Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, D-30173 Hannover, Germany.
J Comp Neurol. 2010 Apr 15;518(8):1157-75. doi: 10.1002/cne.22303.
We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.
我们通过使用各种细胞化学技术，在整体神经系统和脑切片中研究了在蝗虫胚胎发生过程中一氧化氮 - 环鸟苷酸（NO-cGMP）系统的发育。在存在可溶性鸟苷酸环化酶（sGC）激活剂 YC-1 和磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX）的情况下，用 NO 供体孵育后，通过 cGMP 免疫荧光可观察到对 NO 敏感的神经元。中枢神经系统（CNS）细胞早在胚胎发生的 38%时就对 NO 做出反应。通过使用 NADPH 黄递酶技术，我们鉴定了 CNS 中可能合成 NO 的细胞的体细胞和神经突。在胚胎发生的 40%左右，在大脑中首次出现 NADPH 黄递酶阳性细胞体，而在腹神经索中则为 47%。阳性细胞的数量在 80%时达到成年细胞的全部补充。在大脑中，一些结构，例如蘑菇体，仅在胚胎发生后才获得 NADPH 黄递酶染色。L-瓜氨酸的免疫定位证实了 NADPH 黄递酶染色神经元中 NOS 的存在，并且还表明了体内的酶活性。在整体腹神经索中，瓜氨酸免疫标记存在于 NADPH 黄递酶阳性细胞的不同亚群中，但染色非常可变且通常较弱。但是，在一个连接两个神经节的两个连接之一被压碎的再生范例中，早在胚胎发生的 60%时就观察到了强烈可靠的染色。因此，瓜氨酸免疫标记似乎反映了 NOS 的特定活性。然而，在年轻的胚胎中，NOS 可能并非总是组成性活跃，或者可能处于非常低的水平，低于瓜氨酸抗体检测阈值。对于中枢神经系统，NOS 的组织化学标志物不能为该酶的发育作用提供确凿的证据。
