We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

Tüm hayvan deneyleri (2012 revize NIH Yayınları No. 80-23) Laboratuvar Hayvanları Bakım ve Kullanım Sağlık Rehberi National Institutes uyarınca yürütüldü ve çalışmalar Kurumsal Hayvan Bakım tarafından onaylanmış ve Stony Araştırma Komiteleri için kullanın edildi Brook Üniversitesi (# 1618 ve # 1792). 1. vHipp-NACC Sinaptik Co-kültürler <ol><li> Kurban fareleri (doğum sonrası gün 0 - vahşi tip (WT) ya da α7 nAChRs transgenik fare hattı 3) CO2 ile. Her yavru başını kesmek ve post facto genotipleme için 1,5 ml santrifüj tüpüne kuyruk kaydedin. Steril kaputu aşağıdaki adımları uygulayın. </li><li> Tüm beyin ortaya çıkarmak için makas ve forseps ile kafatası üst çıkarın. Birbirini kaudalde serebral korteks bölümü, bir mikroskop altında vHipp 5,21 olmak üzere arka korteks içeren bir koronal bölümü elde noktada başlatılıyor. </li><li> V teşrihGelişmekte olan bir fare beyin atlası 22 (Şekil 1A) göre CA1-subiculum bölgesini dışarı içeren Hipp doku şeritleri. Soğuk kültür ortamı (4 ° C) içeren 35 mm kültür çanak transfer, küçük ince dilim (um 100 civarında × 100 mikron microslices) oyulmuş. </li><li> 37 ° C&#39;de inkübatör içinde en az 30 dakika boyunca kültür ortamı (~ 500 ul) ile 24 oyuklu doku kültürü plakaları içindeki 12 mm poli-D-lisin / laminin ile kaplanmış cam lamelleri önceden inkübe edin. Microslices kaplama önce ortamı çıkarın. </li><li> (Microslices arasında ~ 20 parçalarını içeren ~ 50 ul) ortam az miktarda lamel merkezi bir ateş cilalı Pastör pipeti ile Plaka eksplant bağlanmasını kolaylaştırmak için. Tek bir hayvandan kaynaklanan Plaka vHipp microslices (ya + / + veya - / - transgenik α7 hattının genotip) her lamel. </li><li> Eksplantlar lamel üzerine yerleşmek sonra, yavaşça th ek medya (~ 100 ul) ekleyinE duvar böylece medyanın düzeyi değil, bu lamel merkezinde, tamamen çevre üzerinde eksplantlar karşılamak için yeterince yüksek. </li><li> 37 ° C de bir O / N inkübasyondan sonra,% 5 CO2 kuluçka makinesi, doğum sonrası gün 1 ila farelerde embriyonik gün 18&#39;den NACC nöronlar dağılması ve eksplantlar ekleyin. <ol><li> Gelişmekte olan bir fare beyin atlas 20 (Şekil 1B) göre NACC dokuları dışarı parçalara ayır. </li><li> 15 ml tüp ve transfer (500 500 mikron microslices × um civarında) küçük parçalar halinde kesin. </li><li> 37 ° C&#39;de 15 dakika boyunca% 0.25 tripsin (2 mi) ile tedavi edin. Yıkama doku parçaları soğuk yıkama ortamı ile üç kez (5 mi, 4 ° C) soğuk bir kültür ortamı içinde bir yıkama, ardından (5 mi, 4 ° C). Süspansiyon her yıkama aşamasında 5 dakika dinlenmeye ve sonra dikkatlice süpernatant dökmek için izin verin. </li><li> Hafif ateş cilalı Pastör pipeti ile kültür ortamı 2 ml dikkatlice toz haline getirilerek hücreleri ayırmak. </li><li> Trans5 dakika boyunca 2,000 x rpm&#39;de bir 15 ml tüp ve santrifüj ferin hücre süspansiyonu. Süpernatantı ve 6 yavrular başına 1 ml kültür ortamı hücreleri tekrar süspansiyon. Yumuşak bir ateş cilalı Pastör pipeti ile ortam birkaç kez pipetleme hücreleri dağıtılır. </li><li> VHipp eksplant ile her bir lamel dağılmış NACC hücrelerinin 0.25 ml ilave edilir. </li><li> Nemlendirilmiş bir 37 ° C kültürleri bakımı,% 5 CO2 kuluçka. Mekanik sinaps oluşumunu kolaylaştıran, stabilize ve nemi muhafaza etmek nemlendirilmiş steril gazlı bez üzerine kültür tabak yerleştirin. </li></ol></li></ol> 2. İmmünositokimya <ol><li> Kültürleri saptamak -% 4 paraformaldehid /% 4 sukroz (5 in vitro 7 gün) / PBS (~ lamel 500 ul, 20 dakika, RT). </li><li> % 0.25 Triton X-100 / PBS ile Permeabilize kültürler (~ lamel 500 ul, 5 dakika, RT). </li><li> PBS içinde% 10 normal eşek serumu ile blok kültürleri (~ lamel 500 ul, 30 dakika, RT) Antibod öncesindeY boyama. 4 ° C &#39;de O / N primer antikorlar ile inkübe edin. Anti-veziküler glutamat taşıyıcı 1 (1: 250), anti-GAD65 (1: 100):;: Anti-nAChRs (anti-α 5 alt birimleri, 1 500 500 α 4 -ECD 1:) Aşağıdaki primer antikorlar kullanın. </li><li> (Lamel haftada 3 kez / ~ 500 ul, 5 dakika) PBS ile primer antikor yıkayın ve daha sonra (~ lamel 500 ul, 1: 500), Alexa 488 konjüge edilmiş sekonder antikor kültürleri inkübe ya lamel ortalama Alexa 594 (~ 500 ul , 1: 37 ° C&#39;de 1 saat süre ile 500). </li><li> Önceden etiketlenmesi yüzeyi α7 * nAChR&#39;lerin için tespit için 37 ° C&#39;de 45 dakika boyunca (kültür ortamı 1000 ~ lamel 500 ul, 1) Alexa 594 konjüge αBgTx kültürleri inkübe edin. </li><li> Dağı ve mühür lamelleri. </li><li> Plan-Apochromat hedefleri (0.8 NA veya 1.4 NA ile 63X yağ ile 20X) ile donatılmış bir floresan mikroskop ve CCD kamera ile görüntüleri yakalayın. </li></ol> 3. FM1-43 Tabanlı Veziküler Fusion <ol><li> Bir görüntüleme odasında - (in vitro 7 gün 5) Disk konfokal mikroskop iplik üzerine odasına monte kültürleri korumak. Sürekli oda sıcaklığında HBS kokteyli ile (1 mi / dak) perfüze. </li><li> 90 sn için 56 mM K + ACSF (~ 500 ul) (boyama) içinde 10 uM FM1-43 perfüzyon ve yük kültürleri dur, Ca2 + ADVASEP-7 (0.1 mM) ihtiva eden serbest HBS ile birlikte 15 dakika boyunca uzaklıkta dış boyayı zara bağlı FM1-43 temizlemek için. </li><li> 120 saniye FM1-43 olmadan 56 mM K + ACSF (~ 500 ul) (destaining) ile kültürleri teşvik aynı koşullar kullanılarak FM1-43 ile kültürleri yeniden ve Ca ile tekrar içeren 2+ ücretsiz HBS yıkama ADVASEP-7 için 15 dk. </li><li> CCD kamera ile Plan-Apochromat objektif (1.4 NA ile 60X su, uyarma 488 nm, emisyon 530 nm) ve yakalama görüntüleri ile donatılmış iplik diski konfokal mikroskop ile FM1-43 floresan görüntüler elde. </li><li> 1 dakika boyunca bazal FM1-43 floresan görüntüleri (her 2 sn toplayın) Önceden nikotin kontrol olarak; 1 dakika boyunca hızlı bir perfüzyon (2 ml / dakika) ile nikotin (1 uM) uygulanır, ve daha sonra HBS kokteyli ile nikotin yıkayın. 5 dakika boyunca her 2 saniyede sırasında ve nikotin uygulamadan sonra, daha önce yakalama time-lapse görüntüleri tutun. </li><li> Önce FM1-43 floresan (F boyama) ve sonrası (F destaining) her sinaptik bouton de nikotin uygulamasını ölçmek. </li><li> Öncesi ve nikotin uygulaması (mesafe kadar taşınmış = F boyama -F destaining) sonra FM1-43 floresan yoğunluğu farkını miktarının tarafından vHipp akson boyunca salınabilir FM1-43 floresan toplam miktarını hesaplayın. Aşağıdaki denklemle nikotin sonra floresan yoğunluğu azalma kısmını analiz: F azalma% = mesafe kadar taşınmış / F boyama. </li></ol> 4. Kalsiyum Görüntüleme <ol><li> Daha sonra (5 mcM Fluo-4 Ca 2 + göstergesi ile kültürleri yük normal HBS hızla - (in vitro 7 gün 5) kültürleri durulayınAM ester) ve 37 ° C de 30 dakika karıştırıldı ve% 5 CO2 boyunca HBS&#39;de% 0.02 Pluronic F-127 (2 mi) eklendi. </li><li> (3 kez / 5 dk) HBS ile Flu-4 çözüm yıkayın. (37 ° C /% 5 CO2), en azından 30 dakika boyunca kuluçka kültürler dönün. </li><li> Aynı görüntüleme odası ve FM1-43 göre veziküler füzyon bölümünde tarif edildiği gibi aynı şartlarda kültürleri muhafaza (adım 3.1). </li><li> Bir Plan-Apochromat objektif (1.4 NA ile 60X su, uyarma 488 nm, emisyon 530 nm) ve bir CCD kamera ile vHipp mikro dilim aksonal projeksiyonlar Fluo-4 floresan görüntüleri toplayın. </li><li> 1 dakika boyunca hızlı bir perfüzyon (2 ml / dakika) ile nikotin (1 uM) uygulanır, ön-nikotin kontrol olarak temel Fluo-4 floresans görüntüleri (2 dakika süre ile, her 10 saniye) toplamak, ve daha sonra HBS kokteyli ile nikotin yıkayın. 30 dakika boyunca her 10 saniyede sırasında ve nikotin uygulamadan sonra, daha önce yakalama time-lapse görüntüleri tutun. </li><li> Bir dizi olarak ham fluo-4 floresan görüntüler, ihracat tüm kareleri kaydetDaha fazla analiz için TIFF formatında görüntüler. </li><li> Önce vHipp akson boyunca ve nikotin uygulamadan sonra fluo-4 floresan entegre yoğunluğu toplayın. </li><li> Aşağıdaki denklem ile entegre bir floresan Normalize: kadar taşınmış / F = 0 (FF 0) / F 0 artalan-düzeltilmiş önceden nikotin entegre floresans şiddetidir ve F&#39;den her biri bir zaman noktasında vHipp akson boyunca entegre floresan yoğunluğu F 0, Nikotin uygulaması sonrası. Analiz ve normalize entegre floresan arsa. </li></ol>

<ol>
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ko-kültür hazırlanması açıklanan yeniden taahhüt eder in vitro ventral hipokampal-accumbens devreleri. pre-sinaptik nAChR&#39;lerin aktivasyon glutamaterjik iletimi 5, 21 gelişmiş ortaya çıkaran hangi mekansal ve zamansal profillerin Bu preparat izin oldukça basit ve güvenilir bir sınav. Co-kültürlerinde in vivo benzeri işlev göstermek için, in vitro olarak fizyolojik koşulları sağlayan bir çanak içinde farklı spesifik h...

Devre uyarılabilirliğinin Kolinerjik modülasyon biliş temel yönlerine katkıda bulunur ve değişmiş kolinerjik modülasyonu Alzheimer hastalığı, Parkinson hastalığı, şizofreni ve bağımlılık 1-4 olmak üzere nörodejeneratif ve nöropsikiyatrik bozuklukların bir özelliğidir. CNS&#39;de sinaptik iletim kolinerjik kolaylaştırılması kurulan bir mekanizma, ön-sinaptik olarak lokalize nAChR&#39;lerin doğrudan aktivasyonu yoluyla olmaktadır. Dolaylı olarak bazı nAChR alt tipleri ve nispeten yüksek kalsiyum iletkenlik hem doğrudan, bağlı ile hücre içi sinyal iletim - bu pre-sinaptik reseptörlerinin aktivasyonu, hücre içi Ca + 2 ([Ca2 +] i) ön-sinaptik terminallerindeki sebep olur 5, bu şekilde nörotransmitter salgılanmasını zenginleştirmek. Aslında, pre-sinaptik nAChR&#39;lerin aktivasyonu da dahil olmak üzere, glutamat nörotransmitterlerin çok çeşitli GABA, ACh, serbest değişikliklerle bağlantılı olan birnd dopamin 6-10. Bu işlem, dolaylı, çeşitli sinapslarda elektrofizyolojik yöntemler kullanılarak incelenmiştir, ancak optik habercilerin [Ca2 +] i ve sinaptik vezikül dönüştürme pre-sinaptik olayların daha doğrudan ve zamansal olarak hassas ölçümü sağlar. NAChR&#39;lerin pre-sinaptik lokalizasyon elektron mikroskobik (EM) düzeyinde 11,12 olarak nAChR&#39;lerin doğrudan bağışıklık altın etiketleme ile ikna edici kanıtlanmıştır. Birkaç diğer teknikler de kültürlü nöronlar 13,14 nAChRs subunit- floresan protein kimeralar yerleri tespit da dahil olmak üzere, dolaylı nAChR yerelleştirme hitap için kullanılır olmuştur, sinaptik terminallerde 15,16 yılında nAChR akımların elektrofizyolojik kayıt, [içinde Ca nikotin kaynaklı değişiklikleri izleme sinaptik terminalinde canlı hücre görüntüleme 17 ve nörotransmitter salınımı dolaylı izlenmesi sinaptik sinir terminallerinde 2 +] istiril amfipatik FM boyalar (FM1-43 ve FM4-64) ve / veya synapto-pHluorin ve bu tür glutamat ACH ve iGluSnFr için CNiFERs gibi özel flüoresan nörotransmiter muhabir, tarafından izlendi sinaptik veziküllerin ekzositozu de dahil olmak üzere, floresan göstergesi olan canlı hücre görüntüleme teknikleri 18-20. Genel olarak, nAChR&#39;lerin pre-sinaptik localisation belirlenmesi için, bu mevcut yaklaşımlar karmaşıktır ve güvenilir bir kimlik ve pre-sinaptik aktivitenin fizyolojik izleme sağlamak için özel bir sistem ve teknikler gerektirir. Belirlemek ve sinaptik iletim öncesi ve post-sinaptik bileşenleri hem de analiz doğrudan erişim sağlayan nükleus akumbens (NACC) devre - Burada bir ventral hipokampal (vHipp) in vitro ko-kültür sistemi protokolleri ve ekipmanlar açıklar. Biz pre-sinaptik nAChR&#39;lerin lokalizasyonu ve Ca 2 + sinyalizasyon ve nörotransmitter bırakma alo aracılı nAChR&#39;nin canlı hücre görüntüleme örneklerini göstermektedirng vHipp aksonlar. Burada sunulan protokol doğal (ve basit) uzantısı farklı genotipleri gelen nöronların oluşan öncesi ve post-sinaptik temasların hazırlanmasıdır. Bu şekilde, modülasyon öncesi ve / veya post-sinaptik mekanizma belirli bir gen ürününün katkı, doğrudan tespit edilebilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

kullanılan preparat, in vitro olarak vHipp-NACC devreler gen, kimerik ko-kültürler oluşur. VHipp microslices kaynaklanan Projeksiyonlar, pre-sinaptik akson girdi olarak, post-sinaptik hedefler NACC dağılmış nöronlar ile sinaptik kişileri yapabilirsiniz. VHipp tarafından innerve NACC nöronlardan glutamaterjik iletimin bir sürekli (≥ 30 dk) kolaylaştırma kaynaklı nikotin pre-sinaptik α7 * nAChRs aracılığıyla vHipp aksonlar 5 boyunca sinyal 21 ve uzamış kalsiyum akson...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Nikotin Kaynaklı Kalsiyum Sinyali ve ventral Hipokampal Aksonlar boyunca Nörotransmitter Release Canlı Görüntüleme

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

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8.9K Görüntüleme.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Video: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Nöromodülasyon ve Mitokondrial Ulaştırma: Uzun Süreleri içinde Hipokampal Nöronlar Canlı Görüntüleme

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Nörobilim

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rizotomi sonra Dorsal Kök Aksonlar Canlı Görüntüleme

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Kalsiyum Görüntüleme için akut subventricular dilimi Dilimleri hazırlanması

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Büyüyen Aksonlar boyunca murin Schwann Hücresi Kalkınma Analizi

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Eşzamanlı Elektrofizyolojik Kayıt ve Suprakiazmatik Nucleus Nöronlar Kalsiyum Görüntüleme

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Primer hipokampal nöronlar kalsiyum fosfat transfeksiyonu

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Canlı Görüntüleme<em&gt; Drosophila</em&gt; Larva nöroblastomun

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Endokrin Drosophila içinde sinyal beyin yanıt-e doğru görüntülenmesi için kalsiyum görüntüleme

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Nikotin Kaynaklı Kalsiyum Sinyali ve ventral Hipokampal Aksonlar boyunca Nörotransmitter Release Canlı Görüntüleme

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