Antipsychotic Drug Vesicular Release at Dopamine Synapses

 说明(申请人提供)：抗精神病药物是D2多巴胺受体竞争性拮抗剂。长期以来，人们一直推测，这些药物是穿过血脑屏障的弱碱性胺，通过酸性捕获在包括突触小泡在内的酸性细胞器中积累。以前解决这一假设的实验依赖于不是精神药物的嗜酸性染料和细胞培养，这些细胞培养不能概括D2受体依赖的神经传递。因此，我们确定了一种临床上使用的抗精神病药物，可以通过双光子显微镜直接在脑片上成像。我们的实验证明，这种抗精神病药物容易受到酸性陷阱的影响，包括在中脑多巴胺神经元突触小泡中，这些小泡含有天然的递质。此外，我们还表明，抗精神病药物可以通过动作电位或苯丙胺从囊泡中释放出来，动作电位需要钙离子，苯丙胺需要多巴胺转运体和囊泡单胺转运体。在体外和动物体内全身给药的治疗浓度下，囊泡的积累和释放被观察到。因此，这些结果首次证明了抗精神病药物在突触处可以与多巴胺一起进行囊泡释放。这一发现很耐人寻味，因为它意味着局部抗精神病药物的浓度将与多巴胺准确地在哪里以及何时多巴胺突触活跃的程度相一致。鉴于抗精神病药物的突触囊泡释放已被证实，这一建议确定了抗精神病药物囊泡释放对黑质和纹状体D2受体介导的多巴胺能传递的功能影响。我们的工作假设是，泡囊药物的疗效会随着重复发射而增加，这与行为有关。由于酸性捕获是被动的，如果不抑制多巴胺的囊泡储存就无法破坏，实验利用了泡泡捕获动力学上的显著差异(即，它发生几个小时，然后持续很长时间)和在浴缸中急性应用药物(即，以分钟为单位)，这是双光子显微镜显示的。纹状体实验的重点是电化学测量的多巴胺溢出，它受到D2自身受体的频率依赖抑制。黑质实验利用多巴胺神经元的膜片钳技术来检测由躯体树突状细胞D2受体诱导的自发微小和诱发的抑制电流。总之，这些互补的方法将确定抗精神病药物活性依赖的囊泡释放对突触D2受体功能的影响。

 DESCRIPTION (provided by applicant): Antipsychotic drugs are D2 dopamine receptor competitive antagonists. It has long been speculated that these drugs, which are weak base amines that cross the blood brain barrier, accumulate by acidic trapping in acidic organelles including synaptic vesicles. Previous experiments addressing this hypothesis relied on acidophilic dyes that are not psychiatric drugs and cell cultures that do not recapitulate D2 receptor-dependent neurotransmission. Therefore, we identified a clinically used antipsychotic drug that can be directly imaged in the brain slice by two-photon microscopy. Our experiments established that this antipsychotic is subject to acidic trapping, including in midbrain dopamine neuron synaptic vesicles, which contain the native transmitter. Furthermore, we showed that the antipsychotic drug can be released from vesicles by action potentials, which requires Ca2+, or an amphetamine, which requires the dopamine transporter and the vesicular monoamine transporter. Vesicular accumulation and release were seen at a therapeutic concentration in vitro and with systemic administration in animals. Thus, these results demonstrated for the first time that an antipsychotic drug is subject to vesicular release with dopamine at synapses. This finding is intriguing because it implies that local antipsychotic drug concentration will scale wit dopamine exactly where and when dopamine synapses are active. Given that synaptic vesicular release of an antipsychotic drug has now been established, this proposal determines the functional impact of vesicular antipsychotic drug release on dopaminergic transmission mediated by D2 receptors in the substantia nigra and the striatum. Our working hypothesis is that efficacy of the vesicular drug will increase with repetitive firing, which is relevant for behavior. As acidic trapping is passive and cannot be disrupted without inhibiting vesicular storage of dopamine, experiments make use of the striking difference in kinetics of vesicular trapping (i.e., it occurs over hours and then is long lasting) and acute application of the drug vi the bath (i.e., in minutes) that was revealed by two-photon microscopy. Striatum experiments are focused on electrochemically measured dopamine overflow, which is subject to frequency dependent inhibition by D2 autoreceptors. Substantia nigra experiments utilize patch clamping of dopamine neurons to assay spontaneous miniature and evoked inhibitory currents that are induced by somatodendritic D2 receptors. Together, these complementary approaches will ascertain the impact of activity-dependent vesicular release of antipsychotic drug on synaptic D2 receptor function.