Figure 4. Both FFA2-DREADD-HA and FFA3 are expressed and functional in cells of dorsal root ganglia: the two G-protein-coupled receptors (GPCRs) elevate Ca²⁺ by different mechanisms (A).

Sections of dorsal root ganglia taken from hFFA2-DREADD-HA-expressing mice were immunostained with anti-PGP9.5 to identify neurons (left-hand panel, green) and with anti-HA to detect the receptor (middle panel, red). Merging of such images (right-hand panel) showed modest coexpression (see main text for quantification) but with additional anti-HA staining interspersed between neurons. Inserts: focus on regions in which individual cells express PGP9.5 but not HA (star), express HA reactivity but not PGP9.5 (chevron), or coexpress both PGP9.5 and hFFA2-DREADD-HA (double arrow). (B) Fluorescent X-gal staining (red) of dorsal root ganglia (DRG) sections isolated from mice expressing β-galactosidase reporter gene, which is driven either by the Ffar2 (left-hand panel) or Ffar3 gene promoters (middle panel). DRG sections from wild-type mice were also immunostained with X-gal (right-hand panel). Scale bar = 20 µm. (C, D) Single-cell Ca²⁺ imaging studies were performed on cells isolated from DRGs taken from hFFA2-DREADD-HA-expressing mice. Cells were exposed to 4-methoxy-3-methyl-benzoic acid (MOMBA) (C) or C3 (D). In various examples cells were pre-treated CATPB, with the Gq/G11 inhibitor FR900359 (15 min) or pertussis toxin (24 hr) prior to addition of agonist. CATPB blocked the effect of MOMBA (**p < 0.01) but not C3. FR900359 also blocked the effect of MOMBA (**p < 0.01) but not C3, whilst pertussis toxin treatment blocked the effect of C3 (***p < 0.001) but not MOMBA. One-way analysis of variance followed by Bonferroni’s Multiple Comparison Test. Pertussis toxin treatment blocked the effect of C3 (***p < 0.001) but not MOMBA. One-way analysis of variance followed by Bonferroni’s Multiple Comparison Test. (E) Cells dispersed from DRGs isolated from CRE-MINUS mice were used to assess the ability of ligands to elevate Ca²⁺. No effect of MOMBA was recorded whilst C3 was effective in most of the cells tested (****p <0.0001, unpaired t-test). (F) TUG-1907 (3 µM) was able to elevate Ca²⁺ levels in DRG cells from wild type but not FFA3-knockout mice (***p < 0.001, unpaired t-test).

Figure 4—figure supplement 1. FFA2 is expressed and functional (intracellular calcium response) in nodose ganglia of mice.

(A) Sections of nodose ganglia from hFFA2-DREADD-HA mice were immunostained with anti-PGP9.5 to identify neurons (left-hand panels, green) and with anti-HA to detect the receptor (middle panels, red). Merging of such images (right-hand panels) showed modest coexpression, but with additional anti-HA staining interspersed between neurons in the hFFA2-DREADD-HA-expressing sections. Stars (*) illustrate cells that were positive for PGP9.5 but lacked anti-HA. Inserts: individual cells are illustrated in the boxes to highlight examples of cells expressing PGP9.5 but not HA, cells expressing HA reactivity but not PGP9.5, and cells coexpressing PGP9.5 and hFFA2-DREADD-HA. These are green, red, and yellow, respectively, in the right-hand insert. Scale bar = 20 µm. (B, C) Single-cell calcium imaging studies of dissociation cells from nodose ganglia from either hFFA2-DREADD-HA (B) or wild-type C57BL/6 (C) exposed to C3, 4-methoxy-3-methyl-benzoic acid (MOMBA), or MOMBA + CATPB as indicated. Both C3- and MOMBA-promoted elevation of intracellular [Ca²⁺] in a substantial proportion of cells from hFFA2-DREADD-HA-expressing mice and the effect of MOMBA was prevented by the presence of CATPB (**p < 0.01, ***p < 0.001). In cells from wild-type animals C3 (****p < 0.001) but not MOMBA was effective. Data represent the mean % of cells prepared from tissue of individual mice that responded to ligand treatments (one-way analysis of variance followed by Bonferroni’s Multiple Comparison Test). (D) Individual cells were exposed sequentially to MOMBA (100 µM) and then C3 (1 mM) with washout of MOMBA prior to treatment with C3. A representative example is shown.