Name: stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro
Uploaded: 2019-08-28T13:00:00
Description: Watch this Scientific Journal Video about Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro at JoVE.com

SPS 得到了悉尼大学研究生研究奖学金的支持。

所有实验协议均获得悉尼大学动物伦理委员会的批准（批准协议号：2018/1308）。
1. 动物
注：老鼠是从澳大利亚啮齿动物中心获得的。珀斯，澳大利亚），并在悉尼大学医学基金会建设动物设施举行。
<ol><li>保持小鼠正常12小时光/暗循环与环境富集。</li>
	<li>使用雄性和雌性C57BL/6小鼠（3~5周大）进行所有实验。</li>
</ol>2. 准?...

<ol>
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P., 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=Using+low+levels+of+stochastic+vestibular+stimulation+to+improve+locomotor+stability.">Using low levels of stochastic vestibular stimulation to improve locomotor stability.</a> Frontiers in Systems Neuroscience. 9, 117 (2015).</li><li>Iwasaki, S., 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=Noisy+vestibular+stimulation+increases+gait+speed+in+normals+and+in+bilateral+vestibulopathy.">Noisy vestibular stimulation increases gait speed in normals and in bilateral vestibulopathy.</a> Brain stimulation. 11 (4), 709-715 (2018).</li><li>Serrador, J. M., Deegan, B. M., Geraghty, M. C., Wood, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+vestibular+function+in+the+elderly+with+imperceptible+electrical+stimulation.">Enhancing vestibular function in the elderly with imperceptible electrical stimulation.</a> Scientific Reports. 8 (1), 336 (2018).</li><li>Kim, J., Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+primary+vestibular+neurons+to+galvanic+vestibular+stimulation+(GVS)+in+the+anaesthetised+guinea+pig.">Responses of primary vestibular neurons to galvanic vestibular stimulation (GVS) in the anaesthetised guinea pig.</a> Brain Research Bulletin. 64 (3), 265-271 (2004).</li><li>Flores, 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=Stochastic+resonance+in+the+synaptic+transmission+between+hair+cells+and+vestibular+primary+afferents+in+development.">Stochastic resonance in the synaptic transmission between hair cells and vestibular primary afferents in development.</a> Neuroscience. 322, 416-429 (2016).</li><li>Huidobro, N., 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=Brownian+Optogenetic-Noise-Photostimulation+on+the+Brain+Amplifies+Somatosensory-Evoked+Field+Potentials.">Brownian Optogenetic-Noise-Photostimulation on the Brain Amplifies Somatosensory-Evoked Field Potentials.</a> Frontiers in Neuroscience. 11, 464-464 (2017).</li><li>Goldberg, J., Ferna, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+vestibular-nerve+afferents+in+the+squirrel+monkey+to+externally+applied+galvanic+currents.">Responses of vestibular-nerve afferents in the squirrel monkey to externally applied galvanic currents.</a> Brain Research. 252 (1), 156-160 (1982).</li><li>Baird, R., Desmadryl, G., Fernandez, C., Goldberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vestibular+nerve+of+the+chinchilla.+II.+Relation+between+afferent+response+properties+and+peripheral+innervation+patterns+in+the+semicircular+canals.">The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals.</a> Journal of Neurophysiology. 60 (1), 182-203 (1988).</li><li>Remedios, 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=Effects+of+Short-Term+Random+Noise+Electrical+Stimulation+on+Dissociated+Pyramidal+Neurons+from+the+Cerebral+Cortex.">Effects of Short-Term Random Noise Electrical Stimulation on Dissociated Pyramidal Neurons from the Cerebral Cortex.</a> Neuroscience. 404, 371-386 (2019).</li><li>Paxinos, G., Franklin, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2004).</li><li>Camp, A. J., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+synaptic+transmission+differs+in+mouse+type+A+and+B+medial+vestibular+nucleus+neurons+in+vitro.">Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro.</a> Journal of Neurophysiology. 95 (5), 3208-3218 (2006).</li><li>Camp, 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=Attenuated+glycine+receptor+function+reduces+excitability+of+mouse+medial+vestibular+nucleus+neurons.">Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.</a> Neuroscience. 170 (1), 348-360 (2010).</li><li>Iwasaki, S., 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=Effect+of+Noisy+Galvanic+Vestibular+Stimulation+on+Ocular+Vestibular-Evoked+Myogenic+Potentials+to+Bone-Conducted+Vibration.">Effect of Noisy Galvanic Vestibular Stimulation on Ocular Vestibular-Evoked Myogenic Potentials to Bone-Conducted Vibration.</a> Front in Neurology. 8, 26 (2017).</li><li>Goldberg, J., Smith, C. E., Fernandez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+discharge+regularity+and+responses+to+externally+applied+galvanic+currents+in+vestibular+nerve+afferents+of+the+squirrel+monkey.">Relation between discharge regularity and responses to externally applied galvanic currents in vestibular nerve afferents of the squirrel monkey.</a> Journal of Neurophysiology. 51 (6), 1236-1256 (1984).</li></ol>

电前庭刺激（GVS）对前庭系统的影响在人类体内已经突出3，13，23，豚鼠14，啮齿动物18和非人类灵长类动物24。然而，这些研究都没有评估电噪声对前庭系统中单个神经元敏感性的直接影响。在这里，我们演示了随机噪声首次在体外应用，直接应用于单个内向前庭核（MVN）?...

前庭（或平衡）系统通过整合听觉、自体、躯体感觉和视觉信息来控制我们的平衡感。前庭系统的退化已被证明是作为年龄的函数发生的，并可能导致平衡赤字1，2。然而，针对前庭系统功能的疗法很少。
电前庭刺激（GVS）已被证明改善平衡措施，自主功能和其他感官模式在人类3，4，5，6。这些改进据说是由于随机共振（SR）现象，即通过应用亚阈值噪声7，8来检测非线性系统中较弱的信号。这些研究表明，静态9、10和动态11、12平衡和前庭输出测试（如眼计数器卷（OCR）13）有所改善。然而，许多这些研究使用了不同的刺激参数组合，如白噪声9，彩色噪声13，不同的刺激频率范围和阈值技术。因此，最佳刺激参数仍未知，该协议有助于确定最有效的参数。除了刺激参数，刺激的类型在治疗和实验疗效也很重要。上述工作在人类中是使用电噪声刺激进行的，而大部分体内动物的工作都使用了机械14、15或光遗传学16噪声刺激。该协议将使用电噪声来检查对前庭核的影响。
此前，在松鼠猴体内进行了GVS刺激原发性前庭亲子手术，在松鼠猴17只、钦奇利亚18只、鸡胚胎15只和豚鼠14只身上进行了手术。然而，这些研究中只有两项研究审查了GVS对原位前庭14、15的增益的影响。这些实验是在体内进行的，这意味着不能确定前庭核上施加的确切刺激模式。据我们所知，只有另外一项研究将随机噪声应用于中枢神经系统中个别酶分离的神经元19。然而，在中央前庭核没有进行任何实验来评估适当的刺激参数和阈值技术，使得该协议在确定刺激作用对前庭内单个神经元的影响时更加精确核。
在这里，我们描述如何将正弦和随机（电）噪声直接应用于中前庭核（MVN）中的单个神经元，确定神经元阈值并测量增益/灵敏度的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl</td>
			<td>Scharlau</td>
			<td>CA01951000</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chem-supply</td>
			<td>PA054-500G</td>
			<td>Used for ACSF, sACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847-100G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187-500MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>Chem-supply</td>
			<td>MA00360500</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Na3-GTP</td>
			<td>Sigma</td>
			<td>G8877-100MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-supply</td>
			<td>SO02270500</td>
			<td>Use for ACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O</td>
			<td>Ajax</td>
			<td>AJA471-500G</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761-1KG</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-supply</td>
			<td>SA030-500G</td>
			<td>Used for sACSF</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>1169567762</td>
			<td>Used for anaesthetising mice</td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Warner instruments</td>
			<td>GC150T-7.5</td>
			<td>1.5mm OD, 1.16mm ID, 7.5cm length</td>
		</tr>
		<tr>
			<td>Data acquisition software</td>
			<td>Axograph</td>
			<td></td>
			<td>Used for electrophysiology and analysis</td>
		</tr>
		<tr>
			<td>Friedmen-Pearson Rongeurs</td>
			<td>World precision instruments</td>
			<td>14089</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td>Used for micropipette</td>
		</tr>
		<tr>
			<td>Multiclamp amplifier</td>
			<td>Axon instruments</td>
			<td>700B</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sper scientific</td>
			<td>860033</td>
			<td>Used for internal solution</td>
		</tr>
		<tr>
			<td>Standard pattern scissors</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Sutter micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225/M</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Used for slicing brain tissue</td>
		</tr>
	</tbody>
</table>

stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro

电前庭刺激（GVS）已被证明改善平衡措施的个人与平衡或前庭损伤。建议是由于随机共振 （SR） 现象，该现象被定义为将低电平/子阈值刺激应用于非线性系统，以增加对较弱信号的检测。然而，SR如何表现出其对人类平衡的积极影响，目前仍不得而知。这是正弦和随机噪声对单个神经元影响的首次演示之一。使用全细胞贴片夹电生理学，正弦噪声和随机噪声可以直接应用于C57BL/6小鼠的中庭前庭核（MVN）的单个神经元。在这里，我们演示如何确定 MVN 神经元的阈值，以确保正弦和随机刺激是亚阈值，并因此确定每种类型的噪声对 MVN 神经元增益的影响。结果表明，亚阈值正弦噪声和随机噪声可以调节MVN中单个神经元的灵敏度，而不影响基底发射速率。

初始记录可以提供有关正弦噪声对单个 MVN 神经元基底发射速率的影响以及刺激如何影响神经元增益的信息。图2显示，与对照（无噪声）记录相比，正弦噪声和随机噪声均无变化MVN神经元的基底发射速率。此信息对于确定单个神经元的阈值至关重要。在将电前庭刺激应用于人类过程中，执行感官阈值任务，以确保刺激是亚阈值13。亚阈值刺激...

Watch this Scientific Journal Video about Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro at JoVE.com

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

人类前庭刺激显示前庭功能改善。然而，不知道这些影响是如何发生的。在这里，我们描述如何应用正弦和随机电噪声，并评估C57BL/6小鼠中单个内庭核神经元中适当的刺激幅度。

随机噪声在Vitro内评估中庭前核神经元敏感性的随机噪声应用

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...

该协议是直接将电噪声应用到组织切片中单个神经元的第一批演示之一。为了观察神经元对电刺激的反应。这种技术的主要优点是，我们能够评估单个神经元上一组特定的刺激参数。此技术允许优化刺激参数，以特定功能神经元类型为目标。这对于开发更好的平衡功能治疗具有影响。在开始脑干提取之前，Equilibry s-ACSF 与 CARPOGEN 并冷却溶液在零下 80 摄氏度 25 分钟，直到冰浆形成。溶液冷却后，使用编号22圆剃刀刀片，使三至五周大的C-57黑色六只小鼠的头骨的下垂皮肤切口，并使用一对标准图案剪刀的尖端，使一个小切口，从羔羊，沿着下垂缝合。使用一对浅弯皮尔森冯朱尔斯，仔细反射去修复的肠外骨和外皮骨，并在冰冷的s-ACSF中沐浴大脑。然后，用11号直剃刀刀片将脑干从前脑和骨质包裹中分离开，以切割骨质外肠骨质肉和骨质包裹。将分离的脑干腹和下部安装到可移动切梯形聚苯乙烯块上，并使用一张纸巾从解剖组织周围去除多余的液体。使用氰丙烯酸酯胶水将聚苯乙烯块固定到切割阶段，并附着脑干 rostral 和向下。使用 0.16 毫米/秒的先进速度和 3 毫米的振动振幅获得 200 微米的 MVN 横向切片。然后使用塑料修剪移液器将组织切片转移到过滤纸盘上，在 25 摄氏度下将ACSF移植到过滤上，至少30分钟。对于全细胞贴片夹电生理学，首先拉微移液器的最终电阻范围从三到五兆欧姆时，充满钾为基础的内部溶液，并放置在浴缸。要从 MVN 中的单个神经元获取全细胞贴片夹记录，请将单个组织切片从孵化室转移到标准电生理设置的录音室，并使用 U 形重量上的尼龙螺纹固定切片。连续在 25 摄氏度的流量下用碳酸化 ACSF 在 25 摄氏度下连续灌装记录室，并用内部溶液填充微移液器。使用移液器施加少量正压，将碎屑从移液器尖端推开。使用低功耗目标，在切换到高功率以定位 MVN 内的单个神经元之前，找到 MVN。在用移液器破坏组织之前，施加少量正压，将碎屑从移液器尖端推开，并使用微操纵器将移液器移向所选神经元。神经膜上应形成一个小酒窝。释放正压，施加少量负压。一旦实现了一千兆欧姆密封，通过吸口对移液器支架施加柔和的短压和尖锐的负压，以破裂膜并创建全细胞配置。然后根据标准协议获得全单元电流夹具记录。要将随机噪声和正弦噪声应用于单个中前庭核神经元，请设置从 3 到 24 皮奥放大器的振幅范围，以确定神经元阈值和发射速率，并分组更高和更低的刺激强度来确定感觉阈值。接下来，计算 10 秒期间的平均发射速率，在此期间，将为每个单个电流级别注入极化电流步骤。使用平均触发速率值生成触发速率与当前绘图。然后执行线性回归分析，以确定最佳拟合线的梯度，以确定神经元增益。与控制无噪声记录相比，正弦噪声和随机噪声都未改变MVN神经元的罗勒发射速率。在此实验中，所选的噪声水平为 6 皮科安培是低于阈值的，因为可以观察到，平均发射速率开始从 12 皮科安培的实验阈值增加。此阈值通过将刺激水平分组到实验阈值上方和下方来客观地确定。神经元增益通过使神经元受制于一套从零到 50 皮奥安培的降极电流步数，以 10 皮奥安培的增量（带无噪声）进行评估。事实上，在六皮安培的子阈值振幅下施加的正弦噪声和随机噪声可以改变 MVN 神经元的增益。既定的刺激参数可应用于神经创新单个神经元，以提供更自然的刺激。

Research

JoVE Journal

Neuroscience

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

一个全面协议的内侧颞叶结构的手动分割

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

科研

神经科学

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

录音伽玛乐队在振荡脚桥核神经元

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

小鼠大脑细胞核巴沙里斯中胆碱能纤维长度的立体估计

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...