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 Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

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） <ol><li> 原液的制备 <ol><li>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。 </li><li>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 2 PO 4的。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为117毫摩尔NaCl，4.69毫摩尔的KCl，1.2毫硫酸镁 ，和1.18毫摩尔的NaH 2 PO 4。 </li><li>在4℃冷藏保存长达2周。 </li></ol></li><li> B原液的制备 <ol><li>添加化学品前加入700毫升蒸馏水到一个干净的1升烧杯中。 </li><li>同时连续搅拌下在500毫升体积，添加41.45克D-葡萄糖和41.84克的NaHCO 3。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为11.5毫D-葡萄糖和24.9毫碳酸氢钠 。 </li><li>在4℃冷藏保存长达2周。 </li><li>每个实验混合之前50ml料A的用50ml库存B和带来到1L蒸馏水中，得到最终浓度脑脊液溶液并在RT离开而连续地卡波金（95％O 2 - 5％CO 2的混合物）充氧它至少30分钟。仅在每个实验开始制备最终浓度脑脊液和在一天结束时丢弃。 </li></ol></li></ol> 2.蔗糖人工脑脊液的制备（蔗糖学联） <ol><li> 库存溶液 C 的制备 <ol><li>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。 </li><li>同时连续搅拌500毫升蒸馏水，加入240克蔗糖，6.55克碳酸氢钠的，0.671克氯化钾，4.88克的 MgCl 2，0.22克CaCl 2和0.21g的抗坏血酸。 </li><li>添加更多的蒸馏水中以达到1升的最终体积稀释后，将各化合物的最终浓度为701.1毫蔗糖，78毫碳酸氢钠 ，9毫米氯化钾，24毫氯化镁 ，1.5mM的氯化钙和1.2mM的抗坏血酸。 </li><li>保持股票溶液C在4℃下长达一个星期。 </li><li>每次实验混合之前300毫升50 ml储备下用蒸馏水以获得终浓度的蔗糖学联溶液并在RT离开而连续地卡波金（95％O 2 - 5％CO 2的混合物）充氧它将600ml至少30分钟。 </li></o升&gt; </li></ol> 3.切片准备<ol><li>放置一个干净的烧杯在冰100毫升蔗糖学联溶液中，同时用卡波金充氧，并使用pH计，同时加入0.1M的NaOH溶液几滴7.4固定pH值（在pH &lt;7.4）或0.1M的HCl溶液（当pH值&gt; 7.4）。 </li><li>填补了蔗糖学联一个vibratome的切割室和氧化它。转联接到所述切割室的基于甘油的冷冻装置上，并等待15分钟，以允许它冷却到0 - 4℃。 </li><li>麻醉大鼠幼仔用氯胺酮（成年定时怀孕的Sprague-Dawley大鼠9岁至12）（70毫克/ kg， 腹腔 ;使用&lt;50微升最终体积）。当小狗很平静，仔细检查尾巴捏反射不存在。 </li><li>斩首幼崽。 <ol><li>从前面纵向切割头部皮肤用碳钢手术刀刀片向后，和皮肤拉向两侧使用镊子。切骨保护大脑移动SC横向issors，完全删除它暴露大脑。 </li><li>然后，迅速去除用刮刀（最初置于嗅球下以轻轻地从最喙朝向最尾区压出大脑）的大脑。轻轻推脑入冰冷却的含氧蔗糖人工脑脊液（蔗糖ACSF）。 </li></ol></li><li>使在右侧半球一个矢状切口（除去半球的大约三分之一），和大脑的修整侧胶上，将被磁性固定到vibroslicer的切割室切割含有矢状400微米的部分的金属盘脚桥核（PPN）。保持PPN切片在RT之前全细胞膜片钳记录45分钟。 </li></ol> 4. PPN切片录音伽玛波段振荡<ol><li> 钾细胞内葡萄糖溶液的制备（高K +解决方案） <ol><li>放入冰一个干净的烧杯用10毫升蒸馏水。在不断搅拌下加入:K + -葡萄糖，90.68毫克磷酸迪的Tris盐，47.66毫克HEPES，1.5毫克EGTA，40.58毫克镁离子 -ATP和4毫克的Na + 2 -GTP。 </li><li>调节pH至7.3，用KOH（100毫在蒸馏水中）。加蒸馏水至达到20毫升的最终体积。如果需要的话，调节摩尔渗透压浓度与蔗糖（100mM的蒸馏水中）为280 - 290毫渗透摩尔浓度。稀释后，将各化合物的最终浓度为124毫K + -葡萄糖，10mM的磷酸二的Tris盐，10毫米的HEPES，0.2mM的EGTA，4mM的镁离子 -ATP和0.3毫米的 Na + 2 -GTP。 </li><li>在1ml试管等分试样的细胞内溶液并冷冻在-20℃。使用一个等份每一天，在实验过程中保持在4℃。 </li></ol></li><li> 全细胞膜片钳记录 <ol><li>放置切片中的浸渍室和含氧灌注他们（1.5毫升/分钟）（95％ 的 O 2 - ，5％CO 2）含有下列受体拮抗剂脑脊液:选择性NMDA受体拮抗剂的2-氨基-5- phosphonovaleric酸（APV，40μM），竞争性AMPA /红藻氨酸盐型谷氨酸受体拮抗剂6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX，10μM），甘氨酸受体拮抗剂士的宁（STR，10μM），具体的 GABA A受体拮抗剂gabazine（GBZ，10μM）和钠通道阻滞剂河豚毒素（TTX，3μM） 注:在结果和附图，这些拮抗剂统称为突触阻滞剂或SBS。 </li><li>填记录补丁移液器;与细胞内高-K +溶液使用与市售的膜片吸管填料（电阻2-7兆欧从常规，可商购的厚壁硼硅玻璃1.0毫米外径和0.6内径的毛细管制造）解决方案过滤器。插入其放大器的持有人的吸管。取一小POS机可持续的竞争压力用1ml注射器连接到使用连接到一个三通阀的硅管的吸移管支架。连接吸管保持器到一个膜片钳放大器的背面。 </li><li>移动使用使用4X物镜相结合，近红外微分干涉对比光学的PPN核附近的机械显微记录吸管。 注:PPN核可在背片可以观察到小脑上脚（SCP;观察到轴突的厚束）。录音移液器均位于PPN 的致密部 ，这是直接位于背侧脚的后端。 </li><li>带来与PPN神经元接触记录吸管，同时用40X水浸透镜可视化，并迅速施加负抽吸，以形成与电池的密封。 </li><li>使用电压钳密封使用的软件制造商的协议来监测负压吸引过程中吸管阻力。 <ol><li>当负小​​号，减税缓慢增加，电阻值通过在吸液管的尖端的膜片钳显示器读达到80 - 100兆欧姆，快速改变保持电位至-50毫伏并释放负压。开始连续施加负抽吸直至破裂神经元的膜和电访问在全细胞构造来实现的。 </li><li>如果由电压钳密封软件来测量接入电阻值为10兆欧或更高，然后继续在较小量施加负吸力。 </li></ol></li><li>补偿电容和串联电阻在电压钳模式（ 即 ，慢速和快速瞬变破裂细胞的细胞膜后观察到的）。切换录音模式电流钳，并迅速弥补桥值（ 例如 ，移动放大器旋钮或按使用鼠标computer&#39;s自动补偿菜单上）。 <ol><li> PPN神经元的连续监测静息电位被记录ū唱制造商的协议。如果休息朝去极化或超极化值的膜电位的移位，然后使用少量的直流（高达100帕），以保持最佳-50毫伏最终值。 注:仅保留PPN神经元-48 mV的稳定的静息膜电位（RMP）以上超极化（ 即 RMP &lt;-48 mV的）。 </li></ol></li></ol></li></ol>

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Neurosci. 36 (18), 4917-4929 (2016).</li><li>Fraix, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pedunculopontine+nucleus+area+oscillations+during+stance,+stepping+and+freezing+in+Parkinson's+disease.">Pedunculopontine nucleus area oscillations during stance, stepping and freezing in Parkinson's disease.</a> PLOS ONE. 8 (12), e83919 (2013).</li><li>Luster, B., Hyde, J., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">Mechanisms behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Abstr Soc Neurosci. 38, 257.20 (2014).</li><li>Luster, B., D'Onofrio, S., Urbano, F. J., Garcia-Rill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-threshold+Ca2++channels+behind+gamma+band+activity+in+the+pedunculopontine+nucleus+(PPN).">High-threshold Ca2+ channels behind gamma band activity in the pedunculopontine nucleus (PPN).</a> Physiol. Rep. 3 (6), e12431 (2015).</li></ol>

The authors declare that they have no competing financial interests.

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...

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（&lt;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。

<table><tbody><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;, molecular weight = 342.30</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 84.01</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td>KCl, molecular weight = 74.55</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =&amp;nbsp; 203.30</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>C3881</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O, molecular weight =147.02</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight = 180.16</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;, molecular weight =176.12</td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td>NaCl, molecular weight =&amp;nbsp; 58.44</td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>G4500</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;KO&lt;sub&gt;7&lt;/sub&gt;, molecular weight =&amp;nbsp; 234.25</td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td>C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;P &amp;middot; 2C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;, molecular weight =&amp;nbsp; 453.38</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S, molecular weight = 238.30</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E0396</td><td>[-CH&lt;sub&gt;2&lt;/sub&gt;OCH&lt;sub&gt;2&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;N(CH&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;H)&lt;sub&gt;2&lt;/sub&gt;]&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 380.40</td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td>&amp;nbsp;C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;13&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xMg2+, molecular weight = 507.18</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma-Aldrich</td><td>G8877</td><td>C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;14&lt;/sub&gt;P&lt;sub&gt;3&lt;/sub&gt; &amp;middot; xNa+, molecular weight = 523.18</td></tr><tr><td>Tetrodotoxin citrate</td><td>Alomone Labs</td><td>T-550</td><td>C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;, molecular weight = 319.27</td></tr><tr><td>&amp;nbsp;DL-2-Amino-5-Phosphonovaleric Acid</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;C&lt;sub&gt;5&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P, molecular weight = 197.13</td></tr><tr><td>CNQX disodium salt hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C239</td><td>C&lt;sub&gt;9&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt; &amp;middot; xH2O, molecular weight = 276.12</td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>C&lt;sub&gt;21&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, molecular weight = 334.41</td></tr><tr><td>Mecamylamine hydrochloride</td><td>Sigma-Aldrich</td><td>M9020</td><td>&amp;nbsp;C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;21&lt;/sub&gt;N &amp;middot; HCl, molecular weight = 203.75</td></tr><tr><td>Gabazine (SR-95531)</td><td>Sigma-Aldrich</td><td>S106</td><td>C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;BrN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;, molecular weight = 368.23</td></tr><tr><td>Ketamine hydrochloride</td><td>Mylan</td><td>67457-001-00</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>Pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>Pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>&amp;nbsp;Leica VT1200 S</td><td></td></tr><tr><td>Refrigeration system</td><td>Vibratome Instruments</td><td>900R</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Eclipse E600FN</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>ROE-200</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>MPC-200</td><td></td></tr><tr><td>amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>A/D converter</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>heater</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>pump</td><td>Cole-Parmer</td><td>Masterflex L/S 7519-20</td><td></td></tr><tr><td>pump cartridge</td><td>Cole-Parmer</td><td>Masterflex 7519-85</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>camera</td><td>Q-Imaging</td><td>RET-200R-F-M-12-C</td><td></td></tr></tbody></table>

recording gamma band oscillations in pedunculopontine nucleus neurons

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 。

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...

Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

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。

录音伽玛乐队在振荡脚桥核神经元

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

recording-gamma-band-oscillations-in-pedunculopontine-nucleus-neurons

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.6K 次观看.  University of Buenos Aires. 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。 

视频: Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

科研

神经科学

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

高度组织的Theta振荡在鼠脑的自动检测

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

行为学

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

生产和使用背驮式多管电极<em在体内</em&gt;药理手法的神经反应

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

本地CA1γ振荡通过强直刺激生成

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

视网膜原酸和皮质化突触功能的电生理学研究

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Source: Urbano, F. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

记录脑切片中带蒂核神经元的伽马波段振荡

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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