G-protein-coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope-tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane-associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66 puncta/µm², which corresponds to an average frequency of 1 punctum every 1.50 µm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time, a quantitative estimate of spatial frequency is provided.

多巴胺神经元 (D2R) 上表达的 G 蛋白偶联 D2 自受体抑制轴突末梢和体细胞树突区的递质释放和细胞放电。体细胞树突多巴胺释放的机制细节仍未解决，部分原因是 D2R 的亚细胞分布信息不足。先前定位 D2R 的研究因缺乏在 D2R 敲除动物中验证特异性的抗体而受到阻碍，并且受到电子显微镜成像的小采样区域的限制。本研究利用亚衍射荧光显微镜和电子显微镜检查超黄 pHlourin GFP (SEP) 表位标记的 D2 受体敲入小鼠中的 D2 受体。将活切片与抗 SEP 抗体一起孵育，实现了质膜相关受体的选择性标记，用于大面积黑质致密部 (SNc) 的免疫荧光成像。通过酪氨酸羟化酶 (TH) 抗体观察，SEP-D2R 在多巴胺神经元的树突和体细胞表面呈点状结构。 TH 相关的 SEP-D2R 显示细胞表面密度为 0.66 点/μm ² ，相当于每 1.50 µm 1 个点的平均频率。使用银增强免疫金进行的单独超微结构实验表明，膜结合颗粒占 SNc 内假定多巴胺细胞中总 D2R 的 28%。紧邻树突膜金颗粒的结构是无髓鞘轴突或轴突静脉曲张（40%）、星形胶质细胞（19%）、其他树突（7%）或单个切片中未识别的轮廓（34%）。一些相关的配置文件也表达了 D2R。 荧光和超微结构分析还首次提供了轴突起始段膜 D2R 的可视化，轴突​​起始段是动作电位生成的关键区室。抗 SEP 染色的点状外观表明，沿着质膜在离散信号位点中组织有一群 D2R，并且首次提供了空间频率的定量估计。