The suprachiasmatic nucleus in stationary organotypic culture
The suprachiasmatic nucleus in stationary organotypic culture
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DOI：
10.1016/0306-4522(95)00327-f
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发表时间：
1996-01-01
期刊：
影响因子：
3.3
通讯作者：
Castel, M
中科院分区：
文献类型：
--
作者：
Belenky, M;Wagner, S;Castel, M
新生大鼠的视交叉上核在固定的器官型培养中维持数周。下丘脑切片外植体由Millicell滤过器支撑，在含有血清培养基的培养皿中培养，明显变平，但保留了视交叉上核和周围下丘脑组织的组织。两到三周后，固定培养物，确定三个神经元亚群为血管加压素能，血管活性肠肽或含gaba。培养的视交叉上核gaba能成分特别丰富，广泛地投射到下丘脑切片。单侧切除外植体核显著降低同侧gaba免疫反应性。切口将双侧视交叉上核与室旁核分离，剥夺后者的细口径gaba免疫反应输入。58个培养中有51个获得了视交叉上核的细胞外或细胞内电生理记录。长期培养的视交叉上核的电特性与成年大鼠急性切片记录的电特性相似。在六个培养中，记录延长至10-24小时。在视交叉上核的长期固定器官型培养中，体内核固有的神经元亚群被免疫细胞化学鉴定。病变研究支持了整个下丘脑切片外植体内gaba能神经支配的主要来源似乎是视交叉上核。电生理学研究证实了长期培养细胞核的活力，并揭示了自发电活动的变化可能表明昼夜节律波动。
Suprachiasmatic nuclei, derived from neonate rats, were maintained for several weeks in stationary organotypic culture. Hypothalamic slice explants, supported by Millicell filters and incubated in Petri dishes containing serum-based medium, flattened appreciably, yet preserved the organization of the suprachiasmatic nucleus and the surrounding hypothalamic tissue. After two to three weeks, cultures were fixed, and three neuronal sub-populations were identified as vasopressinergic, vasoactive intestinal peptide-containing, or GABA-containing. The GABAergic component of the cultured suprachiasmatic nucleus was particularly profuse, projecting extensively into the hypothalamic slice. Unilateral ablation of the nucleus in the explant dramatically reduced ipsilateral GABA-immunoreactivity in the slice. Explants in which an incision separated the bilateral suprachiasmatic nucleus from the paraventricular nucleus, deprived the latter of its fine-caliber GABA-immunoreactive input. Extra- or intra-cellular electrophysiological recordings from the suprachiasmatic nucleus were obtained in 51 of 58 cultures. The electrical properties of the long-term cultured suprachiasmatic nucleus were similar to those recorded in acute slices from adult rats. In six cultures recordings were extended for up to 10-24 h.Within long-term stationary organotypic cultures of the suprachiasmatic nucleus, sub-populations of neurons, intrinsic to the nucleus in vivo, were identified immunocytochemically. Lesion studies supported the observation that the main source of the GABAergic innervation within the entire hypothalamic slice explant appeared to be the suprachiasmatic nucleus. Electrophysiological studies confirmed the viability of the long-term cultured nucleus and revealed changes in spontaneous electrical activity that may indicate circadian fluctuation.