我感谢安东尼泰勒在体内的细胞内记录和一个细胞内染色技术的初步发展的帮助，布朗和大卫麦克斯韦的初步培训。我感谢帮助与collocalization宏M.银。我有许多学者与人合作提供洞察到这种技术的发展，包括R.峡谷，M. Moritani，P.罗，R. Ambalavanar。这项技术是由美国国立卫生研究院拨款DE10132，DE15386和RR017971相当大的支持。

1。动物的制备<ol><li>麻醉大鼠用戊巴比妥钠（50mg/kg的IP）和加热垫的地方。剃须皮肤覆动物快船队的后头骨。检查动物，以保证手术的麻醉水平，已撤回反射和发声的情况下获得通过测试时脚趾捏了眼睑的反射的情况下，作为。检查每15分钟的麻醉水平，并保持了anesthetisa注射戊巴比妥钠按15mg/kg，每45分钟的手术水平。 </li><li>使用无菌技术操作，使切口在腹股沟区的远端所形成的腹部和大腿内侧的折痕，然后插入到股静脉导管（直径为1mm，粘土亚当斯）和股动脉。请在颌下区的正中切口，反映舌骨肌肉。在气管的一个小切口插入导管（直径2mm），让空气流通。泰伊缝合cannual place.Monitor通过动脉插管全身的舒张压和收缩压血压，以确保它周围的trachael。现在除了监测撤离和眼睑的反射监测血压，以评估麻醉水平。需要通过静脉插管时，执行额外的麻醉。 </li><li>大鼠放入一个立体框架，进行开颅手术暴露小脑。将气管插管啮齿类动物呼吸机。通风量在加湿空气的100/min率与2厘米3的动物。应该保持一个积极的结束，1厘米的H 2 O呼气压力，以防止肺萎陷。每隔15分钟hyperinflate肺部，以防止肺不张。然后盖上温暖的矿物油（30℃）的大脑表面。要小心，以避免直接下顶叶和interparietal骨骼之间的交界处，位于大静脉窦。 </li><li>氰基丙烯酸酯胶，再加上电磁振动器一杆，杆附着在颚纵裂。振动器移动下颌骨的使用命令信号从一个A / D转换的计算机输出或信号发生器。 </li><li>开颅附近的皮肤下放置一个siliver氯化银接地电极。 </li></ol> 2。电极的制备<ol><li>制造石英或铝硅酸盐玻璃的微电极，使用水平电极拉马。 </li><li>填充5-10％溶解于0.25米的三KCl和0.5M HCl缓冲液（pH值7.6）或2％的盐水tetramethlyrhodamine（pH值6）biotinamide微电极。检查电极测试仪电极阻抗。要记录从大直径的轴突，使电极与一个60 - 80MΩ的阻抗，小轴突和interneurons使电极阻抗为80 -150MΩ。 </li><li>微电极放入头阶段的静电。可视化，通过一个小望远镜（20X与一个封闭的十字线），这是动物的头后面的位置固定在电极。使用的望远镜是非常重要的，因为它允许对电极stereotaxically定位非常精确。 </li></ol> 3。电生理记录和细胞内染色<ol><li>在软脑膜小开放和过度的电容反馈，使电极接触时大脑产生一个反馈信号。这使得大脑表面的准确位置。 </li><li>产生反复下颌位移和提前进入大脑的电极与步进电机。 </li><li>神经impalements承认直流潜在的突然下降和确定intrasomatic神经元突触活动正在进行impalements。嗡嗡过度补偿电容的电极或窃听很少产生一个成功的细胞渗透。由于高阻抗电极，神经元的反应很少观察到渗透前。 </li><li>之后刺穿一个神经细胞，被认为是稳定的渗透，使用的坡道和举行正弦下颌运动特征的神经元的反应，通过探测与非导电探针头部周围的皮肤和口腔内的5地图的神经元的感受野如木棍。如果有关的研究，研究神经元的其他功能，如肌肉收缩和有害刺激的相关刺激的反应，6初级传入痛觉的神经受体伤害性刺激作出反应。如戊巴比妥钠的anesthesetics不阻止在初级传入神经元轴突传导而抑制突触传递。因此，在初级传入的神经元轴突轴突传导被保存下来。 </li><li>电流注入（DC，1 4nA）共注射时间为15 70nA分钟。显示器在电流注入电极的渗透和停止，如果比- 30mV的膜电位变得更加积极。</li></ol> 4。组织处理<ol><li>安乐死的动物过量苯巴比妥（140mg/kg四）和灌注血管冲洗（0.9％氯化钠38 ° C含肝素500个单位和1毫升2％xylocaine 4％多聚甲醛在0.1M磷酸盐缓冲液（pH值7.4） 。 </li><li>删除在50 -100μm的大脑和部分它使用一个vibratome在额叶，矢状面或水平面。 Secions收集，PBS在25℃彗星自由浮动。 </li><li>民建联大脑的过程培育1-2％正常山羊血清和1％的Triton X - 100在0.01M PBS，随后在抗生物素蛋白生物素复合潜伏期（1:50精英Vectastain）。使用镍，民建联与H 2 O 2反应部分。为了处理得克萨斯红，生物素 - 亲和素复合物在PBS（1:50）4℃过夜孵育部分° C，然后在4％得克萨斯红抗生物素蛋白DCS在PBST孵育4℃过夜。染液一个荧光的尼氏染色（NeuroTrace）20分钟23 ° C节</li><li>如果神经元与罗丹明注入，可视化注入神经元直接用荧光显微镜（前545nm）。 </li></ol> 5。逆行标签，免疫细胞化学，共聚焦成像，定量的共定位分析相结合的方法这种技术与其他方法结合起来的成功在很大程度上取决于良好的细胞内的标签。 <ol><li>在这里可以很容易地结合逆行神经标记方法可视化 7-10要做到这一点，麻醉动物和使用无菌技术，注入辣根过氧化物酶（20％的辣根过氧化物酶，西格玛西格玛六）如神经示踪和1％小麦胚芽aggultinated到周边的目标区域，如咬肌的辣根过氧化物酶（Sigma公司）。这种神经细胞示踪将采取到周边的轴突和运动神经元somata通过轴突运输运输。对于咬肌，示踪15μl注入使用无菌的10微升microsyringe肌肉。动物，然后放置在加热垫和监测，直到恢复从麻醉，然后放置在他们的笼子里进行恢复。 24小时后，麻醉动物和生理特征的神经元和细胞内染色。然后灌注所述以上的H​​RP使用四甲基联苯胺（TMB）作为chromagen和钨酸钠作为稳定和加强与钴的存在和过程组织切片的动物。放置节在1％的钠钨溶解于0.1​​M PBS（pH值6.5）和0.0007％TMB无水乙醇和丙酮中溶解，在15 ° C下20分钟。然后加入1.0毫升的0.3％H 2 O 2％的潜伏期为60分钟的解决方案100毫升反应的组织切片。二氨基联苯胺（pH值7.4），0.05％，0.02％钴，和0.01％的H 2 O 2 0.1M PBS液（pH 7.4）10分钟，冲洗0.1M PBS（pH值6.5）的组织和地点。在37 ° C biotinamide注入所述的神经元的可视化过程中的组织和感觉神经元和运动神经元的各种标记之间的关系可视化。 </li><li>这里显示的技术也可以很容易地结合与immnocytochemistry 6作为一个例子，免疫细胞化学过程中的突触脑准确定位标记的神经元突触内（ 图3）。要做到这一点，孵化脑切片，鼠抗突触素抗体（1:10,000）2天在4 ° C，然后在抗鼠FITC（1:400）孵育23小时° C。 </li><li>与荧光标记物标记或荧光成像处理的神经元共焦成像的理想选择。图3是共聚焦显微镜获得通过轴突布顿光学节的一个例子。 （ 图3）。标记神经元的动画生成多个光学部分获得通过标记的神经元（ 图 4 ）。 </li><li>用这种方法标记神经元可用于定量共定位分析。要做到这一点，在与美国国立卫生研究院图像使用公开可用的软件宏（http://phy.ucsf.edu/ ° IDL / colocalization.htm中）。本地化在单一的轴突终端突触相结合，与突触素免疫细胞化学细胞内的标签。 </li></ol> 6。代表性的成果： 可以用这个方法取得的代表性成果的概述如图 1所示。这种单一的脑干神经元electrophysiologically记录下颌骨运动期间，由于可以清楚地看出，这个神经元的反应（图 1左下角，淡蓝色）调制在运动过程中。这个神经元是注射后的电生理特性biotinamide和随后进行可视化处理。重建后的神经元（图1的中间，绿色）可以实时特德解剖标志，在这种情况下指定的三叉神经运动核（红色轮廓）。基于运动和重建过程中的神经元的反应神经元可以被认定为次要肌梭传入神经 ​​元，图2展示了一个有代表性的例子下颚位移过程中神经元的生理反应。代表的神经元的反应是瞬时的发射频率。请注意神经元的反应密切模仿下颌表明这个特定的神经元提供感官反馈有关下颌位置的位移。图3是一个高放大倍率的图像，结合染色突触和一个尼氏染色的细胞染色轴突。注意突触（黄色）的轴突布顿内共定位。 图4是一个单一的，生理特点和神经元细胞标记的动画。 <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="图1"/> 图1概述的方法。左上：下颌位移。从单一的神经元（黄色）中的细胞内记录（绿色）。这种神经元的形态，细胞内记录和注射后重建。红色轮廓显示三叉神经运动核的位置。左下：在下颌运动，这种神经元的生理响应。 <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="图2"/> 图2代表一个单一的肌肉感觉记录下颌骨的运动过程中的神经元在体内的生理反应。注意下颚位移的神经反应的相似性。 <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="图3"/> 图3。终端与一个细胞内染色在肌肉探测回应的感觉神经元突触boutons（红色肿胀）轴突分枝。随后突触的免疫处理，显示了突触内的轴突布顿（黄色）的本地化。绿色是一种荧光尼氏染色。 图4。肌梭初级传入神经 ​​元轴突的生理反应记录下颌运动期间体内的动画，轴突然后被细胞内染色和可视化处理。 <a href="/files/ftp_upload/2650/collateral4.avi">图4的高清晰度视频</a> <a href="/files/ftp_upload/2650/medres.mov">图4的中等分辨率视频</a>

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对动物的实验是在按照guidlines和设置在实验动物护理和使用（1985年修订的美国国立卫生研究院出版号86-23，）指南“规定的调控和马里兰大学的动物护理和使用委员会。

这里说明该方法是一个强大的技术到单个神经元的功能提供重要的启示，以及如何对单个神经元的反应， 有助于神经回路。9这方面的知识，了解感觉功能的根本。这项技术最大的优势就是允许了大量有关的一个包括生理，形态和突触的形态和分布的神经元参数的决心。当与其他技术，如逆行神经标记的其他信息，如神经回路相结合的特点 。7,8这种方法的另一个优点是它可以了解...

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>电磁振动器</td><td>凌动系统</td><td> V101 </td><td></td></tr><tr><td>信号发生器</td><td>反馈系统</td><td> PFG605 </td><td>能够生产梯形输出信号</td></tr><tr><td>电极的玻璃</td><td>萨特仪器</td><td> AF100 - 68 - 10 </td><td>与长丝</td></tr><tr><td>电极拉马</td><td>萨特仪器</td><td>模型P - 2000和P - 80 </td><td></td></tr><tr><td> biotinamide </td><td>媒介实验室</td><td> SP - 1120 </td><td>储存于4 ° C </td></tr><tr><td>德州红抗生物素蛋白DCS </td><td>媒介实验室</td><td>一个- 2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td>分子探针</td><td> D - 3308 </td><td> 3000分子量，赖氨酸可修复</td></tr><tr><td>鼠抗突触素抗体</td><td> Chemicon公司</td><td> MAB5258 </td><td></td></tr><tr><td>荧光尼氏染色</td><td> Neurotrace，分子探针</td><td> N - 21480 </td><td></td></tr><tr><td>电极测试仪</td><td>温斯顿电子</td><td> BL - 1000 - B </td><td>测量电极阻抗</td></tr><tr><td>静电</td><td> Axon仪器</td><td> Axoprobe 1A，2B Axoclamp </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

的单个神经元和神经回路的功能的作用是基本的了解感觉和运动功能神经元的机制。大多数的感觉机制调查依赖于任何的神经元的考试，而动物是静态1,2或在运动的记录外神经元的活动。3,4，虽然这些研究提供了感觉功能的基本背景，他们要么不评价功能的信息发生在运动或在他们的神经解剖学，生理学和神经化学物质的表型的能力，充分体现有限。这里显示的一种技术，它允许在单个神经元在体内的运动广泛的特性。可以使用这种技术，不仅要研究初级传入神经元，但也表征运动神经元和感觉interneurons。最初的单个神经元的响应是使用在其次为神经元的感受野的决心下颌骨的各种动作电生理方法记录。神经示踪细胞内注入的神经和大脑的处理，使神经元可与可视光，电或激光共聚焦显微镜（图1）。详细的形态特征的神经元，然后重建，使神经元的形态可与相关的生理反应的神经元（图2,3）。在这种沟通，提供重要的关键细节，这项技术的成功实施的提示。在研究神经元与其他技术相结合，这种方法可确定有价值的额外信息。逆行神经标签可用于确定神经细胞标记的神经元突触，从而使神经回路的详细决心。这种方法可以结合免疫组织化学内标记神经元的神经递质研究，并确定与该标记的神经元突触神经元的化学表型。标记的神经元也可以处理电子显微镜确定超微结构特征和标记神经元的微型电路。总体来说，这一技术是一种强大的方法来深入刻画在神经元在体内的运动，从而使大量洞察到感觉功能的神经元的作用。

Watch this Scientific Journal Video about Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement at JoVE.com

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

描述量化哺乳动物在运动神经元在体内的生理反应和相关的神经元与神经元的形态，神经化学表型和突触的微型电路的生理的一种技术。

由运动神经元调制生理，形态学和神经化学的表征

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

physiological-morphological-neurochemical-characterization-neurons

Research

JoVE Journal

Neuroscience

13.7K 次观看.  University of Maryland. 描述量化哺乳动物在运动神经元在体内的生理反应和相关的神经元与神经元的形态，神经化学表型和突触的微型电路的生理的一种技术。

视频: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

眼运动监测内存

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

科研

神经科学

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

大脑皮质功能和神经在更改重力条件下的性能之间的一致性

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

总钙在神经元的电子探针X射线显微分析测量

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...