Figure 1.

α,β-Methyleneadenosine 5′-triphosphate activates small and large dorsal root ganglion (DRG) neurons sequentially. (A) Strategy for generating conditional Pirt-GCaMP6s, GFAP-GCaMP6s, and Pirt:GFAP-GCaMP6s mice. (B) Setup for in vivo calcium imaging of L4 DRG in anesthetized mice. (C) Representative images showing changes in fluorescence intensity of DRG neurons after ganglionic administration of α,β-MeATP (500 μM) in Pirt-GCaMP6s mice: baseline [frames (F)1–5, 10 seconds/frame], phase I (F6–7), and phase II post-drug (F8–26). Examples of activated neurons are marked by colored circles and arrows. The red-outlined area in the phase II image is shown at higher magnification to the right. DRG neurons were categorized according to somal area as <450 μm² (small, S), 450 to 700 μm² (medium, M), and >700 μm² (large, L). (D) An example of a color-coded image (temporal code, F1–26) showing response to α,β-MeATP. (E) Fluorescence intensity traces (left, F/F₀) and heat maps (right) of selected neurons activated by α,β-MeATP in (D). Red arrow indicates the time of drug application. (F) Number of neurons in each subpopulation that were activated by α,β-MeATP (5, 50, and 500 μM, n = 6 mice) in phase I (F6–7) and phase II (F8–26). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs 5 μM treatment of the same size group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs indicated group or small-neuron group. Two-way mixed-model ANOVA with Dunnett multiple comparisons test. α,β-MeATP, α,β-methyleneadenosine 5′-triphosphate; ANOVA, analysis of variance; GCaMP, genetically encoded calcium indicator.