Name: prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging
Uploaded: 2017-02-15T15:00:00
Description: Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

We thank Colin Symons and James Wright for technical assistance. This work was supported by seed funding grant (WSU) to Y.B. and the innovation office at Western Sydney University. M.C. is supported by ARC fellowship (DECRA).

下面的协议描述的C57BL / 6和C3H /他（retinally退化）小鼠神经元组织的制备中，但是类似的技术可以应用到其它物种。所有的动物是健康的，并与温度的标准条件，湿度，12小时光照/黑暗周期，自由获得食物和水的处理，并没有任何意图应力刺激。所有实验均获得批准，并按照西悉尼大学的动物护理和伦理委员会，并根据动物的使用和保养指南（动物研究机构＃A9452，＃A10396和＃A8967）进行。 1.?...

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本文介绍了培养法延长急性神经组织的可行性进行成像和电生理实验，从而缩短完成实验目标所需的动物数量。两个主要过程支配神经元组织的劣化随时间：ⅰ）增加细菌水平，并在细菌内毒素伴随增加释放，和ii）随后的创伤切片过程10兴奋性中毒。为急性神经组织是环保手无寸铁，通过pH，温度和细菌水平密切监测，组织环境的紧缩调控对维持细胞活性以及网络的完整性至关?...

电生理和功能成像（钙，电压敏感性染料）是两个在神经科学中最常用的实验技术。脑切片制剂和视网膜wholemount，这将在这里检查，提供检查电生理特性和突触连接不脱离麻醉剂或肌肉松弛剂的污染的装置。脑切片和视网膜wholemount保持其结构的完整性，不同文化或细胞匀浆，允许特定电路和大脑网络1的研究。从孤立的组织录制有超过体内录音与心跳和呼吸相关的运动优势被淘汰。此外，直接可视化使细胞的特定的类来进行有针对性的，和药理学工具2，3本地应用程序。 膜片钳和钙鎓染料加载视网膜wholemount由内的界膜（ILM），它涵盖了视网膜神经节细胞（RGC）层，并防止对细胞的直接访问的存在复杂。通常情况下，该膜被刮掉用玻璃吸管，以允许在单个小区中的千兆欧姆密封件的膜片吸管和形成的直接应用。此外，浴施加钙染料不交叉在ILM和必须或者该膜4下方喷射，逆向转移注射后在视神经5或穿过组织6电穿孔。此外，利用视网膜色素变性的啮齿动物模型时，RD / RD鼠标，ILM是更厚，更令人费解。在这里，我们使用的技术与酶消化7删除ILM，同时允许普遍存在钙染料加载，并直接进入视网膜神经节细胞的膜片钳recordiNGS 8。 无论从大脑切片或视网膜wholemount成功的记录取决于解剖和可行的神经组织的培养。通常情况下，组织被提取的实验的早晨，直到它被用于记录在人工脑脊液（ACSF）温育。通常，组织保持生存能力6 - 8小时，用这个时间窗口以下显著降解。但是，这两个脑切片和wholemount视网膜制剂通常产生比可以从该短的时间内被记录更多的组织。因此，组织通常被丢弃在一天结束和解剖再次完成在随后的几天。这意味着其他动物被利用，并建立和解剖/染色反复〜2小时。以下方案描述了一种用于延长神经元组织的寿命为24小时以上，这意味着较少的动物被利用，以及更多的实验时间是可用的。组织活力W¯¯通过录音电生理特性和钙的动态评估，这些特性均&lt;4小时和&gt; 24小时postdissection之间没有什么区别。 这些结果表明，不仅是单细胞特性完整且功能性延长温育后，但网络活动，通过钙成像和电生理记录的评估，是不变的&gt; 24小时postdissection。此外，我们表明，钙染料可以保留在细胞长时间，而不会造成任何有害的影响。演示此协议的应用程序，它在急性神经组织神经元的功能活性可保持长时间，一旦外部环境是高度调节。另外，作为组织的生存力，由于不同的孵育协议实验室之间变化很大，这种方法建立了应该应用，以减少在急性神经健康变性提供了理想的参数的黄金标准Ronal公司薄纸。

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			<td height="40" style="height:40px;width:315px;">Sodium Chloride</td>
			<td style="width:159px;">Sigma Aldrich VETEC</td>
			<td style="width:135px;">V800372</td>
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			<td height="20" style="height:20px;width:315px;">Potassium chloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">P9333</td>
			<td style="width:225px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;">N-Methyl-D-glucamine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">M2004&#160;</td>
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			<td height="20" style="height:20px;width:315px;">D-glucose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">G5767</td>
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			<td height="20" style="height:20px;width:315px;">Sodium bicarbonate</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">S6014</td>
			<td style="width:225px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">S2554</td>
			<td style="width:225px;"></td>
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		<tr height="31">
			<td height="31" style="height:31px;width:315px;">Magnesium chloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">M9272&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:315px;">Calcium chloride&#160;</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">223506</td>
			<td style="width:225px;"></td>
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			<td height="20" style="height:20px;width:315px;">Papain Dissociation System</td>
			<td style="width:159px;">Worthington&#160;</td>
			<td style="width:135px;">LK003150</td>
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			<td height="20" style="height:20px;width:315px;">Dimethyl sulfoxide anhydrous</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">276855</td>
			<td style="width:225px;"></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:315px;">Pluronic F-127 Low UV absorbance*</td>
			<td style="width:159px;">Life technologies</td>
			<td style="width:135px;">P-6867</td>
			<td style="width:225px;">20% solution in DMSO&#160;</td>
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			<td height="19" style="height:19px;width:315px;"></td>
			<td style="width:159px;"></td>
			<td style="width:135px;"></td>
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			<td height="20" style="height:20px;width:315px;">Fura-2 AM&#160;&#160;</td>
			<td style="width:159px;">Anaspec</td>
			<td style="width:135px;">84017</td>
			<td style="width:225px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Fluo-4 AM&#160;&#160;</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:135px;">F23917</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Fluo-8 AM</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:135px;">Ab142773</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:315px;">Vibrating blade microtome&#160;</td>
			<td style="width:159px;">Leica Biosystem</td>
			<td style="width:135px;">VT1200 S</td>
			<td style="width:225px;">Equiped with Leica&#8217;s Vibrocheck&#8482; measurement device&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Antivibration table</td>
			<td style="width:159px;">Kinetic Systems Vibraplane&#160;</td>
			<td style="width:135px;">Vibraplane 9100/9200&#160;</td>
			<td style="width:225px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Fixed Stage Upright Microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">BX51WI&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">XLUMPlanFLN 20x/1.00w</td>
			<td style="width:225px;">20X&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">LUMPlanFLN 60x/1.00w</td>
			<td style="width:225px;">60X</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">CCD Camera</td>
			<td style="width:159px;">Andor</td>
			<td style="width:135px;">Ixon +</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">High speed wavelength switcher</td>
			<td style="width:159px;">Sutter instrument</td>
			<td style="width:135px;">Lambda DG-4&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Patch clamp amplifier</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">MultiClamp 700B</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Data acquisition system</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">Digidata 1440A</td>
			<td style="width:225px;">Axon Digidata&#174; System</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Borosilicate glass capillaries</td>
			<td style="width:159px;">Sutter Instrument Co</td>
			<td style="width:135px;">BF150-86-10</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microelectrode puller</td>
			<td style="width:159px;">Sutter Instrument Co</td>
			<td style="width:135px;">P-97</td>
			<td style="width:225px;">Flaming/Brown type micropipette puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Recording/perfusion chamber</td>
			<td style="width:159px;">Warner Instruments</td>
			<td style="width:135px;">RC-40LP</td>
			<td style="width:225px;">Low Profile Open Bath Chambers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Software</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">pClamp 10.2</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Andor iQ Live Cell Imaging Software</td>
			<td style="width:159px;">Andor</td>
			<td style="width:135px;">Andor iQ3</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Braincubator</td>
			<td style="width:159px;">Payo Scientific</td>
			<td style="width:135px;">BR26021976</td>
			<td style="width:225px;">www.braincubator.com.au</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peltier-Thermoelectric Cold Plate Cooler</td>
			<td>TE Technology</td>
			<td>CP-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature Controller</td>
			<td>TE Technology</td>
			<td>TC-36-25 RS232</td>
			<td></td>
		</tr>
	</tbody>
</table>

prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

急性神经组织准备，脑切片和视网膜wholemount，通常只能维持6 - 解剖之后8小时。这限制了实验时间，并且增加被每研究利用的动物数量。这种限制影响特别协议，如要求延长预孵育浴用的染料钙成像。 3内指数细菌的生长 - 切片后4小时，紧密与组织健康的减少有关。该研究描述了用于限制细菌的增殖急性制剂维持而不需要抗生素，无菌程序，或组织培养基含有生长因子的长时间（&gt; 24小时）的活的神经元组织的方法。通过循环外液通过紫外线照射，并在15保持组织中一个自定义的保持室 - 16℃下，将组织表示在电生理特性，或钙SI没有差别通过细胞内钙染料在&gt; 24小时postdissection gnaling。这些方法不仅会延长实验时间，使用急性神经元组织的那些，但会减少完成实验目标所需动物的数量，并且将设置为急性神经元组织培养的金标准。

孵化过程中学联的细菌负载和温度的严格监管是必要的，保持神经组织活力。 16℃下（ 图1） -这可以通过与UVC光照射和维持脑脊液的温度在15进行优化。此外，脑脊液参数戳（APS; 图1C）提供实验者使用的环境条件下（pH和温度）的记录，从而培养神经元组织，其中，如果随后将减少之间变异性时，为参数的金标准实验。 <img al...

Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

一旦从主体去除，神经元组织大大受环境条件的影响，从而导致组织的最终降解后6 - 8小时。采用了独特的培养方法，该方法密切监测和调节组织的胞外环境中，组织的生存力可以显著延长&gt; 24小时。

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Research

JoVE Journal

Neuroscience

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

科研

神经科学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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 ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

实时电：采用闭环协议来探测神经动力学和超越

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...