CIRCADIAN SYSTEM AND PRENATAL COCAINE IN ZEBRAFISH

描述（由申请人提供）：拟议项目的目标是建立昼夜节律系统的作用，在胚胎大脑发育的关键时期，在昼夜脊椎动物，斑马鱼可卡因暴露的产前影响。这些研究将基于前3年资助期的结果，该研究建立了可卡因与斑马鱼生物钟系统之间的互惠关系，发现褪黑激素可以抵消可卡因对大脑发育的影响，确定了可卡因对昼夜节律系统和可卡因行为影响的对比效应的两个品系，并描述了可卡因和可卡因戒断对成年和老年两种性别斑马鱼的行为和分子效应。这项研究的继续将特别关注可卡因和生物钟系统对大脑发育的影响的特征，并解决这两个之间的相互作用是由于它们在大脑形成的早期阶段对神经元增殖的对比效应的假设。具体目标将涉及：1）早期可卡因和褪黑激素暴露对斑马鱼大脑发育和基因表达影响的深入表征; 2）早期发现的可卡因昼夜节律反应的遗传变异对产前可卡因和褪黑激素暴露影响的影响; 3）昼夜节律缺陷在产前脑发育中的作用和可卡因的影响，以及外源性褪黑激素是否可以抵消这些影响。斑马鱼将暴露于可卡因皮质（腭）和边缘系统（背内侧和背外侧端脑）的发展，与夜间或白天的昼夜节律相同步的主要时期。褪黑激素将在不同的昼夜节律阶段，在正常和缺乏昼夜节律系统的胚胎中在可卡因之前施用。将在整个发育和成熟过程中评价脑大小、细胞分裂率、多巴胺水平、昼夜节律基因和编码褪黑激素受体、多巴胺转运蛋白、细胞周期蛋白依赖性激酶、细胞周期蛋白A和生长激素的基因的表达。将在具有α-1-微管蛋白和f-spondin荧光标记的转基因鱼中评价神经元集合的发育时间和完整性。可卡因引起的自发活动和睡眠的变化将在出生后早期记录。了解昼夜因素在大脑发育中的作用以及它们减弱可卡因产前效应的机制，应有助于设计预防和治疗策略，以抵消产前可卡因对早期大脑发育或随后大脑功能变化的影响。公共卫生相关性：生物钟系统可以调节可卡因的作用。该项目将解决昼夜因素，包括褪黑激素的作用，在产前可卡因暴露对大脑发育和分子过程的影响，使用昼夜，遗传特征良好的脊椎动物。本研究的结果应提供可卡因和昼夜节律系统的产前效应之间的相互作用的机制的理解，并导致新的预防或治疗策略的发展。

DESCRIPTION (provided by applicant): The goal of the proposed project is to establish the role of the circadian system in the prenatal effects of cocaine exposure during a critical period of embryonic brain development in a diurnal vertebrate, zebrafish. These studies will be based on the results of the previous 3-year funding period, which established a reciprocal relationship between the cocaine and zebrafish clock system, found that melatonin can counteract the effects of cocaine on brain development, identified two strains with contrasting effects of cocaine on circadian system and behavioral effects of cocaine, and characterized the behavioral and molecular effects of cocaine and cocaine withdrawal in adult and aged zebrafish of both genders. The continuation of this research will specifically focus on characterization of the effects of cocaine and circadian clock system on brain development, and address the hypothesis that reciprocal interaction between the two is due to their contrasting effects on neuronal proliferation during early stages of brain formation. The Specific Aims will address: 1) In-depth characterization of the effects of early cocaine and melatonin exposure on zebrafish brain development and gene expression; 2) An impact of the earlier identified genetic variation in circadian response to cocaine on the effects of prenatal cocaine and melatonin exposure; 3) The role of circadian deficits in the prenatal brain development and effects of cocaine, and whether exogenous melatonin can counteract these effects. Zebrafish will be exposed to cocaine during the major periods of cortical (pallium) and limbic system (dorsomedial and dorsolateral telencephalon) development, synchronized with either the nighttime or daytime circadian phase. Melatonin will be administered prior to cocaine at different circadian phases, in embryos with normal and deficient circadian system. The brain size, cell division rate, dopamine levels, expression of the circadian genes and those encoding for the melatonin receptors, dopamine transporter, cyclin-dependent kinases, cyclin A and growth hormone will be evaluated throughout development and maturation. The time of development and integrity of the neuronal ensembles will be evaluated in transgenic fish with fluorescent markers for alpha-1-tubulin and f-spondin. The cocaine-induced changes in the locomotor activity and sleep will be documented during early post-natal period. Understanding the role of the circadian factors in brain development and mechanisms through which they attenuate prenatal effects of cocaine, should help to design preventive and therapeutic strategies to counteract prenatal cocaine impact on early brain development or subsequent changes in brain functions. PUBLIC HEALTH RELEVANCE: The biological circadian clock system can modulate the effects of cocaine. This project will address the role of the circadian factors, including melatonin, in the effects of prenatal cocaine exposure on brain development and molecular processes, using a diurnal, genetically well-characterized vertebrate. The results of this study should provide understanding of the mechanisms involved in reciprocal interaction between the prenatal effects of cocaine and circadian system, and lead to the development of new prophylactic or therapeutic strategies.