This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

注:以下协议是按照卫生指引的国家机构批准的俄勒冈州动物护理和使用委员会的大学。 1.急性手术<ol><li>麻醉用氯胺酮-美托咪定鸡尾酒的动物，通过腹膜内（IP）的注射剂（ 表1）。 注意:在这些实验中使用的小鼠，通过杂交的依赖于Cre的ChR2-EYFP转基因line10到中间神经元驱动线（; SST-iCre12，SOM +;铬iCre12，CR + Pvalb-iCre11，PV +）生成的。病毒递送的ChR2或相关视蛋白的应该同样有效，假设类似的表达水平得到。 </li><li>在开始手术，确保动物展品温柔的脚趾捏没有反应。整个实验过程中重新施用麻醉剂按需要保持此深度麻醉。如果使用注射麻醉剂，可选择植入一个IP导管维护注射。 </li><li>保持水合用生理盐水或乳酸林格氏通过使用适当稀释的麻醉剂鸡尾酒进行维护注射剂（ 表1）在整个试验溶液（大约3毫升/千克/小时），例如动物。 </li><li>将动物在一个立体或其他头部保持装置。确保头骨是安全性很高。这是为维持稳定的单细胞记录必不可少的。 </li><li>适用opthalamic软膏的眼睛，防止干涩。保持体温在36.5-37℃。 </li><li>进行脑池排水的追加记录的稳定性。使用手术刀刀片，从头盖骨的后表面除去组织以暴露小脑延髓池。做一个小切口，在硬脑膜漏脑脊液。使用刀片的唯一的最前端，并避免接触小脑或脑干。 </li><li>使用手术刀，执行一个小开颅手术（〜2 mm 2的）在感兴趣的皮层区域（听觉皮层，大约-2.3毫米后前囟的，中线为4.5mm横向）。 </li><li>去除硬脑膜和覆盖暴露的皮层有一层暖琼脂糖0.5 - 1毫米厚（1.5％琼脂糖中的盐水，0.9％的NaCl;适用于〜37℃）。保持整个实验的琼脂糖湿润通过周期性地施加几滴盐水。 </li></ol> 2.录像设置<ol><li>准备钨电极（7-14MΩ，127微米直径，12°锥尖，环氧涂层）。强力胶将电极在玻璃毛细管中，并添加热缩管为把手（ 图1）。 </li><li>安装在电动（或液压）显微电极，设置在旅游垂直于皮质表面。滑动线接地的皮肤下，对颅骨。避免与在头部的侧面和颈背部的肌肉接触，因为它们可以产生肌电工件。 </li><li>放大使用的细胞外安培的电信号适合于单细胞记录里音响器，优选配备一个阻抗检查模式。监控正在进行的扣球活动（带通300 - 5000赫兹）有两个示波器和一组有源音箱。这里，所记录的数据以10kHz的采样速率连续地进行数字化;尖峰离线提取。 </li><li>通过琼脂糖推进电极直到前端到达皮层的表面上。注意这一步通过显微镜。 "零"的显微这个位置。 </li><li>到最佳近似记录的深度，在视觉上确认，在一个渗透的末端抽出电极时电极尖端退出皮质表面处的深度为零。 注:操纵器读取和观察到的电极位置之间的差异表明它已漂移相对于组织。这可以通过脑或动物，或操纵漂移的不稳定性引起的。 <ol><li>作为一个额外的检查，以确保这个零设定点对应于皮层的表面上，监视刺激诱发电位而穿过组织的第一几百微米前进。当监测场电位，降低细胞外放大器的低带通滤波器的截止到10赫兹。确认该局部场电位的极性反转周围的〜100μm的深度，该层I / II型边界13附近。这是为听觉皮层可靠的参考点，但是它可能不同其它皮层区。 </li></ol></li><li>安装的光纤耦合到蓝色光源（ 图2）上的手动微操纵。使用显微镜，定位纤维尽量靠近琼脂糖尽可能的表面的前端。居中光束在电极将进入组织。 </li><li>调节光源的具有能够提供一个TTL指定的宽度和所需时间的（晶体管 - 晶体管逻辑）脉冲串的一个控制单元的输出正如一个Arduino（ 图3），一台计算机的I / O卡（如图所示），或市售的脉冲发生器。 <ol><li>监测两个示波器的信号。 </li></ol></li><li>测量总的光功率（mW）在光纤使用功率计的前端。使用此值由纤维芯的横截面面积除以计算照度（毫瓦/毫米2）。首先在10的范围内的强度值- 15毫瓦/毫米2，如果需要向下调整，直到工件被消除。 </li><li>检查光文物在琼脂糖电极，准备进入组织。开始搜索的脉冲串（ 例如，30毫秒的光脉冲，500毫秒刺激间隔（ISI））。 <ol><li>消除瞬态光构件（ 图4），通过重新定位纤维相对于电极的光来改变入射光的角度。如果文物仍然存在，请尝试降低光功率。在日Ë极少数情况下的工件不能被完全消除（仅最小化），扩展搜索脉冲的持续时间。这可以识别真正的神经尖峰帮助。 </li></ol></li></ol> 3.直PINP式" <ol><li>通过大脑推进电极慢慢在约1微米/秒的速率。使用一个示波器监测正在进行的活动。另一方面，触发激光脉冲。 </li><li>监听的光诱发尖峰音频监视器上的微弱"散列"，这表明该电极接近的ChR2 +细胞和经常可以感知的电信号是在示波器上明显好之前。放慢前进的速度。 注意:如果该小区直接处于所述电极的路径，光诱发活动将变得更大（响）。作为电极接近单个中间神经元，散列将解析成均匀的尺寸和形状的小而良好定义的峰值。 </li><li>当李向右诱发尖峰大到足以触发单独的示波器，开始这样做。调整在水平轴的缩放来观察尖峰的波形的精确形状。 </li><li>停止并等待组织来解决。抵制诱惑，将电极靠近细胞。本步骤是稳定的记录是至关重要的。如果几分钟之后的信号 - 噪声比并没有得到改善 - 即，组织已经解决，但它并没有使电池的任何靠近电极的前端 - 提前5微米还并再次等待。 <ol><li>重复这个过程，直到该峰或动作电位的槽能够可靠地捕获与设置远高于本底噪声（ 例如，+ 300μV或更大）的电压阈值。 注意:有样品瓶抑制性时的误报或不明确的风险很小。大多数细胞可以很容易地跟随脉冲序列具有30毫秒的持续时间和间普勒本质上间隔500毫秒或更快，具有2-5毫秒（ 图5）的可靠第一穗延迟。多数光伏+细胞，尤其是能够维持烧成整整1秒（ 图6）。因为这些都是抑制性神经元，disynaptic（间接）激活是不是一个大问题。 </li></ol></li><li>在录音时，显示屏上的第一个示波器正在进行的活动。保持的尺寸以及使用所述第二示波器尖峰形状密切关注。注意其对扬声器的声音质量。 <ol><li>倾听的突然变化，这表明该细胞要么是画得太靠近电极（它有可能被刺穿或损坏），或者是渐行渐远。如果尖峰变大和变形，退了出去;如果他们变得更小，推进电极。缓慢移动的2微米的步骤。 </li><li>通过叠加所有尖峰的波形相一致的峰值或波谷确认记录事后的质量。改变电压THRES按住。在很宽的范围内的阈值的，应该永远只能是均匀的高度（ 图5a）中的一个一致的尖峰形状。 </li></ol></li><li>如果在任何点上的信号变成与尖峰从相邻的神经元受到污染，继续前进。前进很慢，就断开的机会，该电极将传递出的相邻小区的范围内，但留在目标神经元的范围。 （这通常是不成功的。） </li><li>通过皮层（900+微米）在各渗透的整个深度移动电极。如果没有光敏感的神经元被许多穿透，或者反过来，录音污染定期与邻国-CHR 2 - 细胞峰值后出现，尝试一种新的电极。 注意:即使在同一个批次，各电极的阻抗和尖几何形状也有很大差别。这两个因素导致了有效的"侦听半径"的电极，其必须足够大，以检测光诱发尖峰的wh搜索ILE，但限于足以使记录孤立的单一中间神经元。 </li><li>如所希望的测试电极的阻抗。为了避免损坏组织，这样与所述电极中的琼脂糖层的上部的前端，以及外面的大脑。内含有7-14MΩ的范围内，确切的阻抗不是性能，或者收率肯定预测，对于这种应用。这就是说，它是听半径的合理代理:低阻抗电极拿起更多的单位;较高的阻抗，更少。 </li><li>在实验结束时，安乐死的动物通过体制认可的方法，例如麻醉剂过量，或麻醉下的深平面颈脱位。 </li></ol>

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	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. 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The authors have no competing financial interests.

虽然PINP是从概念上简单的，也可以是在实践中具有挑战性。成功的一个主要因素是电极的选择。电气听音半径是关键的参数。它必须足够大，以检测光诱发尖峰时的前端还有一段距离远离的ChR2 +细胞，这样就可以相应地调整前进的速度。同时，它必须被限制，足以使良好的单单元隔离。也就是说，电极不能同时拾取尖峰来自相邻-CHR 2 - 单元。达成适当的平衡，听力半径而言将是任何靶细胞类型?...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

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a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

在神经生理学的一个主要挑战是在大脑皮质中表征的许多抑制性细胞类型的响应特性和功能。 在这里，我们分享我们的战略得到稳定的，良好的分离鉴定，从抑制性的单单元记录在麻醉的小鼠皮层利用利马和他的同事开发出1的方法。录音小鼠在特定的神经元亚群表达Channelrhodopsin-2（的ChR2）上进行。该群体的成员是通过它们的响应于蓝光的短暂闪光。这种技术 - 被称为"PINP"，或神经元群体的光刺激辅助识别 - 可以与标准的细胞外记录设备来实现。它可以作为一种廉价的和可替代的钙成像或目视制导修补，用于靶向细胞外记录到确定的转基因细胞中的用途。 ħERE我们提供了一套指引，以优化在日常实践中的方法。我们具体地细化我们的策略用于靶向小清蛋白阳性（PV +）细胞，但已经发现，它适用于其它中间神经元的类型，以及，如促生长素抑制素表达（SOM +）和钙网膜蛋白表达（CR +）的interneurons。

在这里，我们分享我们的战略得到单单元录音来自遗传，分类抑制性的麻醉小鼠皮层，利用，利马等[1]。 表1详细建议的麻醉剂鸡尾酒，氯胺酮美托咪定，乙酰丙嗪（开发的光遗传学方法"密匙"）。 图1示出了钨微电极，用于记录制备。 图2中包含的电路图一个简单的LED控制单元; 图3包含配置和代码为选通光输出与一个Arduino微...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

19.0K 次观看.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

视频: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

科研

神经科学

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

测绘激光扫描Photostimulation抑制神经回路

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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 ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

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. ...

In Vivo Recording of Intracellular Signals from a Single Neuron

Source: Altwegg-Boussac, T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53576">Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of electrical signals from single brain neurons using a fine-tipped glass micropipette and a specialized setup. The micropipette is carefully inserted into the rat&#39;s brain and is buzzed to penetrate the neuron to record internal neuronal electrical signals.

来自单个神经元的细胞内信号的体内记录

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

指南

摘要

探索更多视频

此视频中的章节

单机在体内录音

测绘激光扫描Photostimulation抑制神经回路

单个锥体神经元的高保真光遗传学控制方法

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

同时电生理记录和抑制剂在啮齿动物大脑微注射

在脑片恐惧电路体外光遗传学解剖

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

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

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

来自单个神经元的细胞内信号的体内记录

指南

摘要

探索更多视频

此视频中的章节

单机在体内录音

测绘激光扫描Photostimulation抑制神经回路

单个锥体神经元的高保真光遗传学控制方法

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

同时电生理记录和抑制剂在啮齿动物大脑微注射

在脑片恐惧电路体外光遗传学解剖

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

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

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

来自单个神经元的细胞内信号的体内记录

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