A TISSUE-CULTURE MODEL OF THE HYPOPHYSIOTROPHIC CRF PRODUCING NEURONAL SYSTEM
A TISSUE-CULTURE MODEL OF THE HYPOPHYSIOTROPHIC CRF PRODUCING NEURONAL SYSTEM
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DOI：
10.1159/000126430
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发表时间：
1993-04-01
影响因子：
4.1
通讯作者：
KISS, JZ
中科院分区：
文献类型：
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作者：
BERTINI, LT;KURSNER, C;KISS, JZ
为了研究解剖学特征的促肾上腺皮质激素释放因子-41 （CRF-41）生成神经元的功能和化学特性，采用改进的滚轮培养技术，将6日龄大鼠下丘脑切片在体外培养中维持长达6周。这种技术产生厚的（100微米）切片，平均包含300-400个crf -41免疫染色的神经元。电镜下包埋后免疫细胞化学检测发现，大多数crf -41阳性细胞体积较小（直径为1 ~ 15 μ m），含有直径为100 nm的crf -41标记的致密核囊泡。这些细胞是培养中唯一的crf - 41阳性细胞群。在含血清培养基（SCM）中保存的秋水仙碱处理的培养物的光镜双重免疫标记表明，大约60%的这些crf -41阳性神经元含有可检测水平的加压素相关神经physin （VP-NP）。在无血清、化学定义培养基（SFM）中培养切片导致VP-NP免疫染色增加：在没有秋水仙碱处理的情况下，可以检测到标记为CRF-41和VP-NP的细胞旁神经元，几乎所有CRF-41阳性神经元都表达VP-NP免疫反应性。在电镜水平上，crf -41阳性静脉曲张致密的核心囊泡室中VP-NP标记密度显著增加。在SFM中加入地塞米松（10 nM）恢复了SCM中最初观察到的染色模式。因此，在SFM中培养CRF-41神经元后，VP-NP和CRF-41免疫染色增加很可能是由于缺乏抑制性糖皮质激素。利用免疫分析法评估培养的室旁细胞释放CRF-41的能力。每隔2小时连续取样5次，未受刺激（基础）的CRF-41分泌没有改变，同样的培养物用56 mmol K+刺激后，培养基中CRF-41含量显著增加（2-3倍）。地塞米松（10 nM）在SFM中的存在导致K+刺激的CRF-41释放减少6倍，与不含地塞米松的SFM中维持的培养物相比，组织含量减少5倍。总之，我们已经证明，培养的CRF-41细胞表现出形态和生化特征，以及对糖皮质激素的反应，这与体内的情况相似。因此，该模型非常适合于研究垂体营养性CRF-41细胞的功能。
To investigate functional and chemical properties of anatomically characterized corticotropin-releasing factor-41 (CRF-41) producing neurons in vitro, hypothalamic slices of 6-day-old rats were maintained in culture for up to 6 weeks using a modified roller culture technique. This technique yields thick (100 mum) slices that contained an average of 300-400 CRF-41-immunostained neurons. The majority of CRF-41-positive cells were of small size (I 2-15 mum in diameter), and contained CRF-4 1-labeled dense core vesicles of 100 nm diameter as detected by electron microscopic postembedding immunocytochemistry. These cells represented the only CRF-4 1-positive cell population in the culture. Light microscope double immunolabelling of colchicine-treated cultures kept in a serum-containing media (SCM) indicated that about 60% of these CRF-41-positive neurons contains detectable levels of vasopressin-associated neurophysin (VP-NP). Culturing slices in serum-free, chemically defined media (SFM) resulted in an increased VP-NP immunostaining: parvicellular neurons labeled for both CRF-41 and VP-NP could be detected without colchicine treatment, and practically all CRF-41-positive neurons expressed VP-NP immunoreactivity. At the electron microscopic level there was a significant increase in VP-NP labeling density in the dense core vesicle compartment of CRF-41-positive varicosities. Adding dexamethasone (10 nM) to the SFM restored the staining pattern originally observed in SCM. Hence, the increased VP-NP and CRF-41 immunostaining after culturing CRF-41 neurons in SFM is most likely due to the absence of inhibitory glucocorticoids. The capacity of cultured paraventricular cells to release CRF-41 was assessed using an immunoassay. Unstimulated (basal) secretion of CRF-41 was not altered by five successive samplings at 2-hour intervals and stimulation of the same culture with 56 mmol K+ significantly increased (2-3 times) the CRF-41 content in the medium. The presence of dexamethasone (10 nM) in SFM induced a 6-fold reduction of K+-stimulated CRF-41 release and a 5 times reduction in tissue content in relation to cultures maintained in SFM without dexamethasone. In summary, we have demonstrated that cultured CRF-41 cells display morphological and biochemical features, as well as responsiveness to glucocorticoids, that is reminiscent to the situation in vivo. Thus, the model is well suited for studies of hypophysiotrophic CRF-41 cell functions.