ADENOSINE /DOPAMINE INTERACTIONS IN A2A RECEPTOR KO MICE

描述（来自申请人摘要）：腺苷A2a受体（A2aR）在纹状体状神经元中与多巴胺D2受体（D2R）共表达，并对脑突触后D2R功能产生拮抗作用。然而，我们对a2ar -多巴胺相互作用的理解主要是基于药理学证据，这在本质上受到腺苷能药物缺乏特异性的限制。为了阐明体内A2aR与多巴胺能系统相互作用的细胞机制，我们产生了A2aR敲除（KO）小鼠。我们对A2aR KO小鼠的初步表征揭示了A2aR在突触前的作用，证明了它对纹状体多巴胺释放的促进作用，进而对多巴胺介导的运动活动产生促进作用。因此，我们提出了A2aR与多巴胺能系统之间相互作用的修正模型：A2aR介导的多巴胺释放的突触前促进可能抵消A2aR介导的D2R功能的突触后抑制。因此，A2a腺苷能调节多巴胺能活性可能依赖于a2ar突触前和突触后功能之间的良好平衡。为了实现这一假设，我们将使用突触体制备和体内微透析（SA #1）研究突触前位点的a2ar -多巴胺相互作用，并使用利血平化小鼠和纹状体神经元原代培养（SA #2）研究突触后位点的a2ar -多巴胺相互作用。此外，使用D2R KO和A2aR-D2R双KO小鼠，我们将解决D2R是否介导体内A2aR功能的核心问题（SA #3）。我们将探索A2aR-多巴胺在行为（运动活动）、神经化学（多巴胺释放）和细胞（cAMP形成和c-Fos表达）水平上的相互作用，以深入了解A2aR在体内的综合作用。通过对经典药理学研究的补充和这些受体功能的转基因方法，我们寻求对腺苷生理学的几个基本问题的更精确的答案：(1)在A2aR处作用的内源性腺苷的基础水平是否对脑多巴胺能系统产生紧张性生理作用（抑制性或兴奋性）？(2)突触前和突触后a2ar -多巴胺相互作用的细胞机制是什么？(3) A2aR在体内是否特异性地需要D2R来发挥其神经元功能？这些问题的答案将促进A2aR药物作为帕金森病及相关疾病的替代或辅助治疗的合理发展。

DESCRIPTION (from applicant's abstract): The adenosine A2a receptor (A2aR) is co-expressed with the dopamine D2 receptor (D2R) in striatopallidal neurons and exerts an antagonistic influence on postsynaptic D2R function in brain. Our understanding of A2aR-dopamine interaction, however, is based primarily on pharmacological evidence that is intrinsically limited by the lack of specificity of adenosinergic agents. To elucidate the cellular mechanisms underlying the interaction between A2aRs and the dopaminergic system in vivo, we have generated A2aR knock-out (KO) mice. Our initial characterization of A2aR KO mice reveals a presynaptic role for the A2aR by demonstrating its facilitative effect on striatal dopamine release and, in turn, on dopamine-mediated locomotor activity. Thus, we propose a modified model for the interaction between the A2aR and the dopaminergic system: A2aR-mediated presynaptic facilitation of dopamine release may counterbalance the A2aR-mediated postsynaptic inhibition of D2R function. Hence A2a adenosinergic regulation of dopaminergic activity may depend upon a fine balance between pre- and post-synaptic functions of A2aRs. To pursue this hypothesis, we will study A2aR-dopamine interactions at presynaptic sites using a synaptosomal preparation and in vivo microdialysis (SA #1), and at postsynaptic sites using reserpinized mice and primary cultures of striatal neurons (SA #2). Furthermore, using D2R KO and A2aR-D2R double KO mice we will address the central question whether or not the D2R mediates A2aR functions in vivo (SA #3). We will explore A2aR-dopamine interactions at the behavioral (locomotor activity), neurochemical (dopamine release) and cellular (cAMP formation and c-Fos expression) levels to gain insight into the integrated role of A2aR in vivo. By complementing classical pharmacological studies with these transgenic approaches receptor function, we seek more refined answers to several fundamental questions of adenosine physiology: (1) Do basal levels of endogenous adenosine acting at the A2aR exert tonic physiological effects (inhibitory or excitatory) on the brain dopaminergic system? (2) What are the cellular mechanisms underlying A2aR-dopamine interactions at pre- and post-synaptic sites? (3) Does A2aR specifically require the D2R to exert its neuronal functions in vivo? The answers to these questions will foster the rational development of A2aR agents as an alternative or adjunctive treatment for Parkinson's disease and related disorders.