Bu çalışma, Japonya Uehara Memorial Foundation (ES) yanı sıra Whitehall Vakfı, Ulusal Nörolojik Bozukluklar ve İnme ve Stein-Oppenheimer Endowment Ödülü (BSK) Enstitüsü tarafından desteklendi.

DENEYSEL PROSEDÜRLER Deneysel işlemin bir adım daha akıllıca bir şekilde aşağıda açıklanan iki ana bölümden oluşmaktadır. Bölüm 1: HAZIRLAMA hipokampal astrosit KÜLTÜRLER Kısaca, karışık, hipokampal astrosit nöron kültürleri iyi kurulmuş bir protokol 1,2,3 kullanılarak hazırlanmıştır. Prosedürü, sağlıklı kültürlü astrositler verim için optimize edilmiş. Aşağıda listelenen tüm işlemler, steril bir ortamda böyle bir laminer akış kaputu gibi yapılmalıdır. Hazırlanması lamelleri <ul><li> 22 mm lamelleri (VWR, 48.380-068) </li><li> Poly-D-Lizin (PDL, Sigma P0899), alikotları (1 mg / ml) </li><li> Laminin alikotları (20 mg / ml): 1 mg / ml laminin solüsyonu (Sigma L2020) ile 49 ml steril su ekleyin, -20 ° C&#39;de 1.2 ml alikotları ve mağaza </li></ul><ol><li> (50 mg / ml) steril su PDL çözülür </li><li> Lamelleri Otoklav, PDL steril su ve yeri (100 lamelleri için 20 ml) ile durulayın. Gece boyunca oda sıcaklığında bekletin. </li><li> PDL steril su ile (3 geniş yıkar) değiştirin. Sonra 4 lamelleri ve mağaza ° C kuru </li><li> 6 plaka (Steril Bioscience BD Kapaklar multiwell Düz Tabanlı Tabaklar) steril bir kuyu astrositler, bireysel lamelleri kaplama bir gün önce. Her lamel laminin ul 400 koyun ve buharlaşmasını önlemek için inkübatör, gece inkübe edin. </li></ol> Diseksiyon Diseksiyon medya: <ul><li> 500 ml Earle Dengeli Tuz Çözeltisi (EBSS) (1X), sıvı (Invitrogen, 14.155-063) </li><li> 5 ml Hepes solüsyonu (Sigma, H0887) </li></ul> Hipokampal medya: <ul><li> 412,5 ml Minimum Essential Medium (MEM) (1X), sıvı Earle Kullanıcı tuz içerir, ancak hiçbir L-glutamin veya fenol kırmızısı (Invitrogen, 51.200-038) </li><li> 10 ml glikoz (Sigma, D-(+) GLİKOZ susuz SIGMA ULTRA G7528) 1 M MEM </li><li> 5 ml Penisilin-Streptomisin (Invitrogen, 15.140-122) </li><li> 5 ml sodyum piruvat çözüm (Sigma, S8636) </li><li> 12.5 ml Hepes çözüm 1 M (Sigma, H0887) </li><li> 5 ml N-2 Eki (100X), sıvı (Invitrogen, 17.502-048) </li><li> 50 ml At Serum, Isı-İnaktive (Invitrogen, 26.050-088) </li></ul> Papain çözüm: <ul><li> Worthington papain-022 flakon (LK003178; nihai konsantrasyonu, 20 U / ml), diseksiyon medya 5 ml ve 37 ° C 60 dakika inkübe edin. </li></ul><ol><li> P1 (genellikle 2 yavrular) ulusal düzenlemeler ve yerel Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış ve onaylanan yönergeleri izleyerek yaşlı sıçan yavrular başını kesmek. </li><li> Petri kabındaki soğuk diseksiyon medya ile doldurulmuş deri ve kafatası yer ve beyin çıkarın. </li><li> Meninksler kaldırıldı, her iki yarımkürede incelemek ve hippocampi teşrih. </li><li> Hippocampi ~ 37 ° papain çözüm 1X1 mm parçaları ve hazmı ° C 11-13 dakika süreyle içine doğrayın. </li><li> Doku parçaları yerleştikten sonra, kaldırmak dikkatle papain ve enzim bütün izlerini kaldırmak için 5 ml hipokampal orta eklemek. Hipokampal orta (2 ml) ve bu adımı tekrar süspansiyon tekrarlayın. </li><li> Kümeleri, üç aşamalı olarak küçük delikleri (1000 mm, ~ 500 mm ~ 300 mikron) alev cilalı pipetler sol kalmayana dek hücreleri ~ 5 kez Karışım. </li><li> Triturated sonra, bir hücre süzgeç (gözenek boyutu, 70 mikron, BD Bioscience) aracılığıyla hücrelere geçerler. 10 ul süspansiyon hücreleri saymak. 400.000 hücre / ml sahip amacıyla hipokampal ortamın ses seviyesini ayarlayın. </li><li> Lamelleri kapalı laminin aspire ve lamel başına hücre süspansiyonu kapakları kuru önce, plaka 200 ul. </li><li> Onları inkübatörde 60 dakika ekleyiniz ve sonra iyi ortalama 2 ml hipokampal orta eklemek bırakın. </li><li> Ertesi gün, ölü hücreleri ve enkaz kaldırma ve 2 ml, önceden ısıtılmış taze hipokampal orta eklemek için eski hipokampal orta aspirat. </li></ol> Bakım Neurobasal medya: <ul><li> 500 ml neurobasal (Invitrogen, 12.348-017) </li><li> 10 ml B27 serum serbest eki (Invitrogen, 17.504-044) </li><li> 5 ml Penisilin-Streptomisin (Invitrogen, 15.140-122) </li><li> 1.25 ml L-glutamin (Invitrogen, 25.030-149) </li></ul> Kaplama dört gün sonra başlayan, neurobasal orta astrosit-nöron kültürler haftada iki kez besleyin. Sıcaklık ve CO 2 denge sağlaması için yaklaşık 30 dakika havalandırılan bir balonuna inkübatör medya Preincubate. Bölüm 2: KALSİYUM GÖRÜNTÜLEME Hipokampal kayıt tampon: 110 mM NaCl, 5.4 mMKCl, 1.8 mM CaCl 2, 0.8 mM MgCl 2, 10 mM D-glukoz, 10 mM HEPES (Sigma tüm kimyasallar), pH 7.4 (NaOH ile düzeltilmiş) . Astrositler kalsiyum göstergesi boya yükleme <ol><li> 2 ml hipokampal kayıt tampon DMSO 2.5 mcM Fluo-4, AM (Invitrogen, F-14.217) ve% 0.05 Pluronic ® F-127% 20 çözüm içeren dolu bir 6-plaka (Invitrogen, iyi bir kültür koyun P-3000MP) ve 10-30 dakika süreyle oda sıcaklığında inkübe edin. </li><li> Fluo-4 çözüm çıkarın ve 30 dakika oda sıcaklığında hipokampal kayıt tampon ve inkübe ile lamelleri 3 kere yıkayın. </li><li> Lamel odasına yerleştirin ve lens temizleme sıvı ile lamel diğer tarafında temiz. </li><li> Amaç küçük bir damla immersiyon yağı (Cargille Immersion Yağı TİPİ DF) koyun ve mikroskop (Olympus IX71) sahne odası (25 mm yuvarlak lamel için Warner Instruments Açık odası) koymak. </li><li> Iletim ışık kullanarak hücreleri hücrelerin nasıl göründüğüne bakmak ve onları almak için odak noktası haline bakın. Sonra (Vizyon KADAR Polychrome V) monokromatör gelen 488 nm hücreleri aydınlatmak ve Fluo-4 floresan sinyal üniforma ve astrositler saptanabilir olup olmadığını belirlemek. </li><li> EPI ve TIRF aydınlatma astrositler kalsiyum ölçmek için kullanın. </li></ol> TIRF mikroskopi Kısaca, biz bir Andor IXON DV887DCS EMCCD kamera ile donatılmış bir Olympus IX71 mikroskop kullanıyoruz. Eksitasyon ve görüntü alımı kontrolü TILLVision yazılımı kullanılarak elde edilir. 454/488/515 nm Argon (100 mW) ve 442 nm (45 mW) katı hal lazerleri kirişler kombine ve kontrollü bir KADAR Polyline lazer birleştirici, TIRF çift bağlantı kondansatör ve acoustoptical arasında ayarlanabilir filtre ve denetleyicisi (AOTF tüm TIRF kondenser içine giriş için Kineflex geniş bant fiber içine Fotonik) ve beslenen KADAR. Biz TIRF elde etmek için Olympus 60X 1.45 NA lens kullanın. Kamera kazancı en iyi sinyal-gürültü görüntüleri sağlamak için her astrosit ayarlanır. Arka plan ve TIRF mikroskopi ilkeleri, son zamanlarda, 5 4 gözden geçirilmiştir . Kullandığımız optik bileşenlerin çoğu şimdi Agilent Technologies (http://www.till-photonics.com/) bir parçası Fotonik, KADAR satın alındı. TIR penetrasyon derinliği aşağıdaki denklemler hesaplanabilir. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Denklem" /> d = penetrasyon derinliği n1 = cam kırılma indisi n2 = kırılma indisi hücre insidansı = açısı NAI = sayısal açıklık insidansı Lazer TIRF için optimal aynı hizada olmasını sağlamak için 100 nm floresan boncuk (Invitrogen, F8803) gözlemlemek için yararlı bulabilirsiniz. Biz hala EPI ve TIRF mikroskopi ile boncuk çerçeveleri ve videoları mevcut. Zaman TIRF, bir sinyal-gürültü dramatik bir artış gözlemlemiştir ve boncuk Brown difüzyon gösterilecek. Kritik açı eşit olmasaydı oluşacak tehlikeye eğik aydınlatma yerine daha optimum TIRF oluşur emin olmak için düzenli olarak (haftada ~ kez) TIRF mikroskobu ile 100 nm boncuk davranışlarını gözlemlemek için yararlı buluyorum α (bkz. Şekil 1). G-protein kenetli reseptör agonistleri Uygulama Astrositler Gq-reseptörler 6, 7 metabotropik glutamat reseptörleri ve P2Y reseptörleri (agonist, ATP, ADP) de dahil olmak üzere çeşitli ifade eder. Bu reseptörlerin aktivasyonu astrositler içinde hücre içi kalsiyum düzeylerinde önemli artışlara yol açar. Örneğin, bir hücre içi kalsiyum yükselmeler astrositler 8-10 ATP (30 mcM) uygulama sırasında kolayca izleyebilirsiniz . Warner Instruments switcher VC-77SP Hızlı Adım Perfüzyon Sistemi (http://www.warneronline.com/index.cfm) olarak adlandırılan hızlı bir çözüm kullanın. ~ 10 ms &#39;den daha az bir süre içinde bu yöntemi çözümleri uygulanabilir. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Şekil 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> ŞEKİL 1 Karikatür ve fotoğrafları görüntüleme kurdu. A., (B) lazer montaj, kontrolörler ve kiriş kutuları bir fotoğraf gösterir iken, üzerine monte edilmiş bir airtable mikroskop bir fotoğraf gösterir. C. mikroskop aşamasında bir fotoğraf görüntüleme için monte edilen oda gösterir. Hızlı perfüzyon cihazı (step motor ve teta tüp ile birlikte) görülebilir bıraktı. Sağ tarafta bir Axopatch 200A amplifikatör headstage görülür. Karikatür set ışık yolu schematizes ve nasıl TIRF elde edilir. Lazer, kritik açı α immersiyon ortaya çıkar, böylece merkezi ayarlanır 60X 1.45 NA objektif lens ve konumunu arka odak düzlemi üzerinde duruldu. Bu noktada demiri düşük kırılma indisi orta mesafe λ (ana metin denklemi) ile toplam iç yansıma ve bozunur muzdarip. Bu durumda bu kayıt tampon astrositler ve astrositler kendilerini çevreleyen. Sonuç olarak plazma zarı ~ 100 nm içinde moleküllerin optik uyarım (ve dolayısıyla görüntüleme). Hücre içinde veya hücrenin üst yüzeyinde bu heyecanlı ise hücre karikatür, yeşil &quot;heyecan&quot; membran reseptörleri olarak gösterilir. TIRF mikroskopi tam bir hesap Steyer ve Almers 4 tarafından sağlanmıştır. <img src="/files/ftp_upload/1142/fig2.png" alt="Şekil 2" /> ŞEKİL 2 EPI ve TIRF mikroskobu ile elde edilen 100 nm floresan boncuk Görüntüler. A. birkaç düzine 100 nm floresan boncuk manzaralı bir alan EPI görüntüleri gösterir. Mavi ok ucu suya difüzyon. Boncuk noktası ise, cam lamel üzerine yerleşmiş boncuk kırmızı oklar noktası. B. Bu görüşe göre, sadece kırmızı oklarla gösterilen yapışık boncuk görünür A&#39;da gösterildiği gibi aynı alan TIRF bir görüntü gösterir. Bu cam coverlsip üzerine yerleşti ve böylece ~ 100 nm kaybolan saha içinde vardı çünkü. Boncuk mavi oklarla gösterilen bu bölge içinde değildir ve TIRF görüntüleri böylece görünmez. Düşük araziler görüntülerin 3D render göstermektedir. Bu sinyal-gürültü TIRF mikroskopi tarafından gözlemlenen büyük bir artış kaybolan alanı içinde boncuklar oluşur açıktır. Aslında bu görüntüler için TIRF için 20 iken ± 0.7 EPI için sinyal-gürültü, 7.1 ± 0.6 idi. <img src="/files/ftp_upload/1142/fig3.png" alt="Şekil 3" /> ŞEKİL 3 EPI ve TIRF mikroskobu ile elde Fluo-4 kalsiyum gösterge boyası ile yüklü astrositler Görüntüler. Beş astrositler manzaralı bir alan A. EPI görüntüler. B. A, A ve B görüntüleri anlamlı derecede farklı olduğunu B. Not gösterilen bakış aynı alanda TIRF görüntü. Bu TIRF aydınlatma ile cam lamel yakın apozisyon sadece plazma zarı bölgelerde görüntülü çünkü. Alt panelleri göstermek ATP-uyarılmış hücre içi kalsiyum transientler EPI ve TIRF mikroskobu ile görüntülenmiş.

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Astrositler hücre içi kalsiyum yükselmeler görüntüler çok iyi kurulmuştur. Bu, kendiliğinden meydana gelen nöronal aktivite veya 11 astrosit yüzey reseptörleri aktive agonistleri uygulama tarafından tetiklenebilir. Bir önemli ve tartışmalı bir konu olmadığını astrosit hücre içi kalsiyum yükselmeleri, nöronlar 11, 12 reseptörleri aktive sinyal molekülleri serbest bırakılması tetikleyebilir. Yorum Haydon 7, 13 ve McCarthy 11 laboratuvarları tarafından vurgulanan bu g?...

<table><tbody><tr><td>VWR&amp;#174; Micro Cover Slips, Round, No. 1</td><td>Tool</td><td>VWR international</td><td>48380-068</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Reagent</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Earle&amp;rsquo;s Balanced Salt Solution (EBSS) (1X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>14155-063</td><td></td></tr><tr><td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle&amp;rsquo;s salts, but no L-glutamine or phenol red</td><td>Reagent</td><td>Invitrogen</td><td>51200-038</td><td></td></tr><tr><td>Penicillin-Streptomycin liquid</td><td>Reagent</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Sodium pyruvate solution</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>HEPES solution 1 M</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>N-2 Supplement (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>Horse Serum, Heat-Inactivated</td><td>Reagent</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>PAPAIN-022</td><td>Reagent</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Neurobasal&amp;#8482; Medium (1X) Liquid without Phenol Red</td><td>Reagent</td><td>Invitrogen</td><td>12348-017</td><td></td></tr><tr><td>B-27 Serum-Free Supplement (50X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>25030-149</td><td></td></tr><tr><td>Cell Strainers</td><td>Tool</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td><td>Tool</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>21108</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>HEPES free acid</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Fluo-4, AM 1 mM solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>F-14217</td><td></td></tr><tr><td>Pluronic&amp;#174; F-127 20% solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>P-3000MP</td><td></td></tr><tr><td>Immersion Oil TYPE DF</td><td>Microscope</td><td>Cargill Labs</td><td>16242</td><td></td></tr><tr><td>Open chamber for 25 mm round coverslips, 100 &amp;mu;l volume</td><td>Tool</td><td>Warner Instruments</td><td>64-0362 (RC-21BDW)</td><td></td></tr><tr><td>P-2 platform for Series 20 chambers, non-heater</td><td>Tool</td><td>Warner Instruments</td><td>64-0278 (P-2)</td><td></td></tr><tr><td>FluoSpheres carboxylate-modified microspheres, 0.1 &amp;mu;m, yellow-green fluorescent (505/515) 2% solids</td><td>Reagent</td><td>Invitrogen</td><td>F8803</td><td></td></tr></tbody></table>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

Beyin glial hücreler içerir. Astrositler, glial hücre tipi, uzun nöronlar için pasif bir destekleyici bir rol sağlamak için bilinmektedir. Ancak, artan kanıtlar astrositler nöronlar ile fonksiyonel etkileşim yoluyla da aktif beyin fonksiyonu katılmak olabileceğini düşündürmektedir. Ancak, birçok temel yönleri astrosit biyoloji, tartışmalı, belirsiz ve / veya deneysel keşfedilmemiş kalır. Bir önemli konu astrositlerde hücre içi kalsiyum Transientlerin dinamikleri. Kalsiyum önemli bir ikinci haberci olarak iyi kurulmuş ve çünkü astrosit kalsiyum yükselmeler astrositler gelen vericileri serbest bırakılması tetikleyebilir ileri sürülmektedir çünkü bu geçerlidir. Ancak, yakın plazma membranı kalsiyum astrositler sinyal herhangi bir ayrıntılı ve tatmin edici bir açıklama olmamıştır. Toplam iç yansıma floresan (TIRF) mikroskopi canlı hücrelerinin plazma membranı yaklaşık 100 nm içinde önemli fizyolojik sinyalleme olaylarını analiz etmek için güçlü bir araçtır. Burada, TIRF mikroskobu kullanımı ve neredeyse aynı anda, plazma membranı ve küresel hücre içi kalsiyum dinamikleri yakın izlemek nasıl tarif. Bu yaklaşımın daha rafine ve sistematik bir uygulama astrosit kalsiyum sinyal kesin detaylar hakkında bilgi potansiyeline sahiptir. Astrosit kalsiyum dinamiklerinin ayrıntılı bir anlayış, nasıl, ne zaman ve neden astrositler ve nöron kalsiyum-bağımlı fonksiyonel etkileşimleri tabi anlamak için bir temel sağlayabilir.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Biz toplam iç yansıma ve Epifloresans mikroskopi kullanılarak kültürlü astrositler membran ve küresel hücre içi kalsiyum dinamikleri yakın ölçmek için nasıl açıklar.

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.1K Görüntüleme.  David Geffen School of Medicine, University of California, Los Angeles. Biz toplam iç yansıma ve Epifloresans mikroskopi kullanılarak kültürlü astrositler membran ve küresel hücre içi kalsiyum dinamikleri yakın ölçmek için nasıl açıklar.

Video: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Nörobilim

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivokültür-tarihlerde hızlı, basit ve ucuz AWESAM iletişim kuralını kullanarak fare Astrocytes

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

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 ...

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 ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Genetik Olarak Kodlanmış Ca2+ Göstergeleri (GECI'ler) Kullanarak Astrositlerde ve Nöronlarda Mitokondriyal Ca2+ Alımı Görüntüleme

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

Sıçan Astrositleri ve Mikroglia'nın Birincil Kültürleri ve Amiyotrofik Lateral Skleroz Çalışmalarında Kullanımları

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Hücre içi kalsiyum ve taze izole ve olduğu gibi fare serebral endotel membran potansiyeli eşzamanlı ölçümler

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Görüntüleme hücre içi Ca

Vivokültür-tarihlerde hızlı, basit ve ucuz AWESAM iletişim kuralını kullanarak fare Astrocytes

Yerel Ca^{2 +} sinyalleri membran hedefli Ca^{2 +} göstergeleri tarafından kültürlü hücrelerdeki diseksiyon

Astrositlerde Organeller ve Diğer Kargoların Hücre İçi Taşınmasının Görselleştirilmesi ve Analizi

Genetik Olarak Kodlanmış Ca²⁺ Göstergeleri (GECI'ler) Kullanarak Astrositlerde ve Nöronlarda Mitokondriyal Ca²⁺ Alımı Görüntüleme

Sıçan Astrositleri ve Mikroglia'nın Birincil Kültürleri ve Amiyotrofik Lateral Skleroz Çalışmalarında Kullanımları

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

Hücre içi kalsiyum ve taze izole ve olduğu gibi fare serebral endotel membran potansiyeli eşzamanlı ölçümler