Name: in vivo intracellular recording of type-identified rat spinal motoneurons during trans-spinal direct current stimulation
Uploaded: 2020-05-11T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation at JoVE.com

这项工作得到了国家科学中心第2017/25/B/NZ7/00373号赠款的支持。作者们要表彰汉娜·德日马尼亚-塞利乔夫斯卡和沃齐米尔兹·姆罗奇斯基的作品，他们都为收集和分析本文件介绍的结果做出了贡献。

与本议定书有关的所有程序都已被有关当局（例如地方道德委员会）接受，并遵守关于动物福利和管理的国家和国际规则。
注：参与手术的每个参与者必须接受基本外科手术方面的训练，并且必须拥有进行动物实验的有效许可证。
1. 麻醉和预谋
<ol><li>麻醉大鼠与内丙酮注射五巴比妥钠（初始剂量60毫克+千克-1 为6个月大的雄性威斯塔大鼠体重400\...

<ol>
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V. 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=Effectiveness+of+anodal+transcranial+direct+current+stimulation+to+improve+muscle+strength+and+motor+functionality+after+incomplete+spinal+cord+injury:+a+systematic+review+and+meta-analysis.">Effectiveness of anodal transcranial direct current stimulation to improve muscle strength and motor functionality after incomplete spinal cord injury: a systematic review and meta-analysis.</a> Spinal Cord. , (2020).</li><li>de Paz, R. H., Serrano-Mu&#241;oz, D., P&#233;rez-Nombela, S., Bravo-Esteban, E., Avenda&#241;o-Coy, J., G&#243;mez-Soriano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+transcranial+direct-current+stimulation+with+gait+training+in+patients+with+neurological+disorders:+a+systematic+review.">Combining transcranial direct-current stimulation with gait training in patients with neurological disorders: a systematic review.</a> Journal of Neuroengineering and Rehabilitation. 16 (1), 114 (2019).</li><li>Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+gamma+and+alpha+spinal+motor+neurons+activity+by+trans-spinal+direct+current+stimulation:+effects+on+reflexive+actions+and+locomotor+activity.">Modulation of gamma and alpha spinal motor neurons activity by trans-spinal direct current stimulation: effects on reflexive actions and locomotor activity.</a> Physiological Reports. 4 (3), (2016).</li><li>Bolzoni, F., Jankowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+and+postsynaptic+effects+of+local+cathodal+DC+polarization+within+the+spinal+cord+in+anaesthetized+animal+preparations.">Presynaptic and postsynaptic effects of local cathodal DC polarization within the spinal cord in anaesthetized animal preparations.</a> The Journal of Physiology. 593 (4), 947-966 (2015).</li><li>Cogiamanian, F., 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=Transcutaneous+Spinal+Direct+Current+Stimulation.">Transcutaneous Spinal Direct Current Stimulation.</a> Frontiers in Psychiatry. 3, (2012).</li><li>Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-spinal+direct+current+stimulation+alters+muscle+tone+in+mice+with+and+without+spinal+cord+injury+with+spasticity.">Trans-spinal direct current stimulation alters muscle tone in mice with and without spinal cord injury with spasticity.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 34 (5), 1701-1709 (2014).</li><li>Bolzoni, F., Pettersson, L. G., Jankowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+long-lasting+subcortical+facilitation+by+transcranial+direct+current+stimulation+in+the+cat.">Evidence for long-lasting subcortical facilitation by transcranial direct current stimulation in the cat.</a> The Journal of Physiology. 591 (13), 3381-3399 (2013).</li><li>Manuel, M., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha,+beta+and+gamma+motoneurons:+functional+diversity+in+the+motor+system's+final+pathway.">Alpha, beta and gamma motoneurons: functional diversity in the motor system's final pathway.</a> Journal of Integrative Neuroscience. 10 (3), 243-276 (2011).</li><li>Feiereisen, P., Duchateau, J., Hainaut, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+unit+recruitment+order+during+voluntary+and+electrically+induced+contractions+in+the+tibialis+anterior.">Motor unit recruitment order during voluntary and electrically induced contractions in the tibialis anterior.</a> Experimental Brain Research. 114 (1), 117-123 (1997).</li><li>Van Cutsem, M., Feiereisen, P., Duchateau, J., Hainaut, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+behaviour+of+motor+units+in+the+tibialis+anterior+during+voluntary+contractions.">Mechanical properties and behaviour of motor units in the tibialis anterior during voluntary contractions.</a> Canadian Journal of Applied Physiology = Revue Canadienne De Physiologie Appliquee. 22 (6), 585-597 (1997).</li><li>Gardiner, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+properties+of+motoneurons+innervating+different+muscle+unit+types+in+rat+gastrocnemius.">Physiological properties of motoneurons innervating different muscle unit types in rat gastrocnemius.</a> Journal of Neurophysiology. 69 (4), 1160-1170 (1993).</li><li>Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-spinal+direct+current+stimulation+modifies+spinal+cord+excitability+through+synaptic+and+axonal+mechanisms.">Trans-spinal direct current stimulation modifies spinal cord excitability through synaptic and axonal mechanisms.</a> Physiological Reports. 2 (9), (2014).</li><li>Manuel, M., Iglesias, C., Donnet, M., Leroy, F., Heckman, C. J., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics,+high-frequency+oscillations,+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons.">Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 29 (36), 11246-11256 (2009).</li><li>Liebetanz, D., Koch, R., Mayenfels, S., K&#246;nig, F., Paulus, W., Nitsche, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+limits+of+cathodal+transcranial+direct+current+stimulation+in+rats.">Safety limits of cathodal transcranial direct current stimulation in rats.</a> Clinical Neurophysiology: Official Journal of the International Federation of Clinical Neurophysiology. 120 (6), 1161-1167 (2009).</li><li>B&#261;czyk, M., Jankowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+direct+current+are+reproduced+by+intermittent+depolarization+of+myelinated+nerve+fibers.">Long-term effects of direct current are reproduced by intermittent depolarization of myelinated nerve fibers.</a> Journal of Neurophysiology. 120 (3), 1173-1185 (2018).</li><li>B&#261;czyk, M., Drzyma&#322;a-Celichowska, H., Mr&#243;wczy&#324;ski, W., Krutki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motoneuron+firing+properties+are+modified+by+trans-spinal+direct+current+stimulation+in+rats.">Motoneuron firing properties are modified by trans-spinal direct current stimulation in rats.</a> Journal of Applied Physiology. 126 (5), 1232-1241 (2019).</li><li>B&#261;czyk, M., Drzyma&#322;a-Celichowska, H., Mr&#243;wczy&#324;ski, W., Krutki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lasting+modifications+of+motoneuron+firing+properties+by+trans-spinal+direct+current+stimulation+in+rats.">Long-lasting modifications of motoneuron firing properties by trans-spinal direct current stimulation in rats.</a> European Journal of Neuroscience. , (2019).</li><li>Miranda, P. C., Faria, P., Hallett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+the+ratio+of+injected+current+to+electrode+area+tell+us+about+current+density+in+the+brain+during+tDCS.">What does the ratio of injected current to electrode area tell us about current density in the brain during tDCS.</a> Clinical Neurophysiology: Official Journal of the International Federation of Clinical Neurophysiology. 120 (6), 1183-1187 (2009).</li><li>Rahman, A., 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=Cellular+effects+of+acute+direct+current+stimulation:+somatic+and+synaptic+terminal+effects.">Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.</a> The Journal of Physiology. 591 (10), 2563-2578 (2013).</li><li>Bikson, M., 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=Effects+of+uniform+extracellular+DC+electric+fields+on+excitability+in+rat+hippocampal+slices+in+vitro.">Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro.</a> The Journal of Physiology. 557, 175-190 (2004).</li><li>Jankowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+control+of+motor+outputs+by+intrinsic+and+externally+induced+electric+field+potentials.">Spinal control of motor outputs by intrinsic and externally induced electric field potentials.</a> Journal of Neurophysiology. 118 (2), 1221-1234 (2017).</li><li>Button, D. C., Gardiner, K., Marqueste, T., Gardiner, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-current+relationships+of+rat+hindlimb+alpha-motoneurones.">Frequency-current relationships of rat hindlimb alpha-motoneurones.</a> The Journal of Physiology. 573, 663-677 (2006).</li></ol>

如果执行正确，所述方案的手术部分应在大约三小时内完成。在手术过程中，应特别注意保持动物的生理状况，特别是体温和麻醉深度。除了明显的伦理考虑外，缺乏适当的麻醉可能导致神经解剖或拉明切除术期间肢体过度运动，并导致制剂损伤或实验终止。在用微电子穿透脊髓之前使动物瘫痪后，监测麻醉深度和心率以及应用基于动物体重和肺活量的适当通气参数至关重要。任何偏离所需生理?...

跨脊柱直流刺激（tsDCS）作为一种有效的方法，在健康和疾病1，2，3中改变2脊柱回路兴奋性， 正在得到认可。1在这项技术中，一个恒定电流在位于选定脊髓段上方的有源电极之间传递，参考电极位于呼吸口或更红润的 4。几项研究已经证实，tsDCS可用于管理某些病理状况，如神经病变疼痛5，痉挛6，脊髓损伤7或促进康复8。研究人员认为，tsDCS会唤起细胞内和细胞膜外空间之间离子分布的改变，根据当前方向,9、10、11，,10这可促进或抑制神经元活动。然而，直到最近，还缺乏对莫托龙这种影响的直接证实。
在这里，我们描述了一个详细的方案，用于在体内进行麻醉大鼠腰椎莫酮的电位记录，同时应用 tsDCS，以观察莫托内隆膜的变化和燃烧特性，以应对脊髓神经网络的腺癌或阴极化。细胞内记录打开神经元属性的几个区域调查，不适用于以前使用的细胞外技术9,9，12。例如，可以精确测量由 tsDCS 诱导的莫托龙膜膜电压响应，以指示尖峰生成电压阈值，或分析作用电位参数。此外，该技术还使我们能够确定莫托龙无源膜特性，如输入电阻，并观察细胞内刺激电流与分子节律发射频率之间的关系。基于功能识别神经（即向屈曲器或挤出器提供发源的神经）的抗浪漫性识别，使我们能够进一步识别内向运动单元的类型（快与慢），从而有机会测试极化是否以不同的方式影响成熟的脊柱神经元系统的单个元素。由于在录音前进行了广泛的手术，并且对录音的稳定性和可靠性要求很高，这项技术极具挑战性，但允许对一个motoneuron的电生理特性进行直接和长期的评估：在tsDCS应用之前、期间和之后，这对于确定其急性作用和持续效应至关重要。由于motoneuron直接激活外混肌纤维14，并参与肌肉收缩的反馈控制，并发展力量15，16任何观察到15,的tsDCS对运动单元或肌肉收缩特性的影响可能与motoneuron兴奋性或燃烧特性的调制有关。

<table>
	<tbody>
		<tr>
			<td>Durgs and solutions</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Atropinum sulfuricum</td>
			<td>Polfa Warszawa</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>346351</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Merck</td>
			<td>106329</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pancuronium Jelfa</td>
			<td>PharmaSwiss/Valeant</td>
			<td>-</td>
			<td>Neuromuscular blocker</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Biowet Pu&#322;awy Sp. z o.o</td>
			<td>-</td>
			<td>Main anesthetic agent</td>
		</tr>
		<tr>
			<td>Pottasium citrate</td>
			<td>Chempur</td>
			<td>6100-05-06</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tetraspan</td>
			<td>Braun</td>
			<td>-</td>
			<td>HES solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical equipment</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>21 Blade</td>
			<td>FST</td>
			<td>10021-00</td>
			<td>Scalpel blade</td>
		</tr>
		<tr>
			<td>Cauterizer</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Chest Tubes</td>
			<td>Mila</td>
			<td>CT1215</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dumont #4 Forceps</td>
			<td>FST</td>
			<td>11241-30</td>
			<td>Muscle forceps</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Dura forceps</td>
		</tr>
		<tr>
			<td>Dumont #5F Forceps</td>
			<td>FST</td>
			<td>11255-20</td>
			<td>Nerve forceps</td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>FST</td>
			<td>11252-00</td>
			<td>Pia forceps</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>Blunt forceps</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11053-10</td>
			<td>Skin forceps</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13013-14</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>FST</td>
			<td>16021-14</td>
			<td>For laminectomy</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15000-08</td>
			<td>Vein scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15002-08</td>
			<td>Dura scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14184-09</td>
			<td>For trachea cut</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>104075-11</td>
			<td>Muscle scissors</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14002-13</td>
			<td>Skin scissors</td>
		</tr>
		<tr>
			<td>Tracheal tube</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Vein catheter</td>
			<td>Vygon</td>
			<td>1261.201</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vessel cannulation forceps</td>
			<td>FST</td>
			<td>18403-11</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vessel clamp</td>
			<td>FST</td>
			<td>18320-11</td>
			<td>For vein clamping</td>
		</tr>
		<tr>
			<td>Vessel Dilating Probe</td>
			<td>FST</td>
			<td>10160-13</td>
			<td>For vein dissection</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sugrgical materials</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gel foam</td>
			<td>Pfizer</td>
			<td>GTIN 00300090315085</td>
			<td>Hemostatic agent</td>
		</tr>
		<tr>
			<td>Silk suture 4.0</td>
			<td>FST</td>
			<td>18020-40</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Silk suture 6.0</td>
			<td>FST</td>
			<td>18020-60</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Axoclamp 2B</td>
			<td>Molecular devices</td>
			<td>discontinued</td>
			<td>Intracellular amplifier/ new model Axoclamp 900A</td>
		</tr>
		<tr>
			<td>CapStar-100 End-tidal CO2 Monitor</td>
			<td>CWE</td>
			<td>11-10000</td>
			<td>Gas analyzer</td>
		</tr>
		<tr>
			<td>Grass S-88</td>
			<td>A-M Systems</td>
			<td>discontinued</td>
			<td>Constant current stimulator</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket Systems with Flexible Probe</td>
			<td>Harvard Apparatus</td>
			<td>507222F</td>
			<td>Heating system</td>
		</tr>
		<tr>
			<td>ISO-DAM8A</td>
			<td>WPI</td>
			<td>74020</td>
			<td>Extracellular amplifier</td>
		</tr>
		<tr>
			<td>Microdrive</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement IVM/Scientifica</td>
		</tr>
		<tr>
			<td>P-1000 Microelectrode puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>Microelectrode puller</td>
		</tr>
		<tr>
			<td>SAR-830/AP Small Animal Ventilator</td>
			<td>CWE</td>
			<td>12-02100</td>
			<td>Respirator</td>
		</tr>
		<tr>
			<td>Support frame</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement lab standard base 51601/Stoelting</td>
		</tr>
		<tr>
			<td>Spinal clamps</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made/replacement Rat spinal adaptor 51695/Stoelting</td>
		</tr>
		<tr>
			<td>TP-1 DC stimulator</td>
			<td>WiNUE</td>
			<td>-</td>
			<td>tsDCS stimulator</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1B150-4 glass capillaries</td>
			<td>WPI</td>
			<td>1B150-4</td>
			<td>For microelectrodes production</td>
		</tr>
		<tr>
			<td>Cotton wool</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>flexible tubing</td>
			<td>-</td>
			<td>-</td>
			<td>For respirator and CO2 analyzer connection</td>
		</tr>
		<tr>
			<td>MicroFil</td>
			<td>WPI</td>
			<td>MF28G67-5</td>
			<td>For filling micropipettes</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>-</td>
			<td>-</td>
			<td>For nerve electrodes</td>
		</tr>
	</tbody>
</table>

in vivo intracellular recording of type-identified rat spinal motoneurons during trans-spinal direct current stimulation

体内脊髓腺细胞内记录为确定完整脊柱网络中的细胞电生理特征提供了"黄金标准"，与传统的体外或细胞外记录技术相比具有显著优势。体内细胞内记录的优点是，这种方法可以在神经系统完全成熟的成年动物身上进行，因此许多观察到的生理机制可以转化为实际应用。在本文中，我们描述了这个程序与外部应用的恒定电流刺激相结合，它模仿脊柱神经元网络内发生的极化过程。跨脊柱直流刺激（tsDCS）是一种创新的方法，越来越多地用作各种神经损伤后康复和运动的神经调节干预。tsDCS对神经系统的影响仍然不为人所知，其行动背后的生理机制在很大程度上未知。tsDCS 与细胞内记录同时应用使我们能够直接观察莫托龙膜特性的变化和节律激发的特征，以应对脊柱神经元网络的极化，这对理解 tsDCS 操作至关重要。此外，当提出的协议包括识别内侧肌肉及其功能（柔性与拉伸器）以及生理类型（快速与缓慢）的motoneuron时，它提供了一个机会，有选择地研究tsDCS对已识别的脊柱回路成分的影响，这些成分似乎受到极化的影响不同。所介绍的程序侧重于细胞内记录和刺激的手术准备，重点是实现制备稳定性和结果可重复性所必需的步骤。讨论了阴极或阴极 tsDCS 应用方法的细节，同时关注实际和安全问题。

当确保细胞穿透条件稳定时，可以在细胞内记录的基础上计算作用电位参数和若干膜特性。 图1A 提供了细胞内刺激引起的典型正交作用潜力，它符合数据包含的所有标准（至少为-50 mV的静膜电位，峰值振幅高于50 mV，具有正过冲）。可测量动作电位参数，如尖峰振幅、后白化幅度或后白化半衰减时间（AHP-HDT）。大鼠莫托龙中后一个参数的值是区分快速和慢莫托龙（AHP-HDT > 20...

Watch this Scientific Journal Video about In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation at JoVE.com

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

该协议描述了大鼠腰椎瘤的体内细胞内记录，同时具有跨脊柱直电流刺激。该方法使我们能够测量膜特性，并记录脊髓的腺体或阴极化之前、期间和之后有节奏的分子发射。

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

该协议使用脊柱腺瘤的细胞内记录，直接研究跨脊柱直流刺激对脊柱网络功能的影响。这种技术的主要优点是，它允许在完全成熟的神经系统上进行细胞内录音。促进实验观测结果的翻译到实际应用。在确认对踏板反射缺乏反应后，一只麻醉了六个月大的雄性威斯塔大鼠，使用解剖显微镜和21号刀片，从胸骨到下巴进行纵向皮肤切口。并使用钝解剖来暴露右血管。将两个，4-0连字放在静脉部分下，没有分支点。并在静脉段的近端打一个松动的结。和一个松散的结在段的端。将静脉紧拉至心脏，并夹住静脉的端端。使用虹膜剪刀在夹子和端结字之间进行切口。并且，握住静脉的活瓣，将预填充的导管引入静脉被夹子阻塞的点。然后取下夹子，将导管推入静脉几毫米。对于气管放置，使用钝钳分离覆盖胸腺肌肉的两个腺体。并分离在中线，以暴露气管的胸腺肌肉。将三个，4-0连字放在气管下面，并在气管插入点下方打两个节，在上面打一个节。然后将气管插入第三气管软骨下方的气管。用预先准备的连字固定管子。要解剖后肢神经，请使用21号刀片在左后肢的后侧进行纵向切割，从跟腱到臀部。并且用剪刀在膝关节后部的波普莱特叶的前部和后部区域之间做剪。切两个头的二头肌毛骨，暴露坐骨神经。将横向头部与胃肠肌肉的中脑头部分离，以暴露骨神经及其分支。然后使用数字55钳子，仔细解剖中胃和横向胃和鞋底神经。断开它们与周围组织的连接，同时保持它们与各自肌肉的连接。要进行耳管切除术，请使用数字21刀片，使从囊到胸椎的纵向切口。并识别Th13椎骨，作为一个最低的胸段与肋骨插入。然后使用细的隆格器将旋转过程和层压从 Th13 到 L2 椎骨，以暴露脊髓的腰椎部分。要固定椎柱，请将大鼠放入 37 摄氏度加热垫上的定制框架中，连接到闭环加热系统。并使用皮肤皮瓣在裸露的脊髓上形成一个深池。将金属夹放在 Th12 横向工艺下方和 L3 旋转工艺中，并在池中加注 37 摄氏度的矿物油。使用皮肤皮瓣，使深池矿物油在暴露的三口，中侧胃肠和侧胃和鞋底神经。将神经放在双极银线刺激电极上。然后，使用每个神经的单独刺激通道将电极连接到方形脉冲刺激器。对于表面电极放置，在解剖显微镜下，将银球电极放在暴露脊髓的左侧。在背部肌肉中插入参考电极。将两个电极连接到差分直流放大器。使用恒定电流刺激器刺激中位胃肠和横向胃角神经，以 3 赫兹频率重复的 0.1 毫秒持续时间的平方脉冲，并观察发泡球。在模拟结束时，以玫瑰色方式移动表面电极，并重复刺激，以确定每个神经的振幅最高的脊髓段。确定最大排球的位置后，静脉注射神经肌肉阻滞剂，使大鼠瘫痪。当助手将气管连接到外部呼吸机时，与啮齿动物兼容的帽提名器一样。要打开 dura 和 pia 母体，请使用 55 号钳子轻轻抬起 dura 母体，然后从 L5 段（玫瑰花到 L4 段）切割组织。然后使用一对超薄的5SF钳子，在皮娅中制作一个小补丁，覆盖血管之间的支柱。正好在从中层胃和横向胃和鞋底神经的最大胃排球水平。要放置跨脊柱直流刺激电极，请将盐水浸泡海绵放在Th12椎骨的后侧。并使用精细操作按海绵与有源的跨脊柱直流刺激电极。然后将自定义池式微电子安装到微型操纵器上，允许一到两微米步进移动和立体轴校准。以 15 到 20 度的中向倾斜角将微移管尖端驱动到皮娅中选定的贴片中。为了记录磁膜和发射特性，在细胞内放大器的桥接模式下，刺激各自的神经分支根据全部或无外观识别莫托龙的抗变色作用潜力。在电流开关速率为 4 至 8 千赫的细胞内放大器的不连续电流夹紧模式下，使用 0.5 毫秒的细胞内极化电流脉冲，在磁毒中激发正畸作用电位。要计算细胞输入电阻，请刺激一个莫托龙，用40个短的100毫秒脉冲超极化一个纳米电流。要确定实际基值为引出单个峰值所需的去极化电流的最小振幅，请刺激具有 50 毫秒方波脉冲的motoneuron，以增加振幅。然后注入500毫秒的平方波脉冲去极化电流，在增加振幅。在0.1至两纳米安培的步数中，唤起莫托龙的有节奏放电。对于跨脊柱直流刺激，通过直流应用跨脊柱开始极化过程，同时保持莫托龙的稳定穿透。在这里，一个典型的正交作用潜力，由细胞内刺激引起，满足所有数据包含标准显示。在此分析中，一个细胞响应100毫秒超极化电流脉冲的一毫图。从中可以观察到莫托龙的峰值和高原输入电阻，可以从电压偏转中确定。这种真正基本尖峰的扩展电压轨迹，显示尖峰的电压阈值清晰。指示激活电压门控钠通道以启动作用电位的膜去极化水平。这些图表提供了细胞内电压轨迹的示例。从两个莫托龙，刺激细胞内与500毫秒的平方脉冲去极化电流。跨脊柱直流刺激应用之前、期间和之后。发现阿诺达尔跨脊柱直流刺激对增加的莫托龙兴奋性和更高频率的节奏发射有作用。而阴极跨脊柱直流刺激，作用于对火的抑制。此外，这两种类型的跨脊柱直流刺激的影响，超过极化时期。如果数据包含标准由于细胞穿透不完善而遭到破坏，则可以获取不准确的数据。未能补偿微电位电阻和电容或脊髓不稳定。在解剖过程中，对脊髓不造成损伤是势在必行的，因为损伤可能导致脊髓休克，使得无法进行进一步的记录。可以采集组织样本，以进一步组织学或免疫细胞化学分析。例如，c-fos 表达式的测量可用作神经元活动的代理。

Research

JoVE Journal

Neuroscience

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

经颅直接电流刺激电极定位和蒙太奇

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

科研

神经科学

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

远程监督的经颅直流电刺激: 安全性和耐受的最新进展

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...

The Combined Use of Transcranial Direct Current Stimulation and Robotic Therapy for the Upper Limb

经颅直流电刺激与上肢机器人治疗的联合应用

Neurologic disorders such as stroke and cerebral palsy are leading causes of long-term disability and can lead to severe incapacity and restriction of ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial Direct Current Stimulation tDCS in Mice

经颅直流电刺激 (tDCS) 在小鼠中的作用

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

使用具有高清晰度颅内直接电流刺激的 3D 数字化仪确定刺激位置

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

虚拟现实曝光期间颅外直接电流刺激的同步应用

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through ...

Transcranial Direct Current Stimulation (tDCS) for Memory Enhancement

Transcranial Direct Current Stimulation tDCS for Memory Enhancement

用于增强内存的颅外直接电流刺激 （tDCS）

Memory enhancement is one of the great challenges in cognitive neuroscience and neurorehabilitation. Among various techniques used for memory enhancement, ...