VMAT2 OVEREXPRESSION IN DOPAMINE RELEASE AND BEHAVIOR

神经元以量子单位释放神经递质。 传统上，量子大小（在胞吐过程中由单个囊泡释放的神经递质分子的数量）被认为是不变的，突触前效能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子大小可以被精神兴奋剂可卡因和安非他明改变。这表明多巴胺量子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。 我们现在建议探索多巴胺量子大小的调节底物之一，神经元囊泡单胺转运蛋白VMAT 2。 我们将确定VMAT 2转运蛋白是否提供了一种调节机制来控制培养的中脑多巴胺神经元和表达VMAT 2的海马神经元的异源系统中的多巴胺量子大小和量子频率;然后我们将在自由活动的野生型和转基因VMAT 2过表达小鼠的丘脑核和纹状体中使用微透析来观察基础，苯丙胺和去极化诱导的单胺水平和运动行为。 用携带VMAT 2的腺病毒构建体转染培养的中脑多巴胺神经元使多巴胺量子大小和频率增加6倍，超过内源性VMAT 2水平所提供的。 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，因为我们还观察到用VMAT 2转染并预加载外源性多巴胺的培养的非儿茶酚胺能海马神经元中的刺激依赖性多巴胺量子。 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.