Vaschenko M A, Kotsyuba E P
A.V. Zhirmunsky Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Vladivostok 690041, Russia.
Mar Environ Res. 2008 Aug;66(2):249-58. doi: 10.1016/j.marenvres.2008.03.001. Epub 2008 Mar 18.
NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS) and is widely used to identify nitric oxide (NO) producing cells in the central nervous system (CNS) of both vertebrates and invertebrates. NADPH-d histochemistry was used to quantitatively characterize putative NO-producing neurons in the CNS of the Gray mussel Crenomytilus grayanus subjected to two kinds of stress, environmental pollution and hypoxia, the latter caused by the mollusk transportation in a small volume of water. Mussels were sampled from one relatively clean (reference) and four polluted sites in Amursky and Ussuriysky Bays (Peter the Great Bay, Sea of Japan) in August, 2003. The number of NADPH-d-positive neurons was estimated and enzyme activity was determined from the optical density of the formazan precipitate in the CNS ganglia at 0, 3, and 72 h after sampling. Just after sampling, NADPH-d-positive neurons were found in the cerebropleural, visceral, and pedal ganglia. The number and staining intensity of NADPH-d-positive neurons were significantly higher in the pedal ganglia than the other two ganglia. There were significant differences in the number of NADPH-d-positive neurons and enzyme activity between the mussels from the reference and heavily polluted stations. The proportion and staining intensity of NADPH-d-positive neurons were maximum in the pedal ganglia of the mussels from the heavily polluted station in Amursky Bay. Transportation of mussels in a limited volume of water for 3h resulted in a significant increase in the proportion and staining intensity of NADPH-d-positive neurons in all ganglia. In mollusks from all stations kept in aerated aquaria for 72 h, both the proportion and staining intensity of NADPH-d-positive neurons did not differ significantly from the initial level. However, the differences in the proportion and staining intensity of NADPH-d-positive neurons between the reference and heavily polluted stations were significant. The present results suggest that NO is involved in mollusk nerve cell adaptation to environmental changes.
还原型辅酶Ⅱ黄递酶（NADPH-d）是一氧化氮合酶（NOS）的一种组织化学标记物，广泛用于鉴定脊椎动物和无脊椎动物中枢神经系统（CNS）中产生一氧化氮（NO）的细胞。利用NADPH-d组织化学方法，对遭受两种应激（环境污染和缺氧，后者由贝类在少量水中运输所致）的灰贻贝Crenomytilus grayanus中枢神经系统中假定的产生NO的神经元进行了定量表征。2003年8月，从阿穆尔湾和乌苏里湾（日本海彼得大帝湾）的一个相对清洁（对照）站点和四个污染站点采集贻贝样本。估计NADPH-d阳性神经元的数量，并根据采样后0、3和72小时中枢神经节中azan沉淀的光密度测定酶活性。采样后立即在脑胸膜神经节、内脏神经节和足神经节中发现了NADPH-d阳性神经元。足神经节中NADPH-d阳性神经元的数量和染色强度显著高于其他两个神经节。对照站点和重度污染站点的贻贝在NADPH-d阳性神经元数量和酶活性方面存在显著差异。重度污染的阿穆尔湾站点的贻贝足神经节中NADPH-d阳性神经元的比例和染色强度最高。将贻贝在有限体积的水中运输3小时导致所有神经节中NADPH-d阳性神经元的比例和染色强度显著增加。在充气水族箱中饲养72小时的所有站点的软体动物中，NADPH-d阳性神经元的比例和染色强度与初始水平相比没有显著差异。然而，对照站点和重度污染站点之间NADPH-d阳性神经元的比例和染色强度差异显著。目前的结果表明，NO参与了软体动物神经细胞对环境变化的适应。
