The authors are grateful to G. Barka (SunChrom GmbH, Friedrichsdorf, Germany) for his support and for providing the epoxy glue. Furthermore, the authors acknowledge Fresenius Medical Care (Bad Homburg, Germany) for supplying the Capillary Haemodiafilter FXCorDiax. Funding was obtained from Goethe University of Frankfurt.

1.准备聚四氟乙烯管<ol><li>缩短聚四氟乙烯管进入2.5厘米片。使用纸的规模估算物理尺寸。 （参见图3a） </li><li>切1结束在一个角度，以方便连接出口管。 （参见图3a） </li><li>粗糙化的PTFE管材通过使用砂纸，以允许的环氧树脂胶附着。 </li></ol> 2.准备熔融石英<ol><li>缩短熔融石英到2.25厘米片。 （参见图3a） </li></ol> 3.插入熔融石英进入聚四氟乙烯管<ol><li>插入地下30插管到中空PTFE纤维在从扁平端部的距离为1厘米刺穿它。弯曲聚四氟乙烯管，以简化流程。 （参见图3b） </li><li>将熔融二氧化硅到使用套管为指导的PTFE管材。 （参见图3c） </li><li>轻轻取出套管和安全熔融石英。 （见图3d） </li><li>熔融二氧化硅应该突出为5mm的平端。 （参见图3e） </li><li>胶代替熔融二氧化硅在穿刺部位通过氰基丙烯酸酯胶，并允许它变硬过夜。 （参见图3e） </li></ol> 4.准备环氧胶（双组份胶） <ol><li>加入30份黄色固化剂，以70份黑色环氧树脂胶通过细针的帮助下，轻轻地混合。它直接就可以使用。 </li></ol> 5.运用膜（所有进一步的步骤必须执行在显微镜下和规模纸是用来估计物理尺寸） <ol><li>拉聚砜膜小心在熔融二氧化硅，然后拉膜进入聚四氟乙烯管和用锋利的手术刀调整膜至5.5毫米。 （参见图3f） </li><li>用细笔设置在膜上的标记点定义为2毫米的交换长度从膜的端部。 </li><li>适用于少量环氧树脂胶覆盖用细针的帮助下在膜的尖端。 （参见图3g） </li><li>在步骤5.3中使用的相同细针）与环氧胶的帮助下关闭掉透析膜和PTFE纤维之间的联合。 （参见图3g） </li><li>添加在膜足够环氧树脂胶到标志点，以确保2mm的交换长度。允许它变硬过夜。 （参见图3g）测定的交换长度之前，该膜为5.5毫米。施加环氧树脂胶在膜后，只有2毫米膜可用于扩散。 </li></ol> 6.应用金属套<ol><li>通过弯曲钳帮助削减将25g针头1厘米的金属套管件。 </li><li>拉1厘米金属套筒（25 G）的到熔融二氧化硅的剩余部分。金属套后使进口管道与MICRODIA连接裂解探头。 （ 见图3H） </li><li>金属密封套筒和聚四氟乙烯管，其余环氧胶之间的联合。允许其硬化至少24小时。 （ 见图3I） </li><li>施加热胶围绕探针的分叉为额外的固定和稳定。 注:该探针的入口内部体积是0.045微升和出口内部容积为2.5微升。因此，用1微升/ min流速，需要连接入口管开始的透析液的收集之后约2.5分钟。本协议，探头装配过程产生微透析探头是95％可靠。 </li></ol>的体外回收实验7.性能<ol><li>浸探针与截止的30 kDa的和2mm的交换长度分成含有1mM葡萄糖和1mM乳酸和，在一个单独的实验脑脊液组成的储备溶液，在含有100 CSF的纳米ATP和100nM的乙酰胆碱。 </li><li>灌注探头学联。设定流速为1微升/分钟，进行在室温（22℃）的实验。允许探针平衡1小时，在原料溶液，并收集样品，每30分钟之后，并储存在-80℃直至测量。 </li><li>对于体外回收率的计算，与已知分析物浓度的储备溶液的透析液比较分析物的浓度。表达结果的物质浓度的透析液与原液比例。 </li></ol>

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The authors have nothing to disclose.

图3示出的示例性探针组件。正确内外径尺寸必须被密切油管观察（OD1.6毫米;编号350微米），熔融二氧化硅（外径105微米;编号40微米）和渗析膜（外径245微米;编号210微米）。同样重要的是，保持隔膜和（105微米）和膜和管（105微米）以及之间熔融二氧化硅之间的空间。如果值不同，在探头压力可以上升，并导致该膜的泄漏。 这里我们介绍一种微透析探针以2mm的交换长度，这是适用于微透析在小鼠的大脑区域。然而，尺寸可以很容易地适应较大交换长度在较大的动物中使用。 装配的某些部分可以更换。例如，它是没有必要使用的PTFE管材。聚乙烯管材的应用，也可以用相同的外和旅馆呃直径尺寸。使用聚四氟乙烯管时粗糙化管才是必需的。的替换的透析膜也是可能的。对于本探针组件，一个30kDa的截留膜被使用，但任何其他透析膜可以具有相同的尺寸来使用。因此，我们的定制探针组件具有很大的灵活性，透析物质具有宽分子量范围的优点;较高的分子量截止，较高的物质，其可透析的分子量。 体外回收率通常随分子量截止的透析膜的增加。 以覆盖所述膜的前端，环氧树脂胶，需要以关闭透析膜和聚四氟乙烯管之间的接头，并确定交换长度。施加氰基丙烯酸酯类粘合剂是不可能在此步骤中，因为它影响到透析膜，并导致泄漏。氰基丙烯酸酯胶粘剂胶PLASTIC应适用于熔融二氧化硅与在穿刺部位的管道代替固定。 应当指出的是，该探针设计尚未经过测试具有导向套管。我们的实验室更喜欢植入急性实验中，我们有48-72小时的实验时间前星形胶质细胞增生导致探测失灵直径低探头。对于这种类型的实验中，导向导管不提供一个优点，因为导向插管较大，造成更多的组织损伤。此外，即使有一个导向套管就位，探针始终具有要插入到健康的脑组织，导致在透析的面积急性损伤。重复插入和取出通过导向插管探针导致重复损害，这种情况可以避免由一次性的， 即急性插入。血-脑屏障被证明是完好数小时植入12之后。导向套管是优选的，然而，当连续测量ħ大街做在数天或数周。最后，挥发性麻醉剂应该用于探针植入因为注射麻醉药干扰随后的实验1-2天。 体外回收率为葡萄糖，乳酸，乙酰胆碱和ATP是大约20％， 即对于所有测试的小分子量物质， 在体外回收几乎相等。显然，在较低的范围（高达500道尔顿），分子量对体外回收率没有很大的影响。分子量截断值为30kDa的也能使透析神经肽。由于神经肽的更高的分子量， 在体外回收价值较低。物质P（1348道尔顿）和促生长素抑制素（1638道尔顿）可以透析以相同的程度以2-3％ 的体外恢复值。虽然两种肽具有约300Da的质量位移， 体外回收率保持相同。无论SUBSTANCE P和生长抑素水溶液带正电荷的，并可能表现出相似的物理化学性质。血管紧张素II为1046达分子量展示出高体外回收率高达6％，这比用其他两种神经肽大三倍。因此，分子量高于500达明确影响体外恢复，即使当物质被带电的中性水溶液。在这种情况下， 在体外回收不受透析物质的电荷。然而，与使用其它的膜材料，回收率电荷的化合物可影响8。 总之，一个定制的微透析探针的组件是可以接受的替代商业探头。我们自制探针是坚固的，具有高可靠性和重复性好。

微透析是在神经科学研究中一个常用的技术。在过去的50年中，最小侵入性的微透析技术不断完善，成为一个行之有效的方法来监测外空间的局部浓度的小分子量化合物。几乎每间质液可以自由移动的动物进行调查。 Gaddum介绍，在20世纪60年代推拉技术。他修饰的方法从费尔德等人在其筒箭毒物通过套管终止于侧脑室和通过套管1收集的流出物也灌注。 Gaddum开发，其中套管由两个同心钢针植入在不同脑区和灌注通过溶液而同时除去来自周围的刀片2的神经元释放的神经递质的推挽技术。不幸的是，组织DAMA引起的尖端周围套管戈限于该方法的应用。作为这种方法的一个进一步推进，尾藤和同事提出了透析袋的方法，其中所收集的溶液，从周围组织中通过透析膜隔开。它们注入透析囊到狗脖子的皮下组织。透析袋的含量无蛋白质的，并且可以为离子和氨基酸3许多周后进行分析。接下来的发展是dialytrode，一个原始微透析探针，其最初由Delgado的于1972年4所述。最后，Ungerstedt和同事改善微透析探针的设计，以便它是较小的和位移较少的组织5。 同心微透析探针的行为类似于一个毛细血管。该系统由一个解决方案，包括对周围组织的流体的离子组成，同时缺乏所关注分析物不断灌注。钍Ë透析膜暴露于外部溶液或组织是半渗透性的。它允许的物质的被动扩散进入沿其浓度梯度6探头。渗透率依赖于许多变量，如分子量，形状，电荷和pH的化合物。它也被限制，由于该膜材料的性能，孔膜的尺寸和流速7。 图1和2示出了同心微透析探针。经由入口管导入金属套筒，其围绕熔融二氧化硅的灌注流体进入。内的金属套筒，它流顺着熔融二氧化硅和叶子它在它的前端。在透析膜和聚四氟乙烯（PTFE）-tubing（如特氟隆）之间的空间内的灌注液，然后向上流动。在这里，从组织的物质发生扩散环绕该膜。透析液通过PTF离开探头电子管道，其连接到出口管并可以收集。 微透析技术具有相对于其它体内技术的几个优点。探针构成与透析液不含有酶或细胞的结果的物理屏障。因此，没有必要进行分析前的洗脱物的纯化，以及分析物的无酶促降解发生。氧化降解可发生在管道的分析物的通过期间，但这通常可以通过加入抗氧化剂（ 例如抗坏血酸）的灌注液被阻止。可替换地，氧化性损伤的神经肽，例如，被有效地通过用尖更换出口管路，收集透析液8抑制。透析液可以直接与几乎任何类型的分析方法进行研究和多种分析物可以同时被收集。该系统可在清醒的动物中使用，几乎所有的大脑区域可以是检查。此外，通过探针灌注药物是可能的（retrodialysis）。然而，也有在微透析技术的局限性。偏低的时间分辨率不提供关于神经递质变化的实时信息。因为它是一种侵入性的技术，探针植入引起的手术创伤和麻醉，这会影响神经递质的浓度，在该步骤期间7,9,10是必需的。 在透析液的化合物浓度仅包括少量在细胞外流体中的实际化合物浓度。对于在细胞外流体中的未知化合物浓度的计算中，相对于在体外恢复已被计算。相对于个人在体外回收率为每个探头和每个化合物的测定是在开始体内实验前有必要的。为了这个目的，将探针浸入含的溶液的利益而灌注流体分析物是缺乏的感兴趣的分析物相同的溶液。在透析液测定化合物的浓度后，此数据必须被提到其在周围流体的浓度。几种物质在体外恢复判定可以同时9来实现。 许多神经科学的研究小组利用微透析技术来研究神经递质和代谢物在不同脑区的细胞外空间。因此，商业探针在神经科学研究环境有很大的需求。市售探头一大优点是高功能可靠性。该实验装置用于商业探头是公认和验证众多知名神经递质及代谢产物。然而，尽管可商购的微透析设备价格昂贵，并且在应用程序11的弹性较小，在第是工作一个微透析探针组件中详细介绍，它可以适用于任何应用，并且可以为不到10美元来制造。这种定制的探头是测试在各个脑区8调查同心圆微透析探针。 体外探针回收率的不同分子量（的100-1,600大范围）中，用物质不同的物理化学性质进行了比较。 在体外回收的葡萄糖，乳酸和乙酰胆碱测定，其分子量小于200道尔顿，ATP的具有分子量约500 Da和神经肽血管紧张素II，P物质和生长抑素具有分子量高于1,000达被执行。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Epoxy glue</td>
			<td>SunChrom GmbH, Fiedrichsdorf, Germany</td>
			<td></td>
			<td>&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Fused silica ID 40 &#181;m OD 105 &#181;m</td>
			<td>Ziemer-Chromatographie, Mannheim, Germany</td>
			<td>Art. No: 6.040105</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone membrane (haemodialysis filter FX Cor Diax 600)</td>
			<td>Fresenius Medical Care AG &#38; Co. KGaA, Bad Homburg, Germany</td>
			<td>REF: F00001593</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanacrylate glue (Pattex&#174; superglue plastic)</td>
			<td>Henkel AG&#38;Co. KGaA, D&#252;sseldorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEFLON-tubing 1.6 x 0.35 mm</td>
			<td>SunChrom&#174; GmbH, Friedberg, Germany</td>
			<td>Art. No: 969-195.219</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (Feather&#174; Surgical Blade No. 10)</td>
			<td>pfm medical ag, cologne, Germany</td>
			<td>Art. No: 07310</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>MEIJI Techno&#160;</td>
			<td>EMZ-8TR</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G x 11&#34; (0.3 x 25 mm) cannula&#160; Sterican&#174; Z</td>
			<td>B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>REF: 9324500</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 11/2&#34; (0.5 x 40 mm) cannula 100 Sterican&#174;</td>
			<td>B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>REF: 9186166</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine pen (Stabilo point 88 fine 0.4)</td>
			<td>Schwan-STABILO Schwanh&#228;u&#223;er GmbH &#38; Co. KG</td>
			<td>Art. No: 88/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot glue&#160; (Glue sticks ULTRA Power x 11 mm)</td>
			<td>Steinel&#174; GmbH, Herzebrock-Clarholz, Germany</td>
			<td>Art. No: 4007841046910</td>
			<td></td>
		</tr>
		<tr>
			<td>Sandpaper P60 230 x 280</td>
			<td>Robert Bosch GmbH, Gerlingen-Schillerh&#246;he, Germany</td>
			<td>Catalog Number: 2608605397</td>
			<td></td>
		</tr>
	</tbody>
</table>

custom-made microdialysis probe design

微透析是在神经科学研究中一个常用的技术。因此，商业探头的需求量很大，监测生理，药理和病理变化的脑脊液。不幸的是，商业探头昂贵的研究小组在公共机构。在这项工作中，一个探针组件进行详细说明，以建立一个可靠的，同心的，定制的微透析探针为低于10美元。的微透析探针由聚砜膜具有分子截止为30 kDa的的。探针的体外回收率与不同分子量（在100-1,600大的范围内）和不同的物理化学性质的物质进行比较。探头产生一个体外回收大约20％的小分子化合物的葡萄糖，乳酸盐，乙酰胆碱和ATP。 体外回收率为神经肽与1,000-1,600达量之间的分子量为2-6％。因此，尽管在较高分子量瓦特神经肽八个降低体外恢复值，化合物的透析中的分子量的较低范围（高达500道尔顿）对体外回收率没有很大的影响。本方法允许利用透析膜与其他截止值和膜材料。因此，这种定制的探针组件具有足够的灵活性的优点透析物质在宽的分子量范围。在这里，我们引入一个微透析探针以2mm的交换长度，这是适用于微透析在小鼠和大鼠的大脑区域。然而，探针的尺寸可以很容易地适应较大交换长度在较大的动物中使用。

同心定制微透析探针是由聚砜膜具有分子截止为30 kDa的的。探针组件示于图3。 它显示出在体外恢复为19.10±1.2％和21.2±1.6％的小能量代谢葡萄糖（180.16道尔顿）和乳酸（112.06道尔顿），分别。用于带正电的乙酰胆碱与181.66大的分子量，它表现出22.6±1.4％ 的体外回收价值。 ATP与551.62达和三个负电荷的磷酸残基的分子量，得到的体外恢复到一个可比程度（22.4±0.7％）。 体外回收率小分子的示于图4A中 。 神经肽，其分子量在1000以上大显示出较低的体外回收率相比，更小的分子（&lt;500道尔顿）（见网络连接<html>古尔4B）。在供试的神经肽，血管紧张素II（1,046.18道尔顿）是最小的肽有8个氨基酸。它表现出6.1±0.3％ 的体外恢复。的11个氨基酸残基P物质（1,347.63道尔顿）和14个氨基酸的生长抑素（1,637.88道尔顿） 表现出2.2±0.3％和2.3±0.1％ 体外恢复值大致相等，分别在体外回收率我们的30 kDa的探针是媲美市售微透析探针的体外回收率（CMA微透析，斯德哥尔摩，瑞典）， 例如 。乳酸23％和葡萄糖13％（参见应用笔记没有。1） <img alt="图1" src="/files/ftp_upload/53048/53048fig1.jpg" /> 图1:一个特制的探测器的照片。 8 / 53048fig2.jpg"/&gt; 图2:微透析探针（ 从 Lietsche 等人 ，2014年，重印许可ELSEVIER）的示意图形象。 <img alt="图3" src="/files/ftp_upload/53048/53048fig3.jpg" /> 图3:一个探针组件的示意图图像;艾步有关详细信息，请参阅协议（从Lietsche 等人 ，2014年，重印许可ELSEVIER）。 <img alt="图4" src="/files/ftp_upload/53048/53048fig4.jpg" /> 图4:小分子和神经肽的回收率（A） 在体外回收率的葡萄糖，乳酸盐，乙酰胆碱和ATP。原液含有葡萄糖和乳酸1mM的和，可选择地，100nM的ATP和100nM的乙酰胆碱每个实验条件。结果表示为物质conce的百分比ntration在透析液/原液。测试物质数值是平均值±SD N = 22为葡萄糖，N = 22，乳酸，N = 11的乙酰胆碱和n = 13 ATP。 （二） 在体外回收率的神经肽血管紧张素II，P物质和生长抑素。原液含有100nM的血管紧张素II，P物质和生长抑素每个实验条件。结果表示为在透析液/原液物质浓度的百分比。列的值是平均值±N = 6对每个探头的SD。

Watch this Scientific Journal Video about Custom-made Microdialysis Probe Design at JoVE.com

Custom-made Microdialysis Probe Design

微透析是在神经科学研究中一个常用的技术。因此商用探头有很大demand.In这项工作探针组件进行了详细建立一个可靠的，同心，定做微透析探针为不到10美元解释。

定制的微透析探针设计

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.9K 次观看.  Goethe University of Frankfurt. 微透析是在神经科学研究中一个常用的技术。因此商用探头有很大demand.In这项工作探针组件进行了详细建立一个可靠的，同心，定做微透析探针为不到10美元解释。 

视频: Custom-made Microdialysis Probe Design

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

同时神经刺激，并记录在水迷宫的设计和构建一个具有成本效益探头

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

科研

神经科学

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

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 ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

实时电:采用闭环协议来探测神经动力学和超越

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

体内高分子量截离法从脑间质液中提取大细胞外蛋白的微透析方法

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to altered neurotransmission. Indeed, cerebral microdialysis studies in rodents have demonstrated an excessive extracellular glutamate release in the hippocampus within the first 10 min following trauma. Microdialysis offers the clear advantage of in vivo neurotransmitter continuous sampling while not having to sacrifice the animal. In addition to the aforementioned technique, a closed head injury model that exerts rapid acceleration and deceleration of the head and torso is needed, as such a factor is not available in many other animal models. The Wayne State weight-drop model mimics this essential component of human craniocerebral trauma, allowing the induction of an impact on the head of an unrestrained rodent with a falling weight. Our novel and translational rat model combines cerebral microdialysis with the Wayne State weight-drop model to study, in lightly anesthetized and unrestrained adult rats, the acute changes in extracellular neurotransmitter levels following concussion. In this protocol, the microdialysis probe was inserted inside the hippocampus as region of interest, and was left inserted in the brain at impact. There is a high density of terminals and receptors in the hippocampus, making it a relevant region to document altered neurotransmission following concussion. When applied to adult Sprague-Dawley rats, our combined model induced increases in hippocampal extracellular glutamate concentrations within the first 10 min, consistent with the previously reported post-concussion symptomology. This combined weight-drop model provides a reliable tool for researchers to study early therapeutic responses to concussions in addition to repetitive brain injury, since this protocol induces a closed-head mild trauma.

Neurotransmitter alteration is a mechanism of neural dysfunction that occurs after concussion and contributes to the sometimes-catastrophic long-term consequences. This rat model combines microdialysis, allowing in vivo neurotransmitter quantification, with a weight-drop technique exerting rapid acceleration and deceleration of the head and torso, an important factor of human craniocerebral trauma.

脑震荡结合力与旋转与Vivo脑微透析的新型和转化大鼠模型

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to ...

Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, at a subliminal perceptual level, and related brain mechanisms can be experimentally isolated by presenting the stimuli quickly. Subliminal presentation of faces that belong to different ethnicity groups, races, or gender has been shown to be successful in investigating social implicit responses. However, many implicit responses are based on knowledge previously gained about the faces (e.g., sexual orientation, political views, and socioeconomic status) and not solely on physical appearance. Here, a novel method called post-movie subliminal measurement (PMSM) is presented. When watching a socially engaging movie, a spectator gains knowledge about the protagonist and becomes familiar with his/her identity and world views. When the face of the protagonist is presented subliminally after the movie, it evokes an implicit neural response depending on what is learned about the protagonist. With a vast number of movies available, each depicting a variety of people with different identities, the PMSM method enables investigation of the brain&#39;s complex implicit biases in a manner that resembles real-life social perceptions.

Post-Movie Subliminal Measurement PMSM, for Investigating Implicit Social Bias

This protocol describes the use of movies to investigate brain mechanisms underlying implicit social biases during functional magnetic resonance imaging. When the face of a protagonist is presented after a movie subliminally, it evokes an implicit response based on knowledge of the protagonist gained during the movie.

电影后潜意识测量（PMSM），用于调查隐性社会偏差

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, ...