Organotypic slice culture of the rat suprachiasmatic nucleus: Sustenance of cellular architecture and circadian rhythm-猫眼课题宝

大鼠视交叉上核的器官切片培养：细胞结构和昼夜节律的维持

DOI：

10.1016/0306-4522(94)90303-4

发表时间：

1994

期刊：

Neuroscience

影响因子：

3.3

通讯作者：

中科院分区：

医学3区

文献类型：

作者：

K. Tominaga;Shin;H. Okamura

哺乳动物大脑的视交叉上核被认为是昼夜节律的解剖学场所。为了在体外长时间检查视交叉上核的功能组织和完整的细胞间连接，我们使用滚管技术建立了器官型切片培养系统。含有双侧视交叉上核的脑切片（厚 400 μm）取自 4 至 7 天大的新生大鼠，并在体外保存三周以上。在这三周的时间里，切片变平为一到三层细胞，并形成两个紧密堆积的神经元细胞团（神经元区），直径约为1毫米，周围有神经胶质细胞分散区。在神经元细胞区中，细胞化学发现肽及其信使RNA具有与大脑中的视交叉上核相似的特征模式。原位杂交和免疫细胞化学显示，在视交叉上核切片培养物中，主要在神经元区的腹外侧部分检测到血管活性肠肽信使RNA表达和血管活性肠肽免疫反应性神经元。表达加压素信使 RNA 和加压素免疫反应性细胞位于室管膜细胞区附近的背内侧神经元区。视交叉上核器官切片培养的神经元区中含有这些神经肽及其信使RNA的细胞体和纤维的分布与体内视交叉上核的分布相似。这表明这些器官切片培养物中的视交叉上核保留了这些细胞在体内的生物学特征，因为细胞确实发育、形成紧凑的神经元团并建立了连接。为了检查切片培养物中视交叉上核神经元表现出持续节律活动的可能性，我们还测量了释放到培养基中的加压素的量。每隔4小时采样一次并结合酶联免疫分析结果显示，视交叉上核切片培养物培养基中的加压素浓度以大约24小时的周期波动。目前的研究结果表明，视交叉上核的核内神经元网络在体外维持了很长一段时间，并且视交叉上核的器官型培养物以振荡方式产生和释放生物活性物质。切片培养物中的视交叉上核可能有助于未来体外昼夜节律的分析。

The suprachiasmatic nucleus of the mammalian brain is thought to be the anatomical locus of circadian rhythms. To examine the functional organization of the suprachiasmatic nucleusin vitrowith intact intercellular connections for a prolonged period, we have established an organotypic slice culture system using a roller-tube technique. Brain slices (400 μm in thickness) containing the bilateral suprachiasmatic nuclei, were obtained from newborn rats at four to seven days old and were maintainedin vitrofor more than three weeks. During this three-week period, the slices flattened to one to three cell layers and two tightly packed neuronal cell-masses (neuronal zones), with diameters of about 1 mm were formed, which were surrounded by a peripheral glial cell-dispersed zone. In the neuronal cell zones, peptides and their messenger RNAs were found cytochemically with characteristic patterns similar to the suprachiasmatic nucleus in the brain.In situhybridization and immunocytochemistry showed that vasoactive intestinal peptide messenger RNA expressing and vasoactive intestinal peptide-immunoreactive neurons were detected predominantly in the ventrolateral part of the neuronal zones in the suprachiasmatic nucleus slice culture. Vasopressin messenger RNA-expressing and vasopressin-immunoreactive cells were localized in the dorsomedial neuronal zones near the ependymal cell zone. The distribution of cell bodies and fibers containing these neuropeptides and their messenger RNAs in the neuronal zones of suprachiasmatic nucleus organotypic slice culture were similar to that of the suprachiasmatic nucleusin vivo. This suggests that the suprachiasmatic nucleus in these organotypic slice cultures retains the biological characteristics of these cellsin vivoas the cells did develop, form compact neuronal masses and did establish connections. To examine the possibility that suprachiasmatic nucleus neurons in slice cultures show a persistent rhythmic activity, we also measured the amount of vasopressin released into the culture medium. Sampling at 4-h intervals combined with enzyme immunoassay revealed that vasopressin concentration in the medium embracing suprachiasmatic nucleus slice cultures fluctuated with a period of approximately 24 h.The present findings suggest that the intranuclear neuronal networks of the suprachiasmatic nucleus are maintainedin vitrofor a long duration and that organotypic cultures of the suprachiasmatic nucleus produce and release bioactive substances in an oscillating manner. The suprachiasmatic nucleus in slice cultures may be useful for future analysis of circadian rhythmsin vitro.

Patch-clamp recording of amino acid-activated responses in "organotypic" slice cultures.