资助由NIH P40 OD010952，Korein基金会，迈阿密大学奖学金到SLC和ATK美泰奖学金。作者非常感谢工作人员的海兔 ，以及劳伦Simonitis的汉娜·佩克，谁提供了一个数字显微国家资源。

1。细胞的制备<ol><li>在第1天，体重和麻醉动物。 <ol><li>称取30 G-1公斤动物。麻醉1:1海水量在5-10动物：等渗氯化镁。 6H 2 0 1小时，同时通气诸如电动水族馆空气泵连接的气石。 </li></ol></li><li>准备夹层用品。 <ol><li>组装干净的夹层托盘用不锈钢直针，如面料销。组装几个培养皿中含有人工海水（ASW 见表1和表3）+青霉素/链霉素（P / S）。 </li><li>已经准备好一个低功耗的显微镜。有干净的清扫工具准备好了，，如肋鼻子和精细的手术钳，罚款和大型手术剪刀。喷仪器70％isopropol酒精使用前晾干。 </li></ol></li><li>将动物解剖托盘。 <ol><li>位置动物腹侧下来在解剖托盘。引脚动物通过的体壁边界parapodial，但避免尾部和头部，背表面暴露生殖器槽的边缘</li><li>在慢速运转的冷自来水冲洗背水面。从挤压瓶沿生殖器槽和头顶以上的应用光流70％isopropol酒精，。 </li></ol></li><li>最初的切口。 <ol><li>使用一个低功率显微镜（ 见表2）和反射光观察生殖器槽来源于前壳边缘点，拉紧保持与带肋鼻钳组织褶皱的这个原点的左侧，而剪断一个浅洞体壁光滑，平整面积只是用细剪刀角度生殖器槽右侧的所有的方式通过。 </li><li>应观察淋巴倒孔，有时带着它的腹神经节和胃。 </li></ol></li><li>扩大切口。 <ol><li>使用大剪刀扩大日ë键切口前方之间的点rhinophores的，同时与下刀片上拉，以避免割伤肠道。将观察内部器官，洒出的切口。 </li></ol></li><li>取出神经节。 <ol><li>托盘重新定位和重新聚焦显微镜头区域。 </li><li>使用干净的镊子和细剪刀，取出头节被撞断在一个点形成头部的神经节的神经食管周围成环移除胸膜踏板和脑的神经节为一组。 </li><li>坚持腹侧食道取出颊神经节。 </li><li>取出腹腔神经节，通过削减4大神经连接词的问题，从这个节。 </li></ol></li><li>隔离节利息。 <ol><li>除了修剪头神经节，留下的长度为连接到每个神经节到至少等于直径的神经节还连接词，这会妨碍酶消化过的在下一步骤中感兴趣的细胞。 </li><li>将每个目标腱鞘通过ASW + P / S，用干净的镊子之间移动冲洗冲洗2次。 </li><li>如果针对PVC的细胞，离开的右侧胸腔hemiganglion的附加到右踏板hemiganglion的，，同样离开所附左踏板hemiganglion的左胸腔hemiganglion。 </li><li>丢弃不需要的神经节和动物的其余部分根据公认的研​​究实践。 </li></ol></li><li>酶消化神经节。 <ol><li>对于每一个神经节，准备1毫升酶液组成为3.75毫克1毫克中性蛋白酶，透明质酸酶和0.3克每毫升反潜型胶原酶XI + P / S在15毫升的聚丙烯锥形管。 </li><li>地方冲洗节在酶溶液中，并紧紧地盖上盖子。将管在其一侧旋转振动筛13-5小时通宵（ 见表2），以缓慢的速度在约23℃（室温）。 </li></ol></li><li>准备培养皿。 <ol><li>显微切割前（2日），制备聚-D-赖氨酸（PDL）涂层的培养皿，在组织培养罩。添加约0.5毫升PDL（0.2毫克/毫升的无菌水）〜25分钟35毫米的培养皿的中心。 </li><li>用无菌水冲洗PDL的3倍。使其干燥。紫外线灭菌的板。 </li></ol></li><li>第2天隔离神经元。 <ol><li>填充35 mm培养皿PDL涂层和ASW + P / S约0.5毫升的紫外线消毒，否则干燥的菜里面创建一个小岛媒体的中心。这是可以做到板凳上，组织培养罩外。应准备一个菜，每个细胞群从hemiganglion。 </li><li>准备一个直径100毫米的解剖盘由SYLGARD涂层固化5毫米深的剥离面，配备几个大小被用来作为引脚和探头（ 见表3）精细解剖针。这个菜与70％isopropol酒精冲洗，然后用反潜+ P / S，终于科幻LL反潜+ P / S </li></ol></li><li>准备显微切割技术的消化，神经节。 <ol><li>倒入溶液中含有的酶消化胸腔踏板和颊神经节解剖盘。 </li><li>将菜在舞台上的一个黑色的表面下的反射光显微镜。 </li><li>使用所附的结缔组织，牵制每个胸膜踏板hemiganglion的背侧。该组织将软，不耐拉伸。 </li></ol></li><li>显微切割PVC神经元。 <ol><li>使用2对干净的细镊子，拿着一对作为探针，镊子精细解剖针，隔离PVC细胞从胸腔hemiganglion。酶消化将已删除或破碎的结缔组织鞘覆盖PVC集群，但细胞会坚持到另一个。 </li><li>使用探头分离这些细胞从踏板胸膜结缔组织18，然后让细胞簇下降的底部剥离菜。 </li></ol></li><li>将培养皿中的神经元。 <ol><li>到稍微火抛光一次性玻璃巴斯德吸管的尖端轻轻的吸吮集群，然后分配到媒体岛在培养皿慢慢小心，以避免引入气泡准备好的35毫米培养皿中的细胞转移群集进入吸管。重复并放入一个单独的培养皿的其他hemiganglion PVC集群隔离。 </li></ol></li><li>显微切割和BSC神经元转移。 <ol><li>引脚口腔神经节腹侧，与照顾不遗余力感兴趣的细胞。重复步骤2 BSC细胞簇的颊神经节10 1.12。 </li><li>分离PVC和BSC神经元会产生4个培养皿中的神经元集群：2道菜含BSC集群和2道菜含有PVC集群。丢弃其余的节，并预留100毫米解剖盘。 </li></ol></li><li> Dissoc第十一条细胞簇。 <ol><li>将在舞台上的低功耗显微镜培养皿中的细胞，并取下后盖。 </li><li>游离的PVC轻轻弹培养皿的底部相邻的用细解剖针钳举行的小区集群的集群。轻拂挖针尖端的塑料盘底部仿佛试图挖掘出斑点的塑料。当与塑料摩擦释放针点，敲击波通过介质分裂细胞附近的丛。 </li><li>可能需要5-10笔触，几乎完全分解的细胞，但它是更好地留下小的细胞群而比轻弹太多，免得被破坏的细胞，通过机械纯粹的。 </li><li>更换覆盖和重复与其他文化的菜肴。 </li></ol></li><li>存储细胞培养。 <ol><li>将培养皿包含媒体与分离的细胞在其中心岛屿一个大容器，允许空气流通，如150×25毫米的圆形塑料培养皿中，这个菜放在培养箱中集至17°C。 </li><li>安装在腔室开放的湿度作为源盘水。离开一夜之间不受干扰。细胞将坚持以聚-D-赖氨酸涂层中心的菜。 </li></ol></li><li>洪水培养皿。 <ol><li>第3天，轻轻地涌入2.5毫升反潜菜+ P / S检查并用倒置的细胞培养显微镜计数细胞。存放在17℃培养箱中培养。 </li></ol></li></ol> 2。电的方法是标准膜片钳已经描述的在许多文本（ 如索克曼和内尔，1995）16。此协议将细胞≤100 pF电容，或细胞&lt;60微米直径在3日和4日的协议。细胞没有过程的最佳记录。下面特别的体贴离子适用于： <ol><li>可容纳较大的小区，Axopatch 200B上的放大器设置为β= 0.1的配置设置。可能会导致非常大的电流激活细胞逃避电压钳。取代的ASW的解决方案（ 例如氯化钠取代的一小部分的不渗透，氯化N-甲基-D-葡糖胺盐含量）可用于减少全细胞电流的幅值。 </li><li>纤维填充的硼硅补丁移液管移液管拉出器上拉半回填与450 mM的KCl和其它盐（ 见表1）组成的细胞内的解决方案（ICS）是合适的，并应具有约0.5-1兆欧的电阻。如果所需的特定的离子电流的隔离，例如，Na +的电流的K +，K +电流在该溶液中的情况下，可以与其他离子如Cs ​​+的取代。 ICS以浸透阴离子Cl -的更换也是必要的，如果需要一个更自然的ICS9。 </li><li>细胞&gt; 1小时长的录音有可能在室温强劲。录音3日和4日的协议，是最佳的对应培养24-48小时。 48小时后，有困难形成千兆欧姆海豹，或许是因为在细胞表面的糖被阐述，空间夹紧过程的产物所造成的问题。这些细胞是有用的，但是，非膜片钳毒理学的研究，如在&lt;14天，即可以完成。 </li><li> ASW和重力给料溶液的移动设备被分配的其他解决方案，以及细胞内的溶液中，应进行过滤，通过一个安装在注射器上的0.2微米Acrodisk的过滤器（ 表3），以消除微粒千兆欧姆封接形成干扰。 </li><li>脱敏时，会发生使用如D-天门冬氨酸（D-天冬氨酸）受体激动剂，因此建议〜1-2分钟的应用程序之间的激动剂。反之，增强作用，经常观察到的快速重复应用阳离子如L-谷氨酸（L-Glu浓度）的激动剂。 </li><li>如L-Glu浓度的激动剂激活的电流，可以研究应用激动剂与picospritzer吸管。此吸移管被拉到补丁吸管相同，并包含在ASW的激动剂。在此吸移管的前端时的重力给料溶液的移动设备的流出管的下方，并以45°角从流出管（ 图2F），激动剂会迅速洗涤相差的细胞，将被最小化脱敏。激动剂移液管应建立相对的补丁吸管90°或更大的角度。 </li></ol>

<ol>
	<li>Buttner, N., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+modulation+of+a+hyperpolarization-activated+Cl(-)+current+in+Aplysia+sensory+neurons+by+SCP(B)+and+FMRFamide.">Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide.</a> J. Neurophysiol. 90 (2), 586-598 (2003).</li><li>Byrne, J. H., Castellucci, V. F., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+individual+mechanoreceptor+sensory+neurons+to+defensive+gill-withdrawal+reflex+in+Aplysia.">Contribution of individual mechanoreceptor sensory neurons to defensive gill-withdrawal reflex in Aplysia.</a> J. Neurophysiol. 41 (2), 418-431 (1978).</li><li>Carlson, S. L., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Physiological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> J. Neurophysiol. 106 (4), 1629-1636 (2011).</li><li>Carlson, S. L., Kempsell, A. T., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Pharmacological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> Br. Behav. 2 (4), 391-401 (2012).</li><li>Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Na++and+Ca2++currents+in+bag+cells+of+sexually+immature+Aplysia+californica.">Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica.</a> J. Exp. Biol. 201 (5), 745-754 (1998).</li><li>Fieber, L. .. A., Carlson, S. L., Capo, T. R., Schmale, M. C. <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+D-Aspartate+ion+currents+in+the+Aplysia+nervous+system+with+aging.">Changes in D-Aspartate ion currents in the Aplysia nervous system with aging.</a> Br. Res. 1343, 28-36 (2010).</li><li>Glansman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+in+Aplysia:+The+Cheshire+Cat+of+neurobiology.">Habituation in Aplysia: The Cheshire Cat of neurobiology.</a> Neurobiol. Learn. Mem. 92, 147-154 (2009).</li><li>Hawkins, R. D., Abrams, T. W., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cellular+Mechanism+of+Classical+Conditioning+in+Aplysia:+Activity-Dependent+Amplification+of+Presynaptic+Facilitation.">A Cellular Mechanism of Classical Conditioning in Aplysia: Activity-Dependent Amplification of Presynaptic Facilitation.</a> Science, New. 219 (4583), 400-405 (1983).</li><li>Ichinose, M., Sawada, M., Maeno, T., McAdoo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+acetylcholine+on+ventrocaudal+sensory+neurons+in+the+pleural+ganglia+of+Aplysia.">Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia of Aplysia.</a> Cell Mol. Neurobiol. 9, 233-245 (1989).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cellular basis of behavior. , (1976).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialog+between+nerves+and+synapses.">The molecular biology of memory storage: a dialog between nerves and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Kupfermann, I., Castellucci, V., Pinsker, H., Kandel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+correlates+of+habituation+and+habituation+of+the+gill-withdrawal+reflex+in+Aplysia.">Neuronal correlates of habituation and habituation of the gill-withdrawal reflex in Aplysia.</a> Science. 167 (3926), 1743-1745 (1970).</li><li>Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+an+Aplysia+bag-cell+neuron+cation+channel+by+closely+associated+protein+kinase+A+and+a+protein+phosphatase.">Regulation of an Aplysia bag-cell neuron cation channel by closely associated protein kinase A and a protein phosphatase.</a> J. Neurosci. 24 (30), 6833-6841 (2004).</li><li>R, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transcriptome+of+Aplysia:+neuronal+compartments+and+circuitry.">Neuronal transcriptome of Aplysia: neuronal compartments and circuitry.</a> Cell. 127, 1453-1467 (2006).</li><li>Nick, T. A., Kaczmarek, L. K., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+underlying+developmental+regulation+of+repetitive+firing+in+Aplysia+bag+cell+neurons.">Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons.</a> J. Neurosci. 16 (23), 7583-7598 (1996).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-channel recording. , (1995).</li><li>Tam, A. K., Geiger, J. E., Hung, A. Y., Groten, C. J., Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+Ca2++current+contributes+to+a+prolonged+depolarization+in+Aplysia+bag+cell+neurons.">Persistent Ca2+ current contributes to a prolonged depolarization in Aplysia bag cell neurons.</a> J. Neurophysiol. 102 (6), 3753-3765 (2009).</li><li>Schacher, S., Proshansky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+regeneration+by+Aplysia+neurons+in+dissociated+cell+culture:+modulation+by+Aplysia+hemolymph+and+the+presence+of+the+initial+axonal+segment.">Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment.</a> J. Neurosci. 3 (12), 2403-2413 (1983).</li><li>Walters, E. T., Byrne, J. H., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoafferent+neurons+innervating+tail+of+Aplysia.+I.+Response+properties+and+synaptic+connections.">Mechanoafferent neurons innervating tail of Aplysia. I. Response properties and synaptic connections.</a> J. Neurophysiol. 50 (6), 1522-1542 (1983).</li><li>Wilson, G. F., Richardson, F. C., Fisher, T. E., Olivera, B. M., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+Ca(2+)-sensitive+nonspecific+cation+channel+underlying+prolonged+repetitive+firing+in+Aplysia+neurons.">Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.</a> J. Neurosci. 16 (11), 3661-3671 (1996).</li><li>White, B. H., Nick, T. A., Carew, T. J., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+regulates+a+vesicular+class+of+calcium+channels+in+the+bag+cell+neurons+of+Aplysia.">Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of Aplysia.</a> J. Neurophysiol. 80 (5), 2514-2520 (1998).</li><li>Wilson, G. F., Magoski, N. S., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+a+calcium-sensitive+nonspecific+cation+channel+by+closely+associated+protein+kinase+and+phosphatase+activities.">Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.</a> Proc. Natl. Acad. Sci. U.S.A. 95 (18), 10938-10943 (1998).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J. Vis. Exp. (28), e1355 (2009).</li></ol>

作者宣称，他们有没有竞争经济利益。

这里所描述的解离技术产生感觉神经元的文化含有50-100孤立的神经元穿插着小神经胶质细胞和其他不明身份的细胞数。在协议中最重要的步骤是神经节时，保持在酶溶液，轻弹，打散成单个细胞的群集消化的细胞簇的解离。内切酶消化（步骤1.8），必须在可用的温度下进行了优化。在23℃缓慢摇动，13小时是足以消化的的BSC集群while15小时是最佳的PVC集群。在培养皿中的细胞产量低，是由于没有足?...

的海洋opistobranch软体动物， 海兔 ，几十年来一直是一个有用的神经生物学模型。它最出名的习惯和古典空调7，8作为一种模式。在这个模型中对学习和记忆的研究获得了诺贝尔生理学或医学奖的埃里克·坎德尔在2000年，他与阿尔维德·卡尔松和保罗·格林加德10奖品。研究涉及电气录制减少制剂，这种无脊椎动物的神经系统的元素，从动物解剖与神经和肌肉的左连接，这些都有助于阐明海兔的单个神经元的角色。的确切的分子机制，构成海兔学习识别然而，通常采用的另一种技术，长期共培养的感觉神经元和运动神经元，获得从单个供体动物，并允许以形成突触在培养皿21 。 我们和其他1，3，6，14，15，16利用难易程度，确定在这个模型中的神经元可以有针对性的，以及他们的耐力在长期的实验，使游离短期名义上均匀的神经元集群文化我们研究离子电流电压钳下膜片钳配置。许多海兔的神经元站起来，反复多轮膜片钳技术，以便有时间为持久的实验操作。该技术是非常有用的，如神经内分泌袋腹神经节细胞，和我们在这里描述的解离的胸腔和颊神经节的感觉神经元的神经元，但不适合非常大的神经元大于60微米直径的，如L7或R2腹神经节。我们不采用海兔血清在我们的文化，不同的是感觉，运动神经元共培养别处描述。大多数神经元将不使用此程序获得流程为t他第一次在培养的48小时，方便拍摄​​全细胞电压，但将发芽，约14天死于缺乏的营养物质和/或生长因子的前阐述轴突和其他进程。 该技术生产出每盘50-100个神经元的原代培养的颊和胸膜神经节区域从生理记录。此协议是有用的研究人员在研究单细胞生理学方面的实验，需要无数的实验复制每只动物。这产生一对配对培养由于分离成左，右hemiganglia的靶细胞的解剖，允许匹配的处理的和对照培养物的研究中受益。 该协议的目标颊簇（BSC）的口腔神经节的神经元，并胸膜ventrocaudal（PVC）胸膜神经节神经元。这些细胞是全细胞电压记录和显示ROBUS的适当大小吨谷氨酸回应。讨论的方法是适合大多数在海兔神经系统的神经节。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Artificial seawater ASW</td>
 <td>Sigma-Aldrich</td>
 <td>assorted</td>
 <td>(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6</td>
 </tr>
 <tr>
 <td>Intracellular solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>(mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>penicillin/streptomycin added to ASW at 1:100</td>
 <td>Lonzo Walkersville, Inc.</td>
 <td>17-603E</td>
 <td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td>
 </tr>
 <tr>
 <td>Neutral dispase II</td>
 <td>Roche Diagnostics</td>
 <td>10165859001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hyaluronidase</td>
 <td>Sigma-Aldrich</td>
 <td>H4272</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>collagenase type XI</td>
 <td>Sigma-Aldrich</td>
 <td>C9407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamate (L-Glu)</td>
 <td>Sigma-Aldrich</td>
 <td>49601-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Aspartate (D-Asp)</td>
 <td>Sigma-Aldrich</td>
 <td>11200-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N-methyl-D-aspartate (NMDA)</td>
 <td>Biomol</td>
 <td>100002-268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Asp</td>
 <td>Sigma</td>
 <td>A6683-25G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td>
 <td>Sigma</td>
 <td>A6816-5MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glu R antagonists</td>
 <td>various</td>
 <td>various</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>kynurenate</td>
 <td>Sigma-Aldrich</td>
 <td>61250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APV</td>
 <td>Sigma-Aldrich</td>
 <td>A5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-propanol</td>
 <td>VWRSP</td>
 <td>BDH1133</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloriding solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O</td>
 </tr>
 <tr>
 <td>Sylgard silicone 2-part polymer</td>
 <td>World Precision Instruments (WPI)</td>
 <td>SYL184</td>
 <td>Provides pin-out surface for small dissection dishes</td>
 </tr>
 <tr>
 <td>0-40x zoom magnification microscope for dissections</td>
 <td>Wild</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Techniquip 150 Watts Fiber Optic Illuminator</td>
 <td>Microoptics of Florida</td>
 <td>TQ FOI-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RotoMix 50800 orbital mixer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp; 40x objectives</td>
 <td>SR Research Ltd.</td>
 <td>Eyelink II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tektronix digital oscilloscope</td>
 <td>SR Research Ltd.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pClamp 10 data acquisition and analysis software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PC with Windows XP or higher operating system</td>
 <td>PC Solutions</td>
 <td>&nbsp;</td>
 <td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td>
 </tr>
 <tr>
 <td>Flaming/Brown P87 micropipette puller</td>
 <td>Sutter Instruments, Novato, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>CV203BU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digidata 1200 A/D converter</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picospritzer, powered by N2 adjustable for pressure and duration</td>
 <td>Parker Hannifin, Cleveland, OH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TMC Micro-G Vibration isolation table</td>
 <td>Ametek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>custom manufacture</td>
 </tr>
 <tr>
 <td>Burleigh Piezoelectric Clamshell Micromanipulators</td>
 <td>Burleigh Instruments; Thorlabs</td>
 <td>&nbsp;</td>
 <td>presently PCS-5000; -6000 series + mounts</td>
 </tr>
 <tr>
 <td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td>
 <td>WPI</td>
 <td>1B150-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3 inch length</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl half cell</td>
 <td>WPI</td>
 <td>EP4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Angled Scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumostar Fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 mm falcon tissue culture dishes</td>
 <td>VWRSP</td>
 <td>25382-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>falcon 150 x 25 mm tissue culture dishes; 1013</td>
 <td>VWRSP</td>
 <td>1013</td>
 <td>also can be made into small dissection dishes with sylgard</td>
 </tr>
 <tr>
 <td>sylgard</td>
 <td>WPI</td>
 <td>SYL184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>animal dissection tray</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>For 6-bore gravity-fed perfusion system</td>
 </tr>
 <tr>
 <td>Aluminum clips with screw hole ends</td>
 <td>hardware store</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>23 gauge needles (manually file off points)</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Polyethylene tubing 0.022"ID x 0.042"OD; 427411</td>
 <td>Becton-Dickinson</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>H-7 pipette stand/holder for microcap perfusion array</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>one-way valves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Drummond Microcaps 1 &mu;l</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>18 gauge needles for suction (filed off points)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing</td>
 <td>Cole Parmer</td>
 <td>4.27436E+11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fine dissection pins</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>capillary tubes</td>
 <td>Kimble 71900-100</td>
 <td>&nbsp;</td>
 <td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td>
 </tr>
 <tr>
 <td>modeling clay</td>
 <td>craft store</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dish holder for microscope stage with isolated ground bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Custom manufacture</td>
 </tr>
 <tr>
 <td>pasteur pipettes</td>
 <td>VWRSP</td>
 <td>14672-412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pipette bulbs</td>
 <td>VWRSP</td>
 <td>53283-911</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>acrodisk syringe filters</td>
 <td>VWRSP</td>
 <td>28144-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>thick-walled 1.5 mm diameter borosilicate filament glass</td>
 <td>WPI</td>
 <td>1B150F-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High purity nitrogen cylinder and bifurcating regulator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Tables 1-3. Lists of Reagents, Materials, and Equipment.

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

海洋腹足类软体动物海兔丫有悠长的历史，作为神经系统的功能模型，在学习和记忆研究中具有特别重要的意义。这类研究的典型制剂在其中的感觉和运动神经元保持不变在微创解剖动物，或技术上精心制作的个人感觉和运动神经元的神经细胞共同培养。不太常见的是孤立的小群名义上均匀的神经元是神经制剂，其中分离成单个细胞在短期文化。这种分离的细胞是有用的生物物理特性，使用膜片钳技术，和有针对性的调制这些电导的离子电流。准备这种文化的一种协议。该协议需要容易识别谷氨酸胸膜和颊神经节的感觉神经元中的优势，并介绍了他们的分离和最小的维护，我N培养无血清几天。

在这个协议中的对象内的神经节的感觉神经元的位置，BSC和PVC的神经元是如图1中所示。 BSC的神经元位于口腔神经节的腹侧面向远离的颊质量，完整的神经节（ 图1A），表面上在2个对称的椭圆形集群。 PVC神经元形成双边的，V形的簇，环绕胸神经节的背侧表面向中心轴（ 图1B）。这些感觉神经元的神经节内的刻板位置具有的优点，但缺少的右边或左边的群集?...

Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

我们描述了解剖海洋海兔的神经系统<em&gt;海兔</em麻醉后短期组织培养的神经元，和单细胞离子电流通过膜片钳技术记录的隔离。

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

isolation-sensory-neurons-aplysia-californica-for-patch-clamp

Research

JoVE Journal

Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

12.2K 次观看.  University of Miami. 我们描述了解剖海洋海兔的神经系统海兔

视频: Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

电极的制作及植入海兔californica完整的，自由的行为的动物多通道的神经和肌肉的录音

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

科研

神经科学

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.