SUBCELLULAR MAPPING OF D<SUB >2</SUB>R-A<SUB>2A</SUB>R HETEROMERS IN PARKINSONISM USING A NOVEL IMMUNOGOLD PROXIMITY LIGATION ASSAY

Receptor-receptor interactions critically regulate synaptic signalling, yet current approaches lack the ultrastructural resolution required to map these complexes within specific neuronal compartments. Here we introduce the immunogold proximity ligation assay (IgPLA), a methodological innovation that enables nanometer‑scale visualization of receptor-receptor interactions using electron microscopy. We applied IgPLA to investigate dopamine D₂ receptor (D₂R) and adenosine A_2A receptor (A_2AR) heteromers in the mouse striatum under physiological conditions and in a 6‑hydroxydopamine (6‑OHDA) model of Parkinson’s disease. The conventional proximity ligation assay confirmed the presence of D₂R-A_2AR heteromers in the intact striatum and revealed a marked reduction following dopaminergic denervation. Using IgPLA, we mapped these complexes with subcellular precision, identifying immunogold‑labelled D₂R-A_2AR heteromers primarily at the plasma membrane of dendritic spines, dendritic shafts, and axon terminals, with limited intracellular labelling. In contrast, the 6‑OHDA‑lesioned striatum displayed a redistribution of heteromers to intracellular compartments, consistent with altered trafficking mechanisms under pathological conditions. Quantitative electron microscopy confirmed a reduced surface density of D₂R-A_2AR heteromers in the lesioned hemisphere. Importantly, IgPLA performed in A_2AR knockout mice yielded no signal, validating the specificity and robustness of the method. These findings establish IgPLA as a sensitive tool for detecting and subcellularly mapping receptor-receptor interactions in the brain and reveal nanoscale pathological remodelling of GPCR heteromers in Parkinsonism. This technique provides a powerful platform for dissecting molecular organization within neuronal microcircuits and to understand disease‑related alterations at unprecedented resolution.