1。电极和盐溶液的制备<ol><li>准备生理盐水和电极将动物解剖前。水蛭林格氏液由以下部分组成：115.3毫米氯化钠，1.8毫米氯化钙 ，4.0毫米氯化钾，10毫米的Tris /马来酸或HEPES。将pH调节至7.4。 </li><li>浸清洁的银线放入少量的漂白剂20分钟或直到丝具有光滑的暗灰色涂层准备的Ag-CL线。使用前用蒸馏水洗丝。然后将导线放置成可插入放大器微量持有者。 </li><li>用吸管拉马准备硼硅玻璃的锋利玻璃电极。电极电阻应为20兆欧姆量级。 </li><li>回填移液器用3M乙酸钾溶液，并插入在插入微量支架。 </li><li>对于一个细胞外电极制备如上所述尖锐的玻璃吸管，打破尖端背面由揉附近的锋利边缘再次出现大幅吸管。那么火抛光尖，以便它不会撕裂连接词。塑料外电极也只要是在电极和神经之间的良好接触工作。 </li><li>插入外记录电极插入微量支架（与银氯导线），它配有一橡胶管和注射器进行抽吸。用注射器抽取盐水进入电极至少在银线的电平。 </li><li>通过遵循本手册为您的特定信号记录器的设置硬件（放大器和刺激隔离器）和软件记录参数。见Leksrisawat 等人 22可以看到在下面描述的实验中使用的设备的细节。 </li></ol> 2。腹神经节解剖<ol><li>解剖水蛭在一个大的有机硅弹性体内衬菜水蛭林格氏液。如果动物被纵向拉伸会更容易除去腹神经索（ 见图1A）。 </li><li>与＃10或＃11的大小手术刀，作一纵切口在腹部中线很浅的切口。关于腹侧切口的中间2/3rd示于图1B中 。尽量不要通过黑色长筒丝袜（腹血窦）切，直到动物被完全打开，血窦被拉伸而不扭结。这血窦运行动物沿腹中线的长度。 VNC的包含在此的血管内。 </li><li>后皮肤已被切断动物的全长用细光圈剪刀缺口的血窦（ 图2）。滑的窦下的细虹膜剪一个叶片和稍微提高刀片将其从神经组织中分离出来。切窦VNC的长度，同时避免了神经根，神经节段或神经节之间的连接词。 </li><li>接着，取出腹神经索的一部分远端切到血管连接根部的根。沿腹神经索的长度每节重复此过程。 </li><li>的一个或两个神经节段转移至一个较小的培养皿中。神经节或神经节单需固定绷紧有些呆滞。 </li><li>针的连接词和根部，因此神经节神经胶质鞘稍微拉伸，以防止神经元细胞体通过所述胶质鞘并进入感兴趣的神经元刺穿时走动。该引脚应放在通过黑色窦的根部，两个联结的中间，尽量减少轴突损伤（ 图3）。 </li><li>将培养皿用制剂，在显微镜下，用蜡将其固定在该盘，以防止移动的底部。 </li><li>利用反射镜和聚光看到的神经元， 如图4A小心聚焦光束（示意于图4B中所示</st荣&gt;）。注意两个大Retzius细胞在神经节的中心。 </li><li>使RZ，T，P和N个神经元的细胞内记录时，小心地将电极的尖端在感兴趣的细胞和轻轻降低前端直到一个凹坑被认为是在胶质鞘。 </li><li>点击电极支架或机械手（非常仔细），使电极尖端“啪啪”到感兴趣的细胞。 </li><li>注入正向电流（1-10 NA）的短脉冲（30-40毫秒）进入细胞内，以唤起动作电位。 </li></ol> 3。从腹神经节准备培养神经元<ol><li>脚的神经节腹侧中含有L-15与胎牛血清（FCS）（ 图3）干净的菜。脚根，使神经节稍微拉紧作为细胞内的录音做以上。用细剪刀，尼克胶质囊的一端，滑1剪刀刀片包膜下，然后穿过胶质囊切如图5A。 </li><li>加胶原酶/分散酶的100微升等分试样，以在振荡器上15分钟，在烹调和地点。通过使外露的细胞体周围的培养基中检查细胞。返回的单元格的摇床另外15分钟或更长的时间，直到细胞出来以最小的吸力。 </li><li>使用附连到注射器从神经节轻轻提取细胞的移液管。火抛光该移液管尖端，使得它比细胞体直径稍大。对于RZ细胞在成年水蛭，直径应为约50微米。 </li><li>准备了各种移液器具有不同直径的无灯丝，因为这降低了细胞粘附吸管内的机会。存储移液器在含80％乙醇（EtOH中），使用棉球在底部，以保护技巧从碎裂的瓶子。记住吸细胞前具有中等洗出乙醇吸管。 </li><li>酶处理后，洗出的含酶介质与L-15（有FCS）2倍。找出感兴趣的细胞，轻轻拉动注射器（ 图6A）吸引他们到吸管。放电吸气细胞进入另一道菜，其中包含L-15（带FCS）。 </li><li>到现在为止，所有的程序都是非无菌。执行在无菌罩下面的步骤。将3-4滴大滴无菌的L-15（与FCS）在无菌培养皿内的不同位置。 </li><li>吸取细胞进入前面提到滴之一，在下拉周围吹他们把它们洗干净。在3或4滴介质的重复这个串联。 </li><li>把细胞在一个干净的培养皿中充满了L-15（有FCS）。 </li><li>于室温孵育1-24小时，在室温下进行。事后清洁单元可电镀。将其留在细胞几个小时或隔夜外基质容易脱落。立即板上的细胞来产生较长的过程。 </li><li>使用无菌基材涂层微孔二<html> sh和允许它培养实验前彻底晾干（过夜）。 </li><li>后盘已经涂，用无菌水轻轻冲洗培养皿，然后用少量的L-15培养基中。请记住，使用L-15解决方案，而在FCS加入使细胞附着在菜。该介质可以被轻轻地改变到L-15与FCS后的细胞粘附在基材上。如果细胞要使用镀覆，然后切换到中的L-15与FCS后几个小时是没有必要的。 </li><li>定位单元彼此相邻于镀的时间以诱导快速突触（ 图6B）。后在神经元中的配对：24小时测量的生理活性。 </li><li>利用细胞内电生理技术，以培养对细胞的记录RP或突触反应（1刺激通过神经元从配对的细胞的细胞内电极和记录反应使用的是第二个胞内电极）。 </li></ol>标题“&gt; 4。神经节体墙准备<ol><li>针在如上所述的弹性体衬里的培养皿水蛭背面向下。 </li><li>针在皮肤一侧上被切断的神经节的另一侧根部（ 图7A，放大在图7B），或使一个窗口上的水蛭超过一个神经节（ 图7C）与所有的根到身体的腹侧墙壁完好无损。 </li><li>后获得稳定的细胞内记录中的感觉细胞（T，P，N）的使用已经火抛光，轻轻触碰在同一网段的皮肤小玻璃棒之一。如果从正被记录的神经元是N细胞，然后更多的压力将需要被施加到皮肤上。在大多数情况下，如果皮肤被夹用镊子N个小区将只响应。因为这个原因，这些细胞应当最后尝试因为人们可能损伤皮肤或通过干扰制剂以这样的方式置换细胞内重新编码。 </li><li>每个人都应该能够快速地绘制皮肤和神经节与细节足以说的其中一个触摸可用于确定特定的神经元的感受域的地图。从尽可能多的T和P细胞可能在一节两侧录制完毕后试了N细胞。 </li><li>为了检验是否感觉神经元有通过在延髓或尾的方式，然后在相邻的段皮肤根部的连接词旅行过程中，神经节之间的连接词可切割和感受野复查中问候检测的蔓延。基本上，一个是检查是否有一直在感受野的任何损失。 </li></ol> 5。注射荧光染料<ol><li>准备如上述那样水蛭的神经节。 </li><li>填充微电极与荧光黄-CH（3.0％）和氯化锂（300毫摩尔）的溶液中。 </li><li>如上所述刺穿的神经节的腹侧感觉细胞或RZ小区之一以上。 </li><li>通过使负电流（3nA的）经过15分钟的电极注入的染料进入细胞。设置脉冲的持续时间为100毫秒，并且频率为2赫兹。 </li><li>通过激励染料与水银灯和FITC / GFP滤波器组可视化的填充染料的神经元。 </li></ol>

<ol>
	<li>Fuchs, P. A., Nicholls, J. G., Ready, 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=Membrane+properties+and+selective+connexions+of+identified+leech+neurones+in+culture.">Membrane properties and selective connexions of identified leech neurones in culture.</a> J Physiol. 316, 203-223 (1981).</li><li>Ready, D. F., Nicholls, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+neurones+isolated+from+leech+CNS+make+selective+connections+in+culture.">Identified neurones isolated from leech CNS make selective connections in culture.</a> Nature. 281, 67-69 (1979).</li><li>Blackshaw, S. E., Muller, K. J., Nicholls, J. G., Stent, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurobiology of the Leech. , 51-78 (1981).</li><li>Drapeau, P., Sanchez-Armass, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+and+selection+of+the+responses+to+serotonin+during+reinnervation+of+an+identified+leech+neuron.">Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron.</a> J Neurobiol. 20, 312-325 (1989).</li><li>Garcia, U., Grumbacher-Reinert, S., Bookman, R., Reuter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Na++and+K++currents+in+soma,+axons,+and+growth+cones+of+leech+Retzius+neurones+in+culture.">Distribution of Na+ and K+ currents in soma, axons, and growth cones of leech Retzius neurones in culture.</a> J. Exp. Biol. , 1-17 (1990).</li><li>Cooper, R. L., Miguel Fernandez, d. e., Adams, F., B, W., Nicholls, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+inhibits+expression+of+calcium+currents+in+purely+postsynaptic+cells.">Synapse formation inhibits expression of calcium currents in purely postsynaptic cells.</a> Proc. R. Soc. B-Biol. Sci. , 217-222 (1992).</li><li>Liu, Y., Nicholls, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steps+in+the+development+of+chemical+and+electrical+synapses+by+pairs+of+identified+leech+neurons+in+culture.">Steps in the development of chemical and electrical synapses by pairs of identified leech neurons in culture.</a> Proc. R. Soc. B-Biol. Sci. , 236-253 (1989).</li><li>Fernandez de Miguel, F., Cooper, R. L., Adams, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptogenesis+and+calcium+current+distribution+in+cultured+leech+neurons.">Synaptogenesis and calcium current distribution in cultured leech neurons.</a> Proc. R. Soc. B-Biol. Sci. 247, 215-221 (1992).</li><li>Briggman, K. L., Kristan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+pattern-generating+circuits.">Multifunctional pattern-generating circuits.</a> Annu. Rev. Neurosci. 31, 271-294 (2008).</li><li>Macagno, E. R., Muller, K. J., Deriemer, 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=Regeneration+of+axons+and+synaptic+connections+by+touch+sensory+neurons+in+the+leech+central+nercoys+system.">Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nercoys system.</a> J. Neurosci. 5, 2510-2521 (1985).</li><li>Becker, T., Macagno, 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=CNS+control+of+a+critical+period+for+peripheral+induction+of+central+neurons+in+the+leech.">CNS control of a critical period for peripheral induction of central neurons in the leech.</a> Development. 116, 427-434 (1992).</li><li>Marin-Burgin, A., Kristan, W. B., French, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Synapses+to+behavior:+Development+of+a+sensory-motor+circuit+in+the+leech.">From Synapses to behavior: Development of a sensory-motor circuit in the leech.</a> Dev. Neurobiol. 68, 779-787 (2008).</li><li>Kristan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neurobiology+of+Swimming+in+the+leech.">The Neurobiology of Swimming in the leech.</a> Trends Neurosci. 6, 84-88 (1983).</li><li>Brodfuehrer, P. D., Friesen, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+stimulation+to+undulation:+a+neuronal+pathway+for+the+control+of+swimming+in+the+leech.">From stimulation to undulation: a neuronal pathway for the control of swimming in the leech.</a> Science. 234, 1002-1004 (1986).</li><li>Arbas, E. A., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+oscillations+of+membrane+potential+in+interneurons+that+control+heartbeat+in+the+medicinal+leech.">Slow oscillations of membrane potential in interneurons that control heartbeat in the medicinal leech.</a> J. Neurosci. 7, 3953-3960 (1987).</li><li>Ching, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Synaptogenesis between identified neurons. , (1995).</li><li>Kristan, W. B., Eisenhart, F. J., Johnson, L. A., French, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+neuronal+circuits+and+behaviors+in+the+medicinal+leech.">Development of neuronal circuits and behaviors in the medicinal leech.</a> Brain Res. Bull. 53, 561-570 (2000).</li><li>French, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+expression+is+required+for+normal+chemical+synapse+formation.">Gap junction expression is required for normal chemical synapse formation.</a> J. Neurosci. 30, 15277-15285 (2010).</li><li>Li, Q., Burrell, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+long-term+depression+in+a+polysynaptic+pathway+in+the+leech+CNS:+one+NMDA+receptor-dependent+and+the+other+cannabinoid-dependent.">Two forms of long-term depression in a polysynaptic pathway in the leech CNS: one NMDA receptor-dependent and the other cannabinoid-dependent.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 195, 831-841 (2009).</li><li>Nicholls, J. G., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+modalities+and+receptive+fields+of+sensory+neurons+in+CNS+of+the+leech.">Specific modalities and receptive fields of sensory neurons in CNS of the leech.</a> J. Neurophysiol. 31, 740-756 (1968).</li><li>Yau, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptive+fields,+geometry+and+conduction+block+of+sensory+neurones+in+the+central+nervous+system+of+the+leech.">Receptive fields, geometry and conduction block of sensory neurones in the central nervous system of the leech.</a> J. Physiol. 263, 513-538 (1976).</li><li>Leksrisawat, B., Cooper, A. S., Gilberts, A. B., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+receptor+organs+in+the+crayfish+abdomen:+a+student+laboratory+exercise+in+proprioception.">Muscle receptor organs in the crayfish abdomen: a student laboratory exercise in proprioception.</a> J. Vis. Exp. (45), e2323 (2010).</li><li>Crisp, K. M., Mesce, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+central+pattern+generator:+amine+modulation+of+decision-making+neural+pathways+descending+from+the+brain+of+the+medicinal+leech.">Beyond the central pattern generator: amine modulation of decision-making neural pathways descending from the brain of the medicinal leech.</a> J. Exp. Biol. 209, (2006).</li><li>Puhl, J. G., Mesce, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+activates+the+motor+pattern+for+crawling+in+the+medicinal+leech.">Dopamine activates the motor pattern for crawling in the medicinal leech.</a> J. Neurosci. 28, 4192-4200 (2008).</li><li>Crisp, K. M., Gallagher, B. R., Mesce, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+dopamine+induction+of+crawl-like+bursting+in+leech+motoneurons.">Mechanisms contributing to the dopamine induction of crawl-like bursting in leech motoneurons.</a> J. Exp. Biol. 215, 3028-3036 (2012).</li><li>Macagno, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+medicinal+leech+transcriptome+database+and+its+application+to+the+identification+of+leech+homologs+of+neural+and+innate+immune+genes.">Construction of a medicinal leech transcriptome database and its application to the identification of leech homologs of neural and innate immune genes.</a> BMC Genomics. 11, 407 (2010).</li><li>Kandarian, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medicinal+leech+genome+encodes+21+innexin+genes:+different+combinations+are+expressed+by+identified+central+neurons.">The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons.</a> Dev. Genes Evol. 222, 29-44 (2012).</li><li>Shefi, O., Simonnet, C., Groisman, A., Macagno, 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=Localized+RNAi+and+ectopic+gene+expression+in+the+medicinal+leech.">Localized RNAi and ectopic gene expression in the medicinal leech.</a> J. Vis. Exp. (14), e697 (2008).</li><li>Firme, C. P., Natan, R. G., Yazdani, N., Macagno, E. R., Baker, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+expression+of+select+innexins+in+individual+central+neurons+couples+them+to+pre-existing+neuronal+or+glial+networks+that+express+the+same+innexin.">Ectopic expression of select innexins in individual central neurons couples them to pre-existing neuronal or glial networks that express the same innexin.</a> J. Neurosci. 32, 14265-14270 (2012).</li><li>Siddall, M. E., Trontelj, P., Utevsky, S. Y., Nkamany, M., Macdonald, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+molecular+data+demonstrate+that+commercially+available+medicinal+leeches+are+not+Hirudo+medicinalis.">Diverse molecular data demonstrate that commercially available medicinal leeches are not Hirudo medicinalis.</a> Proc. Biol. Sci. 274, 1481-1487 (2007).</li></ol>

作者什么都没有透露。

这组实验的目的是1）从识别的神经元教，并表现出细胞内记录的基本原则和2）认识到的电信号，可以产生用成年水蛭这些特定类型的神经元的特征形状。有调查采用水蛭的神经生理学研究丰富的历史和研究工作正在进行中处理的神经回路，并在突触的修复和再生10在开发过程中，以及基因调控的问题。神经调节，特别是通过生物胺的途径，也于水蛭是一个成功的研究方向。通过盐溶液加入多巴胺等药物制剂的神经系统，它已经表明，多巴胺调节参与决策23和运动模式的爬行行为24,25神经网络。这种实验方法是简体乐和廉价的方式来把调制成教学实验室，并调节盐溶液的离子构成是教导能斯特和戈德曼霍奇金 - 卡茨方程的有效方法。 为了取得成功与神经节筹备工作有神经节之前，试图戳躯体与细胞内电极拉紧是非常重要的。否则，在神经胶质包推下来时，躯体会移动。此外，desheathing神经节用于培养神经元中除去时，在整个神经节切割时所以胞体不被损坏，因为它们将被删除避免感兴趣区域。此外，过度的温育中的酶可导致神经元太脆弱被删除。它需要一些试验和错误，以达到理想的孵化时间，因为每批酶略有不同在他们的行动。 进一步的调查，以优化培养基可以改善这个技术。变量项因素的影响，如胰岛素，糖，或生长因子的存在下，可以检查对更长的维护文化。以确定是否细胞保持它们的合成发射机，如五羟色胺的合成在RZ细胞的能力，中性红染色可以应用于标记的血清素的过程。还小胶质细胞浸润至受伤和再生组织可以通过核污渍日受伤后进行量化。 虽然这种制剂有很多好处，提供在研究再生与修复，以及神经系统的电路，在突触的药理学和分子鉴定的各种受体亚型还没有得到充分的特点。一个表达序列标签库可用26和医用水蛭基因组中，目前正在组装27，但在水蛭的主要限制相比，一些模型制剂是修改的基因组中选择性基因在特定细胞类型的能力。在胚胎吨他的限制已经解决了注射DNA或双链RNA来调节基因表达28。基因枪技术最近被用于表达无脊椎动物连接蛋白（innexin）的蛋白质在神经元的水蛭。从水蛭verbana（HVE-inx6）的innexin的表情足以异位间隙连接的医用水蛭29的形成。医用水蛭-或H。马鞭草因为它是30是一定要推进我们的生理和电突触的演变，在未来几年的理解。

水蛭制剂是研究识别（感觉，运动和动物的中枢神经系统（CNS）内跨神经元的功能非常有用。水蛭神经元的有用的特性是它们的动作电位和生物物理特性的形状特征的给定的细胞类型（ 例如 P，N，T，RZ）。另外的神经元可以从在适当的培养基中，将细胞保持它们的电特性进行了长达45天的1,2动物和培养被删除。 水蛭神经节学习如何做电生理记录的一个显着优点是细胞特征性和可靠地安排在一个模式，可以让神经节识别不同类型的细胞从神经节神经节，并从动物到动物。独特的地理位置和水蛭神经节神经元的形态进行注意到Retzius早在1891年3。知识次方ë身份和一个神经元的功能是有利的侦查神经回路和用于制备实验用给定的细胞类型。由于神经节它们与解剖显微镜可见，与胞体可与微电极，用于记录它们的电学性能被选择性地刺穿内神经元的相对大的胞体。 染料分子和大分子可注入单个细胞，并研究通过膜片钳信道特性。离子电流，在各种神经元的特征动作电位的各种形状产生已确定4-7。从神经支配的格局和分支中枢神经系统内单个神经元的调查，突触连接已经转载于文化与鉴定神经元2,8。研究中的水蛭神经节提供了洞察功能电路的开发，可以与电测量以及与wholË动物行为的研究。水蛭中枢神经系统还曾担任调查涉及神经元的修复和再生，在整个动物和文化4-6,9-12机制的一个重要前提。 在哺乳动物的大脑是一个艰巨的任务，在属性和个人识别神经元的连接方面解释的行为。原则上我们可以希望，不太复杂的系统，如水蛭的研究可能有助于确定在高等动物中使用的基本机制。所使用的背后游泳图案13,14和15心脏节律的连接得到了很好的特征水蛭。一些水蛭的神经元形成电气和化学通讯的能力是耐人寻味。有些连接是双向的，而有些则是单向的化学性质，但双向电7。了解动物中的突触连接，以及重新创建相同的典型E在培养皿中的连接是调查突触16-18以及19突触药理分析的强大工具。这里所描述的细胞内记录和刺激技术提供了药理实验和研究神经行为学和发展的基础。 此外，该段腹神经索可解剖与神经支配的皮肤补丁。以保持贴剂的皮肤和神经元存活的能力，感官感受域可以通过从在CNS各自腹腔神经节内的感觉神经元的细胞体（ 即胞体）记录的电信号被探测。从而可以通过应用不同程度的力在皮肤上评估感觉神经末梢的敏感性和地图感受野：轻轻一碰，压力和有毒（痛苦的）刺激20,21。 这水蛭神经节皮制剂的优点是，一个CAÑ​​画解剖感受野地图和跟踪神经元的过程来确定的神经元一旦电反应被记录。下述三种水蛭的实验都可以完成多次使用一个单一的标本，这使得这是一个成本有效的教学模块教学实验室。

<table border="0" cellpadding="0" cellspacing="0" width="256">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:64px;">Sylgard</td>
			<td style="width:64px;">Dow Corning</td>
			<td style="width:64px;">&#160;182 silicone kit</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;S7653</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P9333&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C5670&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tris/maleic acid</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bleach&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>To chloride silver wire</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;221465&#160;</td>
			<td>To adjust pH</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;H1758</td>
			<td>To adjust pH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">CULTURING MATERIALS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagenase/Dispase</td>
			<td>Boehringer Mannheim&#160;</td>
			<td>&#160;269-638</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Leibovitz L-15 with L-Glutamine</td>
			<td>Gibco</td>
			<td>041-01415H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;">Fetal calf serum</td>
			<td>Ready Systems Ag&#160;</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">04-001</td>
		</tr>
		<tr height="102">
			<td height="102" style="height:102px;">D-Glucose-Monohydrate</td>
			<td>Merck</td>
			<td>8342</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gentamycin sulfate&#160;</td>
			<td>any&#160; supplier</td>
			<td></td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamine</td>
			<td>Gibco</td>
			<td>043-0503H</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">HEPES</td>
			<td>Sigma</td>
			<td>3375</td>
			<td>Free acid, crystalline</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Concanavalin A Type IV</td>
			<td>Sigma&#160;</td>
			<td>C 2010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Poly-L-Lysine Hydrobromide</td>
			<td>Sigma&#160;</td>
			<td>P 7890</td>
			<td>MW 15-30,000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">&#160;Microwell plate, 60 x 10 &#956;l</td>
			<td>Falcon</td>
			<td>3034</td>
			<td>Flat bottom, 10 in a bag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting tools</td>
			<td>World Precision Instruments</td>
			<td>&#160;assortment</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Intracellular electrode probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Faraday cage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Insect Pins</td>
			<td>Fine Science Tools, Inc</td>
			<td>&#160;26001-60</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting microscope (100X)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fiber optic lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Small adjustable mirror</td>
			<td></td>
			<td></td>
			<td>To direct light beam toward the preparation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glass electrodes</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;CLS7095B5X</td>
			<td>Box of 200, Suction electrodes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>&#160;MD4-M3-R</td>
			<td>&#160;Can fix for base or on a metal rod</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Raised preparation stand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Silver wire (10/1,000 inch)</td>
			<td>A-M Systems</td>
			<td>&#160;782500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Computer</td>
			<td>any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">AC/DC differential amplifier</td>
			<td>A-M Systems</td>
			<td>Model 3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PowerLab 26T</td>
			<td>AD Instruments</td>
			<td>&#160;27T</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Head stage</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Comes with AC/DC amplifier</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LabChart7</td>
			<td>AD Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Electrical leads</td>
			<td>any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glass tools&#160;</td>
			<td>make yourself</td>
			<td></td>
			<td>For manipulating nerves</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cable and connectors</td>
			<td>any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pipettes with bulbs&#160;</td>
			<td>&#160;Fisher Scientific&#160;</td>
			<td>&#160;13-711-7</td>
			<td>Box of 500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Beakers</td>
			<td>any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Wax or modeling clay</td>
			<td>any company or local stores</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Stimulator</td>
			<td>Grass Instruments</td>
			<td>SD9 or S88</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;">Plastic tip for suction electrode</td>
			<td rowspan="2">local hardware store (Watt&#8217;s brand)</td>
			<td rowspan="2">&#188; inch OD x 0.170 inch ID</td>
			<td>Cut in small pieces. Pull out over a flame and cut back the tip to the correct size.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
	</tbody>
</table>

intracellular recording, sensory field mapping, and culturing identified neurons in the leech, hirudo medicinalis

淡水水蛭， 医用水蛭 ，是一个已经被用来解决在神经生理学，神经行为学和发育生物学等领域的科学问题一个多才多艺的模式生物。本报告的目的是为了巩固从水蛭系统实验技术成一个单一的文章，将用来生理学家具有专长的其他神经系统制剂，或生物学的学生很少或根本没有电的经验。我们将演示如何解剖水蛭来自于神经节确定的神经回路在细胞内记录。接下来，我们显示如何已知功能的单个细胞可从神经节在培养皿中进行培养，以及如何从在培养这些神经元来记录被删除。然后，我们演示了如何准备支配的皮肤被用于映射感觉或运动领域的一个补丁。这些水蛭制剂仍然被广泛使用，以解决神经网络结构的基本电性能KS，行为，突触和发展。他们还对神经科学和生理学教学实验室适当的培训模块。

来自水蛭的神经节感觉神经元的细胞内记录的特征是在图4C所示的不同的电压-时间的痕迹。在健康水蛭的神经节细胞的静息细胞的膜电位为-45至-60毫伏。如果电势更小（少负）1应改变玻璃电极，或者如果潜在仍然很低，在移动到神经索的另一部分。自发动作电位的小幅度可能表明该电极是在运动神经元。如果这种情况发生在神经节体壁中的电流注入可以被用来确定该神经支配任何仍附着在皮肤上的肌肉。纤维环竖运动神经元（AE）是在神经节（ 图4B）的腹侧，它会导致年轮（脊或段）通过激活环竖肌上升。 注入电流的感觉细胞，应引起动作电位s的图4C中所示的特性的波形。如果波形看起来不同，它可能是由于不平衡电桥或抵消电容补偿。每个人都应该咨询，伴随着信号采集单元为如何平衡电桥在细胞内详细的说明书。通知的刺激伪像在图4C中的电流脉冲的开始和结束。这是神器怎么看时，电桥平衡。 神经元由神经节水蛭原代培养形成电和化学突触的菜。最初培养的细胞的电特性是不完全，因为它们在体内是相同的。然而，几天后的冲动几乎无法区分这些记录在神经节。这表现在图6C一对​​RZ细胞。 7小时后在文化突触电位的振幅为1〜2毫伏。经过56小时培养SYnaptic势比10毫伏（中上部的曲线图6C）放大。动作电位的形状56hr在培养后也发生变化。根据所描述的条件下，这些细胞是可行的几天。 细胞间通讯可以通过刺激神经根或连接词和从神经节神经元记录来证明。刺激这些神经会产生强大的突触电位和动作电位在不同的神经节细胞。在图8B痕迹从Retzius细胞已被从动物身上取出，并在培养皿中寄托了神经节记录。黑色的痕迹被诱发的应用与细胞外电极的结缔组织阈下刺激。增加刺激电压引起细胞触发一个动作电位（红色曲线）。荧光染料或辣根过氧化物酶可以被注射到这些细胞中，研究它们的形态。荧光黄注射到一个RZ神经元通过使负电流通过电极。另一种方法是利用空气压力的抽吸。 图9A示出在注射开始后不久的染料的染料注入。三十分钟后，将染料已经已经扩散到神经根（ 图9B）。染料的类型取决于应用程序， 如小分子量的染料，可通过间隙连接来识别电突触。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig1.jpg" width="600px" /> 图1。 。腹水蛭清扫术（一）动物被彻底固定腹侧于弹性体内衬菜充满了水蛭生理盐水（B）浅切口跨越前：后轴刚才外侧中线，以避免损坏神经线。黑色的血窦（其中含有多种NS腹神经索）可以很容易地看到。请注意，一个暴露神经节相比，鞘是白色的。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig2.jpg" width="600px" /> 图2：黑色窦夹层的血管（箭头）应仔细切开一侧，露出腹神经索（双箭头）。离开血管段周围的节段性根部用作手柄解剖和记录菜肴之间传输时，操纵腹神经索完好。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3"FO：内容宽度=“3英寸”SRC =“/ files/ftp_upload/50631/50631fig3.jpg”宽度=“400像素”/&gt; 腹神经索的图3。隔离部分由其余的根段血管片段，并在段连接词的两端钢钉腹神经索暴露了神经节的电生理记录。绷紧拉伸神经节使人们看到了可识别的细胞体，将允许用锋利的玻璃电极的胶质鞘更容易渗透。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig4.jpg" width="600px" /> 图4。水蛭神经节（一）理想情况下，Retzius细胞（R）和其他大细胞体（P-细胞按解剖。URE，T型触摸，N-伤害，AE-环竖）将清晰可见，如果在显微镜和聚光设置正确（B）细胞在神经节的示意图。会发生在每个神经节中的胞体位置的微小差异是由于神经节是如何拉伸和固定的拉紧的，从这些神经元的胞 ​​体记录的（C）的动作电位具有特性的波形。这些尖峰是由注入正电流进入细胞（方波脉冲叠加的电压-时间的痕迹）诱发。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig5.jpg" width="600px" /> 图5。Desheathing神经节删除单个的细胞体（A）的胶质鞘应在整个神经节被精心切割，以避免利益损害细胞。红线表示切，以避免击中RZ神经元的胞体。打开外壳给人的消化酶接触到结缔组织基质（B）健康细胞体鼓胀出来的胶质鞘已被打开后，神经节中。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig6.jpg" width="600px" /> 图6：从培养水蛭神经节神经元（A）细胞从神经节使用微量吸并转移至含有培养基一道菜。（b）将多个单元彼此接近会增加突触的识别神经元之间的比率。在这里，STU两个神经元的国会议员在文化表现出略微不同的电性能形成突触（C）神经元。顶部迹线示出通过刺激相邻小区7小时和56小时的细胞铺板后诱发的突触电位。底部迹线是由注入电流直接进入RZ细胞之一诱发。注意增加突触电位的振幅和56小时培养后，改变动作电位波形。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig6highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig7.jpg" width="600px" /> 图7。水蛭神经节-体壁准备映射感受野（A）一种方法是伴随着1-3节清除体内壁的整个部分。在这里，在一边的根有BEEN切割和神经节已经被固定，以一侧。（B）固定，神经节在更高的放大倍率（C）另一种方法是在体壁和记录的完整神经节创建一个小的窗口，同时映射一个感受野（DG）各感觉神经元表现出对机械刺激的特征响应。（D）光触摸唤起了T细胞的活性，但不能在P或N个单元（E）的T细胞应答响应更强关闭刺激，活化在P细胞让路。由于刺激变得更加牢固的N个单元变为激活（FG） <a href="https://www.jove.com/files/ftp_upload/50631/50631fig7highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig8.jpg" width="600px" /> FIGURE 8。 诱发活性的水蛭神经节。连接词的细胞外刺激可以用来驱动突触传递的神经线。（A）这是通过附加抽吸电极的连接词之一，并施加一个小的电压进行。锋利的玻璃电极（箭头）可以看出，在刺穿神经节一Retzius细胞（B）电压-时间的痕迹，显示刺激伪迹后突触电位（黑色）和动作电位（红色）。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig8highres.jpg" target="_blank">点击这里查看大图像</a> 。 <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/50631/50631fig9.jpg" width="600px" /> 图9。染料注入到水蛭的神经元。（一）荧光黄可以通过使电流救援人员到场注入苏摩唉记录微电极。在注射后的水银灯泡和FITC过滤器设置可以被用于可视化荧光（B）染料已扩散到RZ轴突约30分钟。 <a href="https://www.jove.com/files/ftp_upload/50631/50631fig9highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis at JoVE.com

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

本文介绍了三种神经系统准备采用水蛭：细胞内记录从腹侧神经节的神经元，从腹侧神经节神经元培养，并从神经支配的皮肤感觉映射字段的补丁记录。

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 ...

intracellular-recording-sensory-field-mapping-culturing-identified

Research

JoVE Journal

Neuroscience

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

19.5K 次观看.  University of Kentucky. 本文介绍了三种神经系统准备采用水蛭：细胞内记录从腹侧神经节的神经元，从腹侧神经节神经元培养，并从神经支配的皮肤感觉映射字段的补丁记录。 

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

科研

神经科学

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 ...

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. ...

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, ...

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 ...

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

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

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 ...

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 ...

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 ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.