等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em>在体内</em
Summary
在此过程中生理相关的脑状态和正在进行的电活动完全取消后，执行从单个神经元长期在体内的细胞内记录，导致大脑电状态。动物的生理常数过渡到人工昏迷状态期间仔细监测。
Abstract
顺便神经元过程信息既取决于它们的内在膜特性和对传入突触网络的动态。特别是，内源性产生的网络活动，这强烈变化作为警戒的状态的函数，显著调制神经元的计算。探讨自发性脑动态如何影响不同的单个神经元“综合性能，我们开发了包括由高剂量戊巴比妥钠的全身注射的方式在体内抑制所有大脑活动的老鼠了新的实验策略。皮质活动，通过组合电图（脑电图）和细胞内记录连续监测正在逐渐放慢，从而导致稳定的等电轮廓。这种极端的脑的状态，把老鼠进入深度昏迷，小心地通过在整个实验测量动物的生理常数监视。细胞内řecordings允许我们表征和比较嵌入到生理学相关皮质动力学，如在睡眠 – 觉醒周期遇到了同样的神经元的综合性能，并且当大脑完全沉默。
Introduction
在没有任何环境刺激或行为的任务中，“静止”大脑产生可从头皮进行记录的电活动的连续流，如脑电图（EEG）波。此内源性脑活动的细胞内相关成分的特征在于，背景膜的电压波动（也称为“突触噪声”），这是由反映传入网络1,2的正在进行的活动兴奋性和抑制性突触电位的组合。这种自发活动的频率和振幅与警觉的不同状态而变化。阐明网络活动的单神经元的兴奋性和反应能力的影响是神经科学的3,4的主要挑战之一。
许多实验和计算研究探索持续突触活动对综合propertie功能的影响神经细胞的的。然而，由背景突触噪声影响不同的神经元的参数的作用仍然是难以捉摸的。例如，膜去极化的平均等级已经发现与感觉输入触发的动作电位的能力正5,6或负7-9相关。此外，虽然一些调查表明，膜电位的波动，从传入的突触输入的连续变化的数据流得到的，强烈地受到调制的其输入输出关系3,10-13增益影响单个神经元的响应，其他指示通过分流抑制介导的细胞膜输入电导变化是足以调节神经元增益无论膜的波动14,15的幅度。最后，在清醒的动物进行最近的研究强调如何在单个神经元感觉信息的处理需要依靠警惕一个的状态ND当前的行为需求16,17。
阐明一个给定的处理的在高度互连的系统中的功能性作用一个直接的策略是，以确定如何其缺乏特异性改变系统的运作。该方法已在神经科学研究利用实验病变或不同脑区18-21的灭活，或特定的离子通道22,23的药理学阻断广泛使用，例如。值得注意的是，已经在体内施加到推出功能连接和网络动态如何影响单细胞计算24-27。然而，迄今为止本地操作旨在阻断神经元的放电和/或扰乱它们的基本生物物理特性可以是部分有效的，被限制为相对小的脑体积28。
为了克服这些限制，我们开发了体内实验方法进行新的大鼠来比较记录在一个给定的大脑状态单个神经元， 即，嵌入在一个特定的网络的动态，到整个大脑突触活动29的完全抑制后所得的电生理特性。在控制条件下，可能产生两个不同的皮质动态。睡眠就像electrocorticographic（脑电图）模式是由中等剂量戊巴比妥钠注射引起。另外，小振幅的快波脑电图相媲美的大脑皮层的活动清醒状态（醒状图案）基本可以用芬太尼注射产生。接着，同时保持相同的脑电图和细胞内记录时，由高剂量的戊巴比妥钠，其特征在于等电脑电图和细胞内活性的全身注射得到的内源性脑电活性的完全沉默。因为这样一个极端的昏迷诱导可能产生致命的consequen生物功能CES上，生理变量的仔细和连续监测是必不可少的。因此，我们认真随后心脏跳动频率，潮气末CO 2浓度（ETCO 2）时，O 2饱和度（SPO 2）和大鼠的核心温度在整个实验。
我们评估期间使用锋利微电极，其特别适合于在体内长时间稳定记录这些不同状态的单神经元特性。此处描述的方法，可以与其它电和成像方法相结合，并且可以延伸到其他的动物模型。
Protocol
所有的程序均按照欧盟的指导方针进行（指令63分之2010/ EU），并通过动物实验达尔文伦理委员会的批准。在这里我们描述我们在我们的实验室常规使用的方法，但是大多数步骤可以适于以匹配每个人的具体需要。 1.手术准备注：所有的切口和压点应与局部麻醉（利多卡因或布比卡因）反复渗透。本程序是终端，如果需要的无菌制剂几个修改应来实现。 …
Representative Results
诱导和维持的等电脑状态是在体内实验过程的细腻。它已被证明是一种强大的工具，直接研究了神经元兴奋性和传递函数29的皮质的网络活动的影响。 图1示出了（ 图1A之前多参数监控，包括脑电图和重要的常量，动物的生理状态的）和之后（ 图1B）诱导的等电状态的。 <img …
Discussion
我们在这里描述在两个网络和细胞水平在体内自发脑电活动抑制的新方法。这个过程导致了极端的大脑状态，被称为电昏迷41。从临床观点来看，这样的大脑电活动是最严重的异常，可以在脑电图中看到。它主要以不可逆转的昏迷相关，所有患者要么垂死或继续在持续植物人状态42，但可以通过抑制中枢神经系统药物（如硫喷妥钠）的中毒时造成至少部分逆转，一个偶然的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由来自基金会法国，研究所国家德拉桑特的Et德拉RECHERCHE MEDICALE，皮埃尔和玛丽·居里大学和程序“INVESTISSEMENTS德前途报”ANR-10 IAIHU-06的资助。
Materials
| Sodium Pentobarbital | Centravet | Pentobarbital | |
| Ketamine 500 | Merial | Imalgène 500 | |
| Fentanyl | Janssen-Cilag | Fentanyl | |
| Xylocaine | Centravet | Xylovet | |
| Gallamine triethiodide | Sigma | G8134 | |
| ECoG amplifier | A-M Systems | AC amplifier, Model 1700 | |
| Intracellular amplifier | Molecular Devices | Axoclamp 900A | |
| Data acquisition interface | Cambridge Electronic Design | CED power 1401-3 | |
| Data analysis software | Cambridge Electronic Design | Spike2 version 7 | |
| micromanipulator | Scientifica | IVM-3000 | |
| Capillary Puller | Narishige | PE-2 | |
| Borosilicate glass capillaries | Harvard Apparatus | GC150F-10 | |
| Silver wire 0.125mm (intracellular recording) | WPI | AGT0525 | |
| Ag-AgCl reference | Phymep | E242 | |
| Silver wire 0.25mm (ECoG recording) | WPI | AGT1025 | |
| Artificial respiration system | Minerve | Alpha Lab | |
| Physiological parameters monitoring | Digicare | LifeWindow Lite | |
| Heating Blanket | Harvard Apparatus | 507215 | |
| Stereomicroscope | Leica | M80 | |
| Scissors | FST | 15005-08 | |
| Forceps Dumont #5 | FST | 11295-10 | |
| Forceps Dumont #5SF | FST | 11252-00 | |
| IP Polyurethane catheter – 0.43×0.69mm | Instech | BTPU-027 | |
| Silicon elastomere | WPI | KWIK-CAST | |
| Dental drill | NSK | Y1001151 and P496 | |
| Surgical glue | 3M | vetbond |
References
- Fatt, P., Katz, B. Some observations on biological noise. Nature. 166 (4223), 597-598 (1950).
- Brock, L. G., Coombs, J. S., Eccles, J. C. The recording of potentials from motoneurones with an intracellular electrode. J. Physiol. 117 (4), 431-460 (1952).
- Destexhe, A., Rudolph, M., Fellous, J. M., Sejnowski, T. J. Fluctuating synaptic conductances recreate in vivo-like activity in neocortical neurons. Neuroscience. 107 (1), 13-24 (2001).
- Silver, R. A. Neuronal arithmetic. Nat. Rev. Neurosci. 11 (7), 474-489 (2010).
- Azouz, R., Gray, C. M. Cellular mechanisms contributing to response variability of cortical neurons in vivo. J. Neurosci. 19 (6), 2209-2223 (1999).
- Sanchez-Vives, M. V., Nowak, L. G., McCormick, D. A. Membrane Mechanisms Underlying Contrast Adaptation in Cat Area 17 In Vivo. J. Neurosci. 222 (11), 4267-4285 (2000).
- Petersen, C. C. H., Hahn, T. T. G., Mehta, M., Grinvald, A., Sakmann, B. Interaction of sensory responses with spontaneous depolarization in layer 2/3 barrel cortex. Proc. Natl. Acad. Sci. U.S.A. 100 (23), 13638-13643 (2003).
- Sachdev, R. N. S., Ebner, F. F., Wilson, C. J. Effect of Subthreshold Up and Down States on the Whisker-Evoked Response in Somatosensory Cortex. J. Neurophysiol. 92 (6), 3511-3521 (2004).
- Hasenstaub, A., Sachdev, R. N. S., McCormick, D. A. State Changes Rapidly Modulate Cortical Neuronal Responsiveness. J. Neurosci. 27 (36), 9607-9622 (2007).
- Chance, F. S., Abbott, L. F., Reyes, A. D. Gain modulation from background synaptic input. Neuron. 35 (4), 773-782 (2002).
- Shu, Y., Hasenstaub, A., Badoual, M., Bal, T., McCormick, D. A. Barrages of synaptic activity control the gain and sensitivity of cortical neurons. J. Neurosci. 23 (32), 10388-10401 (2003).
- Mitchell, S. J., Silver, R. A. Shunting inhibition modulates neuronal gain during synaptic excitation. Neuron. 38 (3), 433-445 (2003).
- Prescott, S. A., De Koninck, Y. Gain control of firing rate by shunting inhibition: roles of synaptic noise and dendritic saturation. Proc. Natl. Acad. Sci. U.S.A. 100 (4), 2076-2081 (2003).
- Graham, L. J., Schramm, A. In Vivo Dynamic-Clamp Manipulation of Extrinsic and Intrinsic Conductances: Functional Roles of Shunting Inhibition and IBK in Rat and Cat Cortex. Dynamic-clamp: From principles to applications. , (2009).
- Fernandez, F. R., White, J. A. Gain control in CA1 pyramidal cells using changes in somatic conductance. J. Neurosci. 30 (1), 230-241 (2010).
- Polack, P. O., Friedman, J., Golshani, P. Cellular mechanisms of brain state-dependent gain modulation in visual cortex. Nat. Neurosci. 16 (9), 1331-1339 (2013).
- Zhou, M., Liang, F., et al. Scaling down of balanced excitation and inhibition by active behavioral states in auditory cortex. Nat. Neurosci. 17 (6), 841-850 (2014).
- Contreras, D., Destexhe, A., Sejnowski, T. J., Steriade, M. Spatiotemporal Patterns of Spindle Oscillations in Cortex and Thalamus. J. Neurosci. 17 (3), 1179-1196 (1997).
- Charpier, S., Mahon, S., Deniau, J. M. In vivo induction of striatal long-term potentiation by low-frequency stimulation of the cerebral cortex. Neuroscience. 91 (4), 1209-1222 (1999).
- Constantinople, C. M., Bruno, R. M. Effects and Mechanisms of Wakefulness on Local Cortical Networks. Neuron. 69 (6), 1061-1068 (2011).
- Poulet, J. F. A., Fernandez, L. M. J., Crochet, S., Petersen, C. C. H. Thalamic control of cortical states. Nat. Neurosci. 15 (3), 370-372 (2012).
- Hille, B. . Ion Channels of Excitable Membranes, Third Edition. , (2001).
- Sakmann, B., Neher, E. . Single-Channel Recording. , (2009).
- Ferster, D., Chung, S., Wheat, H. Orientation selectivity of thalamic input to simple cells of cat visual cortex. Nature. 380 (6571), 249-252 (1996).
- Paré, D., Shink, E., Gaudreau, H., Destexhe, A., Lang, E. J. Impact of spontaneous synaptic activity on the resting properties of cat neocortical pyramidal neurons In vivo. J. Neurophysiol. 79 (3), 1450-1460 (1998).
- Destexhe, A., Paré, D. Impact of network activity on the integrative properties of neocortical pyramidal neurons in vivo. J. Neurophysiol. 81 (4), 1531-1547 (1999).
- Kara, P., Pezaris, J. S., Yurgenson, S., Reid, R. C. The spatial receptive field of thalamic inputs to single cortical simple cells revealed by the interaction of visual and electrical stimulation. Proc. Natl. Acad. Sci. U.S.A. 99 (25), 16261-16266 (2002).
- Lomber, S. G. The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. J. Neurosci. Meth. 86 (2), 109-117 (1999).
- Altwegg-Boussac, T., Chavez, M., Mahon, S., Charpier, S. Excitability and responsiveness of rat barrel cortex neurons in the presence and absence of spontaneous synaptic activity in vivo. J. Physiol. 592 (16), 3577-3595 (2014).
- Miner, N. A., Koehler, J., Greenaway, L. Intraperitoneal injection of mice. Appl. Microbiol. 17 (2), 250-251 (1969).
- Paxinos, G., Watson, C. . The rat brain in stereotaxic coordinates (2nd edn). , (1986).
- Wolfensohn, S. . Handbook of Laboratory Animal Management and Welfare. , (2013).
- Bester, H., Chapman, V., Besson, J. M., Bernard, J. F. Physiological Properties of the Lamina I Spinoparabrachial Neurons in the Rat. J. Neurophysiol. 83 (4), 2239-2259 (2000).
- Greene, S. A. . Veterinary Anesthesia and Pain Management Secrets. , (2002).
- Morgan, B. J., Adrian, R., Bates, M. L., Dopp, J. M., Dempsey, J. A. Quantifying hypoxia-induced chemoreceptor sensitivity in the awake rodent. J. Appl. Physiol. 117 (7), 816-824 (2014).
- Mahon, S., Deniau, J. M., Charpier, S. Relationship between EEG potentials and intracellular activity of striatal and cortico-striatal neurons: an in vivo study under different anesthetics. Cereb. Cortex. 11 (4), 360-373 (2001).
- Ganes, T., Lundar, T. The effect of thiopentone on somatosensory evoked responses and EEGs in comatose patients. J Neurol Neurosurg Psychiatry. 46 (6), 509-514 (1983).
- Schmid-Elsaesser, R., Schröder, M., Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H. J. EEG burst suppression is not necessary for maximum barbiturate protection in transient focal cerebral ischemia in the rat. J. Neurol. Sci. 162 (1), 14-19 (1999).
- Cummins, T. R., Jiang, C., Haddad, G. G. Human neocortical excitability is decreased during anoxia via sodium channel modulation. J Clin Invest. 91 (2), 608-615 (1993).
- Gu, X. Q., Kanaan, A., Yao, H., Haddad, G. G. Chronic High-Inspired CO2 Decreases Excitability of Mouse Hippocampal Neurons. J. Neurophysiol. 97 (2), 1833-1838 (2007).
- Lehembre, R., Gosseries, O., et al. Electrophysiological investigations of brain function in coma, vegetative and minimally conscious patients. Arch Ital Biol. 150 (2/3), 122-139 (2012).
- Husain, A. M. Electroencephalographic assessment of coma. J Clin Neurophysiol. 23 (3), 208-220 (2006).
- Fink, E. L., Alexander, H., et al. An Experimental Model of Pediatric Asphyxial Cardiopulmonary Arrest in Rats. Pediatr Crit Care Med. 5 (2), 139-144 (2004).
- Lukatch, H. S., McIver, M. B. Synaptic mechanisms of thiopental-induced alterations insynchronized cortical activity. Anesthesiology. 84, 1425-1434 (1996).
- Kroeger, D., Amzica, F. Hypersensitivity of the anesthesia-induced comatose brain. J Neurosci. 27, 10597-10607 (2007).
- Kroeger, D., Florea, B., Amzica, F. Human brain activity patterns beyond the isoelectric line of extreme deep coma. PLoS ONE. 8 (9), e75257 (2013).
- Margrie, T. W., Brecht, M., Sakmann, B. In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch. 444 (4), 491-498 (2002).
- DeWeese, M. Whole-Cell Recording In Vivo. Current Protocols in Neuroscience. , (2007).
- Schramm, A. E., Marinazzo, D., Gener, T., Graham, L. J. The Touch and Zap Method for In Vivo Whole-Cell Patch Recording of Intrinsic and Visual Responses of Cortical Neurons and Glial Cells. PLoS ONE. 9 (5), e97310 (2014).
- Mahon, S., Charpier, S. Bidirectional Plasticity of Intrinsic Excitability Controls Sensory Inputs Efficiency in Layer 5 Barrel Cortex Neurons in Vivo. J. Neurosci. 32 (33), 11377-11389 (2012).
- Destexhe, A., Rudolph, M., Paré, D. The high-conductance state of neocortical neurons in vivo. Nat. Rev. Neurosci. 4 (9), 739-751 (2003).
Cite This Article
Altwegg-Boussac, T., Mahon, S., Chavez, M., Charpier, S., Schramm, A. E. Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo. J. Vis. Exp. (109), e53576, doi:10.3791/53576 (2016).