Abstract
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.
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Acknowledgements
This work was supported by National Institutes of Health Grants DA004523 and DA007262 (Williams), and and P30 NS061800 (Aicher). The authors gratefully acknowledge the generous provision of beta IV-spectrin antibody by Dr. Matthew Rasband from the Baylor College of Medicine and the kind gift of ankyrin-G antibody from Paul Jenkins at the University of Michigan. We acknowledge expert technical assistance by staff in the Advanced Light Microscopy Core, supported by Grant P30NS061800.
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This study was funded by National Institute on Drug Abuse grant nos. DA004523 and DA007262 (Williams), and National Institutes of Health grant no. P30 NS061800 (Aicher).
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JRB created the superecliptic pHluorin-tagged D2 receptor knockin mouse. JRB, SRS, and JTW designed the studies. JJL, JRB, BR, and SDLT performed and analyzed the fluorescent imaging experiments. MT, JJBP, SH, SA, and SRS performed and analyzed the electron microscopy experiments. The paper was written by JJL and MT with editorial contributions from JRB, SA, SRS, and JTW. All authors read and approved the final manuscript.
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Lebowitz, J.J., Trinkle, M., Bunzow, J.R. et al. Subcellular localization of D2 receptors in the murine substantia nigra. Brain Struct Funct 227, 925–941 (2022). https://doi.org/10.1007/s00429-021-02432-3
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DOI: https://doi.org/10.1007/s00429-021-02432-3