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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 保持小鼠正常12小时光/暗循环与环境富集。 使用雄性和雌性C57BL/6小鼠（3~5周大）进行所有实验。 2. 准?…
Representative Results
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…
Discussion
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）?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SPS 得到了悉尼大学研究生研究奖学金的支持。
Materials
| CaCl | Scharlau | CA01951000 | Used for ACSF and sACSF |
| D-(+)-Glucose | Sigma | G8270 | Used for ACSF and sACSF |
| EGTA | Sigma | E0396-25G | Used for K-based intracellular solution |
| HEPES | Sigma | H3375-25G | Used for K-based intracellular solution |
| KCl | Chem-supply | PA054-500G | Used for ACSF, sACSF and intracellular solution |
| K-gluconate | Sigma | P1847-100G | Used for K-based intracellular solution |
| Mg-ATP | Sigma | A9187-500MG | Used for K-based intracellular solution |
| MgCl | Chem-supply | MA00360500 | Used for ACSF and sACSF |
| Na3-GTP | Sigma | G8877-100MG | Used for K-based intracellular solution |
| NaCl | Chem-supply | SO02270500 | Use for ACSF and intracellular solution |
| NaH2PO4.2H2O | Ajax | AJA471-500G | Used for ACSF and sACSF |
| NaHCO3 | Sigma | S5761-1KG | Used for ACSF and sACSF |
| Sucrose | Chem-supply | SA030-500G | Used for sACSF |
| Isoflurane | Henry Schein | 1169567762 | Used for anaesthetising mice |
| EQUIPMENT | |||
| Borosilicate glass capillaries | Warner instruments | GC150T-7.5 | 1.5mm OD, 1.16mm ID, 7.5cm length |
| Data acquisition software | Axograph | Used for electrophysiology and analysis | |
| Friedmen-Pearson Rongeurs | World precision instruments | 14089 | Used for dissection |
| Micropipette puller | Narishige | PP-830 | Used for micropipette |
| Multiclamp amplifier | Axon instruments | 700B | Used for electrophysiology |
| pH meter | Sper scientific | 860033 | Used for internal solution |
| Standard pattern scissors | FST | 14028-10 | Used for dissection |
| Sutter micromanipulator | Sutter | MP-225/M | Used for electrophysiology |
| Upright microscope | Olympus | BX51WI | Used for electrophysiology |
| Vibratome | Leica | VT1200 | Used for slicing brain tissue |
References
- Amiridis, I. G., Hatzitaki, V., Arabatzi, F. Age-induced modifications of static postural control in humans. Neuroscience Letters. 350 (3), 137-140 (2003).
- Iwasaki, S., Yamasoba, T. Dizziness and imbalance in the elderly: age-related decline in the vestibular system. Aging and disease. 6 (1), (2015).
- Fujimoto, C., et al. Noisy galvanic vestibular stimulation induces a sustained improvement in body balance in elderly adults. Scientific Reports. 6, 37575 (2016).
- Breen, P. P., et al. Peripheral tactile sensory perception of older adults improved using subsensory electrical noise stimulation. Medical Engineering & Physics. 38 (8), 822-825 (2016).
- Yamamoto, Y., Struzik, Z. R., Soma, R., Ohashi, K., Kwak, S. Noisy vestibular stimulation improves autonomic and motor responsiveness in central neurodegenerative disorders. Annals of Neurology. 58 (2), 175-181 (2005).
- Soma, R., Nozaki, D., Kwak, S., Yamamoto, Y. 1/f noise outperforms white noise in sensitizing baroreflex function in the human brain. Physical Review Letters. 91 (7), 078101 (2003).
- Wiesenfeld, K., Moss, F. Stochastic resonance and the benefits of noise: from ice ages to crayfish and SQUIDs. Nature. 373 (6509), 33-36 (1995).
- Moss, F., Ward, L. M., Sannita, W. G. Stochastic resonance and sensory information processing: a tutorial and review of application. Clinical Neurophysiology. 115 (2), 267-281 (2004).
- Goel, R., et al. Using low levels of stochastic vestibular stimulation to improve balance function. PloS one. 10 (8), e0136335 (2015).
- Inukai, Y., et al. Effect of noisy galvanic vestibular stimulation on center of pressure sway of static standing posture. Brain Stimulation: Basic, Translational, and Clinical Research in Neuromodulation. 11 (1), 85-93 (2018).
- Mulavara, A. P., et al. Using low levels of stochastic vestibular stimulation to improve locomotor stability. Frontiers in Systems Neuroscience. 9, 117 (2015).
- Iwasaki, S., et al. Noisy vestibular stimulation increases gait speed in normals and in bilateral vestibulopathy. Brain stimulation. 11 (4), 709-715 (2018).
- Serrador, J. M., Deegan, B. M., Geraghty, M. C., Wood, S. J. Enhancing vestibular function in the elderly with imperceptible electrical stimulation. Scientific Reports. 8 (1), 336 (2018).
- Kim, J., Curthoys, I. S. Responses of primary vestibular neurons to galvanic vestibular stimulation (GVS) in the anaesthetised guinea pig. Brain Research Bulletin. 64 (3), 265-271 (2004).
- Flores, A., et al. Stochastic resonance in the synaptic transmission between hair cells and vestibular primary afferents in development. Neuroscience. 322, 416-429 (2016).
- Huidobro, N., et al. Brownian Optogenetic-Noise-Photostimulation on the Brain Amplifies Somatosensory-Evoked Field Potentials. Frontiers in Neuroscience. 11, 464-464 (2017).
- Goldberg, J., Ferna, C., Smith, C. Responses of vestibular-nerve afferents in the squirrel monkey to externally applied galvanic currents. Brain Research. 252 (1), 156-160 (1982).
- Baird, R., Desmadryl, G., Fernandez, C., Goldberg, J. The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals. Journal of Neurophysiology. 60 (1), 182-203 (1988).
- Remedios, L., et al. Effects of Short-Term Random Noise Electrical Stimulation on Dissociated Pyramidal Neurons from the Cerebral Cortex. Neuroscience. 404, 371-386 (2019).
- Paxinos, G., Franklin, K. B. . The mouse brain in stereotaxic coordinates. , (2004).
- Camp, A. J., Callister, R. J., Brichta, A. M. Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro. Journal of Neurophysiology. 95 (5), 3208-3218 (2006).
- Camp, A., et al. Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons. Neuroscience. 170 (1), 348-360 (2010).
- Iwasaki, S., et al. Effect of Noisy Galvanic Vestibular Stimulation on Ocular Vestibular-Evoked Myogenic Potentials to Bone-Conducted Vibration. Front in Neurology. 8, 26 (2017).
- Goldberg, J., Smith, C. E., Fernandez, C. Relation between discharge regularity and responses to externally applied galvanic currents in vestibular nerve afferents of the squirrel monkey. Journal of Neurophysiology. 51 (6), 1236-1256 (1984).
Cite This Article
Stefani, S. P., Breen, P. P., Serrador, J. M., Camp, A. J. Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro. J. Vis. Exp. (150), e60044, doi:10.3791/60044 (2019).