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><tbody><tr><td colspan="4">1. Culture Media (50 ml)</td></tr><tr><td>Neurobasal&amp;nbsp;</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 colspan="4">2. washing media (HBSS, 100 ml)</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red&amp;nbsp;</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 colspan="4">3. HEPES buffered saline&amp;nbsp; (HBS)&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">4. HBS Cocktail for live imaging pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td>tetrodotoxin&amp;nbsp;</td><td>Tocris</td><td>1078</td><td>2 &amp;micro;M</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>10 &amp;micro;M</td></tr><tr><td>D-AP-5</td><td>Tocris</td><td>105</td><td>50 &amp;micro;M</td></tr><tr><td>CNQX</td><td>Tocris</td><td>1045</td><td>20 &amp;micro;M</td></tr><tr><td>LY341495</td><td>Tocris</td><td>1209</td><td>10 &amp;micro;M</td></tr><tr><td colspan="4">5. Calcium-free&amp;nbsp; HBS&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">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&amp;nbsp;</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 ...

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9.0K 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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Nörobilim

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Kronik Nikotin İdaresi fareler Knock-in içinde floresan etiketli Nikotinik Reseptör Spektral Konfokal Görüntüleme

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Canlı fareler içinde dorsal hipokampal CA1 uzunlamasına Iki foton görüntüleme

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Davranış

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Fare beyin dilim Photoactivatable nikotin lazer Flash Photolysis üzerinden nikotinik asetilkolin reseptör işlevinde sondalama

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Behaving Farelerinin Hipokampusunda Nöronal Aktiviteleri Görselleştirmek için Kraniyotomi Prosedürü

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

İn Vivo Bozulmamış Dorsal Kök Ganglialarında Primer Duyusal Nöronların Ağlarında Nöronal Toplulukların Kalsiyum Görüntülenmesi

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Source: Zhong, C., et al. <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

Ventral Hipokampal ve Nucleus Accumbens'in birlikte kültürlenmesi

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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Nöronal Kültür Teknikleri

Çoklu kültür ve Etkileşim Çalışmaları

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

Sinaptik Ko-Kültürlerde Ventral Hipokampal Aksonlar Boyunca Nikotin Kaynaklı Kalsiyum Sinyalinin Görüntülenmesi

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

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