Purinergic P2Y₁ and P2Y₁₂ receptors control enteric nervous system activity through neuro-glia-macrophage crosstalk

Purines are important mediators of intercellular communication in the enteric nervous system (ENS) that participate in physiological gut functions and disease. Purinergic transmission is prominent in mechanisms of crosstalk between enteric neurons and glia where enteric glia exhibit high responsiveness to adenosine diphosphate (ADP) through P2Y₁ receptors and neurons to adenosine triphosphate (ATP) through P2X₃ receptors. Despite functional data suggesting that enteric glia are the primary site of P2Y₁ expression in the ENS, gene sequencing suggests that P2Y₁ expression is more enriched in neurons than glia. The reason for the mismatch between genomic and functional data is unclear but could involve co-expression of inhibitory P2Y₁₂ receptors in neurons. We addressed this issue by studying the expression and function of P2Y₁ and P2Y₁₂ receptors in the mouse ENS using live immunolabeling and calcium imaging techniques. The data show that ADP drives activity among enteric glia and neurons in the myenteric plexus. Interestingly, inhibiting P2Y₁₂ activity increased neuron responses to ADP and overall spontaneous activity among enteric neurons and glia while decreasing the magnitude of glial responses to ADP. Investigating the location of the receptors involved revealed P2Y₁ receptor expression by both neurons and glia, while P2Y₁₂ receptor expression was minimal in the ENS. Instead, P2Y₁₂ expression was enriched in the surrounding muscularis macrophages. Macrophages positive for P2Y₁₂ overlapped with CD163 positive subsets that have known inhibitory influences over myenteric neurocircuits. Together, these data suggest that macrophage P2Y₁₂ pathways act to constrain activity in the ENS, which could have implications in mechanisms that contribute to enteric hyperexcitability following disease.