Molecular microdomains in a sensory terminal, the vestibular calyx ending

doi:10.1523/JNEUROSCI.0521-11.2011

J Neurosci. Author manuscript; available in PMC 2012 February 9.

Published in final edited form as:

J Neurosci. 2011 July 6; 31(27): 10101–10114.

doi: 10.1523/JNEUROSCI.0521-11.2011

PMCID: PMC3276652

NIHMSID: NIHMS309840

Molecular microdomains in a sensory terminal, the vestibular calyx ending

Anna Lysakowski,¹ Sophie Gaboyard-Niay,²^* Irina Calin-Jageman,³^* Shilpa Chatlani,⁴ Steven D. Price,¹ and Ruth Anne Eatock⁵

¹Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL 60612

²INSERM U583, Hôpital Saint Eloi - Bâtiment INM, 80 rue Augustin Fliche, Montpellier, France

³Department of Biological Sciences, Dominican University, River Forest, IL 60305

⁴Committee on Neurobiology, University of Chicago, Chicago, IL 60637

⁵Department of Otology and Laryngology, Harvard Medical School, Boston, MA 02115; Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, MA 02114

Corresponding Author: Anna Lysakowski, Ph.D., Dept. of Anatomy and Cell Biology, Univ. of Illinois at Chicago, 808 S. Wood Street, Chicago, IL 60605, Phone: 312-996-5990, FAX: 312-413-0354, Email: alysakow/at/uic.edu

^*These authors contributed equally to this work.

The publisher's final edited version of this article is available free at J Neurosci

See other articles in PMC that cite the published article.

Abstract

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent’s central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

Keywords: potassium channel, sodium channel, cell adhesion molecule, scaffolding protein, extracellular matrix protein, node of Ranvier, axon initial segment

Introduction

In vestibular sensory epithelia of mammals and other amniotes, primary afferents form expanded calyx terminals around type I hair cells in addition to conventional bouton terminals on type II hair cells (Wersäll, 1956). Unlike well-known calyces of the auditory brainstem and ciliary ganglion, the vestibular calyx is postsynaptic: its inner face is postsynaptic to the type I hair cell. Its outer face is continuous with the initial segment leading to a hemi-node where spikes are initiated, and is postsynaptic to cholinergic terminals of efferent neurons in the brainstem. This combination of functions provides an opportunity to investigate how nodal proteins interact with synaptic proteins in a compact ending, in contrast to such nodal models as motor neurons and sciatic nerve (Schafer and Rasband, 2006; Lorincz and Nusser, 2008). We find that the calyx expresses novel combinations of ion-channel and tethering proteins, including some that are characteristic of axonal nodes or initial segments (Salzer, 2003; Lai and Jan, 2006).

Type I hair cells release glutamate onto calyces from vesicles arrayed around synaptic ribbons (Bonsacquet et al., 2006; Holt et al., 2007; Dulon et al., 2009). A single calyx ending may receive input from tens of ribbons; in contrast, each bouton ending on a type II cell is generally driven by vesicular release from a single ribbon (Lysakowski and Goldberg, 1997, 2008; Holt et al., 2007). Type I-calyx transmission also includes a non-quantal component of unknown mechanism (Yamashita and Ohmori, 1990; Holt et al., 2007), and retrograde vesicular transmission has been suggested (Scarfone et al., 1988; Devau et al., 1993; Scarfone et al., 1996; Chen and Eatock, 2000; Lysakowski and Singer, 2000). Cholinergic efferent terminals on calyx outer faces may provide positive feedback on afferent activity (Holt et al., 2011).

In mammals, afferents from central and peripheral zones of vestibular epithelia form two physiologically distinct populations. Central-zone, “irregular” afferents have variable spike timing and comparatively phasic response dynamics; peripheral-zone “regular” afferents have highly stereotyped spike timing and more tonic dynamics (reviewed in Goldberg, 2000; Eatock and Songer, 2011). The two populations do not relate in a simple way to the two types of hair cells and afferent terminals: type I and II hair cells are distributed throughout both zones and supply calyces and boutons of both populations. More subtle zonal differences must be at work. Here we describe zonal variations in calyceal ion channels that may contribute.

Ion channels of interest include three potassium channel families [KCNQ (K_V7), erg (K_V11), and K_V1] that include low-voltage-activated channels, which may help set spike timing (Iwasaki et al., 2008; Kalluri et al., 2010). KCNQ and erg channels are present in calyces (Kharkovets et al., 2000; Hurley et al., 2006; Rennie and Streeter, 2006), together with an unusual Na channel, Na_V1.5 (Wooltorton et al., 2007). We report localization of these channels to specific calyceal sub-domains. To investigate how sub-domains are established, we also localized contactin-related cell adhesion molecules, scaffolding proteins and an extracellular matrix protein.

Materials and Methods

Procedures involving animals were approved by the Institutional Animal Care and Use Committees at the University of Illinois at Chicago.

Choice of antigens

We chose ion channels from those known to be expressed at nodes and initial segments (K_V1, KCNQ, Na_V1) and/or implicated by previous studies on vestibular calyces or eighth-nerve neurons (K_V1, KCNQ, erg, Na_V1.5 and Na_V1.6). K_V1.1 and 1.2 subunits form channels that carry sub-threshold currents near nodes (Arroyo and Scherer, 2000) and K_V1 channels are expressed by vestibular ganglion neurons (Iwasaki et al., 2008; Kalluri et al., 2010). KCNQ (K_V7) channel subunits contribute to M currents, are expressed at nodes of Ranvier and initial segments of central neurons (Arroyo and Scherer, 2000), and are present in vestibular calyces and neuronal cell bodies (Kharkovets et al., 2000; Hurley et al., 2006; Rennie and Streeter, 2006). Evidence for ether-a-go-go related (erg, KCNH, K_V11) channel subunits has been obtained from immature calyceal terminals (Hurley et al., 2006). Because nodes express Na_V channels at high density, we used a pan-Na_V antibody. We also chose antibodies to two specific Na_V channels: Na_V1.5, known to be present in immature vestibular calyces (Wooltorton et al., 2007), and Na_V1.6, a nodal ion channel that Hossain et al. (2005) localized to the initial segments of auditory afferents.

For scaffolding proteins, we chose those known to tether Na_V and K_V channels at or near nodes of Ranvier (Hedstrom et al., 2007; Ogawa and Rasband, 2008): ankyrins B and G, βIV spectrin, dystrophin, ezrin, and neurofascin 186. We investigated the cell adhesion molecules (CAMs), contactin and Caspr1 and 2, because they are paranodal or juxtaparanodal in other neurons (Lai and Jan, 2006). We also used an antibody to tenascin-C, an extracellular matrix molecule (ECM) associated with synaptic clefts (Dityatev and Schachner, 2003, 2006) and type I hair cells (Swartz and Santi, 1999; Warchol and Speck, 2007).

Antibodies

For details on primary antibody provenances, see Table 1. Antibodies were obtained from several vendors, including Chemicon (Temecula, CA), Alomone (Jerusalem, Israel), Sigma, Inc. (St. Louis, MO), US Biological (Swampscott, MA), Covance, Inc. (Princeton, NJ), UC Davis/NIH NeuroMab Facility, (Davis, CA). We also obtained antibodies as gifts from Matthew Rasband and Edward Cooper of Baylor College of Medicine, Elior Peles of the Weizmann Institute of Science, Thomas Jentsch of the Max-Delbrück-Centrum for Molecular Medicine, S. Rock Levinson of University of Colorado at Denver, Vann Bennett of Duke University, Álvaro Villarroel of Instituto Cajal and Universidad del País Vasco (Leioa, Spain), and Michel Roux of Université de Strasbourg. The rabbit polyclonal antiserum against pan-Na_V was made to an epitope common to all subunit subtypes of the voltage-gated Na⁺ channel. Secondary antibodies were made in donkey against rabbit, goat and mouse IgGs (obtained from Chemicon, AP182R, AP180F, and AP192S, respectively), or similar Alexa dye-labeled secondary antibodies obtained from Molecular Probes (Eugene, OR). Goat anti-mouse IgG isotype-specific Alexa dye-labeled secondary antibodies were obtained from Invitrogen (Carlsbad, CA).

Table 1

Antibody Provenances

Immunohistochemistry and Microscopy

Adult Long-Evans rats of both sexes were deeply anesthetized with Nembutal (80 mg/kg), then perfused transcardially with 100 ml physiological saline containing heparin (1000 IU), followed by 2 ml/g body weight of fixative (4% paraformal-dehyde, 1% acrolein, 1% picric acid and 5% sucrose in 0.1 M phosphate buffer (PB) at pH 7.4). Vestibular epithelia were dissected in PB. Some endorgans were fixed by immersion and dissection of the temporal bones in 100% EtOH. In some experiments with Na_V antibodies, rats were perfused with only 4% paraformaldehyde and 5% sucrose in 0.1M PB. Otoconia were dissolved with Cal-Ex (Fisher Scientific, Pittsburgh, PA) for 10 min. Background fluorescence was reduced by incubating organs and ganglia in a 1% aqueous solution of sodium borohydride for 10 min and tissue was cryo-protected in 30% sucrose-PB overnight at 4°C. Frozen sections (35 µm) were cut with a sliding microtome. Immunocytochemistry was performed on free-floating sections. Tissues were first permeabilized with 4% Triton X-100 in phosphate-buffered saline (PBS) for 1 hr at RT, then incubated with 0.5% Triton X-100 in a blocking solution of 0.5% fish gelatin and 1% BSA in PBS for 1 hr at RT. Ethanol-fixed tissues had no permeabilization pre-treatment, although 0.5% Triton X-100 was sometimes included in the antibody diluent. Sections were incubated with a cocktail of two, or in some cases three, primary antibodies for 72 hrs at 4°C. Most primary antibodies were diluted to 1:200 in the blocking solution, except for Caspr1 (1:250), βIV spectrin (1:400), ankyrinB (1:2000) and the Sigma pan-Na_V (1:100). We used calretinin antibody as a marker of type II hair cells and calyx afferents. Specific labeling was revealed by incubating sections in a cocktail of two secondary antibodies (fluorescein-conjugated donkey anti-goat IgG and rhodamine-conjugated donkey anti-rabbit IgG, diluted 1:200 in blocking solution) for 24 hrs at 4°C. If a third primary antibody was used, then typically Cy5-conjugated donkey anti-mouse was used with fluorescein- or Alexa 488-conjugated donkey anti-goat, and rhodamine- or Alexa 594-conjugated donkey anti-rabbit. To be able to combine two different mouse monoclonal antibodies in the same experiment, we used IgG subtype-specific secondary antibodies (Invitrogen, Carlsbad, CA). Sections were rinsed with PBS between and after incubations and mounted on slides in Mowiol (Calbiochem, Darmstadt, Germany).

Controls

In addition to the various controls listed in Table 1, we performed “no primary antibody” controls, which revealed a lack of non-specific staining. We also confirmed expression of each of the Na_V subunits and erg subunits by performing RT-PCR or by quantitative RT-PCR (qPCR) on whole vestibular ganglia and endorgans.

Image Analysis

Slides were examined on a laser scanning confocal microscope (LSM 510 META, Carl Zeiss, Oberköchen, Germany). Final image processing and labeling was done with Adobe Photoshop (San Jose, CA). Some whole macular organs were processed for various combinations of antibodies; Z stacks of optical sections were then obtained on the confocal microscope, with the thickness of the optical sections varying from 0.4 – 1.0 µm. The Z-series were deconvolved and reconstructed in 3D with VOLOCITY software (v. 3.7, Improvision, Lexington, MA).

Immunogold Electron Microscopy (EM)

We investigated ultrastructural localization of some markers with immuno-gold EM and we present results here for Na_V1.5, tenascin-C, and βIV spectrin. Vestibular epithelia were sectioned at 40 µm with a Vibratome 2000 (Technical Products International, St. Louis, MO). Free-floating sections were permeabilized with 0.5% Triton X-100 for 1hr, then blocked in a solution consisting of 0.5% fish gelatin and 1% BSA for 1 hr. Sections were incubated in primary antibody (1:50 dilution) for 72 hrs, rinsed, then incubated for 24 hrs in secondary antibody (1:40 dilution), tagged with ultra-small (0.8 nm) colloidal gold-labeled F (ab) goat anti-mouse IgG and goat anti-rabbit IgG (Aurion Cat. No. 25413, distributed in the U.S. by Electron Microscopy Sciences, Hatfield, PA). Colloidal gold staining was silver-enhanced (4–8 min; IntenSE M kit; Amersham Biosciences, Piscataway, NJ). Sections were dehydrated in a graded series of alcohols and propylene oxide, embedded in Araldite (Fluka Durcupan, Ronkonkoma, NY) on glass slides with plastic coverslips, and polymerized at 55°C for 48 hrs. The section of interest was cut free from the slide, glued on top of a blank Araldite block, sectioned with a diamond knife (Diatome, Biel/Bienne, Switzerland), and stained with uranyl acetate and lead citrate. Sections were examined and photographed with a JEOL 1220X transmission electron microscope.

Results

Co-localization of various ion channels and candidate tethering proteins revealed three to four contiguous domains of the calyx. We have labeled these as Domains 1 to 4, progressing smoothly from the inner face opposite the pre-synaptic ribbons (Domain 1) up and around the apex of the calyx (Domain 2, not always present) to the outer face (Domain 3) and ultimately the heminode adjacent to the first myelin wrapping (Domain 4). We present results from each domain in sequence. Throughout, we also note zonal variations in immunolocalization because afferent physiology and synaptic and afferent morphology vary with zone. Note that we often used calretinin to mark afferent type and epithelial zone. Among afferents, calretinin selectively labels calyx-only afferents, which have distinctive physiology and whose calyceal terminals delineate the central zones of cristae and the striolar zones of the utricular and saccular maculae (Desai et al., 2005a, b). In rats and mice, calretinin also labels type II hair cells (Desai et al., 2005a, b). Because striolar and extra-striolar zones of maculae resemble the central and peripheral zones, respectively, of cristae, we have simplified the text by referring to zones in both cristae and maculae as “central” and “peripheral”. Bouton-only afferents, which innervate only type II cells, are restricted to peripheral zones, while dimorphic afferents, which make both calyceal endings on type I cells and bouton endings on type II cells, are found throughout the epithelia.

KCNQs, K_V1.1, Caspr1, tenascin-C and Na_V1.5 delineate Domain 1 on the calyx inner face

In type I hair cells, synaptic ribbons and their associated vesicles are arrayed next to the basolateral cell membrane in the basal part of the cell and extending part way up the sides. The number and extent of the ribbons are larger in central type I cells than in peripheral type I cells (Lysakowski and Goldberg, 1997). Previous work on immature rat epithelia (Hurley et al., 2006) showed KCNQ4 (K_V7.4) immunoreactivity on the calyx inner face opposite the synaptic ribbons of the hair cell — Domain 1, illustrated in the inset of Figure 1. Here we show that KCNQ4 labeling of Domain 1 is more intense in adult calyces (P60–P180) and that it overlaps with strong immunoreactivity for other voltage-gated K and Na channel subunits.

Figure 1

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 (more ...)

Figure 1(A–C) shows labeling of the calyx inner face with antibodies to KCNQ2 (Fig. 1A) and KCNQ5 (Fig. 1B,C), in about the same location as previously reported for KCNQ4 (Kharkovets et al., 2000; Hurley et al., 2006). KCNQ4 immunoreactivity shows zonal variation that mirrors the extent of pre-synaptic ribbons: it is both more intense and extends further apically in central calyces than peripheral calyces (Lysakowski and Price, 2003). In contrast, KCNQ2 appeared not to vary with zone or afferent type, while KCNQ5 immunoreactivity did not vary with zone but did vary with afferent type. As shown in Figure 1(B,C), KCNQ5 staining was seen in the calyx endings of dimorphic afferents, which are found in both zones and are calretinin-negative. KCNQ5 was not localized to calretinin-positive calyx-only afferents, which are found only in central zones and have distinctive physiology (reviewed in (Lysakowski and Goldberg, 2004).

To provide a more complete picture of KCNQ4 immunolabeling, we generated three-dimensional reconstructions of confocal stacks of optical sections (Fig. 1D). These pictures reveal “holes” in the intense KCNQ4 label: 1–3 large holes (1–3 µm diameter) at the bottom of each calyceal cup, and smaller holes (200–400 nm) at calyceal invaginations, where the calyx membrane pushes into the hair cell and the synaptic cleft narrows (Spoendlin, 1966). The invaginations are especially numerous in the large synaptic domains of central-zone calyces (Lysakowski and Goldberg, 1997) and may serve to fasten the hair cell and calyx together, like punctae adherens in the calyx of Held. The small holes in KCNQ4 immunolabel are reminiscent of freeze-fracture material showing that invaginations are devoid of membrane particles (Gulley and Bagger-Sjöbäck, 1979).

The low-voltage-activated channel, K_V1.1, is, like KCNQ2, expressed in Domain 1 in all calyces, both central and peripheral (Fig. 1E). K_V1.1 is thus a candidate for the dendrotoxin-sensitive conductance of some vestibular ganglion somata, which has a strong impact on firing pattern (Iwasaki et al., 2008; Kalluri et al., 2010).

Na_V1.5, a tetrodotoxin-insensitive subunit best known as the cardiac Na channel, is also concentrated in Domain 1 (Fig. 1F). The localization strongly overlaps that of KCNQ4 and, like KCNQ4 immunoreactivity, is seen in all calyces but is more intense in central zones.

What molecules bind these voltage-gated ion channels to Domain 1? Sousa et al. (2009) showed that localization of KCNQ4 subunits in the calyx inner face requires the cell adhesion molecule Caspr1. Caspr1 is strongly expressed in Domain 1 (Fig. 1A2), as is a related CAM, contactin (see Domain 4, below). Although both KCNQ2 and Na_V subunits have a binding motif for the scaffolding protein ankyrinG (Cooper, 2011), we found no ankyrinG in Domain 1. This negative finding was strengthened by staining for ankyrinG in the same tissue sections at heminodes (Domain 4, described below), where it did co-localize with KCNQ2 and Na_V subunits. The ECM protein, tenascin-C, has been described as a marker of type I hair cells (Swartz and Santi, 1999; Warchol and Speck, 2007). In confocal material (Fig. 1G,H), tenascin-C is closely associated with Domain 1. In ultrastructural material, however, we show that tenascin-C is in the synaptic cleft, congruent with a dense cleft substance (Fig. 1I) that may contain other proteins. Like the pre-synaptic ribbons and the calyceal immunoreactivity for Na_V1.5 and KCNQ4, tenascin-C label showed regional variation in extent, reaching further up from the base of the calyceal cup in central/striolar than peripheral/extrastriolar zones (compare panels G and H in Fig. 1). Tenascin-C has been linked in neurons to synaptic plasticity involving L-type calcium channels (Dityatev et al., 2010). L-type channels are expressed by vestibular hair cells (Bao et al., 2003; Dou et al., 2004) where they mediate transmitter release, and by primary vestibular neurons (Chambard et al., 1999). Alternatively or additionally, tenascin-C might help stabilize the Na_V channels (Srinivasan et al., 1998).

In summary, Domain 1, which is co-extensive with synaptic ribbons in the type I hair cell, includes multiple KCNQ and K_V1 subunits in addition to Na_V1.5. KCNQ2, K_V1.1 and K_V1.2 are expressed in calyces regardless of type (calyx-only and dimorphic) or zone (central or peripheral). KCNQ4 and Na_V1.5 are also expressed in all calyces but staining is most intense and extensive in central calyces. KCNQ5 is restricted to dimorphic afferents of both zones. Caspr1, contactin and tenascin-C are all candidates for localizing specific proteins and functions to this domain. Differential expression of these proteins with zone (KCNQ4, Na_V1.5 and tenascin-C) or afferent type (KCNQ5) may contribute to known physiological differences between afferent fiber populations.

Caspr2 demarcates Domain 2, an apical domain of the calyx membrane

The high density of K_V channel subunits and Caspr1 prompted us to probe the calyx inner face for additional membrane proteins found near nodes of Ranvier. On the calyces of dimorphic afferents, Caspr2, a CAM typically expressed in axonal juxtaparanodes (Peles and Salzer, 2000; Salzer, 2003), labeled the apical part of the inner face, extended around the apex and about one-third to half-way down the outer face (Fig. 2A, right hair cell). We refer to this domain of intense Caspr2 labeling as Domain 2. Less intense label was seen on the outer face at the base. High-magnification views of cross-sections through the apical half of the calyx revealed two concentric circles, indicating surface labeling on both the inner and outer membranes (Fig. 2B, inset). The labeling is much stronger in the peripheral (extrastriolar) zone (Fig. 2B), partly because many of the calyces of central, calyx-only afferents, lack the apical part, or neck, of Domain 2. Figure 2A illustrates this difference: Domain 2 is labeled by Caspr2 in the right calyx, but is truncated in the left calyx. Calretinin labeling shows that the left calyx belongs to a calyx-only (calretinin-positive) afferent while the right calyx belongs to a dimorphic (calretinin-negative) afferent. In a count of calretinin-positive calyces, about two-thirds (57/86) were truncated in this way, and most of the remainder had small neck regions. This striking anatomical difference may also play a role in the physiological differences between calyx-only and dimorphic afferents.

Figure 2

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 (more ...)

Caspr2 staining overlaps with staining for erg1 (K_V11.1) subunits (Fig. 2C) and erg2 (K_V11.2; not shown). The same concentric pattern of circles shown for Caspr2 was seen with erg staining in peripheral zone calyces (not shown), indicating that erg channels are present on both faces. Thus, Caspr2 is a candidate for tethering erg subunits in place, just as Caspr1 does for KCNQ4 subunits in Domain 1 (Sousa et al., 2009). Domain 2 in dimorphic afferents also has an apical band of immunoreactivity for βIV spectrin (Fig. 2D,E), a nodal scaffolding protein (Schafer and Rasband, 2006).

Domain 2 is defined not just by the presence of Caspr2, but also by the absence of other proteins. For example, dystrophin expression in Domains 1 and 3 stops abruptly at the border with Domain 2 (Fig. 2F). Dystrophins are cytoskeletal glycoproteins frequently associated with Na_V channels and certain K_V subunits, e.g., KCNQ3 (Byers et al., 1991, 1993; Saito et al., 2003). The absence of dystrophin from Domain 2 may account for the lack of pan-Na_V labeling in this domain (Fig. 3A, see below). KCNQ3 immunoreactivity was strongly present in Domains 3 and 4, as described below, but was absent from Domain 2.

Figure 3

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 (more ...)

Na_V channels, dystrophin, KCNQ3, and ankyrinB define Domain 3 on the calyx outer face

Domain 3 comprises the basal outer face of the calyx and most of the unmyelinated part of the fiber immediately below the calyx (Fig. 3, inset). This domain was labeled by pan-Na_V antibody made against an epitope common to all Na_V α subunits (Fig. 3A). The complex pattern of labeling, which also included Domains 1 and 4 and hair cells, is likely to reflect expression of multiple Na_V isoforms. For example, pan-Na_V labeling of Domain 1 may correspond to Na_V1.5 given its similarity to the Na_V1.5-like immunoreactivity in Figure 1F. The pan-Na_V staining in hair cells may correspond to Na_V1.5 and/or Na_V1.2, based on reports from younger rats (Chabbert et al., 2003; Wooltorton et al., 2007) and our unpublished observations with subunit-specific antibodies in adult rats (S. Gaboyard and A. Lysakowski).

We saw significant differences between afferent types (dimorphic vs. calyx-only) in labeling of Domain 3 for certain ion-channel-associated proteins, which again suggests differences in their ion channel expression. Pan-dystrophin antibody labeled both faces (Domains 1 and 3) of the calyces of dimorphic afferents (Fig. 3B) but not the calyces of calyx-only afferents (Fig. 3C). Thus, Na_V subunit expression may differ between calyx-only and dimorphic calyces. Furthermore, given the strong staining for Na_V1.5 in Domain 1 of calyx-only afferents (Fig. 1F and (Wooltorton et al., 2007)), it appears that Na_V1.5 is not coupled to dystrophin.

Expression in Domain 3 also differed between zones. Staining with an antibody to KCNQ3, a marker of nodes of Ranvier and axonal initial segments (Chung et al., 2006; Schwarz et al., 2006), was much more intense in Domain 3 of peripheral calyces than in central calyces (Fig. 3D). Domain 2 was not labeled (see dashed outlines of Domain 2 in Fig. 3E). Weak KCNQ4 immuno-reactivity was also present in Domain 3, but was much less intense than in Domain 1 (Fig. 1D).

An antibody to ankyrinB, a paranodal scaffolding protein associated with K_V channels (Scotland et al., 1998; Ogawa et al., 2006), labeled the unmyelinated terminal segments of dimorphic afferents but not calyx-only afferents (Fig. 3D). In dimorphic calyces, ankyrinB immunofluorescence formed a thin shell along the outer face, continuing below the calyx (long arrows, Fig. 3E) and becoming more intense near the heminode. For calyx-only afferents, ankyrinB labeling was only seen at the heminode (short arrows, Fig. 3E).

In summary, Domain 3 in dimorphic afferents is defined by expression of Na_V and specific K_V channels and associated proteins. The staining for pan-dystrophin, ankyrinB and KCNQ3 differs between zones, in part because Domain 3 of the calyx-only afferents is not stained and these constitute a significant fraction of central afferents (≈50% in crista, 25% in macula of chinchilla; (Fernández et al., 1988, 1990). These zonal variations in Domain 3 ion channel expression may influence firing pattern differences between afferent populations.

Nodal and ion channel proteins label Domain 4, the heminodal membrane next to the myelin

Domain 4 is the heminode, a stretch of about 1 µm on the afferent fiber adjacent to the first internode. The first internode can be visualized by staining its myelin sheath with antibody against myelin basic protein (blue label, Fig. 4A). The heminode and nodes further along the afferent were selectively labeled by antibodies against the nodal proteins Na_V1.6 (Fig. 4A), βIV spectrin (Fig. 4B), neurofascin 186 (NF186, Fig. 4C), pan-dystrophin (Fig. 4D), ezrin (Fig. 3D, inset), and ankyrinG (not shown). Labeling for these nodal markers generally did not extend distally along the unmyelinated fiber toward the calyx, justifying the delineation of this portion of the axon as a separate domain. Restriction of Na_V1.6 staining to the heminode and nodes (Fig. 4A and inset) in dimorphic, but not calyx, afferents indicates that other Na_V subunits are responsible for the pan-Na_V staining of Domains 3 and 4 (Fig. 3A).

Figure 4

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 (more ...)

KCNQ staining of Domain 4 differed strongly between calyx-only and dimorphic afferents. Heminodes of calyx-only afferents stained intensely for KCNQ4 (Fig. 4E,F). Three-dimensional reconstructions of nerve fibers showed that KCNQ4 immunoreactivity formed a ring around the cytosolic calretinin label of calyx-only fibers (long arrows in Fig. 4E,F), consistent with membrane localization of the channels. In contrast, heminodes below the calyces of dimorphic afferents expressed KCNQ3, as shown by co-localization of KCNQ3 and ezrin staining (yellow, inset in Fig. 3D).

Caspr1 (Fig. 4A), contactin (Fig. 4G) and ankyrinB (Fig. 3D) antibodies labeled the paraheminodal membrane distal to the heminode. The paraheminodal contactin staining (arrowheads, Fig. 4G) shows that the peripheral-zone heminodes are well below the basement membrane of the epithelium, much further from the hair cells than are central-zone heminodes (long arrows, Fig. 4F).

Antibodies to K_V1.1 (Fig. 1E), K_V1.2 (not shown), and Caspr2 (not shown) labeled the juxtaparaheminodal membrane beyond Domain 4 (beneath the myelin), as seen at juxtaparanodes in other neurons.

Discussion

Functional significance of different microdomains

The vestibular calyx integrates afferent signals from hair cells and efferent signals from the brain into spike activity representing head motions. Calyces in different zones contribute to afferents with distinct discharge patterns and response properties. The localization of ion channels and various associated proteins suggests that these large synaptic cups, previously thought of as uniform, are divided into several discrete domains (Fig. 5), from a postsynaptic Domain 1 deep in the calyceal cup to the heminodal Domain 4 adjacent to the first myelin wrapping (Table 2 and Fig. 5). In addition to ion channels, the domains express a number of scaffolding, CAM and ECM proteins characteristic of axonal nodes and initial segments. Our results support the hypothesis that differences in molecular organization of these domains, between zones and between calyx-only and dimorphic afferents, may help differentiate the discharge patterns of irregular afferents and regular afferents (Smith and Goldberg, 1986; Baird et al., 1988; Goldberg et al., 1990b; Kalluri et al., 2010).

Figure 5

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 (more ...)

Table 2

Comparison of Calyx Proteins to Proteins in Nodes of Ranvier and Initial Segments

Domain 1 comprises much of the calyceal inner face and is contiguous with a dense substance in the intervening synaptic cleft, apposing the synaptic ribbons. Indeed, the extent of the immunolabeled cups (Fig. 5A–F) co-varies with hair cell membrane devoted to transmitter release, as reflected by adjacent synaptic ribbons. Ribbons cluster in the bottom third in peripheral-zone type I hair cells but in the bottom two-thirds of central-zone type I hair cells (Lysakowski and Goldberg, 1997). Therefore, in addition to the proteins shown here, Domain 1 also expresses glutamate receptors – principally AMPA-type, but also NMDA receptors (Matsubara et al., 1999; Bonsacquet et al., 2006).

Domain 1 has an impressively dense expression of K_V and Na_V channels. K_V channels include multiple KCNQ (K_V7) and erg (K_V11) channel isoforms as well as K_V1.1 and K_V1.2. Their expression closely matches that of the CAM Caspr1, known to occur at paranodes (Salzer, 2003). Sousa et al. (2009) showed that Caspr1 is necessary for retention of KCNQ4 in the calyx membrane and for maintaining appropriate synaptic cleft width. We report that contactin labeling is comparable to the Caspr1 pattern. Both CAMs may together stabilize other K_V channels in this domain, such as KCNQ2, KCNQ5, K_V1.1, and K_V1.2.

Several proteins localized to Domain 1 are reported to associate with Na_V subunits in other tissues: contactin (Kazarinova-Noyes et al., 2001; Rush et al., 2005), tenascin-C (Srinivasan et al., 1998; Evers et al., 2002; Dityatev and Schachner, 2006; Ullian and Dityatev, 2006), and dystrophin (Byers et al., 1991, 1993; Kim et al., 1992; Saito et al., 2003; Occhi et al., 2005). Thus, Na_V1.5 subunits in Domain 1 may associate with any or all of these proteins. Contactin may regulate current density and expression of Na_V1.5 channels, as it is thought to do for Na_V1.8 and 1.9 subunits (Kazarinova-Noyes et al., 2001; Isom, 2002; Rush et al., 2005). Tenascin-C contributes to the electron-dense cleft material in the synaptic cleft of Domain 1 and could interact with Na_V β subunits and/or contactin in Domain 1 to stabilize the Na channels (Zisch et al., 1992; Srinivasan et al., 1998). Dystrophin forms a complex with dystroglycan that connects the actin cytoskeleton to the extracellular matrix, and is found where Na_V channels are present in high density, e.g., the deep folds of the neuromuscular junction (Byers et al., 1991), nodes (Byers et al., 1993; Occhi et al., 2005), and postsynaptic densities (Kim et al., 1992).

Thus, a complex involving contactin, tenascin-C, dystrophin, and Caspr1 may hold both K_V and Na_V channels at high density and in specific sub-domains within the calyceal synaptic cleft. Analogous roles are proposed for agrin and laminin at the neuromuscular junction and for reelins and integrins at forming synapses (Dityatev and Schachner, 2003; Ullian and Dityatev, 2006).

The between-zone differences we report in ion channel expression in Domain 1 may play a role in differentiating afferent firing patterns between epithelial zones (Smith and Goldberg, 1986; Baird et al., 1988; Goldberg et al., 1990a; Kalluri et al., 2010). Recent work has specifically proposed that low-voltage-activated channels (Kv1 and KCNQ) help make the firing of central afferents irregular (Iwasaki et al., 2008; Kalluri et al., 2010). KCNQ4 immunolabeling correlates with irregular spike timing: it is more extensive and intense in the central-zone calyces of irregular afferents than in the peripheral-zone calyces of regular afferents. Furthermore, M-current through KCNQ channels in calyces might be modulated by G-protein coupled receptors activated by acetylcholine, ATP, CGRP, opioid peptides or GABA released from efferent terminals onto calyx outer faces. In that case, the greater expression of KCNQ4 in central calyces could contribute to the greater effect of efferent activation on irregular afferents (Holt et al., 2011).

KCNQ4 is present at high density on the calyx inner face (Domain 1) and much lower density on the outer face (Domain 3; see Fig. 1D and (Lysakowski and Price, 2003). In heterologous expression systems, KCNQ4 can form homomeric channels (Søgaard et al., 2001; Xu et al., 2007) and can heteromultimerize with KCNQ2, KCNQ3 and KCNQ5 (Howard et al., 2007; Xu et al., 2007; Bal et al., 2008). Our results suggest that KCNQ4 could partner with KCNQ2 in Domain 1 of any calyx (Fig. 1A). In peripheral dimorph calyces, KCNQ4 could also partner with KCNQ5 on the inner face (Domain 1, Fig. 1C) and KCNQ3 on the outer face (Domain 3, Fig. 3D).

Immunoreactivity for erg subunits was much stronger in calyces of dimorphic afferents than in calyces of calyx-only afferents. This difference explains the reduced erg staining previously noted in central zones in immature epithelia (Hurley et al., 2006). Again, such zonal differences in M-like channel expression have the potential to shape afferent discharge regularity and/or efferent responses.

Na_V1.5, localized to Domain 1, includes an amino acid sequence that shifts activation and inactivation voltage ranges negative relative to tetrodotoxin-sensitive Na_V isoforms (Camacho et al., 2006). In this way, the voltage range of Na_V channel activation in calyces may be matched with that of nearby KCNQ and erg channels (Hurley et al., 2006). Either Na_V1.5 or Na_V1.6 could contribute to a persistent Na_V current (Holt et al., 2007; Wooltorton et al., 2007), which could shape spike timing. In the rat utricular macula, Na_V1.5 immunoreactivity is strongest in the striola, at least up to P21 (Wooltorton et al., 2007), thus Na_V1.5 is also a candidate to contribute to zonal differences in discharge regularity.

Domain 2, the apical calyx membrane, is defined both by what is expressed and what is not expressed. Antibodies against βIV spectrin, Caspr2, erg1, and erg2 labeled Domain 2 more than other zones. Antibodies that labeled Domain 3 and not Domain 2 include those against Na_V channels, dystrophins, KCNQ3, and ankyrinB. Since βIV spectrin associates with Na_V channels at nodes, and Caspr2 associates with K_V channels at juxtaparanodes (Arroyo and Scherer, 2000), Domain 2 may express yet-to-be-identified voltage-gated channels.

Domain 2 has been proposed as a possible source of retrograde feedback from calyces to hair cells, based on immunolocalization of such pre-synaptic proteins as rab3A (Dechesne et al., 1997), synaptophysin (Scarfone et al., 1988; Dechesne et al., 1997), synapsin I (Scarfone et al., 1988), syntaxin, SNAP25 and synaptotagmin (Dememes et al., 2000). Calyces might release neuropeptides (Scarfone et al., 1996) or glutamate (Devau et al., 1993) onto the type I hair cell. Detailed ultrastructural studies in rodents have revealed no post-synaptic densities in hair cell membranes adjacent to Domain 2, but clusters of dense-cored vesicles are seen in Domain 2 in squirrel monkey calyces (A. Lysakowski, personal observations). Dense-cored vesicles are consistent with the neuropeptide hypothesis, and because they release their contents away from active zones (Lysakowski et al., 1999; Shakiryanova et al., 2005), post-synaptic densities are unnecessary.

Domain 2 appears to be lacking in most calyx-only afferents (Figs. 1H, 2A–C, ,5E),5E), another zonal difference that could influence afferent physiology. In particular, retrograde feedback from the calyx to the hair cell might be missing at calyx-only synapses, which are principally found in central and striolar zones.

In Domain 3, we identified many proteins characteristic of axonal initial segments but there are also some differences. To our knowledge, the combination of ion channels does not match any of those described in central neurons, although some diversity of expression has been reported for axonal initial segments (Lorincz and Nusser, 2008). For example, olfactory mitral cells express K_V1.2, layer 5 pyramidal cells also express K_V1.1, and Purkinje cells express neither (Lorincz and Nusser, 2008). Domain 3 of the vestibular calyx resembles the axonal initial segment in that it expresses a gradient of ion channels and scaffolding proteins. In addition to ion channels, Domain 3 contains ankyrinB, which anchors Na⁺-K⁺ ATPases and Na⁺-Ca²⁺ exchangers in heart (Mohler et al., 2005) and retina (Kizhatil et al., 2009). Na⁺-Ca²⁺ exchangers and the α3β1 isoform of Na⁺-K⁺ ATPase have been reported within the vestibular sensory epithelium (ten Cate et al., 1994; Boyer et al., 1999), but resolution was not sufficient to localize either to pre- or post-synaptic membranes. Unlike some axonal initial segments, Domain 3 does not express βIV spectrin and neurofascin 186.

Finally, Domain 4 contains many known nodal proteins (Peles and Salzer, 2000; Salzer, 2003; Dityatev and Schachner, 2006; Ullian and Dityatev, 2006), and resembles in its organization one-half of a full node (Fig. 4A, inset). Many vestibular heminodes were recognized by the juxtaposition of Na_V1.6, the most common nodal Na_V channel, with the paranodal protein Caspr1 and the internodal protein myelin basic protein (MBP). Scaffolding proteins at nodes of Ranvier (βIV spectrin and neurofascin 186) also label vestibular afferent heminodes intensely (Fig. 4B,C). The expression of K_V7 subunits (KCNQ3, but not KCNQ2, at dimorphic heminodes, and KCNQ4 at calyx-only heminodes) differs from the combination of KCNQ2 and KCNQ3 expression reported at other axonal nodes (Pan et al., 2006; Bennett and Healy, 2009).

The spike trigger zone in eighth nerve afferents has been a subject of speculation (Goldberg, 1996; Hossain et al., 2005). The presence of many known nodal proteins in Domain 4 suggests that the heminode immediately below calyces may be a spike trigger zone, but does not rule out a more distant locus (Palmer and Stuart, 2006; Bean, 2007). Furthermore, the location of spike initiation may vary with zone, given that peripheral dimorphic afferents have more extensive arbors with longer unmyelinated portions (Fig. 4A,F,G).

In summary, the calyx terminal combines unique features of dendrites and initial segments in a highly ordered, novel arrangement. The large size of the ending has allowed us to define several regions, or microdomains, each characterized by a distinct set of ion channels, neurotransmitter receptors, and accessory proteins. None of the antibodies we used produced detectable labeling of bouton terminals on type II cells. The much smaller surface areas of bouton endings (5–10 µm²) compared to calyx endings (~1000 µm²) may be less able to accommodate microdomains.

ACKNOWLEDGEMENTS

This study was supported by NIH (R01 DC-02521, R01 DC-02058, R01 DC-02290). The Electron Microscopic Facility of the Research Resources Center of the University of Illinois at Chicago provided equipment and assistance to conduct this study. We are grateful to Drs. Matt Rasband (Na_V1.6, Na_V1.2, Caspr1, βIV spectrin, neurofascin 186 and pan-neurofascin), James Trimmer (pan-Na_V), Elior Peles (Caspr3 and Caspr4), Edward Cooper (KCNQ2 and KCNQ3), Thomas Jentsch (KCNQ4 and KCNQ5), Rock Levinson (pan-Na_V), Vann Bennett (ankyrinG and ankyrinB) and Michel Roux (H4, pan-dystrophin) for generous gifts of antibodies, and to Mr. Marcin Klapczynski for technical assistance. We thank Drs. Jay M. Goldberg, D. Kent Morest and Matt Rasband for helpful discussions.

Footnotes

Conflicts of Interest: None.

References

Abriel H, Kass RS. Regulation of the voltage-gated cardiac sodium channel Nav1.5 by interacting proteins. Trends Cardiovasc Med. 2005;15:35–40. [PubMed]
Arroyo EJ, Scherer SS. On the molecular architecture of myelinated fibers. Histochem Cell Biol. 2000;113:1–18. [PubMed]
Baird RA, Desmadryl G, Fernández C, Goldberg JM. The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals. J Neurophysiol. 1988;60:182–203. [PubMed]
Bal M, Zhang J, Zaika O, Hernandez CC, Shapiro MS. Homomeric and heteromeric assembly of KCNQ (Kv7) K⁺ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis. J Biol Chem. 2008 Nov 7;283(45):30668–30676. Epub 2008 Sep 11. PMID: 18786918. [PubMed]
Bao H, Wong WH, Goldberg JM, Eatock RA. Voltage-gated calcium channel currents in type I and type II hair cells isolated from the rat crista. J Neurophysiol. 2003 Jul;90(1):155–164. Erratum in: J Neurophysiol. 2004 Jan;91(1):589. PMID: 12843307. [PubMed]
Bean BP. The action potential in mammalian central neurons. Nat Rev Neurosci. 2007;8:451–465. [PubMed]
Bennett V, Healy J. Membrane domains based on ankyrin and spectrin associated with cell-cell interactions. Cold Spring Harb Perspect Biol. 2009;1:a003012. [PMC free article] [PubMed]
Black JA, Renganathan M, Waxman SG. Sodium channel Na(v)1.6 is expressed along nonmyelinated axons and it contributes to conduction. Brain Res Mol Brain Res. 2002;105:19–28. [PubMed]
Bonsacquet J, Brugeaud A, Compan V, Desmadryl G, Chabbert C. AMPA type glutamate receptor mediates neurotransmission at turtle vestibular calyx synapse. J Physiol. 2006 Oct 1;576(Pt 1):63–71. Epub 2006 Aug 3. [PubMed]
Boyer C, Sans A, Vautrin J, Chabbert C, Lehouelleur J. K⁺-dependence of Na⁺-Ca²⁺ exchange in guinea-pig type I vestibular sensory cells. Eur J Neurosci. 1999;11:1955–1959. [PubMed]
Byers TJ, Kunkel LM, Watkins SC. The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J Cell Biol. 1991;115:411–421. [PMC free article] [PubMed]
Byers TJ, Lidov HG, Kunkel LM. An alternative dystrophin transcript specific to peripheral nerve. Nat Genet. 1993;4:77–81. [PubMed]
Camacho JA, Hensellek S, Rougier JS, Blechschmidt S, Abriel H, Benndorf K, Zimmer T. Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region. J Biol Chem. 2006;281:9498–9506. [PubMed]
Chabbert C, Mechaly I, Sieso V, Giraud P, Brugeaud A, Lehouelleur J, Couraud F, Valmier J, Sans A. Voltage-gated Na⁺ channel activation induces both action potentials in utricular hair cells and brain-derived neurotrophic factor release in the rat utricle during a restricted period of development. J Physiol. 2003;553:113–123. [PubMed]
Chambard JM, Chabbert C, Sans A, Desmadryl G. Developmental changes in low and high voltage-activated calcium currents in acutely isolated mouse vestibular neurons. J Physiol. 1999;518(Pt 1):141–149. [PubMed]
Chen JWY, Eatock RA. Major potassium conductance in type I hair cells from rat semicircular canals: characterization and modulation by nitric oxide. J Neurophysiol. 2000;84:139–151. [PubMed]
Chung HJ, Jan YN, Jan LY. Polarized axonal surface expression of neuronal KCNQ channels is mediated by multiple signals in the KCNQ2 and KCNQ3 C-terminal domains. Proc Natl Acad Sci USA. 2006;103:8870–8875. [PubMed]
Cooper EC. Made for "anchorin": Kv7.2/7.3 (KCNQ2/KCNQ3) channels and the modulation of neuronal excitability in vertebrate axons. Semin Cell Dev Biol. 2011;22:185–192. [PMC free article] [PubMed]
Cooper EC, Aldape KD, Abosch A, Barbaro NM, Berger MS, Peacock WS, Jan YN, Jan LY. Colocalization and coassembly of two human brain M-type potassium channel subunits that are mutated in epilepsy. Proc Natl Acad Sci U S A. 2000;97:4914–4919. [PubMed]
Cooper EC, Harrington E, Jan YN, Jan LY. M channel KCNQ2 subunits are localized to key sites for control of neuronal network oscillations and synchronization in mouse brain. J Neurosci. 2001;21:9529–9540. [PubMed]
Dechesne CJ, Kauff C, Stettler O, Tavitian B. Rab3A immunolocalization in the mammalian vestibular end-organs during development and comparison with synaptophysin expression. Brain Res Dev Brain Res. 1997;99:103–111. [PubMed]
Demêmes D, Seoane A, Venteo S, Desmadryl G. Efferent function of vestibular afferent endings? Similar localization of N-type calcium channels, synaptic vesicle and synaptic membrane-associated proteins. Neuroscience. 2000;98:377–384. [PubMed]
Desai SS, Zeh C, Lysakowski A. Comparative morphology of rodent vestibular periphery. I. Saccular and utricular maculae. J Neurophysiol. 2005 Jan;93(1):251–266. Epub 2004 Jul 7. [PubMed]
Desai SS, Ali H, Lysakowski A. Comparative morphology of rodent vestibular periphery. II. Cristae ampullares. J Neurophysiol. 2005 Jan;93(1):267–280. Epub 2004 Jul 7. [PubMed]
Devau G, Lehouelleur J, Sans A. Glutamate receptors on type I vestibular hair cells of guinea-pig. Eur J Neurosci. 1993;5:1210–1217. [PubMed]
Devaux JJ, Kleopa KA, Cooper EC, Scherer SS. KCNQ2 is a nodal K⁺ channel. J Neurosci. 2004;24:1236–1244. [PubMed]
Dityatev A, Schachner M. Extracellular matrix molecules and synaptic plasticity. Nat Rev Neurosci. 2003;4:456–468. [PubMed]
Dityatev A, Schachner M. The extracellular matrix and synapses. Cell Tissue Res. 2006;326:647–654. [PubMed]
Dityatev A, Schachner M, Sonderegger P. The dual role of the extracellular matrix in synaptic plasticity and homeostasis. Nat Rev Neurosci. 2010;11:735–746. [PubMed]
Dodd J, Morton SB, Karagogeos D, Yamamoto M, Jessell TM. Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron. 1988;1:105–116. [PubMed]
Dou H, Vazquez AE, Namkung Y, Chu H, Cardell EL, Nie L, Parson S, Shin HS, Yamoah EN. Null mutation of alpha1D Ca²⁺ channel gene results in deafness but no vestibular defect in mice. J Assoc Res Otolaryngol. 2004;5:215–226. [PMC free article] [PubMed]
Dulon D, Safieddine S, Jones SM, Petit C. Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses. J Neurosci. 2009;29:10474–10487. [PMC free article] [PubMed]
Eatock RA, Songer JE. Vestibular hair cells and afferents: Two channels for head motion signals. Annual Review of Neuroscience. 2011;34:501–534. [PubMed]
Evers MR, Salmen B, Bukalo O, Rollenhagen A, Bosl MR, Morellini F, Bartsch U, Dityatev A, Schachner M. Impairment of L-type Ca²⁺ channel-dependent forms of hippocampal synaptic plasticity in mice deficient in the extracellular matrix glycoprotein tenascin-C. J Neurosci. 2002;22:7177–7194. [PubMed]
Fernández C, Baird RA, Goldberg JM. The vestibular nerve of the chinchilla. I. Peripheral innervation patterns in the horizontal and superior semicircular canals. J Neurophysiol. 1988;60:167–181. [PubMed]
Fernandez C, Goldberg JM, Baird RA. The vestibular nerve of the chinchilla. III. Peripheral innervation patterns in the utricular macula. J Neurophysiol. 1990;63:767–780. [PubMed]
Goldberg JM. Theoretical analysis of intercellular communication between the vestibular type I hair cell and its calyx ending. J Neurophysiol. 1996;76:1942–1957. [PubMed]
Goldberg JM, Desmadryl G, Baird RA, Fernández C. The vestibular nerve of the chinchilla. IV. Discharge properties of utricular afferents. J Neurophysiol. 1990 Apr;63(4):781–790. [PubMed]
Goldberg JM, Desmadryl G, Baird RA, Fernández C. The vestibular nerve of the chinchilla. V. Relation between afferent discharge properties and peripheral innervation patterns in the utricular macula. J Neurophysiol. 1990 Apr;63(4):791–804. [PubMed]
Gulley RL, Bagger-Sjöbäck D. Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system. J Neurocytol. 1979;8:591–603. [PubMed]
Hedstrom KL, Xu X, Ogawa Y, Frischknecht R, Seidenbecher CI, Shrager P, Rasband MN. Neurofascin assembles a specialized extracellular matrix at the axon initial segment. J Cell Biol. 2007;178:875–886. [PMC free article] [PubMed]
Holt JC, Chatlani S, Lysakowski A, Goldberg JM. Quantal and nonquantal transmission in calyx-bearing fibers of the turtle posterior crista. J Neurophysiol. 2007;98:1083–1101. [PMC free article] [PubMed]
Holt JC, Lysakowski A, Goldberg JM. The efferent vestibular system. In: Ryugo DD, Fay RR, Popper AN, editors. Auditory and Vestibular Efferents. New York: Springer; 2011. pp. 135–186.
Hossain WA, Antic SD, Yang Y, Rasband MN, Morest DK. Where is the spike generator of the cochlear nerve? Voltage-gated sodium channels in the mouse cochlea. J Neurosci. 2005;25:6857–6868. [PMC free article] [PubMed]
Howard RJ, Clark KA, Holton JM, Minor DL., Jr Structural insight into KCNQ (Kv7) channel assembly and channelopathy. Neuron. 2007;53:663–675. [PMC free article] [PubMed]
Hurley KM, Gaboyard S, Zhong M, Price SD, Wooltorton JR, Lysakowski A, Eatock RA. M-like K⁺ currents in type I hair cells and calyx afferent endings of the developing rat utricle. J Neurosci. 2006;26:10253–10269. [PubMed]
Huss D, Navaluri R, Faulkner KF, Dickman JD. Development of otolith receptors in Japanese quail. Dev Neurobiol. 2010;70:436–455. [PMC free article] [PubMed]
Isom LL. The role of sodium channels in cell adhesion. Front Biosci. 2002;7:12–23. [PubMed]
Iwasaki S, Chihara Y, Komuta Y, Ito K, Sahara Y. Low-voltage-activated potassium channels underlie the regulation of intrinsic firing properties of rat vestibular ganglion cells. J Neurophysiol. 2008;100:2192–2204. [PubMed]
Kalluri R, Xue J, Eatock RA. Ion channels set spike timing regularity of mammalian vestibular afferent neurons. J Neurophysiol. 2010;104:2034–2051. [PubMed]
Kazarinova-Noyes K, Malhotra JD, McEwen DP, Mattei LN, Berglund EO, Ranscht B, Levinson SR, Schachner M, Shrager P, Isom LL, Xiao ZC. Contactin associates with Na⁺ channels and increases their functional expression. J Neurosci. 2001;21:7517–7525. [PubMed]
Kharkovets T, Hardelin J-P, Safieddine S, Schweizer M, El-Amraoui A, Petit C, Jentsch TJ. KCNQ4, a K⁺ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway. Proc Natl Acad Sci USA. 2000;97:4333–4338. [PubMed]
Kharkovets T, Dedek K, Maier H, Schweizer M, Khimich D, Nouvian R, Vardanyan V, Leuwer R, Moser T, Jentsch TJ. Mice with altered KCNQ4 K⁺ channels implicate sensory outer hair cells in human progressive deafness. EMBO J. 2006;25:642–652. [PubMed]
Kim TW, Wu K, Xu JL, Black IB. Detection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy. Proc Natl Acad Sci USA. 1992;89:11642–11644. [PubMed]
Kizhatil K, Sandhu NK, Peachey NS, Bennett V. Ankyrin-B is required for coordinated expression of beta-2-spectrin, the Na/K-ATPase and the Na/Ca exchanger in the inner segment of rod photoreceptors. Exp Eye Res. 2009;88:57–64. [PubMed]
Kordeli E, Lambert S, Bennett V. AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J Biol Chem. 1995;270:2352–2359. [PubMed]
Lacas-Gervais S, Guo J, Strenzke N, Scarfone E, Kolpe M, Jahkel M, De Camilli P, Moser T, Rasband MN, Solimena M. BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments. J Cell Biol. 2004;166:983–990. [PMC free article] [PubMed]
Lai HC, Jan LY. The distribution and targeting of neuronal voltage-gated ion channels. Nat Rev Neurosci. 2006;7:548–562. [PubMed]
Leterrier C, Brachet A, Dargent B, Vacher H. Determinants of voltage-gated sodium channel clustering in neurons. Semin Cell Dev Biol. 2011;22:171–177. [PubMed]
Lorincz A, Nusser Z. Cell-type-dependent molecular composition of the axon initial segment. J Neurosci. 2008;28:14329–14340. [PMC free article] [PubMed]
Lysakowski A, Goldberg JM. A regional ultrastructural analysis of the cellular and synaptic architecture in the chinchilla cristae ampullares. J Comp Neurol. 1997;389:419–443. [PubMed]
Lysakowski A, Goldberg JM. Morphophysiology of the vestibular sensory periphery. In: Highstein SM, Fay RR, Popper AN, editors. The Vestibular System. New York: Springer-Verlag; 2004. pp. 57–152.
Lysakowski A, Price SD. Potassium channel localization in sensory epithelia of the rat inner ear. Assoc Res Otolaryngol Abstr. 2003;26:1534.
Lysakowski A, Singer M. Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry. J Comp Neurol. 2000;427:508–521. [PubMed]
Lysakowski A, Figueras H, Price SD, Peng YY. Dense-cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: implications for exocytosis and calcium buffering by intraterminal organelles. J Comp Neurol. 1999;403:378–390. [PubMed]
Matsubara A, Takumi Y, Nakagawa T, Usami S, Shinkawa H, Ottersen OP. Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs. Brain Res. 1999;819:58–64. [PubMed]
Mohler PJ, Davis JQ, Bennett V. Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain. PLoS Biol. 2005;3:e423. [PMC free article] [PubMed]
Ng FL, Davis AJ, Jepps TA, Harhun MI, Yeung SY, Wan A, Reddy M, Melville D, Nardi A, Khong TK, Greenwood IA. Expression and function of the K⁺ channel KCNQ genes in human arteries. Br J Pharmacol. 2011;162:42–53. [PubMed]
Occhi S, Zambroni D, Del Carro U, Amadio S, Sirkowski EE, Scherer SS, Campbell KP, Moore SA, Chen ZL, Strickland S, Di Muzio A, Uncini A, Wrabetz L, Feltri ML. Both laminin and Schwann cell dystroglycan are necessary for proper clustering of sodium channels at nodes of Ranvier. J Neurosci. 2005;25:9418–9427. [PMC free article] [PubMed]
Ogawa Y, Rasband MN. The functional organization and assembly of the axon initial segment. Curr Opin Neurobiol. 2008;18:307–313. [PubMed]
Ogawa Y, Schafer DP, Horresh I, Bar V, Hales K, Yang Y, Susuki K, Peles E, Stankewich MC, Rasband MN. Spectrins and ankyrinB constitute a specialized paranodal cytoskeleton. J Neurosci. 2006;26:5230–5239. [PubMed]
Palmer LM, Stuart GJ. Site of action potential initiation in layer 5 pyramidal neurons. J Neurosci. 2006;26:1854–1863. [PubMed]
Pan Z, Kao T, Horvath Z, Lemos J, Sul JY, Cranstoun SD, Bennett V, Scherer SS, Cooper EC. A common ankyrin-G-based mechanism retains KCNQ and Na_V channels at electrically active domains of the axon. J Neurosci. 2006;26:2599–2613. [PubMed]
Peles E, Salzer JL. Molecular domains of myelinated axons. Curr Opin Neurobiol. 2000;10:558–565. [PubMed]
Pilgram GS, Potikanond S, Baines RA, Fradkin LG, Noordermeer JN. The roles of the dystrophin-associated glycoprotein complex at the synapse. Mol Neurobiol. 2010;41:1–21. [PMC free article] [PubMed]
Rasband MN, Trimmer JS. Subunit composition and novel localization of K⁺ channels in spinal cord. J Comp Neurol. 2001;429:166–176. [PubMed]
Rasband MN, Kagawa T, Park EW, Ikenaka K, Trimmer JS. Dysregulation of axonal sodium channel isoforms after adult-onset chronic demyelination. J Neurosci Res. 2003;73:465–470. [PubMed]
Rasband MN, Peles E, Trimmer JS, Levinson SR, Lux SE, Shrager P. Dependence of nodal sodium channel clustering on paranodal axoglial contact in the developing CNS. J Neurosci. 1999;19:7516–7528. [PubMed]
Rennie KJ, Streeter MA. Voltage-dependent currents in isolated vestibular afferent calyx terminals. J Neurophysiol. 2006;95:26–32. [PubMed]
Rivier F, Robert A, Hugon G, Bonet-Kerrache A, Nigro V, Fehrentz JA, Martinez J, Mornet D. Dystrophin and utrophin complexed with different associated proteins in cardiac Purkinje fibres. Histochemical Journal. 1999;31:425–432. [PubMed]
Rush AM, Craner MJ, Kageyama T, Dib-Hajj SD, Waxman SG, Ranscht B. Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons. Eur J Neurosci. 2005;22:39–49. [PubMed]
Rush AM, Cummins TR, Waxman SG. Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons. J Physiol. 2007;579:1–14. [PubMed]
Saito F, Moore SA, Barresi R, Henry MD, Messing A, Ross-Barta SE, Cohn RD, Williamson RA, Sluka KA, Sherman DL, Brophy PJ, Schmelzer JD, Low PA, Wrabetz L, Feltri ML, Campbell KP. Unique role of dystroglycan in peripheral nerve myelination, nodal structure, and sodium channel stabilization. Neuron. 2003;38:747–758. [PubMed]
Salzer JL. Polarized domains of myelinated axons. Neuron. 2003;40:297–318. [PubMed]
Salzer JL, Brophy PJ, Peles E. Molecular domains of myelinated axons in the peripheral nervous system. Glia. 2008;56:1532–1540. [PubMed]
Scarfone E, Dememes D, Jahn R, De Camilli P, Sans A. Secretory function of the vestibular nerve calyx suggested by presence of vesicles, synapsin I, and synaptophysin. J Neurosci. 1988;8:4640–4645. [PubMed]
Scarfone E, Ulfendahl M, Lundeberg T. The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study. Neuroscience. 1996;75:587–600. [PubMed]
Schafer DP, Rasband MN. Glial regulation of the axonal membrane at nodes of Ranvier. Curr Opin Neurobiol. 2006;16:508–514. [PubMed]
Schafer DP, Custer AW, Shrager P, Rasband MN. Early events in node of Ranvier formation during myelination and remyelination in the PNS. Neuron Glia Biol. 2006;2:69–79. [PMC free article] [PubMed]
Schroeder BC, Hechenberger M, Weinreich F, Kubisch C, Jentsch TJ. KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents. J Biol Chem. 2000;275:24089–24095. [PubMed]
Schwarz JR, Bauer CK. Functions of erg K⁺ channels in excitable cells. J Cell Mol Med. 2004;8:22–30. [PubMed]
Schwarz JR, Glassmeier G, Cooper EC, Kao TC, Nodera H, Tabuena D, Kaji R, Bostock H. KCNQ channels mediate IKs, a slow K⁺ current regulating excitability in the rat node of Ranvier. J Physiol. 2006;573:17–34. [PubMed]
Scotland P, Zhou D, Benveniste H, Bennett V. Nervous system defects of AnkyrinB (−/−) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons. J Cell Biol. 1998;143:1305–1315. [PMC free article] [PubMed]
Shakiryanova D, Tully A, Hewes RS, Deitcher DL, Levitan ES. Activity-dependent liberation of synaptic neuropeptide vesicles. Nat Neurosci. 2005;8:173–178. [PubMed]
Smith CE, Goldberg JM. A stochastic afterhyperpolarization model of repetitive activity in vestibular afferents. Biol Cybern. 1986;54:41–51. [PubMed]
Søgaard R, Ljungstrøm T, Pedersen KA, Olesen SP, Jensen BS. KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology. Am J Physiol Cell Physiol. 2001;280:C859–C866. [PubMed]
Sousa AD, Andrade LR, Salles FT, Pillai AM, Buttermore ED, Bhat MA, Kachar B. The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells. J Neurosci. 2009;29:3103–3108. [PMC free article] [PubMed]
Spiegel I, Salomon D, Erne B, Schaeren-Wiemers N, Peles E. Caspr3 and caspr4, two novel members of the caspr family are expressed in the nervous system and interact with PDZ domains. Mol Cell Neurosci. 2002;20:283–297. [PubMed]
Spoendlin H. Some morphofunctional and pathological aspects of the vestibular sensory epithelium; Second Symposium on The Role of the Vestibular Organs in Space Exploration, NASA SP-115; Washington, D.C.: U.S. Government Printing Office; 1966. pp. 99–116.
Srinivasan J, Schachner M, Catterall WA. Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R. Proc Natl Acad Sci USA. 1998;95:15753–15757. [PubMed]
Swartz DJ, Santi PA. Immunolocalization of tenascin in the chinchilla inner ear. Hear Res. 1999;130:108–114. [PubMed]
ten Cate WJ, Curtis LM, Rarey KE. Na,K-ATPase alpha and beta subunit isoform distribution in the rat cochlear and vestibular tissues. Hear Res. 1994;75:151–160. [PubMed]
Tzingounis AV, Heidenreich M, Kharkovets T, Spitzmaul G, Jensen HS, Nicoll RA, Jentsch TJ. The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus. Proc Natl Acad Sci U S A. 2010;107:10232–10237. [PubMed]
Ullian EM, Dityatev A. Extracellular matrix molecules and formation of CNS synapses. In: Dityatev A, El-Husseini A, editors. Molecular Mechanisms of Synaptogenesis. New York, NY: Springer; 2006. pp. 163–179.
Warchol ME, Speck JD. Expression of GATA3 and tenascin in the avian vestibular maculae: normative patterns and changes during sensory regeneration. J Comp Neurol. 2007;500:646–657. [PubMed]
Wersäll J. Studies on the structure and innervation of the sensory epithelium of the cristae ampullaris in the guinea pig. A light and electron microscopic investigation. Acta Otolaryngol Suppl. 1956;126:1–85. [PubMed]
Wooltorton JR, Gaboyard S, Hurley KM, Price SD, Garcia JL, Zhong M, Lysakowski A, Eatock RA. Developmental changes in two voltage-dependent sodium currents in utricular hair cells. J Neurophysiol. 2007 Feb;97(2):1684–1704. Epub 2006 Oct 25. [PubMed]
Xu T, Nie L, Zhang Y, Mo J, Feng W, Wei D, Petrov E, Calisto LE, Kachar B, Beisel KW, Vazquez AE, Yamoah EN. Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels. J Biol Chem. 2007;282:23899–23909. [PubMed]
Yamashita M, Ohmori H. Synaptic responses to mechanical stimulation in calyceal and bouton type vestibular afferents studied in an isolated preparation of semicircular canal ampullae of chicken. Exp Brain Res. 1990;80:475–488. [PubMed]
Yus-Najera E, Munoz A, Salvador N, Jensen BS, Rasmussen HB, Defelipe J, Villarroel A. Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation. Neuroscience. 2003;120:353–364. [PubMed]
Zisch AH, D'Alessandri L, Ranscht B, Falchetto R, Winterhalter KH, Vaughan L. Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains. J Cell Biol. 1992;119:203–213. [PMC free article] [PubMed]