King's College London, Wolfson CARD, London, United Kingdom.
Nevro Corporation, Redwood City, CA, United States.
J Neurosci Methods. 2020 Jan 15;330:108479. doi: 10.1016/j.jneumeth.2019.108479. Epub 2019 Nov 6.
Using in and ex vivo preparations, electrophysiological methods help understand the excitability of biological tissue, particularly neurons, by providing microsecond temporal resolution. However, for in vivo recordings, in the context of extracellular recordings, it is often unclear precisely which type of neuron the tip of the electrode is recording from. This is particularly true in the densely-populated central nervous system, such as the spinal cord dorsal horn at both superficial and deep levels.
Here, we present a detailed protocol for the identification of superficial dorsal horn spinal cord neurons that receive peripheral input and project to the brain, using multiple surgical laminectomies and the careful placement of electrodes. Once a superficial projection unit was found, quantification to electrical peripheral stimulation was performed using a Matlab algorithm to form a template of projection neuron response to controlled C2 stimulation and accurately match this to the responses from peripheral stimulation.
These superficial spinal projection neurons are normally activated by noxious peripheral stimuli, so we adopted a well-characterised wind-up protocol to obtain a neuronal excitability profile. Once achieved, this protocol allows for testing specific interventions, either pharmacological or neuromodulatory (e.g., spinal cord stimulation) to see how these affect the neuron's excitability. This preparation is robust and allows the accurate tracking of a projection neuron for over 3-h.
COMPARISON WITH EXISTING METHOD(S): Currently, most existing methods record from dorsal horn neurons that are often profiled based on their excitability to different peripherally-applied sensory modalities. While this is well-established, it fails to discriminate between interneurons and projection neurons, which is important as these two populations signal via distinctly different neuronal networks. Using the approach detailed here will result in studies with improved mechanistic understanding of the signal integration and processing that occurs in the superficial dorsal horn.
The refinements detailed in this protocol allow for more comprehensive studies to be carried out that will help understand spinal plasticity, in addition to many considerations for isolating the relevant neuronal population when performing in vivo electrophysiology.
使用在体和离体标本，电生理方法通过提供微秒时间分辨率来帮助理解生物组织（尤其是神经元）的兴奋性。然而，对于在体记录，特别是在细胞外记录的情况下，电极尖端所记录的神经元类型往往并不清楚。在密集的中枢神经系统中，如浅层和深层脊髓背角，情况更是如此。
在这里，我们提出了一个详细的方案，使用多次椎板切除术和仔细放置电极，来鉴定接收外周输入并投射到大脑的浅层背角脊髓神经元。一旦发现一个浅层投射单位，就使用 Matlab 算法对电外周刺激进行量化，以形成投射神经元对受控 C2 刺激的反应模板，并准确地将其与外周刺激的反应相匹配。
这些浅层脊髓投射神经元通常被有害的外周刺激激活，因此我们采用了一种特征明确的“wind-up”方案来获得神经元兴奋性谱。一旦实现，该方案允许测试特定的干预措施，无论是药理学的还是神经调节的（例如，脊髓刺激），以观察这些措施如何影响神经元的兴奋性。该方案稳健，可准确跟踪投射神经元超过 3 小时。
目前，大多数现有的方法记录来自背角神经元，这些神经元通常根据其对不同外周感觉模态的兴奋性进行分类。虽然这已经得到了很好的证实，但它不能区分中间神经元和投射神经元，而这两种神经元通过截然不同的神经元网络进行信号传递，这一点很重要。使用这里详细描述的方法将导致进行更全面的研究，有助于理解浅层背角中的信号整合和处理机制，此外，在进行在体电生理学时，还有许多考虑因素需要隔离相关的神经元群体。
本方案详细描述的改进允许进行更全面的研究，有助于理解脊髓可塑性，此外，在进行在体电生理学时，还有许多考虑因素需要隔离相关的神经元群体。
