Name: preparation of acute spinal cord slices for whole-cell patch-clamp recording in substantia gelatinosa neurons
Uploaded: 2019-01-18T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons at JoVE.com

这项工作得到了国家自然科学基金的资助 (81560198、31660289号)。

所描述的所有实验方案均获得南昌大学动物伦理委员会 (南昌, 中国, 道德号 2017-010号)。所有的努力都是为了最大限度地减少实验动物的压力和痛苦。在室温下进行的电生理记录 (rt, 22–25°c)。
1. 动物
<ol><li>使用 sprague-dawley 大鼠 (3-5周大) 的任何性别。将这些动物以12小时的光暗循环居住, 让它们获得足够的食物和水。</li>
</ol>2. 溶液和材料的制备
<ol>...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciatic+chronic+constriction+injury+produces+cell-type-specific+changes+in+the+electrophysiological+properties+of+rat+substantia+gelatinosa+neurons.">Sciatic chronic constriction injury produces cell-type-specific changes in the electrophysiological properties of rat substantia gelatinosa neurons.</a> Journal of Neurophysiology. 96 (2), 579-590 (2006).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-regulated+kinase+(ERK)+activation+is+required+for+itch+sensation+in+the+spinal+cord.">Extracellular signal-regulated kinase (ERK) activation is required for itch sensation in the spinal cord.</a> Molecular Brain. 7, 25 (2014).</li><li>Kopach, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+alters+trafficking+of+extrasynaptic+AMPA+receptors+in+tonically+firing+lamina+II+neurons+of+the+rat+spinal+dorsal+horn.">Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn.</a> Pain. 152 (4), 912-923 (2011).</li><li>Takasu, K., Ono, H., Tanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+hyperpolarization-activated+cyclic+nucleotide-gated+cation+channels+at+primary+afferent+terminals+contribute+to+chronic+pain.">Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain.</a> Pain. 151 (1), 87-96 (2010).</li><li>Iura, A., Takahashi, A., Hakata, S., Mashimo, T., Fujino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductions+in+tonic+GABAergic+current+in+substantia+gelatinosa+neurons+and+GABAA+receptor+delta+subunit+expression+after+chronic+constriction+injury+of+the+sciatic+nerve+in+mice.">Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor delta subunit expression after chronic constriction injury of the sciatic nerve in mice.</a> European Journal of Pain. 20 (10), 1678-1688 (2016).</li><li>Alles, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+injury+increases+contribution+of+L-type+calcium+channels+to+synaptic+transmission+in+spinal+lamina+II:+Role+of+alpha2delta-1+subunits.">Peripheral nerve injury increases contribution of L-type calcium channels to synaptic transmission in spinal lamina II: Role of alpha2delta-1 subunits.</a> Molecular Pain. 14, 1-12 (2018).</li><li>Santos, S. F., Rebelo, S., Derkach, V. A., Safronov, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+interneurons+dominate+sensory+processing+in+the+spinal+substantia+gelatinosa+of+rat.">Excitatory interneurons dominate sensory processing in the spinal substantia gelatinosa of rat.</a> The Journal of Physiology. 581 (Pt 1), 241-254 (2007).</li><li>Lu, Y., Perl, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+organization+of+excitatory+circuits+between+neurons+of+the+spinal+superficial+dorsal+horn+(laminae+I+and+II).">Modular organization of excitatory circuits between neurons of the spinal superficial dorsal horn (laminae I and II).</a> The Journal of Neuroscience. 25 (15), 3900-3907 (2005).</li><li>Hantman, A. W., van den Pol, A. N., Perl, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+physiological+features+of+a+set+of+spinal+substantia+gelatinosa+neurons+defined+by+green+fluorescent+protein+expression.">Morphological and physiological features of a set of spinal substantia gelatinosa neurons defined by green fluorescent protein expression.</a> The Journal of Neuroscience. 24 (4), 836-842 (2004).</li><li>Yasaka, T., Tiong, S. Y., Hughes, D. I., Riddell, J. S., Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+inhibitory+and+excitatory+interneurons+in+lamina+II+of+the+adult+rat+spinal+dorsal+horn+revealed+by+a+combined+electrophysiological+and+anatomical+approach.">Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.</a> Pain. 151 (2), 475-488 (2010).</li><li>Yin, H., Park, S. A., Han, S. K., Park, S. 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H., Hu, T., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+enhances+inhibitory+transmission+to+substantia+gelatinosa+neurons+of+the+rat+spinal+dorsal+horn.">Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn.</a> Neuroscience. 319, 183-193 (2016).</li><li>Peng, S. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+ascorbate+in+the+preparation+and+maintenance+of+brain+slices.">Use of ascorbate in the preparation and maintenance of brain slices.</a> Methods. 18 (2), 144-149 (1999).</li><li>Takasu, K., Ogawa, K., Minami, K., Shinohara, S., Kato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-specific+functional+alteration+of+N-type+voltage-gated+calcium+channels+in+synaptic+transmission+of+primary+afferent+C-fibers+in+the+rat+spinal+superficial+dorsal+horn.">Injury-specific functional alteration of N-type voltage-gated calcium channels in synaptic transmission of primary afferent C-fibers in the rat spinal superficial dorsal horn.</a> European Journal of Pharmacology. 772, 11-21 (2016).</li><li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+synaptic+transmission+in+the+spinal+substantia+gelatinosa+is+under+an+inhibitory+tone+of+endogenous+adenosine.">Excitatory synaptic transmission in the spinal substantia gelatinosa is under an inhibitory tone of endogenous adenosine.</a> Neuroscience Letters. 477 (1), 28-32 (2010).</li><li>Funai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+dexmedetomidine+augments+inhibitory+synaptic+transmission+in+the+superficial+dorsal+horn+through+activation+of+descending+noradrenergic+control:+an+in+vivo+patch-clamp+analysis+of+analgesic+mechanisms.">Systemic dexmedetomidine augments inhibitory synaptic transmission in the superficial dorsal horn through activation of descending noradrenergic control: an in vivo patch-clamp analysis of analgesic mechanisms.</a> Pain. 155 (3), 617-628 (2014).</li><li>Yamasaki, H., Funai, Y., Funao, T., Mori, T., Nishikawa, K. <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+tramadol+on+substantia+gelatinosa+neurons+in+the+rat+spinal+cord:+an+in+vivo+patch-clamp+analysis.">Effects of tramadol on substantia gelatinosa neurons in the rat spinal cord: an in vivo patch-clamp analysis.</a> PLoS One. 10 (5), e0125147 (2015).</li><li>Furue, H., Narikawa, K., Kumamoto, E., Yoshimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+of+rat+substantia+gelatinosa+neurones+to+mechanical+but+not+thermal+stimuli+revealed+by+in+vivo+patch-clamp+recording.">Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording.</a> The Journal of Physiology. 521 (Pt 2), 529-535 (1999).</li><li>Ting, J. 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该协议详细介绍了准备脊髓切片的步骤, 我们在 sg 神经元18、19、20、21上进行全细胞膜片时成功地使用了脊髓切片。通过实施这种方法, 我们最近报告说, 米诺环素, 第二代四环素, 可以显著提高抑制突触传递通过突触前机制在 sg 神经元19。此外, 该制剂可降低 ih 的振幅, 进一?...

胶质 (sg, 脊髓背角的层 ii) 是一个无可争辩的重要中继中心, 用于传输和调节感官信息。它由兴奋和抑制间神经元组成, 接收来自初级传入纤维、局部神经元间和内源性下降抑制系统1的输入。近几十年来, 急性脊髓切片制剂的发展和全细胞膜片夹具记录的出现, 使 sg2的内在电生理和形态特性的各种研究,3 个,4并且地方电路的研究在 sg5,6。此外, 通过体外脊髓切片制剂, 研究人员可以解释神经元兴奋性的变化 7,8, 离子通道9,10, 和突触活动11,12在各种病理条件下。这些研究加深了我们对 sg 神经元在慢性疼痛和神经病理性瘙痒的发展和维持中的作用的理解。
从本质上讲, 实现神经元 soma 的清晰可视化和理想的全细胞修补使用急性脊髓切片的关键先决条件是确保切片的优良质量, 从而获得健康和可修补的神经元。然而, 准备脊髓切片涉及几个步骤, 如进行腹侧椎板切除术和去除皮-蜘蛛核细胞膜, 这可能是获得健康切片的障碍。虽然准备脊髓切片并不容易, 但在体外对脊髓切片进行录音有几个优点。与细胞培养制剂相比, 脊髓切片可以部分保存生理相关条件下的固有突触连接。此外, 使用脊髓切片的全细胞膜片夹具记录可与其他技术相结合, 如双膜片钳13、14、形态学研究15、16和单细胞 rt-pcr17. 因此, 这项技术提供了更多关于特定区域内解剖和遗传多样性特征的信息, 并可以调查当地电路的组成。
在这里, 我们提供了一个基本和详细的描述, 我们的方法准备急性脊髓切片和获取全细胞膜片夹从 sg 神经元。

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Used for the preparation of ACSF and PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Sigma</td>
			<td>71500</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF and PBS</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Used for the preparation of ACSF</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>A7631</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td>Used for the preparation of sucrose-ACSF</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Wako</td>
			<td>169-11835</td>
			<td>Used for the preparation of K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P1937</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>Sigma</td>
			<td>G5884</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>CsMeSO4</td>
			<td>Sigma</td>
			<td>C1426</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Sigma</td>
			<td>C3011</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>Sigma</td>
			<td>T2265</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Neurobiotin 488</td>
			<td>Vector</td>
			<td>SP-1145</td>
			<td>0.05% neurobiotin 488 could be used for morphological studies</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>3% agar block was used in our protocol</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>4% paraformaldehyde was used for immunohistochemical processing</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Hengxing Chemical Reagents</td>
			<td></td>
			<td>Used for the preparation of PBS</td>
		</tr>
		<tr>
			<td>Mount Coverslipping Medium</td>
			<td>Polyscience</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>Urethan</td>
			<td>National Institute for Food and Drug Control</td>
			<td>30191228</td>
			<td>1.5 g/kg, i.p.</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW150F-4</td>
			<td>1.5 mm OD, 1.12 mm ID</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>Used for the preparation of micropipettes</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Technical Manufacturing Corporation</td>
			<td>63544</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared CCD camera</td>
			<td>Dage-MIT</td>
			<td>IR-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Y stage</td>
			<td>Burleigh</td>
			<td>GIBRALTAR X-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Advanced</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Mettler Toledo</td>
			<td>FE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocol microscope</td>
			<td>Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of acute spinal cord slices for whole-cell patch-clamp recording in substantia gelatinosa neurons

最近的全细胞膜片钳研究从明胶 (sg) 神经元提供了大量的信息, 有关脊柱机制的基础感觉传递, 诱发电位调节, 慢性疼痛或瘙痒的发展。在急性脊髓切片的效用基础上, 实施电生理记录和形态学研究, 进一步提高了我们对 sg 中神经元特性和局部电路组成的认识。在这里, 我们提出了一个详细和实用的指南, 为脊髓切片的制备, 并显示具有代表性的全细胞记录和形态学结果。该协议允许理想的神经元保存, 并能在一定程度上模拟体内条件。总之, 能够获得脊髓切片的体外准备, 可以稳定的电流和电压夹具记录, 从而有助于对固有膜特性、局部电路和神经元结构使用不同的实验方法。

根据图 1所示的图表制备了急性脊髓切片。切片和恢复后, 脊髓切片被转移到记录室。利用 ir-dic 显微镜, 根据 soma 的外观鉴定了健康的神经元。其次, 当神经元在 rmp 上保持时, 一系列的去极化电流脉冲 (1秒) 激发了 sg 神经元的动作电位。如图 2所示, 在 sg 神经元中观察到的发射模式包括扁桃体发射、延迟发射、杂波发射、初始爆...

Watch this Scientific Journal Video about Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons at JoVE.com

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

在这里, 我们描述了由体外脊髓切片中的明胶 (sg) 神经元制成的全细胞膜片夹记录的基本步骤。该方法可对 sg 神经元的固有膜特性、突触传递和形态表征进行研究。

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

这种方法有助于澄清感觉传播、异常调节和慢性疼痛或疼痛发展背后的脊髓机制。首先使用毛细管拉拔器从硅酸盐玻璃微胶囊中拉出记录电极。然后，用熔融的加糖填充模具，准备一个1.2厘米1.5厘米由2.0厘米的加糖块。如果规划横向部分，修剪块以具有中央通道。或具有抛物线部分块边缘的通道肉。在转心性灌注和脊髓提取之前，准备500毫升的蔗糖为基础的ACSF。使用 95%O2 和 5%CO2 冷却冰冷溶液并均衡。冷却冰上所有的解剖工具。在背部皮肤上做一个纵向五厘米切口，从肌科达尔到罗斯特拉。然后，切开脊柱和两侧肋骨之间的肋骨笼。用剪刀在脊柱的骨端做切口。然后切开周围的组织，并迅速分离脊柱的隆起部分。将 lumbosacral 段转移到含有冰冷蔗糖的玻璃盘中。与腹侧向上，使用精细剪刀切开椎骨，并小心地暴露脊髓。用凸体扩大分离脊髓的两厘米长部分，将脊髓部分转移到另一个装满冷蔗糖ACSF的玻璃盘中。在解剖显微镜下工作，去除梅宁和皮亚蜘蛛膜。尽快切开所有心根和后根。切中时注意避免脊髓受伤，尤其是后节角。将脊髓放在先前修剪的胡须块上。要准备横向切片，请将脊髓的腹侧连接到带侧朝刀片的 agar 上。要准备寄生虫片，请将带超级胶水的腹侧垂直连接到阿加。然后，将阿加块安装到具有超级胶水的振动器的平台上。并准备300至500微米横向或副卫星切片，高级速度为0.025毫米/秒，振动频率为80赫兹。脊髓应被切入多索-呼吸。为了获得最佳效果，应在 15 到 20 分钟内准备切片。脊柱片的厚度不应超过600微米，以满足细胞的可见度。使用塑料修剪移液器将切片转移到 32 摄氏度的连续氧化 ACSF 的存储室中的尼龙网上。要进行来自亚斯坦蒂娅胶质酶或SG神经元的全细胞贴片夹记录，请使用基于钾离子的细胞内溶液进行记录，同时仅应用基于钙的阳离子溶液进行抑制性后突触电流的记录。轻轻地将脊髓切片移动到记录室。然后用带尼龙线的 U 形铂线牢固地稳定住，以实现最佳的切片稳定性。在室温下用气泡ACSF稳定地将切片进行，将灌注速率设置为每分钟2至4毫升，以实现足够的氧合。然后，使用低分辨率显微镜透镜，将SG神经元区域识别为半透明带。选择一个健康的神经元，其特征是三维形状，以明亮光滑的膜作为目标细胞，使用高分辨率目标，并调整到视频监视器屏幕的中心。用适当的钾离子或基于钙离子的细胞内溶液填充微移液器。然后将微移液器插入电极支架，并确保细胞内溶液与支架内的银线接触。将微型移液器对焦，并使用微操纵器将它浸入 ACSF 中。施加温和的正压，以强制清除任何污垢和碎屑。逐渐将微移液器移向目标神经元。一旦移液器接近神经元，神经元膜上形成非常小的酒窝，释放正压形成千兆酶。将保持电位改变为负70毫伏，接近细胞的生理静膜电位。然后，对微移液器施加瞬态和温和的吸力，以破裂膜，并创建良好的全细胞配置。通过测试当前夹钳中每个神经元的点火模式，用一秒去极化电流脉冲 25 到 150 皮安，在静膜电位下以 25 皮安增量记录点火性能。要记录体电位电位，在电压夹紧模式下将膜电位保持在负 50 毫伏，然后应用一系列超极化电压脉冲，持续时间从负 60 毫伏到负 120 毫伏，并降低 10 毫伏。在负70毫伏的保持电位下，在电压夹紧模式下，使用基于钾离子的细胞内溶液记录突触后兴奋电流。记录 IPSC 使用基于 caesium 离子的细胞内溶液记录 IPSC 的。将膜电位保持负70毫伏约5分钟后，逐渐将保持电位更改为零毫伏。等待几分钟以稳定，然后开始记录 IPSC 事件。通过将一系列一秒去极化电流脉冲注入休息膜电位上的 SG 神经元而确定的点火模式可分为补品激发、延迟射击、间隙激发、初始爆发、磷爆炸、单尖峰和不情愿的激发。此图显示了电压夹中低于额定电流的唤起协议。这些代表性跟踪显示了对分类为 IH、IA 和 IT 的超极化电流注入的反应。这是SEPSC在50微摩尔 APV 和 20 微摩尔 CNQX 不存在的情况下从 SG 神经元以负 70 毫伏记录的代表踪迹。此跟踪显示在扩展时间刻度上添加 APV 和 CNQX 之前记录的 SEPC。遵循这个程序是方法，如配对补丁夹状或光遗传学可以执行回答其他问题，如描述特定神经元微电路在亚斯坦蒂娅胶质。

Research

JoVE Journal

Neuroscience

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

科研

神经科学

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 ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

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 ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

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

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

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 ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

下丘脑Kisspeptin神经元作为全细胞膜片钳记录的靶标

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...