Bertini L T, Kursner C, Gaillard R C, Corder R, Kiss J Z
Department of Morphology, University of Geneva Medical School, Switzerland.
Neuroendocrinology. 1993 Apr;57(4):716-28. doi: 10.1159/000126430.
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 microns) slices that contained an average of 300-400 CRF-41-immunostained neurons. The majority of CRF-41-positive cells were of small size (12-15 microns in diameter), and contained CRF-41-labeled dense core vesicles of 100 nm diameter as detected by electron microscopic postembedding immunocytochemistry. These cells represented the only CRF-41-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.
为了在体外研究经解剖学特征鉴定的促肾上腺皮质激素释放因子41（CRF - 41）产生神经元的功能和化学特性，采用改良的转瓶培养技术，将6日龄大鼠的下丘脑切片培养长达6周。该技术可产生厚度为100微米的切片，平均含有300 - 400个经CRF - 41免疫染色的神经元。大多数CRF - 41阳性细胞体积较小（直径12 - 15微米），通过电子显微镜包埋后免疫细胞化学检测发现，这些细胞含有直径为100纳米的CRF - 41标记的致密核心囊泡。这些细胞是培养物中唯一的CRF - 41阳性细胞群体。在含血清培养基（SCM）中对秋水仙碱处理的培养物进行光学显微镜双重免疫标记表明，这些CRF - 41阳性神经元中约60%含有可检测水平的血管加压素相关神经垂体素（VP - NP）。在无血清、化学成分明确的培养基（SFM）中培养切片会导致VP - NP免疫染色增加：在未用秋水仙碱处理的情况下，可检测到同时标记CRF - 41和VP - NP的小细胞神经元，并且几乎所有CRF - 41阳性神经元都表达VP - NP免疫反应性。在电子显微镜水平上，CRF - 41阳性曲张体的致密核心囊泡区室中VP - NP标记密度显著增加。向SFM中添加地塞米松（10 nM）可恢复最初在SCM中观察到的染色模式。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。使用免疫测定法评估培养的室旁细胞释放CRF - 41的能力。以2小时间隔连续5次取样，未刺激（基础）的CRF - 41分泌未发生改变，用56 mmol K⁺刺激同一培养物可使培养基中的CRF - 41含量显著增加（2 - 3倍）。与在不含地塞米松的SFM中培养的培养物相比，SFM中存在地塞米松（10 nM）可使K⁺刺激的CRF - 41释放减少6倍，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性，这与体内情况相似。因此，该模型非常适合用于研究促垂体性CRF - 41细胞的功能。
