ADENOSINE /DOPAMINE INTERACTIONS IN A2A RECEPTOR KO MICE

描述（来自申请人的摘要）：腺苷A2 a受体（A2 aR）与多巴胺D2受体（D2 R）在纹状体苍白球神经元中共表达，并对脑中的突触后D2 R功能产生拮抗作用。然而，我们对A2 aR-多巴胺相互作用的理解主要基于药理学证据，其本质上受到腺苷能药物缺乏特异性的限制。为了阐明A2 aRs与体内多巴胺能系统之间相互作用的细胞机制，我们产生了A2 aR敲除（KO）小鼠。我们的A2 aR基因敲除小鼠的初步表征揭示了突触前的A2 aR的作用，证明其促进作用纹状体多巴胺的释放，反过来，多巴胺介导的运动活动。因此，我们提出了一个修改后的模型A2 aR和多巴胺能系统之间的相互作用：A2 aR介导的突触前促进多巴胺释放可能会抵消A2 aR介导的突触后抑制D2 R功能。因此，多巴胺能活性的A2 a腺苷能调节可能依赖于A2 aRs的突触前和突触后功能之间的精细平衡。为了追求这一假设，我们将研究A2 aR-多巴胺相互作用在突触前网站使用突触体制备和体内微透析（SA #1），并在突触后网站使用利血平小鼠和原代培养的纹状体神经元（SA #2）。此外，使用D2 R KO和A2 aR-D2 R双KO小鼠，我们将解决D2 R是否在体内介导A2 aR功能的中心问题（SA #3）。我们将探索A2 aR-多巴胺在行为（自发活动），神经化学（多巴胺释放）和细胞（cAMP形成和c-Fos表达）水平的相互作用，以深入了解A2 aR在体内的综合作用。通过补充经典的药理学研究与这些转基因方法受体功能，我们寻求更精细的答案腺苷生理学的几个基本问题：（1）基础水平的内源性腺苷作用于A2 aR发挥紧张性生理效应（抑制或兴奋）对大脑多巴胺能系统？(2)在突触前和突触后位点，A2 aR-多巴胺相互作用的细胞机制是什么？(3)A2 aR是否特别需要D2 R才能在体内发挥其神经元功能？这些问题的答案将促进A2 aR药物作为帕金森病及相关疾病的替代或替代治疗的合理开发。

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.