实时电：采用闭环协议来探测神经动力学和超越
Summary
Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.
Abstract
实验神经学正在经历的发展和应用新颖的和往往是复杂的，闭环协议，其中的刺激施加实时取决于系统的响应的兴趣增加。最近的应用范围从虚拟现实系统的实施为研究运动 反应无论是在小鼠1和2斑马鱼，以控制使用以下3光遗传学皮质中风发作。闭环技术的关键优点在于，探测更高维属性不能直接访问或依赖于多个变量，如神经元兴奋4和可靠性，而在同一时间最大化实验吞吐量的能力。在这方面的贡献，并在细胞电的情况下，我们将描述如何各种闭环协议适用于锥体皮层神经元，REC响应特性的研究在从幼年大鼠躯体感觉皮层急性脑片膜片钳技术orded细胞内。由于没有市售或开源软件提供了所有必需的用于高效执行这里所描述的实验的特征，开发了一种新的软件工具箱称为LCG 5，其模块化结构最大化的计算机代码的重用，并且容易实现新的实验范例的实施。刺激波形使用的是紧凑元说明中指定和完整实验方案在基于文本的配置文件中描述。此外，LCG具有适合于反复试验和实验规程自动化的命令行界面。
Introduction
近年来，细胞电已从在电压和电流钳实验采用现代闭环协议传统的开环模式演变而来。最有名的闭环技术也许是动态钳6,7，这使合成注射人工电压门控离子通道，以确定神经元膜电压8中的非确定性闪烁的影响的深入研究离子通道在神经反应动力学9，以及娱乐的现实体外接近体内的突触背景活动10。
已经提出的其它闭环范例包括反应性夹具11，以研究在体外自我维持的持久性活动的生成，并且响应夹紧4,12，调查细胞机制底层神经元兴奋。
内容】“>这里，我们描述了允许应用各种闭环电协议中的急性脑切片进行全细胞膜片钳记录的上下文中，功能强大的框架。我们将展示如何通过膜片钳记录装置来记录体膜电压从幼年大鼠的躯体感觉皮层和锥体神经元使用申请LCG，在理论和神经生物学的神经工程实验室开发的一个命令行的软件工具箱三种不同的闭环协议。简要地，所描述的协议中，首先在自动注射的一系列电流钳刺激波，相关的一大组有源和无源膜性能的表征。这些已被建议来捕获的细胞的电生理学表型在其响应性质方面至刻板一系列刺激波形。称为小区的电子代码 （ 例如 ，参见＆＃160; 13,14），电反应这样的集合所使用的几个实验室的它们的电学性能的基础上，以客观地分类的神经元。这包括在固定输入输出传递关系（FI曲线），通过创新的技术，包括通过一个比例 – 积分 – 微分（PID）控制器的装置触发速率的闭环，实时控制的分析的，现实的体内样背景突触活动的第二体外制剂10和，由虚拟GABA能interneuron，这是由计算机模拟的装置在两个同时记录锥体细胞的实时第三人工连接的娱乐。
此外，LCG实现了被称为活跃的补偿电极（AEC）15，它允许使用实现单电极钳动态协议技术。这使得补偿的不良影响（一当它被用于输送细胞内的刺激出现的记录电极的rtifacts）。该方法是基于在记录电路的等效电特性的非参数估计。
在本文中描述的技术和实验方案可以在常规开环电压和电流钳实验可容易地提供，并且可以延伸到其他制剂，如胞外4,16或细胞内记录体内 17,18。设置为全细胞膜片钳电的精心组装是稳定的，高品质的录音非常重要的一步。在下文中，我们假设这样一个实验装置已经提供给实验者，和我们的注意力集中于描述LCG的使用。请读者指出，19-22关于优化和调试的其他提示。
Protocol
Representative Results
Discussion
在该文本的完整协议的实时实施，闭环单细胞电生理实验进行了说明，使用膜片钳技术和最近开发的软件工具箱称为LCG。要优化录音的质量是至关重要的录音设置正确接地，屏蔽和无振动：这可确保稳定持久的全细胞进入细胞，这与自动化的刺激方案整个路段的可能性在一起允许对实验的吞吐量最大化。
两种情况，即LCG可以应用已经提出，一个单元的即在其电生理特性（ <str…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Financial support from the Flanders Research Foundation FWO (contract n. 12C9112N to DL), the 7th Framework Programme of the European Commission (Marie Curie Network “C7”, contract n. 238214; ICT Future Emerging Technology “ENLIGHTENMENT” project, contract n. 306502), the Interuniversity Attraction Poles Program initiated by the Belgian Science Policy Office (contract n. IUAP-VII/20), and the University of Antwerp is kindly acknowledged.
Materials
| Tissue slicer | Leica | VT-1000S | |
| Pipette puller | Sutter | P-97 | |
| Pipettes | WPI | 1B150F-4 | 1.5/0.84 mm OD/ID, with filament |
| Vibration isolation table | TMC | 20 Series | |
| Microscope | Leica | DMLFS | 40X Immersion Objective |
| Manipulators | Scientifica | PatchStar | |
| Amplifiers | Axon Instruments | MultiClamp 700B | Computer controlled |
| Data acquisition card | National Instruments | PCI-6229 | Supported by Comedi Linux Drivers |
| Desktop computer | Dell | Optiplex 7010 Tower | OS: real-time Linux |
| Oscilloscopes | Tektronix | TDS-1002 | |
| Perfusion Pump | Gibson | MINIPULS3 | Used with R4 Pump head (F117606) |
| Temperature controller | Multichannel Systems | TC02 | PH01 Perfusion Cannula |
| Manometer | Testo | 510 | Optional |
| Incubator | Memmert | WB14 | |
| NaCl | Sigma | 71376 | ACSF |
| KCl | Sigma | P9541 | ACSF, ICS |
| NaH2PO4 | Sigma | S3139 | ACSF |
| NaHCO3 | Sigma | S6014 | ACSF |
| CaCl2 | Sigma | C1016 | ACSF |
| MgCl2 | Sigma | M8266 | ACSF |
| Glucose | Sigma | G7528 | ACSF |
| K-Gluconate | Sigma | G4500 | ICS |
| HEPES | Sigma | H3375 | ICS |
| Mg-ATP | Sigma | A9187 | ICS |
| Na2-GTP | Sigma | 51120 | ICS |
| Na2-Phosphocreatine | Sigma | P7936 | ICS |
References
- Saleem, A. B., Ayaz, A., Jeffery, K. J., Harris, K. D., Carandini, M. Integration of visual motion and locomotion in mouse visual cortex. Nature neuroscience. 16, 1864-1869 (2013).
- Ahrens, M. B., Li, J. M., et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature. 485 (7399), 471-477 (2012).
- Paz, J. T., Davidson, T. J., et al. Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury. Nature neuroscience. 16 (1), 64-70 (2013).
- Wallach, A., Eytan, D., Gal, A., Zrenner, C., Marom, S. Neuronal response clamp. Frontiers in neuroengineering. 3 (April), 3 (2011).
- Linaro, D., Couto, J., Giugliano, M. Command-line cellular electrophysiology for conventional and real-time closed-loop experiments. Journal of neuroscience. 230, 5-19 (2014).
- Sharp, A., O’Neil, M., Abbott, L. F., Marder, E. Dynamic clamp: computer-generated conductances in real neurons. Journal of neurophysiology. 69 (3), 992-995 (1993).
- Robinson, H. P., Kawai, N. Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons. Journal of neuroscience methods. 49 (3), 157-165 (1993).
- Vervaeke, K., Hu, H., Graham, L. J., Storm, J. F. Contrasting effects of the persistent Na+ current on neuronal excitability and spike timing. Neuron. 49 (2), 257-270 (2006).
- White, J. A., Klink, R., Alonso, A., Kay, A. R. Noise from voltage-gated ion channels may influence neuronal dynamics in the entorhinal cortex. Journal of neurophysiology. 80 (1), 262-269 (1998).
- Destexhe, a., Rudolph, M., Fellous, J. M., Sejnowski, T. J. Fluctuating synaptic conductances recreate in vivo-like activity in neocortical neurons. 신경과학. 107 (1), 13-24 (2001).
- Fellous, J. -. M. Regulation of Persistent Activity by Background Inhibition in an In Vitro Model of a Cortical Microcircuit. Cerebral Cortex. 13 (11), 1232-1241 (2003).
- Gal, A., Eytan, D., Wallach, A., Sandler, M., Schiller, J., Marom, S. Dynamics of excitability over extended timescales in cultured cortical neurons. The Journal of neuroscience. the official journal of the Society for Neuroscience. 30 (48), 16332-16342 (2010).
- Wang, Y., Toledo-Rodriguez, M., et al. Anatomical, physiological and molecular properties of Martinotti cells in the somatosensory cortex of the juvenile rat. The Journal of physiology. 561 (Pt 1), 65-90 (2004).
- Wang, Y., Gupta, A., Toledo-Rodriguez, M., Wu, C. Z., Markram, H. Anatomical, physiological, molecular and circuit properties of nest basket cells in the developing somatosensory cortex). Cerebral cortex (New York, N.Y). 12 (4), 395-410 (1991).
- Brette, R., Piwkowska, Z., et al. High-resolution intracellular recordings using a real-time computational model of the electrode. Neuron. 59 (3), 379-391 (2008).
- Rutishauser, U., Kotowicz, A., Laurent, G. A method for closed-loop presentation of sensory stimuli conditional on the internal brain-state of awake animals. Journal of neuroscience. 215 (1), 139-155 (2013).
- Margrie, T., Brecht, M., Sakmann, B. In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Archiv European Journal of Physiology. 444 (4), 491-498 (2002).
- Graham, L., Schramm, A. In Vivo Dynamic-Clamp Manipulation of Extrinsic and Intrinsic Conductances: Functional Roles of Shunting Inhibition and I BK in Rat and Cat Cortex. Dynamic Clamp: From Principles to Applications. , (2008).
- Sakmann, B., Neher, E. . Single-channel recording. , (1995).
- Molleman, A. . Patch Clamping. , (2002).
- Davie, J. T., Kole, M. H. P., et al. Dendritic patch-clamp recording. Nature Protocols. 1 (3), 1235-1247 (2006).
- Gold, R. . The Axon Guide for Electrophysiolog., & Biophysics Laboratory Techniques... , (2007).
- Mainen, Z. F., Sejnowski, T. J. Reliability of spike timing in neocortical neurons. Science. 268 (5216), 1503-1506 (1995).
- Buzsáki, G. Action potential threshold of hippocampal pyramidal cells in vivo is increased by recent spiking activity. 신경과학. 105 (1), 121-130 (2001).
- Koch, C., Segev, I. . Methods in Neuronal Modeling: From Synapses to Networks. , (1988).
- Silberberg, G., Markram, H. Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells. Neuron. 53 (5), 735-746 (2007).
- Berger, T. K., Silberberg, G., Perin, R., Markram, H. Brief bursts self-inhibit and correlate the pyramidal network. PLoS biology. 8 (9), (2010).
- Tsodyks, M., Pawelzik, K., Markram, H. Neural networks with dynamic synapses. Neural computation. 10 (4), 821-835 (1998).
- Kapfer, C., Glickfeld, L. L., Atallah, B. Supralinear increase of recurrent inhibition during sparse activity in the somatosensory cortex. Nature. 10 (6), 743-753 (2007).
