Surface D2 Autoreceptor Expression on Substantia Nigra Dopamine Neuron Dendrites

Abstract G-Protein coupled D2 receptors expressed on dopamine neurons, i.e. D2-autoreceptors (D2Rs), negatively regulate transmitter release and cell firing in axonal terminals and somatodendritic compartments. However, as the membrane distribution of D2Rs at somatodendritic signaling sites has not been fully characterized, functional mechanistic details remain uncertain. This study utilized a knockin mouse to examine surface D2Rs linked at the N-terminus to a superecliptic pHlourin green fluorescent protein epitope (SEP) in dopamine neurons of the substantia nigra. By incubating live slices with a highly specific anti-SEP antibody, the selective labeling of plasma membrane associated receptors was achieved. The SEP-D2Rs appeared as puncta like structures apposed to the surface of dendrites and soma of dopamine neurons. The percentage of D2R expression apposed to TH positive structures varied linearly with the density of TH fibers observed. TH-associated SEP-D2Rs displayed a cell surface density of 0.180 puncta per µm2, which corresponds to an average frequency of 1 punctum every 1.326 µm using nearest neighbor analysis. The distinct punctate appearance of the anti-SEP staining indicates there is a population of D2Rs organized in discrete clusters along the plasma membrane, an arrangement that can spatially localize signaling and influence GPCR efficacy. The results also indicate that D2Rs are spatially distributed on the plasma membrane at a frequency higher than previously reported.