该研究项目得到了一个的ARRA补充，5 R01 NS056380。通过病毒载体核心和埃默里大学神经科学NINDS核心设施补助，P30NS055077显微镜核心提供额外的支持。我们感谢埃默里大学的转基因小鼠和基因打靶核心的鼠标线从GENSAT的派生格雷格帕佐尔SSTR3-GFP的稳定IMCD3细胞株; SSTR3-GFP慢病毒布拉德利尤德构建。单克隆抗体从发展研究杂交瘤细胞银行，开发的的NICHD的主持下，获得和维护由美国爱荷华大学生物科学系，爱荷华州爱荷华市52242。所有动物的程序批准由IACUC和埃默里大学生物安全委员会。

成年小鼠安乐死机械颈椎脱位。所有动物的程序批准由IACUC和埃默里大学生物安全委员会。 1。胚胎生成<ol><li>过他莫昔芬诱导表达Cre重组酶行CAGGCreER，dsRedCre记者线（玻璃转化温度（CAG-BGEO的，红色荧光蛋白* MST）1Nagy）（ 图1）5,6。要监视的Shh的响应中的子细胞，跨CAGGCreER和DsRed的配合的OLIG2-eGFP的BAC转基因小鼠（Tg的（OLIG2-EGFP）EK23Gsat）（ 图1）7,8间的相对定时。 </li><li>他莫昔芬100％的乙醇溶解，并执行到怀孕的女性在胚胎6.5（E6.5）腹腔注射2.5毫克每40克体重的他莫昔芬诱导Cre表达中的一小部分细胞和DsRed标记。 </li><li> 48小时后胚胎解剖，查明DsRed的阳性胚胎使用荧光显微镜，他们转移到培养皿中观测E单细胞分裂过程中体外成像（见下文）。 </li></ol> 2。全小鼠胚胎文化<ol><li>解剖E8.5胚胎在预热的的洗涤培养基中的DMEM/F12（1:1）中添加10％新生小牛血清和1％青霉素/链霉素（P / S）9。 </li><li>直接解剖后，放置在37℃加热阶段的胚胎在荧光显微镜下识别GFP和/或DsRed的阳性（见下文）。 </li><li>传输多达2个胚胎成500μl的预平衡补充L-谷氨酰胺的无酚红培养基中含有50％从只Sprague-Dawley雄性大鼠血清和50％的DMEM/F12（1:1）和1％的颗粒在0.85％NaCl和P / S 9 1 M HEPES。 </li><li>涂抹薄薄的一层（0.1厘米）平衡过的轻质矿物油以防止蒸发，含有胚胎的培养皿转移到37℃，5％CO 2培养箱中在中。 </li></ol> 3。病毒感染<ol><li>在以神经上皮纤毛标签，添加5-10微升的SSTR3-GFP慢，约2万病毒颗粒，到500微升平衡下降培养基含有E8.5胚胎。 </li><li>培养18小时后，感染的胚胎转移到几滴洗涤介质洗出病毒，并准备神经管片。 </li></ol> 4。神经管片制备实时成像<ol><li>解剖神经管预热的洗涤介质中，使用一个微型刀尺寸0.025毫米，在1％的琼脂涂层菜E8.5胚胎。 </li><li>将孤立的神经管腹侧平衡培养基150μL降不含酚红35毫米的聚-L-赖氨酸镀膜玻璃底菜。 </li><li>将少量的混合物（1:1）由100％纯凡士林和融化的蜡烛（蜡烛宜家）神经管周围安装，并轻轻按压，以一个狭窄的一块玻璃盖玻片固定样本。 </li><li>量烹调平衡轻质矿物油（ 图2）有一薄层（约0.1厘米）。 </li></ol> 5。实时成像和时间推移共聚焦显微镜<ol><li>地方菜下尼康的A1R激光的扫描共聚焦显微镜倒置显微镜配备环境室调节温度，设定为37℃，5％CO2。 </li><li>使用油浸物镜60X记录GFP标记的纤毛和DsRed的阳性细胞，而40倍的油浸物镜使用监控OLIG2 GFP和DsRed阳性细胞的。 </li><li>打开NIS Elements软件成立时间推移成像条件。每10分钟获得的z叠至25μm，间隔为1.5μm（40倍物镜）和最多8微米，间距为0.4μm的（60倍物镜）。如果需要使用488和561 nm的激发波长和传输通道。收购512×512大小的图像。设置多个用户定义的int地区erest进行同步录制。 通过使用低激光强度（最多11％561 mCherry EGFP的488分之405高达4.5％），扫描速度1，线路2和平均大小的销孔（61获得最佳曝光的激光功率和图像的亮度的调整红色荧光蛋白微米，28.1微米为OLIG2; 72微米为SSTR3GFP;为红色荧光和SSTR3GFP的61.3微米，58微米DsRed和OLIG2的）。基因编码的荧光记者的使用使我们能够检测到的亮度与激光功率低。 </li><li>分析记录的数据使用Imaris里三维重建软件。 </li></ol> 6。免疫<ol><li>修复胚胎或孤立的神经管在4％多聚甲醛/ 0.1 M磷酸缓冲液冰（4℃），在玻璃盘1小时。 </li><li>洗净的样品2小时，在冰上在PBS（4℃）（变化PBS了几次），并把在30％蔗糖/ 0.1 M的磷酸盐缓冲液在夜间或直至胚胎下沉。 </li><li>嵌入在华侨城，冻结干冰上并储存在-80°C。 Perform切片在低温恒温器（10毫米）。 </li><li>洗10分钟，在洗涤溶液中含有1％热灭活的羊血清和0.1％的Triton X-100的PBS中的幻灯片。 </li><li>后浓度稀释在洗涤溶液中的初级抗体:鼠单克隆抗RFP（5F8）1:200;兔抗血清1:1500 Arl13b和小鼠单克隆抗Arl13b 1:5（295B/54）;兔抗OLIG2中1:300;鼠标单克隆抗体，Shh和Nkx2.2所有1:10 PAX6和多克隆Ki67的1:500。新增约150微升平加湿室每张幻灯片，用封口膜覆盖，以避免干燥和遗留晚上，在4°C </li><li>洗三次，每次20分钟，在室温下在洗涤溶液中的幻灯片。 </li><li>在洗涤解决方案1:200浓度稀释第二抗体的Alexa Fluor 488，568，350。使用赫斯特1:3,000或TO-PRO-3 1:500染色细胞核。每张幻灯片中加入150μl，离开1小时，在室温下避光加湿室。 </li><li>在洗涤溶液总重量洗净幻灯片O次数，每次30分钟，在室温下。 </li><li>安装滑动在80％，，延长黄金抗褪色试剂和视图的24小时内。 </li></ol>

<ol>
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没有利益冲突的声明。

我们的体外系统，使我们在发展中的神经上皮实时直接观察单个细胞分裂。作为一个例子，我们研究小鼠胚胎神经管内的细胞分裂和监控无论是纤毛形成或嘘响应。我们确认我们的成像效果组（n = 24）与固定部分（N = 178），表明我们的技术提供有关生理数据的结果是一致的。 我们的技术依赖于被限制酶Cre诱导的细胞的一个子集，因此，他莫昔芬的剂量必须精确。我们制定了剂量单细胞标记。我们相信，这将是很容易有轻微的增加剂量的基础上初步分析，呈剂量依赖性反应，他莫昔芬6 CAGGcreER线标记小细胞克隆。此外，胚胎培养条件是至关重要的，因为如果出现发育异常的条件是关闭。我们证实，发展正常进行培养条件下我们使用，这是众所周知的工作至少36小时11。 诱导Cre重组酶Cre记者行我们使用的是公开可用的，所以使得这个技术相当适应各种问题的研究人员可以很容易获得。我们的技术的真正威力是在单细胞基因标记，我们的方法可以用来审问单细胞行为的许多现有的和未来的突变小鼠线。了解什么是真正的错在许多哺乳动物胚胎发育过程中的细胞内直接观察这些线已经得到了广泛的研究，这将是至关重要的。我们的基因编码的荧光记者incorporaton检测激光功率低亮度，提供了一个真正的优势，这样的观察。这是很容易想象荧光标记的蛋白质标记特定的细胞器或其他TRanscriptional记者集成的未来。 我们感兴趣的是相对初级纤毛的形成和Shh子细胞分裂细胞反应时间。但是，转基因株系，使我们能够监视响应的Shh表达OLIG2-eGFP的整个细胞质中，禁止我们看到在同一个细胞的的主要纤毛，SSTR3-GFP标记。因此，我们会大大受益于核限制OLIG2-EGFP或光谱上不同的的SSTR3荧光蛋白融合。 除了使用的针对性和转基因株系中，我们发现，我们的技术可以使用病毒结构调整，并感染神经上皮文化提供了有用的数据。这将是有用的调查提供了一个更快的方法比一代的转基因小鼠线。此外，该方法可以验证这样一条线的产生;的确，我们的数据认为一个跨基因线表达SSTR3-GFP将是很有用处的领域。 我们的方法提供了与该领域可以更直接监测细胞行为的工具。小鼠突变再加上丰富的资源，我们期望这个方法来检查数组原位细胞行为，尤其是强大的。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J</td>
			<td>Jackson Laboratory</td>
			<td>005438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd</td>
			<td>MMRRC,</td>
			<td>010555-UCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CAGGCreER</td>
			<td>Jackson Laboratory</td>
			<td>003724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Lonza</td>
			<td>14-416F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat Serum SD male</td>
			<td>Harlan Bioproducts</td>
			<td>4520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1M Hepes</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sstr3-GFP lentivirus</td>
			<td>Emory Viral Core</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-knife, size 0.025 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-L-lysine coated glass bottom dish</td>
			<td>MatTek</td>
			<td>P35GC-0-10-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100% petroleum jelly</td>
			<td>Kroger</td>
			<td>FL9958c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1R Laser Scanning Confocal Inverted Microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Imaris 3D software</td>
			<td>Bitplane AG</td>
			<td>Imaris 7.2.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat-inactivated sheep serum</td>
			<td>Invitrogen</td>
			<td>16210-072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafolmaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Lab made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-RFP (5F8)</td>
			<td>Chromotek</td>
			<td>110411</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Arl13b serum</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Arl13b 1:5</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2</td>
			<td>Chemicon</td>
			<td>AB9610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Pax6</td>
			<td>Developmental Hybridoma Bank</td>
			<td>Pax6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonalsShh</td>
			<td>Developmental Hybridoma Bank</td>
			<td>5E1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Nkx2.2</td>
			<td>Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Ki67</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568</td>
			<td>Molecular Probes</td>
			<td>A11031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350</td>
			<td>Molecular Probes</td>
			<td>A11046</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Fisher</td>
			<td>AC22989</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of single cell divisions in mouse neuroepithelium

我们开发了一个系统，集成了实时成像的荧光标记物和小鼠胚胎神经上皮的培养切片。我们利用现有的鼠标线的遗传细胞谱系追踪:他莫昔芬诱导的Cre线和红色荧光蛋白的酶Cre记者表达Cre重组酶介导的重组后。通过使用他莫昔芬的相对较低的水平，我们能够在一个小数量的细胞以诱导重组，允许我们按照个别的细胞分裂。此外，我们观察到的转录反应刺猬（SHH）信号使用OLIG2-EGFP转基因株系1-3，我们监测纤毛的形成与病毒感染培养的片表达的纤毛标记，SSTR3-GFP 4。为了图像的神经上皮，我们收获胚胎E8.5，分离出神经管，安装在适当的培养条件下成神经切片成像室和执行时间推移共聚焦成像。我们的前活体内成像方法，使我们能够跟踪单细胞分裂，初级纤毛的形成和Shh响应有关生理的方式评估的相对时序。这种方法可以很容易地适应用不同的荧光标记物，并提供该字段，用以监测细胞行为的原产地和实时的工具。

在这里，我们进行体外实时成像单细胞分裂E8.5小鼠神经上皮内。要标记单个细胞，诱导Cre重组酶在细胞内含有一个的酶Cre记者行表达红色荧光蛋白重组后，5,6（ 图3A）的一个子集。因此，48小时后，我们能够观察到在体外成像（ 图4A-D）的单细胞分裂。随之而来的是，我们监测标记细胞成为嘘响应的包括修改BAC转基因OLIG2-EGFP线（ 图3C-D;图4G-N）1-3嘘转录记者。要观察纤毛的形成，我们产生SSTR3-GFP慢病毒感染培养的胚胎（ 图3B，图4A-D）4。免疫荧光法进行，以确认在体内的结果（ 图4E-F）。 时间圈记者dsRedCre表达OLIG2-EGFP的胚胎在体外培养期间与SSTR3-GFP慢或感染É共聚焦成像观察显示在图5 10。为了SSTR3-GFP的表达，我们使用可视化纤毛确保不会干扰任何潜在的生物学过程，我们佐证了我们的实时成像数据与固定野生型胚胎部分。因为我们发现了同样的百分比的异步纤毛形成和Shh响应，它表示的的SSTR3-GFP病毒并不会影响这些进程（ 图5A）。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/4439/4439fig1highres.jpg" src="/files/ftp_upload/4439/4439fig1.jpg" /> 图1。鼠标的最后交叉和tamoxi的流程图体外实时成像程序使用鼠标神经上皮。芬治疗描绘整个小鼠胚胎培养方法。表达荧光标记蛋白的胚胎挑选和准备实时成像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4439/4439fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5.5in" fo:src="/files/ftp_upload/4439/4439fig2highres.jpg" src="/files/ftp_upload/4439/4439fig2.jpg" /> 图2。神经管片编制实时成像。）E8.5千方百计胚胎的首次亮相体节对B）神经管解剖预热介质用微型刀。）神经管片安装倒在腹侧聚-L-赖氨酸涂布的玻璃底菜D）的量小，从凡士林油和蜡的混合物，应用神经管周围，轻轻地覆盖由一个缺rrow一块玻璃盖玻片。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4439/4439fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4439/4439fig3.jpg" /> 图3。可视化荧光标记的蛋白质在神经管活细胞共聚焦显微镜A）红色荧光蛋白标记B）的 SSTR3-GFP的慢病毒表达形式（黄色箭头）初级纤毛。CD）E8.5胚胎携带OLIG2-GFP单个细胞。显示GFP表达神经上皮内。酒吧（A，D）为30微米，15微米和1.5微米（C）（B）。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/4439/4439fig4highres.jpg" src="/files/ftp_upload/4439/4439fig4.jpg" /> 图4。纤毛形成监测和Shh信号在分裂的细胞发展神经管AD）单DsRed的阳性细胞分裂显示了一个女儿细胞纤毛形式在之前的其他细胞（C，白色箭头）。电影成像的一帧的速率，每10分钟，EF）免疫荧光法使用抗RFP在绿色（DsRed的谱系追踪）和红色Arl13b（纤毛）。盒装区（E），无赫斯特（F）。GN）扩大DsRed的阳性细胞进行分裂（IM）。该OLIG2-GFP的表达只有一个女儿（J，N）。在记录成像的一帧的每10分钟的速率。酒吧是5微米（AD），50微米（F），25微米（GN）。 <img alt="图5" fo:content-width="6in"FO:SRC ="/ files/ftp_upload/4439/4439fig5highres.jpg"SRC ="/ files/ftp_upload/4439/4439fig5.jpg"/&gt; 图5。红色荧光蛋白阳性细胞纤毛和Shh的外观计数A）红色荧光蛋白的细胞进行分裂和纤毛本地化数。符号*表示，实时成像纤毛的形成是同步的。B）红色荧光蛋白和OLIG2-GFP阳性细胞进行分裂和Shh信号的数量。

Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

在这里，我们开发所需的工具<em&gt;体外</em&gt;实时成像跟踪单E8.5的小鼠神经上皮细胞分裂

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

ex-vivo-live-imaging-of-single-cell-divisions-in-mouse-neuroepithelium

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

10.3K 次观看.  Emory University School of Medicine. 在这里，我们开发所需的工具体外实时成像跟踪单E8.5的小鼠神经上皮细胞分裂

视频: Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.