Supported by APA to RJH, NHMRC program grant 400121 to SP, and NMHRC fellowship 1005050 to SP. CAR acknowledges the support of the ARC (FT0990628) and the DOWD fellowship scheme. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds.

在老鼠身上所有实验均批准弗洛里学院动物伦理委员会。 1.设置切削脑片<ol><li>制备由（mM计）125胆碱氯，2.5氯化钾，0.4氯化钙2，6的MgCl 2，1.25的NaH 2 PO 4，26的NaHCO 3的切削液，20 D-葡萄糖饱和卡波金气（95％O 2 -5由（毫米）125氯化钠，2.5氯化钾，2-氯化钙2，2的MgCl 2，1.25的NaH 2 PO 4，26的NaHCO 3％的CO 2）和人工脑脊液（ACSF）记录溶液，10 D-葡萄糖，饱和卡波。置于冰上切割解决方案，以保持低温。 </li><li>冻结大约400毫升切削溶液，并用100毫升解冻切削溶液混合在一起以创建一个冰浆。泡沫与卡波（95％O 2 -5％CO 2）以约0.5升/分钟通过小C流aliber管或烧结玻璃产生气泡的稳定，但水流平缓。 </li><li>准备的250ml烧杯带有凸起尼龙筛插入在其上的大脑切片将被放置。与脑脊液填充以覆盖大约2厘米网眼和气泡与卡波金，确保气泡不会直接破坏片保持区域。同样重要的是不存在任何气泡在尼龙网，因此，如果存在的话将其删除。这将是保持室和保持在室温（20 - 25℃）。 </li><li>布局解剖工具，包括一把大剪刀，一把小剪刀，小和大微铲，和大，小双钳的肃杀他们在冰上。放置在冰上vibratome组织切割块上铝箔的正方形。获得2个6厘米的滤纸，单个边缘剃刀刀片，和25毫升的烧杯中填充有切削液浆为好。 </li><li>填写第二容器用冰，躺在一块组织人民行动党呃在冰面上，并放置在顶部12 cm的培养皿。填写培养皿切割液冰浆和气泡与卡波。这是在其中的大脑解剖与要执行的容器。 </li><li>准备vibratome。除去新鲜双刃刀片从它的包装（用新鲜叶片每次）和喷雾用80％乙醇然后用去离子水。切有3毫升的塑料移液管在它开始锥度的点。这将被用于传送脑切片。弯曲一个27G的针在基由大约45°和附加到1ml注射器。这将用于在切割期间操作片。 </li></ol> 2.切断脑片<ol><li>麻醉鼠标（P16 - P18）用2％异氟醚或本地认可的方法。以下归纳，斩首用大剪刀的动物和拖放头进12 6cm培养皿包含冒泡切割液浆。浆料必须完全沉浸在头快速冷却。 </li><li>保持头部的前部，用一只手剥离皮肤和结缔组织向前朝向鼻子。采用切开结缔组织的小剪刀，露出底层的头骨。然后取出肌肉覆盖在头骨和颈部背侧。 </li><li>首先确保头骨的前部与大镊子取下头盖骨的大 ​​脑，然后通过使枕骨大孔的骨两边用小剪刀（ 图1，标记为削减A1和A2）两个横向切口。 <ol><li>让眼睛之间的另一个切（只是前的前囟门的）（ 图1，切标记为B），然后小心地沿矢状缝前方切反映切割颅骨部分揭示大脑（ 图1，切口标记为C）。 </li><li>用小铲舀出大脑和地点到培养皿。要注意的CRAN的上，这将需要大脑的劣方面胶质神经被切断，这可以通过使用较小尺寸的微抹刀来完成。使用较大的刮刀脑转移到25毫升烧杯中填充有切削液浆料。 </li></ol></li><li>准备大脑半球切片。 <ol><li>放置一块的6厘米滤纸在一个新鲜培养皿的底部然后装满新鲜切割溶液浆和泡沫。使用较大的刮刀向下定位大脑，腹侧，到滤纸上。采取新的清洗和单边刀片和削减大脑取出小脑，然后进行沿中线切大脑分成两个半球。 </li></ol></li><li>准备vibratome组织块。 <ol><li>取冷冻vibratome组织块，表面干燥，并放置在中间一滴氰基丙烯酸酯胶和均匀地散布到大脑的近似大小。用小铲操纵之一大脑半球到大微锅铲使内侧大脑的下跌。 </li><li>触摸抹刀的边缘处的第二片滤纸大脑的接口以除去尽可能多的溶液，作为可能的。滑动大脑关闭使用较小的锅铲引导它到胶水的大锅铲。 </li><li>固定切割块到vibratome室并填写切削液浆和气泡室，保证了大脑完全浸入。转动切割块，使脑的腹侧朝向刀片。 </li></ol></li><li>切片的大脑。 注意:每个vibratome是唯一的，从而按照制造商的说明。 <ol><li>设置切片厚度450微米，并确保刀片振动，因为它通过大脑移动以大约0.3mm / s的速度。通过从腹侧的皮质表面的大脑完全切断，这将产生全脑矢状切片，可以bË用于电生理记录。片会产生横向内侧到，通常3 - 4片可以从每个半球被削减。 </li><li>如果切片升降机底座采用弯曲27G的针头要轻柔按片回落。由于每个切片切断，用移液管将其移动到所述平台中的保持室，其中它们是可行的长达8小时。 </li></ol></li></ol> 3.外电录音<ol><li>安装在记录室中的切片。 <ol><li>使用移液管，将脑切片成灌注脑脊液流动在1浸入式录音室 - 2毫升/分钟，并加热至32℃。用于录像的脑脊液不同于在作为镁离子的浓度是从2毫米增加到4毫所述保持室。 </li><li>固定片以"竖琴"（半圆形不锈钢带在2拉伸尼龙对面股 - 3毫米间距）。地方竖琴从而使股并行运行CA1。增加灌注速度至8 - 10ml /分钟，在32℃。 </li></ol></li><li>在解剖显微镜下的地方有刺激电极和CA1区（ 图2A）的辐射层的表面上的记录电极（填充有脑脊液记录溶液玻璃电极）。放置刺激电极第一然后将记录电极。 </li><li>刺激谢弗络带有120 - 150微安振幅和0.1毫秒持续时间的测试脉冲，并观察所得到的场兴奋性突触后电位（fEPSP）的波形，以确定片健康（ 图2B）。的刺激和记录的电极可能需要被移动到片约50 - 从切片表面100微米获得fEPSP记录具有小纤维凌空和大振幅。该谢弗抵押品诱发fEPSPs是定型，并提供切片健康的良好指标。 Healthy片通常表现出纤维凌空fEPSP小于0.3的振幅比。 </li><li>重新定位的电极。 <ol><li>使用解剖显微镜，移动刺激电极对地层东方明珠Oriens中间并移动记录电极的锥体细胞层尽可能靠近记录电极尽可能如图3A所示。的刺激和记录的电极可能需要大约被推入片50 - 100微米，使得fEPSP响应幅值到120 - 150微安测试脉冲约为1毫伏。 </li></ol></li><li>产生γ振荡。 <ol><li>为了产生γ振荡刺激与组织在200赫兹交付20×0.1毫秒脉冲序列。交付时，每5分钟这强直刺激可以产生可重复的响应。 </li></ol></li><li>使用以下记录的参数。 <ol><li>缩放的输出信号的增益来匹配的输入电压范围模拟到数字转换器。确保最大期望信号偏移使用最少的的输入电压范围的30％。设定收益高信号可能剪辑时要小心。需要现场录音的典型带宽为500赫兹的交流耦合在0.1赫兹，除去基线漂移。 </li><li>数字化至少4 - 比低通滤波器，以避免信号混叠的拐点频率快5倍。 </li></ol></li><li>数据分析。 <ol><li>在5 Hz的高通滤波器的数据，以消除任何基线漂移。识别用衍生物阈尖峰，以最小的事件间隔为10毫秒，一个典型的事件峰值时间和7毫秒回归间隔的〜100毫伏/毫秒的阈值，和100％的分离谷。计算等待时间取为第一所识别尖峰时的刺激伪迹的结束之后发生。这种分析可以用于尖峰，延迟，事件之间的时间间隔的数量的表征，和持续时间计算为如下: </li><li>计算尖峰的数目为每振荡尖峰对于每次扫描的总数被确定。 </li><li>计算振荡延迟。对于每次振荡，减去从所述刺激伪迹的端部与第一所识别尖峰的时间。 </li><li>计算平均事件之间的间隔（ISI）。确定多个探测到的峰值和平均值之间的时间段。 </li><li>计算振荡时间。测量每个振荡中的第一个和最后检测尖峰之间的时间。 </li></ol></li></ol>

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Methods. 183 (2), 107-113 (2009).</li><li>Boido, D., Jesuthasan, N., de Curtis, M., Uva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+Dynamics+During+the+Progression+of+Seizure-Like+Events+in+the+Hippocampal-Parahippocampal+Regions.">Network Dynamics During the Progression of Seizure-Like Events in the Hippocampal-Parahippocampal Regions.</a> Cereb Cortex. 24 (1), 163-173 (2012).</li><li>Antuono, M., K&#246;hling, R., Ricalzone, S., Gotman, J., Biagini, G., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiepileptic+drugs+abolish+ictal+but+not+interictal+epileptiform+discharges+in+vitro.">Antiepileptic drugs abolish ictal but not interictal epileptiform discharges in vitro.</a> Epilepsia. 51 (3), 423-431 (2010).</li><li>Stenkamp, K., 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=Enhanced+temporal+stability+of+cholinergic+hippocampal+gamma+oscillations+following+respiratory+alkalosis+in+vitro.">Enhanced temporal stability of cholinergic hippocampal gamma oscillations following respiratory alkalosis in vitro.</a> J. Neurophysiol. 85 (5), 2063-2069 (2001).</li></ol>

作者什么都没有透露。

一个强大的方法来产生急性脑片CA1γ振荡描述。出现所产生的振动从本地电路能够控制和了解网络振荡12的神经生理学基础上更好的机会。 AMPA受体，GABA A受体，I H和T型Ca 2+通道所需的所有在此模型γ振荡。而这里介绍的地方CA1振荡可以产生强劲，这是依赖于确保脑片是健康的。一个关键步骤是快速去除从头骨的大脑，小心去除，然后迅速浸入冰冷的溶液中不会穿透脑。在理想情况下断头和去除从头骨大脑之间的时间应该不超过30 - 60秒保持片健康21。 通过使用强直性刺激本地CA1γ振荡可不依赖于药物制剂的标准生理录音解决方案产生。这是有利的，对于在疾病模型，其中另外的药物制剂可能混淆解释表征的病症。例如，利用该模型本地CA1活性和敏感性抗癫痫药物中增强了人类基因癫痫22的小鼠模型。利用该模型，调查网络活动没有限制癫痫，并且可以很容易地应用于其他疾病，如阿尔茨海默氏病，自闭症和精神分裂症。而药剂不需要生成本地CA1振荡足够的组织氧合是，由于脑切片23的代谢需求。高灌注率提高切片创造一个更符合生理环境，为脑切片24,25内的氧气和pH值平衡。 使用强直刺激发电机密封的另一个优点e网振荡是一个偶发的实验设计可以用来使网络活动的参数，如延迟发作，持续时间和穗数目更多准备定量，以可重复的方式。与此相反，化学诱导的网络活动是自发13,15,16,26-28和更难以量化。强直性刺激，但是，可以引起变化的NMDA受体依赖性突触可塑性，这可能会导致的变化的网络活动一段时间。为了控制这一点，并能够进行稳定的振荡产生在多个强直性刺激可塑性变化可以通过提高镁离子水平的限制。虽然这个协议增强了可重复性是不透明的NMDA受体的功能没有任何影响。 接口录音室可以提高身体片，更好的信号的刺激较小的器物额外的好处，产生的噪声比和多个协调低强度的刺激。穿孔室使用r拍摄方法也提高长期的实验片的健康。使用微电极阵列录音室或膜片钳使广阔的领域，单个神经元功能中的记录进行测定，以更好地调查潜在的γ振荡及其对更广泛的网络功能的影响机制。掺入的电压和钙传感器以及光遗传学方法将在两个记录和刺激范式引入附加的实验灵活性。类似的协议开发，可能是更相关的所研究的疾病病理等脑区。该协议的最终用途说明这里可能是为建设，并结合实地录音与单个神经元膜片钳和影像学检测振荡的计算模型。

这关联到行为状态不同频带内的脑网络的振荡出现。在啮齿类动物，海马θ振荡（5 - 10赫兹）在探索行为1,2观察，而γ振荡（20 - 80赫兹）与相关联的各种认知过程，包括感知和关注3,4。同步γ网络活动也牵连在病症如癫痫和精神分裂症5,6的病理。例如，γ振荡被认为对应于皮质癫痫病灶5,7,8的领域，并可以作为pharmacosensitivity或电阻，调查在癫痫研究9的两个重要方面的标记。 海马脑切片是已经广泛地用于研究网络活动10-12的模型。各种协议已被开发，以产生在脑切片γ振荡通常我nvolve药理调节，如低镁离子，4-氨基吡啶（4AP），荷包牡丹和红藻氨酸12-17。药理触发振荡的缺点是，他们随机出现后用药，不可靠产生或保持稳定的时间。电触发γ振荡克服许多这样的问题，也有被暂时锁定到刺激事件允许偶发记录和分析的优点。这里的协议描述为通过提供强直刺激的地层东方明珠Oriens海马切片CA1产生γ振荡。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>4-(N-Ethyl-N-phenylamino)-1,2- dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288)</td>
			<td>Sigma-Aldrich</td>
			<td>Z3777</td>
			<td></td>
		</tr>
		<tr>
			<td>Biuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14340</td>
			<td></td>
		</tr>
		<tr>
			<td>6-cyano-7-nitroquinoxa- line-2,3-dione (CNQX)</td>
			<td>Sigma-Aldrich</td>
			<td>C127</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel</td>
			<td>Sigma-Aldrich</td>
			<td>266965</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbamazepine</td>
			<td>Sigma-Aldrich</td>
			<td>C4024</td>
			<td></td>
		</tr>
		<tr>
			<td>(2R)-amino-5-phosphonopentano-ate (APV)</td>
			<td>Tocris Bioscience</td>
			<td>0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Retigabine</td>
			<td>ChemPacific</td>
			<td>150812-12-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline-Cl</td>
			<td>Sigma Aldrich</td>
			<td>C1879-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>S9638-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>Sigma Aldrich</td>
			<td>223506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma Aldrich</td>
			<td>M2670-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode glass</td>
			<td>Harvard Apparatus&#160;</td>
			<td>GC150F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentric bipolar stimulating metal electrode&#160;</td>
			<td>FHC</td>
			<td>CBBPF75</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Isolator</td>
			<td>Getting Instruments</td>
			<td>Model BJN8-9V1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 1800 amplifier</td>
			<td>A-M systems</td>
			<td>Model 1800 amplifier</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>National Intruments</td>
			<td>NI USB-6211</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of local ca1 &#947; oscillations by tetanic stimulation

神经网络振荡是在健康和疾病的大脑活动的重要特征，并且可以通过一系列临床使用药物进行调制。的协议提供生成模型为研究CA1γ振荡（20 - 80赫兹）。这些γ振荡是稳定至少30分钟，取决于兴奋性和抑制性突触活动除了激活起搏器电流。 Tetanically刺激振荡具有许多重复且容易量化的特性，包括穗计数，振荡持续时间，延迟和频率时的网络状态报告。的电刺激振荡的优点包括稳定性，再现性和偶发采集使网络功能的鲁棒表征。可用于CA1γ振荡的这种模型来研究细胞机制，并系统地研究网络如何神经元活动的改变在疾病和毒品。疾病状态药理可通过使用脑切片的从转基因或介入的动物模型可容易地并入到能够选择的药物特异性靶向疾病的机制。

地层东方明珠Oriens的强直刺激产生的可再生的γ振荡（35.4±2.2赫兹）， 见图3B。为了证明是本地网络CA1区CA3从投入是用弯曲32克针切割片在CA2区域内切断产生的振动。在切割片的振荡性质没有从未切割切片差异（P = 0.85;切割片6.16±1.1尖峰，每组6;未切割切片5.89±0.8尖峰，N = 6），表明该振荡机产生的。 这种方法的一个重要优点是记录的稳定性。当破伤风交付在5分钟间隔的振荡是稳定的（在15分钟与30分钟p值= 0.26为尖峰数）为至少30分钟，对于任何给定的片。有一个振荡和其他参数内尖峰的总数片之间的可变性。当d配对实验地毯研究可以这样做，在基线属性差异是次要的因素。对于帧内切片对比的变化可以是一个问题，可以通过充分供电的比较来克服。 接着，所需要的这些tetanically刺激CA1γ振荡的产生的离子通道和受体进行了调查。 α-氨基-3-羟基-5-甲基-4-异唑丙酸（AMPA）药理学阻断受体与20μM的6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）（ 图4A，B）中 ，GABA的A受体具有20μM的荷包牡丹碱（ 图4C），I H的电流与20μM的4-（N-乙基-N-苯基氨基）-1,2-二甲基-6-（甲基氨基）嘧啶氯（ZD7288）（ 图4D），和T型Ca 2+通道与100微米的Ni 2+（ 图4E）分别独立地能够减少产生的尖峰的个数（P &lt;0.05; CNQX N = 6;荷包牡丹N = 7; ZD7288 N = 6; 镍离子N = 6）。 （2R） - 氨基-5- phosphonopentanoate（AP5，20μM）的应用没有影响的尖峰的个数（p值= 0.22; N = 5; AP5，5.9±3.1尖峰;控制，8.3±2.2尖峰），这表明NMDA受体不参与γ振荡生成在此制备。 该制剂是理想的，用于确定药品的网络规模的操作。瑞替加滨是打开钾通道，降低了膜兴奋18-20临床使用抗癫痫药物。瑞替加滨的浴应用产生的剂量依赖性降低振荡的尖峰的数量和持续时间（ 图5）。 <img alt="图1" src="/files/ftp_upload/52877/52877fig1.jpg" /> 图1.原理的头骨和上覆的皮肤呈现重新切割的位置<html>小牛肉大脑。 A1，A2，B和C标记需要作出开拓头骨移除大脑切口的位置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52877/52877fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52877/52877fig2.jpg" /> 图2.放置在CA1谢弗的刺激和记录电极的络脉测试片的健康。 （a）所示的刺激电极（兴奋剂），并记录电极（REC）。（B）一fEPSP由一个120μA刺激（标记*纤维抽射）诱发的代表性例子安置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52877/52877fig2large.jpg" target="_blank">请点击此处查看大图版本这个数字。</a> jove_content"FO:保持together.within页="总是"&gt; <img alt="图3" src="/files/ftp_upload/52877/52877fig3.jpg" /> 图3.配置用来唤起振荡记录和刺激电极。（A）在CA1区的锥体层地层东方明珠Oriens和记录电极（REC）的刺激电极（兴奋剂）的位置。（B）的代表性例子诱发强直性刺激的γ振荡（神器标有"兴奋剂"）。代表跟踪显示计量产出。 ISI跨穗间隔。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52877/52877fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52877/52877fig4.jpg" /> tetanically赫内尔图4.药理特性atedγ振荡。（A）代表一丝展示CNQX的效果。图表展示（B）的 CNQX的效果（20μM; N = 6），（C）的荷包牡丹（20μM; N = 7），（D）的 ZD7288（20μM的; N = 6）和（E）的Ni 2+ （100μM; N = 6）上的尖峰数。数据表示为成对比较。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52877/52877fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52877/52877fig5.jpg" /> 关于网络性能图5瑞替加滨的效果。表示感应振荡（A）中在控制条件下和（B）和的减少网络活动代表性实例&lt;STRONG&gt;（C）与瑞替加滨。在（D）的穗计数，（E）的振荡的持续时间，（F），延迟到振荡开始时，和（G）间的尖峰间隔（ISI）所示的效果的总结。数据表示为成对比较。 * P &lt;0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52877/52877fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Local CA1 ÃŽÂ³ Oscillations by Tetanic Stimulation at JoVE.com

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

振荡是基本的网络性能和疾病和药物调节。研究脑切片振荡允许在受控条件下隔离网络的表征。提供用于制备急性脑片的CA1唤起γ振荡协议。

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 ...

generation-of-local-ca1-oscillations-by-tetanic-stimulation

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.1K 次观看.  University of Melbourne. 振荡是基本的网络性能和疾病和药物调节。研究脑切片振荡允许在受控条件下隔离网络的表征。提供用于制备急性脑片的CA1唤起γ振荡协议。 

视频: Generation of Local CA1 γ Oscillations by Tetanic Stimulation

A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical stimulation of the occipital cortex, but also by non-invasive Transcranial Magnetic Stimulation (TMS)1 of the same cortical regions. The intensity at which a phosphene is induced (phosphene threshold) is a well established measure of visual cortical excitability and is used to study cortico-cortical interactions, functional organization 2, susceptibility to pathology 3,4 and visual processing 5-7. Phosphenes are typically defined by three characteristics: they are observed in the visual hemifield contralateral to stimulation; they are induced when the subject s eyes are open or closed, and their spatial location changes with the direction of gaze 2. Various methods have been used to document phosphenes, but a standardized methodology is lacking. We demonstrate a reliable procedure to obtain phosphene threshold values and introduce a novel system for the documentation and analysis of phosphenes. We developed the Laser Tracking and Painting system (LTaP), a low cost, easily built and operated system that records the location and size of perceived phosphenes in real-time. The LTaP system provides a stable and customizable environment for quantification and analysis of phosphenes.

Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.

一个新颖的方法记录经颅磁刺激诱发幻视

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical ...

科研

神经科学

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书"

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

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 ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

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 ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...