Domain 1 of the calyx ending, facing the zone of pre-synaptic ribbons in the type I hair cell, is strongly immunoreactive for voltage-gated K and Na channels and associated proteins. Schematics in the upper left corner of Figures 1–4 delineate the successive domains in red. (A) KCNQ2 immunostaining (A1, green) on the inner surface of the calyx ending, where it co-localized with Caspr1 (A2, red). (B, C) KCNQ5 antibody (red) labeled the inner surface of calyces of both central-zone (CZ) and peripheral-zone (PZ) dimorphic (D) afferents (B, and C1, C2, calretinin-negative), but not calyx-only (C) afferents (B and C1). Calretinin antibody (green, B, C1) selectively labels type II hair cells (arrows) and calyx-only (C) afferents, not dimorphic (D) afferents. Localization of the KCNQ5 label to the calyx membrane (rather than, e.g., the hair-cell membrane) was determined by immunogold EM (not shown). (D) Top view of a whole-mount utricular macula, stained with KCNQ4 antibody (red). The antibody stained the calyx membrane intensely on its inner face (Domain 1) and lightly on its outer face (arrowheads, Domain 3, see below). Interruptions in the KCNQ4 immunostaining at the bottom of each calyx appear as black holes (arrows). (E) Antibodies to K_V1.1 (red) and K_V1.2 (similar pattern but not shown) also label Domain 1 (arrow) in all calyces, in addition to labeling membrane of the nerve fiber near the first internode (the juxtaparaheminodal membrane, arrowhead). (F) Immuno-reactivity for Na_V1.5 subunit was restricted to the calyx inner membrane, as shown here for a calyx (Cal) surrounding a type I hair cell (I); the calyx belonged to a dimorphic afferent in the peripheral zone of a crista. (G) Antibody to tenascin-C (red) formed cup-like labeling at the base of type I hair cells, but not calretinin-positive type II hair cells (green) in the extrastriolar (peripheral) zone of the utricular macula. (H) Tenascin-C forms much larger cups on these striolar calyces than on the extrastriolar calyces of G. Staining for tenascin-C (red) and calretinin (green) overlaps in confocal images, producing a yellow color in Domain 1 (arrows) of calyx-only afferents in the striolar (central) zone. Tenascin-C antibody also labeled extracellular matrix in the tissue stroma below the sensory epithelium (see red stain below right-hand calyces). (I) The higher resolution of immunogold EM revealed that tenascin-C is neither in the hair cell nor in the calyx, but rather in the synaptic cleft between the two (thus, the overlap in tenascin-C and calretinin in H reflects proximity of the antigens rather than co-localization, an under-appreciated likelihood in most confocal studies). Shown are tenascin-C-immuno-labeled gold particles in the synaptic cleft of a calyx in a crista peripheral zone. Such particles were not seen in the synaptic clefts of bouton synapses on type II hair cells (not shown). Scale bars: A, D, G, H, 10 µm; B, 20 µm; C, E, 5 µm; F, I, 500 nm.

Domain 3, comprising the basolateral calyx and the stretch of axon leading to the heminode, selectively expressed Na_V channels, KCNQ3, and certain scaffolding proteins. (A) Pan-Na_V antibody produced a complex pattern of label, including Domains 1 and 3 (arrows) and the heminode (Domain 4), as well as type I hair cells (arrowheads) and adjacent type II hair cells (asterisk). (B, C) Pan-dystrophin (Pan-dystr, red) labeled Domain 3 of dimorphic (calretinin-negative), but not calyx-only (calretinin-positive, green) afferents (compare B with C1 and C2). (D) Domain 3 in peripheral-zone dimorphic afferents was intensely immunoreactive for KCNQ3, as shown by this flattened projection of a stack of confocal images. KCNQ3 staining (arrows) extended throughout Domain 3 from the basolateral outer surface of the calyx to the heminode. Note the lack of stain in Domain 2, marked by dashed white lines. The inset shows a higher magnification single image of a dimorphic afferent with a “complex” calyx ending that envelops two adjacent type I hair cells. Antibody to ezrin (green) labeled the apical microvilli of the sensory epithelium and the heminode, where it combined with the red KCNQ3 immunolabel to form a yellow band. (E) AnkyrinB antibody labeled Domain 3 in dimorphic afferents (long arrows) but not calyx-only afferents (arrowheads). Scale bars: A, D, E, 10 µm; D, inset, 5 µm; B, C, 2 µm.

Ion channels and their associated proteins form calyceal microdomains in patterns that vary with afferent class and epithelial location. Most calyx endings belong to dimorphic afferents, which comprise 70–80% of vestibular afferents and innervate both central and peripheral zones of the epithelia. Domain 1 in peripheral/extrastriolar dimorphic afferents (A–D) neither extends as high nor contains as many synaptic ribbons (Lysakowski and Goldberg, 1997) as Domain 1 in central/striolar afferents, whether calyx-only (E) or dimorphic (F). Domain 2 of peripheral dimorphic calyces is more extensive, corresponding to the longer, thinner necks of peripheral type I hair cells (Lysakowski and Goldberg, 1997). For the peripheral dimorphic calyces (A–D), we separately summarize calyceal microdomains (A), scaffolding proteins (B), K_V channels (C), and Na_V channels (D). (E) Calyx-only afferents, which are restricted to central zones, appear to have a truncated Domain 2 and do not express many of the proteins found in dimorphic calyces. (F) In central dimorphic afferents, the synaptic zone (Domain 1) extends further upwards than in peripheral dimorphic afferents. Central afferents of both types (E,F) express more KCNQ4 and tenascin-C than do peripheral dimorphic afferents.

Domain 2, the apical part of the calyx, was selectively labeled by antibodies to the cell adhesion molecule, Caspr2, and to erg K_V subunits, and lacked several proteins found in Domain 3. (A, B) Caspr2 antibody (red) outlines Domain 2: the apical part of the calyx (above the dashed line in A). These central calyces belong to a calyx-only (calretinin-positive) and a dimorphic (calretinin-negative) afferent. Domain 2 extends higher in dimorphic afferents (arrows, in A and B) than in most calyx-only afferents (arrowhead, in A), as shown in confocal transverse sections (A) and 3D reconstructions of whole-mount utricular maculae (B). In B, the striola (Str) had much less Caspr2 staining (red) than did the extrastriolar region (ExtStr). Arrowheads in B indicate Caspr2 label of three calyces of striolar dimorphic afferents, which extended to the apical surface of the epithelium. A transverse optical section of Domain 2 (inset), taken above the level of the dotted line in A, shows concentric circles of Caspr2 label, demonstrating that it is present on both the inner and outer calyx surfaces. (C) Domain 2 was labeled by antibodies to erg1 and erg2, but not by any of the other K_V antibodies we used (KCNQ2, 3, 4, 5; K_V1.1, 1.2). Erg1 antibody labeled Domain 2 in calretinin-negative dimorphic afferents (arrows) but not in calyx-only afferents, as shown by the calretinin-positive (green) calyx on the left. Erg2 (not shown) had a similar distribution. (D, E) Immunoreactivity for βIV spectrin, which is usually found at nodes (arrowheads in D point to heminodes on the afferent fibers) and axonal initial segments, is also found in Domain 2 (arrows), shown by both confocal immunohistochemistry (D) and EM immuno-gold localization (E). Note the gold particles (E, arrows) in the calyx (Cal) surrounding the upper part of a type I (I) hair cell below the cuticular plate (CP). (F) Domain 2 is also defined by the absence of several Domain 3 proteins, including dystrophin (red); dashed white lines outline the unlabeled Domain 2 in several calyces of peripheral-zone (PZ) dimorphic afferents. Dimorphic afferents are calretinin-negative, unlike central-zone (CZ) calyx-only afferents (green, Cal) and peripheral-zone (PZ) type II hair cells (II). Scale bars: A, B inset, 5 µm; B–D, F, 10 µm; E, 500 nm.

Domain 4, the heminodal membrane adjacent to the first internode, was marked by several ion channel and scaffolding proteins that mark nodes or initial segments in other tissues. (A) Flattened projection of a confocal stack of images of the utricular epithelium and underlying stroma, where distal afferent fibers course before penetrating the basement membrane to approach the hair cells. Two heminodes (arrows) were labeled with the nodal markers Caspr1 (red) and Na_V1.6 (green). They are located next to the first inter-nodes, labeled by myelin basic protein (MBP, blue). Inset shows higher magnification of a full node (arrowhead, between two paranodes) from within the stroma, labeled with the same markers. Another full node is indicated in main panel (arrowhead). Heminodes (arrows) were also immunoreactive to antibodies against such other nodal markers as βIV spectrin (B), neurofascin186 (NF186, C), and pan-dystrophin (D). Note that the pan-dystrophin label in D encircles the cytoplasmic calretinin label of a calyx-only afferent, consistent with a membrane association. (E) Three-dimensional reconstruction showing the underside of a whole-mount utricular macula. Striolar calyx-only afferents and extrastriolar type II hair cells (asterisk) are calretinin-positive (green). KCNQ4 antibody (red) labeled calyces and the heminodes (arrows) of striolar calyx-only afferents. The remaining structures are the inner calyx surfaces (Domain 1), marked with dense KCNQ4 label (red) and viewed from below. (F) A longitudinal section through striolar calyces from the same whole-mount utricular macula shown in Fig. 1D, showing holes (arrowheads) in the bottom of the intense KCNQ4 label, and several KCNQ4-positive heminodes (arrows). (G) Contactin antibody labeled paraheminodes (arrowheads) in addition to Domain 1. Comparison with panel F shows that heminodes are further from the hair cell in these peripheral-zone afferents than in central-zone afferents. Scale bars: A–G, 10 µm; A, inset, 2.5 µm.