Investigating Purinergic Receptor Signaling in Meningeal Afferents

We used topical drug application to target local purinergic receptor signaling at the afferents’ meningeal RF level. Drug doses were based on previous studies using local administration and pilot studies in our laboratory. We used Pyridoxalphosphate-6-antagonist, and 2’,3’-O-(2,4,6-Trinitrophenyl)adenosine-5’-triphosphate tetra(triethylammonium) salt (TNP-ATP) to inhibit P2X₃ and P2X_2,3 receptors. N-[2-[[2-[(2-Hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride (AZ 10606120) was used to inhibit P2X7_, and (3β,20β)-3-(3-Carboxy-1-oxopropoxy)-11-oxoolean-12-en-29-oic acid disodium (carbenoxolone) was employed to inhibit Panx1. All pharmacological agents were purchased from Tocris and diluted in SIF for meningeal application. We first established the level of baseline ongoing activity and mechanosensitivity for 60 min in the presence of the vehicle (SIF). We then evaluated the change in the activity and mechanosensitivity of the afferents in the presence of the drugs for 60 min to ensure a lack of direct inhibitory effect on the afferents before CSD triggering. Post-CSD data was then collected in the presence of the drug or SIF (Figure 1B).