该项目是由MINECO资助授予BFU201343608-P和PSI2013-49348-EXP和JCYL授予SA343U14男男性行为者和MRC核心资金ARP。 YAA举行了国家科学技术委员会（216106）和SEP奖学金。

所有试验程序都在萨拉曼卡大学与批准进行，使用符合标准的方法，萨拉曼卡动物保健委员会的大学，以及欧盟的标准（指令63分之2010/ EU）对在神经科学研究中使用动物。 1.钨电极注意:钨电极的制造是基于在美林和安斯沃思27和安斯沃思等人 28所述的原技术和使用布洛克等人 17所述的工作站设置进行。 <ol><li>以记录的IC神经元的单单元外活性，使用电极1.5的尖端阻抗 - 2.5兆欧。这里，所涉及的钨电极的制造步骤仅简要地描述的，由于一个详细描述在上述参考文献中找到。 </li><li>将钨丝在定制校准工具（ 图1A），以协助它们切割成所要求的长度。对准工具包括以2毫米的间隔在一端胶合并联，止损30针（25克）来限制电线〜40毫米的长度。认真重视用胶带的电线有始有终，砍电线的其余部分具有较强的剪刀。 </li><li>从磁带轻轻拉动以从对准工具移除钨丝，并确保所有的导线保持附着到磁带上。滚动磁带到抛光黄铜锭（ 图1B，C），抱着电线坚决反对主轴所以有良好的电气连接。 </li><li>主轴连接到工作站5的转速电动机，浸入突出导线提示到蚀刻液（KNO 2，每80毫升90克）。角主轴轴线相对于蚀刻液的表面45°。浸入线材提示，使得导线的〜1/3接触的溶液。 </li><li> IMMERSE碳棒电极浸入蚀刻浴以闭合电路。使用弹簧铜编织刷而主轴旋转的蚀刻供给适用于主轴。调节是通过在电极和蚀刻溶液250 mA的通过的电流。停止尖端蚀刻时，电流下降到200毫安。该线提示将被蚀刻成很细的点。 </li><li>传递一个削尖线通过火焰，以消除任何油脂和残胶。放置电线（钝端在前）到高硼硅玻璃毛细管（1.5 mm外径，0.86毫米内径） 与它的下端封闭橡皮泥。垂直夹住吸管在顶部和底部。拉使用加热线圈（直径为5毫米，深度5毫米）和悬浮柱塞吸管。当导线被柱塞的重量拉下，熔融玻璃形成围绕它非常精细涂层。 </li><li>除去覆盖尖端在电极的尖锐端与钠的熔融珠的帮助下在玻璃四硼酸。以形成胎圈，以水（约0.5毫升）少量混合的四硼酸钠（〜0.25g）和缓慢将其置于一个加热元件上，直到它融化成珠〜2毫米的直径。加热元件的温度是由一个电位器控制。放置在固定于表一个操纵臂胎圈和加热元件，并移动，直到胎圈是在低放大倍数的显微镜下聚焦。 </li><li>固定玻璃包线到幻灯片，并把它放在显微镜下。移动滑板与阶段旋钮直到尖端和四硼酸珠处于焦点。热火珠，直到它稍微融化，然后将丝尖端〜10 - 15微米。关闭供暖电位器了。作为珠子冷却下来时，它收缩并从电极的前端去掉玻璃，暴露下面的钨。钨电极就可以使用了。 </li></ol> 2.多管玻璃吸管制造<ol><li>保障多的两端桶玻璃毛细管（以H-配置五桶）用热缩管，让更好的抓地力，车夫夹具和防止破损，并放置一个在垂直拉拔。 </li><li>要获取〜15毫米尖的长度，设置起拔器以下参数:热火:84分磁铁力:49，主磁铁力:38微调这些参数，根据特定的设置，变更以及后加热元件。 </li><li>拉毛细管获得两个移液器闭塞的提示。安装在使用橡皮泥滑动吸管和在显微镜下打破枪头直到外径为约20 - 30微米，用细剪刀或镊子或手术刀的扁平侧。如果破碎的边缘过于粗糙，使用步骤1.7中所述的四硼酸珠技术，完善它。 </li><li>放置一个钨电极（从第1部分）在一个3轴微型微定位器的端部与一个20G的针制成的支持器安装在显微镜雄鹿即使用镊子，弯曲电极5的端部 - 10°，〜从末端30毫米。 </li><li>在显微镜下，小心地将在多管尖钨电极。一旦对准，降低电极直到它接触到多管，在两个上部桶之间的槽嵌合。 20微米从多管的前端远 - 直到它突出〜15滑动电极的尖端。使在电极和多管尽可能小，以获得更好的粘合和更薄的合奏之间的角度。 </li><li>从技巧应用的光固化胶粘剂〜5mm的小降了。小心胶水没有达到尖端避免阻塞多管通道。 </li><li>治用蓝光LED灯的胶水。如果需要重复上胶/固化，获得更好的粘接。 </li><li>轻轻移动显微镜载物台，直到钨电极的端部从其架上释放。取下幻灯片背驮式合奏。 </li><li>第裹在长度较短的背驮式电极电子中段热收缩管和用于玻璃的钨电极，以进一步固定到多管应用快速固化环氧树脂为宜。 </li></ol> 3.药物对微电泳注意:在水中溶解时使用的微电泳的药物必须具有的电荷。检查文献以查看是否感兴趣的药物是适合此过程。这里，对于gabazine的程序，GABA A受体的拮抗剂，进行说明。 <ol><li>使用0.2微米的注射器过滤器进行消毒的水并固定不含有溶质过滤蒸馏水。准备〜1000微升20毫米的使用这种蒸馏水gabazine。 </li><li>使用细尖pH电极调节稀释的PH值至4以0.2微米过滤的NaOH。 </li><li>在-20ºC200微升等分 - 存储100个。除霜在记录的那天。这天，在此之前的动物是克制版，填充桶 - 的多管电极（3 5微升）用的药物，使用装配有柔性塑料针100微升微量。 </li></ol> 4.手术和Headpost植入<ol><li>在两名男性CBA / J小鼠（ 小家鼠 ）为27和30克的重量进行实验。按照科比和他的同事29的手术和登记程序，Portfors和合作者30-32，和杜克和马尔米耶卡26。 </li><li>在手术前的日子里，处理动物，让他们自由地探索他们habituate到录音室。执行每天三节，每一个至少持续30分钟。这样，录音过程中，动物会平静和舒适。 </li><li>麻醉用氯胺酮（50毫克/千克）和赛拉嗪（10毫克/千克）的混合鼠标肌内注射。与该剂量的动物为约1小时深度麻醉。提供补充剂量的1锡尔的根据需要初始剂量d的。 </li><li>放置动物上的加热毯设定为保持38±1℃的高温和通过使用两个耳棒和咬的酒吧在立体定位框架稳定动物的头。要小心，不要刺穿与耳棒鼓膜。 </li><li>通过应用眼用凝胶一滴保护眼睛。 </li><li>用剪刀刮头皮，并应用碘伏消毒皮肤。 </li><li>使用手术刀，做一个切口沿中线，露出头骨和缩回覆盖顶和枕部的骨头更延髓部分骨膜。 </li><li>胶水一个轻量级的杜拉铝headpost（重量〜0.65克，长30毫米， 图2A）与颅骨圆平底（直径为5毫米）。胶银线到headpost离开其端部自由的中间。银线将被用作用于电生理记录的参比电极。 </li><li>应用的LIG一薄层在头骨HT固化性粘合剂和headpost的基础。等待10秒，然后将headpost在顶骨的喙区域，沿中线。 </li><li>为一个有效的接合强度，光使用蓝色光源20秒固化粘合剂。 </li><li>钻三个孔周围使用电钻和小钻头headpost的基地后面。 </li><li>放置在钻孔两位制表螺钉（〜2毫米长），以服务于头骨和探头之间的附加的接触点。螺丝需要1.5变为实现紧密贴合。测试用钳子螺丝，以确保他们没有松动。 </li><li>将银接地线的尖端插入第三孔，确保它接触硬脑膜。申请耐水氰基丙烯酸酯胶的一小滴的导线的头骨结合。 </li><li>应用光固化复合材料加强与颅骨headpost的债券。覆盖headpost的基础上，所述银线的低部分，和螺钉，小心不要延长背后的lambda，允许在录制过程中访问。固化使用蓝色光源。 </li><li>缩回肌肉在颈部的其插入邻近于头骨后部。用一个小环钻（直径2.35毫米），钻一个圆窗下方的lambda缝合和横向的中线，暴露下丘（IC）。当边界松动，拉起覆盖骨细镊子。如果发生出血，用冷无菌生理盐水冲洗，以阻止它。 </li><li>使窗口周围的小（约1毫米宽，约1毫米高）墙复合光固化它。 </li><li>覆盖暴露的头骨和肌肉用抗生素软膏。然后，弄湿了IC的暴露表面用无菌盐水并用凡士林覆盖它，填充使记录窗口周围的壁之后阱产生的。 </li><li>注入丁丙诺啡皮下（0.03毫克/公斤，在无菌盐水中1:10稀释）作为镇痛剂。 </li><li>保持T上的动物他电热毯加热，直到它醒来，并返回到其房屋笼从手术中恢复了之前的记录会话三天。房子动物个体rat&#39;s住房笼笼防止队友清理出果冻和headpost触摸金属网罩。放置一些富集在笼子里时，动物的单装。此外，每日更换木屑，防止感染，并仔细检查动物的正常恢复并没有显示不舒服的迹象。还要检查是否凡士林是在记录窗口保护暴露大脑。 </li></ol> 5.电生理记录和微电泳<ol><li>恢复后，给动物的时间来适应环境的录音环境，并具有其头部约束。通过在鼠标坐在雕花其机身尺寸（ 图2B）定制的缓冲泡沫阻挡器headpost固定到支架驯化动物。这将LIM它鼠标的移动，将有助于它的宁静。 </li><li>开始10分钟的会议，并逐步增加持续时间长达120分钟。在会议期间，在给定的时间间隔33奖励甜液体动物（炼乳，用水稀释30:70）。后来，记录会话期间，给予同样的奖励在启动，并在会议结束时或者在没有神经元被隔离。 </li><li>如果动物是非常之前记录激发，注入轻度镇静乙酰丙嗪（2mg / kg的，腹膜内）。根据感兴趣的神经系统，镇静会影响你的成绩。 </li><li>让动物自由探索录音室5 - 10分钟。 </li><li>将动物的尸体进入泡沫定制的缓冲阻挡（ 图2B）。 </li><li>固定headpost到附着到立体定位框架（ 图2C）的保持器。这是给动物液体奖励的好时机。 </li><li>有限公司版本的动物的身体用棉毯和松散耦合固定使用塑料hemicylinder及胶粘带它。 </li><li>从记录窗口中删除凡士林用温无菌生理盐水冲洗。 </li><li>记录神经活动和gabazine离子电渗应用。 <ol><li>放置在其支架上的多管电极，由一个微驱动控制，并连接到一个显微。 </li><li>连接线记录，用小鳄鱼夹。连接正极端子连接到钨电极的端，和接地端子的银导线接触硬脑膜。 </li><li>放置一个未涂覆的银线每个桶的内部，所以一端接触溶液，而另一端是可访问的。这些目的连接到微电泳装置中，按照制造商的说明书。 </li><li>移动多管电极直到尖端接触大脑的表面上。套用温暖无菌生理盐水，以防止日的干燥Ë脑组织。在这一点上施加保持电流（-10 nA的用于gabazine，检查文献对于特定的药物），以避免从筒体前端的药物漏出，同时寻找单个单位活动。 </li><li>使用标准技术分离单个神经元细胞外的活动。获得神经元的频率响应区域（FRA）， 即 ，频率和能够因神经元是这些声音34-36敏感唤起的响应的强度的组合。 </li><li>选择两个频率（F1和F2），该唤起一个类似的燃烧速率和图案。目前古怪范式下的各频率的稀有和重复的刺激。 注意:简单地说，在古怪范例，一个频率（F1）是作为与发生的概率高的重复刺激，而第二（F2）是很少发生的异常刺激，重复那些中无规地散布。在第二古怪序列，相对两个刺激概率逆转。刺激范式的细节先前已经描述22,37。 </li><li> （ - 20 nA的10）当前转向正值释放gabazine。再次获得FRA和gabazine应用下重复古怪的范例。保持的喷射，直到在被观察到的燃烧率的可见效果;通常需要5 - 20分钟，这取决于具体的药物，其浓度，和喷射电流的幅度。重复记录协议，以获得药物的作用下的值。 </li><li>通过返回电流到保留值停止注射。等待药物的作用通​​过监测的FRA或响应于古怪范例返回到控制值，以洗去。 注意:记录会话可以持续2 - 每天3小时，而如果动物显示不适或挣扎的任何迹象应提前结束。给予奖励液体并覆盖录音通道凡士林琥珀色。 </li></ol></li></ol> 6.数据分析<ol><li>测量前后gabazine注入以及SSA水平期间FRA。 </li><li>由共SSA指数（CSI），并如先前19,22,23,38,39所描述的特定频率的SSA指数（SI）量化SSA响应的强度。的CSI和SI反映到罕见刺激神经元响应，并重复一个响应之间的归一化。正CSI和SI值表明神经反应（每穗刺激）较反复的音调更高罕见。 </li><li>比较从控制和药物喷射条件数据。 </li></ol>

The authors have nothing to disclose.

的神经活性物质在清醒的动物的微电泳是一个功能强大的技术来探测并解剖上单个神经元40,41的活性的特定的突触输入的作用。更重要的是，这个过程可以对神经回路的神经递质和调质无麻醉剂的潜在干扰的影响的决心。这里，我们表明，在清醒小鼠的集成电路gabazine的应用鲁棒改变频率调谐（ 图4A），时间的反应模式（ 图4B）和SSA（ 图5）。 <p cla...

神经兴奋和抑制的相互作用是感觉信息1的处理的基础。它也已知麻醉对皮层激活和突触输入2,3的时间模式的动态的强烈冲击。例如，已经观察到，麻醉剂改变的视觉诱发反应在皮质神经元3,4的持续时间。此外，兴奋性和抑制性突触输入之间的比例是在麻醉和清醒动物4,5-不同，改变既诱发和自发活动率6,7。通过测量突触电导，海德尔和他的同事发现4在振幅抑制匹配激励麻醉下而清醒时，抑制作用大于激发更强。这些发现提示的实验程序的开发，研究具体的突触输入感官处理在清醒动物的影响。 &lt;P类="jove_content"&gt;通过施加小电流注射的（NA的数量级上）带电神经活性物质的受控喷射已被广泛用于研究的突触输入的贡献，并推定细胞受体的感觉处理8-13的作用。这种技术被称为微电泳，允许药物在记录神经元，这有助于快速和密闭效果附近的应用。这个过程是更适合于学习的神经活性物质局部作用，相比于其它实验操作如全身注射，微透析或使用光遗传学技术引起了广泛的影响。通常，一个背驮式电极结构14,15被用于同时记录所述目标神经元，并提供所关注的神经活性物质。它由附接到承载神经活性物质的多管吸移管的记录电极。邻的修改通过Havey和Caspary 14描述riginal程序已付诸实施。例如，钨电极，而不是玻璃之一，可以用来记录神经活动16。对钨电极17,18的制造先前公布的方法包括三个基本步骤:钨丝提示，玻璃绝缘，并且末端曝光调整电解蚀刻，以满足记录的要求。 听觉神经科学的一个有趣的和紧急字段是特定刺激的适应（SSA 19）的研究。 SSA是在给不推广到其他，很少呈现声音重复声音的神经反应的特定下降。 SSA的重要性在于它在听觉大脑神经机制底层偏差检测，以及对听觉的后期失匹配负成分的可能相关的神经元的潜在作用诱发电位20,21。 ØSSA从IC ccurs到听觉皮层19,22-24。 GABA A介导的抑制已被证实作为对SSA 7,16,25的增益控制机制，这也被证明是由麻醉26的影响。这里，我们提出，结合先前描述的方法之前和清醒小鼠的GABA A -受体的选择性拮抗物的应用过程中记录的IC神经元的单单元活动的协议。首先，我们描述背驮式电极和下，手术和记录方法中的用途。为了测试药物释放的功效，我们比较了感受域以及集成电路神经元SSA之前和gabazine的微电泳喷射期间的水平。

<table>
	<tbody>
		<tr>
			<td>Tungsten wire</td>
			<td>Harvard Apparatus LTD</td>
			<td>33-0099</td>
			<td>0.005 inches x 3 inches</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary&#160;</td>
			<td>Harvard Apparatus LTD</td>
			<td>30-0053</td>
			<td class="xl66" height="21" style="height:15.75pt;width:494pt" width="658">Borosilicate standard wall without filament, 1.5 mm OD, 0.86 mm ID, 100 mm long</td>
		</tr>
		<tr>
			<td>Multibarrel glass capillaries&#160;</td>
			<td>World Precision Instruments</td>
			<td>5B120F-4&#160;</td>
			<td>5-barrel capillary, 4 inches long, 1.2 mm OD, with filament</td>
		</tr>
		<tr>
			<td>Diaplus</td>
			<td>DiaDent</td>
			<td>2001-2101</td>
			<td>Light-curing adhesive, used to attach the tungsten electrode to the glas multibarrel pipette</td>
		</tr>
		<tr>
			<td>G-Bond</td>
			<td>GC Corporation</td>
			<td>2277</td>
			<td>Light-curing adhesive, used to attach the headpost to the animal&#39;s skull</td>
		</tr>
		<tr>
			<td>Charisma</td>
			<td>Heraeus Kulzer</td>
			<td>66000087</td>
			<td>Light-curing composite, used to reinforce the bond of the headpost with the skull</td>
		</tr>
		<tr>
			<td>Araldit Cristal</td>
			<td>Ceys</td>
			<td></td>
			<td>2-component expoxy, used to further secure the attachment of the tungsten electrode to the glass multibarrel pipette</td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>Cibertec</td>
			<td>RTC1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Narishige</td>
			<td>SR-6N</td>
			<td>Modified for mice</td>
		</tr>
		<tr>
			<td>Microiontophoretic device</td>
			<td>Harvard Apparatus LTD</td>
			<td>Neurophore BH-2</td>
			<td>Including IP-2 iontophoresis pumps (one for each drug delivery channel) and a balance module</td>
		</tr>
		<tr>
			<td>Multibarrel glass pipette puller</td>
			<td>Narishige</td>
			<td>Model PE-21</td>
			<td></td>
		</tr>
		<tr>
			<td>LED lamp</td>
			<td>Technoflux</td>
			<td>CV-215</td>
			<td>5 W, 430-485 nm</td>
		</tr>
		<tr>
			<td>MicroFil</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Flexible plastic needle, 34 AWG</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Merial</td>
			<td></td>
			<td>Ketamine, 100 mg/mL</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, 20 mg/mL</td>
		</tr>
		<tr>
			<td>Gabazine / SR-95531</td>
			<td>Sigma</td>
			<td>S106</td>
			<td>Prepare ~ 1000&#181;l of 20 mM gabazine in distilled water and adjust the pH to 4</td>
		</tr>
	</tbody>
</table>

extracellular recording of neuronal activity combined with microiontophoretic application of neuroactive substances in awake mice

神经递质和调质，因此不同的神经反应的活性差异，可以麻醉和清醒的动物之间找到。因此，允许突触系统的清醒动物的操作方法，需要在以确定的突触输入的神经元处理由麻醉剂的影响的贡献。这里，我们提出的方法用于电极的结构，以同时记录细胞外神经活动和在清醒小鼠的记录点的附近释放多个神经活性物质。通过结合这些过程，我们进行了gabazine的微电泳注射，以选择性阻断GABA A受体头抑制小鼠的下丘神经元。 Gabazine成功修改神经反应性能如频率响应区域和特定刺激的适应。因此，我们证明我们的方法适用于recordin克单单元活动和解剖在听觉处理特定神经递质受体的作用。 所描述的过程的主要限制是在相对较短的记录时间（〜3小时），这是由动物到记录会话的习惯的水平来确定。另一方面，多个记录会话可以在相同的动物中进行。这种技术在用于操纵神经传递或神经调节（如全身注射或使用光遗传学模型）的水平的其他实验程序的优点是，药物的效果被限制在本地突触输入到目标神经元。此外，电极的定制制造允许根据神经结构和感兴趣（如用于提高记录的信噪比的尖端电阻）神经元的类型的具体参数的调整。

我们记录4以及隔离IC的神经元的单单元的活动。期间在清醒小鼠外记录获得的典型的信号-噪声比示于图3B。 图4A示出每个神经元的频率响应区域（FRA）之前和期间的GABA A的封锁与gabazine受体拮抗剂。未观察到响应强度（峰值/刺激）以及光谱调谐的扩大的增量。在诱发反应的增加也很明显在从显示（ 图4B）对所有的频率和强度?...

Watch this Scientific Journal Video about Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice at JoVE.com

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

extracellular-recording-neuronal-activity-combined-with

Research

JoVE Journal

Neuroscience

9.9K 次观看.  University of Salamanca. We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

视频: Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

科研

神经科学

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

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

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

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

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

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

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

外导线四极管记录在行走自如昆虫大脑

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

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