This work was supported by Ministry of Science and Technology (MOST) grants MOST104-2410-H-002-053 &amp; NSC99-2320-B-002-026-MY3. This manuscript was edited and proofread by Mr. Brian Chang, who has experience revising professional documents.

所有实验方案由国立台湾大学的机构动物护理和使用委员会（IACUC）的批准。 1，立体定向手术植入耳蜗电图电极，电极肌电图，脑热敏电阻和注射引导套管<ol><li> 对于大鼠（250 - 350克，6至8周龄的Sprague-Dawley大鼠） <ol><li>麻醉大鼠腹腔注射50毫克/公斤zoletil。按捏后肢后观察缺少响应确认麻醉合适的深度。应用眼膏，刮胡子的皮毛，和消毒用聚维酮碘溶液和75％乙醇的皮肤。注射抗生素（青霉素G），以防止感染。 </li><li>准备手术刀，剪刀，止血钳，纱布和烧灼的机器进行手术。由高压釜中并通过75％乙醇的烧灼消毒外科齿轮和纱布。 </li><li>将耳杆进入耳道和老鼠安装到立体定向。 </li><li>运用手术刀，使头骨大约2厘米的正中切口沿着两个耳朵之间的直线，尾部移动。剪辑皮瓣具有止血，露出颅骨，取出组织过用手术刀头骨。 </li><li>钻头八孔（F1，F2，P1，P2，O1，O2，R 1，和R 2），每个约0.7mm的直径，在具有旋转工具的头骨。拧上额叶，顶叶，枕叶8脑电图电极和在左和右半球都小脑。这些电极用于局灶性癫痫检测。 <ol><li>用于记录电极以下坐标:从中线2.0毫米前方前囟和2.5毫米，顶叶（P1和P2）:额叶（F1和F2）-2.0毫米前方前囟和3.0毫米从中线和枕骨（O1和O2）:-5.5毫米前方前囟以及从中线3.0毫米。 </li><li>放置两个参比电极（R1和R2）在小脑（-11.0毫米前方前囟并从midli4.0毫米NE）。 </li></ol></li><li>在大鼠中，钻三个孔并放置在右额（F2）中，用相同的坐标的皮质顶叶（P2）的两个螺旋EEG电极的单独的基团，在步骤1.1.5.1中所述。将在左小脑（R1），其作用是地面动物，减少信号假象第三脑电图电极。这些电极被用于确认全身性癫痫和分析警惕阶段。 </li><li>分离颈部皮肤和肌肉，并插入两个EMG电极插入颈部肌肉。 </li><li>钻头骨另一个孔和一个放置注射引导套管插入左侧杏仁（AP，从囟门2.8毫米; DV，相对于前囟门7.8毫米; ML，4.2毫米）的大鼠。坐标将被改编自Paxinos和Watson鼠图谱10。 </li><li>钻取头骨一个更大的孔（直径为1.6毫米）和顶叶皮层的表面上，这将在后面凝成插入校准的30千伏热敏电阻器，以MONITOR大鼠皮层温度。 </li><li>用纱布和烧灼停止当它发生出血。 </li><li>路线从脑电图电极和电极肌电绝缘导线为底座和热敏电阻连接到系绳。水泥基座和引导插管用牙科丙烯酸头骨。 </li><li>与polysporin（杆菌肽锌/多粘菌素B），以防止感染局部治疗的切口。给一周中的水的动物都布洛芬和青霉素G手术后。 </li></ol></li><li> 对于小鼠（20 - 30克，6至8周龄的C57BL / C小鼠） <ol><li>麻醉通过IP注射小鼠用50mg / kg的zoletil和按捏后爪后观察缺乏响应的确认麻醉的适当深度。应用眼膏。剃毛发后，消毒用聚维酮碘溶液和75％的乙醇的皮肤。注射抗生素（青霉素G），以防止感染。 </li><li>将耳杆插入耳道安装鼠标的立体定位。 </li><li>使用手术刀，使在头骨的近似1.5厘米正中切口沿两个耳朵之间的线路，尾部移动。剪辑皮瓣具有止血，露出颅骨，取出组织过用手术刀头骨。 </li><li>戳与手术剪颅骨两个孔并放置两线​​脑电图电极上的右额叶（F2:2.0毫米到前囟和+1.5到中线）和左顶叶（P1:-3.0毫米到前囟和-2.5毫米到中线）。 </li><li>分离颈部皮肤和肌肉，并插入两个EMG电极插入颈部肌肉。 </li><li>从金属丝脑电图电极和EMG电极绝缘导线水泥连接到阴端子和一2.54毫米连接器，然后用牙科用丙烯酸系的头骨。 </li><li>与polysporin（杆菌肽锌/多粘菌素B），以防止感染局部治疗的切口。给在水中的动物都布洛芬和青霉素G一凌晨手术后ķ。 </li></ol></li><li>允许所有的动物，以之前的实验的开始恢复七天。 </li><li>房屋分别大鼠或小鼠，在各个记录笼，在隔离室，其中的温度保持在23±1℃，和光:暗（L:D）的节奏被控制在12:12小时循环（40瓦×4管照明）。提供食物和水随意 。 </li><li>通过系绳1周连接脑电图，肌电图，和热敏电阻到放大器手术后开始录音。 </li></ol> 2.局灶性癫痫TLE，SE和自发性癫痫复发的建立<ol><li> 对于大鼠 <ol><li>通过使用微量注射泵注射引导套管辖毛果芸香碱（2.4毫克/μL）进入左东航0.5微升。喷射率应在0.2微升/分钟设定为诱导焦TLE。 </li><li> IP注射300毫克/公斤的毛果芸香碱诱导广义外延复发性SE lepsy。 </li></ol></li><li> 对于老鼠 <ol><li>在一个特定的ZT点IP管理PTZ（0.035毫克/克鼠标体重），隔日1次。五个连续注射会导致自发和循环地全身性癫痫的发展。 </li></ol></li><li> 对于大鼠和小鼠 <ol><li>由5000放大脑电图信号，0.1和40赫兹之间过滤模拟带通。 </li><li>使用一个A / D转换板将模拟ECoGs信号转换成数字信号，与一个128赫兹的采样率。 </li><li>使用视觉得分软件和分析发作和癫痫的持续时间。测量，以便表示的持续时间的时间比例。 </li><li>由癫痫尖峰的外观比振幅2 mV的更大，拥有超过30秒13的持续时间定义脑电图癫痫。 </li></ol></li></ol> 3.警惕的状态分类<ol><li> 对于大鼠<ol><li>通过使用记录的12-S集内ECoGs，肌电图，脑温度和运动获取的参数确定警惕状态。通过系绳1周连接脑电图，肌电图，和热敏电阻到放大器手术后开始录制。 3.1.7 - 根据步骤3.1.5使用自定义国产软件评分的状态。 </li><li>测量使用红外运动检测器的运动活动，整合的信号每隔1秒，并存储信号。 </li><li>测量皮质温度并存储信号。 </li><li>根据我们先前定义的标准分类，14警惕的状态。 </li><li>通过使用下面的特征评分觉醒:（ - 4.0赫兹0.5）和下部THETA功率高频频谱小振幅ECoGs，更高的增量功率（6.0 - 9.0赫兹）;占主导地位的自主活动;高肌电活动;逐步增加对皮质的温度。 </li><li>通过跟随着分数NREM睡眠摹特点:三角波主导ECoGs大的振幅，谢绝肌电活动，减少皮质温度，并没有自主活动。 </li><li>通过使用下面的特征得分REM睡眠:ECoGs的振幅与主导THETA频率，突然增加皮质温度，最小EMG活动，和低自发活动与身体抽搐降低。 </li></ol></li><li> 对于老鼠 <ol><li>重复警惕状态的分类中如在大鼠中进行，除有来自热敏电阻没有记录皮质温度小鼠。 </li></ol></li></ol> 4.性能大鼠手册针灸和EA的<ol><li>大鼠被麻醉了EA协议。 </li><li>通过定位解剖穴位和穴位确认低皮肤阻抗。当检测到低皮肤阻抗的指示灯闪烁。注:风池穴取穴位于3毫米远离后正中线上是吐温两个耳朵，脖子上的。 </li><li>以2mm的深度插入不锈钢针入穴。 </li><li>抽搐插入针每隔5分钟10次。 </li><li>使用功能性电刺激，用1毫安通过针穴位强度双相传送脉冲（150微秒的时间每个）的一列火车。 </li><li>执行假针灸或假EA作为对照。 </li></ol>

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The authors have nothing to disclose.

选择一个可行癫痫的动物模型为每个实验目的是至关重要的。我们的目标之一是阐明电针对癫痫抑制的作用。 EA是一种替代药物，可能在癫痫表现出的治疗效果，并在中国古代文献中已有记载。然而，有一个缺乏科学证据来证明这一点。为了确定EA对癫痫的影响，我们主要侧重于EA的影响轻度局灶性癫痫，而不是严重的癫痫大发作或SE。我们以前的研究中使用大鼠的焦点TLE模型来证明对毛果芸香碱?...

癫痫是在整个病人的寿命反复发作会出现一种常见的神经系统疾病。大多数癫痫复发可以通过抗癫痫药物（AEDs）良好的控制。然而，癫痫患者约30％的开发难治性癫痫1。癫痫原因睡眠干扰，这可能进一步加剧复发。证据表明，癫痫患者既可以在夜间扰乱睡眠或导致白天过度嗜睡2,3。我们先前的研究进一步表明，癫痫在时间授时（ZT）0，即发生在光的光周期的开始:黑暗循环，减少睡眠;这是由促肾上腺皮质激素释放激素（CRH），一个稳态因子介导的。癫痫在ZT13（暗期的开始）增强的另一稳态因子，白介素-1（IL-1），这增加睡眠的表达。当癫痫发生在ZT6，的中间睡眠昼夜节律发生改变光周期4,5。另一方面，睡眠问题进一步加剧癫痫6的进展和复发。根据上述证据，我们力图揭示一个最佳的治疗方法同时控制癫痫和防止干扰睡眠癫痫患者。我们以前发现，电针与10赫兹的刺激频率，其中的电流在一定量通过一个不锈钢针递送到穴位，成功地抑制了电图（脑电图）癫痫活动和癫痫引起的睡眠障碍7 。 EA用100赫兹的刺激频率进一步降低癫痫活动和大鼠8,9-睡眠中断。这个实验成功取决于三个因素:首先，一个可行的癫痫动物模型;其次，对于在啮齿类动物睡眠记录和分析的方法;第三，针灸的准确性能和穴位位置的服务的准确性附件。 癫痫病一直分为两大类型:局灶性癫痫和全身性癫痫。我们感兴趣的是焦距颞叶癫痫（TLE），全身性癫痫，癫痫持续状态（SE），和自发全身性癫痫的复发。因此，不同的操作应用到我们的实验，创造出适合癫痫模型。建立联络的TLE，毛果芸香碱的低剂量给予到杏仁核（CEA）的左中央核。为了验证此模型，六脑电图电极植入在额叶（F1＆F2），顶叶（P1＆P2）和枕骨（O1＆O2）裂片在左和右半球两者，和另外两个参考电极（R 1 R2）被放置在两个半球，小脑。一个附加的显微注射导向插管手术植入到左杏仁（AP，来自前囟2.8毫米; ML，4.2毫米; DV，相对于前囟7.8毫米）。坐标适于从Paxinos和Watson鼠图谱10。如果焦点TLE被成功地诱导，只从（P1）上左顶叶的电极，其是邻近左侧杏仁记录，应获得主导癫痫ECoGs，与来自其他脑电图电极记录无显著痫ECoGs。毛果芸香碱的腹膜内（IP）注射入大鼠诱发全身性癫痫和SE，但是这可能是致命的。五戊四氮（PTZ）每次注射之间的为期一天的间隔注射IP诱导成功自发全身性癫痫小鼠和也保证了小鼠的生存。两线脑电图电极植入在小鼠额叶和顶叶皮质接受脑电图信号，并核实自发反复癫痫。 多导睡眠图（PSG）是记录睡眠过程中发生的生理变化的综合方法，它可以客观地划分成睡眠非快速眼动（NREM）和r的不同阶段APID眼动（REM）睡眠。 PSG记录的身体功能参数，包括脑电波（脑电图，EEG），眼球运动（眼电，EOG），骨骼肌音（肌电图，EMG），心脏节律（心电图，心电图）和血氧水平和呼吸参数。在大鼠中，我们记录ECoGs，肌电图，皮质下，和运动活动的警惕状态分为清醒，NREM睡眠和快速眼动睡眠。在小鼠睡眠分析使用ECoGs，肌电图，和自主活动的结果进行。大鼠手术有三个脑电图立体定向手术拧在额叶，顶叶电极，对侧小脑皮层植入。警戒状态（觉醒，NREM睡眠和REM睡眠）的采集后判断根据来自ECoGs，肌电图，脑温，和运动活动所获得的参数进行。进行分类在大鼠和小鼠的动物的行为的详细条件在叔描述他协议。 大鼠和小鼠需要用低剂量zoletil的（25毫克/千克），它是立体手术过程中通常施用麻醉剂的一半剂量被麻醉，执行手动针刺或EA之前。这一剂量可以向动物注射后醒来30至35分钟。无论手动针刺或EA在黑暗时期的开始进行的，以30分钟的恒定的时间段，并且每个动物被连续为两到三天处理。刺激EA电流通过被插入到穴的不锈钢针递送到特定穴位。刺激电流是双相方脉冲序列，其中脉冲持续时间为150毫秒和刺激强度为1毫安。如果干针用于手动针刺，插入穴位针头抽动每隔5分钟10次。手动针刺或EA的困难的部分是定位取穴在啮齿类动物。组委会在大鼠或小鼠穴位通货膨胀类似于在人体内的解剖学位置。例如，双侧凤池穴位于从颈部后部中线3毫米远，两个耳朵，这类似于在人体11的解剖位置之间。此外，在皮肤上的低阻抗的穴位，可以进一步证实。假针灸或假EA操作是必要的针灸或EA实验。假针刺或假的EA应在靠近穴，如腋下12附近的非穴位进行。 要成功地观察针刺或电针的癫痫和癫痫引起的睡眠中断的影响，以下因素必须到位:一个可行的癫痫动物模型，癫痫ECoGs的精准分析和癫痫的复发，要警惕状态进行分类的方法和在啮齿类动物针刺或EA的精确性能。

<table><tbody><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zoletil</td><td>Virbac</td><td></td><td>50 mg/kg i.p.</td></tr><tr><td>pilocarpine</td><td>Sigma-Aldrich</td><td>P6503</td><td>300 mg/kg i.p.; 1.2 mg microinjection</td></tr><tr><td>PTZ</td><td>Sigma-Aldrich</td><td>P6500</td><td>0.035 mg/mouse</td></tr><tr><td>polysporin</td><td>Pfizer</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ECoG electrode</td><td>Plastics One</td><td>E363/20</td><td>screw electrode for rats</td></tr><tr><td>Pedestal</td><td>Plastics One</td><td>MS363</td><td></td></tr><tr><td>Cannula</td><td>Plastics One</td><td>C315G/spc</td><td></td></tr><tr><td>Thermistor</td><td>Omega Engineering</td><td>44008</td><td></td></tr><tr><td>Dental acrylic</td><td>Tempron</td><td></td><td></td></tr><tr><td>Stereotaxic Instrument</td><td>Stoelting</td><td></td><td>Dural arms</td></tr><tr><td>&lt;strong&gt;Recording equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ECoG amplifier</td><td>Colbourn Instruments</td><td>V75-01</td><td></td></tr><tr><td>A/D Board</td><td>National Instruments</td><td>NI PCI-6033E</td><td></td></tr><tr><td>Infrared-based motion detectors</td><td>Biobserve GmbH</td><td>custom-made</td><td></td></tr><tr><td>ICELUS</td><td>G-System</td><td></td><td></td></tr><tr><td>AxoScope 10 Software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Acupuncture needs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stainless needles</td><td>Shanghai Yanglong Medical Articles Co.</td><td>32 gauge x 1&amp;rdquo;</td><td></td></tr><tr><td>Functions Electrical Stimulator</td><td>I.T.O., Japan</td><td>Trio 300</td><td></td></tr><tr><td>AcuPen</td><td>Lhasa OMS</td><td>Pointer Excel II</td><td></td></tr></tbody></table>

manipulation of epileptiform electrocorticograms (ecogs) and sleep in rats and mice by acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

有不同的大鼠和小鼠模型，以满足不同的癫痫类型的需求。为了诱导焦点TLE，毛果芸香碱0.5μL（2.4毫克/μL）给予到左侧杏仁核。和稀有癫痫活动由脑电图电极的其余部分拾取（ 图1A:A，C，D，E:主要的癫痫样ECoGs从脑电图电极上的左半球的顶叶（ 二图1A）获得，和f）当毛?...

Watch this Scientific Journal Video about Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture at JoVE.com

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

癫痫Electrocorticograms（ECoGs）的操作，并睡在针刺大鼠和小鼠

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

manipulation-epileptiform-electrocorticograms-ecogs-sleep-rats-mice

Research

JoVE Journal

Behavior

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

9.5K 次观看.  Aletheia University, Tainan Campus. This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

视频: Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

科研

神经科学

Polygraphic Recording Procedure for Measuring Sleep in Mice

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.

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.

印刷业记录程序在小鼠体内测量睡眠

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

量化睡眠小鼠的光谱功率和心率的慢速动力学

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

使用腺相关病毒在小鼠中瞄准科菲林操纵的大脑皮层区域的电皮质记录

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

使用 3D 打印支架对小动物进行体电针

Bulk Electroacupuncture Operation for Mice or Young Rats

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering its promotion. The mechanistic study requires mouse or rat models to address this issue. However, these animals are not obedient to the experimental process, which is time-consuming. To solve these problems, we designed a 3D-printed small animal body bulk fixator to improve the efficiency of EA's animal experiments. This video shows in detail how to use the fixator to perform bulk EA on mice or young rats. For the selection of acupoints, the anterior oblique line of vertex temporal (MS6 head) and Tianshu point (ST25 belly) were chosen to verify the effect of the fixation device with prone positioning and supine positioning. Using the 3D-printed small animal holder allows multiple rodents to be immobilized and treated simultaneously, reducing the time and resources required for the experiment. This technique could be applied to other animal models by 3D printing different sizes and could potentially be used for various fixing conditions. The device is beneficial for the promotion of experimental scientific research in EA.

作者聚焦:使用 3D 打印支架同时进行多次电针手术

Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

小鼠或幼鼠的批量电针操作

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...

癫痫Electrocorticograms（ECoGs）的操作，并睡在针刺大鼠和小鼠

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小鼠或幼鼠的批量电针操作

小鼠或幼鼠的批量电针操作

小鼠或幼鼠的批量电针操作