作者希望感谢以下组织: 中国国家自然科学基金 (31171018, 31171355), 广东省自然科学基金 (2013KJCX0054), 广东科学与技术部 (2014A030313418、2014A030313440) 和广州科技局 (201607010320)。

动物住房和所有动物实验都是按照华南师范大学动物研究伦理委员会批准的程序进行的, 根据国家研究所设立的动物保育指南。健康. 
 1. 准备解决方案 <ol> <li> 准备500毫升标准人工脑脊液 (ACSF), 其中包含我们发布的工作中所描述的组件 7 中列出的 表 1 。
	<ol> <li> 使用干净的玻璃烧杯与去离子水, 准备一个新的 ACSF 解决方案。用去离子水和干前的清洁铲子添加氯化钠, 氯化钾, NaH 2 PO 4 , NaHCO 3 , MgSO 4 , d-葡萄糖, 并添加 CaCl 2 后冒泡的解决方案与 95% O 2 /5% CO 2 混合物为〜20分钟 (详见表 1) 到500毫升的去离子水。搅拌直到完全溶解. </li>
		<li> 确保 ACSF 完全清除, 无需任何降水。然后将 pH 值调整到 7.2-7.4。气泡的气体混合物由 95% O 2 和 5% CO 2 组成, 用于 ~ 20 min. </li>
	</ol> </li> <li> 在 表 2 中描述了包含化学品的250毫升切片切割解决方案 7 。
	<ol> <li> 添加氯化钾, 氯化镁 2 , NaH 2 PO 4 , NaHCO 3 , 胆碱和 #183; Cl, d-葡萄糖, 并添加 CaCl 2 最后, 如1.1.1 中所示, 到250毫升的去离子水和溶解, 在1.1.1 和1.1.2 中描述. </li>
		<li> 冒泡后 95% O 2 /5% CO 2 , 将解决方案放在冷冻库中15-20 分钟, 以便切割解决方案变得 ice-cold 和部分冻结. 
		注意: 我们在1-2 月大的小鼠制作切片时使用这种切削液;该公式可能不同于2月以上的小鼠. </li>
		<li> 在无菌内毒素-游离等渗盐水 (0.9% 氯化钠) 中溶解, 制备 LPS 溶液。LPS 是管理腹腔 (0.5 毫克/千克), 并开始记录2小时后. </li>
	</ol> </li> </ol> 2。前额叶皮质切片的制备 <ol> <li> 从冰箱中取出切削液, 然后质, 以获得一层泥浆。以 95% O 2 /5% CO 2 将解决方案放在冰和气泡上。
	<li> 使用大而锋利的剪刀, 快速斩首描述的鼠标 9 , 并立即将头放入一个充满 ice-cold (0-4 和 #176; C) 切割液的烧杯中. </li>
	<li> 用细剪刀把头骨的顶端沿中线 caudal-to-nasal, 去掉侧面的颅骨部分。用细刮刀取出大脑, 然后滴入 ice-cold 切割液中. 
	注: 步骤 2.2-2.3 必须迅速完成 (理想情况下, #60; 1 分钟). </li>
	<li> 将大脑放在 9 cm 培养皿的盖子上, 装满冰块。用 ice-cold 切削液冲洗大脑。用刀片切断小脑和嗅球. </li>
	<li> 将氰胶水应用于 vibratome 的标本持有人。小心地将脑块放在胶水滴上, 侧一侧向上, 并立即浸入 ice-cold 切割液中. </li>
	<li> 将 vibratome 的刀片式服务器调整到15和 #176 的角度; 参照水平平面, 准备350和 #181; m 冠状脑切片 (叶片振动频率 = 85 赫兹, 速度 = 0.1 毫米/秒). </li>
	<li> 使用吸管, 将切片转移到充满 ACSF 的恢复腔中, 孵育1小时 (在 RT), 持续冒泡 95% O 2 /5% CO 2 。
</ol> 3。全细胞贴片记录 <ol> <li> 根据拉拔器操作手册中的特定指南, 将玻璃硼硅 micropipettes 用作记录电极或粉扑 micropipettes。对于记录电极, 尖端电阻是在范围4-6 米和 #8486;, 而粉扑 micropipettes 有尖端直径的范围2-5 和 #181; M. </li>
	<li> 添加 Na + 通道拦截器 (TTX, 1 和 #181; M), 以阻止快速 Na + 和 #160; 通道和因此行动电位, ACSF 和保持这个解决方案的恒定流速2-3 毫升/分钟在记录室, 由蠕动泵入房间和愿望。气泡的 ACSF 与 95% O 2 /5% CO 2 不断确保切片的可行性. </li>
	<li> 使用改良的巴斯德吸管将脑切片转入记录室, 其尖端被切割以适合切片大小。用带有平行尼龙线的铂片锚片覆盖切片, #8805; 2 毫米分开, 以保持在平台上的切片. </li>
	<li> 用内部解决方案填充记录微 7 ( 表 3 ), 用 GABA 在10和 #181; m、50和 #181; m 和100和 #181; m (溶解于 ACSF), 或 ACSF 作为车辆控制. </li>
	<li> 为了防止碎片堵塞, 在将记录电极浸入 ACSF 中之前, 使用1毫升注射器应用光正压。使用显微镜下的微 (4x 物镜), 定位记录电极和粉扑微, 使提示出现在显示器的视频图像的中心 ( 图 1 a ). </li>
	<li> 调整显微镜焦距 (40x 物镜), 同时逐渐降低粉扑微, 并将其置于记录神经元的45和 #176 的角度;。保持微和20-40 和 #181 之间记录的神经元之间的距离; m ( 图 1 B )。慢慢地和小心地接近神经元与记录电极, 然后释放正压。通过连接到电极托架的油管上的微弱和短暂的吸力来制造一个千兆欧姆的密封. </li>
	<li> 保持0毫伏的电压。在千兆欧姆密封形成后, 手动或自动补偿快、慢的电容。然后应用一个简短的和强吸力通过上述油管进入全细胞模式. </li>
	<li> 在电压钳模式下记录 eIPSC。使用 Picospritzer 控制的低气体压力 (氮气, 4-6 psi) 到粉扑微采用主8电压步进发生器提供单或配对 GABA 粉扑 ( 图 2 ). </li>
	<li> 更改粉扑持续时间以获得最佳的 eIPSC 结果 ( 图 3 ) 并测量 LPS 诱发的抑郁症模型 ( 图 4 ) 中的 eIPSC. </li>
</ol>

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作者没有什么可透露的。

粉扑应用广泛用于评价突触后受体功能3,4,7, 但在每个实验中都需要精确的控制。我们在这里描述了一个涉及全细胞贴片钳夹的过程, 它显示了 GABA-粉扑诱导的前额皮质脑切片中的干细胞 (即 eIPSCs)。记录电极的电阻约为5ω, 而粉扑 micropipettes 的尖端直径约为2-5 µm. 粉扑压力介于 4-6 psi 之间: 这一范围是至关重要的, 因为较高的压?...

膜片钳技术是研究神经元电信号的主要工具, 在二十世纪七十年代1,2中得到了发展。这项技术的一个主要优点是它提供了有关特定治疗 (例如, 药理学) 如何能够实时改变神经元功能或通道的知识3。脑切片全细胞记录中神经元功能的药理学评价需要应用药物, 如激动剂或特定受体的拮抗药, 来记录神经元。此方法允许识别在应用特定药物后发生的神经元改变, 从而更好地理解神经元的生理和病理特性4。虽然药理学管理可以通过灌注5或粉扑6进行, 后者是优势技术。特别是: (i) 粉扑应用迅速增加了在记录的神经元周围的药物浓度到一个水平, 使膜受体的脱敏被阻止;(ii) 药物膨化量极低, 因此对沐浴液的影响不大, 因而减少了被管理的化学药品对脑切片的不良影响;(iii) 粉扑协议可以设定和保存, 使实验非常精确地重现;(iv) 粉扑的应用代表了激动剂/拮抗药的经济用途, 特别是在这种试剂昂贵或难以获得的情况下。
在这里, 我们将专注于记录在急性制备的脑切片中, 由 GABA 引起的全细胞电流, 这种制备具有相对保存完好的脑回路的优点。我们如何进行粉扑诱导的抑制电流7将在本文中介绍。通过使用铯 (Cs+) 的内部解决方案, 并持有 0 mV 的神经元, 我们将引入 GABA-粉扑诱发抑制突触后电流 (eIPSCs) 与适当的技术细节。应用脂多糖 (lps) 注射液诱发的小鼠抑郁症模型, 结果表明, 与汽车控制相比, lps 注射的小鼠 eIPSC 振幅明显降低.我们的意图是, 这篇文章应该显示如何粉扑技术是广泛适用于研究, 以评估的影响, 任何化学物质, 化合物或药物的神经元活动的脑切片。

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	<colgroup>
		<col style="width:161pt" width="214" />
		<col style="width:140pt" width="187" />
		<col style="width:155pt" width="206" />
		<col style="width:210pt" width="280" />
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	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Glass Borosilicate micropipettes</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">BF150-86-10</td>
			<td class="xl18" style="width:210pt" width="280">1.50 mm outer diameter; 0.86 mm inner diameter</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micropipette Puller</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MODEL P-97&#160;</td>
			<td class="xl18" style="width:210pt" width="280">Flaming/Brow Micropipette Puller</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micromanipulators</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MP-285</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Computer controlled Amplifier&#160;&#160;&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Multiclamp 700B</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Digital Acquisition system&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Digidata 1440A</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Imaging Camera</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl18" style="width:155pt" width="206">2115001</td>
			<td class="xl18" style="width:210pt" width="280">Inspection equipment</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Microscopy&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl20" style="width:155pt" width="206">Eclipse FN1&#160;</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Master 8</td>
			<td class="xl20" style="width:140pt" width="187">A.M.P.I.</td>
			<td class="xl20" style="width:155pt" width="206">Master-8 Pulse stimulator</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Vibratome Slicer</td>
			<td class="xl20" style="width:140pt" width="187">Leica</td>
			<td class="xl20" style="width:155pt" width="206">VT 1000S</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Picospritzer &#8546;</td>
			<td class="xl20" style="width:140pt" width="187">Parker Hannifin</td>
			<td class="xl20" style="width:155pt" width="206"></td>
			<td class="xl18" style="width:210pt" width="280">Pressure Systems for Ejection of 
			Picoliter Volumes in Cell Research</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Razor blade</td>
			<td class="xl20" style="width:140pt" width="187">Gillette</td>
			<td class="xl20" style="width:155pt" width="206">74-S</td>
			<td class="xl20" style="width:210pt" width="280">FLYING EAGLE</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video monitor</td>
			<td class="xl20" style="width:140pt" width="187">Panasonic</td>
			<td class="xl20" style="width:155pt" width="206">WV-BM 1410</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">502 Glue</td>
			<td class="xl20" style="width:140pt" width="187">Deli</td>
			<td class="xl18" style="width:155pt" width="206">7146</td>
			<td class="xl20" style="width:210pt" width="280">Cyanoacrylate Glue</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Peristaltic pump</td>
			<td class="xl20" style="width:140pt" width="187">Shanghai JIA PENG Corporation</td>
			<td class="xl20" style="width:155pt" width="206">BT100-1F</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video Camera</td>
			<td class="xl20" style="width:140pt" width="187">Olympus America Medical</td>
			<td class="xl20" style="width:155pt" width="206">OLY-150</td>
			<td class="xl20" style="width:210pt" width="280"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">Transfer Pipets</td>
			<td class="xl18" style="width:140pt" width="187">Biologix</td>
			<td class="xl18" style="width:155pt" width="206">30-0138A1</td>
			<td class="xl20" style="width:210pt" width="280"></td>
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whole-cell currents induced by puff application of gaba in brain slices

在脑切片中进行全细胞膜膜片钳记录时, 常用的是药理学管理方法。在电生理记录中, 药物应用的最佳方法之一是粉扑技术, 可用于研究药理试剂对脑切片神经元活动的影响。粉扑应用的最大优点是在记录部位周围的药物浓度迅速增加, 从而防止了膜受体的脱敏。粉扑应用的成功使用需要注意以下因素: 药物的浓度、粉扑微的参数、粉扑微的尖端与记录的神经元之间的距离、持续时间和压力驱动粉扑 (磅每平方英寸, psi)。本文介绍了一个 step-by 步骤的过程, 记录全细胞电流诱导的膨化γ-丁酸 (GABA) 的一个神经元的前额皮质切片。值得注意的是, 同样的程序可以适用于其他脑区, 如海马和纹状体, 以及不同的准备, 如细胞培养。

这种粉扑技术可以让研究人员研究药物对特定神经元表面特殊受体的影响。在这篇文章中, 我们关注的电流介导 GABA 受体。图 1显示了粉扑和记录 micropipettes。为了消除对 ACSF 和蔗糖泡芙 (图 2A) 所产生的物理压力的记录神经元的任何影响, 将其用作控件。gaba 粉扑 (黑色) 引起的反应可以被 gaba 受体抑制剂阻断, 如 picro...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

粉扑诱导的全细胞电流在脑切片中的应用

我们描述的粉扑技术, 其中的药理试剂可以在整个细胞膜片钳记录, 并突出了各方面的特点, 这是至关重要的成功。

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Research

JoVE Journal

Neuroscience

9.3K 次观看.  South China Normal University. 我们描述的粉扑技术, 其中的药理试剂可以在整个细胞膜片钳记录, 并突出了各方面的特点, 这是至关重要的成功。

视频: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

在大鼠脑片的单通道和补品GABA激活电流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

科研

神经科学

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...