INTRACELLULAR CA2+ OSCILLATIONS IN LHRH NEURONS FROM EMBRYONIC OLFACTORY PLACODE

目的：测定促肾上腺皮质激素释放激素神经元的振荡模式 在文化上。结果1)培养的LHRH 猴胚胎嗅觉定位区神经元的释放 LHRH以大约50分钟为间隔的脉动方式，以及2) 单个LHRH细胞表现出[Ca~(2+)]i振荡。为了 了解LHRH脉冲的产生机制，我们进一步 描述了LHRH神经元[Ca~(2+)]_i振荡的模式。 嗅觉定位器和LHRH神经元的迁移途径 在E35-37的猴子胚胎被解剖并在玻璃上培养 生长介质中的盖片。两到五周后培养的细胞 用钙染料Fura-2标记，并检测[Ca2+]i 震荡。在30秒内，每10秒记录一次[CA2+]i变化 使用视频成像系统到152分钟。记录后，细胞被 LHRH固定并免疫组织化学染色。在16种培养中，359个LHRH细胞 (4-51个细胞/培养)显示[Ca~(2+)]i振荡。 在16种培养物中，有13种细胞的[Ca~(2+)]i振荡同时发生在 50-100%的单个细胞，而[Ca~(2+)]i 其余3种培养物中的细胞振荡不同步。 引人注目的是，在13种培养物中，有8种细胞的[Ca~(2+)]i同步 在47.3q3.4分钟完全一致地重复发生振荡， 这与观察到的LHRH释放周期相似，而在5 在13种培养物中，[Ca~(2+)]i振荡的同步性较差 反复出现在23.4q4.2min。脉冲间隔(IPI， 紧张型青光眼细胞[Ca~(2+)]_i振荡6.9q0.8min，n=134 紧凑程度较低的小区中的同步和IPI(9.2q1.0分钟，n=130) 同步与IPI没有显著不同(8.5q0.4min， N=95)在没有同步的信元中。既不是文化的时代，也不是 腔室类型(有或无中层灌流)与 振荡的同步。这项研究的结果表明 猴嗅觉胎盘来源的LHRH神经元具有 [Ca~(2+)]_i振荡同步的内生机制 这可能与LHRH释放的周期性有关。未来 说明我们将研究同步的机制。钥匙 LHRH神经元，钙振荡，LHRH脉冲产生，嗅觉 Placode

OBJECTIVE: To determine the oscillatory pattern of LHRH neurons in culture. RESULTS Previously, we have shown that 1) cultured LHRH neurons derived from the olfactory placode of monkey embryos release LHRH in a pulsatile manner at approximately 50-min intervals, and 2) individual LHRH cells exhibit [Ca2+]i oscillations. In order to understand the mechanism of LHRH-pulse generation, we further characterized the pattern of [Ca2+]i oscillations in LHRH neurons. The olfactory placode and the migratory pathway of LHRH neurons from monkey embryos at E35-37 were dissected out and cultured on glass coverslips in a growth medium. Two to five weeks later cultured cells were labeled with the calcium dye, fura-2, and examined for [Ca2+]i oscillations. [Ca2+]i changes were recorded every 10 seconds for 30 to 152 min using a video imaging system. After recording, cells were fixed and immunostained for LHRH. In 16 cultures, 359 LHRH cells (4-51 cells/culture) exhibiting [Ca2+]i oscillations were recorded. In 13 of 16 cultures, [Ca2+]i oscillations occurred synchronously in 50-100% of the individual cells in a culture, whereas [Ca2+]i oscillations in cells in the remaining 3 cultures did not synchronize. Strikingly, in 8 of 13 cultures the synchronization of [Ca2+]i oscillations repeatedly occurred in complete unison at 47.3q3.4 min, which is similar to the period observed for LHRH release, whereas in 5 of 13 cultures the less tight synchronization of [Ca2+]i oscillations repeatedly occurred at 23.4q4.2 min. Interpulse intervals (IPI, 6.9q0.8 min, n=134) of [Ca2+]i oscillations in cells with tight synchronization and IPI (9.2q1.0 min, n=130) in cells with less tight synchronization did not differ significantly from IPI (8.5q0.4 min, n=95) in cells without synchronization. Neither age of cultures, nor type of chambers (with or without media perifusion) was related to synchronization of oscillations. Results from this study indicate that LHRH neurons derived from monkey olfactory placode possess an endogenous mechanism for synchronization of [Ca2+]i oscillations, which may be related to the periodicity of LHRH release. FUTURE DIRECTIONS We will examine the mechanism of the synchronization. KEY WORDS LHRH neurons, Ca2+ oscillation, LHRH-pulse generation, olfactory placode