VMAT2 OVEREXPRESSION IN DOPAMINE RELEASE AND BEHAVIOR

神经元以量子单位释放神经递质。经典理论认为，量子大小（单个囊泡在胞吐过程中释放的神经递质分子数量）是不变的，突触前效应的变化被认为是量子频率（每次刺激释放的量子数量）变化的结果；然而，我们最近发现，中脑神经元释放多巴胺的量大小可以被精神兴奋剂可卡因和安非他明改变。这表明多巴胺量大小的变化可能有助于精神兴奋剂诱导的神经元功能和行为的改变。我们现在提议探索多巴胺量大小的调节底物之一，神经元囊泡单胺转运蛋白VMAT2。我们将确定在培养的中脑多巴胺神经元和表达VMAT2的海马神经元异种系统中，VMAT2转运体是否提供了一种调节机制来控制多巴胺量子大小和量子频率；然后，我们将对自由运动的野生型和转基因vmat2过表达小鼠的伏隔核和纹状体进行微透析，观察基础、安非他明和去极化诱导的单胺水平和运动行为的差异。携带VMAT2的腺病毒构建体转染培养的中脑多巴胺神经元，使多巴胺的量子大小和频率比内源性VMAT2水平增加6倍。这种调节可能不是由于驱动酪氨酸羟化酶活性的反馈机制，因为我们也在培养的非儿茶酚胺能海马神经元中观察到刺激依赖性多巴胺量，这些神经元转染了VMAT2并预加载了外源性多巴胺。我们计划在活体大脑中扩展这些见解，为理解突触前机制在精神兴奋剂诱导的奖励、行为敏感化、药物强化和成瘾中的作用提供基础。

Neurons release neurotransmitter in quantal units. Classically, quantal size (the number of neurotransmitter molecules released by a single vesicle during exocytosis) was thought to be invariant and changes in presynaptic efficacy were thought to occur as a result of changes in quantal frequency (the number of quanta released per stimulation); however, we have recently found that the quantal size of dopamine release from midbrain neurons can be altered by the psychostimulants cocaine and amphetamine. This suggests that changes in dopamine quantal size may contribute to psychostimulant-induced alterations in neuronal function and behavior. We now propose to explore one of the regulatory substrates of dopamine quantal size, the neuronal vesicular monoamine transporter VMAT2. We will determine if the VMAT2 transporter provides a regulatory mechanism to control dopamine quantal size and quantal frequency in cultured midbrain dopamine neurons and a heterologous system of VMAT2-expressing hippocampal neurons; we will then use microdialysis in the nucleus accumbens and striatum of freely moving wild-type and transgenic VMAT2-overexpressing mice to observe differences in basal, amphetamine- and depolarization-induced monoamine levels and locomotor behavior. The transfection of cultured midbrain dopamine neurons with a VMAT2-carrying adenoviral construct increases dopamine quantal size and frequency by 6-fold, over that provided by endogenous levels of VMAT2. This regulation is probably not due to feedback mechanisms driving tyrosine hydroxylase activity because we have also observed stimulation-dependent dopamine quanta in cultured non- catecholaminergic hippocampal neurons transfected with VMAT2 and preloaded with exogenous dopamine. We plan to extend these insights in the living brain to provide a basis for understanding the roles of presynaptic mechanisms in psychostimulant-induced reward, behavioral sensitization, drug reinforcement and addiction.