这项工作是由NSERC补助BK和RG资助。作者还要感谢科和谢和罗素Hosain他们高尔基 - 考克斯染色的专业知识。

所有实验均符合动物保护的加拿大议会批准通过莱斯布里奇大学，动物护理委员会。 1。育种和触觉刺激<ol><li>订单孕鼠从普通动物的供应商，或使用标准的实验室繁育程序内部滋生的幼崽。 </li><li>房子在一个温度控制饲养室（21℃），保持在12:12小时的光所有的动物：暗周期，并提供获得食物和水随意 。 </li><li>当幼仔出生后，房子雌性大鼠分别与它们的后代。 </li><li>为了避免额外的压力与出生后立即处理，幼仔开始在P3的触觉刺激。 </li><li>触觉刺激应该从P3〜P21进行三次，每天（上午9:00，下午1:00和4:00 PM）和幼犬应在P21的母亲断奶。 </li><li>当使用交叉垃圾设计，一半的幼仔FR嗡每窝将经历触觉刺激的另一半用作对照。随机将雄性和雌性幼仔数目相等各组。为了区分幼崽接受触觉刺激和控制，对他们的后腿和尾巴记号笔标注一组幼崽。标记必须被重新应用每隔一天。 </li></ol>触觉刺激<ol start="7"><li>从他们的家笼坝移除，并放置在一个临时笼子里的食物和水。保持在养殖/屋室大坝和临时笼。 </li><li>称取幼鼠作为一个群体之前，早上触觉刺激会（上午9:00）。对于每个会话，运输幼鼠饲养笼到一个单独的试验室触觉刺激。将笼子上，它被设置为24°C的加热垫</li><li>使用硬板来划分家笼成两半。将幼鼠的触觉刺激组一半，控制幼崽中的另一半。设置一个计时器到15分钟。 </li><li>使用柔软的羽毛般的掸子，刷触觉刺激组中的所有幼崽的同时，连续15分钟。这位年轻的幼鼠（〜P3-P12）后容易挤在一起，似乎进入深睡眠周期使它很容易刺激所有的幼崽一次。随着年龄的幼崽，他们变得​​更加活跃，一些幼崽行走，并在会议期间调查。实验者应连续移动的小狗闲逛进组，以确保每一个小狗获得相等的刺激。 </li><li>一旦15分钟会话结束后，运送幼仔回到饲养室和母亲回到笼子里。 </li><li>重复此过程每天三次对19天触觉刺激的。在P21的最后触觉刺激会后，幼崽应该从他们的母亲断奶。幼崽然后应安置在笼子里与同性的5或6其他断奶的动物。 </li><li>如果大鼠进行任何额外的测试，他们应保留不做不安，在正常光线：暗周期与获取食物和水libitm（除了定期笼清洗和处理），直到他们达到大约100天的年龄。如果发生其他实验操作，应该这样对待动物试验或按照这些实验室的协议。 </li></ol> 2。牺牲和高尔基 - 考克斯染色<ol><li>通过IP注射在约P100，管理的戊巴比妥钠过量的大鼠和用大约100ml的0.9％盐水灌注心内。 </li><li>提取时，大脑灌注完成。从头骨中取出大脑，切断视神经下方与努力保持小脑完好，研究人员可以选择保留或删除嗅球。把提取的大脑中的不透明的Nalgene瓶用20ml高尔基考克斯溶液14。 </li><li>保持大脑中高尔基体考克斯解决方案14天。 14天后，更换高尔基-C牛溶液用30％的蔗糖溶液。保持大脑在2-5天的蔗糖溶液切片之前。 </li></ol>注意：如果大脑不能内转移到蔗糖溶液后14天内切片，研究人员需要用30％蔗糖的新股票取代蔗糖。研究者可与染色无不良影响取代蔗糖每两周为4个月。 <ol start="4"><li>为了部分大脑，大脑应吸干并固定在切片阶段cyanocacrylic胶水。为了防止撕裂或不均匀切片，研究人员必须保持谨慎，确保整个大脑被牢固地固定在舞台。 </li><li>在vibratome水库必须充满6％蔗糖溶液，以覆盖该切片刀片的水平。设置vibratome参数的速度和幅度为5，（在两个尺度上的中点）。切片脑入200微米的部分，然后将切片用2％凝胶化的微范围滑动。请务必切片的过程中，保持部分湿润。 </li><li>当所有感兴趣的部分已收集，通过施加压力与湿润吸水纸的幻灯片按路段上的幻灯片。保存幻灯片在湿度室的幻灯片机架，直到他们准备被染色。他们应该留在潮湿的房间至少12小时，但不超过4天以上。幻灯片需要是潮湿和不应该被允许干燥。 </li><li>在开始染色过程前准备染色团。标签12玻璃染色皿（10.7×8.5×6.8厘米）和过程以下面的方式： <ol><li>蒸馏水 - 1分钟</li><li>氢氧化铵 - 30分钟（在黑暗中） </li><li>蒸馏水 - 1分钟</li><li>柯达修复的电影 - 30分钟（在黑暗中） </li><li>蒸馏水 - 1分钟</li><li> 50％酒精 - 1分钟</li><li> 70％酒精 - 1分钟</li><li> 95％酒精 - 1分钟</li><li> 100％Alcoh醇 - 5分钟</li><li> 100％酒精 - 5分钟</li><li>溶液（1/3氯仿，1/3 HemoDe，1/3 100％酒精） - 15分钟</li><li> HemoDe - 15分钟</li></ol></li></ol> *二甲苯可用于替换HemoDe的。为了确保一致的染色质量，新的解决办法应该用于处理每张幻灯片机架。 <ol start="8"><li>继上次15分钟再现的HemoDe，盖玻片Permount的幻灯片。 </li><li>检查用显微镜才允许的幻灯片风干。 </li></ol>

<ol>
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作者宣称，我们没有竞争的经济利益。

由于发育中的大脑的可塑性明显，但重要的是，涉及早期经历的实验步骤进行彻底检讨，并尝试都是为了控制所有中介变项。出于这个原因，一个横垫料设计的触觉刺激过程期间使用，以确保幼仔被接收在发展的所有其他域类似的经历。此外，它也是重要的，从多个窝幼仔多个被选择用于分析，以避免影响来自于单一小狗​​或垫料的偏压导致的可能性。 触觉刺激被认为是模仿天然存在的母体行为，舔和修饰，这被认为是给子代发育有利。虽然之前的研究强调生命的第一周（P0-P7）是为舔和梳理16的关键时期，变革的庞大规模确定以下18天TA的ctile刺激可能意味着，虽然敏感时期存在的，较长的曝光优越。同样重要的是要注意，接受触觉刺激在这个实验范式的幼仔也经历舔和修饰由他们的母亲，因此触觉刺激是除了正常的刺激由母亲管理。最后，我们还必须认识到的区域依赖性变化。虽然在开发过程中的触觉刺激前额叶皮质树突增加的复杂性，这并不能保证这种性质的结构性变化将体现在所有大脑区域。这可能是在其他脑区如顶叶皮层神经元形态，会在一个截然不同的方式，以同样的经历作出回应。然而，由于触觉刺激程序是容易施用并不会带来任何危险的后代或水坝，它具有以作为许多研究，旨在改善发展的有价值的工具的潜在人的结果。 相对于脑组织的高尔基体考克斯染色，是对神经元细胞的成功的可视化的关键步骤如下：1）必须有大脑用生理盐水溶液充分灌注。脑组织导致血管文物，使其难以实际上是通过血管的迷宫可视化的神经元细胞，同时也复杂拍摄染色神经元的能力，不当或不充分灌注。 2）灌注的大脑应该存储在高尔基体考克斯溶液并在黑暗中的蔗糖溶液。存储所述的脑组织在黑暗中减少了组织的背景染色，再增加成功可视质量的神经元细胞的机会。 3）脑组织被存储在以下第14天存储在高尔基体考克斯溶液的蔗糖溶液。当组织浸没在2-5天的30％蔗糖溶液，大脑更柔韧，防止粉碎和撕裂部分的时切割。它以防止残留在增加的时间段的蔗糖溶液的脑组织（除蔗糖溶液连续用新鲜溶液替换）因为长期贮存中蔗糖降低了染色质是重要的。 4）最后，一旦幻灯片已染色和盖下滑，就应该给他们足够的时间可视化前的干燥用显微镜。如果幻灯片是不允许的充分风干，该组织可能会变暗，降低皮层神经元的成功可视化。 在心理功能和行为反应发生在回应经验稳定的变化被认为是由神经元形态和突触连接17的重组提供便利。由于这些结构变化的经验依赖性可塑性提供了一个可衡量的资源，使用可靠染色过程中是非常重要的。这vibratome基于高尔基 - 考克斯染色过程提供可靠的污点ING精分支和树突棘，可能不会很明显与其它协议如火棉胶嵌入的。在高尔基体考克斯程序的独特性源于其容量仅染色的神经元元件（1-10％），其允许单个神经元的跟踪长距离的一小部分。尽管神经元的只有一小部分被浸渍有污垢，所呈现的可见细胞，维持所有的功能，包括细胞体，树突，树突棘和轴突。此外，当染色过程是否正确执行，染色细胞中脱颖而出清楚而明确地对一个透明的背景，因为其他的皮层结构仍然未染色和透明。由于认识到在向外运作持续变化，必须进行相关的神经系统的可塑性， 即神经元来修改它们的结构和连接能力，高尔基-考克斯染色过程为研究人员提供一个可靠的技术来可视化和量化这种可塑性。

虽然有许多报道和利用技术的负面早期经验对大脑的成熟，如围产期应激1,2，感觉剥夺3，药物的毒副作用4考试，有来检查的积极经验的影响非常少的方法这个时间段。除了 ​​丰富环境，触觉刺激是几大脑加强与展示效果5疗程之一。触觉刺激的感官刺激，模仿母亲的行为鼠，舔和修饰皮肤的方法。其普遍接受作为一个积极的操作来自研究表明触觉刺激改善早产儿和新生儿大鼠6成熟。此外，研究在实验室米尼7表明，较高水平的孕产妇舔和仪容产品都与后代的积极成果。由于到T灰褐色的积极影响，触觉刺激迅速演变成一个补救战略，旨在减少焦虑8，改善与脑损伤有关的9-11的结果，并减弱药物过敏12。因此，触觉刺激是一个有价值的技术，以促进积极的早期经验，具有公认的能力大幅改组神经元形态和发育中的大脑的突触连接。 为了检查和定量改变神经元的形态，它是必要的可视化完好的神经元。高尔基-考克斯染色过程是刊登在1800年代后期卡米洛高尔基的技术，提供少数完整的神经元13离散染色的修改。虽然过程会出现染色神经元随机和再现性普遍提及的重大缺陷，整个神经元的小浸渍速度许可证可视化，包括CELL体，树突，树突棘和轴突。同样，浸渍给定的大脑与高尔基体解决方案所需的时间也被认为是一个垮台。不过，由于一旦染色完成组织可以无限期保存，和神经元用显微镜的大脑和可视化的灌注之间的时间不到21天完成，时间周期是没有道理的。此外，稍加修改的协议，高尔基 - 考克斯染色可有效地用于所有年龄段浸渍啮齿动物的大脑。由于在结构层面的变化都与执着的修改行为和心理功能，高尔基 - 考克斯染色技术为研究人员提供了一个宝贵的工具来测量神经可塑性。

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	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Potassium Dichromate</td>
			<td>Fisher</td>
			<td>P188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mercuric Chloride</td>
			<td>Fisher</td>
			<td>M1561</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium Chromate</td>
			<td>Fisher</td>
			<td>P220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Fisher</td>
			<td>A669-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kodak Rapid Fix</td>
			<td>Vistek</td>
			<td>Kodak 146 4016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EtOH-95 &amp; Anhydrous</td>
			<td>Commercial Alcohols</td>
			<td>No Cat Numbers</td>
			<td>All other Et-OH are dilutions</td>
		</tr>
		<tr>
			<td>Swiffers- Soft Feather-Like dusters</td>
			<td>Safeway</td>
			<td>&nbsp;</td>
			<td>Can be found at most grocery stores</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S-9378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HemoDe</td>
			<td>Electron Microscopy Sciences</td>
			<td>23410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>Sp15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>160004-365</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>062011-9</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

visualizing the effects of a positive early experience, tactile stimulation, on dendritic morphology and synaptic connectivity with golgi-cox staining

为了产生行为的长期变化，经验必须在生产神经元形态和突触连接的稳定变化。触觉刺激是一个积极的早期经验，模仿产妇舔和修饰大鼠。暴露幼鼠这个积极的经验，可以通过使用非常方便的材料，如一个家庭掸子轻松且经济高效地完成。使用跨垃圾设计，幼仔要么抚摸或静置，15分钟，每天三次在整个围产期。测量与此相关的阳性早期经验的神经可塑型变化，脑组织中的高尔基体考克斯染色被利用。由于该高尔基考克斯浸渍染色离散神经元的数目，而不是所有的细胞的事实，所述啮齿动物的脑与高尔基体考克斯溶液的染色使得整个神经元素，包括细胞体，树突，轴突的可视化，并树突棘。在染色过程中，我s进行了数天，并要求研究人员密切关注细节。然而，一旦染色完成后，整个大脑已经浸渍和可无限期保存正在进行的分析。因此，高尔基体考克斯染色是研究经验依赖性可塑性的宝贵资源。

当这个染色过程一直遵循适当，会产生枝晶的一致和统一的染色和刺。参见图1中高尔基体考克斯神经元染色的表示。此过程会产生染色相媲美的新开发的体外方法和可允许的树突状字段是不间断以下组织包埋的可视化。在vibratome基于技术高尔基考克斯染色已经发现产生终末分支与锥体神经元更广泛的染色，比用染色或火棉胶包埋和低温恒温器剖组织，15。此外，由于染色的这个方法浸渗到整个大脑，各段可立即或在将来分析。作为形态学改变的位置是生成原始假设当它并不总是明显的，这个过程允许额外的大脑区域的探索一个D可预防处置的组织，可能在未来会很有用的。 这种染色方法可用于任何老年大鼠大脑（P0-老年）的可靠的染色。然而，染色年轻人的头脑（&lt;1克）时，大脑应该只停留在高尔基体考克斯解决方案6天，而不是推荐用于成人的大脑14天，所有其他程序维持不变。可能在这个高尔基 - 考克斯染色过程中遇到的主要困难是无法产生的中央脑区，包括丘脑的高品质的染色。作为整个大脑中浸渍的同时，中央区域可以不接受染色的理想浓度。尽管这一发现，高尔基 - 考克斯技术生产皮质下区域如海马和纹状体的特殊染色。该过程的最后一个限制来源于不完全清除血液从大脑中灌注过程。血液可能在脑组织产生伪影这使得它很难拍摄和跟踪的神经元。 高尔基 - 考克斯染色技术提供树突形态学和解剖学是为旨在探讨在神经元水平变化的研究范围广泛有用的可靠措施。当在前额皮层以下的触觉刺激在早期产后检查的神经元结构，高尔基 - 考克斯染色显示急剧增加树突的复杂性。与早期触觉刺激的解剖结构变化可以直观地通过比较锥体神经元棘密度从对照大鼠从鼠是经历触觉刺激锥体神经元（参见图1）来证明。此外，参数如树突分支，树突长度和脊柱密度，可以计算和统计分析，以产生可以在动物相比连续数据。为了产生可靠的结果，分析应进行对时每个治疗组三只动物，每个半球大约五神经元的最小值应在分析中随机选取每个大脑区域。 图2显示了树突的复杂性所收到的触觉刺激和动物，没有动物（刺/枝晶微米×微米） 。同时在心尖和基底从内侧前额叶皮质（CG3）分析了神经元的字段，触觉的刺激导致的神经细胞增殖的参数。 <img alt="图1" src="/files/ftp_upload/50694/50694fig1.jpg" /> 图1。高尔基-考克斯A.代表的照片在开发过程中收到的触觉刺激大鼠染色锥体神经元从区域CG3的第三层。B.代表放大在CG3的基底领域的终端树突树突棘（1,000倍）的照片;左侧较浅的图像是对照组的老鼠并在里较深的图像GHT是谁收到触觉刺激老鼠。 <img alt="图2" src="/files/ftp_upload/50694/50694fig2.jpg" /> 图2。的平均顶端和基底树突复杂性（棘/微米×微米），用于从该接收的任一触觉刺激在开发期间或没有（* P &lt;0.01）成年大鼠的CG3区域神经细胞说明性表示。

Watch this Scientific Journal Video about Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining at JoVE

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

本文介绍的程序幼鼠的触觉刺激和神经元形态的后续高尔基 - 考克斯染色。触觉刺激是受抚摸幼崽与家用除尘器投在围产期一个积极的经验。高尔基体的cox染色是一种可靠的方法，允许整个神经元的可视化。

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

visualizing-effects-positive-early-experience-tactile-stimulation-on

Research

JoVE Journal

Neuroscience

17.9K 次观看.  University of Lethbridge. 本文介绍的程序幼鼠的触觉刺激和神经元形态的后续高尔基 - 考克斯染色。触觉刺激是受抚摸幼崽与家用除尘器投在围产期一个积极的经验。高尔基体的cox染色是一种可靠的方法，允许整个神经元的可视化。 

视频: Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

厚脑切片使用高尔基考克斯方法中成像神经元

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

在培养的小鼠神经元中可视化轴突生长锥塌陷和早期淀粉样β效应

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...