We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

在本协议中所述的所有实验程序批准了希伯来大学的动物护理和使用委员会。 1.准备的解决方案和工具切片<ol><li>制备1升标准生理溶液（SPS）的含有表1中记载的离子。 <ol><li>制备含有该盐的最终浓度10倍的原液中预先存储玻璃瓶填充有1000毫升去离子水（0.055μS/厘米电导）。添加的盐（氯化钠，氯化钾，KH 2 PO 4，和硫酸镁 ），并搅拌至完全溶解。存储原液在4℃。 </li><li>使在实验当天最后的SPS溶液。加入100 ml的储备液至700毫升纯水中，用磁力搅拌棒溶解葡萄糖（3.6克）和碳酸氢钠 （2.18克）和然后添加精制水至1L。 </li><li>平衡的soluti在通过用95％氧气脱气它4/10％CO 2的〜20分钟。在此之后，加入2毫升的1M的CaCl 2溶液。 </li></ol></li><li>装配以下工具（ 图1）。 <ol><li>制备大剪刀或者其他工具，断头，小外科剪刀用于打开头骨，细尖镊子用于提升颅骨以暴露脑，手术刀用刀片解剖脑的所需部分被切下的小抹刀，用于操纵解剖的大脑部位。 </li><li>此外，制备小片滤纸，用于从大脑除去过量的液体，两个小的培养皿为大脑中两个玻璃500毫升烧杯被解剖用于容纳加热的纯净水和SPS，及氰基丙烯酸酯胶胶合脑到切片的阶段。 </li><li>准备一个小刷子用于切片和一个广口的玻璃巴斯德移液管，用于移动切片从切片浴到恢复茶时的处理的切片MBER。消毒工具，以防止细菌生长在温暖的切片浴场。此外，使用清洁的实验室手套。 </li></ol></li><li>所描述的吉布和爱德华兹15准备淹没切片复苏室等。连续气SPS它与95％O 2/5％CO 2，并保持在36℃。确保该冲气不会导致被困在可能损坏切片浴的小气泡。 </li><li>暖〜300毫升的SPS至36℃，在加热板有磁搅拌器，或在水浴中。如果一个加热板的情况下，要小心防止过热的溶液中，这可能导致离子沉淀。如果它发生，温暖清新的SPS，而不是冷却旧的。 </li><li>带来〜500毫升的纯净水通过一个电热水壶煮沸并且在一个烧杯中，将结合300毫升它用冷水，得到稍暖的水比切片温度。用断头术在此温热纯水并使用remai宁开水后维持切片沐浴的生理温度。 </li><li>通过注入0.1毫升戊巴比妥（60毫克/毫升）腹腔内麻醉的小鼠。几分钟后检查该动物不强脚趾或尾巴捏响应，以确定缺少感觉。 </li></ol> 2.解剖大脑<ol><li>迅速斩首鼠标通过切割颈部附近与头骨后部的大剪刀，让头部落入与温热纯水冲洗掉多余的血液的烧杯中。 </li><li>暴露在颅骨的枕骨大孔下颈部肌肉和皮肤，去除可能更颈部的小剪刀。 </li><li>拉颅骨顶部的皮肤能够清楚地看到颅骨缝合，以引导开放的头骨。 </li><li>通过枕骨大孔中插入的小剪刀下尖，马上把他们往切开颅骨开放侧面。沿顶骨到眼后和额顶缝合位置的横向边缘轻轻地切割;然后，把对颅骨的中心，跨越中线的头骨的另一边（见绿色虚线图2A）。 </li><li>用细尖镊子，解除从额顶角落颅骨和拉对角线向上和向侧面露出的脑（参见图2A中的黄色虚线箭头）。确保该头骨不附着到头部的任何骨或皮肤，使大脑将留在原地，当颅骨被提起。的情况下，大脑是移动与颅骨，使用小剪刀除去头骨和大脑之间的任何结缔组织。 </li><li>当大脑暴露出来，不要让大脑保持干燥。因而，执行下一个步骤，在保持头骨和大脑在培养皿中填充有温热，充气的SPS。 </li><li>分离的大脑用于电子零件从大脑的使用手术刀和刮勺其余xperiment。在使用小脑，来自前脑分离，通过中脑和脑桥单切（沿红线在图2B-D）和它移动到一个小的培养皿中，包含新鲜，充气的SPS。 </li><li>除去脑干以形成直链和宽基胶合大脑切片在水平面小脑当切割阶段。简要地放置小脑在湿滤纸，以便使被来自前脑切的面朝下。 <ol><li>切脑干用手术刀以形成基极（与蓝线在图2B中所示），和小脑返回到所述SPS。当需要切割的其他飞机，修剪小脑相应的块（ 图2C＆D蓝线）。 </li></ol></li></ol> 3.切片脑<ol><li>通过确定它是d准备切割阶段RY，将一小滴强力胶在舞台中间前。 </li><li>解除从所述SPS使用具有正确的一面朝上抹刀脑块，并使用小片滤纸大脑除去任何过量的液体。然后，用一个单一的运动，滑动距离锅铲大脑上的胶水在舞台上跌落。 </li><li>为了防止干燥的大脑，并从上所述组织的侧面跑上胶水，申请几滴的SPS用巴斯德移液管，并放置在切割阶段中的切片室。对准脑块，使得所感兴趣的区域（ 例如，小脑皮层）所面对的叶片，从而将经受最少的机械压力被切片之前。 </li><li>根据制造商的说明削减每个切片的同时，不断脱气所述SPS在切割室以及保持在34-37℃的温度。保持切割室的温度保持在该范围内通过填充的extern用温热的纯净水并更换水时的温度下降得太低的限幅器的人的腔室中。 注：该限幅参数必须通过实验发现，对于每种类型的组织和神经元的正在审查。万一平卡姆登SMZ 700切片机用陶瓷刀片和小脑高尔基细胞，用0.75毫米振幅在65赫兹和推进为0.05毫米/秒的速度;对于小脑核神经元更高的振幅和频率（1毫米和90赫兹，分别地）和较慢的前进速度（0.01-0.02毫米/秒）被推荐。为高尔基体和小脑核神经元，切片厚度应为300微米。 </li><li>在切片，不允许片折叠自己。用软毛刷轻轻支持他们，最好是触摸只有在那些对实验不感兴趣区域的切片防止这种情况。请注意，以防止刷接触到的vibratome刀片;特别是在壳体的陶瓷刀片，即使是光接触可能损坏叶片。 </li><li>移动从切片室每个切片，以回收/储存室用广口，火抛光的玻璃巴斯德吸管。 </li><li>让在开始实验前的切片休息在室中至少1小时。这允许损坏或垂死的组织和细胞的退化，从而导致清洁片表面。在第一个小时内，保持所述SPS的温度在34-36℃的范围内的腔室;确保切片不被气泡，会损坏片或使其浮触摸。 </li></ol> 4.实验<ol><li> 1小时后，让该回收室的温度冷却至室温（17-25℃）。 注意：这可能会延长，在此期间片可用于通过减缓细菌的生长以及细胞代谢的时间段。这个周期取决于大脑区域和细胞类型。例如，某些类型的高尔基体细胞可以被认为是健康的切片切而其他类型后8小时仅持续6小时。 </li><li> 1小时后，由限幅传递，使用切片关于各种电生理实验。 </li></ol>

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The authors declare that they have no competing financial interests.

我们证明了在生理，而不是冰冷的温度筹备急性脑切片的小鼠的方法。 已经显示11，当与制备的那些与冷的条件下相比在温暖条件下获得的切片的质量是优异的，其前提是该切片机叶片具有最小的垂直振动。切片在生理温度可以防止生理伪影引起的低温，例如那些涉及到改变在代谢过程17-19引起可在单细胞的像差以及网络行为。此外，即使在切片过程显然应避...

该方法的目的是为了得到高质量的急性脑切片的体外电生理实验，尤其是当使用成人或甚至老的动物。 急性脑切片的方法，因为两个优雅的句子描述Skrede和Westgaard 1，已成为现代神经科学研究的基础之一，并在全球范围内采用无数的变化。切片的质量反映在活每片神经元的数目，该时间周期，在此期间细胞保持它们的电生理学和形态学特性，以及在组织中的完整性。此外，最大持续时间为稳定记录依赖于切片的质量。因此，沿几十年来，原来切断方法有了进一步的发展由个体研究团体切割2-10，常常通过切割的组合物的复杂的修改后，提升片恢复öR恢复解决方案（例如，添加抗坏血酸，硫脲或甚至H 2 O 2），以及心内预灌注动物用冷却的生理溶液。 正如最近的研究显示11，切片过程中的生理温度似乎比冷却到神经元的健康更有益;与成年（2-8月）的啮齿动物工作时的改善是最引人注目的。避免剧烈的温度变化时防止工件因温度依赖性过程中的细胞，如可塑性13和离子通道动力学13,14。这些变化可能会影响膜电位和细胞内钙信号，穗门槛，穗形。 “热”急性切片制备这里介绍的方法是用于获得高品质的急性脑切片从任何脑区域，包括小脑，皮层，海马，脑干晶核16的一般步骤&lt;/ SUP&gt;以及嗅球，无论是在大鼠和小鼠。 值得注意的是，在生理温度下的切片过程要求的切削刀片的振动几乎完全水平，是没有任何结构上的缺陷。这样的精确度可能无法实现与老款切片;在这种情况下，我们建议在进行切片准备在冷冻 - 寒冷的条件下作为低温度似乎使机械损伤，在代谢异常的费用的组织更耐即使。

<table border="0" cellpadding="0" cellspacing="0" width="574">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

在这里，我们提出了一个协议，准备对急性脑片。这个程序是用于电生理膜片钳实验的关键元件，其在很大程度上决定结果的质量。它已经显示，省略了切割过程中的冷却步骤是有利的，获得健康的切片和细胞，特别是当与从成熟动物的高度髓脑结构处理。虽然确切的机制，使高温下支持神经健康时只能推测，它按理说，只要有可能，在切片时的温度应接近生理条件，以防止温度有关的文物。该方法的另一个重要的优点是该过程的简单性，因此，制备时间短。在证明方法成年小鼠进行使用，但相同的过程可以与年轻小鼠以及大鼠施用。此外，下面的补丁CL安培实验是在水平的小脑切片进行的，但同样的过程也可以用在其它的面以及大脑的其他后部区域内使用。

在所描述的方式制备的切片可用于各种电和光遗传学实验。在图3A和图3C，我们显示一个水平小脑切片和冠状的大脑皮质切片，分别根据微分干涉（DIC）的光学观察的一个代表性的例子。在小脑切片，几种类型的小脑的神经元可以容易地由他们的位置和细胞体的形状识别，允许有针对性的电生理记录。在图3B和3D中 ，例如从在电流钳模式全细胞膜?...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门：成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

25.6K 次观看.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

视频: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的：一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

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 ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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 ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...