Fig 2: HCN channel co-expression in SGNs. Co-expression of HCN1, HCN2, and HCN4 shown in spiral ganglion neurons (SGNs) of 33-day-old CBA/J mice and in human. Confocal microscopy was used to unravel the distribution of these channels at the neuronal membrane in basal–middle turn (a–r). Colorimetric immunohistochemistry showed co-expression in human SGNs in the apical–middle turn (e–u). a–d, HCN1 and HCN2 co-expression in CBA/J. a, Co-expression of HCN1 and HCN2 channels at the neuronal membrane. b, c, Single channel expression for HCN1 (b) and HCN2 (c). d, High magnification of the neuronal membrane showed a spotted distribution without color overlapping suggesting homomeric channels. e–g, Co-expression of HCN1 and HCN2 in human showed a more balanced expression of these subunits, asterisks mark putative type II cells. HCN1 (DAB, f) and HCN2 (AEC, g) channels after color deconvolution. h–k, HCN1 and HCN4 co-expression revealed a patchy distribution of staining without color overlaps (h). i–j, Single channel staining for HCN1 (i) and HCN4 (j). k, High magnification of the membrane suggested predominant presence of homomeric channels. l–n, HCN1 and HCN4 co-expression in human (l). Single channel staining for HCN1 (DAB, m) and HCN4 (AEC, n) after color deconvolution showed single-stained neurons for HCN4 with a large central axon initial segment (asterisk). o–r, HCN2 and HCN4 co-expression in mice showed higher correlation of staining with partial overlapping fluorescent light (o). Single channel images for HCN2 (p) and HCN4 (q). r, High magnification of the membrane showed overlapping emission spectra (white color). The presence of heteromeric channels cannot be excluded here. s–u, HCN2 and HCN4 co-expression in human showed a very heterogeneous distribution pattern (s). Single channel staining for HCN2 (DAB, t) and HCN4 (AEC, u) after color deconvolution. v–x, HCN fluorescent staining intensities from different sections in each cochlear turn were plotted as XY-diagrams with its relative mean intensities of HCN1 versus HCN2 (v), HCN1 versus HCN4 (w) and HCN2 versus HCN4 (x)

Fig 3: Differential expression of HCN channels in spiral ganglion neuron membrane with aging. HCN staining intensities at the perisomatic membrane of CBA/J and C57Bl/6N strains was semi‐quantified for HCN1, HCN2, and HCN4. Age groups spanned 3 months starting from the onset of hearing until 19‐month‐old mice. a–c, C57Bl/6N semi‐quantification of HCN1 (a), HCN2 (b), and HCN4 (c). d–g, CBA/J semi‐quantification of HCN1 (d), HCN2 (e), and HCN4 (f). Evoked auditory brainstem responses results are plotted as a second y axis representing pure‐tone hearing thresholds (compare to Figure 9). The nonparametric Kruskal–Wallis test and Dunn's post hoc test with Bonferroni correction was applied andp < 0.05 was considered statistically significant

Fig 4: HCN2 and HCN4 postnatal expression until the onset of hearing. HCN2 and HCN4 were expressed in spiral ganglion neurons (SGNs) and the organ of Corti of C57Bl/6N mice as early as P1 and changed expression during maturation until onset of hearing. a–l, HCN2 expression at 1, 7, 9, and 16 days after birth. a–c, HCN2 was highly expressed at P1 without any visible tonotopical gradient (a). Staining located at the soma (b) as well as in the organ of Corti at afferent fibers underneath the inner hair cell (IHC), (c). d–f, At P7 the staining decreases in neurons (d, e) as well as sensory epithelium (f). g–i, at P9, neurons presented similar staining compared to P7 (g, h) but the intensity underneath the IHC increased and a faint staining underneath the outer hair cells (OHCs) appeared (i). j–l, around the onset of hearing, SGNs presented a more intense staining at the perisomatic neuron membranes compared to P9 and P7 (j, k). The now functional organ of Corti expressed HCN2 in putative type I afferents as well as in the efferent fibers underneath the OHCs (l). m–x: HCN4 expression in 1-, 7-, 9-, and 16-day-old C57Bl/6N. m–o, High intensity staining of HCN4 was present at P1 at the soma membrane of SGNs in each turn (m–n). Prominent staining was visible underneath the IHCs and OHCs corresponding to afferent fibers at that developmental stage (o). p–r: at P7 a tonotopical gradient was visible with most intense reactivity in apical clusters (p). Staining intensity decreased at the neuron membrane (q) but prominent staining was still present in the afferent fibers of the organ of Corti (r). s–u, similar staining pattern was present in P9 neurons (s, t) and organ of Corti (u). v–w, after the onset of hearing, the tonotopical gradient faded with similar levels of staining in all turns (v), while overall staining in single neurons remained constant (w). No visible specific staining was detectable in the organ of Corti (x)

Fig 5: HCN2 expression in the adult mammalian sensory epithelia. HCN2 was present at afferent and efferent nerve fibers innervating the organ of Corti. a, Differential interference contrast image (DIC) of DAB visualized immunostaining showed presence in C57Bl/6N neurons, osseous spiral lamina (OSL), and underneath the inner hair cell (IHC). The inset with a magnified view of the organ of Corti exposed staining at the inner spiral plexus (isp) underneath the IHC, boutons and nerve fibers between outer hair cells (OHCs) and supporting cells (arrows) and the efferent fibers of the tunnel spiral bundle (tsb). b, Similar staining in CBA/J in the organ of Corti with expression underneath IHC and OHCs (arrows). Positive bigger caliber tunnel crossing fibers (tcf) account for the medial efferent innervation. c, DIC of a CBA/J horizontal orientation, inset: high magnification clearly showed staining in the large efferent synapses (asterisk), while smaller putative afferent type II terminals (arrow) were void of immunoreactivity. d, Ly5.1 mice HCN2 staining underneath the IHC, fibers traveling at the base of the tunnel (basilar fibers, bf) and thin caliber tcf (arrows) represent type II afferent fibers. Large boutons underneath OHCs correspond to the efferent innervation. Guinea pig (e) and cat (f) organ of Corti showed the same HCN2 staining pattern like Ly5.1 mice with type I and type II afferent and efferent fibers stained. In cat, the inset shows staining in the large synapses underneath OHCs from the medial efferent fibers (asterisk) and smaller putative type II terminals (arrow). Human sensory epithelia presented high levels of HCN2 expression at equal localization as in animals (g‐m) emphasized in the massive staining in outer spiral bundles (osb) typical for human. g, Immunopositive isp and bf (type II afferents), as well as medial efferent fibers of the tsb. The prominent osb comprises type II afferent as well as efferent fibers shown in (h) in transmission electron microscopy. The figure expose the different nerve fibers of the osb that are difficult to distinguish at light microscopic level. Big caliber efferent (medial olivocochlear fibers, MOC) intermingle with small‐sized type II afferents that are numerous in human. i, Bigger efferent nerve terminals (asterisk) adhere around the basal pole of OHCs together with smaller putative type II afferent terminals (arrow, DC, Deiters cell). j, Horizontal orientated sections from the human organ of Corti confirmed HCN2 staining in isp, osb and fibers crossing Corti's tunnel, (OP, outer pillar; IP, inner pillar). k, Scanning electron microscopy of human OHCs depicts thin caliber nerve fibers (colored nerve fibers) that climb up OHCs as far as the reticular lamina (yellow‐colored fiber). These fibers are positively labeled for HCN2 in human (l, arrow) and mice (d) marked with arrows. The function of these fibers is unknown. l, Another human specimen with positive HCN2 staining m, High magnification of human IHCs identified type I afferents nerve terminals positive for HCN2. B6, C57Bl/6N; CBA, CBA/J; P, postnatal day