树突分支中的海马区对小鼠的齿状回评
Summary
We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.
Abstract
Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.
Introduction
动态改变在突触的数量和结构的发展，老化的标志，以及众多的神经变性疾病1-3。神经元的接收和整合突触信息的能力取决于树枝状形态和动态的改变在突触连接。事实上，一个正相关树突棘和突触数目，这既影响认知功能4之间存在。因此，这并不奇怪，在树突棘数目递减已与认知功能障碍相关的一些神经障碍5-7，促使在树突棘量化了极大的兴趣。然而，脊柱密度的量化仍然是一个耗时且繁琐的任务失败，以产生与通过该树突树突触的地形和分布的有用信息。幸运的是，染色方法（ 如高尔基-考克斯和doublecortin（DCX））结合与先进的成像技术可以被用来克服目前的障碍，并以可靠和迅速的方式产生树突分支的高清晰度图像。而高尔基-考克斯染色法可以部署到评估所有神经元树突8树枝状的状态，DCX可以部署鉴于神经发生在这两个标签新生神经元特别是在齿状回和脑室下区9，一个重要的考虑因素这些地区的整个生命周期10,11。
染色后，两个成像方法被部署，以评估树突特性：ⅰ）实时成像（RTI）和场成像（EDFI）的ⅱ）延伸的深度。的RTI技术提供的平均来跟踪和量化树枝状的长度和顺序沿个体树突段和分支。因此，它使一个估算的总面积和每个树突树占据的体积。多个Specifically，在该RTI方法的用户连续地识别段和迭代地重新聚焦为神经元的跟踪软件收集在x，y和z的树枝状结构的坐标和重构树枝状结构在三维的轨迹。相比之下，EDFI方法提供了一种相当简单的，快速的用于通过产生合成图像，从而提供对整个z轴信息评估相当厚的组织标本树突密度的手段。这样做时，用户记录的高清晰度视频文件贯穿部的厚度，然后使用软件来搜索视频帧以识别点，其中一个像素是完全聚焦。随后，将聚焦像素被合并和集成到一个高分辨率，复合2D图像。这种复合图象包含分别在焦，不论其在z轴位置的所有像素。这些2D图像的定性和定量分析随后可以用来确定密度的树枝状分枝中的每个字段。
最后，我们提出了一个全景方法用于产生极高分辨率的图像进行分析和树突的评估在感兴趣的整个区域。这种技术可以被部署到克服缺乏接入的到非常高的分辨率和昂贵的数码相机。使用这种方法，因此一个捕获在沿x轴和y轴的不同位置的序列的图像，然后自动缝合在一起使用免费软件（ 例如 ，图片复合编者）。值得注意的是，可以在一个相当宽的区域被用于树突分支的定性和定量评估此方法。
Protocol
Representative Results
Discussion
这里，两个方法被描述以量化在使用中与RTI和EDFI结合常规染色方法成熟和新生神经元树突分支的程度。采集的神经元的高分辨率图像提供了一个非常有效的方法，用于测试的神经变性疾病的有害影响，并反过来，提供了一种方法来评估靶向海马神经元的治疗策略。
而RTI方法用于捕获深度数据有关树枝状和地形的程度，所述EDFI方法不能提供有关该个体的段或树木的复杂度信息…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by grants from the LuMind Foundation, Research Down Syndrome, and the Alzheimer’s Association (AS). CP was partially supported by a faculty development grant from the College of Nursing and Health Professions at Arkansas State University.
Materials
| Name | Company | Catalog Number | Comments |
| Modified Golgi-cox staining solution | Weill Cornell Medical College | NA | store at 4°C till use |
| 1x Developing Solution (Stock 10x) | Weill Cornell Medical College | NA | store at 4°C till use |
| 30% Sucrose, | Sigma | CAS # 57-50-1 | make fresh in ddH2O |
| 0.3% Gelatin | Sigma | CAS # 9000-70-8 | NA |
| Graded Ethanol Solutions (20%, 30%, 40%, 50%, 80%. 90%, 95%. 100%) | Sigma | CAS 603-003-00-5 | NA |
| Xylene | Sigma | CAS # 1330-20-7 | NA |
| DPX Medium | EMS | #13510 | NA |
| Superfrost (+) white | Electron Microscopy Sciences | 71869-10 | NA |
| Coverslip 22x50mm (VWR #48393-059) | VWR | #4811-703 | NA |
| DCX Antibody | Santa Cruz Biotechnology | sc-8066 | 4 C |
| DAB | Sigma | CAS Number 91-95-2 | -20 |
| OCT | Tissue-tek | 4583 | NA |
| Tris | Sigma | CAS Number 77-86-1 | NA |
| ABC Lite | Vector | PK4000 | NA |
| Name | Company | Catalog Number | Comments |
| Microscope | Nikon | Eclipse 80i | |
| Digital Camera | Nikon | DS-Ri1 | |
| 12 bit Camera | QImaging | 01 MBF2000RF-CLR-12 | |
| Neurolucida System | MBF Bioscience | V.10 | |
| Image Composite Editor | Microsoft | 1.4.4.0 | |
| NIS Elements | Nikon | F 3.0 | |
| Image Pro Plus | Mediacy | Versin 7.00 |
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