这项工作是由耳聋研究基金会研究资助支持安永和NIDCD DC006476 EG和NIDCD DC005211听觉和平衡的中心，约翰霍普金斯大学。作品版权添菲尔普斯，美国约翰霍普金斯大学。 LG写了最初的手稿，安永和LG拍摄解剖和录音。所有的作者提供了典范人物，写的稿子做出了贡献。

1。解决方案<ol><li>解决方案，可提前做好准备。在4 ° C外解决方案，可存储长达一个月。内部解决方案等分可以储存冷冻（-20℃）。确定的解决方案，在室温下开始实验前。 </li><li>剖析解决方案和细胞外记录解决方案（毫米）:5.8氯化钾; 144氯化钠， 氯化镁 2 0.9; 1.3氯化钙0.7的NaH 2 PO 4 5.6葡萄糖; 10 HEPES; 1丙酮酸钠，pH值7.4（氢氧化钠），300 mOsm 。对于免疫组化的Ca 2 +电流隔离外的解决方案可以修改显示:5.8氯化钾; 114氯化钠，氯化镁2 0.9; 1.3氯化钙0.7 的NaH 2 PO 4 5.6葡萄糖; 10 HEPES 30茶氯离子，pH值7.4 （氢氧化钠）; 300 mOsm。 </li><li> 1-2μM河豚毒素（TTX）可以添加到细胞外液块电压门控钠离子通道和隔离兴奋性突触后电流或电位（EPSCs或EPSPS）。 </li><li>细胞内记录解决方案（毫米）:20氯化钾; 110 K -磺酸; 5 MgCl 2的0.1氯化钙; 5 EGTA; 5 HEPES; 5 NA 2 ATP; 0.3 NA 2的GTP; 5 NA 2 phoshocreatine，pH值7.2（氢氧化钾） ， 290 mOsm;或135氯化钾，氯化镁2 3.5 0.1 氯化钙 ; 5 EGTA; 5 HEPES; 4 NA 2 ATP; 0.2 NA 2的GTP; pH值7.2（KOH），290 mOsm。 IHC胞内溶液的Ca 2 +电流隔离:135 CsMeSO3 13茶氯; 氯化镁 2 3.5 2.5 NA 2 ATP; 1 EGTA，pH值7.2（CsOH）; 290 mOsm 。这个解决方案会阻止一个大得多的K +电导数额巨大，目前剩下的留下的Ca 2 +。 </li></ol> 2。为持有解剖组织<ol><li>修改盖玻片使用期间举行录音准备到位，也可以提前做好准备。 </li><li>应用Sylgard（道康宁，美联，MI）的下降对一个圆形的玻璃盖玻片（8-12毫米）边缘。将盖玻片（FST，项目没有26002-10）罚款昆虫针粗末。昆虫的脚紧紧地握住反对用钳玻璃和一个加热线圈设置sylgard附近举行。 </li></ol> 3。制作电极<ol><li>准备新鲜的电极上每一个实验的当天和储存在密封盒。十到二十的电极制造为每个准备。 </li><li>选择电极的玻璃，我们用1毫米的高硼硅玻璃毛细管（1B100F - 4 WPI的，萨拉索塔，佛罗里达州）。 </li><li>设计上一个多步骤的拉马拉电极的尖端直径约为2-3微米（〜6MΩ在上文所述的解决方案）的方案。我们使用一个萨特的P - 87多步的水平拉拔或一个Narishige垂直PC - 10拉马。 </li><li>仔细大衣Sylgard电极柄接近尽可能的一角。这降低吸管电容和录音噪音降到最低。 </li><li>消防波兰电极使用，如加热灯丝，从WPI的一个microforge。消防抛光电极应该有一个外尖端直径约1微米（在上文所述的解决方案10-15MΩ）。吸管壁的厚度大约是1 / 3微米。外径大小要修补传入布顿。 </li><li>在实施同步录音的情况下:IHC吸管应构建以同样的方式（相同的玻璃，拉程序相同）的区别，火抛光应该留下一个较大的尖端直径〜3微米（6 - 8MΩ）。 </li></ol> 4。设置实验<ol><li>填写商会与重力美联储灌注系统外的解决方案和测试解决方案，含有药物或毒素的利益。 </li><li>设置灌注，使浴体积约2毫升，不断灌注在一个速率为1.5 mL min - 1的顺序。 </li><li>如果与局部灌注系统的实验过程中应用不同的解决方案，填补水库的解决方案。通过系统运行的解决方案，并确保没有气泡。 </li></ol> 5。夹层和样品制备<ol><li>所附的影片，说明了三个星期的老年大鼠（只Dawley，查尔斯河），这是较早期产后阶段的困难Corti器的夹层。记录从不同年龄大鼠的优点和缺点是审查讨论。 </li><li>深刻异氟醚吸入麻醉大鼠。缺席时撤回反射和角膜反射严重抑郁，杀头。由约翰霍普金斯大学的动物护理和使用委员会已批准了这些程序。 </li><li>删除从口鼻和皮肤的断头头。对开的头，并取出大脑暴露颞骨。 </li><li>颞骨和地方清洁解剖菜含有标准细胞外液中删除。 </li><li>删除颞骨骨封装，以揭露耳蜗。颞骨保持牢固的基地一双镊子。确定的圆形和椭圆形的窗户。定位耳蜗卵圆窗和螺旋式侧耳蜗朝上，使围绕耳蜗中删除多余的骨。 </li><li>取出的骨封装耳蜗感觉上皮暴露，同时注意保护根尖线圈，将用于实验。使用第二对的罚款钳直接芯片关闭耳蜗的骨，开始与该地区比其他骨更透明。这里的骨较薄和容易消除。继续删除从根尖线圈周围的骨头。 </li><li>使用微解剖剪刀穿过下方顶转的modiolus。然后断开从根尖耳蜗较低轮流转。 </li><li>再次使用微解剖剪刀，如果有必要，以确保根尖又是完全切断。注意保护根尖线圈，它不应该拉或拉伸。用细镊子哄其余耳蜗根尖转。 </li><li>取出根尖转两侧的骨休息。 </li><li>小心取出血管纹，位于外毛细胞的区域（示意图1）组织的光亮带钢。请一定要避免删除的感觉毛细胞，它可以很容易脱落，血管纹沿。 </li><li>用细镊子分离盖膜，有光泽，半透明膜，坐在上面的感觉毛细胞。 </li><li>现在，修剪多余的组织和骨和扁平镊子准备。这是必要的，这样的组织可以放在下针均匀。 </li><li>下放置的引脚连接到盖玻片（先前准备），照顾引脚的位置，离毛细胞的准备。 </li><li>使用镊子盖玻片转移到录音室。确保人工耳蜗组织是完全下降而细胞外液转移盖玻片覆盖。按盖玻片下来到会议厅的玻璃底部牢固，以确保它在录制过程中不动。 </li><li>立即着手与细胞外液灌注，以确保更好地生存的准备。 </li></ol> 6。记录<ol><li>找到准备通过显微镜目镜使用的10倍和40倍水浸泡DIC的目标。定位的准备记录电极可以使正交方法综合家居照顾服务的IHC侧壁。 </li><li>如果准备卷曲，限制的综合家居照顾服务的基础地区的能见度，使用记录电极反推玻璃盖玻片的外缘下来的准备，照顾，以避免按综合家居照顾服务自己。 </li><li> *如果使用幼鼠，在这个阶段厚厚的一层以上的毛细胞，支持细胞，可以用清洁吸管（与尖端直径约10-20微米），以获得感官细胞1列，12中删除。 </li><li>使用的监控，以评估是否是健康的组织。使用显微镜和NC70 Newvicon相机的4倍的放大倍率镜头，进一步放大图像和项目，面积约4800μm 的监视器上2。应拉长毛细胞具有完整的发束。当组织恶化，毛细胞膨胀并变得更加透明和颗粒感。 </li><li>围绕基地的综合家居照顾服务本地化传入boutons。 Boutons是球形或椭圆形，直径约1微米，在一个闪亮的外观颜色的光。 boutons多数本地化细胞核水平以下。当组织，是不健康的boutons膨胀到正常大小约4倍，成为透明的，而不是有光泽，。 </li><li>填写记录电极与细胞内的解决方案，适用于正压和使用的显微操纵电极的准备。 </li><li>进行编制的免疫组化基地与电极水平的切口。按电极之间的一层薄薄的支持细胞和综合家居照顾服务，使用正压伪造通道在该地区的综合家居照顾服务的基础。这一步是为达到传入布顿和不支持紧密ensheath传入神经末梢的细胞，记录的"钥匙"。 </li><li>小心地取出电极和一个充满新鲜的电极更换。 </li><li>使用积极的压力，保持一个干净的电极尖端，通过访问孔机动电极膜的免疫组化。正压应协助在周围的支持细胞周围的综合家居照顾服务的移动。 </li><li>电极周围相邻的支持细胞对传入布顿在所附的影片所示（移动，另一种方法是方法通过移动传入布顿长期的免疫组化膜电极尖端触碰，直至布顿）。确保电极尖端在前面的传入布顿（仍然是一个小的差距之间的布顿和电极由于正压移液器）。 Boutons提供超过支持细胞的抵抗力和免疫组化膜吸管的运动，它们可以"感觉"实验者。 </li><li>移动吸管向上和向下推，以确保，布顿移动。这表明，枪头和布顿在同一Z平面。 </li><li>对传入布顿按的电极，同时释放出积极的压力，适用于吸，形成GΩ的密封。形成一个传入布顿GΩ的密封是到毛细胞相似。印章形成可迅速出现或可能出现缓慢。 </li><li>应用温柔的阵阵吸内的电极膜破裂，并进入全细胞记录配置。 </li><li>如果细胞修补传入布顿，小库容瞬变将出现在广场密封形成过程中监测的测试脉冲（见图1）。我们估计电容传入的结束为0.4 - 1.8 PF（ 见3，4） 。如果修补细胞是免疫组化，瞬态将取决于接入电阻的顺序上的3-5倍以上。 </li><li>为了确认该单元格传入布顿，极化和去极化电压步骤运行的协议。传入纤维及综合家居照顾服务（经常打补丁误时，针对传入布顿）的电流电压关系（IVS）是个别的细胞类型的特征，如图2所示。 </li><li>监视器的膜电位;传入纤维，它通常是-60至-65 mV的周围。 </li><li>如果细胞是不是一个传入布顿，一个充满新鲜的电极取出电极，然后重复步骤8 - 13。每次尝试使用新的电极。 </li><li>监控整个串联电阻施加电压的步骤，以确保密封不打烊的记录。如果串联电阻的增加，它可以帮助申请吸温和的脉冲或稍向后移动电极。串联电阻通常是大约30兆欧。分析突触活动时，我们放弃串联电阻大于50MΩ的录音。 </li><li>现在，您可以记录突触活动（见图3）。无论是展品的传入纤维自发活动，或毛细胞需要去极化激活递质释放。应用具有较高的钾浓度（例如40毫米）的细胞外液的毛细胞去极化，往往激活或增加递质释放的速度。 </li><li>对于同步录音的综合家居照顾服务和传入boutons 2，程序应以下列方式进行修改:继续通过步骤1至5。 IHC吸管填写与细胞内相应的解决方案对Ca 2 +电流隔离，并按照6。离开的IHC记录在"等待的立场"接近的IHC记录吸管。继续步骤6 - 14传入记录。当传入的吸管，在整个单元的配置，继续与IHC吸管。 Manoeuvre对相应的免疫组化的侧壁第二移液器，始终保持积极的压力。 IHC应显示侧壁压力缩进，它需要得到保证，支持细胞分离从。步骤12 - 13为免疫组化以及可用于。正如在14日的传入纤维相比，更大的电容瞬变的IHC录制的一个标志，除了特征IV关系。一旦建立了同步录音，应该允许一段时间（3 - 5分钟，根据串联电阻）为细胞内的解决方案，洗成的免疫组化。这将导致更大的Ca 2 +电流由于增加更大的K +电导块。 </li><li>为了执行宽松的密封外录音，在第12步，而不是一个GΩ的密封，到传入布顿一个宽松的密封约30至50MΩ。这可以通过申请，同时释放正压少吸。传入布顿从P21的大鼠细胞外记录的一个例子，请参阅图5。 </li></ol> 7。故障排除<ol><li>如果密封可以形成，但细胞贴附过渡到全细胞记录无法实现，吸管内径可能过于狭窄。 </li><li>如果无法形成严密密封，内吸液管直径可能太大，整个布顿可以吸进电极。 </li><li>如果所有的突触事件是小而均匀，录音外传入。测试事件积极的膜电位逆转;细胞内记录EPSCs将扭转积极的潜力。 </li><li>如果串联电阻是一贯的高修补传入终端时，尝试前试图打入整个单元的配置通过吸管向后移动。这有助于防止吸管堵塞和高访问性。 </li><li>无所畏惧的移液器的正确位置。这是可能的电极移动，没有"松动"传入的印章，以减少高串联电阻或吸管堵塞。在建立同步录音，IHC通常是"推"对布顿。传入吸管的位置，可以予以更正。 </li></ol> 8。代表性的成果<img src="/files/ftp_upload/2442/2442fig1.jpg" alt="图1" /> 图1。 AB型 。记录传入纤维（A）和免疫组化（二）典型的瞬态极化从-94毫伏的控股电压电压命令到10毫伏。由于狭窄的吸管直径和高访问性，免疫组化录音（B）是不理想的全细胞免疫组化录音。记录显示，这里只说明从综合家居照顾服务和传入神经纤维之间的库容瞬变的区​​别。这有助于形成全细胞配置时的细胞类型之间的区别。从综合家居照顾服务的全细胞库容瞬变的幅度大于传入纤维的5倍。光盘。从A和B的瞬态显示在一个扩大的时间表。 C.传入的反应衰变可配合两个指数函数。电容传入的结局，估计从快的组成部分。 <img src="/files/ftp_upload/2442/2442fig2.jpg" alt="图2" /> 图2四传入布顿（A）和免疫组化（二）关系。从保持电位-84 MV从-124 mV时，以10 mV递增（额定电压）+ 36毫伏的电压步骤IV关系记录。电压显示一些痕迹的权利。这些录音进行了在室温下用5.8毫米外氯化钾。规模为:500 PA，200毫秒。 。IV关系，从出生后第19天的传入纤维。 EPSCs目前在大多数的电压步骤; EPSCs 6 mV的反向积极。此记录进行了在TTX的存在阻断电压门控Na +电流。请注意在极化电压（I H）慢慢激活的内向电流。此电流在综合家居照顾服务或支持细胞中不存在，并提供了一个很好的迹象，记录从细胞传入纤维（见3）， B。四，从P19的免疫组化关系。由于狭窄的吸管直径和高访问性，记录不理想的表征IHC电流和电流小于预期。录音显示，在这里可以清晰地分辨出，企图传入纤维录音时，只展示综合家居照顾服务和传入纤维的IV关系。注意积极的潜力，其次是延迟整流钾电流13（箭头）的快速激活外向K +电流。 <img src="/files/ftp_upload/2442/2442fig3.jpg" alt="图3" /> 图3。突触电流传入纤维在40毫米的细胞外K +释放率增加IHC存在从一个在出生后第21天录得的典范。室温TTX，应用于块电压门控Na +电流。规模200 PA，5毫秒，请注意变量的大小和形状的EPSCs。对于EPSC特性的详细描述见 4，B 。 （:多相，O:＃单相）标记的两个EPSCs规模的不断扩大显示:刻度100 PA，1毫秒。 <img src="/files/ftp_upload/2442/2442fig4.jpg" alt="图4" /> 图4。尔蒂，产后10天（也见2）切除大鼠器官的免疫组化和联系传入布顿的同步录音。一个电压阶跃去极化的IHC引发神经递质的释放和激活传入布顿EPSCs 上跟踪:IHC去极化电压的协议。控股的潜力:-79 mV时，50毫秒-29毫伏的步骤中跟踪 :L -型钙 2 IHC 记录电流通常表现出小的失活和负电位激活下部曲线:反应传入纤维突触电流。 IHC去极化。请注意在50毫秒IHC去极化的突触的抑郁症。 <img src="/files/ftp_upload/2442/2442fig5.jpg" alt="图5" /> 图5。 。传入布顿在产后21天的细胞外记录的典范。这是记录在室温下TURE，用5.8毫米的细胞外K + 。这个录音有一个典型的信号噪声比为记录在准备从3周龄大鼠，B。从P20的传入布顿记录外事件的平均波形。这是从10272事件的平均波形。 <img src="/files/ftp_upload/2442/2442sche.jpg" alt="示意图1" /> 示意图1。穿越说明内层和外层的毛细胞，螺旋神经节，血管纹和盖膜之间的解剖关系的大鼠耳蜗转剖视图。

<ol>
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D., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+calcium+dependence+of+transmitter+release+at+a+single+ribbon+synapse.">Time course and calcium dependence of transmitter release at a single ribbon synapse.</a> Proc Natl Acad Sci U S A. 104 (41), 16341-16346 (2007).</li><li>Yi, E., Roux, I., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+HCN+channels+shape+excitatory+postsynaptic+potentials+at+the+inner+hair+cell+afferent+synapse+in+the+mammalian+cochlea.">Dendritic HCN channels shape excitatory postsynaptic potentials at the inner hair cell afferent synapse in the mammalian cochlea.</a> J Neurophysiol. 103 (5), 2532-2543 (2010).</li><li>Grant, L., Yi, E., Glowatzki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+release+shape+the+postsynaptic+response+at+the+inner+hair+cell+ribbon+synapse.">Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse.</a> J Neurosci. 30 (12), 4210-4220 (2010).</li><li>LoGiudice, L., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ribbons+at+sensory+synapses.">The role of ribbons at sensory synapses.</a> Neuroscientist. 15 (4), 380-391 (2009).</li><li>Singer, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+multivesicular+release+at+a+mammalian+ribbon+synapse.">Coordinated multivesicular release at a mammalian ribbon synapse.</a> Nat Neurosci. 7 (8), 826-833 (2004).</li><li>Keen, E. C., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+characteristics+of+the+hair+cell's+afferent+synapse.">Transfer characteristics of the hair cell's afferent synapse.</a> Proc Natl Acad Sci U S A. 103 (14), 5537-5542 (2006).</li><li>Li, G. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+spontaneous+activity+in+the+developing+auditory+system.">The origin of spontaneous activity in the developing auditory system.</a> Nature. 450 (7166), 50-55 (2007).</li><li>Kros, C. J., Ruppersberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+potassium+current+in+inner+hair+cells+during+development+of+hearing+in+mice.">Expression of a potassium current in inner hair cells during development of hearing in mice.</a> Nature. 394 (6690), 281-284 (1998).</li><li>Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+of+frequency+representation+in+the+rat+cochlea.">Developmental changes of frequency representation in the rat cochlea.</a> Hear Res. 56 (1-2), 1-7 (1991).</li><li>Seal, R. 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在此过程中的关键步骤是剥离。如果组织被拉长，或在夹层中损坏，传入神经纤维将无法生存。年轻的大鼠组织更多的弹性和宽容。我们发现，产后10至11天是最简单的解剖和实验的成功率较高。一个人工耳蜗成熟的重要程度发生出生后，开始听到来自12〜14日龄大鼠。因此，在年龄夹层是最容易的地方，突触可能无法完全成熟。 这里描述的大鼠解剖基本上是小鼠，主要的区别是更小的尺寸小鼠耳蜗相同。这种技术可以在小鼠15 transgenically修改审查带状突触的属性。这种技术的进一步修改包括:标签纤维 3到细胞内的解决方案中加入荧光染料;与突触前的内毛细胞和突触后传入布顿配对录音，让前和突触后细胞之间的传递函数 ，以确定2和宽松密封外录音传入boutons细胞的完整性，以避免损失。外记录的配置比整个单元的配置更容易实现和实验一般持续时间更长。

最重要的是在我们的电设置用于记录传入终端设备。

postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse

之间的内毛细胞（IHC）和听觉神经纤维传入突触提供一个electrophysiologically访问网站的记录 1-4单一的带状突触的突触后活动。感觉细胞带状突触释放的神经递质不断，这是调制在IHC膜电位的梯度变化的响应率。带状突触已被证明经营，多泡释放，可以释放多个水泡，同时唤起人们的兴奋性突触后电流（EPSCs）不同振幅1，4，6-11 。无论是突触前的色带，也不多泡释放的内在机制的作用，是目前很好的理解。 10-20听觉神经纤维，并与每一个结束，形成一个单一的带状突触的无髓单纤维接触的免疫组化免疫组化是支配。传入boutons联系综合家居照顾服务（直径约1微米）的小尺寸，使具有特殊时空分辨率进行录制。此外，该技术可以适应记录前和突触后细胞的同时，允许在突触传递函数进行研究，直接2。因此，这种方法提供了一个由神经传递的基本方面，可以研究的方法，从多泡释放的感觉细胞剪彩难以捉摸的功能。

Watch this Scientific Journal Video about Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse at JoVE.com

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

全细胞膜片钳从听觉神经纤维在哺乳动物的耳蜗内毛细胞带状突触树突。

与耳蜗内毛细胞传入树突:多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

postsynaptic-recordings-at-afferent-dendrites-contacting-cochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.4K 次观看.  The Johns Hopkins School of Medicine. 全细胞膜片钳从听觉神经纤维在哺乳动物的耳蜗内毛细胞带状突触树突。

视频: Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

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  ...

科研

神经科学

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

结状神经节注射液只SD大鼠的方法

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

调频染料循环在突触: 高钾偏极化, 电和 Channelrhodopsin 刺激的比较

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

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

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

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

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

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

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

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...