Işin çoğunluğu ve ilgili personel NIH hibe NS060677 ve kısmen NIH MH099559 ve (BSK kadar) MH104069 verir tarafından desteklenmiştir. Eserin de CHDI Vakfı tarafından desteklenmiştir.

Tüm hayvan protokoller Laboratuvar Hayvanları Bakım ve Kullanım Sağlık Rehberi, ABD Ulusal Sağlık Enstitüleri göre ve UCLA Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1.1) Mikropipet, AAV2 / 5 Virüs Yükleme hazırlayın <ol><li> Virüsün enjeksiyon için iyi borosilikat cam mikropipetler kullanın. Dikey bir çekici ile iki aşamalı bir çekme programı kullanılarak mikropipet çekin. Konik bir pipet öğütücü kullanılarak 40 ° &#39;lik bir açı ile pipet. 40 um ve 6 bir şaft uzunluğu - - 7 mm kullanım için ideal olan pipet 20 bir uç çapına sahip olacaktır. Pipet ve cerrahi aletler otoklav. Tatbikatta,% 70 etanol ile silinerek sterilize edilir. </li><li> 10 ul alikolar içinde -80 ° C virüsü AAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40 (1.5 x 10 ila 13 GC / ml) depolayın. Cerrahi öncesinde, dondurucu üzerinden hacimde çıkar ve th doldurmak için kullanılan kadar buz üzerinde saklayıne mikropipet. </li><li> Virüs, bir şırınga pompası kullanılarak mikropipet doldurun. Sıkıca takviye edilmiş bir pistonu olan bir cam şırıngaya, uygun uygun büyüklükte bir hortum sıkıştırma yoluyla hortumun bir parçasının bir ucunu. Montaj için uygun boyutta bir çıkarılabilir bir iğne sıkıştırma yoluyla boru parçasının diğer ucuna otoklavlanmış mikropipet takın. </li><li> Viral vektör yüklemeden önce, bir iğne ile 1 ml şırınga içinde Sudan Kırmızı IV (~ 1 mg / 50 mi) ile boyanmış, mineral yağ ile sistemin (27 g doldurarak boru, şırınga ve mikropipet de dahil olmak üzere pompa sisteminin hava kabarcıklarını çıkarmak ). </li><li> Cam mikropipet altında parafilm temiz bir parça yerleştirin ve 5 dağıtmak - parafilm üzerine viral vektörün 10 ul. </li><li> Geri 0.5 ml / dk&#39;da, enjektörün pistonu hareket ettirerek viral vektör Suck ve cam pipet ile ilgili vektör ve yağ arasındaki sınırı. </li></ol> 1.2) Anestezi, Saç Kesme, HKraniotomi için ead Tespit ve Hazırlık <ol><li> N2 O ve O 2, ve% 5 izofluran ile dolu odacık içine bir fare (P49-63, C57BL / 6) koyun. Maksatlı hareket kaybı ve yavaşlatılmış solunum hızı (- 90 / dk ~ 60) tarafından anestezi derinliğini değerlendirin. Fare anestezi sırasında kafasına saçlarını kesti. </li><li> Başını bir anestezi ve havalandırma sisteminin içine yerleştirilen künt kulak çubukları ve burun tarafından güvenli ile, stereotaktik çerçeve içinde fareyi takın. % 3 - 2 sürekli izofluran koruyun. Fareler anestezi altında göz kırpma refleksi kaybetmek beri her iki göze yapay gözyaşı merhem sürün. </li><li> Ağrı tedavisi için bir deri altından enjeksiyon ile Buprenorfin (0.1 mg / ml), 0.05 ml uygulayın. </li><li> % 10 povidon iyot ve% 70 alkol ile üç kez temiz bir cerrahi alan, iki çözümleri arasında değişen ve povidon iyot ile başlayan. Önden başlar ve herhangi bir önceden kesilmiş tüyleri çıkarmak için arkaya doğru silin. </li><li> Üst o bir cilt kesi yapmakgözler arasında başlar ve kulakları arasındaki biter kafa f. </li></ol> 1.3) Kraniyotomi ve Microinjections <ol><li> Bir pamuklu çubukla kaydırarak periost çıkarın ve sonra diş pamuk topları ile kafatası yüzeyini kurulayın. Enjeksiyon yeri çevresinde bir işareti yapmak, ve bir yüksek devirli tarafından desteklenmektedir küçük bir çelik çapak kullanarak işaretinin kenarına etrafında matkap. </li><li> Kemik çıkarın ve tuzlu su ile yüzeyi temizleyin. </li><li> Ön-arka 0,8, medial-lateral: şu koordinatlara (mm) striatumdaki yanal sol dorsal içine pipet yerleştirin 2,0, dorsal-ventral pial yüzeyinden: -2,4 13. Bu koordinatlar mikroenjeksiyon iğne ucu sadece biraz bu çekirdeğin içinde nüfuz anlamı sadece dorsolateral striatum (Şekil 1A, B) Yukarıda oturur anlamına unutmayın. Zararlı kan damarları kaçının. Pipet bir kan damarına doğru olması için olursa, hafifçe ayarlayın0.2 mm - 0.1 ile koordine eder. </li><li> 0.2 ml / dk&#39;da ayarlanmış enjeksiyon oranı ile enjeksiyon başlayın ve tipik olarak virüs 1-1,5 ul enjekte edilir. </li><li> 10 dakika boyunca, enjeksiyondan sonra yerinde pipet bırakın ve daha sonra yavaş yavaş pipet çıkarın. </li><li> Sürekli dikiş dış naylon sütür ile cerrahi yara kapatarak bitirin. </li></ol> 1.4) Kurtarma <ol><li> 24 saat için bir ısıtma pedi üzerine temiz bir kafes içinde fare koyun. Tamamen iyileşene kadar diğer hayvanların şirkete ameliyatı geçirmiş fare iade etmeyin. Bu sternal yatma korumak için yeterli bilince kavuşana kadar fare gözetimsiz bırakmayın. </li><li> Trimetoprim Sulfamethoxiazole ekleme, bir hafta boyunca su içinde (40 mg trimetoprim 200 mg sulfamethoxiazole ihtiva eden 500 ml su başına 5 mi). Ameliyat sonrası Buprenorfin kadar 3 gün boyunca günde iki kez uygulayın. </li><li> , A beslenen bir 12 saat ışık-karanlık döngüsü üzerinde fareyi korumak ve günlük sulanannd ameliyattan sonra 3 gün sonra günde başına düşen ağırlıklı. Bu 3 gün içinde,% 10&#39;dan daha fazla bir vücut ağırlığı kaybı ile herhangi bir fare tehlikeye, ve bunun yerine bir deneye tabi kalma ötenazi olacaktır olarak kabul edilir. </li><li> Ayrı, haftada iki kez fare kafes değiştirmek ve en az günde bir kez genel sağlık izlemek. Stereotaksik mikroenjeksiyon sonrası 3 hafta - Tipik olarak, fare 2 içinde kullanılır. </li></ol> Konfokal Ca 2+ Görüntüleme 2. Akut Beyin Dilim Hazırlık Kesme ve Kayıt Çözümleri 2.1) hazırlanması. <ol><li> (MM) ihtiva eden çözelti, kesme hazırlayın: 194 sukroz, 30 NaCl, 4.5 KCl, 10 D-glikoz, 1 MgCl2, 1.2 NaH 2 PO 4 ve% 95 O2 ve% 5 CO2 ile doymuş 26 NaHCO 3. </li><li> Içeren kayıt solüsyonu (mM olarak) hazırlayın: 124 NaCl, 4.5 KCl, 1 MgCl2, 10 D-glikoz, 2 CaCl2, 1.2 NaH 2 PO 4 </sub&gt; ve 26 NaHCO 3; pH 7,3-7,4, 290 -% 95 O2 ve% 5 CO2 ile doymuş 295 mOsm. Kayıt çözümü ile beyin dilim tutarak beher doldurun ve 34 ° C&#39;de tutmak. </li></ol> 2.2) Dilimleme <ol><li> İki üç hafta stereotaksik mikroenjeksiyon sonra, derin ve ölümcül fare uyutmak, ve sonra başını kesmek. </li><li> Beyin ayıklayın ve uninjected sağ yarımkürede kaldırmak için bir bıçak kullanın ve süper yapıştırıcı kullanarak Vibratome tepsiye bıraktı birini monte edin. Buz gibi soğuk kesim tamponu ile Vibratome tepsiyi doldurun, ve% 95 O 2 ve% 5 CO 2 ile doyurarak tutun. </li><li> 300 mikron kalınlığında koronal veya sagittal striyatal beyin dilimleri kesin. Genellikle, 4-5 koronal veya 6-7 sagital striyatal dilim alınabilir. </li><li> 34 ° C&#39;de ısıtıldı dilim tutarak behere dilimleri transferi ve daha sonra recordi oda sıcaklığında depolamadan önce, 30 dakika boyunca orada tutmakng. </li><li> [Ca2 +] görüntüleme önce en az 30 dakika oda sıcaklığında oksijenli bir kayıt tamponu içinde beyin dilimleri inkübe edin. </li></ol> 3. İmmünohistokimya (İHK) <ol><li> İki hafta virüsünün, enjeksiyondan sonra, transkardiyal PBS ile fare PBS içinde% 10 formalin ve ardından serpmek. </li><li> Kaldır gecede sonrası düzeltmek ve en az 2 gün tamponlu% 30 sakaroz beyin cryoprotect. </li><li> Bir kriyostat mikrotom kullanılarak 40 um bölümler hazırlayın. </li><li> 10 dakika aralıkla kesitler, PBS ile 3 kez yıkayın. </li><li> % 10 normal keçi serumu ile PBS içinde 1 saat süre ile ve% 0.5 Triton X-100 ile bölümleri inkübe edin. </li><li> % 0.5 Triton X-100 içeren PBS içinde hazırlanan primer antikor içinde 4 ° C&#39;de bir gece boyunca bölümleri inkübe edin. Bu kapsamda kullanılan primer antikorlar: tavuk anti-GFP, 1: 1000 fare anti-S100β 1: 400, fare anti-neun 1: 600, fare anti-glutamin sentetaz 1: 300 arasındadır. </li><li> 10 m bölümlerinin PBS ile 3 kez durulayınaralıklarla. </li><li> Oda sıcaklığında 2 saat boyunca,% 10 normal keçi serumu ile PBS içinde hazırlanan ikincil antikorlarla bölümleri inkübe edin. Aşağıdaki gibi, ikinci antikorlar: keçi anti-fare Alexa546 1: 500, keçi anti-tavuk Alexa488 1: 500. </li><li> Daha sonra yavaş yavaş, bir cam lamel bölümleri kapsayacak, kuru bir cam slaytlar üzerinde bölümleri monte ve antifade montaj orta birkaç damla uygulanır. 4 ° C&#39;de slaytlar saklayın. </li><li> 1.3 sayısal diyafram ile 40X yağ daldırma lens ile bir konfokal mikroskop görüntüleri almak. </li></ol> 4. Konfokal [Ca 2 +] i Görüntüleme NOT: [Ca 2+] i 0.8 sayısal diyafram ile 40X su daldırma objektif lens ile bir konfokal mikroskop kullanılarak yapıldı görüntülenmesi. <ol><li> 2 ml başına - sa 1 ila 5 kayıt bölümü içinde, bir yer dilim dilimleme ve 1 akış hızında oda sıcaklığında oksijenli bir kayıt tamponu superfuse sonraMin. Sonra deney sırasında hareketini en aza indirmek için dilimin üstüne naylon dizeleri ile bir platin arp yerleştirin. </li><li> Görselleştirmek ve 10X suya daldırma hedefi altında parlak bir alan luminesansın ile dorsal striatum bulun. </li><li> 40X suya daldırma objektif lens geçmek ve Argon lazer 488 nm hattı açın. Odak düzlemi değiştirerek, soma, şube ve ağızları bazal floresan görüntüleme, yaklaşık 20 mikron dilim yüzeyinin altında bulunan hücreleri bulmak. Notun tehlikeye hücreler genellikle kaçınılmalıdır ortalamanın üzerinde floresan seviyesi, gösterir. </li><li> Taramayı başlatmak için &#39;çerçeve tarama&#39; modunu kullanın. Genellikle, 0.5 ayarlanmış lazer yoğunluk - maksimum çıkış% 5 [Ca2 +] sito-GCaMP3 ile görüntü yeterlidir. Astositlerin bütün topraklarında [Ca 2 +] i dinamiklerini izlemek için, çerçeve ya da daha hızlı başına 1 sn tarama hızı ile &#39;Çerçeve Tarama&#39; recomme olduğunuNDED, ancak bu durum, sözkonusu deneye bağlı olarak karar verilmesi gereklidir. </li><li> Görüş tüm alanında 2 astrositler - 1 izlemek için 3 kat - Gerekirse, görüntü için [Ca 2 +] i süreçlerinde, 2 dijital büyütme kullanmak. </li><li> Kayıt sırasında doymuş piksel önlemek için, hücreleri PseudoColor için &#39;Hilo&#39; arama modunu kullanın. Her zaman kırmızı renk doygunluğu göstergesi olarak görünür olmadığını test etmek için, yaklaşık 5 dakika boyunca bir &quot;pilot kayıt&quot; ile başlar. Eğer öyleyse, lazer çıkış gücünü düşürecek, ya da PMT alt / Kazanç / ofset değerleri ve piksel değerleri olmayan doymuş aralıkta test etmek için bir başka pilot kayıt yapmak. - 5% (10 mW), PMT 550-650 Volt, Kazanç 2.0 - lazer gücü 0,5 3.5x ve ofset 0: Tipik haliyle, bir araya getirilen parametrelerin görüntüleme astrosit için kullanılan [Ca2 +] striatumda I aşağıdaki gibidir -% 5. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Burada tarif edilen yöntemler, US yerinde daha sonra [Ca2 +] görüntüleme için in vivo striatal astrositlerde sito-GCaMP3 ifade etmek için izin vermektedir. Bu yöntem transgenik veya sağlam hedeflenen protein, hızlılık ifade ve deneysel uygulama ve anatomik özgüllük esnekliği dahil knock-fareler kullanılarak üzerinde avantajlara sahiptir. AAV2 / 5 ile GCaMP3 ekspresyonu spesifik ve güçlü olduğu bulundu. Serotip 5 AAV ile GFAP GfaABC 1 D ilerleticinin...

Astrositler beynin her yerde ve bol miktarda glial hücreler bulunmaktadır. İyi astrocytes hayati destek ve hücre dışı boşlukta K + konsantrasyon tamponlama, alımı nörotransmitterlerin sağlamanın yanı sıra besinlerin de dahil olmak üzere homeostatik rolleri hizmet ettiğini kurulmuştur. Ancak son çalışmalar onlar da kendiliğinden ortaya çıkar ve nöronal aktivitenin 1 artmıştır [Ca 2+] i sinyallerini görüntülemeye olduğunu göstermektedir. Astrosit varlığı [Ca 2 +] i sinyal giderek nöronlar ile iletişimi tetiklemek için düşünülmüş, ve gibi astrositlerden içinde &quot;Ca 2+ uyarılabilirliğinin&quot; bir formu olarak yorumlanmıştır. Son iki yılda mevcut veriler astrocytes ve nöronlar belki iki yönlü bir tarzda, iletişim kurabileceğini iki ayarları öneririz. İlk olarak, astrositlerin sıklıkla nörotransmiterler ve aktive [Ca2 +] artışa yanıtnöronlar 2 serbest nöromodülatörler. İkinci olarak, [Ca2 +] astrositler içine dahil artışlar da nöronlar ve kan damarlarını etkileyen astrositlerden sinyal molekülleri salınımına neden olur. Kanıt astrositlerden salınan moleküllerin astrosit-to-nöronun sinyalizasyonu ile sinaps, devreler ve sonuçta davranış 3-5 işlevlerinde değişikliklere yol açtığını göstermektedir. Ancak, bu hızla gelişmekte olan bir araştırma alanı kalır ve astrosit daha iyi ve ayrıntılı bir anlayış [Ca 2 +] i mevcut belirsizliklerin 6 bazı çözmek için gerekli olduğu ileri sürülmüştür. Geçmiş çalışmalarında, astrositlerde içine organik Ca 2+ gösterge boyaların toplu yükleme güvenilir algılamak için başarısız olduğu gösterilmiştir [Ca 2+] kültür ve yerinde 7-10 tüm astrositlerde içinde i sinyaller. Bu bulgular bize ve diğerleri 6,11,12 tarafından tartışılmıştır. Emerging resim [Ca 2 +] i nöronlar ve kan damarları ile etkileşimleri için birincil siteleri astrocyte süreçler (örneğin, şube ve ağızları), içinde sinyaller, nadiren ayrıntılı olarak incelenmiştir olmasıdır. Son zamanlarda, örneğin sitosolik GCaMP3, GCaMP5G ve GCaMP6 ve plazma zar olarak bulunan genetik olarak kodlanmış kalsiyum göstergeleri (Geçiş) &#39;nin kullanımı, bağlı versiyonlar (örneğin Lck-GCaMP3) astrositlerin bu küçük bölümlerinde [Ca2 +] sinyalleri çalışma izin vermiştir gibi ince süreçler, plazma zarı yakın ve tüm toprakları 7,8 içinde. Bununla birlikte, Geçiş organik Ca 2 + göstergesi boyalar üzerinde bir dezavantajı vardır ve Geçiş uygun olarak ifade etmek için genetik yöntemler haftalık dönemler için in vivo olarak astrositleri seçici kodlayan genlerin sunmak için bu gerekliliktir. In vivo ifadesi tipik knock-fareler veya virüs bazlı teslimat uygulaması ile, transgenik fareler kullanılarak elde edilirHamam böcekleri. Mevcut jove makalede, adeno-bağlantılı virüsler kullanarak çizgili astrositlere Geçiş sunmak için kullanılan yöntem ve prosedürler rapor etmektedir. Biz örnek olarak cyto-GCaMP3 odaklanmak, ancak aynı temel prosedürü diğer Geci veya floresan protein bazlı muhabiri herhangi için çalışır.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries</td>
			<td >World Precision Instruments</td>
			<td >1B100-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Narishige</td>
			<td >PC-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette grinder</td>
			<td >Narishige</td>
			<td >EG-40</td>
			<td></td>
		</tr>
		<tr >
			<td >pZac2.1 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I mL syringe</td>
			<td >BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe needle</td>
			<td >BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr >
			<td >AAV2/5 virus</td>
			<td >UPenn vector core</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan red IV</td>
			<td >Sigma-Aldrich</td>
			<td >67386</td>
			<td></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >CVS Pharmacy</td>
			<td>152355</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Leica</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
			<td >900LS</td>
			<td></td>
		</tr>
		<tr >
			<td >High Speed Rotary Micromotor Kit</td>
			<td >FOREDOM</td>
			<td >K.1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Santa cruz biotechnology</td>
			<td >sc-281692</td>
			<td></td>
		</tr>
		<tr >
			<td >Super Glue</td>
			<td>Krazy&#174;Glue</td>
			<td>KG925</td>
			<td></td>
		</tr>
		<tr >
			<td >Microslicer</td>
			<td >Ted Pella</td>
			<td >DTK-Zero 1</td>
			<td></td>
		</tr>
		<tr >
			<td >Confocal microscopes</td>
			<td >Olympus</td>
			<td >FV300 and FV1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal goat serum</td>
			<td >Vector</td>
			<td >S-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >chicken anti-GFP</td>
			<td >Abcam</td>
			<td >ab13970</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-s100&#946;</td>
			<td >Sigma-Aldrich</td>
			<td >S2532</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-NeuN</td>
			<td >Millipore</td>
			<td >MAB377</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-glutamine synthetase</td>
			<td >Millipore</td>
			<td >MAB302</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-mouse-Alexa546</td>
			<td >Invitrogen</td>
			<td >A11003</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken-Alexa488</td>
			<td >Invitrogen</td>
			<td >A11039</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Glass</td>
			<td >Fisher Scientific</td>
			<td >12-548-5J</td>
			<td></td>
		</tr>
		<tr >
			<td >Mounting Medium</td>
			<td >Vector</td>
			<td >H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Striatumda sito-GCaMP3 arasında astrosit spesifik ekspresyonu sağlamak için, daha önce, sağlam GCaMP3 ve raportör geni tahrik etmek için gösterilmiştir, adeno-bağlantılı virüs (AAV) 5 serotip ve GFAP GfaABC 1 D yükseltici (Şekil 1A), kullanılan hipokampal ve kortikal astrositlerde 8,14 anlatım. İki hafta fare striatumu içine, virüs mikroenjeksiyon sonra fare (~ 10 haftalık), perfüze edilmiş ve IHC striatum (Şekil 1B) sito-GCaMP3 ekspresyonun...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

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 ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

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

14.7K Görüntüleme.  University of California Los Angeles. 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. 

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

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 ...

Nörobilim

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Belirlenen Nöronlar ve Beslenme Davranışı Kalsiyum sinyaller aynı anda kayıt Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelleme Astrositom Patogenez<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Kortikal Astrositler veya Şartlı, Genetiği fareler ile Nöral Kök Hücreler kullanma

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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.

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

Genetik Kodlanmış Floresan Gerilim Sensörleri İki Türleri ile Potansiyel görüntüleme Membran

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

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 ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Yerel Ca2 + sinyalleri membran hedefli Ca2 + göstergeleri tarafından kültürlü hücrelerdeki diseksiyon

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Normal ve Huntington Farelerden Akut Beyin Dilimlerinde Glutamat Salınımı ve Alımının Tek Sinaps Göstergeleri

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrositlerde Organeller ve Diğer Kargoların Hücre İçi Taşınmasının Görselleştirilmesi ve Analizi

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Yetişkin ve Yaşlanan Farelerden Hipokampal Dilimlerde Hipoksinin En Aza Indirilmesi

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.