这项工作是由来自基金会法国，研究所国家德拉桑特的Et德拉RECHERCHE MEDICALE，皮埃尔和玛丽·居里大学和程序"INVESTISSEMENTS德前途报"ANR-10 IAIHU-06的资助。

所有的程序均按照欧盟的指导方针进行（指令63分之2010/ EU），并通过动物实验达尔文伦理委员会的批准。在这里我们描述我们在我们的实验室常规使用的方法，但是大多数步骤可以适于以匹配每个人的具体需要。 1.手术准备注:所有的切口和压点应与局部麻醉（利多卡因或布比卡因）反复渗透。本程序是终端，如果需要的无菌制剂几个修改应来实现。 <ol><li>麻醉与两个局部分离的腹膜内（IP）注射戊巴比妥钠（40mg / kg）和氯胺酮（50毫克/千克）大鼠。 </li><li>让全身麻醉下的动物去反复验证手术的麻醉平面达到（从头到脚没有反应捏）。放置老鼠在FEedback加热毯，并插入直肠探头保持37℃左右的核心温度。 </li><li>放置导管在腹腔，以促进麻醉剂的随后注射，避免反复针穿刺30射孔器官。 <ol><li>夹在头发上的小（〜2 - 3毫米），位于胃的下部右侧或左侧象限内的区域之上的区域。 注意:脱毛霜也可使用。 </li><li>做一个2 - 用锋利的剪刀或手术刀表层3毫米的切口。使用钝性分离，直至腹腔观察去除脂肪和肌肉层。 </li><li>插入约1 - 2厘米空腔小导管，并用手术胶水闭合伤口。 （ 如 ，2法语-对应于内径0.043毫米）:注意的直径和导管的总长度应是最小的，以减少死体积。聚氨酯管是最适合。 </li><li>做一个循环，并缝合Ť他导管皮肤固定到位。 </li></ol></li><li>安装气管插管人工呼吸过程中控制通风。 <ol><li>作为前一步骤，制备感兴趣的区域（1 - 2厘米在气管右柄以上），除去头发和切开皮肤。 </li><li>钝剖析脂肪和肌肉的第几层，然后移动唾液腺放在一边，轻轻解剖肌肉的最后一层暴露气管。 </li><li>小心地取出了气管组织和使用小镊子下方滑动线程。做一个外科医生的结与线程，但不紧张呢。 </li><li>切开2软骨环之间的横向气管。拭子血液在气管如果有的话。 </li><li>插入带有适当的直径的气管导管，拧紧线程的结，以确保管稳定。为了更稳定的线程可以进一步在更高点气管管的连接。 </li><li> SuturE中的伤口闭合或使用手术缝合钉。这里避免手术胶水如果气管套管打算重复使用。 </li></ol></li><li>在立体框架安装动物和仔细监视以下生理变量:脑电图， 血氧饱和度，ETCO 2，心脏率（通过心电图，ECG）和内部温度。监控和​​调整这些变量来麻醉和生理状态的适当深度。具体地，补充麻醉用戊巴比妥钠的必要时小剂量（10毫克/千克）。 </li><li>上双眼应用眼膏，以避免干燥。剪辑过的头发头皮，做一个纵行切口（〜2厘米），切除结缔组织覆使用手术刀或刮匙头骨。 </li><li>请在感兴趣的地区的一个小（1.5〜毫米直径）与开颅牙钻。 注:在这里，初级躯体感觉皮层每桶领域的目标是（7 - 8 mm产品前到双耳线，4.5- 5.5毫米横向中线31）。反复冲洗散热。 </li><li>使用额外的细镊子轻轻地使硬膜一个小洞。颅环锯术来放置脑电图电极内保留一个为0.5 mM区域（见下一步）。永久保留皮层湿润用0.9％NaCl溶液（或人造脑脊髓液）。 </li><li>放置一个低阻抗（〜60千欧）银电极（脑电图电极）上硬脑膜，避免未包括的脑膜皮层区域，并将参考电极上的头部的另一侧的头皮肌肉。 </li><li>在这个阶段（最后戊巴比妥钠注射后30分钟），保持通过经由IP导管戊巴比妥钠（10至15毫克/千克/小时）或芬太尼（3-6微克/千克/小时）的重复注射麻醉。前者将导致缓慢振荡，睡眠状，脑电图图案而后者将导致不同步，苏醒状，​​皮层轮廓。 </li><li>调整人工ventilation系统使得呼吸频率和音量类似于鼠的自发呼吸（正常范围70 - 115呼吸/分钟32）。然后，连接呼吸机的气管插管，并验证正确的胸廓膨胀（两侧）。如果不是这样，调整在气管轴的通风管道的位置。如果需要的话，吸存在于与连接到注射器或真空泵的导管气管的分泌。 </li><li>如果所有的生理变量32-35和脑电图29,36模式反映麻醉的稳定外科平面，做加拉明三乙基的肌内注射在每条腿瘫痪了大鼠，40毫克/公斤的第一注射，然后20毫克/千克每2小时19,29,36,50。 </li></ol> 2.细胞内记录<ol><li>拉玻璃微（微电极锐）用〜0.2微米的尖端比如它的电阻范围betweeñ50和80MΩ一次充满2M乙酸钾（醋酸钾）。 </li><li>放置在一个特定的保持器的吸管用银/氯化银（银/氯化银）丝（通过头部阶段）的移液管溶液到细胞内放大器连接。保持器应附到显微操纵。放置在大鼠的颈部肌肉一个Ag / AgCl参考电极。 </li><li>慢慢插入在大​​脑中的吸移管下到感兴趣的区域并通过监视电压验证其电阻下降响应于电流的步骤。使用动态更新（或扎普）按钮放大器，如果需要清除吸管。 </li><li>用棉签或合成吸收三角形干开颅（注意不要触碰脑电图或细胞内的电极）与硅橡胶或4％琼脂糖覆盖它来降低脑运动之前。 </li><li>降低1吸移管 - 2微米的步骤，直到其电阻增大接近的小区时。然后使用放大器的嗡嗡声功能penetra忒成神经元。 </li></ol> 3.诱导等电国家<ol><li>在这个阶段执行相应的实验方案（ 例如 ，射击反应，细胞内注入电流，电流-电压关系，感官刺激的反应），同时监视神经元的膜电位的脑电图和生理变量。 </li><li>确保细胞内电极是稳定的细胞通过在整个记录会话分析两个膜电位和动作电位特性的稳定性。如果没有，不去下一个步骤，并再次等待，直到记录变得稳定或查询其他神经元。 </li><li>注入戊巴比妥钠的高，但红外致死量（〜90毫克/千克，可低至从戊巴比妥初始条件35毫克/公斤，并为从所述芬太尼初始条件155毫克/千克）经由IP线路。 注意:在15 - 20分钟细胞内和脑电图WAveforms应减速慢行间歇电气沉默以瞬时达到所谓的"突发抑制"配置文件37,38，其折叠逐步向一个完整的等电位状态。据预计，心脏率显著减慢（由约10 - 20％），但血氧饱和度和ETCO 2应该保持相对稳定。 </li><li>如果未达到等电状态，注入戊巴比妥钠的少量（〜步骤3.1剂量的10％）。如果仍不能达到国家电增加更多的麻醉剂之前，请等待15分钟。 </li><li>重复实验方案进行比较的网络动态特性所记录的神经元的综合性能的影响。 注意事项:注射麻醉剂后停止，电气大脑活动应充分3至4小时内恢复。 </li><li>在实验结束时，注入戊巴比妥钠致死剂量（200毫克/千克，IP）来安乐死大鼠。 </li></ol>

The authors have nothing to disclose.

我们在这里描述在两个网络和细胞水平在体内自发脑电活动抑制的新方法。这个过程导致了极端的大脑状态，被称为电昏迷41。从临床观点来看，这样的大脑电活动是最严重的异常，可以在脑电图中看到。它主要以不可逆转的昏迷相关，所有患者要么垂死或继续在持续植物人状态42，但可以通过抑制中枢神经系统药物（如硫喷妥钠）的中毒时造成至少部分逆转，一个偶然的?...

在没有任何环境刺激或行为的任务中，"静止"大脑产生可从头皮进行记录的电活动的连续流，如脑电图（EEG）波。此内源性脑活动的细胞内相关成分的特征在于，背景膜的电压波动（也称为"突触噪声"），这是由反映传入网络1,2的正在进行的活动兴奋性和抑制性突触电位的组合。这种自发活动的频率和振幅与警觉的不同状态而变化。阐明网络活动的单神经元的兴奋性和反应能力的影响是神经科学的3,4的主要挑战之一。 许多实验和计算研究探索持续突触活动对综合propertie功能的影响神经细胞的的。然而，由背景突触噪声影响不同的神经元的参数的作用仍然是难以捉摸的。例如，膜去极化的平均等级已经发现与感觉输入触发的动作电位的能力正5,6或负7-9相关。此外，虽然一些调查表明，膜电位的波动，从传入的突触输入的连续变化的数据流得到的，强烈地受到调制的其输入输出关系3,10-13增益影响单个神经元的响应，其他指示通过分流抑制介导的细胞膜输入电导变化是足以调节神经元增益无论膜的波动14,15的幅度。最后，在清醒的动物进行最近的研究强调如何在单个神经元感觉信息的处理需要依靠警惕一个的状态ND当前的行为需求16,17。 阐明一个给定的处理的在高度互连的系统中的功能性作用一个直接的策略是，以确定如何其缺乏特异性改变系统的运作。该方法已在神经科学研究利用实验病变或不同脑区18-21的灭活，或特定的离子通道22,23的药理学阻断广泛使用，例如。值得注意的是，已经在体内施加到推出功能连接和网络动态如何影响单细胞计算24-27。然而，迄今为止本地操作旨在阻断神经元的放电和/或扰乱它们的基本生物物理特性可以是部分有效的，被限制为相对小的脑体积28。 为了克服这些限制，我们开发了体内实验方法进行新的大鼠来比较记录在一个给定的大脑状态单个神经元， 即，嵌入在一个特定的网络的动态，到整个大脑突触活动29的完全抑制后所得的电生理特性。在控制条件下，可能产生两个不同的皮质动态。睡眠就像electrocorticographic（脑电图）模式是由中等剂量戊巴比妥钠注射引起。另外，小振幅的快波脑电图相媲美的大脑皮层的活动清醒状态（醒状图案）基本可以用芬太尼注射产生。接着，同时保持相同的脑电图和细胞内记录时，由高剂量的戊巴比妥钠，其特征在于等电脑电图和细胞内活性的全身注射得到的内源性脑电活性的完全沉默。因为这样一个极端的昏迷诱导可能产生致命的consequen生物功能CES上，生理变量的仔细和连续监测是必不可少的。因此，我们认真随后心脏跳动频率，潮气末CO 2浓度（ETCO 2）时，O 2饱和度（SPO 2）和大鼠的核心温度在整个实验。 我们评估期间使用锋利微电极，其特别适合于在体内长时间稳定记录这些不同状态的单神经元特性。此处描述的方法，可以与其它电和成像方法相结合，并且可以延伸到其他的动物模型。

<table><tbody><tr><td>Sodium Pentobarbital</td><td>Centravet</td><td>Pentobarbital</td><td></td></tr><tr><td>Ketamine 500</td><td>Merial</td><td>Imalg&amp;egrave;ne 500</td><td></td></tr><tr><td>Fentanyl&amp;nbsp;</td><td>Janssen-Cilag</td><td>Fentanyl</td><td></td></tr><tr><td>Xylocaine</td><td>Centravet</td><td>Xylovet</td><td></td></tr><tr><td>Gallamine triethiodide</td><td>Sigma</td><td>G8134</td><td></td></tr><tr><td>ECoG amplifier</td><td>A-M Systems</td><td>AC amplifier, Model 1700</td><td></td></tr><tr><td>Intracellular amplifier</td><td>Molecular Devices</td><td>Axoclamp 900A</td><td></td></tr><tr><td>Data acquisition interface</td><td>Cambridge Electronic Design</td><td>CED power 1401-3&amp;nbsp;</td><td></td></tr><tr><td>Data analysis software</td><td>Cambridge Electronic Design</td><td>Spike2 version 7</td><td></td></tr><tr><td>micromanipulator</td><td>Scientifica</td><td>IVM-3000</td><td></td></tr><tr><td>Capillary Puller</td><td>Narishige</td><td>PE-2</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>GC150F-10</td><td></td></tr><tr><td>Silver wire 0.125 mm (intracellular recording)</td><td>WPI</td><td>AGT0525</td><td></td></tr><tr><td>Ag-AgCl reference</td><td>Phymep</td><td>E242</td><td></td></tr><tr><td>Silver wire 0.25 mm (ECoG recording)</td><td>WPI</td><td>AGT1025</td><td></td></tr><tr><td>Artificial respiration system</td><td>Minerve</td><td>Alpha Lab</td><td></td></tr><tr><td>Physiological parameters monitoring</td><td>Digicare</td><td>LifeWindow Lite</td><td></td></tr><tr><td>Heating Blanket</td><td>Harvard Apparatus</td><td>507215</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>15005-08</td><td></td></tr><tr><td>Forceps Dumont #5</td><td>FST</td><td>11295-10</td><td></td></tr><tr><td>Forceps Dumont #5SF</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>IP Polyurethane catheter - 0.43x0.69 mm&amp;nbsp;&amp;nbsp;</td><td>Instech</td><td>BTPU-027</td><td></td></tr><tr><td>Silicon elastomere</td><td>WPI</td><td>KWIK-CAST</td><td></td></tr><tr><td>Dental drill</td><td>NSK</td><td>Y1001151 and P496</td><td></td></tr><tr><td>Surgical glue</td><td>3M</td><td>vetbond</td><td></td></tr></tbody></table>

induction of an isoelectric brain state to investigate the impact of endogenous synaptic activity on neuronal excitability in vivo

顺便神经元过程信息既取决于它们的内在膜特性和对传入突触网络的动态。特别是，内源性产生的网络活动，这强烈变化作为警戒的状态的函数，显著调制神经元的计算。探讨自发性脑动态如何影响不同的单个神经元"综合性能，我们开发了包括由高剂量戊巴比妥钠的全身注射的方式在体内抑制所有大脑活动的老鼠了新的实验策略。皮质活动，通过组合电图（脑电图）和细胞内记录连续监测正在逐渐放慢，从而导致稳定的等电轮廓。这种极端的脑的状态，把老鼠进入深度昏迷，小心地通过在整个实验测量动物的生理常数监视。细胞内řecordings允许我们表征和比较嵌入到生理学相关皮质动力学，如在睡眠 - 觉醒周期遇到了同样的神经元的综合性能，并且当大脑完全沉默。

诱导和维持的等电脑状态是在体内实验过程的细腻。它已被证明是一种强大的工具，直接研究了神经元兴奋性和传递函数29的皮质的网络活动的影响。 图1示出了（ 图1A之前多参数监控，包括脑电图和重要的常量，动物的生理状态的）和之后（ 图1B）诱导的等电状态的。 <img ...

Watch this Scientific Journal Video about Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo at JoVE.com

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

在此过程中生理相关的脑状态和正在进行的电活动完全取消后，执行从单个神经元长期在体内的细胞内记录，导致大脑电状态。动物的生理常数过渡到人工昏迷状态期间仔细监测。

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em&gt;在体内</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

induction-an-isoelectric-brain-state-to-investigate-impact-endogenous

Research

JoVE Journal

Neuroscience

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

8.0K 次观看.  Inserm U1127. 在此过程中生理相关的脑状态和正在进行的电活动完全取消后，执行从单个神经元长期在体内的细胞内记录，导致大脑电状态。动物的生理常数过渡到人工昏迷状态期间仔细监测。 

视频: Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

科研

神经科学

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

啮齿类动物和人体组织急性 ictal 活性的产生及按需启动

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

绘制脑血流和电场图:小鼠的电极放置

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

作者聚焦:基于刺激的方法改善阿尔茨海默病模型中的脑血流 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

摘要

探索更多视频

同时电生理记录和抑制剂在啮齿动物大脑微注射

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

啮齿类动物和人体组织急性 ictal 活性的产生及按需启动

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

摘要

探索更多视频

同时电生理记录和抑制剂在啮齿动物大脑微注射

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

啮齿类动物和人体组织急性 ictal 活性的产生及按需启动

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

麻醉小鼠颅外刺激电极的放置和脑血流量和颅内电场的测量

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问