sonra, bir anti-solma tepkin maddesi ile monte edilmiştir. transfeksiyon koşulları optimize sonra, varyasyon sonraki kaynak analiz edilecek faiz belirleme bölgesi geliyor. En yüksek floresans değişikliği ile hücrenin bölgelerinin belirlenmesi için kare çıkarma kullanılması genellikle kadar taşınmış / F değeri maksimize etmek için kullanılır. Alternatif hücresinden ışığı alan tüm pikselleri seçmektir. Bu gürültünün azaltılması ile sonuçlanan piksel sayısını artıran, ancak yanıt olmayan iç floresan dahil beri sinyal boyutunu azaltır. Her iki yöntem de sürece deneyci tutarlı kalır gayet iyi. <pfo class = &quot;jove_content&quot;: keep-together.within-page = &quot;1&quot;&gt; Şekil 4A tek FP tabanlı GeVi, Bongwoori ifade eden bir HEK hücre floresan değişimi gösterir. Bu basamaklı voltaj darbeleri yanıt olarak, tipik bir floresan değişikliktir. Bu verilerden yola çıkarak bir GeVi gerilim hassasiyeti arsa ve üzerinde ve tek veya çift üstel çürüme işlevi takarak farklı gerilimlerde τ sabitleri kapalı belirler. Küçük gerilim adımları SNR geliştirmek ve kayıt sırasında herhangi bir potansiyel ağartma tespit etmek HEK hücreleri görüntüleme sırasında 16 denemeler genellikle ortalaması alınır. 100 mV güçlü bir sinyal vermek, bu sondalar ayrışmış hipokampal nöronlar (Şekil 4C) test edilebilir. hipokampal nöron floresan iz, tek bir çalışmadır. <img alt="Şekil 4," src="/files/ftp_upload/53566/53566fig4.jpg" /> Tek FP Tabanlı GeVi Şekil 4. Gerilim Görüntüleme ifadeHEK 293 Hücreler ve Hipokampal İlköğretim Nöronlar. yüksek hızlı CCD kamera ile 1 kHz kaydedildi basamaklı voltaj darbeleri tepkileri gösteren tek bir FP tabanlı GeVi, Bongwoori (A) kadar taşınmış / F iz, içinde. (B) yüksek hızda CCD kamera ile görüntülenmiş bir HEK 293 hücre; Bongwoori ve floresans değişikliği gözlenmiştir piksel gösteren (sağ) çerçeve çıkarma görseli eksprese eden bir hücrenin (sol) istirahat ışık yoğunluğu (RLI). (C) Bongwoori ifade eden fare hipokampal birincil nöronlardan uyarılan aksiyon potansiyellerinin Optik kaydı. aksiyon potansiyelleri tüm hücre akım kelepçe modunda altında uyarılmış bulundu. Kadar taşınmış / F iz soma korelasyon piksel seçildi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Bir temsilci fluoverici ve alıcı kromoforlarının rescence saati Şekil 5A&#39;da gösterilmiştir. Oranlı metrik analiz sağlayarak donör ve alıcı ilişkili gürültü kaynaklarını, örneğin., Hareketi azaltmak için floresan değişimi polaritesi tersidir. Örneğin, hareket korelasyon gürültü hem donör ve akseptör floresan aynı yönde floresan bir değişiklik gösterecektir. FRET prob göre rasyometrik görüntüleme kapasitesine sahip olmakla birlikte, sadece daha parlak kromoforu analiz genellikle daha iyidir. Dimer kromofor esas kayıt gürültü artırabilir olmasıdır. Şekil 5B, bu etkiyi göstermektedir. Optik sinyal hücrede olduğu parlak Clover gelen Şekil 5B çerçeve çıkarma açıkça göstermektedir. Bunun aksine, mRuby2 floresans çerçevesi çıkartma hücre boyunca gürültü yüksek derecelerde göstermektedir. Bu nedenle, Clover sadece donör sinyali Fi bir hipokampal nöron gösterilmiştirşekil 5C. <img alt="Şekil 5," src="/files/ftp_upload/53566/53566fig5.jpg" /> GeVi dayalı bir FRET ile Şekil 5. Gerilim görüntüleme HEK 293 hücre ve hipokampal birincil nöronlar olarak ifade edilmiştir. (A) yüksek hızlı CCD kamera ile 1 kHz kaydedildi basamaklı voltaj darbeleri iki dalga boylarında yanıtları gösteren bir FRET tabanlı GeVi, Nabi2.242, bir mesafe kadar taşınmış / F iz. (B) Üst; Donör (Clover-RLI, Yonca-kare çıkarma) ve alıcı (mRuby2-RLI, mRuby2 çerçeve çıkarma) her FP, Bottom için iki farklı eşikleri ile işlenmiş görüntüler; aynı eşik mRuby2 göreli loşluğu göstermek için, her iki vericiden (yonca RLI, yonca çerçeve çıkarma) ve alıcı (mRuby2-RLI, mRuby2 çerçeve çıkarma) görüntüler için kullanılmıştır. Tüm görüntüler Nabi2.242 ifade aynı HEK 293 hücre alınmıştır. (C) 520 nm dalga boyu trNabi2.244 sensörü ifade eden bir fare hipokampal birincil nöron kaynaklı aksiyon potansiyelleri ası. Kadar taşınmış / F iz soma korelasyon piksel seçildi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig5large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 6," src="/files/ftp_upload/53566/53566fig6.jpg" /> Mesafe kadar taşınmış ve mesafe kadar taşınmış / F değerleri için Değişen Işık Seviyeleri Şekil 6. Sonuçları. (A) tek FP tabanlı GeVi, Bongwoori ifade eden bir HEK 293 hücre bir görüntü, Dinlenme Işık Şiddeti gösterilen (RLI), (B) floresan izleri gösteren çekirdek ortalama Parlak iç floresan ile bir bölge, bir: iyi lokalize floresan sinyali, bölge 2 membran bölgesi: mesafe kadar taşınmış üç farklı bölgelerde, bölge 1 değerleri (F -F 0 x)d bölgesi 3:. optik sinyalin uzak bölge, (C) çekirdek gösteren floresan izleri (B) Aynı bölgelerinden kadar taşınmış / F değerleri ortalama <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53566/53566fig6large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> kavramlar </td><td> denklemler </td><td> dikkat </td></tr><tr><td> Fraksiyonel floresan değişimi (mesafe kadar taşınmış / F) </td><td><img alt="denklem 2" src="/files/ftp_upload/53566/53566eq2.jpg" /></td><td> Bir zaman noktasında ölçülen F 1 = ışık yoğunluğu, K = 0 ışık şiddeti tutma potansiyeli ölçülür </td></tr><tr><td> Boltzmann fonksiyonu </td><td><img alt="denklem 3" src="/files/ftp_upload/53566/53566eq3.jpg" /></td><td> y = mesafe kadar taşınmış / F, mV V = zar potansiyeli, V 1/2 yarı-maksimum mesafe kadar taşınmış / F, A mV membran potansiyeli <sub&gt; 1 = minimum değer, A 2 = maksimum değer, dx = eğim </td></tr><tr><td> Çift üstel çürüme işlevi </td><td> Y = y, 0 + A 1 E - (T - t = 0) / τ 1 + A 2E - (T - t = 0) / τ 2 </td><td> τ 1, τ 2 = zaman sabitleri, A 1, A 2 = genlikleri, T, t = 0, iki zaman noktası, y, 0 = ofset </td></tr></tbody></table> Tablo 2. Denklemler

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

Genetik Kodlanmış Floresan Gerilim Sensörleri İki Türleri ile Potansiyel görüntüleme Membran

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.6K Görüntüleme.  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.

Gerilim duyarlı Boyalar ile Yengeç Stomatogastric Ganglion Nöronlar Optik Görüntüleme

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

Nörobilim

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.

Mitokondrial Membran Potansiyeli ve Reaktif Oksijen Türleri Canlı Rat Kortikal nöronlar Belirlenmesi

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.

FM Boyalar Kültür Serebellar Granül Nöronlar Synaptic veziküllü Havuzu Yenileme Kantitatif Analiz

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.

Görüntüleme Nöronal cevaplar Vomeronasal Organ Dilim hazırlıklar Genetiği Kodlanmış Kalsiyum Sensör ifade

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.

Eşzamanlı Gerilim-kelepçe ve Amperometri ile Dopamin Film Tek Hücre Ölçümü

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.

Membran Yetişkin farelerin ventromedial Hipotalamus Nöronlar Potansiyel Boya Görüntüleme Glikoz Algılama Çalışması için

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.

Görüntüleme hücre içi Ca<sup&gt; 2+</sup&gt; Genetik olarak kodlanmış Kalsiyum Göstergelerini Kullanma Yetişkin fareler ile Striatal Astrositler Sinyalleri

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.

Fare Omurilik Hücresel Dynamics İki foton Görüntüleme

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.

Nöronlar bir Genetiği kodlanmış doğal olmayan Amino Asit kullanarak Nöronal Protein Optik Kontrol

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.

Genetik Kodlanmış Floresan Gerilim Sensörleri İki Türleri ile Potansiyel görüntüleme Membran

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