Name: whole-cell currents induced by puff application of gaba in brain slices
Uploaded: 2017-10-12T14:00:00
Description: Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

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?...

<ol>
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J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. 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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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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Glass Borosilicate micropipettes</td>
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			<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>
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			<td class="xl18" style="width:155pt" width="206">2115001</td>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Microscopy&#160;</td>
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			<td class="xl20" style="width:140pt" width="187">A.M.P.I.</td>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Vibratome Slicer</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video Camera</td>
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			<td class="xl18" style="width:155pt" width="206">30-0138A1</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 ...

Research

JoVE Journal

Neuroscience

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

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

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

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

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

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 ...

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

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) ...

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

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 ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...