Name: in situ ca2+ imaging of the enteric nervous system
Uploaded: 2015-01-29T15:00:00.000+00:00
Duration: 11 min 26 s
Description: Watch this Scientific Journal Video about In Situ Ca2+ Imaging of the Enteric Nervous System at JoVE.com

This work was supported by grants from the Pharmaceutical Research and Manufacturers Association of America (PhRMA) Foundation (to B. Gulbransen), National Institutes of Health (Building Interdisciplinary Research Careers in Women&#8217;s Health) grant K12 HD065879 (B. Gulbransen) and start-up funds from Michigan State University (B. Gulbransen).

注:涉及实验动物组织中的下列程序与动物2013年安乐死的AVMA准则一致，是由美国密歇根州立大学IACUC批准。 1.组织准备<ol><li>麻醉动物研究中含有氧或通过将3-5毫升液体异氟烷到吸收材料上的腔室的地板上，以确保物理屏障防止动物从与异氟烷直接接触2.5％异氟烷的腔室。测试麻醉按捏足垫的深度。 注:麻醉深度被认为是合适的时候有后肢没有撤回反射。一旦适当麻醉，安乐死鼠标颈椎脱位</li><li>将动物仰卧位置和清洁腹部皮肤，用70％的乙醇。使用镊子夹住腹部皮肤在中线，用手术剪刀做出6厘米媒体升切口，沿白线，露出内部的消化器官。 </li><li>使用钝钳找到并揭露腹膜内回肠。切回肠/结肠肠系膜用剪刀，开始解开肠道。 </li><li>一旦肠的长度被充分地解开，切开肠远侧到胃和近端至盲肠为回肠制备。对于大肠的准备，切开结肠远端盲肠和近端到直肠。 </li><li>迅速取出肠段，并将其放置在与补充有3微米尼卡地平盐酸化物和1μM的东莨菪碱盐酸盐的DMEM / F12介质的烧杯中（以下简称为"介质"）上的冰。添加这些抑制剂被麻痹肠道平滑肌有利于显微切割和随后的成像。 </li><li>感兴趣的基础上建立的解剖标记切段（ 例如 ，空肠，回肠，远端或近端结肠）。通常情况下市利用Ë组织从远端回肠和结肠。然而，使用相同的基本程序来分离，负载和图像肌间神经元和神经胶质细胞中的所有肠道地区。 </li><li>在一个的Sylgard涂覆培养皿填充有冷却介质除去所需肠段和地点的一小部分（4-6厘米）。 </li><li>固定的近端和肠段与昆虫销的远端并且通过使直打开肠管，沿肠系膜边界纵向切割。 </li><li>针组织在轻张力粘膜侧扁了，仔细用细镊子（＃5，＃四十五分之五工作最好的）和很细的弹簧剪刀解剖走粘膜层。 注:粘膜的除去可以相当创伤为ENS如果没有正确完成。对于质量的准备，照顾通过剥离或刮限制突然去除粘膜。最好的做法是解除粘膜切下的细剪刀。 </li><li>切割组织成更小的准备S（约为0.5 平方厘米）和引脚到成像菜（4个角有圆形肌肉层朝上）置于冰上新鲜的媒体。 </li><li>仔细剖析走环形肌与精细镊子戏弄除了揭露肌间神经丛。避免过度伸展。 </li><li>将成像菜重回冰变化的解决方案，新的设备。 </li><li>准备每盘2毫升酶混合物[分散酶1单位/毫升（4.48毫克/ 8毫升）中，胶原酶II 150 U / ml的（5.45毫克/ 8毫升）中的介质。 </li><li>从冰中取出的菜肴和步骤1.13添加酶混合物。 </li><li>孵育菜在RT 15分钟，用5％的CO 2/95％空气。 </li><li>洗涤组织标本与媒体的3倍，并重新销角落。 </li></ol> 2.装载荧光4染料注:避免用有限的光，同时处理装有指示剂染料荧光染料和组织工作的漂白。 <ol><li>准备4μM荧光4加载的​​解决方案。 <ol><li>添加1.5毫升媒体和1.2微升250mM的丙磺舒股票4毫的Fluo-4的库存1.5微升等分试样。到50的Fluo-4，AM微克; 4毫的Fluo-4的库存通过加入11.4微升尼克F-127的（补充有0.25％cremaphor-EL在DMSO中的20％）来制备。 </li></ol></li><li>在37℃下在黑暗培养箱孵育的Preps中的Fluo-4加载溶液45分钟。 </li><li>从孵化器中取出并清洗与媒体3倍的准备。 </li><li>交换介质为含有介质200μM的丙磺舒孵育15分钟，在37℃前成像。 注:丙磺舒是一种药物，抑制多药耐药转运体中的神经元。此外，利用这种药物的抑制神经元的挤出染料的能力并增强神经元标记。在没有丙磺舒批量装载的钙指标染料生产的主要胶质细胞负荷。加入丙磺舒允许对神经元和神经胶质反应可视化。 </li><li>制备改性贵安BS缓冲区。 <ol><li>使修饰的Krebs缓冲液，使得所述组分的终浓度（以mM计）是如下:121氯化钠，氯化钾5.9，2.5 氯化钙 ，1.2的MgCl 2，1.2的NaH 2 PO 4，10 HEPES，21.2 碳酸氢钠 ，1丙酮酸酸和8个葡萄糖（pH值调节至7.4，用NaOH）。加入3μM尼卡和1μM东莨菪碱钙在抑制肌肉收缩2+成像和整个安装解剖。 </li></ol></li></ol> 3.成像和分析注:使用至少一个基本的成像钻机用荧光光源，显微镜，品质CCD摄像头和适当的采集软件。改变加法取决于光源和特定应用的其他组件。滤光轮和快门必须使用传统的氙弧光源。然而，LED光源和照明系统不需要那些组件。 <ol><li>定位REC奥尔丁显微镜下腔室，并用重力流灌注系统具有多个加热注射器储层建立的在37℃的Krebs缓冲液的2-3毫升/分钟的连续灌注速率。要确保防止在输入和吸入管线连接到真空捕集空气气泡的形成。 </li><li>将所需丛成在明亮的视场照明的重点。避免过度曝光的组织，这可能会导致光漂白性。 </li><li>检查神经节内的荧光4加载和选择健康的神经节成像。不健康的/损坏的神经节将呈现自发荧光或点状的形态，而不应被用于成像。 </li><li>一旦神经节被选中，疏导光路摄像头，并获得与图像采集软件的实时图像。确保节是重点，设定图像采集速度和曝光时间。 注:图像采集速度和时间将取决于调查人员希望记录的事件各不相同。对于大多数实验中，图像传统上，在收购0.5-1赫兹胶质细胞和高达2-10赫兹的神经元，因为胶质的Ca 2+的反应并不像快速的钙瞬变的神经元。 </li><li>开始试验并建立基线活动，持续30秒。 </li><li>应用预热药物的兴趣，例如使用重力流灌注系统以2-3毫升/分钟的速率受体激动剂和拮抗剂。按照应用的激动剂/拮抗剂通过返回到正常缓冲的灌注，并允许至少10分钟的洗涤/恢复期。 注:该药物的施用时间将取决于个体化合物和试验设计而变化。一般来说，激动剂20-30秒的应用是足以激活G-蛋白偶联受体的神经元和神经胶质细胞。然而，配体门控离子通道（如烟碱乙酰胆碱受体）要求不超过5-10秒施用时间。而且持续时间曝光会导致配体门控离子通道迅速DESEnsitize。拮抗剂应适用于约3-15分钟，以确保受体途径的完整封锁。然而，这是一个总的概括和研究者应该总是优化任何实验性药物在其特定的模式。 </li><li>停止实验记录和查看时间推移电影。感兴趣的精心选择的区域（ROI的）使用适当的图像分析软件。 </li><li>使用软件来规范和比较投资回报率的荧光强度对初始基线荧光值。变化标准化荧光成正比变化的[Ca 2+]。 <ol><li>使用的方法的使用ΔF/ F =（（F 1 - f 0的）/ F 0）的变形例前面所述的ROI 26 - （（F 1 - f 0的）/ F 0）的背景下，其中，F1是所述荧光在任何给定点和F0是基线荧光，以改善评估的准确性。此修改辅助从组织筹备荧光变化表现出的肌间神经丛背后的肌肉层的运动降低了噪声。 </li></ol></li></ol>

<ol>
	<li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+of+neurons+in+the+enteric+nervous+system.">Types of neurons in the enteric nervous system.</a> Journal of the Autonomic Nervous System. 81 (1-3), 87-96 (2000).</li><li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+autonomic+nervous+system:+peripheral+connections.">The organization of the autonomic nervous system: peripheral connections.</a> Neuroscience. 130 (1-2), 1-5 (2006).</li><li>Pham, T. D., Gershon, M. D., Rothman, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+of+origin+of+neurons+in+the+murine+enteric+nervous+system:+sequence+in+relation+to+phenotype.">Time of origin of neurons in the murine enteric nervous system: sequence in relation to phenotype.</a> Journal of Comparative Neurology. 314 (4), 789-798 (1991).</li><li>Nasser, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+enteric+glia+in+intestinal+physiology:+effects+of+the+gliotoxin+fluorocitrate+on+motor+and+secretory+function.">Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function.</a> Am J Physiol Gastrointest Liver Physiol. 291, G912-927 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+in+the+gut.">Glial cells in the gut.</a> Neurogastroenterology &amp; Motility. 17 (6), 777-790 (2005).</li><li>Broadhead, M. J., Bayguinov, P. O., Okamoto, T., Heredia, D. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++transients+in+myenteric+glial+cells+during+the+colonic+migrating+motor+complex+in+the+isolated+murine+large+intestine.">Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.</a> J. Physiol. 590, 335-350 (2012).</li><li>Gulbransen, B. D., Bains, J. S., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+are+targets+of+the+sympathetic+innervation+of+the+myenteric+plexus+in+the+guinea+pig+distal+colon.">Enteric glia are targets of the sympathetic innervation of the myenteric plexus in the guinea pig distal colon.</a> J. Neurosci. 30, 6801-6809 (2010).</li><li>McClain, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++Responses+in+Enteric+Glia+Are+Mediated+by+Connexin-43+Hemichannels+and+Modulate+Colonic+Transit+in+Mice.">Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice.</a> Gastroenterology. 146 (2), 497-507 (2014).</li><li>Chandrasekharan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+motor+dysfunction+in+human+diabetes+is+associated+with+enteric+neuronal+loss+and+increased+oxidative+stress.">Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress.</a> Neurogastroenterology &amp; Motility. 23 (2), e131-e126 (2011).</li><li>Abdo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cells+protect+neurons+from+oxidative+stress+in+part+via+reduced+glutathione.">Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione.</a> FASEB J. 24, 1082-1092 (2010).</li><li>Aube, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+enteric+neurone+phenotype+and+intestinal+functions+in+a+transgenic+mouse+model+of+enteric+glia+disruption.">Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption.</a> Gut. 55, 630-637 (2006).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nature Reviews Molecular cell biology. 1 (1), 11-21 (2000).</li><li>Gulbransen, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+neuronal+P2X7+receptor-pannexin-1+mediates+death+of+enteric+neurons+during+colitis.">Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis.</a> Nat Med. 18, 600-604 (2012).</li><li>Bayguinov, P. O., Hennig, G. W., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+activity+in+different+classes+of+myenteric+neurons+underlying+the+migrating+motor+complex+in+the+murine+colon.">Calcium activity in different classes of myenteric neurons underlying the migrating motor complex in the murine colon.</a> J Physiol. 588, 399-421 (2010).</li><li>Okamoto, T., Bayguinov, P. O., Broadhead, M. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+transients+in+submucous+neurons+during+the+colonic+migrating+motor+complex+in+the+isolated+murine+large+intestine.">Ca(2+) transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine.</a> Neurogastroenterol Motil. 24 (8), 769-778 (2012).</li><li>Kunze, W. A., Clerc, N., Furness, J. B., Gola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soma+and+neurites+of+primary+afferent+neurons+in+the+guinea-pig+intestine+respond+differentially+to+deformation.">The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation.</a> J Physiol. 526, 375-385 (2000).</li><li>Schemann, M., Michel, K., Peters, S., Bischoff, S. C., Neunlist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+the+gastrointestinal+tract:+mapping+the+human+enteric+nervous+system.">Imaging and the gastrointestinal tract: mapping the human enteric nervous system.</a> Am J Physiol. 282, G919-G925 (2002).</li><li>Hennig, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spread+of+pacemaker+activity+in+through+ICC+in+guinea-pig+antrum.">Visualization of spread of pacemaker activity in through ICC in guinea-pig antrum.</a> Neurogastro Motil. 14, 575 (2001).</li><li>Stevens, R. J., Publicover, N. G., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+organization+of+Ca2++waves+by+enteric+neural+reflexes.">Induction and organization of Ca2+ waves by enteric neural reflexes.</a> Nature. 399, 62-66 (1999).</li><li>Stevens, R. J., Publicover, N. G., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+and+neural+regulation+of+calcium+waves+in+longitudinal+and+circular+muscle+layers+of+guinea-pig+small+intestine.">Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea-pig small intestine.</a> Gastroenterology. 118, 982-984 (2000).</li><li>Jessen, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-like+glia+in+the+peripheral+nervous+system:+an+immunohistochemical+study+of+enteric+glia.">Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia.</a> Journal of Neuroscience. 3 (11), 2206-2218 (1983).</li><li>Gulbransen, B. D., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+functional+roles+for+enteric+glia+in+the+gastrointestinal+tract.">Novel functional roles for enteric glia in the gastrointestinal tract.</a> Nat Rev Gastroenterol Hepatol. 9, 625-632 (2012).</li><li>Gomes, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-dependent+paracrine+communication+between+enteric+neurons+and+glia+in+a+primary+cell+culture+derived+from+embryonic+mice.">ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.</a> Neurogastroenterology &amp; Motility. 21 (8), e870-e862 (2009).</li><li>Gulbransen, B. D., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+neuron-to-glia+signaling+in+the+enteric+nervous+system.">Purinergic neuron-to-glia signaling in the enteric nervous system.</a> Gastroenterology. 136, 1349-1358 (2009).</li><li>Ren, J., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+receptors+and+synaptic+transmission+in+enteric+neurons.">Purinergic receptors and synaptic transmission in enteric neurons.</a> Purinergic Signal. 4, 255-266 (2008).</li><li>Takahashi, A., Camacho, P., Lechleiter, J. D., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+calcium.">Measurement of intracellular calcium.</a> Physiol Rev. 79, 1089-1125 (1999).</li><li>Dongcheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+regionalisation+for+enteric+nervous+system+formation:+implications+for+Hirschsprung's+disease+and+stem+cell+therapy.">Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy.</a> Developmental Biology. 339 (2), 280-294 (2010).</li><li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behind+an+enteric+neuron+there+may+lie+a+glial+cell.">Behind an enteric neuron there may lie a glial cell.</a> J Clin Invest. 121, 3386-3389 (2011).</li><li>Boesmans, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuron-glia+interactions+in+the+enteric+nervous+system.">Imaging neuron-glia interactions in the enteric nervous system.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li></ol>

The authors have nothing to disclose.

The methodologies described in this manuscript provide a consistent approach to effectively study neurons and enteric glia in the ENS. Although imaging neurons and enteric glia in culture has yielded a wealth of insight into the function of individual cells, studying these cells in their native, multi-cellular environment is crucial for our understanding their physiology and pathophysiology. Fluorescence microscopy is a crucial technique for assessing multidirectional interactions of cells in the ENS. Loading cells of the ENS with selective fluorescent markers and image acquisition with high-resolution microscopy permits quantitative observations of cellular activity in the ENS. Imaging live tissue samples of the ENS is performed relatively quickly and generates large amounts of in-depth functional and spatial data. Mouse myenteric and submucosal plexus preparations used in these experiments allow for molecular and genetic manipulation approaches. Ca2+ imaging in whole-mount preparations provides a useful tool for the assessment of neuron-glia interactions.
In advanced experimental paradigms, several probes can be combined to obtain information about different events within the cells. Fluorescence microscopy can record images with enhanced contrast of specific molecules, if an appropriate fluorescent label is used. Fluo-4 was chosen because it possesses a large dynamic range. Sufficient incubation time is vital when using the AM dyes in ENS. Dye concentration and loading method may need to be adjusted to achieve best results. Ideal preparations should be loaded with sufficient dye to visualize changes in fluorescence but not so much so that the dye chelates the target ions and interferes with intracellular signaling. Exposure to fluorescent light should be limited to prevent phototoxicity in cells and photobleaching of dyes. 
Investigators must be careful with several steps of this experiment, especially solution and tissue preparation. Particular care has to be taken during processing and dissection of ENS tissue in order to maintain cellular functions. The GI tract contains several layers and tissue varieties, which pose challenges for dissection and imaging quality in these whole-mount preparations 27. Furthermore, the interconnecting fiber tracts of the MP are wider and ganglia are larger than those of the SMP 2. The neuronal density of the myenteric plexus is higher compared to that of the submucosal plexus 28. Slow and imprecise dissections will have detrimental effects on the quality of the plexus preparations and thus the overall success of the experiments. Therefore, clean/undamaged tools, practice and manual dexterity are critical to this procedure. 
In whole-mount tissue preparations, careful consideration should be taken when drawing the regions of interest (ROI) to correctly assess the kinetics and degree of observed change in fluorescence intensity of the desired cell type. As the ganglia are located on a contractile muscle layer, motion artifacts caused by gut motility are a primary concern during in situ imaging. Thus, suppressing these motion disturbances through re-pinning tissue preparations after incubation with enzymes and the addition of pharmacological inhibition (nicardipine/scopolamine) to buffers permits clear and reliable image acquisition. Aside from pharmacology and mechanical approaches to prevent tissue movement, recent studies illustrate the application of advanced software methodologies and cell type response characteristics to correct for residual tissue movement in the recordings and improve the accuracy of analysis 29. Barring these technical hurdles, this method provides physiologically relevant conditions to assess morphologic and quantitative characteristics of neurons and enteric glia in the ENS.

肠神经系统（ENS）被组织成嵌入消化道1的壁内的两个神经节丛。这些肌肉的神经回路中，肠肌丛（MP）和粘膜下丛（SMP），是由神经元和神经胶质肠溶（ 图1）2。在MP和SMP调节胃肠（GI）功能，例如肠能动性和上皮的吸收和分泌，分别为3。肠溶胶质位于靠近内神经节的神经元，但肠溶胶质细胞种群内互连纤维束和肠壁3,4的外神经节部也存在。肠神经胶质细胞原本被认为只提供给神经元营养支持。然而，最近的研究有力地表明，神经元的神经胶质细胞的相互作用是必不可少的ENS功能5,6。例如，数据显示，肠溶胶质"收听"神经元活动7和调节神经元电路6,8，保护肠神经元免受氧化应激9，并且能够响应于损伤10,11产生新的肠神经元的。在此技术审查提出的协议提供了一种简单而可靠的方法来检查使用原位细胞内Ca 2+成像的神经元和神经胶质肠溶之间复杂的相互作用。 钙离子是在可兴奋细胞中普遍存在的信号分子，并起着突触信号事件在神经系统12中起重要作用。神经元或神经胶质细胞肠道激发无论是通过大量涌入的Ca 2+ -permeable通道或钙诱发细胞质中钙离子浓度的升高，从细胞内钙库离子释放 。成像钙瞬变的神经元和神经胶质细胞与荧光染料是既定的和广泛使用的技术，研究功能组织和动态在ENS 13-17。的Ca 2+成像已经被证明是在研究完整的GI组织片段通过ICC起搏器网络18和肠平滑肌19,20阐明兴奋性传播的重要工具。它使研究人员能够探测生理参数的广谱，并提供有关他们俩的空间分布和时空动态的信息。细胞可以通过使用膜可渗透荧光指标和优化的染色方案21被有效地染色以微创方式。这提供了机会，以监视大量的神经元和神经胶质肠溶在功能上保留制剂14-16,22，以及在体内 23。整个安装组织标本是散装装载有高亲和力的Ca 2+指示剂染料如荧光4当结合的Ca 2+，增加其荧光。变化的荧光用CCD照相机和ANA记录裂解数字6。钙离子的出现提供了机会以监测神经元和神经胶质细胞的相互作用，响应于各种刺激，而这些细胞类型的胃肠道的过程中实时的参与。 在原位 钙成像已经取得了巨大的洞察肠道神经元和神经胶质细胞的信号传导机制和过细胞培养模型6,24具有几个明显的优势。首先， 在原地准备维持神经元和神经胶质细胞的原生基质环境，让他们的大部分连接到靶组织完好无损。二，遗传学和培养肠道神经胶质细胞的形态是显著已发生变化相比， 在体内 6,24。第三，许多异型相互作用丢失在原代细胞培养，这限制评估细胞 - 细胞相互作用。虽然培养细胞非常适合于调查基本性质，它们的usefulneSS学习肠神经胶质细胞和神经元之间复杂的相互作用是有限的。使用原位方法研究神经元的神经胶质细胞相互作用更生理有关的突触途径保持不变25。相比于细胞培养方法， 原位方法提供了改进的条件，系统地了解神经元和神经胶质细胞肠道之间错综复杂的相互作用。此外，在整个安装制剂的神经节丛的平面组织是理想的细胞内Ca 2+瞬变的荧光成像和该技术提供了用于评估在ENS神经元-神经胶质细胞活性的简单的方法。

<table><tbody><tr><td>BubbleStop Syringe Heater&amp;nbsp;</td><td>AutoMate Scientific</td><td>10-4-35-G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C3306</td><td></td></tr><tr><td>Collagenase, Type II, powder</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Sigma-Aldrich</td><td>42613-33-2</td><td></td></tr><tr><td>Dissection tools</td><td>Roboz&amp;nbsp;</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Fixed-stage microscope</td><td>Olympus&amp;nbsp;</td><td>BX51WI</td><td></td></tr><tr><td>Fluo-4 AM dye</td><td>Invitrogen</td><td>F-14201</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>Minutien Pins</td><td></td></tr><tr><td>iQ Live Cell Imaging Software</td><td>Andor</td><td>Andor iQ3</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S8282</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>Neo sCMOS camera</td><td>Andor</td><td>Neo 5.5 sCMOS</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma</td><td>N7510</td><td></td></tr><tr><td>Perfusion chamber</td><td>Custom</td><td></td><td></td></tr><tr><td>Peristaltic pump</td><td>Harvard Apparatus</td><td>Model 720</td><td></td></tr><tr><td>Pluronic F-127&amp;nbsp;</td><td>Invitrogen</td><td>P3000MP</td><td></td></tr><tr><td>Probenecid</td><td>Molecular Probes</td><td>P36400</td><td></td></tr><tr><td>Scopolamine</td><td>Sigma</td><td>S1013&amp;nbsp;</td><td></td></tr><tr><td>Sutter Lambda DG-4</td><td>Sutter</td><td>DG-4</td><td></td></tr><tr><td>Sylgard&amp;nbsp;</td><td>Dow Corning</td><td>184</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments</td><td>TC-344C</td><td></td></tr></tbody></table>

in situ ca2+ imaging of the enteric nervous system

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

正确使用这种技术可以让研究人员准确地测量细胞内钙的[Ca 2+] i的瞬态肠神经元和神经胶质细胞在整个安装组织准备。激动剂诱发的Ca 2+在神经胶质细胞内从小鼠结肠肌间神经节反应的代表性实例示于视频1。下面的结果是为了说明，我们使用这种方法获得的一些有代表性的结果。首先， 图2显示了一个实验测量肠神经胶质结果的[Ca 2+] i变化的范围内豚鼠结肠纵肌间神经丛的肌肉（LMMP）准备应对刺激ATP。具体地，该图显示了该方法用于上述包括所分析的肌间神经节和星号表示肠神经元的位置的轮廓中列出的实验方案的适当分析。这些结果还我llustrate的百微摩尔ATP最佳剂量的动员[ 钙 ]在我豚鼠肌间神经胶质细胞。此响应可以被用于校准肠溶胶质刺激和标准化的响应测试刺激。接下来， 图3阐明了如何正确选择感兴趣（投资回报）周围的神经胶质细胞的区域，显示与周围的黄色圆圈。这些结果还表明在基础条件下，并且响应于药理刺激所需的荧光变化。最后， 图4展示的空间考虑选择肠神经胶质细胞和神经元的[Ca 2+] i的整个安装准备响应。 <img alt="图1" src="/files/ftp_upload/52506/52506fig1highres.jpg" /> 图1.组织的ENS的 ENS包含两个主要的神经节神经丛。肌间神经丛位于升之间ongitudinal和圆形肌肉层。黏膜下神经丛位于粘膜和圆形肌肉层之间。该ENS仅仅由神经元和神经胶质细胞肠道的。神经纤维束连接神经节。 <a href="https://www.jove.com/files/ftp_upload/52506/52506fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52506/52506fig2highres.jpg" /> 图2.肠道神经胶质细胞在豚鼠结肠肌间神经丛响应到ATP 原位 。 （A）的肌间神经节荧光4荧光基础条件下（虚线所述）。箭头指向厚interganglionic纤维束（B）的刺激后用100微摩尔/ L的ATP，神经胶质细胞，但不是神经元，迅速 ​​增加的Fluo-4的荧光指示增加的[Ca 2+]&gt;我。需要注意的是响应细胞体积小，周围的大得多的神经元（标记星号黑暗的空间）。（C）肠神经胶质细胞具有剂量依赖的方式与1 mmol / L的引发最大反应24回应ATP。 <a href="https://www.jove.com/files/ftp_upload/52506/52506fig2large.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a> <img alt="图3" src="/files/ftp_upload/52506/52506fig3highres.jpg" /> 图3.小鼠的S-100-GFP +细胞在结肠肠肌丛响应原位到ATP。（A）的 S-100-GFP +的小鼠结肠肌间神经节的神经胶质细胞（绿色）（由虚线画出的）。六个地区的利益（投资回报）​​周围神经节内的神经胶质细胞显示为黄色圆圈。箭头表示厚的纤维束领先进入神经节。星号ð2肠神经元的刺激后用100微摩尔/ L的ATP enote位置。（B）中相同的神经节表示基础条件下RHOD-2荧光。（℃），神经胶质细胞具有增加的[Ca 2+] i的如通过增加Rhod-所示响应2荧光。（D）的痕迹对应于A-C中所示的每个投资回报率。（E）的平均值24 D所示的6 ROI的反应（平均值±SEM）。 <a href="https://www.jove.com/files/ftp_upload/52506/52506fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52506/52506fig4highres.jpg" /> 图4. 在肠神经元对胶质细胞通信的现场影像。从钙成像实验（A）代表的图像（伪彩色），其中整体式安装准备肌间神经丛的神经元P2X7受体激动剂BzATP（100微米，30秒）的挑战。需要注意的是神经激动剂导致前周边肠溶胶质细胞（A"） ​​的增加Fluo4荧光的神经元（A&#39;）。（B）的荧光随时间的变化中的神经胶质细胞（蓝色）和神经元分析（红）以下应用神经激动剂BzATP（℃）的神经元和神经胶质反应BzATP在正常缓冲液（实线），并在缓冲液含低的Ca 2+和Mg 2+（虚线），以使可能的神经性P2X 7受体13。 <a href="https://www.jove.com/files/ftp_upload/52506/52506fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在肠神经胶质视频1激动剂诱发的Ca 2+响应现场，该视频显示，从装有钙指示剂染料，荧光4鼠标远端结肠肌间神经节。胶质细胞激动剂，ADP添加到浴中时表示。 ADP引发2+在荧光4荧光性升高观察到增加细胞内钙在肠道神经胶质细胞。 <a href="https://www.jove.com/files/ftp_upload/52506/JOVE.mp4" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about In Situ Ca2+ Imaging of the Enteric Nervous System at JoVE.com

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

in-situ-ca2-imaging-of-the-enteric-nervous-system

Research

JoVE Journal

Neuroscience

In Situ Ca2+ Imaging of the Enteric Nervous System

17.1K 次观看.  Michigan State University. The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging. 

视频: In Situ Ca2+ Imaging of the Enteric Nervous System

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

科研

生物化学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

神经科学

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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.

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

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

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

大鼠三叉神经节与钙指示剂的体内可视化

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

作者聚焦：探索三叉神经损伤中神经性疼痛的外周机制 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

体内 大鼠三叉神经节感觉神经元的钙成像

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

在肠神经系统的原位 ^钙成像

摘要

探索更多视频

此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内小鼠劣质橄榄中的钙成像

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

体内神经元钙处理的亚细胞成像

体内大鼠三叉神经节感觉神经元的钙成像

体内大鼠三叉神经节感觉神经元的钙成像

体内大鼠三叉神经节感觉神经元的钙成像

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

在肠神经系统的原位 钙成像

摘要

探索更多视频

此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

在体内 小鼠劣质橄榄中的钙成像

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

体内神经元钙处理的亚细胞成像

体内 大鼠三叉神经节感觉神经元的钙成像

体内 大鼠三叉神经节感觉神经元的钙成像

体内 大鼠三叉神经节感觉神经元的钙成像

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

在肠神经系统的原位 ^钙成像

在体内小鼠劣质橄榄中的钙成像

体内完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

体内大鼠三叉神经节感觉神经元的钙成像

体内大鼠三叉神经节感觉神经元的钙成像

体内大鼠三叉神经节感觉神经元的钙成像