Name: electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording
Uploaded: 2015-11-19T13:00:00.000+00:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

此方法需要使用动物对象，由拉瓦尔大学动物伦理委员会（协议＃2012-170）所允许的。 1.设置和准备<ol><li>解决方案<ol><li>根据下列配方7,17制备脑脊液原液。其他食谱浓度变化是在文献中提供。商店储备溶液在4℃最多一个月。 <ol><li>盐溶液:加75.39克氯化钠（129毫米决赛）; 2.5克氯化钾（3.35毫最终）; 0.81克磷酸二氢钠的（0.58毫最终）; 2.33克氯化镁（1.15毫最终）; 1.85克氯化钙（1.26毫米）。在800毫升蒸馏水溶解，然后填充到1升用蒸馏水。 </li><li>碳酸氢钠溶液:加17.65克碳酸氢钠 （21毫米决赛）的。 900毫升蒸馏水溶解，然后填充到1升蒸馏水。碳酸氢盐浓度的变化将导致pH值的变化。 </li><li>葡萄糖溶液:增加54.06克葡萄糖（30毫米决赛）。在400ml的蒸馏水溶解然后填充到500毫升蒸馏水。 </li></ol></li><li>通过稀释百毫升盐溶液，100毫升的碳酸氢钠溶液，和50毫升在1升蒸馏水的葡萄糖溶液准备脑脊液。储存在4℃直至使用。 </li><li>通过加热和拉伸玻璃管直至其断裂制成玻璃电极。落砂，使用前尖。所述电极可以，只要它保持清洁重复使用许多时间。 </li></ol></li><li>实验装置 注:的建立的细节在图2中呈现。 <ol><li>打开放大器，移动平均器，数据采集系统，温度控制器和泵。检查信号放大（增益= 10,000），过滤（低阈值，10赫兹;高阈值，5千赫兹），以及模拟的采样率对原始信号（2.5千赫）的数字转换。 </li><li>填写一瓶与学联和气泡用卡波（95％O 2 </sub&gt;下，5％的CO 2）。在4毫升/分钟诱导bubbled-和温热-脑脊液流动录音室（体积5毫升）的。持在26的录音室中的温度（±1）℃。 pH值应在这些标准条件7.4。 </li><li>请50毫升注射器解剖室附近50毫升RT和卡波鼓泡学联。 </li><li>打开电脑，启动录音软件。 </li></ol></li></ol> 2.解剖<ol><li>称量并直观地判断动物的性别（男性有更长的肛门 - 生殖器距离，而女性有一个短暂的肛门 - 生殖器距离;男性生殖器往往暗而女性的生殖器是粉红色）。 </li><li>通过下列选项之一麻醉新生的老鼠:cryoanesthesia（完全沉浸在冰上动物4 - 5分18）（4毫升/公斤equitensine），注射19或吸入挥发性麻醉剂（异氟烷20或醚21）。 CONFIRM麻醉由缺乏缩足反射足够的飞机。 </li><li>将动物在板凳上，腹部朝下。部的头冠的用手术刀在囟水平（通过皮肤和颅骨可见）延髓一部分。执行此步骤动物麻醉后立即使用。 </li><li>段体与冠下的前成员手术刀。 </li><li>除去皮肤，肌肉，脂肪组织和内脏手术和薄剪刀和钳子。由于骨头是软的，在这个年龄段，谨慎，不损害神经系统。执行板凳上这一步。 </li><li>将制剂在清扫室。使用存储在注射器中的脑脊液充氧的制备。从这个点到记录结束，则使用显微镜。 </li><li>关于制剂的背面，切从延髓颅骨和椎体沿着与手术薄剪刀和钳子的介质轴的尾部。打开切头骨而为了椎体暴露的神经组织。使用strored在注射器中的脑脊液充氧的制备。 </li><li>用钳子，取出蛛网膜膜，薄的组织覆盖神经组织的表面上。保留软脑膜和血管对神经组织。使用存储在注射器中的脑脊液充氧的制备。 </li><li>放置制剂与背面朝下，并小心地通过切割神经和结缔组织用剪刀，同时保持制剂代替打倒脑干和脊髓。一背的做法也是可以的。保持根和神经尽可能长的时间。取出骨头分离脑干和脊髓。使用存储在注射器中的脑脊液充氧的制备。 </li><li>除去小脑和通过用解剖刀切片他们剩余喙结构。使用存储在针的脑脊液充氧的制备。 </li><li>可选取决于experimenTAL设计，由冠状部分前取出用手术刀脑桥的小脑后下动脉22。需要注意的是保持整个脑桥将大鼠放慢节奏和充分抑制其在小鼠中。准备工作包括脑桥只有尾部分显示与在C4根频率稳定的呼吸样活性。 </li></ol> 3.记录<ol><li>将制剂在记录室中，腹侧面朝上。固定的制备与在脊髓和脑干延髓-大部分的最下部销。 </li><li>使用链接到由它的针电极的注射器，诱导电极中的凹陷（玻璃尖端直径:150 - 225微米）通过拔出注射器活塞，以部分地与脑脊液填充电极。 </li><li>使用显微操作，小心地将电极靠近第四腹侧根源之一。其他前根也可能呈现出呼吸般的活动（e。克，十二对脑神经，C1神经前根）。 </li><li>经由注射器通过以轻轻抽吸神经支根拉出活塞诱导电极槽的凹陷。然后小心地将电极，将其应用对脊髓。 注意:理想的是，支根的大小相匹配的电极开口的尺寸，从而造成在电极的内部和外部隔室之间的密封。由于差分放大器通常用于这种记录，电极的记录室中的内部和之间的任何开口降低了信号的质量，并使得难以消除背景噪音。 </li><li>开始录制。 </li><li>记录由含氧量正常条件下的制备产生的节律（即，脑脊液鼓泡与卡波金:95％O 2和5％的CO 2）至少20分钟，以确定制剂的基线参数。 </li><li>从卡波鼓泡学联切换灌注刺激脑脊液（即，鼓泡用95％的N 2和5％CO 2的低氧刺激）15分钟。改变根据实验方案的曝光时间。记录在记录和动物数据表中的曝光时间。使用不锈钢管中的脑脊液瓶并尽可能避免气体扩散到管的外部的腔室之间。 </li><li>切换灌注回标准卡波鼓泡学联至少15分钟的恢复录制。记录本上的记录和动物的数据表。 </li><li>结束录制。 </li></ol> 4.统计分析<ol><li>从合并的信号，计算出的频率为每分钟（表示在脉冲串/分钟）脉冲串的数量，振幅为基线和突发（以mV为单位）的峰值之间的差，该脉冲串持续时间作为从时间开始到脉冲结束（以s）时，脉冲串区域作为CUR下的面积已经对积分信号（以mV·秒）（图3）的脉冲串的。 </li><li>计算的频率，振幅，脉冲串持续时间和脉冲串区域下含氧量正常（基线），缺氧（刺激）和后缺氧（刺激后）的恢复条件的最后5分钟的录音为每个条件的平均值。不分析每种条件的第一部分，因为有切换灌注和脑脊液均质录音室中之间的延迟。 </li><li>然后表达的刺激（即，缺氧 ）和刺激后（即，后缺氧）恢复值作为基准值的相应记录的百分比。快递结果均值±标准差</li></ol>

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S., Denavit-Saubie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+NMDA+receptors+in+the+respiratory+phase+transition+is+different+in+the+adult+guinea+pig+in+vivo+and+in+the+isolated+brain+stem+preparation.">Involvement of NMDA receptors in the respiratory phase transition is different in the adult guinea pig in vivo and in the isolated brain stem preparation.</a> J Neurophysiol. 74, 770-778 (1995).</li><li>Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+volatile+anesthetics+on+respiratory+activity+and+chemosensitivity+in+the+isolated+brainstem-spinal+cord+of+the+newborn+rat.">Effects of volatile anesthetics on respiratory activity and chemosensitivity in the isolated brainstem-spinal cord of the newborn rat.</a> Hokkaido Igaku Zasshi. 73, 117-136 (1998).</li><li>Gestreau, C., 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=Task2+potassium+channels+set+central+respiratory+CO2+and+O2+sensitivity.">Task2 potassium channels set central respiratory CO2 and O2 sensitivity.</a> PNAS. 107, 2325-2330 (2010).</li><li>Caravagna, C., Soliz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+and+MEK+molecular+pathways+are+involved+in+the+erythropoietin-mediated+regulation+of+the+central+respiratory+command.">PI3K and MEK molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command.</a> Respir Physiol Neurobiol. 206C, 36-40 (2014).</li><li>Tree, K., Caravagna, C., Hilaire, G., Peyronnet, J., Cayetanot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anandamide+centrally+depresses+the+respiratory+rhythm+generator+of+neonatal+mice.">Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.</a> Neuroscience. 170, 1098-1109 (2010).</li><li>Arata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+activity+of+the+neonatal+dorsolateral+pons+in+vitro.">Respiratory activity of the neonatal dorsolateral pons in vitro.</a> Respir Physiol Neurobiol. 168, 144-152 (2009).</li><li>Onimaru, H., Homma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+functional+neuron+group+for+respiratory+rhythm+generation+in+the+ventral+medulla.">A novel functional neuron group for respiratory rhythm generation in the ventral medulla.</a> J Neurosci. 23, 1478-1486 (2003).</li><li>St-John, W. M., Paton, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizations+of+eupnea,+apneusis+and+gasping+in+a+perfused+rat+preparation.">Characterizations of eupnea, apneusis and gasping in a perfused rat preparation.</a> Respir Physiol. 123, 201-213 (2000).</li></ol>

The authors have no competing financial interests or conflicts of interests to disclose.

呼吸活动的准确定量是很有挑战性的。的确，呼吸是一个函数，既可以是自动和自愿的，即根据环境，人体的需要，情绪状态和行为调制。这种技术的优点是负责产生呼吸命令神经元件的隔离。因此，脑干脊髓制剂和体积描记法电生理记录是互补的技术，研究了整个神经网络呼吸体外和体内分别。在脑干脊髓制备的膜片钳记录（例如，从腹面接近）也可以设想，并允许在一保存呼吸网络 24的特定神经元的研究。 该协议包括一些关键步骤，主要是在夹层中。为了避免神经组织损伤，所有的清扫措施应迅速，准确地执行。神经组织不应该被损坏，并且在学联不断浸泡谨慎保守。如前面提到的，神经支根和电极之间的连接的质量是第二关键步骤。在小根的吸能带来极接近的准备。而在电极和脑干之间的接触可以提高密封件的质量，​​就必须避免使一个凹陷或物理损坏的制备。 所述积分信号应始终用于分析，但原始信号可以是有用的，以区别于背景噪声适当的脉冲串。此外，扬声器可以被添加到设置听节奏和确定的信号质量和背景噪声存在由试听。的确，在背景基线一些变型可以存在由于电干扰和导致基线不稳定。这种INTERFerences应该通过检查电缆连接并根抽吸通过电极来防止。另外，因为突发幅度取决于两个背景噪声和根与电极之间的连接，频率是一个更可靠的参数。因此，脉冲串幅度应该总是被表示为基线值的百分比，以避免由于设置个体间差异。 该技术的局限性是由从该制剂可实现动物的年龄来确定。这允许中央呼吸驱动的出生时设立，这是非常有趣和临床上相关的一个精确的研究，但它着重于这些早期年龄和不能扩展到以后的年龄。老年阶段可以在动脉灌注工作心脏-脑干制剂25中使用，但有大量的协议的修改（见下文）。从成年豚鼠分离的脑干，脊髓准备已经发展由桑色素-苏润等人 26，其中，脑干经由基底动脉灌注和节奏被记录在十二脑神经编另一个限制是在实验的持续时间。制备不能保持超过7小时，在先前描述的条件6。因此，这种技术不适合于长期实验中，即使从蝌蚪（见下文）的制剂一直保持24小时，在实验室中。 各种修改可以应用到这种技术。这里，缺氧挑战已经执行，但其它气体变化也是可想到（例如，高碳酸血症27以及pH值变化28）。以相同的方式，该制剂可灌注脑脊液，其中药物已经溶解和施加到脑干作为预处理29或30中所述记录。在这种情况下，节奏可以加速29或减慢陶氏根据实验药物Ñ30。此外，与一个隔间记录室中，不同的人工脑脊液可以在制备6和温度变化9可以采用选择性份施加。这种划分使选择的脑干部位的刺激诱导作用的含义的研究。而提出的技术使用新生鼠，它也可以适用于其它动物模型。使用类似于应用于大鼠一个协议，小鼠可用于从妊娠第16天（E16），直到P4 12。主要的区别是，需要在解剖温热和卡波金气泡的脑脊液。海龟，蝌蚪和成人青蛙也可以使用;然而，这些种类需要不同的解剖技术以及调整脑脊液的组合物。 这种技术也可以加上膜片钳记录的制备31的喙面，允许网络输出信号的同时可视化和该网络中的一个特定信元的活性。电压敏感的染料成像也可以在新生大鼠脑干脊髓制剂施加到对脑干32的腹侧面的激活区定位。的c-fos表达的制剂分析也可以实现，以鉴定参与特定呼吸道反应的神经种群。最后，该制剂可被修改，以使其他器官，如心脏25，肋6，或生理动脉灌注与其他生理节律33。然而，这些非常重要的修改会要求其他解剖协议。

呼吸是一个复杂和生命活动由大脑控制，允许双氧（O 2）的摄取和二氧化碳（CO 2）的消除。中枢性呼吸的驱动器是由一个复杂的网络，位于脑干的两个哺乳动物1，两栖类2类，爬行类3，4鸟类和鱼类5生成。即使呼吸的研究可在体内进行处理，精密机械研究需要直接访问的呼吸控制网络。为此，Adrian和Buytendijk开发出减少的金鱼制剂，其中电极放置在脑干表面记录用鳃通风5相关联的产生的节奏。这一做法后来被Suzue于1984年6适应新生老鼠用。这种制剂的出现，导致呼吸神经生物学显著的进步。由于它是比较简单的，该技术呈现ħERE是适合于各种有节奏的运动行为和他们的新生啮齿动物起源的基本调查。 该方法的总的目标是记录吸气活动的神经相关，一个呼吸状节律称为假想呼吸，通过呼吸网络产生。这种方法可以用在广泛的研究目标，目标吸气响应呼吸变化或药理学在野生型7和转基因动物的8。鉴于实验关于记录的信号的生理相关性在低温下进行的，没有感觉传入，并且其中葡萄糖和 O 2的脑脊液内的浓度高的条件下，问题已经被提出。虽然有在体内和体外条件差别明显（例如，吸气突发的频率）的事实仍然是存在呼吸网络 6的核心元素使得能够研究相关 ​​的一个重要的体内平衡功能9,10一个健壮的节奏。 后面的发展和使用该技术的基本原理是，以促进直接进入呼吸网络的脑干元素，这是在体内难以进入，特别是在新生儿。脑干被置于严格控制的条件下:录制的节奏不被从肺部或颈动脉体周围神经传入调制，使研究专注于呼吸中枢驱动器本身11上。因此，该访问被用来施加激励和记录的输出信号。在对比体积描记法记录，呼吸节律是通过其所有组件整个身体调制（例如，肺腹胀，外围化学传感器），使之难以应用于精确的刺激。 在ewborn大鼠，该协议包括上记录的分离的脑干第四​​前根信号和截短脊髓，保持在人造脑脊髓液（ACSF）中。由脑干脊髓制剂产生的节奏由链接到吸气信号 9个体慢突发。隔离脑干，脊髓制剂很容易在大鼠0产后天可记录到4（P0 - P4）7。这种方法通常被用来评估呼吸道网络的低氧反应，并且还响应于高碳酸血症，酸中毒或药物。急性缺氧协议这里介绍。被撤销的学联O 2获得此刺激;这种方法通常用于评估耐受性和响应性缺氧的侮辱。该协议导致从第一分钟节奏抑郁直到缺氧暴露的端部（ 图1）12。这个凹陷反转在后低氧恢复12。关于实验设计，它必须注意到，脑桥，位于脑干延髓部分，对节律发生器8的抑制作用是重要的。因此，全脑干延髓和脊髓的筹备工作显示较低的节奏。用于记录分离的样品中的脑桥夹杂物根据实验13的目标确定;延髓网络上脑桥影响的研究将需要记录有和没有脑桥比较结果14。此外，这种技术的优点之一是延伸制剂的喙部，以包括中脑和/或间脑区域15,16，使得可以评估这些区域的脑桥延髓呼吸网络的影响的可能性。

<table><tbody><tr><td>Sylgard</td><td>Sigma Aldrich</td><td>761036-5EA</td><td>Use under hood</td></tr><tr><td>NaCl</td><td>Bioshop</td><td>SOD002</td><td></td></tr><tr><td>KCl</td><td>Bioshop</td><td>POC888</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>CCL444</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>MAG510</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Bioshop</td><td>SPM306</td><td></td></tr><tr><td>D-glucose</td><td>Bioshop</td><td>GLU501</td><td></td></tr><tr><td>Carbogen</td><td>Linde</td><td>343-02-0006&amp;nbsp;</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments, Hamden, CT, USA</td><td>TC-324B</td><td></td></tr><tr><td>Suction electrode</td><td>A-M Systems, Everett, WA, USA</td><td>model 573000</td><td></td></tr><tr><td>Differential AC amplifier</td><td>A-M Systems, Everett, WA, USA</td><td>model 1700</td><td></td></tr><tr><td>Moving averager</td><td>CWE, Ardmore, PA, USA</td><td>model MA-821</td><td></td></tr><tr><td>Data acquisition system</td><td>Dataq Instruments, Akron, OH, USA</td><td>model DI-720</td><td></td></tr><tr><td>LabChart software</td><td>ADInstruments, Colorado Springs, CO, USA</td><td></td><td></td></tr><tr><td>Prism sofware</td><td>Graphpad, La Jolla, CA, USA</td><td></td><td></td></tr><tr><td>Dissection chamber</td><td>Plastic box (e.g. petri box) will do</td><td></td><td></td></tr><tr><td>Recording chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instrument Inc, Sarasota, FL, USA</td><td>KITE-R</td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson, Middleton, WI, USA</td><td>MINIPULS 3</td><td></td></tr><tr><td>Faraday Cage</td><td>Home made</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr></tbody></table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

如在引言中提到，在此技术的最重要的优点之一是可直接进入脑干应用各种刺激。作为一个例子，缺氧在这里使用。 图1。AB显示一个完整的协议记录，既常氧和缺氧的条件。 图1.CE显示记录在含氧量正常条件（即节奏，脑脊液入95％O 2和5％ 的 CO 2在26℃）。如先前在此确切制剂11证明的，频率大约为8个 ​​脉冲串/分，振幅为约0.800毫伏。需要注意的是幅度是因为其重要的跨制剂变化仅供参考。上方的轨迹表示积分信号而下部曲线代表的原始信号。 图1.DF显示记录在低氧条件（即，脑脊液入95％的N 2和5％的CO节奏&lt;子&gt; 2，在26°C）。正如前文所表征23，频率急剧缺氧下降。 图1.GH显示单个突发显示在常氧和缺氧条件下的典型模式。 <img alt="图1" src="/files/ftp_upload/53071/53071fig1.jpg" /> 录音从脑干，脊髓准备工作取得的图1的例子。从下常氧，缺氧和后低氧恢复条件（（A）集成信号和（B）原始信号）从P4大鼠制剂的C4前根记录呼吸输出。对于每个条件，扩大录音（（c）综合常氧信号;（d）综合缺氧的信号;（五）原常氧信号;（六）原缺氧信号）和单脉冲（（G）常氧爆和 （H）低氧突发）被示出。正如文中提到，请注意，幅度必须谨慎解释。 <a href="https://www.jove.com/files/ftp_upload/53071/53071fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53071/53071fig2.jpg" /> 安装图2概括性架构的起泡学联被加热，并通过录音室中开泵发送。脑脊液过量录音室中通过泵吸处置。记录室被连接到地和温度控制器。该电极输出被直接中继到放大器，然后向移动平均器和数据采集系统，最后的计算机。 <a href="https://www.jove.com/files/ftp_upload/53071/53071fig2large.jpg" target="_blank">请按此吨</a>Ø查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53071/53071fig3.jpg" /> 图3.分析突发参数，频率被计算为每分钟（表示在脉冲串/分钟）脉冲串的数量，振幅为基线和突发（以mV为单位）的峰值之间的差，该脉冲串持续时间作为从年初到突发的结束时间（以秒表示），突发面积上集成的信号突发的曲线下面积（单位:毫伏·秒）。 <a href="https://www.jove.com/files/ftp_upload/53071/53071fig3large.jpg" target="_blank">请点击此处查看本一个更大的版本数字。</a>

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Nanyang Technological University

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Neuroscience

8.5K 次观看.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

视频: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

生物学

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em&gt;在体内</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

脑干中的数据采集与分析 在小鼠中引发响应音频测量

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

<meta charset="utf8"></head><body>小鼠深部脑干结构的倾斜硅多电极记录

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>作者聚焦：研究小鼠麻醉诱导的睡眠通路和神经元兴奋性

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

来自头部固定小鼠脑干的急性单单位多电极记录

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

摘要

探索更多视频

此视频中的章节

脊髓电二：外吸电极的制作

脊髓电

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

使用新颖的方法在自由移动的新生大鼠中记录脑电图

急性

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

头脊髓和薄片体外制备心律失常活性

脑干中的数据采集与分析在小鼠中引发响应音频测量

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

来自头部固定小鼠脑干的急性单单位多电极记录

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

摘要

探索更多视频

此视频中的章节

脊髓电二：外吸电极的制作

脊髓电

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

使用新颖的方法在自由移动的新生大鼠中记录脑电图

急性

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

头脊髓和薄片体外制备心律失常活性

脑干中的数据采集与分析 在小鼠中引发响应音频测量

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

来自头部固定小鼠脑干的急性单单位多电极记录

脑干中的数据采集与分析在小鼠中引发响应音频测量