我们感谢孟博士林青女士远东的技术援助。这项工作是从美国国立卫生研究院HD053767（转包NLW），由授出从生理和副校长办公室的研究，美国加州洛杉矶大学（NLW）部的资金支持。

1。成人脑解剖鳉<ol><li>麻醉成年男性或女性（ 图1A）浸泡5毫升的MS-222（150毫克/升，pH值7.4），等待一两分钟后，鳃变动之前已经停止斩首。加州洛杉矶大学的机构动物护理和使用委员会批准的所有程序。 </li><li>杀头在盐水鱼的鱼鳃盖的尾端用剪刀在一个直径为60毫米的培养皿。 </li><li>传输鱼头一个直径为35毫米的培养皿中，一半充满了盐水鱼及内衬SYLGARD有抑郁症，它为大脑解剖定位和固定头部。位置和固定头背的一面朝上，昆虫针。 </li><li>小心切断背侧部围绕其周边的​​头骨与细剪刀，轻轻取出镊子，露出大脑完全包括前部的脊髓背侧部分。 </li> &lt;LI&gt;轻轻抬起脊髓细尖镊子和断绝双边结缔组织的细尖的剪刀:颅神经，脊神经，视神经，大脑腹侧神经，嗅觉神经的前部大脑。 </li><li>将充分解剖的大脑在一个充满盐水鱼的新鲜的培养皿中，并在显微镜下仔细检查，以确保没有任何切口或穿刺，全脑是否完好（包括脑垂体）。受损的大脑不用于实验。 </li></ol> 2。在录音室中安装的大脑<ol><li>瞬干胶或其他一些快熔胶用针进入录音室的中心放置一小滴和传播胶约1厘米2。 </li><li>迅速转移大脑和胶水中腹的一面朝上位置。 </li><li>添加1毫升的盐水鱼，使其充满录音室，并涵盖了大脑。不断灌注的大脑与曝气鱼生理盐水至少为200微升/分钟的速率。 </li><li>使用细尖钳子小心地取出脑膜的大脑的表面上的记录区域。 </li></ol> 3。电<ol><li>松散的膜片钳记录动作电位射击<ol><li>玻璃微电极（6-10MΩ）构建的硼硅玻璃吸管（1B150F-4，世界精密仪器）使用电极拉马（ 如萨特的P-87），和背面充满了半路上了松散的膜片录音解决方案电极。 </li><li>使用一个直立的荧光显微镜用冷却的电荷耦合器件（CCD）摄像机，发现GFP-表达TN-GnRH3的神经元通过10倍（ 图1B），然后40倍的水浸泡的目标（ 图1C）。它们存在于嗅球，只是前端脑腹侧表面处或附近。 </li><li>切换到视频显示器提供从显微镜的实时图像定位的TN-GnRH3的神经元集群（ 图1C）。 </li><li>微电极定位入熔池，使尖端的电​​极上方的神经元靶。适用于恒定的轻微的正压力，以便它不被堵塞，并轻轻地接近目标神经元与电极尖端的微电极。 </li><li>检查和监控电压钳模式的使用AxoGraph软件和的放大器Axoclamp 200B与Axograph软件（Axon仪器）的电极电阻。的时候可以看到，电极尖端从视频监视器的表面上的神经元和从的AxoGraph显示的电阻略有变化，释放的正压力，并如果有必要施加轻微的负压力，使一个低电阻的密封件的神经元（&lt;100MΩ）。 </li><li>切换电流钳模式下，连续记录膜电压没有任何电流输入，调整规模电压和如果有必要的记录率。 PowerLab系统软件（的ADInstruments公司）的数据收集和分析。 </li><li>记录基线电活动的正常鱼的盐水任何治疗（ 图2A）的前5分钟左右。浴灌注药物，激素，肽等 。添加到鱼盐水继续上面的速率为200微升/分钟，然后用洗脱期在正常鱼生理盐水5，6，11。 </li></ol></li><li>全细胞记录膜电位 膜片钳全细胞电生理过程是相同的如上述外松补丁录制过程中，从步骤3.1.1 - 3.1.4。然后程序相应的偏离: <ol><li>检查和监控电压钳模式的电极电阻。的时候可以看到，电极尖端从视频监视器的表面上的神经元，并从示波器的电阻变小，释放的正压力和施加一点点的负面压力，使高电阻印章上的神经元（〜3千兆Ω）。轻轻吸口破裂修补膜，你会看到一个突然下降的阻力约120-250MΩ。 </li><li>切换电流钳模式下，连续记录膜电压没有任何电流输入，调整电压和如果有必要的记录速率的规模。 </li><li>记录基线电活动的正常鱼生理盐水任何治疗前，约5分钟，如上述3.1.7中（ 图2B）。 </li></ol></li></ol>

<ol>
	<li>Kah, O., Lethimonier, C., Lareyre, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin-releasing+hormone+(GnRH)+in+the+animal+kingdom.">Gonadotrophin-releasing hormone (GnRH) in the animal kingdom.</a> J. Soc. Biol. 198 (1), 53-60 (2004).</li><li>Ramakrishnan, S., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+from+conspecifics+alter+electrical+activity+of+gonadotropin-releasing+hormone+neurons+in+the+terminal+nerve+via+visual+signals.">Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (1), R135-R141 (2009).</li><li>Abe, H., Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neuromodulation+by+a+nonhypophysiotropic+GnRH+system+controlling+motivation+of+reproductive+behavior+in+the+teleost.">Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost.</a> 57 (6), 665-674 (2011).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+persistent+Na++current+underlying+pacemaker+potentials+of+fish+gonadotrophin-releasing+hormone+neurones.">Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones.</a> J. Physiol. 482 (Pt. 1), 1-6 (1995).</li><li>Zhao, Y., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Kisspeptin1+on+Electrical+Activity+of+an+Extrahypothalamic+Population+of+Gonadotropin-Releasing+Hormone+Neurons+in+Medaka.">Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka.</a> PLoS One. 7 (5), e37909 (2012).</li><li>Wayne, N. 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=Whole-cell+electrophysiology+of+gonadotropin-releasing+hormone+neurons+that+express+green+fluorescent+protein+in+the+terminal+nerve+of+transgenic+medaka+(Oryzias+latipes).">Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).</a> Biol. Reprod. 73 (6), 1228-1234 (2005).</li><li>Okubo, K., 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=Forebrain+gonadotropin-releasing+hormone+neuronal+development:+insights+from+transgenic+medaka+and+the+relevance+to+X-linked+Kallmann+syndrome.">Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.</a> Endocrinology. 147 (3), 1076-1084 (2006).</li><li>Okubo, K., 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=A+novel+form+of+gonadotropin-releasing+hormone+in+the+medaka,+Oryzias+latipes.">A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes.</a> Biochem. Biophys. Res. Commun. 276 (1), 298-303 (2000).</li><li>Ramakrishnan, S., 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=Acquisition+of+spontaneous+electrical+activity+during+embryonic+development+of+gonadotropin-releasing+hormone-3+neurons+located+in+the+terminal+nerve+of+transgenic+zebrafish+(Danio+rerio).">Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).</a> Gen. Comp. Endocrinol. 168 (3), 401-407 (2010).</li><li>Abraham, E., 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=Targeted+gonadotropin-releasing+hormone-3+neuron+ablation+in+zebrafish:+effects+on+neurogenesis,+neuronal+migration,+and+reproduction.">Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.</a> Endocrinology. 151 (1), 332-340 (2010).</li><li>Wayne, N. L., Kuwahara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-endorphin+alters+electrical+activity+of+gonadotropin+releasing+hormone+neurons+located+in+the+terminal+nerve+of+the+teleost+medaka+(Oryzias+latipes.">Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes.</a> Gen. Comp. Endocrinol. 150 (1), 41-47 (2007).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+types+of+gonadotrophin-releasing+hormone+neurones+and+steroid-sensitive+sexually+dimorphic+kisspeptin+neurones+in+teleosts.">Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts.</a> J. Neuroendocrinol. 21 (4), 334-338 (2009).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. , (2003).</li></ol>

什么都没有透露。

激素3:提供了独特的绿色荧光蛋白转基因鱼模型来研究的神经生理机制神经集成中央控制和调节的行为，都直接和间接地参与再现3 8-10。这个模型系统的显着优势之一是，许多GnRH3表达GFP神经元的大脑腹面，可以相对容易获得的神经元电生理记录，不破坏神经回路6，9，11，12（ 图图1B和图1C，图3A和3B）。在这段视频中，我们已经表明了松散的膜片和鳉鱼的完整的成人大脑中从TN-GnRH3的神经元的全细胞记录。 TN-GnRH3神经元表现出一种补品或跳动模式的自发动作电位发放频率为0.5-6Hz.There自发放电没有检测到的性别差异率5，但也有戏剧性的发展变化GnRH3神经元的电活动，在胚胎发育过程中9。松散的膜片和全细胞电生理有不同的方法的优点和缺点。松散的膜片在技术上更容易比全细胞记录，但只显示了神经元放电模式和频率。另一方面，全细胞记录，可以提供更多的信息:静息膜电位，放电模式和频率，动作电位的分析，膜电容和电阻。因为松散的膜片录音不破坏细胞膜，破坏细胞内的离子和第二信使的浓度，它是理想的长期记录的神经元的活动。全细胞记录细胞膜破裂，并最终导致细胞内分子的透析，从而有可能改变神经元的性能与更长时间的录音。由于两个electrophysi的优点和缺点ological的方法，也可以是重要的研究神经元的属性，使用这两种技术。由于pH值和同渗容摩神经元的存活和功能是非常重要的，在使用中的所有的解决方案需要适当调整。 所记录的神经元的身份确认，施加负压的记录完成后，然后在电极尖端荧光证实使用显微荧光成像。小分子染料或探针（如染料或生物胞素的Alexa Fluor 568）可以被包括在全细胞记录标记的记录，可进一步的形态学分析神经元的细胞内的解决方案。 要成功记录目标完整的大脑中的神经元，神经元的位置的深度是至关重要的 - 更深层次的神经元，就越难补丁（松散的膜片和全细胞）。一般情况下，成功修补可行的深度是从表面约5-6细胞层。医学又名的TN-GnRH3神经元通常是在大脑的腹面，而斑马鱼的下丘脑GnRH3神经元可以是高达6个细胞层的表面。正压力是非常有帮助的，以防止堵塞而接近细胞记录电极。这种类型的实验方法可用于任何荧光蛋白基因标记的神经元。要在成人大脑研究GnRH神经元的活动，我们经常使用的鳉鱼和斑马鱼的4-6个月的年龄。年轻的鱼，更小的大脑或以上的鱼的头骨钙化，将使解剖更具挑战性。 TN:GnRH3神经元电从成人鳉脑方便，但是这是不是成年斑马鱼的情况下，TN:GnRH3神经元装在一个艰难的结缔组织鞘。在另一方面，GnRH3的小一入学统筹办法，VT，和下丘脑的神经元更容易获得电在成年斑马鱼脑不是从青鳉。有没有性行为差异基础上的困难，无论是物种的解剖及电生理记录。 虽然这不是一个体内实验方法，记录相对完整的神经元电路，以最小的损伤是一个有吸引力的方式来探索的生理功能，调节神经系统的兴趣。完整的大脑准备持续了几个小时，和良好的记录可以持续6小时。此协议是适合于研究谁已经有基本知识和经验的电生理技术13，以及专门的设备，需要做的工作。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>BX50W (Upright)</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axoclamp 200B </td>
 </tr>
 <tr>
 <td>A-D converter</td>
 <td>Computer Interference Corp.</td>
 <td></td>
 <td>Digidata ITC-18 </td>
 </tr>
 <tr>
 <td>Cooled CCD camera</td>
 <td>PCO Computer Optics </td>
 <td></td>
 <td>Sensicam</td>
 </tr>
 <tr>
 <td>Xenon lamp</td>
 <td>Sutter Instruments Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GFP filter set</td>
 <td>Chroma Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging Software</td>
 <td>Intelligent Imaging Innovations</td>
 <td></td>
 <td>Slidebook software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axograph software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>AD Instruments Inc.</td>
 <td></td>
 <td>PowerLab </td>
 </tr>
 <tr>
 <td>Headstage for electrophysiology</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>CV 203BU </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument Co </td>
 <td>MP-285 </td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Chamber Platform</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>P1 </td>
 </tr>
 <tr>
 <td>Recording Chamber</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>RC-26G </td>
 </tr>
 <tr>
 <td>Electrode Puller</td>
 <td>Sutter instruments</td>
 <td></td>
 <td>P87 </td>
 </tr>
 <tr>
 <td>Filament for electrode puller</td>
 <td>Sutter Instruments</td>
 <td>FB330B</td>
 <td>3.0 mm wide trough filament</td>
 </tr>
 <tr>
 <td>1.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B150-4</td>
 <td>Microelectrode for recording</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309586</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>MS-222 </td>
 <td>Sigma</td>
 <td>E10521-10G</td>
 <td>Ethyl 3-aminobenzoate methanesulfonate salt</td>
 </tr>
 <tr>
 <td>Fish saline</td>
 <td></td>
 <td></td>
 <td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES </td>
 </tr>
 <tr>
 <td>Electrode solution (loose-patch) </td>
 <td></td>
 <td></td>
 <td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose </td>
 </tr>
 <tr>
 <td>Electrode solution (whole-cell patch) </td>
 <td></td>
 <td></td>
 <td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td>
 </tr>
 </tbody>
</table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

了解细胞的生理调节复杂的行为的神经回路是通过使用模型的系统，在该系统中，可以进行这项工作，在一个完整的大脑制备的中枢神经系统的神经电路保持不变大大增强。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑中的绿色荧光蛋白基因的标签。鱼GnRH神经元有多个种群，它们的功能是依赖于他们的位置在大脑和GnRH基因，他们表达1。我们集中示范GnRH3在终端转基因青鳉鱼（ 图1B和C）使用完整的大脑嗅球神经（田纳西州）的神经元位于。研究表明，青鳉的TN-GnRH3神经元是神经调节，中枢神经系统作为信息发射器从外部环境; T嘿，不要直接发挥作用，调节垂体-性腺功能，做知名GNRH1丘脑神经元2，3。进补动作电位射击TN-GnRH3神经元的自发模式的内在属性4-6，频率是调制的同种2和神经肽kisspeptin 1 5的视觉线索。在这段视频中，我们使用了一个稳定的转基因青鳉这TN-GnRH3的神经元表达了含转基因的启动子区域Gnrh3向您展示如何识别神经元，并监察他们在整个大脑的电活动与增强型绿色荧光蛋白7制备例6。

GFP标记的TN-GnRH3神经元从切除大脑的青鳉鱼的双边集群的一个例子示于图1B和图1C。每个集群包含大约8-10 GnRH神经元。自发的神经元活动的目标TN-GnRH3的被记录在电流钳模式（I = 0），与典型的发射率0.5-6赫兹。动作电位射击模式是一个典型的补药或跳动模式，一个相当普通的峰峰间隔。样品的痕迹显示在图2（2A:宽松的补丁; 2B:全细胞）。 这种实验的方法也是成功的记录GFP标记GnRH神经元位于在成年斑马鱼（ 图3A和3B）的前脑。切除，完整的大脑中的神经元的电活动记录在上述类似的方法在青鳉松散的膜片（ 图3C）和全细胞记录（ <strong&gt;的图3D）。不像与青鳉TN-GnRH神经元，斑马鱼视前区，腹前脑和下丘脑GnRH神经元的自发动作电位射击模式往往是不规则的。 <img alt="图1" src="/files/ftp_upload/50312/50312fig1.jpg" /> 图1。动物模型，用于TN-的神经元GnRH3研究在这个视频。答:成人青鳉GnRH3:GFP转基因鱼，左:男，右:女，B:鉴于前脑腹侧共聚焦图像。 OB:嗅球; TN:终端神经GnRH3神经元; TELE:端脑，视神经C:高倍率的TN-GnRH3神经元显示面板B.白盒（比例尺:A:5毫米; B:100微米，C:20微米）。 <img alt="图2" src="/files/ftp_upload/50312/50312fig2.jpg" /> 图2。斯波ntaneous动作电位在电流钳模式（I = 0）从TN-GnRH3的神经元的记录。 答:样品一丝松散的膜片记录，B:样品从全细胞记录的痕迹。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50312/50312fig3highres.jpg" src="/files/ftp_upload/50312/50312fig3.jpg" /> 图3。记录GnRH3:EMD（翡翠绿色荧光蛋白）成年转基因斑马鱼9完整的大脑准备使用相同的实验方法所描述的青鳉脑制剂的神经元活动。答:GnRH3:EMD神经元位于前脑腹侧（VT）和视前区成年斑马鱼的前脑区（POA），B:GnRH3:EMD神经元位于下丘脑（海波）。标尺:100微米。C:样品一丝松散的膜片录音GnRH3:EMD神经元VT D:样品跟踪从VT全细胞膜片记录。

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

在这段视频中，我们将演示如何记录保留复杂的神经回路在整个大脑的准备，从确定单个神经元的电活动。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑制备一种荧光蛋白基因的标签。

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

recording-electrical-activity-from-identified-neurons-intact-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of California, Los Angeles. 在这段视频中，我们将演示如何记录保留复杂的神经回路在整个大脑的准备，从确定单个神经元的电活动。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑制备一种荧光蛋白基因的标签。

视频: Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

科研

神经科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...