概要

印刷业记录程序在小鼠体内测量睡眠

Published: January 25, 2016
doi:

概要

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Abstract

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

Introduction

技术的进步往往沉淀飞跃的神经生物学过程的理解。例如,汉斯Berger的发现于1929年,从人的头皮记录电势采取正弦波的形式,其频率直接与受试者的觉醒状态的水平,导致在睡眠觉醒的理解迅速进展法规,在动物和人类一样的。1为了这一天的electroencephlogram(EEG),与肌电图(EMG), 相结合,由骨骼肌产生的电活动,代表了几乎所有的实验和临床数据“脊梁”评估,即寻求相关行为和生理与皮质神经元的行为动物,包括人类的活动。在最基本的睡眠研究实验室这些脑电图记录是通过使用一个基于电缆的系统图1),其中获得ð执行ATA进行离线到图案和频谱分析[例如,施加快速傅立叶变换(FFT)算法]来确定受试者的警觉状态被记录。2,第3睡眠由快速眼动(REM)和非快速眼动(NREM)睡眠。 REM睡眠的特征在于快速低压脑电图,随机眼球运动,和肌肉弛缓,一种状态,其中的肌肉得到有效瘫痪。 REM睡眠也被称为异相睡眠的,因为大脑活动类似于觉醒,而体是来自大脑的主要断开,似乎是在深度睡眠。与此相反,运动神经元期间NREM睡眠刺激但没有眼睛运动。人NREM睡眠可分为4个阶段,其中第4阶段被称为深睡眠或慢波睡眠,并与0.5之间的三角活动确定大的,缓慢的脑电波 – 4赫兹的脑电图。另一方面,在较小的动物NREM睡眠的相之间的细分,象大鼠次的小鼠,还没有建立,主要是因为它们在人类可见没有睡眠的长综合时间。

多年来,和脑电图解释的基础上,几种模式的睡眠觉醒调节,既电路交换和体液为基础,已经被提出。神经和睡眠的需要,或者细胞基础“睡眠驱动器,”仍然没有得到解决,但已被概念化为衡压力在清醒周期的建立,是由睡眠消散。有一种说法是,在觉醒,他们的点滴积累是睡眠衡压力托底内源性somnogenic因素的累积。虽然睡眠是通过体液因素的调控的第一个正式假说已记入出版罗森鲍姆的工作,于1892年4,这是石森5,6和 Pieron 7谁独立,并在100多年前,展现了促睡眠的化学物质的存在。这两个研究人员提出,事实上证明,这hypnogenic物质或“hypnotoxins”存在于睡眠剥夺狗的脑脊液(CSF)8近百年来牵连的睡眠自我平衡过程中的几个额外的假定hypnogenic物质已被确定(综述见文献9),包括前列腺素(PG)的D 2,10细胞因子,11腺苷,12花生四烯酸乙醇胺,13和尾加压素II肽。14

在初期和中期,20 世纪产生的结果,鼓舞睡眠和清醒和电路为基础的理论在一定程度上由Economo 15,16,Moruzzi和Magoun 17,和其他人的实验工作,盖过了当时的体液学说睡觉。到目前为止,几个“电路模型”已经提出,每一个通知不同的质量和数量的数据(综述见参考文献18)。一种模式例如提出,慢波睡眠是通过从胆碱能神经元在基底前脑,区域主要consisiting布罗卡的对角线频带和质inominata的水平肢的细胞核的腺苷介导的抑制乙酰胆碱的释放产生的。睡眠/觉醒 19另一种流行的模型描述的触发器开关机构的睡眠诱发的神经元之间的腹外侧视前区和下丘脑和脑干相互抑制相互作用唤醒诱导的神经元基础上的。18,20,21此外,对于在进出REM睡眠的开关,一个类似的往复抑制相互作用已提出在脑干区域,即腹侧导水管周围灰质,横向脑桥被盖,和sublaterodorsal核。22总的来说,这些模型已经证明有价值启发式和提供重要的解释性框架,在睡眠的调查研究;然而,一个叶的分子机制和电路调节睡眠 – 觉醒周期的叔更全面的理解将需要其组件的更完整的认识。该系统为印刷业记录下文详述应该有助于这一目标。

Protocol

伦理声明:涉及动物主题程序已经批准在筑波大学的体制动物实验委员会。 1.准备电极和电缆脑电图/肌电图记录按照下列程序准备脑电/肌电图记录电极。 注意:电极是一次性的,并且只能用于1动物。认真规划所有连接器的接线配置。将标记的连接器正确的方向。 焊接一个4针接头的每个引脚到2厘米的不锈钢丝。简言之,把导线到销的一个端部,放置一个?…

Representative Results

图1B示出的鼠标脑电图在不同警觉状态的例子。如表 1所示,信号出现时间被分类为NREM睡眠如果脑电图显示缓慢的大脑电波低于4 Hz的δ节奏和肌电图仅具有弱或无信号。时期被列为REM睡眠,如果脑电图显示6和10赫兹之间的θ范围内迅速低电压脑电波和肌电图显示低振幅。其他历元应归类为觉醒(即,低到中等电压EEG和发生EMG活动的)。 <p…

Discussion

这个协议描述一个建立​​脑电/肌电图记录,允许睡眠和清醒的下低噪声,低成本,以及高通量条件的评估。由于脑电图/ EMG电极头组件的小尺寸,本系统可与其它植入物用于帧内脑实验,包括光遗传学(光纤植入),或结合同步插管植入,微注射的药物进入鼠标进行组合脑31。此外,电极头组件相对于多针头的设计提供了灵活性在记录信道数,如果需要的额外的电信号例如,对…

開示

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestlé Nutrition Council, Japan (to M.L.).

Materials

4-pin header Hirose A3B-4PA-2DSA(71)
Ampicillin Meiji Seika N/A
Analog-to-digital converter Contec AD16-16U(PCIEV)
Caffeine Sigma C0750
Carbide cutter Minitor B1055
Crimp housing Hirose DF11-4DS-2C
Crimp socket Hirose DF11-30SC
Dental cement (Toughron Rebase) Miki Chemical Product N/A
Epoxy adhesive Konishi #16351
FFC/FPC connector Honda Tsushin Kogyo FFC-10BMEP1(B)
Flat cable Hitachi Cable 20528-ST LF
Instant glue (Aron Alpha A) Toagosei N/A
Meloxicam Boehringer Ingelheim N/A
Pentobarbital Kyoritsu Seiyaku N/A
Signal amplifier Biotex N/A
Sleep recording chamber APL N/A
SleepSign software Kissei Comtec N/A for EEG/EMG recording/analysis
Slip ring Biotex N/A
Stainless steel screw Yamazaki N/A φ1.0×2.0
Stainless steel wire Cooner Wire AS633

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記事を引用
Oishi, Y., Takata, Y., Taguchi, Y., Kohtoh, S., Urade, Y., Lazarus, M. Polygraphic Recording Procedure for Measuring Sleep in Mice. J. Vis. Exp. (107), e53678, doi:10.3791/53678 (2016).

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