录音伽玛乐队在振荡脚桥核神经元
Summary
脚桥核（PPN）位于脑干和神经元在清醒和快速眼动（REM）睡眠大脑状态最大限度地激活。这部作品描述了实验方法在 PPN神经元体外伽玛带阈下膜振荡记录。
Abstract
从PPN突触传出已知的几种调节丘脑板内核区域的神经元活动（ 例如 ，centrolateral /束旁;氯/ PF核）。无论是PPN 或体内氯/ Pf的核的活化已经描述以诱 导动物的觉醒和伽玛频带活性皮质脑电图（EEG）的增量。在网状活化系统伽马频带的振荡的产生的细胞机制（RAS）的神经元的那些相同发现生成其他大脑原子核伽玛波段振荡。期间PPN神经元的电流钳记录（从9矢状切片 – 40日龄鼠），使用去极化平方步骤的快速激活电压依赖性钾通道，阻止PPN神经元被去极化超出-25毫伏。
注射1 – 2秒长的去极化电流逐渐逐渐去极化PPN膜电位资源婷对0毫伏值。然而，注射去极化方形脉冲产生的膜电位的伽玛波段振荡即表明要与由斜面所产生的振动的振幅变小。所有实验都在电压 – 门控钠通道和快速突触受体阻断剂的存在下进行。它已经表明，高阈值电压依赖性钙通道的活化背后伽马波段振荡活性PPN神经元。具体的方法和药物干预在这里描述的，提供了必要的工具来诱导和维持PPN阈下伽玛波段振荡体外 。
Introduction
PPN核被解剖列入尾脑被盖。该PPN是RAS 1的重要组成部分。该PPN参与维护行为激活状态（ 即清醒，REM睡眠）2。 体内 PPN电刺激诱 发的快速振荡（20 – 40赫兹）在皮层脑电图3，而双侧病变PPN在大鼠中减少或消除REM睡眠4。虽然大多数PPN神经元开火β/γ波段频率动作电位（20 – 80赫兹），一些神经元呈现自发放电率较低（<10赫兹）5。此外，PPN似乎参与的行为其他方面，如动机和关注6。直接高频（40 – 60赫兹）在去大脑动物PPN核7电刺激可以促进运动。近年来，深部脑刺激（DBS）PPN的已被用于治疗来回患者涉及步态缺陷米障碍，例如帕金森氏病（PD）8。
以前的报告显示，几乎所有的神经元PPN可以用平方电流脉冲去极化9时在伽马射线波段频率发射动作电位。因为电压 – 门控钾通道的过程中方形脉冲去极化到或在-25毫伏的急剧的活化。其结果是，阻止使用河豚毒素10动作电位产生之后没有观察到鲁棒伽马振荡。在努力绕过这样的问题，1 – 2秒长的去极化电流斜坡中使用。坡道从休息值高达0毫伏逐渐去极化膜电位，而部分失活的电压 – 门控钾通道。清除伽玛带膜振荡高门槛钙通道的电压依赖性窗口内是明显的（ 即 -25 mV至-0之间毫伏）10。总之，γ-带活性状况TY在PPN神经元9观察到的，和两个P / Q-和N型电压门控钙通道需要，以便产生在PPN 10伽玛波段振荡被激活。
一系列的研究确定了PPN神经元的高门槛钙通道的位置。注射染料的组合， 比例荧光成像通过在不同的树突激活使用电流斜11去极化时的电压门控钙通道表明钙瞬变。
PPN神经元的固有特性已建议允许唤醒和REM睡眠，从而诱发RAS和丘脑皮层环路之间高频振荡神经元活动中这些细胞的同时激活。这种长期深远的互动被认为是支持能够在连续的基础上12可靠地评估我们周围的世界的大脑状态。这里，我们描述了实验必要人条件，以产生和维持伽马频带振荡PPN细胞在体外 。该协议还没有被先前所描述，并有助于若干组研究固有膜性能在其他脑区介导伽马带活性。此外，目前的步骤可能会导致错误的结论伽马射线波段活动不能在这些细胞中产生。
Protocol
所有的实验方案得到阿肯色大学医学科学（协议号＃3593）的机构动物护理和使用委员会批准，均符合卫生准则的国家机构的协议照顾和使用实验动物。 1.标准人工脑脊液的制备（学联） 原液的制备 添加化学品前加入700毫升蒸馏水至一个干净的1升烧杯中。 同时连续搅拌下在500毫升体积，添加136.75克的NaCl，6.99克氯化钾，2.89克硫酸镁 ，并2.83克的NaH <…
Representative Results
最初，伽马振荡采用方形电流脉冲引起的。突触阻滞剂和TTX的存在PPN神经元的电流钳记录连续监测，以确保静息膜电位在约-50 mV的（ 图1A）保持稳定。两个第二长方形的电流脉冲是由通过记录吸管膜片钳放大器细胞内注射，增加其幅度从200 pA的600 PA（ 图1A）。膜电位去极化电压等级接近-20 mV的时候600 pA的，造成小幅度的伽马振荡。伽玛振荡的权力…
Discussion
PPN神经元有内在属性，使他们在从动物醒着REM睡眠过程中，而不是在慢波睡眠2,3,5,13-17 体内录音开火β/γ频段频率动作电位。其他作者发现，脑干横断在更前水平在脑电图记录比减少PPN伽玛频率。然而，当脑干病变后路到这个核心所在，PPN的直接刺激所允许的EEG 2,3,5,18-21皮质伽玛活动的表现。伽马带神经活动已经报道在小鼠PPN 体外 22，在大鼠REM睡眠诱导区域…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promociòn Cientìfica y Tecnològica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).
Materials
| Sucrose | Sigma-Aldrich | S8501 | C12H22O11, molecular weight = 342.30 |
| Sodium Bicarbonate | Sigma-Aldrich | S6014 | NaHCO3, molecular weight = 84.01 |
| Potassium Chloride | Sigma-Aldrich | P3911 | KCl, molecular weight = 74.55 |
| Magnesium Chloride Hexahydrate | Sigma-Aldrich | M9272 | MgCl2 · 6H2O, molecular weight = 203.30 |
| Calcium Chloride Dihydrate | Sigma-Aldrich | C3881 | CaCl2 · 2H2O, molecular weight =147.02 |
| D-(+)-Glucose | Sigma-Aldrich | G5767 | C6H12O6, molecular weight = 180.16 |
| L-Ascorbic Acid | Sigma-Aldrich | A5960 | C6H8O6, molecular weight =176.12 |
| Sodium Chloride | Acros Organics | 327300025 | NaCl, molecular weight = 58.44 |
| Potassium Gluconate | Sigma-Aldrich | G4500 | C6H11KO7, molecular weight = 234.25 |
| Phosphocreatine di(tris) salt | Sigma-Aldrich | P1937 | C4H10N3O5P · 2C4H11NO3, molecular weight = 453.38 |
| HEPES | Sigma-Aldrich | H3375 | C8H18N2O4S, molecular weight = 238.30 |
| EGTA | Sigma-Aldrich | E0396 | [-CH2OCH2CH2N(CH2CO2H)2]2, molecular weight = 380.40 |
| Adenosine 5'-triphosphate magnesium salt | Sigma-Aldrich | A9187 | C10H16N5O13P3 · xMg2+, molecular weight = 507.18 |
| Guanosine 5'-triphosphate sodium salt hydrate | Sigma-Aldrich | G8877 | C10H16N5O14P3 · xNa+, molecular weight = 523.18 |
| Tetrodotoxin citrate | Alomone Labs | T-550 | C11H17N3O8, molecular weight = 319.27 |
| DL-2-Amino-5-Phosphonovaleric Acid | Sigma-Aldrich | A5282 | C5H12NO5P, molecular weight = 197.13 |
| CNQX disodium salt hydrate | Sigma-Aldrich | C239 | C9H2N4Na2O4 · xH2O, molecular weight = 276.12 |
| Strychnine | Sigma-Aldrich | S0532 | C21H22N2O2, molecular weight = 334.41 |
| Mecamylamine hydrochloride | Sigma-Aldrich | M9020 | C11H21N · HCl, molecular weight = 203.75 |
| Gabazine (SR-95531) | Sigma-Aldrich | S106 | C15H18BrN3O3, molecular weight = 368.23 |
| Ketamine hydrochloride | Mylan | 67457-001-00 | |
| Microscope | Nikon | Eclipse E600FN | |
| Micromanipulator | Sutter Instruments | ROE-200 | |
| Micromanipulator | Sutter Instruments | MPC-200 | |
| Amplifier | Molecular Devices | Multiclamp 700B | |
| A/D converter | Molecular Devices | Digidata 1440A | |
| Heater | Warner Instruments | TC-324B | |
| Pump | Cole-Parmer | Masterflex L/S 7519-20 | |
| Pump cartridge | Cole-Parmer | Masterflex 7519-85 | |
| Pipette puller | Sutter Instruments | P-97 | |
| Camera | Q-Imaging | RET-200R-F-M-12-C | |
| Vibratome | Leica Biosystems | Leica VT1200 S | |
| Refrigeration system | Vibratome Instruments | 900R | |
| Equipment | |||
| microscope | Nikon | Eclipse E600FN | |
| micromanipulator | Sutter Instruments | ROE-200 | |
| micromanipulator | Sutter Instruments | MPC-200 | |
| amplifier | Molecular Devices | Multiclamp 700B | |
| A/D converter | Molecular Devices | Digidata 1440A | |
| heater | Warner Instruments | TC-324B | |
| pump | Cole-Parmer | Masterflex L/S 7519-20 | |
| pump cartridge | Cole-Parmer | Masterflex 7519-85 | |
| pipette puller | Sutter Instruments | P-97 | |
| camera | Q-Imaging | RET-200R-F-M-12-C |
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Cite This Article
Urbano, F. J., Luster, B. R., D’Onofrio, S., Mahaffey, S., Garcia-Rill, E. Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons. J. Vis. Exp. (115), e54685, doi:10.3791/54685 (2016).