CIRCADIAN SYSTEM AND PRENATAL COCAINE IN ZEBRAFISH

描述(由申请人提供)：拟议项目的目标是确定昼夜节律系统在昼夜脊椎动物斑马鱼胚胎大脑发育的关键时期暴露于可卡因的产前影响中的作用。这些研究将基于前三年资助期的结果，该三年资助期建立了可卡因和斑马鱼时钟系统之间的相互关系，发现褪黑素可以抵消可卡因对大脑发育的影响，鉴定了可卡因对昼夜节律系统和可卡因行为影响的两个相反的菌株，并表征了可卡因和可卡因戒断对成年和老年斑马鱼的行为和分子影响。这项研究的继续将特别侧重于描述可卡因和生物钟系统对大脑发育的影响，并解决这一假说，即两者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影响。具体目标将涉及：1)深入描述早期接触可卡因和褪黑素对斑马鱼大脑发育和基因表达的影响；2)早期发现的对可卡因的昼夜反应的遗传变异对产前暴露的可卡因和褪黑素影响的影响；3)昼夜节律缺陷在产前大脑发育和可卡因的影响中的作用，以及外源褪黑素是否可以抵消这些影响。斑马鱼在大脑皮层(大脑皮层)和边缘系统(背内侧和背外侧端脑)发育的主要时期暴露于可卡因，与夜间或白天的昼夜节律阶段同步。在昼夜节律系统正常和缺陷的胚胎中，褪黑激素将在不同的昼夜节律阶段先于可卡因使用。大脑的大小、细胞分裂率、多巴胺水平、昼夜节律基因的表达以及褪黑激素受体、多巴胺转运体、细胞周期蛋白依赖性激酶、细胞周期蛋白A和生长激素的编码基因将在整个发育和成熟过程中进行评估。在带有α-1-微管蛋白和f-响应素荧光标记的转基因鱼中，将评估神经元集合的发育时间和完整性。可卡因引起的运动活动和睡眠的变化将在出生后早期被记录下来。了解昼夜节律因素在大脑发育中的作用及其减弱可卡因产前影响的机制，应有助于制定预防和治疗策略，以抵消产前可卡因对大脑早期发育或随后大脑功能变化的影响。与公共健康相关：生物生物钟系统可以调节可卡因的影响。该项目将利用一种具有良好遗传特征的昼夜节律脊椎动物，研究包括褪黑激素在内的昼夜节律因素在产前可卡因暴露对大脑发育和分子过程的影响中的作用。这项研究的结果将有助于了解可卡因的产前效应和昼夜节律系统之间相互作用的机制，并有助于开发新的预防或治疗策略。

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.