SPINE MORPHOLOGY IN AN ANIMAL MODEL OF FRAGILE X SYNDROME

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。对人体解剖组织和脆性X智力迟钝综合征小鼠模型的高尔基浸染研究表明，树突棘具有不发达的外观-倾向于延长，扭曲的脊柱形态。脆性X型精神发育迟滞是一种遗传性疾病，其原因是无法产生FMRP蛋白。FMRP表达的缺乏与人类死后组织和疾病小鼠模型中树突棘形态的改变有关。此外，脊柱形状的稳定性已被证明是依赖于肌动蛋白的丝状形式，f -肌动蛋白。在这项研究中，我们比较了FMRP基因敲除（KO）小鼠与野生型（WT）对照小鼠的脊柱形态。我们使用phalloidin共轭荧光探针进行光氧化，在光镜和电子显微镜水平上对F-actin染色的脊柱进行相关性分析，并在路西法黄填充细胞和光转化后进行高级结构成像断层扫描。所有层析成像均在大阪大学的3MeV UHVEM显微镜上进行，切片厚度为4-5 μ m。较厚的切片是必要的，以便对合理长度的树突进行取样，并完整地捕获较大的棘。我们报告三个主要观察结果。首先，KO小鼠的带针脊柱比WT小鼠更容易被f -肌动蛋白染色。其次，KO小鼠的f -肌动蛋白染色脊柱的平均脊柱长度比WT小鼠长。第三，断层扫描数据使我们能够更好地将这些发现和其他发现联系起来，更准确地表征这种疾病中表达的形态表型。这些发现与以前使用高尔基浸渍法的报道一致，而断层扫描的结果为准确有效地阐明该疾病的形态特征提供了巨大的潜力

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Golgi-impregnation studies of human autopsy tissue and of a mouse model of fragile X mental retardation syndrome suggest that dendritic spines have an underdeveloped appearance ¿ a tendency towards elongated, tortuous spine morphology. Fragile X mental retardation, a genetically-linked disorder, results from the inability to produce the protein FMRP. The lack of FMRP expression has been associated with altered dendritic spine morphology in human post-mortem tissue and a mouse model of the disorder. Moreover, stability of spine shape has been shown to be dependent upon the filamentous form of actin, F-actin. In this study we compared spine morphology in mice with knockout (KO) of the FMRP gene to that of wild-type (WT) controls. We used of phalloidin conjugated flurophores followed by photooxidation for correlative analysis of F-actin stained spines at the light and electron microscope levels and advanced structural imaging tomography after cell filling with Lucifer yellow and photoconversion. All tomography was performed on the 3MeV UHVEM microscope in Osaka Univerisity on sections 4-5 ¿m thick. The thicker sections were necessary in order to sample a reasonable length of dendrite and to capture larger spines in their entirety. We report three primary observations. First, pedunculated spines in KO mice are much more likely to stain for F-actin than in WT. Second, the mean spine length of F-actin stained spines in KO mice is longer than WT. Third, tomography data enable us to better correlate these findings, and others, to more accurately characterize the morphological phenotype expressed in this disorder. These findings are consistent with previous reports using Golgi impregnation while those from the tomography offer tremendous potential to accurately and efficiently elucidate the morphological profile of this dis