Department of Neurosurgery, New York University Grossman School of Medicine, New York, New York 10016.
Department of Cell Biology, New York University Grossman School of Medicine, New York, New York 10016.
J Neurosci. 2022 May 11;42(19):3919-3930. doi: 10.1523/JNEUROSCI.2416-21.2022. Epub 2022 Mar 31.
The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca concentration ([Ca]). Here we tested the hypothesis that the high-affinity Ca sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca] in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca], pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca] than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons. The respective Ca dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca sensor, underlies phasic somatodendritic DA release and its Ca sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca sensors subserving different aspects of somatodendritic DA release processes.
尽管多巴胺（DA）树突-胞体释放的分子机制自发现以来已经过去了几十年，但仍未得到解决。我们之前的工作表明，在亚毫摩尔细胞外钙浓度 ([Ca]) 下，会出现强烈的树突-胞体 DA 释放。在这里，我们测试了以下假设：高亲和力钙传感器突触结合蛋白 7（Syt7）是树突-胞体 DA 释放及其钙依赖性的关键决定因素。使用来自雄性和雌性小鼠的中脑切片中的全细胞记录来评估 SNc DA 神经元中的树突-胞体 DA 释放，以监测诱发的 DA 依赖性 D2 受体介导的抑制性电流 (D2ICs)。单细胞应用 Syt7 抗体 (Syt7 Ab) 可减少脉冲串诱发的 D2ICs，表明 Syt7 具有功能作用。脉冲串诱发的 D2ICs 的钙依赖性评估证实了野生型 (WT) 神经元在亚毫摩尔 [Ca] 中存在强大的 DA 释放，但在细胞内 Syt7 Ab 或 Syt7 敲除 (KO) 小鼠中，这种敏感性丧失。在毫摩尔 [Ca] 下，Syt7 KO 中的脉冲串诱发的 D2ICs 在降低 [Ca] 下的减少幅度大于在 WT 小鼠中看到的减少幅度；然而，两种基因型之间在单脉冲诱发的 DA 释放方面没有差异。在 WT SNc DA 神经元中，单细胞应用 Syt1 Ab 对列车诱发的 D2ICs 没有影响，但在 Syt7 KO 中引起 D2IC 幅度降低，表明 Syt1 对 Syt7 的功能替代。此外，Syt1 Ab 降低了 WT 细胞中单脉冲诱发的 D2ICs，表明 Syt1 参与了 tonic DA 释放。因此，Syt7 和 Syt1 在 SNc DA 神经元中的树突-胞体 DA 释放中发挥互补作用。树突-胞体和轴突多巴胺 (DA) 释放的各自钙依赖性不同，导致亚毫摩尔钙浓度下的树突-胞体 DA 释放持续存在，而这种钙浓度太低，不足以支持轴突释放。我们证明，高亲和力钙传感器突触结合蛋白 7（Syt7）是突触小体 DA 释放的时相和其在黑质致密部中的钙敏感性的基础。相比之下，我们发现，轴突 DA 释放的钙传感器突触结合蛋白 1（Syt1）在野生型小鼠中发挥作用，但在树突-胞体 DA 释放的时相而非 tonic 中发挥作用。然而，Syt7 缺失后，Syt1 可以促进时相 DA 释放。因此，我们表明 Syt1 和 Syt7 都可以作为钙传感器，用于不同方面的树突-胞体 DA 释放过程。
