Fig 2: CFA‐induced inflammation alters HCN2 protein expression on 1 day post‐CFA. (a) Representative images of HCN2 IR in contralateral and ipsilateral L5 sections 1 day post‐CFA injection. Scale bars are 100 μm. (b) HCN2 expression is elevated in experimental compared to control DRG. Average mean pixel intensities ± SEMs are plotted for three size classes of DRG neurons. A one way ANOVA with a Tukey's post‐hoc test that makes all possible comparisons shows HCN2 IR is significantly elevated in small cells for experimental versus control treatment groups (F(2,17) = 8.468; p = 0.0028). Asterisks indicate which two‐way comparisons were significant. A one‐way ANOVA with a Tukey's post‐hoc shows HCN2 IR is significantly elevated in medium cells for ipsilateral versus control treatment groups (F(2,17)=3.748; p = 0.0448). Asterisks indicate which pairwise comparisons were significant: *p < 0.05; **p < 0.01. A one‐way ANOVA shows that HCN2 IR was not significantly different between large cells from experimental and control DRG (F(2,17) = 1.794; p = 0.1963). (c) HCN2 frequency is unaltered 1 day post‐CFA injection. Bar plots of the frequency of HCN2 positive neurons in each size category ([HCN2‐expressing cells per category ÷ total number of cells in all categories] × 100). Statistical tests show there were no significant differences between experimental and control animals for any cell type (small; KW(2,17) = 3.603; p = 0.1682; medium; F(2,17) = 0.1860; p = 0.8317; large; KW(2,17) = 1.999; p = 0.3824). CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; IR, immunoreactivity; KW, Kruskal–Wallis

Fig 3: Measuring HCN2 channel SUMOylation. (a) Verification of SUMO antibodies. Antibodies were (right) or were not (left) preabsorbed with the corresponding peptide for SUMO2/3 (upper panel) or SUMO1 (lower panel). SUMO is predominately expressed in nuclei. Note the loss of nuclear staining following preabsorption, for example, white arrows in bottom panel. Scale bars are 100 µm. (b) Method for quantifying HCN2 channel SUMOylation. In situ PLA was performed on DRG cryosections with (experimental) or without (control) antibodies against SUMO2/3 and HCN2. Upper panel shows a 5 µm projection of confocal optical slices through representative cells from control and experimental treatment groups. Each image represents a projection of five slices in continuous succession that together encompass the centre of the cell. Each optical section is 0.9 µm with a z‐stack interval of 1 µm. Note that the red puncta indicating SUMOylated HCN2 channels were largely absent when antibodies were omitted. Scale bar is 10 µm. The cells were circle and thresholded, and the resulting image is shown in the lower panel. Black puncta within the circled region were counted using the analyse particle tool on imageJ and normalized by the area (µm2) of the circle. Note that all black puncta, regardless of size were counted. (c) HCN2 channels are SUMOylated in DRG neurons. Plots of puncta per µm2 show there are significantly more puncta when primary antibodies for HCN2 and SUMO2/3 were included (0.08 ± 0.01 vs. 0.16 ± 0.02, p = 0.0149, paired t‐test, n = 3, 70 and 62 cells analysed in total) *, p < 0.05. The lines indicate that the cells were from the same experiment, that is, alternate sections from a single DRG on the same slide receiving the same PLA reagents and treated in an identical fashion. DRG, dorsal root ganglia; HCN2, hyperpolarization‐activated, cyclic nucleotide‐gated 2; PLA, proximity ligation assays; SUMO, small ubiquitin like modifier

Fig 4: HCN2 expression in the adult mammalian SGNs. HCN2 presented staining at the neuronal soma membrane and axons. a, Midmodiolar view exposed immunostaining in the organ of Corti (OC) and a rather homogeneous staining along the tonotopical axis in CBA/J across the spiral ganglion in the apical (SGa), middle (SGm), and basal (SGb) turn. b, Super-resolution confocal imaging with calretinin as neuronal counterstaining and DAPI nuclear stain. b1, direct-conjugated HCN2 antibody signal, b2, calretinin immunoreactivity. HCN2 was present only in perisomatic SGN membranes, axons (arrows) are void of any HCN2-LI. c, High intensity of the staining was visible in a Ly5.1 mouse in the apical turn within a neuron cluster (arrow), along the osseous spiral lamina (OSL) and in the organ of Corti (OC). d–i, Comparison of neuronal staining in three inbreed mouse strains identified similar expression levels of HCN2 in CBA/J (d, g) and C57Bl/6N (e, h) but higher intensity of staining in Ly5.1 mice (f, i). HCN2 in type I neurons of a guinea pig (j), type I and peripherin (PRPH)-positive type II neurons in cat (k) and human (l–p); k1 DAB channel (HCN2), k2, AEC channel (PRPH), after color deconvolution. l, Peripheral nerve fibers (PNF) appear weakly positive in human. m, Small human type II neurons were heavily stained for HCN2 (arrows). n, High magnification of human type I SGNs showed intense staining at the soma membrane, and weaker in the cytoplasm; postsomatic segment (asterisks) and the satellite glia cell (SGC) were void of reactivity. Double staining with PRPH was positive for HCN2 in human type II neurons (arrows) (o, p), but negative in CBA/J mice (q) and C57Bl/6N (r). B6, C57Bl/6N; CBA, CBA/J; P, postnatal day

Fig 5: HCN2-shRNA corrects the excessive autophagy induced by TGCI in rats. A Representative photomicrographs of immunohistochemical staining with anti-LC3 antibody in the hippocampal CA1 area (scale bar, 100 μm). B Quantitative analysis of the LC3 immunoreactivity (n = 4 per group). C Protein expression of LC3-II in CA1 of each group (n = 4 per group). ●P < 0.05 versus HCN2-shRNA group, **P < 0.01 versus Con-shRNA group; ##P < 0.01 versus Con-shRNA+TGCI group; &&P < 0.01 versus HCN2-shRNA+TGCI group.