我们要感谢他们的协助迈克尔Feyder和特雷​​尔霍洛威在初始设置的技术和二三大野博士的实验室，他们共聚焦显微镜的访问。这项研究是由美国国立卫生研究所通过N​​IAAA和NINDS壁间方案。

1。应做好准备DII /钨珠子弹 ：DII（1 - 1&#39; -双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐）子弹提前削减大脑切片。这个过程大约需要2-4小时一次准备多少子弹。 <ol><li>如果从一个新一批钨珠的开始，准备等份，每次300毫克。等分准备暂停钨6克，在2毫升二氯甲烷。然后，吸液管100μL到离心管珠的解决方案，以生产分装的300毫克钨珠在室温店等分（ 注 ：二氯甲烷蒸发速度非常快的酒精也可用于减少蒸发 。）。 。 </li><li>当开始从珠等份，每等份（300毫克钨珠）重悬在300μL的二氯甲烷，快速和覆盖，以防止水分蒸发。这个数量是足够的3个批次的子弹。 </li><li>准备重达13.5毫克的亲脂性染料DII和加入二氯甲烷450μL（终浓度=3mg/100μl）染料溶液。 </li><li>沾上染料珠。一个载玻片上放置了一块蜡涂层纸张和吸液管100μL珠重达到幻灯片。加入100μLDII的解决方案，调匀使用枪头，空气干燥，直到珠转浅灰色。 </li><li>用刀片刮去载玻片上的珠子和骰子珠成细粉（注 ：使用新的刀片，如果做一个以上的子弹，因此不会污染染料的颜色）。 </li><li>到刮珠重达15 ml锥形管纸和漏斗珠。在室温为10-30分钟，加入3毫升的水，在水浴超声（ 注 ：粉和超声切割应用，以尽量减少涂珠，会破坏稀疏标签的单个神经元形成团块 ） 。 </li></ol> 2。准备聚乙烯吡咯烷酮（PVP）解决方案和管 ：为了提高珠附件子弹管（TEZFEL管），聚乙烯吡咯烷酮（PVP）的解决方案，它的外衣。 <ol><li>准备10毫升PVP水溶液中，在浓度10毫克PVP /毫升。 </li><li>使用与管适配器12 ml注射器（软管直径略大）穿过的子弹管PVP的解决方案，然后驱逐出另一端。大衣更子弹管重用的解决方案，同时准备几批。使用后丢弃的PVP解决方案。 </li><li>要添加珠子弹管，珠产生同质性的解决方案和使用的注射器，通过管子拉珠的解决方案，使珠均匀分布整个油管的旋涡。 （ 注：尽量减少管路中的气泡数量 ）。 </li><li>通过预习站进油管和允许的珠子解决。使用的注射器，轻轻地去除水中的，因此，只有珠保持。 </li><li>自旋准备站和干燥的氮气管，直到水滴不再可见。这一步将需要约10-20分钟。 </li><li>管切割机切成13毫米长度和地方在闪烁瓶含有Drierite或某种无水硫酸钙的子弹。包装在保护光箔的小瓶。 </li></ol> 3。 DII染色：这种标签技术可用于染色来自不同物种的脑组织中的神经元，在这个特殊的情况下，从鼠标（3-6月龄）和非人类灵长类动物（9-15岁）。 最好是切片获得transcardiac灌注固定液固定脑组织的准备，因为预计将在此条件下的最佳组织保存。然而，通过灌注固定并不总是可行的（如猴组织的情况下）和DiOLISTIC标签仍然可以成功地组织编制不同的程序下应用。在这里，我们描述了三个不同的程序组织编制： <ol class="lalpha"><li>修复transcardiac灌注大脑→大脑→后缀删除10分钟→切割片→染色</li><li>移除大脑→切成块状组织修复→块→洗的PBS→切割片→染色</li><li>移除大脑→切割片→修复→清洗→染色</li></ol>在本研究中，猴子大脑获得transcardially，与冰冷的人工脑脊髓液（学联）前脑收获和组织块（4毫米厚的），含有尾一个灌注科目ND壳固定在室温为60分钟。对于所有的程序，固定液含有4％多聚甲醛和4％蔗糖PBS（准备新鲜或使用之日起15日内）。重要的是要注意，固定的时间是染色成功的关键。 Overfixation可以影响破坏质膜的完整性，造成泄漏的细胞（见结果下的更多信息）的染料染色。 <ol><li>对于程序A和 B，被切断，从固定的脑组织块和切片置于24孔板用PBS片（200微米） 。对于程序 C，新鲜急性脑切片（200微米），切断与一个冷切割解决方案vibratome（毫米）110胆碱CL，25 碳酸氢钠 3，1.25的NaH 2 PO 4 0.5，2.5氯化钾，氯化钙，7氯化镁2，25葡萄糖，抗坏血酸11.6，3.1丙酮酸（渗透压= 310 mOsmols）和后，立即片放置在24孔板和固定固定液在室温为30分钟。 </li><li>洗净切片，用PBS 3次。 </li><li>拍摄前片，从水井中删除的PBS，用画笔，地点以及中心的切片。 </li><li> DII子弹射穿3.0微米的过滤器使用一个赫利俄斯基因枪系统文件放置在一个1.5厘米之间的样品和年底的每桶的距离的枪在120-180 PSI氦气压力。 （注意 ：只有50毫米的片表面内神经元将标记用这种方法，所以重要的是保持在清洗和安装朝上片相同的一面，这样 ，您将确保标记神经元时，将焦距内共聚焦显微镜成像） 。 </li><li>洗片3次，用PBS和存储几个小时，他们在PBS让DII沿着树突传播。 </li><li>到载玻片延长Gold抗淬灭山片和一个18 × 18毫米的第一盖玻片覆盖。 （ 注：程序C，最好是安装在同一天切片切片的完整性没有很好地维持隔夜） 。 </li><li>安装后，媒体的透明指甲油密封干燥。 </li><li>另外，片可与抗体染色，在安装前，如下所述。 （ 注：幻灯片，可以使用至少6个月到一年，如果在黑暗中储存在4 ° C. DII是光敏感和长期暴露在光线下会导致色斑，褪色） 。 </li></ol> 4。抗体染色：免疫染色步骤3.4后，在安装前应进行。 <ol><li> 通透：孵育切片在0.01％的Triton X - 100的PBS在室温下15分钟（每孔300-500微升）解决方案。 注意：不要使用较高浓度的清洁剂，因为这将解散DII和显着降低荧光染色。尽量减少洗涤剂中的广泛孵化也将减少DII标签。如果延长孵化是必要的，而不是保持在PBS片阻塞的解决方案。 </li><li> 阻塞：在含10％山羊血清封闭液孵育切片，在PBS的0.01％的 Triton X - 100在室温为30分钟。 </li><li> 主要抗体：添加所需的阻塞解决方案（如兔抗GFP抗体1:1000稀释）稀释抗体。每孔加入300-500μL孵育1-2小时。 （ 注 ：如果隔夜孵化是必要的，封闭液中删除洗涤剂 ） 。 </li><li>切片用PBS清洗5 - 15分钟，3倍。 </li><li> 二次抗体与二级抗体在室温下30分钟的孵育。准备好所需的封闭液稀释（如Alexa的福陆公司的488标记的抗体1:1000稀释）二次抗体溶液。 </li><li>洗净切片用PBS为5-15分钟，4次。 </li><li>幻灯片片山延长Gold抗淬灭，并覆盖18 × 18毫米第一盖玻片。用指甲油密封安装后，媒体已经干涸。 </li></ol> 5。代表性的成果： <ol><li> 正确的标签检查 ：部分可以配备一个水银灯泡和红色荧光过滤立方体确认成功DiOLISTIC标签下的荧光解剖范围的可视化。图1显示了在低倍率得到很好的标记大脑部分的模范形象。寻找的两个重要特征是稀疏的标签模式（图1A）和识别能力，个人的细胞成分（图1B - C）。 </李&gt; <li> 故障排除DiOLISTIC标签 ：图2显示部分的例子DiOLISTIC标签不正确的工作。根据我们的经验，为低效的标签有三个主要原因： <ul><li> 标签过于稀疏或密集 ：在一种情况下，极少数细胞标记每节，或过多的脑细胞被标记为防止单细胞成分的分离和研究。解决方案：调整用于涂料TEFZEL子弹油管（1.6节）的最终解决方案的包被的磁珠浓度。减少或增加体积的水用来溶解的珠子，以正确的过疏或太密的标签（3毫升的初始值），分别。此外，可能会造成低效率的标签不到最佳的气体压力时拍摄到的部分涂珠。确保TEFZEL子弹油管已发布的涂层珠的大部分区域，并出现清晰的拍摄后，这种可能性可以排除。否则，拍摄的气体压力应增加。 </li><li> 坏子弹 ：子弹准备过程中形成大的团块或染料涂层的钨珠集群。路段将类似于图2A - B的例子。解决方案：新的子弹，要特别注意第1.5和/或超声时间延长1.6节。 </li><li> 在固定 ：组织多聚甲醛溶液中保存的时间比优化所需的时间固定为200-300微米的部分（30分钟，1小时4毫米）。这种妥协质膜的完整性，并产生标记的部分看起来像图2C - D所示的标签似乎重点由于染料分散细胞。解决方案：减少固定时间。 </li></ul></li><li> 共焦成像：图像堆栈收购共聚焦显微镜（蔡司LSM 510 META）使用20倍的目标与整个细胞和枝蔓段63X水浸泡的目的。 DII从二级抗体的存保计划的561 nm激光线和绿色荧光，使用488 nm激光线很兴奋很兴奋。使用共聚焦显微镜时取得的标记样品的光学切片进一步允许单标记细胞样品中的隔离。图3A显示了从小鼠大脑和复杂的树突分支模式的纹状体中刺神经。这些神经元的树突分支从啮齿类动物（图3B）和非人类灵长类动物（图3C）的高倍率图像证明树突和树突棘，使用这种技术在这两个物种在染色的程度。树突和突起（棘）出现良好定义的，即使考虑到长，薄的脊柱元首如此丰富的中型刺神经元。 与免疫相结合DiOLISTICS时，共焦成像，也有利于明确本地化污渍同一焦平面和相同的细胞。图4显示了一个DII的标签，在单细胞绿色荧光蛋白的免疫共定位的例子。该图像是一个栈（Z -节0.7微米）从下D1多巴胺受体启动子的荧光蛋白GFP表达的转基因小鼠的纹状体切片获得的。 </li></ol><img src="/files/ftp_upload/2081/2081fig1.jpg" alt="图1" /> 图1。 DII的神经元在非人类灵长类动物的大脑切片的标签。 （一）低倍率影像显示稀疏DII的神经元标签从食蟹猴，尾状核的大脑包含了染色片（BC）的隔离介质刺神经元可以很容易地用荧光解剖范围确定。 <img src="/files/ftp_upload/2081/2081fig2.jpg" alt="图2" /> 图2。故障排除DiOLISTIC标签。从背侧纹状体的猴子（A）和鼠标染色片（AB），（b）显示的染料涂层珠子的大团块，作为一个后果，没有个别的细胞成分可以区分（CD）的图像，体现了申请DiOLISTIC标签，在保持长时间的时间或地方组织保存的固定液的部分失败的猴子（C）和鼠标（四）组织。 <img src="/files/ftp_upload/2081/2081fig3.jpg" alt="图3" /> 图3。刺的纹状体中神经元的共焦影像与DII染色 。 （一），整个细胞图像堆栈允许树突分支的形态分析。（BC）枝晶段（二）从小鼠和猴（三）中等刺神经元枝蔓和棘明确标示。 <img src="/files/ftp_upload/2081/2081fig4.jpg" alt="图4" /> 图4。结合免疫DiOLISTIC标签。 （一）DiI标记<s仲&gt;中多刺的BAC转基因小鼠表达GFP D1多巴胺受体启动下纹状体神经元（B）使用抗GFP抗体的免疫染色改编自Lee等人的方法。 （2006年）。 A.（三）红色和绿色荧光的复合图像相同的字段标识为GFP阳性神经元的直接投资收益标记神经元。

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	<li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+"DiOLISTIC"+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor "DiOLISTIC" labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Gan, W. B., Grutzendler, J., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+delivery+of+dyes+for+structural+and+functional+studies+of+the+nervous+system.">Ballistic delivery of dyes for structural and functional studies of the nervous system.</a> Cold Spring Harb Protoc. , pdb.prot5202-pdb.prot5202 (2009).</li><li>Grutzendler, J., Tsai, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+labeling+of+neuronal+populations+by+ballistic+delivery+of+fluorescent+dyes.">Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes.</a> Methods. 30, 79-85 (2003).</li><li>Lee, K. W., Kim, Y., Kim, A. M., Helmin, K., Nairn, A. C., Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine-induced+dendritic+spine+formation+in+D1+and+D2+dopamine+receptor-containing+medium+spiny+neurons+in+nucleus+accumbens.">Cocaine-induced dendritic spine formation in D1 and D2 dopamine receptor-containing medium spiny neurons in nucleus accumbens.</a> Proc. Natl. Acad. Sci. U S A. 103, 3399-3404 (2006).</li><li>Matsubayashi, Y., Iwai, L., Kawasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+double-labeling+with+carbocyanine+neuronal+tracing+and+immunohistochemistry+using+a+cholesterol-specific+detergent+digitonin.">Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin.</a> J Neurosci Methods. 174, 71-81 (2008).</li><li>Neely, S. t. a. n. w. o. o. d., Deutch, G. D., Y, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+diOlistic+labeling+with+retrograde+tract+tracing+and+immunohistochemistry.">Combination of diOlistic labeling with retrograde tract tracing and immunohistochemistry.</a> J Neurosci Methods. 184, 332-336 (2009).</li><li>O'Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diolistics:+incorporating+fluorescent+dyes+into+biological+samples+using+a+gene+gun.">Diolistics: incorporating fluorescent dyes into biological samples using a gene gun.</a> Trends Biotechnol. 25, 530-534 (2007).</li><li>Shen, H. W., Toda, S., Moussawi, K., Bouknight, A., Zahm, D. S., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+dendritic+spine+plasticity+in+cocaine-+withdrawn+rats.">Altered dendritic spine plasticity in cocaine- withdrawn rats.</a> J. Neurosci. 29, 2876-2884 (2009).</li></ol>

NIAAA和俄勒冈国家灵长类动物研究中心的动物护理和使用委员会的指导，所有动物的程序进行。作者们没有透露。

DiOLISTIC标签是最通用的技术可用于细胞荧光标签之一，因为它可以从不同的物种的组织切片，广泛的年龄，和组织获得新鲜或从固定液灌注动物（见也甘等，2009）。这个过程是比较快的，因为它需要1-2天，它可以与其他更经典的标签的方法，如免疫（Lee 等 ，2006）相结合。特别应重视避免过度固定和孵化解决方案使用高洗涤剂浓度，因为这些将包括的亲脂膜的完整性，并导致染料泄漏的细胞。低浓度促进抗体的渗透可以通过TRITON X - 100（Lee 等 ，2006），以及毛地黄皂苷或皂苷洗液（松林等，2008）。这种技术可以应用到的一些修改，包括与甘等人（2000）或改变抗体曝光（ 尼利等，2009）的长度和类型的多个染料使用的子弹。 传统上，DII已经用于跟踪在大脑中的神经元的预测。 DiOLISTIC标签扩展描述一个有用的方法来研究细胞的形态及其应用。极大的兴趣，因为在大脑中发现的大型多样性和猜测，细胞的形状可能反映在中枢神经系统的神经元群体的功能多重的神经元的形态。这样的一个例子是，事实上，在许多哺乳动物的中枢神经系统显示的小突起神经元称为树突棘是谷氨酸突触的网站。通过这种方式，可以到一个细胞的谷氨酸突触的密度相关的树突棘密度，使用此处所述的标记技术，可以测量。此外，如树突总长度，分枝格局，树突棘的形状和密度等形态参数可量化和研究。 使用荧光标记，为研究神经元的形态有许多优点相比，更多的传统技术依赖亮视野显微镜（如高尔基染色），因为它允许更高的分辨率共焦成像。使用DII作为一个旨在测量树突棘密度的实验中的荧光团和形态的另一个优点是其亲脂性的属性。 DII到质膜分区，并提供一个明确界定的神经元突起和树突状突起的轮廓。鉴于大多数树突棘（小于1 femtoliter）体积小，染色膜是更有效的，并允许更好的可视化小，薄的突起比胞质染色。

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<td>1-1&#x2019;-dioctadecyl-3,3,3&#x2019;,3&#x2019;- tetramethyl-indocarbocyanine perchlorate (DiI)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-282</td>
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<td>Methylene chloride</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>H485-06</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten beads</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2269</td>
<td>1.7 &mu;m diameter</td>
</tr>
<tr>
<td>Polyvinylpyrrolidone </td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>529504</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel bullet tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
<tr>
<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2431</td>
<td>&nbsp;</td>
<tr>
<td>Isopore membrane filter paper</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>TSTP02500</td>
<td>3.0 &mu;m pore size</td>
</tr>
<tr>
<td>ProLong Gold Antifade</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>30525-89-4</td>
<td>(16% w/v)</td>
</tr>		</tbody>
		</table>
		</div>

diolistic labeling of neurons from rodent and non-human primate brain slices

DiOLISTIC染色采用基因枪，引入神经 ​​元脑片（甘等人，2009年; O&#39;Brien和Lummis，2007年，甘等，2000）。荧光染料，如直接投资收益，。在这里，我们提供了一个好的和坏的结果，这将有助于设立的技术，当模范的图像所需的每一步的详细说明。根据我们的经验，几步证明的成功应用DiOLISTICS的关键。这些考虑因素包括DII涂层子弹的质量，固定液暴露程度，和孵化解决方案中使用的洗涤剂浓度。提供提示和常见问题的解决。 这是一个多功能的标签技术，可以适用于广泛的年龄多种动物物种。 DiOLISTIC标签，准备从年轻的动物或限制，因为他们依靠一个荧光的转基因表达对小鼠与其他荧光标记技术，可以适用于所有年龄段，物种和基因型的动物，它可以结合使用免疫识别特定的细胞亚群。在这里，我们展示了使用标签神经元的DiOLISTICS量化枝蔓分支和树突棘形态的目的从成年小鼠和成人的非人类灵长类动物的大脑切片。

Watch this Scientific Journal Video about DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices at JoVE.com

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

我们展示了基因枪的使用，引入神经元在不同年龄段的啮齿类动物和非人类灵长类动物的大脑切片的荧光染料，如直接投资收益，。在这种特殊情况下，我们使用成年小鼠（3-6个月以上）和成人cynomologus猴子（9-15岁）。这项技术，最初是由实验室博士李奇曼（甘<em&gt;等</em&gt;，2000年），是非常适合的分支的树突和树突棘形态的研究，可与传统的免疫相结合，如果保持在低浓度的洗涤剂。

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

diolistic-labeling-neurons-from-rodent-non-human-primate-brain

Research

JoVE Journal

Neuroscience

23.8K 次观看.  NIH. 我们展示了基因枪的使用，引入神经元在不同年龄段的啮齿类动物和非人类灵长类动物的大脑切片的荧光染料，如直接投资收益，。在这种特殊情况下，我们使用成年小鼠（3-6个月以上）和成人cynomologus猴子（9-15岁）。这项技术，最初是由实验室博士李奇曼（甘等，2000年），是非常适合的分支的树突和树突棘形态的研究，可与传统的免疫相结合，?...

视频: DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...