Antipsychotic Drug Vesicular Release at Dopamine Synapses

 描述（由申请人提供）：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，通过酸性捕获在酸性细胞器（包括突触囊泡）中积累。以前的实验解决这一假设依赖于嗜酸性染料，而不是精神药物和细胞培养，不重演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，可以直接在脑切片成像的双光子显微镜。我们的实验确定，这种抗精神病药是受酸性捕获，包括在中脑多巴胺神经元突触囊泡，其中含有天然的递质。此外，我们发现，抗精神病药物可以通过动作电位从囊泡中释放，这需要Ca2+，或安非他明，这需要多巴胺转运蛋白和囊泡单胺转运蛋白。在体外治疗浓度和动物全身给药时观察到囊泡蓄积和释放。因此，这些结果首次证明了抗精神病药物在突触处与多巴胺一起进行囊泡释放。这一发现是有趣的，因为它意味着当多巴胺突触活跃时，局部抗精神病药物浓度将与多巴胺精确地成比例。 鉴于突触囊泡释放的抗精神病药物，现在已经建立，这个建议确定囊泡抗精神病药物释放多巴胺能传递介导的黑质和纹状体中的D2受体的功能影响。我们的工作假设是，囊泡药物的功效将随着重复放电而增加，这与行为有关。由于酸性捕获是被动的，并且在不抑制多巴胺的囊泡储存的情况下不能被破坏，因此实验利用囊泡捕获动力学的显著差异（即，其发生超过数小时并且持续时间长）和在浴中急性施用药物（即，这是通过双光子显微镜发现的。纹状体实验集中在电化学测量的多巴胺溢出，这是受D2自身受体的频率依赖性抑制。黑质实验利用多巴胺神经元的膜片钳来测定由体树突D2受体诱导的自发性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的活性依赖性囊泡释放对突触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.