Desensitization and recovery of D2 autoreceptors

项目摘要/摘要 多巴胺系统在中枢神经系统中有多种功能，包括自主运动。 控制和奖励。抑制和兴奋输入的混合叠加在中脑的起搏器放电上 多巴胺神经元控制电路功能。一个这样的控制因素是由 躯体树突向邻近的下丘脑和下丘脑室旁核多巴胺神经元释放多巴胺。多巴胺激活 与GIRK通道偶联的抑制性D2-自身受体。D2敏化水平控制着 多巴胺释放产生的抑制程度。对D2受体的作用机制知之甚少 除钙外的脱敏作用已被证实可加速脱敏。这是 最近的工作强调了G蛋白偶联受体(GPCR)的非规范机制 脱敏。这项建议寻求进一步表征D2受体的调节，具有两个特定的目的，使用 急性脑片SNC多巴胺神经元的全细胞电压钳记录。 首先，脱敏恢复的时间进程和钙敏感性是什么？初步 研究结果表明，从脱敏中恢复是一个快速的过程，信号在大约 五分钟。这与使用类似受体进行的实验形成对比，如µ-阿片受体，后者 需要30分钟或更长时间才能恢复。然而，还需要更多的数据来揭示钙依赖。 以及确定脱敏程度与时间之间的关系(如果有的话 恢复的过程。 目标二集中于D2受体信号的受体占用要求；个体 受体需要结合激动剂才能使下游信号脱敏吗？这一目标将由三个人实现 子问题以及钙可能如何改变结果将在整个过程中进行测试 实验。低浓度的多巴胺是否会预先脱敏对高浓度的 多巴胺？在初步实验中，低浓度的多巴胺(~500 NM)会使反应减敏 到高浓度测试脉冲。其他GPCRs的激活是否会使D2受体失敏？虽然结果是 必须推广到其他GPCR，初步实验表明明显的异源脱敏 通过激活抑制性GPCRGABAB受体。最后，脱敏会不会从 最初的位置是什么？D2信号在特定间隔的研究仍然无法获得，因为 技术上的限制。最近生产的CyHQ-O-DA，一种可光激活的笼式多巴胺，具有 能够被双光子发射光解，允许在空间上定义的多巴胺释放。这将是 允许通过在焦点上重复取消以及探测到什么程度来诱导脱敏 脱敏是通过沿树突和胞体的不同位置的不同去势来定位的。

Project Summary/Abstract The dopamine system has a diverse repertoire of functions in the CNS including in voluntary motor control and reward. Mixtures of inhibitory and excitatory inputs overlaid on pacemaker firing of midbrain dopamine neurons controls circuit function. One such controlling factor is lateral inhibition by somatodendritic release of dopamine onto neighboring SNc and VTA dopamine neurons. Dopamine activates inhibitory D2-autoreceptors which couple to GIRK channels. The level of D2 sensitization controls the magnitude of inhibition generated by dopamine release. Little is known about the mechanism of D2 receptor desensitization except that calcium entry has been demonstrated to accelerate desensitization. This is highlighted by recent work which implicates non-canonical mechanisms of G-protein-coupled-receptor (GPCR) desensitization. This proposal seeks to further characterize D2 receptor regulation with two specific aims using whole-cell voltage-clamp recordings of SNc dopamine neurons in acute slice. First, what is the time course and calcium sensitivity of recovery from desensitization? Preliminary findings indicate recovery from desensitization is a rapid process, with signaling returning to baseline in about five minutes. This contrasts with experiments done with similar receptors, such as the µ-opioid receptor, which takes 30 or more minutes to recover. However, additional data are needed to uncover any calcium-dependence of desensitization as well as determine the relationship, if any, between degree of desensitization and time course of recovery. Aim two focuses on the receptor occupancy requirements for D2 receptor signaling; does an individual receptor require a bound agonist for downstream signaling to desensitize? This aim will be pursued by three sub-questions and, once again, how calcium might modify outcomes will be tested throughout these experiments. Does a low concentration of dopamine pre-desensitize the response to a high concentration of dopamine? In preliminary experiments, low concentrations of dopamine (~500 nM) desensitize the response to a high concentration test pulse. Does activation of other GPCRs desensitize D2 receptors? Though the results must be extended to other GPCRs, initial experiments have indicated pronounced heterologous desensitization with by activation of the inhibitory GPCR GABAB receptors. And finally, can desensitization spread from an initial location? The study of D2 signaling in specific compartments has remained inaccessible due to technological limitations. The recent production of CyHQ-O-DA, a photoactivatable caged dopamine with the ability to be photolyzed by 2-photon emission, allows for the spatially defined release of dopamine. This will allow for the induction of desensitization by repeated uncaging on a focal point and the probing to what degree the desensitization is localized by uncaging at various locations along dendrites and soma.