监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作
Summary
在这里,我们描述了在监测小鼠睡眠/觉醒状态期间对特定类型神经元的光遗传学操作方法,并举例说,我们最近对纹状体末期骨细胞核的床核进行了实验。
Abstract
近年来,光遗传学在神经科学研究领域得到了广泛的应用。在许多情况下,蛋白酶,如通道红蛋白2(ChR2),由病毒载体在各种Cre-驱动程序小鼠的特定类型的神经元细胞中表达。这些蛋白酶的激活由激光或LED通过光缆传递的光脉冲应用触发,激活效果以非常高的时间分辨率观察到。实验者能够敏锐地刺激神经元,同时监测小鼠的行为或其他生理结果。光遗传学可以启用有用的策略来评估神经元回路在调节小鼠睡眠/觉醒状态的功能。在这里,我们描述了一种技术,用于检查在脑电图(EEG)和肌电图(EMG)监测期间具有特定化学特性的神经元的光遗传学操作的影响,以评估小鼠的睡眠阶段。例如,我们描述了在纹状体末期性(BNST)的床核中GABAergic神经元的操纵。这些神经元的急性光遗传学激发在NREM睡眠期间应用时触发快速过渡到觉醒。光遗传学操作以及EEG/EMG记录可以应用于破译调节睡眠/觉醒状态的神经元回路。
Introduction
睡眠对于最佳的认知功能至关重要。最近的发现还表明,睡眠障碍与各种疾病1,2,3有关。虽然睡眠的功能尚未基本解决,但最近在理解控制睡眠/觉醒状态的神经回路和机制4方面取得了实质性进展。在哺乳动物中,有三种警惕状态:觉醒、非快速眼动(NREM)睡眠和快速眼动(REM)睡眠。觉醒性的特点是低振幅的快速脑电图振荡(5-12 Hz),具有有目的和持续的电机活性。NREM 睡眠由高振幅(增量波)的缓慢振荡(1-4 Hz)定义,缺乏意识和有目的的运动活动。REM睡眠的特点是低振幅相对快速的振荡(6-12赫兹),几乎完成双边肌肉肌张力5。
Borbely提出了一种睡眠觉醒调节理论,称为两个过程模型6,7。静温过程,也称为过程S,表示在觉醒期间积累并在睡眠期间消散的睡眠压力。另一个过程,称为过程C,是一个昼夜过程,这解释了为什么警戒水平在24小时周期中波动。除了这两个过程,等静因素对于调节睡眠/觉醒8,9也很重要。过敏因素包括营养状态和情绪。恐惧和焦虑通常伴随着觉醒的增加,以及自主和神经内分泌反应10,11,12。边缘系统被认为在调节恐惧和焦虑方面发挥作用,自主和神经内分泌反应背后的机制已经被广泛研究,但边缘系统影响睡眠/觉醒状态的途径尚未尚未透露。最近大量使用光遗传学和药理遗传学的研究表明,调节睡眠/觉醒状态的神经元和神经元回路分布在整个大脑中,包括皮质、基底前脑、丘脑、下丘脑、和脑干。特别是,光遗传学的最新进展使我们能够刺激或抑制具有高空间和时间分辨率的特定神经回路i n vivo。这项技术将允许我们了解睡眠和觉醒的神经基质,以及睡眠/觉醒状态如何受到昼夜过程、睡眠压力和包括情绪在内的同感因素的调节。本文旨在介绍如何使用光遗传学操作与睡眠/觉醒记录相结合,这有可能更新我们对大脑中在调节NREM睡眠、REM睡眠、REM睡眠方面起着一定作用的连体和机制的理解。和觉醒。理解这种机制,通过边缘系统调节睡眠/觉醒状态是对健康至关重要的,因为失眠通常与焦虑或恐惧无法入睡(失眠恐惧症)相关。
BNST被认为在焦虑和恐惧中起着至关重要的作用。GAD 67-表达GABAergic神经元是BNST 12,13的主要种群。我们研究了光遗传学操纵这些神经元(GABABNST)对睡眠/觉醒状态的影响。近年来神经科学的最大进步之一是能够操纵体内具有特定化学特性的神经元,具有高空间和时间分辨率。光遗传学对于证明神经活动和特定行为反应之间的因果关系非常有用14。我们将光遗传学描述为一种在调节睡眠/觉醒状态时检查已定义神经回路的功能连接性的方法。通过利用这一技术,在理解调节睡眠/觉醒状态的神经元回路15,16,17,18,19方面取得了很大进展。.在许多情况下,蛋白酶通过Cre-驱动程序小鼠和可诱导的AAV介导基因转移的组合,在选择性大脑区域具有特殊化学特性的神经元中具体引入。此外,光敏蛋白酶(如通道性多氯环素 2 (ChR2)20或古黄素 (ArchT)21与 Cre-loxP 或 Flp-FRT 系统结合使用,可让我们操纵选择性神经元总体和特定神经通路22.
我们在这里以BNST中GABAergic神经元的实验为例。为了在指定的神经元种群中表达蛋白酶,最常使用适当的 Cre 驱动程序小鼠和 Cre 依赖性病毒载体。转基因或敲线,其中蛋白酶表达在特定神经元群体也很有用。在以下实验中,我们使用GAD67-Cre敲撞小鼠23,其中只有GABAergic神经元表达具有C57BL/6J遗传背景的Cre重组酶,以及含有ChR2(hChR2 H134R)与EYFP或EYFP融合的AAV载体作为对照带有”FLEx(翻转切除)开关”24 。该程序特别描述了在监测睡眠/觉醒状态25期间,BNST中GABAergic神经元的光遗传学激发。
Protocol
Representative Results
Discussion
我们提出了一种评估具有特定化学特性的神经元的光遗传学刺激对睡眠/觉醒状态过渡的影响的方法,并给出了GABABNST神经元操作的例子。我们的数据表明,GABABNST神经元的光遗传学激发导致从NREM睡眠到觉醒的即时过渡。
由于多种光遗传学工具的开发,各种实验设计都可用。可以使用不同类型的蛋白酶(如ChR2、SSFO、哈洛多普辛、ArchT和iChloC27)激活?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了默克调查员研究计划(#54843、创新领域科学研究援助卡肯希助学金、”威尔动力”(16H06401)和KAKENHI创新领域探索性研究资助资助(T.S.)的支持。(18H02595)。
Materials
| 1×1 Fiber-optic Rotary Joints | Doric | FRJ 1×1 FC-FC | for optogenetics |
| 6-pin header | KEL corporation | DSP02-006-431G | |
| 6-pin socket | Hirose | 21602X3GSE | |
| A/D converter | Nippon koden | N/A | Analog to digital converter |
| AAV10-EF1a-DIO-ChR2-EYFP | 3.70×1013(genomic copies/ml) | ||
| AAV10-EF1a-DIO-EYFP | 5.82×1013(genomic copies/ml) | ||
| Ampicillin | Fuji film | 014-23302 | |
| Amplifier | Nippon koden | N/A | for EEG/EMG recording |
| Anesthetic vaporizer | Muromachi | MK-AT-210D | |
| Automatic injecter | KD scientific | 780311 | |
| Carbide cutter | Minitor | B1055 | φ0.7 mm. Reffered as dental drill, used with high speed rotary micromotor |
| Cyanoacrylate adhesion (Aron alpha A) and acceleration | Konishi | #30533 | |
| Dental curing light | 3M | Elipar S10 | |
| Epoxy adhesive | Konishi | #04888 | insulation around the solder of 6-pin and shielded cable |
| Fiber optic patch cord (branching) | Doric | BFP(#)_50/125/900-0.22 | |
| Gad67-Cre mice | provided by Dr. Kenji Sakimura | Cre recombinase gene is knocked-in in the Gad67 allele | |
| Hamilton syringe | Hamilton | 65461-01 | |
| High speed rotary micromotor kit | FOREDOM | K.1070 | Used with carbide cutter |
| Interconnecting sleeve | Thorlab | ADAF1 | φ2.5 mm Ceramic |
| Isoflurane | Pfizer | 871119 | |
| Laser | Rapp OptoElectronic | N/A | 473nm wave length |
| Laser intesity checker | COHERENT | 1098293 | |
| Laser stimulator | Bio research center | STO2 | reffered as pulse generator in text |
| Optic fiber with ferrule | Thorlab | FP200URT-CANNULA-SP-JP | |
| pAAV2-rh10 | provided by PennVector Core | ||
| pAAV-EF1a-DIO-EYFP-WPRE-HGHpA | Addgene | plasimid # 20296 | |
| pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA | provided by Dr. Karl Deisseroth | ||
| Patch cord | Doric | D202-9089-0.4 | 0.4m length, laser conductor between laser and rotary joint |
| pHelper | Stratagene | ||
| Photocurable dental cement | 3M | 56846 | |
| Serafin clamp | Stoelting | 52120-43P | |
| Shielded cable | mogami | W2780 | Soldering to 6-pin socket for EEG/EMG recording |
| Sleep recording chamber | N/A | N/A | Custum-made (21cm× 29cm × 19cm) with water tank holder |
| Sleep sign software | KISSEI COMTEC | N/A | for EEG/EMG analysis |
| Slip ring | neuroscience,inc | N/A | for EEG/EMG analysis |
| Stainless screw | Yamazaki | N/A | φ1.0 x 2.0 |
| Stainless wire | Cooner wire | AS633 | 0.0130 inch diameter |
| Stereotaxic frame with digital console | Koph | N/A | Model 940 |
| Syringe needle | Hamilton | 7803-05 | |
| Vital recorder software | KISSEI COMTEC | N/A | for EEG/EMG recording |
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