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<...

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 border="0" cellpadding="0" cellspacing="0">
	<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>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</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&#160;</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...

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

Research

JoVE Journal

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

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 ...

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

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

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

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

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

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

Neural Circuit Recording from an Intact Cockroach Nervous System

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

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

神经电路记录从一个完整的蟑螂神经系统

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

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

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 ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

使用微电极阵列记录脊髓伤害感受回路中的网络活动

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

小鼠轻度创伤性脑损伤的电磁控制闭头模型

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

了解运动神经元对脊髓刺激的反应

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

作者聚焦:推进脊髓刺激 - 通过膜片钳电生理学探索运动神经元的细胞反应 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髓切片 的离体 制备，用于脊髓刺激过程中运动神经元的全细胞膜片钳记录

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...