“ERA - NET神经元”的框架，这项工作是由寿险公司“诺瓦利斯Taitbout”，巴黎高等DES神经科学（ENP），法新社国立德拉Recherche“的ANR - 09 - NEUR - 004”的支持由欧洲委员会第七框架计划，“基金会争取RECHERCHE Medicale”，Letten基金会和巴斯德基金会。

1。立体定向注射<ol><li>在这些实验中使用的病毒是一个艾滋病毒的复制缺陷型慢病毒载体来表达Channelrhodopsine2 - YFP（黄色荧光蛋白）融合构建一个突触子 6驱动。质粒是慷慨提供K. Deisseroth的研究组（ <a href="http://www.stanford.edu/group/dlab/optogenetics/index.html">http://www.stanford.edu/group/dlab/optogenetics/index.html</a> ）。慢病毒可以从商业渠道获得，或在实验室中根据既定协议 7 。平均载体滴度均在10转导单位/ ml的顺序。病毒分装储存于-80 ° C和移液器装载前应解冻。应避免多个冻结/解冻周期。从未曝光的病毒解决方案，直接干冰。 </li><li>准备玻璃移液器使用标准移液器拉马（萨特P硼硅玻璃毛细管-97吸管拉马）。拉动设置应正确校准，以产生长期的和僵化的的吸管。接下来，将使用一把剪刀，获得了直径30-40微米的尖端吸管尖。存放在无尘箱拉移液管。 </li><li>安装与制造商的指示交付50 NL Nanoject二微量。准备回填土与矿物油的注射针，并把它插入到微量柱塞。连接到一个立体框架的微量。从玻璃吸管填充病毒解决方案之前，部分空油</li><li>随着微，转移到一个封口膜无菌，2μL病毒解决方案。轻轻地降低到病毒解决方案中的玻璃吸管，并填写1-2μL移液器。确保有移液器无气泡。 </li><li> 100毫克/公斤氯胺酮和10毫克/公斤甲苯噻嗪，在无菌生理盐水稀释麻醉鼠标。删除从头皮的头发动物用老鼠的快船，剃刀，剪刀，或化学depilitory剂。头皮消毒三交70％的乙醇和优碘的申请。所有手术器械也应进行高压灭菌，然后用70％乙醇消毒。放置在动物耳朵酒吧和鼻子吧的立体框架，确保头部安全。应用眼膏，防止角膜干燥。 </li><li>用手术刀，切开头皮，眼睛之间的两耳之间。一边拉扯皮肤暴露的头骨和一双夹具锚皮肤。清洁表面的头骨。前囟门玻璃吸管提示立体定位仪是零。然后提示是定位在注射部位（安特罗后路，3.30;德尔梅迪奥侧面，± 0.82，左，右半球，分别）。鼻栏的位置进行调整，根据立体坐标的计算。在我们的实验中，鼻栏调整üntil前囟及注射部位对齐到相同的高度。 </li><li>确定注射部位。使用高速的手术钻，小心地钻入颅骨的孔。迷上尖端弯曲的注射器针，瘦骨取出残余，暴露硬脑膜。检查该洞是在正确的位置为中心。在钻井过程中大脑的表面不小心破裂的血管。如果血管破裂，轻轻按到用小棉签或组织几秒钟的大脑表面，直到血液流动停止。 </li><li>下移液器和零背腹的高度时，吸管尖触及大脑表面。然后，降低到正确的背腹深（背腹，脑表面2.9），并等待30秒，压力平衡。注资共200 NL病毒的4倍50 NL。等待30秒之间的每次注射。 </li><li>一分钟最后一次注射后，慢慢退出管TTE。另一半球的重复点1.6-1.8。从立体定位仪中取出的动物，清洁切口和缝合切开皮肤，用手术线程。让动物恢复变暖垫，然后再返回到笼。此外，动物可以后立即给予非甾体类消炎镇痛药（如卡洛芬4mg/kg），手术后3天。 </li></ol> 2。慢性LED植入体内 optogenetic刺激嗅球<ol><li>与慢病毒颗粒注射动物双边未来长期植入了微型蓝色LED（OSRAM LED CMS 4.6W蓝色）。焊接的LED引脚，一个女的微型电机连接，使连接器上的LED对面定位。检查有没有电的捷径，并确定连接器上的正面和负面的的引脚。通过它连接到一个电流控制器LED虽然是正确的电缆测试的LED。 </li><li>为了测量和/或设置绝对光功率的LED，装入一个显微和位置上的LED的LED与功率计的一个峰值在470 nm的光强度的光探测器直接接触。钻在一个不透明的PVC板3毫米直径的针孔。贴上这个针孔功率计和建设标准曲线的光功率与输入电流来计算每平方毫米的电源。 </li><li>要通过大脑的测量光的传播，减少新鲜脑组织300块，500，1000，1500和2500在0-4 ° C学联使用一个vibratome 14微米厚度。放置一个开颅手术在活体小鼠下的LED相同的一块头骨。首先，没有对LED为中心的组织测量光功率。然后，将每个组织块之间的LED和功率计的光电探测器，并测量功率。建立一个组织厚度与相对传输分数的曲线，按比例计算通过组织和无组织所测得的功率之间。 </li><li> 100毫克/公斤氯胺酮和10毫克/公斤甲苯噻嗪，在无菌生理盐水稀释麻醉鼠标。清洗和消毒用70％乙醇的LED。取出头发从头皮的动物和消毒头皮有三个交70％的乙醇和优碘的申请。在立体框架，然后将动物。应用眼膏，防止角膜干燥。将连接器连接到一个立体的持有人。嗅球以上的头骨表面，直到完全水平和平行于表面的微型LED，鼻栏的位置应调整。 </li><li>用手术刀，切开头皮，从3毫米前到3毫米，后耳朵眼睛。一边拉扯皮肤和一双夹具锚。刮膜，在使用刀片的头骨表面。清洁和干燥的头骨。应用氰基丙烯酸酯胶粘剂的薄盖，以便随后的广告粘附的牙齿丙烯酸。 </li><li>使用一个高速的手术演练，执行一个长方形的开颅手术在嗅球（尾椎1毫米和3毫米的横向5.1毫米尾鳍为本，从囟外侧为0毫米）。要做到这一点，仔细薄的头骨，直到只有一层薄薄的骨遗骸。小心取出剩余的骨弯曲的注射器针头的帮助。应采取特别小心，以避免刺破硬脊膜问题和破裂在于两个半球之间的嗅球的尾部边界的主要血管。在大脑表面的积血和凝血排除最佳的光线穿透进入组织。冲洗开颅手术，并用无菌生理盐水的大脑表面，然后干的头骨。 </li><li>将LED连接器的缩影。下顶部的嗅球上的微型LED。微型LED的主轴线应鼠标喙尾椎轴平行，用无菌生理盐水和D清洁RY的头骨。然后安全的LED头骨，第二层的牙科丙烯酸与丙烯酸的第一层（即，聚羧酸水泥） 。另外两个小螺丝，可能需要修复的LED。螺丝应插在颅骨（后LED的位置）和两项丙烯酸涵盖。虽然丙烯酸仍然是液体，拉回来和胶水牙科丙烯酸表面上的自由边缘的头皮。注意不要将连接器适用于任何丙烯酸。丙烯酸硬化后，删除从立体定位仪上的动物，让恢复变暖垫，然后再返回到笼。动物离开隔离笼从手术中恢复过来，至少7天。此外，动物可以后立即给予非甾体类消炎镇痛药（如卡洛芬4mg/kg），手术后3天。 LED应终止后，植入前和实验测试。 </li><li>光电气CAL电缆是用来系绳的动物加上一个脉冲发生器（主8）控制的时间和强度的LED为LED电流控制器。细缆允许完全的行动自由。照顾方面的连接器的极性。反相极性可能打破LED。 </li></ol> 3。代表性的成果： ChR2 - YFP转约48小时后注射神经元表示，和几个连续7个月保持稳定。图1是一个代表了一个多聚甲醛固定的动物，15天注射后矢状切面画面。注意茂密的标签和注射部位在RMS的大小。大量的新生细胞迁移沿RMS拓殖嗅球的不同层次（图1）。融合蛋白表达在所有的神经细胞车厢，包括胞体，树突棘。 小型的LED impla<html> ntation允许大规模和廉价的一个兴趣广泛的区域的光刺激。动物是拴在光电缆，允许完全的行动自由的LED控制器。通过描绘在脑组织和ChR2激活阈值的光扩散，我们估计，光活化可触发〜2毫米低于LED的表面（图2A）的深度。要监视功能的激活，表达ChR2的神经元在体内表达的c - fos，神经元活动的标志，可以分析重复性的光刺激后（图2B）。作为一种替代方法， 在体外表达ChR2，再加上焦光刺激神经 ​​元的电生理记录可以执行 14 。最后，双光子针对性的宽松的补丁录音已被用于研究ChR2表达皮层神经元功能激活 体内 9 LED类似的缩影。 ntent“&gt; <img alt="图1" src="/files/ftp_upload/3380/3380fig1.jpg" /> 图1。 ChR2 - YFP的表达慢病毒注射到延髓的迁徙流 。鼠标在喙RMS的慢病毒注射后15天嗅球一个矢状部分代表共聚焦图像。注意分散YFP的新生神经元迁移的RMS年底OB的不同层次的存在。大多数（95％），新生细胞颗粒细胞，它在颗粒细胞层（GCL）和树突的SOMA在于外部网状层（EPL）的arborize。一个新生神经元的小的人口迁移OB的最表面的一层，小球层，（GL）。比例尺，100微米。插图，高倍率新生儿的颗粒细胞，树突以及在刺膜表达ChR2 - YFP。比例尺，20μm的。 <img alt="图2" src="/files/ftp_upload/3380/3380fig2.jpg" /&gt; 图2。图植入嗅觉灯泡和ChR2 - YFP光诱发c - fos表达，表达新的颗粒细胞的顶部的LED。 一个示意图上述嗅球植入LED。 B，双标ChR2 - YFP的代表共聚焦图像显示+ c - fos的一小时后，光刺激嗅球颗粒细胞。 c - fos表达显露使用兔抗- c - fos基因抗体（Calbiochem，1:5000）和A568标记的抗兔二抗（Invitrogen公司，1:1000）。比例尺为10μm。

<ol>
	<li>Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+and+functional+plasticity+in+neuronal+circuits.">Adult neurogenesis and functional plasticity in neuronal circuits.</a> Nat. Rev. Neurosci. 7, 179-193 (2006).</li><li>Alonso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+astrocytes+from+the+rostral+migratory+stream+into+neurons:+a+role+for+the+olfactory+sensory+organ.">Turning astrocytes from the rostral migratory stream into neurons: a role for the olfactory sensory organ.</a> J. Neurosci. 28, 11089-11102 (2008).</li><li>Mouret, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+survival+of+newly+generated+neurons:+when+time+matters.">Learning and survival of newly generated neurons: when time matters.</a> J. Neurosci. 28, 11511-11516 (2008).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How,+when,+and+where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">How, when, and where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> J. Neurosci. 30, 17023-17034 (2010).</li><li>Haubensak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+an+amygdala+microcircuit+that+gates+conditioned+fear.">Genetic dissection of an amygdala microcircuit that gates conditioned fear.</a> Nature. 468, 270-276 (2010).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+fast+optical+interrogation+of+neural+circuitry.">Multimodal fast optical interrogation of neural circuitry.</a> Nature. 446, 633-639 (2007).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+Production.">Lentivirus Production.</a> J. Vis. Exp. (32), e1499-e1499 (2009).</li><li>Cardin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+optogenetic+stimulation+and+recording+of+neurons+in+vivo+using+cell-type-specific+expression+of+Channelrhodopsin-2.">Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2.</a> Nature protocols. 5, 247-254 (2010).</li><li>Huber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+optical+microstimulation+in+barrel+cortex+drives+learned+behaviour+in+freely+moving+mice.">Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice.</a> Nature. 451, 61-64 (2010).</li><li>Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+light-induced+activation+of+neural+circuitry+in+transgenic+mice+expressing+channelrhodopsin-2.">In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.</a> Neuron. 54, 205-218 .</li><li>Albeanu, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+arrays+as+cost+effective+and+efficient+light+sources+for+widefield+microscopy.">LED arrays as cost effective and efficient light sources for widefield microscopy.</a> PLoS ONE. 3, 2146-2146 (2008).</li><li>Slotnick, B., Bodyak, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+Discrimination+and+Odor+Quality+Perception+in+Rats+with+Disruption+of+Connections+between+the+Olfactory+Epithelium+and+Olfactory+Bulbs.">Odor Discrimination and Odor Quality Perception in Rats with Disruption of Connections between the Olfactory Epithelium and Olfactory Bulbs.</a> J. Neurosci. 22, 4205-4216 (2002).</li><li>Gradinaru, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+approaches+for+diversifying+and+extending+optogenetics.">Molecular and cellular approaches for diversifying and extending optogenetics.</a> Cell. 141, 154-165 (2010).</li><li>Valley, M., Wagner, S., Gallarda, B. W., Lledo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Affordable+LED+Arrays+for+Photo-Stimulation+of+Neurons.">Using Affordable LED Arrays for Photo-Stimulation of Neurons.</a> J. Vis. Exp. (57), e3379-e3379 (2011).</li></ol>

没有利益冲突的声明。

Optogenetic工具最近增加了我们对选择性的神经元的人群，包括出生于成人的嗅觉系统神经元的控制。在这里，我们描述了如何执行的慢病毒载体表达ChR2 - YFP的成人出生的神经元的具体人口的立体注射。通过仔细监测期间注射后，我们能够分析和处理出生于一个相对同质化时代的成人神经元群活动。 由于他们的低扩散蔓延，其高转导效率，其相对良好的RMS神经母细胞向性，慢?...

<table border="1"><tbody><tr><td> 材料 </td><td> 公司 </td><td> 目录号 </td></tr><tr><td>氯胺酮</td><td>梅里亚</td><td> Imalgène 1000 </td></tr><tr><td>甲苯噻嗪</td><td>拜耳医药保健</td><td> Rompum 0.2％ </td></tr><tr><td>移液器拖轮</td><td>萨特</td><td> P - 97 </td></tr><tr><td>玻璃毛细管（3.5“） </td><td>德拉蒙德科学</td><td> 3 - 000 - 203 - G / X </td></tr><tr><td> Nanoject二微量</td><td>德拉蒙德科学</td><td> 3-000-204 </td></tr><tr><td>高速手术钻头</td><td>精细科学工具</td><td> 18000-17 </td></tr><tr><td>微型钻头钢毛刺0.7毫米尖端直径</td><td>精细科学工具</td><td> 19008-07 </td></tr><tr><td> LED CMS的蓝色4.6 W </td><td>欧司朗</td><td> LBW5SN </td></tr><tr><td>女电连接器（2.54毫米） </td><td>菲舍尔Elektronik公司</td><td> BL5.36Z </td></tr><tr><td>用于LED电流控制器（最大1A） </td><td> A1W Electronik </td><td>天文台- KL50 - 1000 </td></tr><tr><td>脉冲发生器</td><td> AMPI </td><td> MASTER - 8 </td></tr><tr><td>光功率计</td><td>托尔实验室</td><td> PM100 </td></tr><tr><td>瞬干胶</td><td>乐泰</td><td> 454 </td></tr><tr><td>聚羧酸水泥（第一层水泥） </td><td> Septodont </td><td> Selfast（＃0068Q） </td></tr><tr><td>甲基丙烯酸水泥（第二层水泥） </td><td>气相色谱InternationalCorp。 </td><td> Unifast繁体 - 液（＃339292） Unifast繁体 - 粉（＃339114） </td></tr><tr><td>卡洛芬</td><td>辉瑞公司</td><td> Rimadyl </td></tr></tbody></table>

selective viral transduction of adult-born olfactory neurons for chronic in vivo optogenetic stimulation

当地interneurons不断再生1-3成年啮齿动物的嗅球。在这个过程中，被称为成年神经发生，神经干细胞在侧脑室壁引起神经母细胞迁移沿喙迁徙流（RMS）达到并纳入到嗅球几毫米。为了研究不同的步骤和成人出生的神经元整合到预先存在的嗅觉电路的影响，这是必要的选择性标签和操纵的这种神经元的具体人口的活动。 optogenetic技术最近的发展提供了机遇，利用光，正是这个特定的神经元激活，而不会影响周围神经元 4,5队列。在这里，我们目前在神经母细胞的时间限制在RMS队列一个维拉利表达Channelrhodopsin2系列程序（ChR2）YFP的，在他们到达嗅球之前，成为成人出生NEurons。此外，我们将展示如何植入和校准为慢性LED 在体内刺激的ChR2表达神经元的一个缩影。

Watch this Scientific Journal Video about Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation at JoVE.com

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

成人出生在嗅球的神经元可optogenetically控制使用Channelrhodopsin2表达慢病毒注射在延髓的迁徙流和植入微型慢性photostimulation的LED。

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

selective-viral-transduction-adult-born-olfactory-neurons-for-chronic

Research

JoVE Journal

Neuroscience

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

15.4K 次观看.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). 成人出生在嗅球的神经元可optogenetically控制使用Channelrhodopsin2表达慢病毒注射在延髓的迁徙流和植入微型慢性photostimulation的LED。

视频: Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

科研

神经科学

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...