Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

注:所有程序均按照加拿大理事会动物保护和蒙特利尔大学的伦理审查委员会的指示进行的。 1.大会记录，注射针的<ol><li>拉用吸管拉马大约7厘米长的玻璃毛细管（1毫米外径的）。 </li><li>打破毛细管的尖端和检查光显微镜下的光圈。确认该内径为30微米至40微米。 </li><li>插入一个7厘米长的银线与从玻璃吸管的非锥形端伸出约1cm的玻璃毛细管。 </li><li>正交弯曲过量灯丝的玻璃毛细管。 </li><li>在地下30皮下注射针的轴施加的柔性塑料粘合剂的液滴。 </li><li>根据在图1中呈现的示意图插入地下30皮下注射针在玻璃吸管</stroNG&gt;。 </li><li>添加胶水的第二涂层，以确保从玻璃吸管和皮下注射针头之间的连接处的适当的密封。 </li><li>离开吸管干燥与尖端朝上约12小时，以确保胶的适当固化。成品结果示于图2。 </li></ol>注:此过程是在急性实验和枪头灭菌完成不是必需的。 2.动物的制备<ol><li>将大鼠在麻醉箱。 </li><li>诱导使用4％异氟烷进行5至10分钟麻醉。 </li><li>放在立体定位表与加热垫和直肠探头动物保持37℃的体温。使用鼻锥维持麻醉，用2％异氟醚。使用耳酒吧和牙齿固定器固定老鼠的头部。 </li><li>应用眼药膏或眼药水，其中包括1％阿托品滴在扩瞳，以帮助。为了防止干燥，适用润滑降š大约每30分钟。 </li><li>剃的头部，并与10％的聚维酮 - 碘清洁。 </li><li>的局部麻醉，通过抬起皮肤和插入针的尖端注入0.5毫升2％利多卡因由2-3位置的头皮下。 </li><li>通过执行脚趾捏和观察，缺乏运动的确认麻醉适当水平。另外，监测心脏速率，以确保它是正常值（300至400次/分）内。 </li><li>切割沿的中间的直线头皮用＃10解剖刀刀片露出两个冠状和矢状缝线。 </li><li>揭示Lambda和前囟门点被推开被覆盖颅骨用手术铲组织。 </li><li>平颅骨以便前囟门和Lambda位置是在同一平面上。 </li><li>要设置基准点，采用立体设备与安装玻璃管设置是正确的前囟门上方。这将是"零"的前 - 后和配有IAL-横向测量坐标。 </li><li>通过移动立体安装到所需的坐标值设定的兴趣点，请注意立体坐标和绘制目标区域，标志着其中开颅将执行绕方。 </li><li>使用无压力的外科钻沿缓缓的显着广场上演练消毒慢慢去除骨材料。要小心，不要钻得长在同一区域，因为它会产生热量，导致病变的皮层。 </li><li>当骨划定开颅手术已经变得足够薄，仔细用镊子取出颅部揭露皮层。 </li><li>经常灌溉用人工脑脊髓液的露出皮质，以防止组织脱水。 注:硬脑膜去除不必要的大鼠作为injectrode尖足够牢固，能够穿透。 </li></ol> 3.填充和安装喷射系统<ol><li>填写5-10微升微量吸用矿物油。 </li><li>填充皮下注射针与0.5％的芝加哥天蓝（CSB）和300μMγ氨基丁酸（GABA）或2％利多卡因和0.5％CSB 9的溶液中的溶液中固定玻璃吸管。稀释用生理盐水所有的解决方案。 <ol><li>在一个丰富的物质的情况下，填使用常规注射器和使用一般的预防技术，以避免产生气泡的形成。 </li><li>在更昂贵的物质的情况下，可以使用矿物油，以填补injectrode和化学试剂可以然后通过抽吸被引入。矿物油和水之间的密度差是比较高的，这种物质是一种很好的候选注射水溶液。 注:染料可以添加到确认两种液体之间的分离。 </li></ol></li><li>填补了注射针，填充1ml注射器用溶液通过吸出，然后慢慢地注入该溶液入注射针。 </li><li>请特别注意在区域泄漏通过擦拭这些区域的清洁和通过进一步注入用注射器将溶液慢慢观察泄漏在图1中表示。 </li><li>除去1ml注射器的。这样做时，一定要保持光压在柱塞上，使得真空不会删除从注射针的解决方案。 </li><li>吸填补与矿物油的微量，坚决将其附加到填充注射针，然后小心地擦去组装injectrode用纱布任何多余的解决方案。 </li><li>验证该尖端不会被阻止通过注入体积非常小，足以看到一小滴在玻璃吸管的尖端成形。 </li><li>装入微型泵系统上的injectrode并确保它被固定好。 </li><li>仔细定位injectrode尖端在目标坐标和降低尖皮层的表面上。 </li><li>慢慢放下injectrODE使用使用适当的解剖坐标立体定位装置到目标结构（在这种情况下丘）。 </li><li>盖上温暖的琼脂暴露的皮质，以防止组织脱水。 </li></ol> 4.注射灭活可逆<ol><li>设置显微注射泵注入400至800 NL在40升/分钟，按Run开始注射。注意，穗率将显示在注射过程中减少。 注:在实验设置中，神经活性的GABA传递结束后一小时内恢复。任何校准的机械装置可用于在为了进行注射适用于显微注射注射器内压。 </li><li>采集的数据电后，使用安乐死批准由当地动物伦理共同体的方法。 </li></ol>

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We have nothing to disclose.

该协议旨在解决与当前可逆灭活方法所带来的挑战。具体而言，该项目旨在改进用于物质调节神经活动，特别是在脑深部结构的化学显微注射方法。一个技术难题摆脱这种类型的设置是需要两个探头将在共定位在体内同样有限的空间，以便在注射部位，以获得精确的记录。这个问题可以通过使用设备，如一个在这里提出，其能够注与记录在同一部位来克服。替代的方法包括使用根据气体的压力脉冲装置。这些工具已经存在多年，但使用的可压缩中间减少了注射速度和容量，两个参数是很重要的控制，以保证可逆性的控制。其它方法如离子电渗注射systems也可用，但在液体中的扩散动力学是不同的与推注，减少失活的潜在范围。这些方法有具有球形散射图案，而不是观察到的微注射7的椭圆图案的优点。因此，灭活方法的选择应根据目标区域与实验设计计划。尽管商业替代品存在，该协议提供了一种监测药物的物质递送，以及允许定制的程度高的成本效益的方式。在注射装置的草拟这样有利于自由大范围的实验灵活性和调整的具体应用环境。 至于所提出的协议中，关键的步骤是填充玻璃吸管的过程。应避免气泡，空气压缩将使喷射的监测卷棘手。当通过手动移液器推的液体，确认系统中的自由流动，一个很小的阻力应该也能感受到。不存在的液体与手动注射可指示在系统中或不正确的吸移管制备从而导致阻塞尖端泄漏。移液管的阻抗也应以获得期望的类型电生理记录仪（LFP，诱发电位，多单位活动等 ），作为较大的提示大小将导致较低的阻抗。 如果注入成功，的300微米的GABA溶液的400至800 NL或2％利多卡因溶液的体积是足以取消扣球活性。有喷射分散在空间和时间的一个想法，琼脂可以用来模拟神经组织。注射的传播就可以很容易地与一个CSB溶液观察到。后的模拟，必须通过使用Ò表征注射传播组织学˚F染料如CSB，使用放射性药物放射自显影或通过代谢途径，如葡萄糖放射自显影间接代理来衡量激活神经活性1或失活。 同样重要的是要注意，快速注射（≥100升/分钟）将有可能导致损害充分可逆性达不到的。所提出的协议的主要优点是用软件，将反馈控制的喷射率的一组神经元活动水平整合喷射系统的潜力。这样的实施使得研究者着眼于失活（或活化）的参数，而不是同时提供药物仅适量对于所考虑的应用的技术参数，例如注入速度或卷。这将通过优化所需的药量减少探头位移，允许药物输送更多的时间敏感的控制，有利于重现性和所有流直接成对比较的数据。 这种技术结合用于电生理信号的物质传递和记录的系统。我们通过使用利用闪光灯诱导多单位活动的列车功能定位上丘我们吸管的记录容量证明其疗效刺激11。在失活，多单位活动减少，注射后偏移逐渐恢复。可逆失活的技术，如这里介绍，提供相当大的优势超过机械或化学性病变的技术，提供不存在或差恢复3。可逆失活的技术加强实验的统计显着性，因为配对比较是可能的3，从而避免特质的差异。我们已开发了一种成本有效的和可定制的技术，其允许精确控制的物质递送和健壮的持续时间探测目标脑区。

皮质区和副皮质细胞核失活是在不同脑结构间的2-4个功能关系的研究非常重要。最近的文献采用功能丧失的化学或低温技术来研究大脑结构2,5的作用。关于药理显微注射，小体积的药物可以控制的速率施用到脑区域，同时最小化对周围组织6,7的附带损害。这种技术可以用于递送特异性激动剂，反向激动剂或拮抗剂来研究不同的药理学靶上的神经元活动的影响。这样的效果也可以通过测量变化从遥远地点神经元的反应，使研究人员研究了不同的皮质和皮质下结构之间的关系的研究。 在这里，我们展示了一个设备的装配，injectrode，可博个记录电生理信号，并在目标位置输送少量的药物。我们证明该系统的功能，通过注入的GABA，神经元活性的共同抑制剂，在大鼠上丘。这个地区是视觉刺激，使我们能够利用视觉诱发多组活动确认injectrode本地化敏感。失活的可逆性通过正常神经元活性的GABA注射结束后恢复进行评估。 从注射部位监测多单位活动的能力允许为达到所需药效应答所需的注射速率和体积的微调。因此，这种技术的优点是由微灌组织损伤的限制性的潜力，因为最小的有效量被注入。所提出的协议提供了一种成本有效的方法，用于产生所述一次性硬件necessary代表进行实验，其中药物输送和本地的神经元活动的记录是需要的。

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Name of the reagent</td>
			<td style="width:209px;">Company</td>
			<td style="width:184px;">Catalogue number</td>
			<td style="width:308px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection pump (UltraMicroPump III)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#UMP3</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection console (Micro4 Controller)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#SYS-MICRO4</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Hamilton syringe</td>
			<td style="width:209px;">Hamliton</td>
			<td style="width:184px;">(80301) 701LT 10 &#181;L SYR</td>
			<td style="width:308px;">Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Gel cyanoacrylate adhesive</td>
			<td style="width:209px;">Krazy Glue</td>
			<td style="width:184px;">KG86648R</td>
			<td style="width:308px;">The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Glass pipettes</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#TW100F-4</td>
			<td style="width:308px;">Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">720 Needle Pipette Puller</td>
			<td style="width:209px;">Kopf</td>
			<td style="width:184px;">720</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Silver wire</td>
			<td style="width:209px;">A-M Systems, Inc.</td>
			<td style="width:184px;">782500</td>
			<td style="width:308px;">Bare 0.010&#8221;</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

在这里，我们描述了使用商业上获得和负担得起份的单次使用"injectrode"的结构的方法。甲探测系统的开发，其允许注射药物的同时记录来自受影响的神经元群的电生理信号。此方法提供了一种简单且经济的替代的商业解决方案。玻璃移液管将其与一皮下注射针和银长丝组合改性。该injectrode附着到商业微量注射泵用于药物递送。这导致了一种技术，通过多单元外信号从药物递送位点发端提供实时药效反馈。作为概念证明，我们记录了神经元活动从上丘通过光的闪烁大鼠引起，与之同时通过injectrode递送药物。该injectrode记录容量允许每次注射的功能分析现场挠度利于精确控制药物释放的本地化。这种方法的应用也远远超出这里所展示，作为化学物质装入injectrode的选择是巨大的，包括跟踪标记解剖实验。

该injectrode的结构示于图1，一种银线（C）的供入玻璃吸管（D）中与导线弯曲的一部分，并且从所述开口伸出。甲地下30针（B）的连接并密封到用胶水玻璃吸管的开口。后的吸移管被填充有喷射的物质，玻璃微量注射器（A）的附着在针。重要的是，有一个很好的密封，其中所述微注射器与针（E）和其中银线突出于玻璃吸管（F）的连接。 图2显示的是什么injectrode样子完成组装后的照片。 视觉诱发多组活性在上丘， 如图3以下一个300毫秒闪光对侧眼获得的。当注射的GABA，扣球活性响应于闪光刺激被抑制。视觉诱发多组活动通常在45回60分钟注射后已经停止。 图4示出了显微注射系统的安装。喷射泵控制器允许指定设置用于注射的用户。弹簧的电连接器连接的银线突出的玻璃吸管。所述的连接器导致头部阶段与接地电极和参考电极，然后插入一个放大器。模拟/数字（A / D）的接口被用于获取电生理数据，和一个扬声器，用于神经元活动的互补音频监视。 <img alt="图1" src="/files/ftp_upload/52271/52271fig1highres.jpg" /> 图1:该injectrode组件的示意图的微型注射器（A）被连接到记录-注射针其中包括一个30 G Hypodermic针（B）的附着于银线（C）的玻璃吸管（D）的内侧。区域盘旋（E - F），可能容易受到漏洞的亮点区域。 <img alt="图2" src="/files/ftp_upload/52271/52271fig2highres.jpg" /> 图2:采用地下30针（B），防水粘接胶（F），银线（C）和玻璃吸管（ 四）构建吸管的照片。 <img alt="图3" src="/files/ftp_upload/52271/52271fig3highres.jpg" width="600" /> 图3:注射GABA（300μM）上在上丘视觉诱发多单位活动的抑制效果的示图中，箭头表示闪光发作 。电用一个带通滤波器30和3000赫兹之间设置人信号进行过滤。 <img alt="图4" src="/files/ftp_upload/52271/52271fig4highres.jpg" width="600" /> 图4:完整的微喷射系统的示意图。

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain at JoVE.com

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

simultaneous-electrophysiological-recording-micro-injections

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.0K 次观看.  Université de Montréal. Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

视频: Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

同时神经刺激，并记录在水迷宫的设计和构建一个具有成本效益探头

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

在健康志愿者的气体麻醉剂氙和一氧化二氮的管理中记录大脑电磁活动

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...