A study of putative brain circadiom oscillators in amphibians and fish

1.为了深入了解两栖动物和鱼类生物钟的分子机制，本研究从日本鳗中克隆了编码PER2的部分基因，从AYU中克隆了编码PER2、PER3、CLOCK和Bmall的部分基因。用放射免疫法测定了金枪鱼、金枪鱼和虹鳟鱼的血浆褪黑激素水平，在光暗循环下表现出显著的日变化。在蝾螈中，血浆褪黑激素水平的昼夜变化可以通过松果体切除或双眼摘除来消除。恒定黑暗条件下，乌鱼血浆褪黑激素水平呈现昼夜节律变化，而虹鱼则无此变化。我们研究并定位了脑中的褪黑素受体。褪黑素受体是褪黑素的高度特异性受体，具有皮摩尔亲和力。放射自显影研究显示脑内分布不均，以视顶盖密度最高。利用…中的突触带数目此外，松果体光感受器作为松果体生物钟的相位标记，我们发现每天注射褪黑素都会改变松果体生物钟以及新的昼夜运动活动节律。我们用铝箔覆盖了蝾螈的头骨，同时颠倒了明暗循环。实验动物的运动节律进入反的明暗周期，而松果体突触带数目的节律与原始节律基本相同。这些数据表明，在明暗循环下，视网膜感知到的光可以在没有松果体生物钟帮助的情况下将生物钟带到大脑中。研究了松果体切除对虹鳟鱼摄食活动昼夜节律的影响。松果体摘除既不影响明暗条件下的需求摄食活动，也不影响恒定光照条件下的摄食活动，表明松果体器官不是维持大脑生物钟功能所必需的。较少

1. To obtain the insight on the molecular mechanisms of the circadian clock in amphibians and fish, we isolated partial cDNAs encoding Per2 from the Japanese newt (Cynops pyrrhogaster) and Per2, Per3, Clock and Bmall from the ayu (Plecoglossus altivels).2. Plasma melatonin levels in the newt, ayu and rainbow trout (Oncorhynchus mykiss) determined by radioimmuno-assay exhibited significant daily variation under light-dark cycles. In the newt, the day-night change in plasma melatonin level was abolished by either pinealectomy or bilateral ocular enucleation. Plasma melatonin levels exhibit circadian variation under constant darkness in the ayu but not in the rainbow trout.3. We characterized and localized melatonin receptors in the newt brain. The melatonin receptors are highly specific for melatonin with picomolar affinity. Autoradiographic studies revealed uneven distribution in the brain with the highest density observed in the optic tectum.4. By using the number of synaptic ribbon in … More the pineal photoreceptor as a phase marker of the pineal circadian clock, we showed that daily melatonin injections entrained the pineal circadian clock as well as circadian locomotor activity rhythm in the newt.5. We covered the newt's skull with aluminum foil and simultaneously reversed the light-dark cycle. Locomotor activity rhythm of the head-covered animals was entrained to the reversed light-dark cycle while the rhythm of pineal synaptic ribbon number remained almost the same phase as the original one. These data suggest that under light-dark cycle retinally perceived light can entrain the circadian clock in the brain without help from the pineal circadian clock.6. The effects of pinealectomy on circadian rhythms in the demand-feeding activity of the rainbow trout were examined. Pinealectomy affected the demand-feeding activity under neither light-dark nor constant light conditions, indicating that the pineal organ is not essential to maintain the function of the circadian clock in the brain. Less