同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作
Summary
一个过程的描述，操纵大脑皮质锥体神经元的活动optogenetically而脑电图，肌电图，脑乳酸浓度监测。进行实验记录，电缆绳系小鼠，而他们自发的睡眠/觉醒周期。 optogenetic设备被组装在我们的实验室;记录设备是市售的。
Abstract
尽管大脑表示小于5质量％的身体时，它利用身体所利用的葡萄糖在休息1的约四分之一。非快速眼动睡眠（NREMS），睡眠时间的最大部分的功能是不确定的。然而，一个显着的特点NREMS是一个显着的减少的速率相对于觉醒2-4脑葡萄糖利用率。这与其他研究结果已广泛持有的信念，睡眠提供相关的脑代谢的功能。然而，仍有待澄清NREMS期间脑葡萄糖代谢的降低机制。
与的NREMS可能影响脑代谢率相关联的一种现象是慢波，在小于4赫兹的频率的振荡的发生，在脑波5,6。这些慢波在颅骨或大脑皮质表面的水平反映了检测相关的神经元去极化/状态和超极化/下的状态7之间振荡。在向下状态，细胞不发生动作电位到几百毫秒的间隔。后续动 作电位的离子浓度梯度的恢复是一个重要的代谢负荷对细胞8，动作电位与NREMS在掉电状态的情况下可能会有助于减少代谢相关唤醒的。
为了要测试这个假设的关系，必须解决两个技术挑战。首先，它是必要的测量脑糖酵解代谢与脑EEG的动态反射的时间分辨率（即，超过秒，而不是分钟）。要做到这一点，我们测得的乳酸浓度，有氧糖酵解的产物，因此，在老鼠的大脑中的葡萄糖代谢率的读数。乳酸是采用基于乳酸氧化酶的实时传感器嵌入的额叶皮层。感测机构包括由乳酸氧化酶分子的层包围的铂 – 铱电极。由乳酸氧化酶的乳酸代谢产生的过氧化氢，其产生的电流中的铂 – 铱电极。因此，一个加速脑糖酵解提供乳酸氧化酶，然后被反射在增加的电流在检测电极中的底物浓度的增加。这是另外需要这些变量，而操纵大脑皮质的兴奋性，以找出这个变量的NREMS从其他方面来衡量。
我们设计了一个实验系统的神经元活动的同时测量通过大脑皮质神经元的活动的elecetroencephalogram，通过乳酸生物传感器的糖酵解流量测量，操作通过optogenetic激活的金字塔midal神经元。我们已经利用本系统之间的睡眠相关的脑电图波形和在大脑皮质的乳酸浓度的时刻到时刻动态文件的关系。该协议可能是有用的任何个人感兴趣，学习，在自由活动的啮齿动物，在脑电图能量大脑内的水平和细胞之间的关系测定的神经元活性。
Protocol
1。动物的外科下游 1。实验的主题使用小鼠的Tg（Thy1-COP4/eYFP）的的B6.Cg 18Gfng / J转线9; JAX应变＃7612）或其他小鼠表达的蓝色光敏感的阳离子通道，Channelrhodopsin-2，在大脑皮质的神经元。应用到大脑皮质的B6.Cg-TG（Thy1-COP4/eYFP）的18Gfng / J，转基因株系的蓝色光会导致锥体神经元去极化，并进行动作电位9,10表达Channelrhodopsin-2。作为一个结果，锥?…
Representative Results
正如在图2中所示， 经历了自发的睡眠/唤醒状态转换，同时鼠标配备optogenetic刺激和乳酸/ EEG / EMG数据收集的脑电图，肌电图和脑乳酸浓度连续监测。目前在乳酸传感器增加了在低振幅脑电图的时期，期间，高振幅EEG下降。正如在图3中所示，两个通道的脑电图响应于optogenetic刺激交付的额叶皮层。 <img alt="…
Discussion
这里介绍的方法允许一个以前是不可能实现的时间尺度上测量大脑中的浓度之间的关系的睡眠和变化的糖酵解中间乳酸。动物自发唤醒，的NREMS和REMS之间的转换。此外，我们当动物接受这些转变可以申请optogenetic刺激。最新收集到的数据表明，自发和诱发波影响的乳酸氧化酶为基础的生物传感器的读出。
人们可以构建实验系统，类似于此处所述的设备和软件从其他来源。多导…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
由国防部（国防高级研究计划局，青年教师奖，项目编号N66001-09-1-2117）和NINDS（R15NS070734）的研究。
Materials
| Component | Company | Catalogue number | Comments (optional) |
| BASi Mouse Guide Cannula | Pinnacle Technology/BASi Inc | 7032 | |
| Lactate Biosensor | Pinnacle Technology | 7004 | |
| Head Mount | Pinnacle Technology | 8402 | |
| Sleep/Biosensor Recording system | Pinnacle Technology | 8400-K1-SL | 2 EEG channels, 1 EMG channel, & 1 biosensor |
| Tethered Mouse in-vitro Calibration kit | Pinnacle Technology | 7000-K1-T | |
| Fiber Optic Guide Cannula | Plastics One | C312G | 21 Gauge Guide Cannula |
| Dummy Cannula | Plastics One | C312DC | 21 Gauge Dummy |
| Diamond Fiber Scribe | Thorlabs | S90W | |
| Fiber Connector Crimp Tool | Thorlabs | CT042 | |
| Furcation Tubing | Thorlabs | FT030 | 03.0 mm |
| Thorlabs | T10S13 | Max Dia. 0.012 | |
| Furcation Tube Stripper | Thorlabs | FTS3 | |
| Bare Hard Cladding Multimode Fiber | Thorlabs | BFL37-200 | 200 μm Core, 0.37 NA |
| Wire Snips/Kevlar Shears | Thorlabs | T865 | |
| Fiber Optic Epoxy | Thorlabs | F112 | |
| Fiber Stripper Tool | Thorlabs | ||
| Glass Polishing Plate | Thorlabs | CTG913 | |
| Rubber Polishing Pad | Thorlabs | NRS913 | |
| Eye Loupe | Thorlabs | JEL10 | |
| Kim Wipes | Thorlabs | KW32 | |
| Compressed Air | Thorlabs | CA3 | |
| Polishing Puck | Thorlabs | D50-xx | |
| Fiber Inspection scope | Thorlabs | CL-200 | |
| Polishing Films | Thorlabs | LFG5P, LFG3P, LFG1P, LFG03P | |
| FC/PC connector end | Thorlabs | 30126G2-240 | 240 μm Bore, SS Ferrule |
| MC Stimulus Unit | Multi-Channel Systems | STG-4002 | |
| MC Stimulus Software | Multi-Channel Systems | MC-Stimulus V 2.1.5 | |
| Blue Laser | CrystaLaser | CL473-050-0 | |
| Laser Power supply | CrystaLaser | CL2005 | |
| Fiber Optic Rotary Joint | Doric Lenses | FRJ-v4 | |
| Table 2. Supplies and equipment. |
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Clegern, W. C., Moore, M. E., Schmidt, M. A., Wisor, J. Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex. J. Vis. Exp. (70), e4328, doi:10.3791/4328 (2012).