使用微电极阵列记录脊髓伤害感受回路中的网络活动
概要
概述了微电极阵列技术和4-氨基吡啶诱导的化学刺激相结合的使用，以研究脊髓背角的网络级伤害感受活性。
Abstract
脊髓背角（DH）内特定类型神经元的作用和连接性正在快速描绘，以提供支撑脊柱疼痛处理的电路的越来越详细的视图。然而，这些连接对DH中更广泛的网络活动的影响仍然不太清楚，因为大多数研究集中在单个神经元和小微电路的活动上。或者，使用微电极阵列（MEAs）可以监测许多细胞的电活动，提供神经活动的高空间和时间分辨率。在这里，描述了使用具有小鼠脊髓切片的MEA来研究由化学刺激4-氨基吡啶（4-AP）的DH回路诱导的DH活性。由此产生的节律活性仅限于浅表DH，随时间稳定，被河豚毒素阻断，并且可以在不同的切片方向上进行研究。总之，该制剂提供了一个平台来研究来自幼稚动物，慢性疼痛动物模型和具有遗传改变的伤害感受功能的小鼠组织中的DH回路活性。此外，4-AP刺激脊髓切片中的MEA记录可用作快速筛选工具，以评估新型抗伤害感受化合物破坏脊髓DH活性的能力。
Introduction
特定类型的抑制性和兴奋性中间神经元在脊髓 DH 中的作用正在以1，2，3，4 的速度被发现。中间神经元共同占DH中神经元的95%以上，并参与感觉处理，包括伤害感受。此外，这些中间神经元回路对于确定周围信号是否上升神经轴到达大脑并有助于疼痛感知5，6，7非常重要。迄今为止，大多数研究已经使用体外细胞内电生理学，神经解剖学标记和体内行为分析1，3，8，9，10，11，12，13，14的组合，研究了DH神经元在单细胞或全生物体水平上的作用。.这些方法显着推进了对特定神经元群体在疼痛处理中的作用的理解。然而，在理解特定细胞类型和小宏观回路如何在微电路水平上影响大量神经元以随后塑造DH的输出，行为反应和疼痛体验方面仍然存在差距。
可以研究宏电路或多细胞级功能的一种技术是微电极阵列（MEA）15，16。几十年来，MEAs一直用于研究神经系统功能17，18.在大脑中，它们促进了神经元发育，突触可塑性，药理筛选和毒性测试的研究17，18。它们可用于 体外 和 体内 应用，具体取决于MEA的类型。此外，多边环境协定的发展发展迅速，现在有不同的电极编号和配置19.MEAs的一个关键优点是它们能够通过多个电极同时评估许多神经元的电活动，具有很高的空间和时间精度15，16。这提供了神经元如何在控制条件下以及在局部应用的化合物存在下在电路和网络中相互作用的更广泛读数。
体外DH制剂的一个挑战是持续的活性水平通常较低。在这里，使用电压门控K +通道阻滞剂4-氨基吡啶（4-AP）在脊髓DH电路中解决了这一挑战，以化学刺激DH电路。该药物先前已用于在急性脊髓切片的DH中以及在急性体内条件下建立节律性同步电活动20，21，22，23，24。这些实验使用单细胞贴片和细胞外记录或钙成像来表征4-AP诱导的活性20，21，22，23，24，25。总之，这项工作已经证明了兴奋性和抑制性突触传递和电突触对于有节奏的4-AP诱导活性的需求。因此，4-AP反应被视为一种揭示具有生物学相关性的天然多突触DH回路的方法，而不是药物诱导的偶发现象。此外，4-AP诱导的活性表现出与神经性疼痛状况相似的镇痛和抗癫痫药物的反应谱，并已被用于提出新的基于脊柱的镇痛药物靶标，例如连接蛋白20，21，22。
这里描述了一种将MEA和脊柱DH的化学活化与4-AP相结合的制剂，以在宏观回路或网络水平分析中研究这种伤害性回路。这种方法为研究幼稚和神经性“疼痛样”条件下的伤害性回路提供了一个稳定且可重复的平台。该制剂也容易地应用于测试已知镇痛药的回路级作用，并在过度活跃的脊髓中筛选新型镇痛药。
Protocol
对3-12个月的雄性和雌性c57Bl / 6小鼠进行了研究。所有实验程序均按照纽卡斯尔大学动物护理和伦理委员会（协议A-2013-312和A-2020-002）进行。 1. 体外 电生理学 脊髓切片制备和记录溶液的制备 人工脑脊液注意：人工脑脊液（aCSF）用于界面培养室，切片被储存直到记录开始和实验期间作为药物的灌注液和稀释剂。详细组成见 表1 。 </l…
Representative Results
脊髓背角网络活动模型4-AP的应用可靠地诱导脊髓DH中的同步节律活动。这些活动表现为EAP和LFPs增加。后面的信号是低频波形，这在之前的MEA记录30中已经描述过。药物应用后EAP和/或LFP活性的变化反映了神经活动的改变。EAP 和 LFP 的示例如图 3B 和图 4 所示。这里的重点是EAP / LFP数据的以下参数或特征：频率，总数，活?…
Discussion
尽管脊髓DH在伤害性信号传导，处理以及由此产生的表征疼痛的行为和情绪反应中的重要性，但该区域内的回路仍然知之甚少。研究这个问题的一个关键挑战是构成这些电路的神经元群体的多样性6，31，32。以光遗传学和化学遗传学为首的转基因技术的最新进展开始解开这些重要的联系，并定义了处理感官信息的微电路<sup class=…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作由澳大利亚国家卫生和医学研究委员会（NHMRC）资助（拨款631000，1043933，1144638，1184974给B.A.G.和R.J.C.）和亨特医学研究所（拨款给B.A.G.和R.J.C.）。
Materials
| 4-aminopyridine | Sigma-Aldrich | 275875-5G | |
| 100% ethanol | Thermo Fisher | AJA214-2.5LPL | |
| CaCl2 1M | Banksia Scientific | 0430/1L | |
| Carbonox (Carbogen – 95% O2, 5% CO2) | Coregas | 219122 | |
| Curved long handle spring scissors | Fine Science Tools | 15015-11 | |
| Custom made air interface incubation chamber | |||
| Foetal bovine serum | Thermo Fisher | 10091130 | |
| Forceps Dumont #5 | Fine Science Tools | 11251-30 | |
| Glucose | Thermo Fisher | AJA783-500G | |
| Horse serum | Thermo Fisher | 16050130 | |
| Inverted microscope | Zeiss | Axiovert10 | |
| KCl | Thermo Fisher | AJA383-500G | |
| Ketamine | Ceva | KETALAB04 | |
| Large surgical scissors | Fine Science Tools | 14007-14 | |
| Loctite 454 Instant Adhesive | Bolts and Industrial Supplies | L4543G | |
| MATLAB | MathWorks | R2018b | |
| MEAs, 3-Dimensional | Multichannel Systems | 60-3DMEA100/12/40iR-Ti, 60-3DMEA200/12/50iR-Ti | 60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in an 8×8 square grid. Electrodes are 12 µm in diameter, 40 µm (100/12/40) or 50 µm (200/12/50) high and equidistantly spaced 100 µm (100/12/40) or 200 µm (200/12/50) apart. |
| MEA headstage | Multichannel Systems | MEA2100-HS60 | |
| MEA interface board | Multichannel Systems | MCS-IFB 3.0 Multiboot | |
| MEA net | Multichannel Systems | ALA HSG-MEA-5BD | |
| MEA perfusion system | Multichannel Systems | PPS2 | |
| MEAs, Planar | Multichannel Systems | 60MEA200/30iR-Ti, 60MEA500/30iR-Ti | 60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in either a 8×8 square grid (200/30) or a 6×10 rectangular grid (500/30). Electrodes are 30 µm in diameter and equidistantly spaced 200 µm (200/30) or 500 µm (500/30) apart. |
| MgCl2 | Thermo Fisher | AJA296-500G | |
| Microscope camera | Motic | Moticam X Wi-Fi | |
| Multi Channel Analyser software | Multichannel Systems | V 2.17.4 | |
| Multi Channel Experimenter software | Multichannel Systems | V 2.17.4 | |
| NaCl | Thermo Fisher | AJA465-500G | |
| NaHCO3 | Thermo Fisher | AJA475-500G | |
| NaH2PO4 | Thermo Fisher | ACR207805000 | |
| Rongeurs | Fine Science Tools | 16021-14 | |
| Small spring scissors | Fine Science Tools | 91500-09 | |
| Small surgical scissors | Fine Science Tools | 14060-09 | |
| Sucrose | Thermo Fisher | AJA530-500G | |
| Superglue | cyanoacrylate adhesive | ||
| Tetrodotoxin | Abcam | AB120055 | |
| Vibration isolation table | Newport | VH3048W-OPT | |
| Vibrating microtome | Leica | VT1200 S |
参考文献
- Smith, K. M., et al. Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn. eLife. 8, 49190 (2019).
- Smith, K. M., et al. Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing. The Journal of physiology. 593 (19), 4319-4339 (2015).
- Boyle, K. A., et al. Defining a spinal microcircuit that gates myelinated afferent input: Implications for tactile allodynia. Cell Reports. 28 (2), 526-540 (2019).
- Browne, T. J., et al. Transgenic cross-referencing of inhibitory and excitatory interneuron populations to dissect neuronal heterogeneity in the dorsal horn. Frontiers in Molecular Neuroscience. 13, 32 (2020).
- Graham, B. A., Hughes, D. I. Rewards, perils and pitfalls of untangling spinal pain circuits. Current Opinion in Physiology. 11, 35-41 (2019).
- Todd, A. J. Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews Neuroscience. 11 (12), 823-836 (2010).
- Hughes, D. I., Todd, A. J. Central nervous system targets: inhibitory interneurons in the spinal cord. Neurotherapeutics. 17 (3), 874-885 (2020).
- Duan, B., et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell. 159 (6), 1417-1432 (2014).
- Hachisuka, J., Chiang, M. C., Ross, S. E. Itch and neuropathis itch. Pain. 159 (3), 603 (2018).
- Foster, E., et al. Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch. Neuron. 85 (6), 1289-1304 (2015).
- Bourane, S., et al. Identification of a spinal circuit for light touch and fine motor control. Cell. 160 (3), 503-515 (2015).
- Cheng, L., et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nature neuroscience. 20 (6), 804-814 (2017).
- Peirs, C., et al. Mechanical allodynia circuitry in the dorsal horn is defined by the nature of the injury. Neuron. 109 (1), 73-90 (2021).
- Huang, J., et al. Circuit dissection of the role of somatostatin in itch and pain. Nature Neuroscience. 21 (5), 707-716 (2018).
- Obien, M. E. J., Deligkaris, K., Bullmann, T., Bakkum, D. J., Frey, U. Revealing neuronal function through microelectrode array recordings. Frontiers in Neuroscience. 8, 423 (2015).
- Nam, Y., Wheeler, B. C. In vitro microelectrode array technology and neural recordings. Critical Reviews in Biomedical Engineering. 39 (1), 45-61 (2011).
- Johnstone, A. F., et al. Microelectrode arrays: a physiologically based neurotoxicity testing platform for the 21st century. Neurotoxicology. 31 (4), 331-350 (2010).
- Stett, A., et al. Biological application of microelectrode arrays in drug discovery and basic research. Analytical and Bioanalytical Chemistry. 377 (3), 486-495 (2003).
- Xu, L., et al. Trends and recent development of the microelectrode arrays (MEAs). Biosensors and Bioelectronics. 175 (1), 112854 (2020).
- Chapman, R. J., Cilia La Corte, P. F., Asghar, A. U. R., King, A. E. Network-based activity induced by 4-aminopyridine in rat dorsal horn in vitro is mediated by both chemical and electrical synapses. The Journal of Physiology. 587, 2499-2510 (2009).
- Ruscheweyh, R., Sandkühler, J. Epileptiform activity in rat spinal dorsal horn in vitro has common features with neuropathic pain. Pain. 105 (1-2), 327-338 (2003).
- Kay, C. W., Ursu, D., Sher, E., King, A. E. The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro. Physiological Reports. 4 (14), 12852 (2016).
- Jankowska, E., Lundberg, A., Rudomin, P., Sykova, E. Effects of 4-aminopyridine on synaptic transmission in the cat spinal cord. Brain Research. 240 (1), 117-129 (1982).
- Semba, K., Geller, H. M., Egger, M. D. 4-Aminopyridine induces expansion of cutaneous receptive fields of dorsal horn cells. Brain Research. 343 (2), 398-402 (1985).
- Ruscheweyh, R., Sandkühler, J. Long-range oscillatory Ca2+ waves in rat spinal dorsal horn. European Journal of Neuroscience. 22 (8), 1967-1976 (2005).
- Egert, U., et al. A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays. Brain Research Protocols. 2 (4), 229-242 (1998).
- Thiebaud, P., De Rooij, N., Koudelka-Hep, M., Stoppini, L. Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures. IEEE Transactions on Biomedical Engineering. 44 (11), 1159-1163 (1997).
- Rey, H. G., Pedreira, C., Quiroga, R. Q. Past, present and future of spike sorting techniques. Brain Research Bulletin. 119, 106-117 (2015).
- Satuvuori, E., et al. Measures of spike train synchrony for data with multiple time scales. Journal of Neuroscience Methods. 287, 25-38 (2017).
- Mendis, G. D. C., Morrisroe, E., Reid, C. A., Halgamuge, S. K., Petrou, S. Use of local field potentials of dissociated cultures grown on multi-electrode arrays for pharmacological assays. 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. , 952-956 (2016).
- Hughes, D. I., et al. Morphological, neurochemical and electrophysiological features of parvalbumin-expressing cells: a likely source of axo-axonic inputs in the mouse spinal dorsal horn. The Journal of Physiology. 590 (16), 3927-3951 (2012).
- Peirs, C., Seal, R. P. Neural circuits for pain: recent advances and current views. Science. 354 (6312), 578-584 (2016).
- Li, J., Baccei, M. L. Developmental regulation of membrane excitability in rat spinal lamina I projection neurons. Journal of Neurophysiology. 107 (10), 2604-2614 (2012).
- Li, J., Baccei, M. L. Pacemaker neurons within newborn spinal pain circuits. Journal of Neuroscience. 31 (24), 9010-9022 (2011).
- Sandkühler, J., Eblen-Zajjur, A. Identification and characterization of rhythmic nociceptive and non-nociceptive spinal dorsal horn neurons in the rat. 神経科学. 61 (4), 991-1006 (1994).
- Lucas-Romero, J., Rivera-Arconada, I., Roza, C., Lopez-Garcia, J. A. Origin and classification of spontaneous discharges in mouse superficial dorsal horn neurons. Scientific Reports. 8 (1), 9735-9735 (2018).
- Antonio, L., et al. L. al. In vitro seizure like events and changes in ionic concentration. Journal of Neuroscience Methods. 260, 33-44 (2016).
- Avoli, M., Jefferys, J. G. Models of drug-induced epileptiform synchronization in vitro. Journal of Neuroscience Methods. 260, 26-32 (2016).
- Taccola, G., Nistri, A. Low micromolar concentrations of 4-aminopyridine facilitate fictive locomotion expressed by the rat spinal cord in vitro. 神経科学. 126 (2), 511-520 (2004).
- Mitra, P., Brownstone, R. M. An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse. Journal of Neurophysiology. 107 (2), 728-741 (2012).
- Egert, U., Heck, D., Aertsen, A. Two-dimensional monitoring of spiking networks in acute brain slices. Experimental Brain Research. 142 (2), 268-274 (2002).
記事を引用
Iredale, J. A., Stoddard, J. G., Drury, H. R., Browne, T. J., Elton, A., Madden, J. F., Callister, R. J., Welsh, J. S., Graham, B. A. Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays. J. Vis. Exp. (180), e62920, doi:10.3791/62920 (2022).