Fig 1: HCN4 expression in the adult mammalian cochlea. a, Mid-modiolar view of a section from a C57Bl/6N mouse presented visually a tonotopical apex to base gradient. Neurons of the apical turn, especially in neuron clusters were intensely stained. b, Confocal microscopy confirmed the high expression of HCN4 at the neuronal membrane, especially between neurons of a cluster. c–e, HCN4 presented higher intensity of staining in Ly5.1 (e), compared to CBA/J (c) and C57Bl/6N (d). f–h, Confocal microscopy showed HCN4 to be present at the neuronal membrane of type I neurons as well as the peripheral axon initial segment (pAIS) and central axon initial segment (cAIS) in guinea pig (f). CASPR 1 was used as a marker for the distal end of the AISs. AISs lengths differed considerably (f, h). f1, h1, HCN4 staining, f2, h2, CASPR 1 staining, f3, nuclear staining (DAPI). Cat type II neurons presented immunoreactivity in single (i, arrow) and double-stained sections with additional peripherin (PRPH) staining (j). j1, DAB channel (HCN4), j2, AEC channel (PRPH). Human sections (k–m) presented staining at the perisomatic membrane as well as the cAIS and pAIS. Type II neurons with their delicate unmyelinated axons in humans are positively stained for HCN4 (k). l–m, cAIS length in human varied from very short (l) up to more than 30 µm here (m, arrows). SGN membranes were immunopositive especially in the neuron clusters (arrow, l). n, Double staining with PRPH in a CBA/J mouse showed no co-expression with HCN4 above background level, n1, DAB channel (HCN4), n2, AEC channel (PRPH) after color deconvolution. o–v, HCN4 expression in the sensory epithelia differed among species: in adult mice, no staining is visible in CBA/J and C57Bl/6N while in Ly5.1 showed staining in young (o) as well as old individuals (p, q). Guinea pig sections presented HCN4 mainly at type I afferents (r), while for cat mainly type II fibers (s, t) were positive. Standard immunohistochemistry methods were not sensitive enough to detect HCN4 in the human organ of Corti (u), but highly sensitive visualization methods using alkaline phosphatase enzymes exposed weak staining underneath the IHC (v). B6, C57Bl/6N; CBA, CBA/J; P, postnatal day