这项工作得到了三菱基金会，武田科学基金会，JST PRESTO和JSK KAKENHI Grant Number 16H06144的支持。

所有实验程序均经东京大学动物实验伦理委员会和东京大学批准实验动物护理和使用指南进行。 1.准备解决方案<ol><li>准备0.01M磷酸盐缓冲盐水（PBS）:将PBS片剂（0.14M NaCl，0.0027M KCl，0.010M PO 4 3- ，pH 7.4）加入到1L蒸馏水中，并在室温下搅拌直到其完全溶解。 </li><li> 在PBS中制备4％多聚甲醛（PFA）:将4g PFA加入到100mL PBS中。将溶液在60℃下搅拌直至完全溶解。 <ol><li>将PFA溶解在PBS中后，用1N氢氧化钠（NaOH）将溶液的pH调节至7.4。然后，在冰上冷却并通过滤纸过滤溶液以除去任何颗粒物。储存于4°C。 注意:使用这些试剂时，请妥善保管。处理小心使用手套，安全护目镜和化学罩下的实验室外套。 注意:使用2 d内制备的新鲜4％PFA溶液。 </li></ol></li><li>准备补充有0.3％Triton X-100（PBST）的0.01M PBS:加入3mL Triton X- 100至997mL PBS。在室温下搅拌该溶液直到其完全溶解。 </li><li>制备1％和5％封闭溶液:加入10g脱脂乳至200 mL PBST，制备5％封闭溶液。在室温下搅拌直至完全溶解。用PBST稀释5％封闭溶液5次以制备1％封闭溶液。 </li></ol> Parasagittal OB部分的准备<ol><li>使用1-2周龄的动物。这些年龄组适用于以下实验，因为肾小球清晰可见，可以用头骨切割大脑。 </li><li>用腹膜内注射戊巴比妥（50mg / kg体重）麻醉动物。用冰冷的4％P经皮灌注动物FA在PBS。小心处理，使用手套，安全护目镜和化学罩下的实验室外套。 </li><li>灌注后，用剪刀切开头部，小心地取出皮肤。然后，将剪刀刀片插入上下齿之间的空间，水平切割以去除下颚骨。用镊子和剪刀剪掉多余的组织，以留下嗅觉组织，包括OB和OE。 注意:不要去除OB周围的头骨，因为它保留了内侧肾小球垂直排列。 </li><li> 将嗅觉组织浸泡在PBS中以去除鼻腔中的空气。垂直切除脑后部，将嗅觉组织放置在嵌入模具的底部，切割面朝下。用最佳切割温度（OCT）复合物填充模具，然后将模具浸入液氮中。 <ol><li>化合物中的组织完全冷冻后，将其孵育在冷冻液中tat在-20℃下1小时。 </li></ol></li><li>用低温恒温器制作OB的串联旁路截面（10μm），并通过粘贴到MAS涂覆的玻璃片上收集。粘贴后，立即用吹风机干燥载玻片。 </li></ol>第1天:OB切片的四次免疫染色<ol><li>在室温下用PBS洗涤载玻片5分钟。重复3次。 </li><li>通过在RT下用5％封闭溶液孵育载玻片1小时来阻断非特异性结合位点。 </li><li>在1％的封闭溶液中，将载玻片O / N与初级抗体混合（每个载玻片400μL）孵育。使用以下一抗:豚鼠抗Kirrel2抗体（1:1000），山羊抗Semaphorin-7A（Sema7A）抗体（1:500），大鼠抗OL-原螯合素（OLPC）抗体（1:500），和小鼠抗囊泡谷氨酸转运蛋白2（VGLUT2）抗体（1:500）。 </li></ol> 4.第二天:OB的四次免疫染色片<ol><li>弃去一级抗体溶液，并在室温下用PBST洗涤载玻片5分钟。重复3次。 </li><li>在PBS中，用PBS中的二次抗体（每个载玻片400μL）混合1小时。使用以下二抗:驴抗小鼠Alexa Fluor 405（1:400），驴抗山羊Alexa Fluor 488（1:400），驴抗豚鼠Alexa Fluor 555（1:400）和驴抗 - 大鼠Alexa Fluor 647（1:400）。 注意:所有二级抗体应来自相同的宿主物种。为每个一级抗体选择不同波长的二级抗体荧光，以确保波长不重叠。 </li><li>弃去溶液，并在室温下用PBS洗涤载玻片5分钟。重复3次。 </li><li>在载玻片上安装盖玻片与安装介质。为此，在每张幻灯片上涂2滴安装介质，然后通过去除气泡放置盖玻片。 </li></ol>强度我asurements <ol><li>用荧光显微镜获取荧光图像。使用以下过滤器立方体:对于Alexa Fluor 405信号的DAPI过滤器立方体（360/40nm激发，460/50nm发射和400nm分色镜），GFP滤光镜立方体（470/40nm激发，525/50nm发射，和用于Alexa Fluor 555的Alexa Fluor 488，TRITC滤色器立方体（545/25nm激发，605/70nm发射和565nm分色镜）和Cy5滤光器立方体（620/60nm激发，700 / 75nm发射和600nm二向色镜）用于Alexa Fluor 647。 注意:调整曝光时间以获得图像的荧光信号而不饱和。 </li><li>通过VGLUT2的免疫荧光信号定义肾小球结构。用ImageJ测量肾小球内轴突分选分子的染色强度。 </li></ol>数据分析<ol><li>从轴突分选分子的染色强度中减去背景信号。 </li><li>准备e表达数据矩阵，其具有由轴突分选分子和由肾小球组成的行组成的列。 </li><li>使用准备的表达式数据集执行主成分分析（PCA）。然后，获得每个主成分的PCA分数，贡献率和因子负荷。 </li></ol>

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作者声明没有竞争的经济利益。

旁分泌OB部分的四次免疫染色使得能够在大量肾小球中同时观察和定量多达四个轴突分选分子的表达水平。通过用PCA分析这些多变量数据，可以推测这些分子表达的特征。 为了成功染色，组织样品制备至关重要。一些方案表明组织应用4％PFA后固定，并用30％蔗糖处理以进行冷冻保护。但是，在本协议中省略了这些步骤。这是因为在许多情况下，这些步骤减少染色信号强度并增加背景。此外，切片不应该在任何步骤干燥，以避免染色时的高背景。 根据所使用的组织和抗体的类型，最佳染色条件是不同的。所以是必须的y确定每个实验的具体染色条件。此外，一抗的浓度也是重要的。当浓度过高时，会增加背景。然而，当浓度太低时，会降低信号。因此，重要的是确定适当的一级抗体浓度以获得高质量的染色结果。一个限制是该方法在很大程度上取决于一级抗体和一级抗体组合相对于动物物种的质量。 OB的冠状切片已经常规用于免疫染色。然而，在该方案中，使用OB的内侧的旁侧部分能够将更多的肾小球放置在单个部分上。由于这些部分保留了沿背腹和前后轴的肾小球的地形顺序，它们也可用于分析轴突指导分子，其确定OSN轴突的投影位点。 在本研究中，准备了OB的旁矢状切片以使更多的肾小球放置在单个部分上。此外，水平部分也可用于研究人员揭示沿内侧或前后轴的差异。随着可用于多种免疫染色的抗体的发展，这种四重染色方法不仅可以应用于OB，而且可以应用于其他脑区域。

在发育过程中，神经元彼此精确连接，形成适当的神经回路，这对正常脑功能至关重要。由于大脑中的异常神经回路被认为是自闭症和精神分裂症等精神障碍的原因，因此了解神经回路形成的机制是神经科学领域的主要挑战之一。 在小鼠嗅觉系统中，嗅觉上皮（OE）中的每个嗅觉感觉神经元（OSN）仅表达一个功能性嗅觉受体（OR）基因，并且表达相同OR的OSN在其定型位置收敛其轴突到特定的一对肾小球嗅球（OB） 1，2 。小鼠嗅觉系统是研究神经回路形成的分子机制的优秀模型系统，因为研究人员可以利用OR表达来识别特定的sOSN的ubtype和可视化OSN轴突的投影位点作为清晰的肾小球结构。 OSN投射的一个显着特征是ORs在将OSN轴突投影到OB 3，4，5，6等方面发挥了指导作用。更具体地说，在OSN轴突被引导到近似目标区域之后，它们以OR依赖的方式被分离以形成肾小球。以前的研究表明，OR分子控制轴突分选分子的表达，调节肾小球分离7,8 。此外，积累的证据表明OR分子通过轴突分选分子9的独特组合产生神经元识别码。因此，为了理解OR依赖性肾小球分离的机制，有必要表征轴突分选的表达谱OSN中的ecules。 荧光免疫染色是可视化特定基因表达的常用方法。由于轴突分选分子的蛋白质主要定位于OSN轴突，研究人员需要使用OB部分来表征其在OSN中的表达模式。 OB的冠状切片常规用于免疫染色。然而，该制剂在相同的OB部分中沿着前后轴线丢失了地形信息。因此，我们开发了OB的内侧的旁侧制剂，其可以在相同的OB部分上安装尽可能多的周围的肾小球。结合使用多种抗体的免疫染色，该制剂允许比较和分析轴突分选分子的表达模式，而在OB部分之间没有染色变异。 此外，已经提出了免疫组织化学染色方法，而不需要用PFA a进行固定nd蔗糖处理。这种方法允许研究人员获得足够高质量的染色数据进行多变量数据分析。本文介绍的方案将为研究嗅觉神经电路形成的研究人员提供有力的方法。

<table><tbody><tr><td>Phosphate-Buffered Saline (PBS) Tablets, pH7.4</td><td>TAKARA BIO</td><td>T9181</td><td></td></tr><tr><td>Skim Milk</td><td>nacalai tesque</td><td>31149-75</td><td></td></tr><tr><td>goat anti-Sema7A antibody</td><td>R&amp;amp;D Systems</td><td>AF2068</td><td></td></tr><tr><td>rat anti-OLPC antibody</td><td>Merck Millipore</td><td>MABT20</td><td></td></tr><tr><td>mouse anti-VGLUT2 antibody</td><td>Merck Millipore</td><td>MAB5504</td><td></td></tr><tr><td>goat anti-BIG-2 antibody</td><td>R&amp;amp;D Systems</td><td>AF2205</td><td></td></tr><tr><td>gunea pig anti-Kirrel2 antibody</td><td>Operon Biotechnologies</td><td></td><td>Anti-Kirrel2 antibodies were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides (644-673aa): CRLYRARAGYLTTPHPRAFTSYMKPTSFGP</td></tr><tr><td>donkey anti-mouse Alexa Fluor 405</td><td>Abcam</td><td>ab175658</td><td></td></tr><tr><td>donkey anti-goat Alexa Fluor 488&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>705-545-003</td><td></td></tr><tr><td>donkey anti-guinea pig Alexa Fluor 555</td><td>Thermo Fisher Scientific</td><td>A21432</td><td></td></tr><tr><td>donkey anti-rat Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>712-605-153</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Wako</td><td>162-16065</td><td></td></tr><tr><td>MAS coated slide glasses</td><td>MATSUNAMI</td><td>MAS-01</td><td></td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>11253-27</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>dissecting scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>fluorescent microscope</td><td>KEYENCE</td><td>BZ-X700</td><td></td></tr><tr><td>DAPI filter cube</td><td>KEYENCE</td><td>OP-87762</td><td></td></tr><tr><td>GFP filter cube</td><td>KEYENCE</td><td>OP-87763</td><td></td></tr><tr><td>TRITC filter cube</td><td>KEYENCE</td><td>OP-87764</td><td></td></tr><tr><td>Cy5 filter cube</td><td>KEYENCE</td><td>OP-87766</td><td></td></tr><tr><td>filter paper</td><td>ADVANTEC</td><td>00011185</td><td></td></tr><tr><td>O.C.T compound</td><td>Sakura Finetek</td><td>M71484</td><td></td></tr></tbody></table>

quadruple immunostaining of the olfactory bulb for visualization of olfactory sensory axon molecular identity codes

由于其简单的解剖结构，小鼠嗅觉系统通常用于研究神经回路形成的机制。嗅觉神经元（OSN）是具有单个枝晶和单个无分支轴突的双极细胞。 OSN仅表达一个嗅觉受体（OR）基因，表达给定类型的OR的OSN将其轴突收敛于嗅球（OB）中的几组不变的肾小球。 OSN预测的一个显着特征是表达的ORs在轴突投影中起到指导作用。 ORs调节多个轴突分选分子的表达，并在OSN轴突末端产生轴突分选分子的组合分子代码。因此，要了解OR特异性轴突引导机制的分子机制，在同一肾小球中的OSN轴突末端表征其表达谱是至关重要的。本文的目的是介绍收集尽可能多的肾小球的方法e在单个OB部分上，并使用多个抗体进行免疫染色。这将允许比较和分析轴突分选分子的表达模式，而在OB部分之间没有染色变异。

嗅肾小球图由初始全局靶向和随后的OSN轴突1,2的肾小球分离形成。通过由表达水平由表达的OR分子决定的轴突分选分子介导的粘附/排斥轴突相互作用调节肾小球分离7 。涉及肾小球偏析的轴突分选分子在OB 9中以位置无关的镶嵌方式表达。在本研究中，我们选择了以下基因: Kirrel2 ， Sema7A ， OLPC ， BIG-2和PCDH17 。这些轴突分选分子中的一些以活性依赖性方式表达7,8,10。 四倍免疫这里所示的方法允许同一部分同时显示四个分子的表达模式。这些轴突分选分子显示位置无关的镶嵌表现，但它们的图案不相同（ 图1A，B ）。肾小球结构由VGLUT2的荧光信号定义（ 图2A ）。轴突分选分子（Kirrel2，Sema7A，OLPC）在每个肾小球中差异表达;然而，用作肾小球标记的VGLUT2在肾小球中均匀表达（ 图2B，2C ）。 单个肾小球可以被认为是由多个变量表示的数据单元。为了分析该多变量表达数据，进行主成分分析（PCA）。 PCA定义了一个新的坐标系，使得第一个坐标具有最大的方差数据集，并且每个坐标系都成功坐标具有最大的残差方差。表达式数据集经受PCA并绘制在第一和第二主成分（PC1和PC2）的空间中（ 图3A ）。每个主成分的贡献率表示每个主成分在变量的总趋势中代表多少百分比。 PC1，PC2，PC3，PC4和PC5的贡献率分别为33.4％，28.2％，19.8％，17.3％和6.3％（ 图3B ）。 PCA还揭示了每个主要组成部分的因素负荷。平方因子加载指示原始变量中的方差百分比多少由一个因素解释。在PC1中，Sema7A，OLPC和Kirrel2的因子负载正负载，而BIG-2和PCDH17的因子载量不是（ 图3C ）。以前的研究分析了这些分子在缺陷型环核酸突变小鼠中的表达<html> （CNG）通道基因，其是嗅觉信号转导的组成部分11 ，并且显示出表达水平中CNG依赖性变化的模式类似于PC1 9中的因子负载。这些结果表明，这些分子的多种表达是由CNG通道介导的神经活动产生的。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55893/55893fig1.jpg" /> 图1:旁路OB部分的四次免疫染色。 （ A ）OE＆OB示意图。 （ B ）来自用针对vGLUT2（白色），Kirrel2（红色），Sema7A（绿色）和OLPC（蓝色））的抗体免疫染色的2周龄小鼠的旁矢状面。 Kirrel2（红色），Sema7A（绿色）和OLPC（蓝色）的合并图像显示在右侧。刻度棒=100μm。/files/ftp_upload/55893/55893fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55893/55893fig2.jpg" /> 图2:轴突分选分子的表达分析。 （ A ）用VGLUT2（紫色），Kirrel2（红色），Sema7A（绿色）和OLPC（蓝色）的抗体免疫染色的旁侧嗅球（OB）切片。每个肾小球由VGLUT2（突触前突触）的荧光信号定义。肾小球结构被虚线包围。 （ B ）肾小球＃1，＃3，＃4和＃6中Kirrel2，Sema7A和OLPC的表达水平。在每个肾小球中测量轴突分选分子的染色强度。 （ C ）在肾小球之间比较轴突分选分子和VGLUT2的表达水平。这些分子的信号强度用ea测量ch肾小球图表上的灰色条表示表达水平的平均值。刻度棒=100μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55893/55893fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55893/55893fig3.jpg" /> 图3:轴突分选分子的表达数据的PCA。 （ A ）五个轴突分选分子（Kirrel2，Sema7A，OLPC，BIG-2和PCDH17）的表达数据集的主成分分析（PCA）分数（PC1Vs.PC2）。共分析了1799例肾小球。 （ B ）所有五个主要组成部分的缴款比率和累积缴款比率。 PC1，PC2，PC3，PC4和PC5的贡献率分别为0.334,0.282,0.198,0.173和0.063。 （ C ）Kirrel的因子载荷2，PC1-3中的Sema7A，OLPC，BIG-2和PCDH17。 PC1，PC2和PC3的特征值分别为1.67,1.16和0.99。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55893/55893fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes at JoVE.com

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

嗅觉神经元表达各种各样的轴突分选分子，建立适当的神经电路。该方案描述了一种免疫组织化学染色方法来观察嗅觉神经元轴突末端的轴突分选分子的组合表达。

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

quadruple-immunostaining-olfactory-bulb-for-visualization-olfactory

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.0K 次观看.  The University of Tokyo. 嗅觉神经元表达各种各样的轴突分选分子，建立适当的神经电路。该方案描述了一种免疫组织化学染色方法来观察嗅觉神经元轴突末端的轴突分选分子的组合表达。 

视频: Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

科研

神经科学

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

一种用于果蝇嗅觉回路组装延时成像研究的外植体系统

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

发育生物学

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

摘要

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此视频中的章节

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

嗅觉受体神经元在全山免疫标记

嗅觉系统作为模型来研究轴突生长的模式和形态

纤毛对小鼠的主要嗅觉系统整装标签

一种用于果蝇嗅觉回路组装延时成像研究的外植体系统

通过的嗅球钙监控录像的变化温度引起的神经元电活动

活的嗅觉途径的功能评价

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization