Distribution of the Na,K-ATPase α Subunit in the Rat Spiral Ganglion and Organ of Corti
Processing of sound in the cochlea involves both afferent and efferent innervation. The Na,K-ATPase (NKA) is essential for cells that maintain hyperpolarized membrane potentials and sodium and potassium concentration gradients. Heterogeneity of NKA subunit expression is one mechanism that tailors physiology to particular cellular demands. Therefore, to provide insight into molecular differences that distinguish the various innervation pathways in the cochlea, we performed a variety of double labeling experiments with antibodies against three of the α isoforms of the NKA (NKAα1–3) and markers identifying particular subsets of neurons or supporting cells in whole mount preparations of the organ of Corti and spiral ganglion. We found that the NKAα3 is abundantly expressed within the membranes of the spiral ganglion somata, the type I afferent terminals contacting the inner hair cells, and the medial efferent terminals contacting the outer hair cells. We also found expression of the NKAα1 in the supporting cells that neighbor the inner hair cells and express the glutamate transporter GLAST. These findings suggest that both the NKAα1 and NKAα3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-inner hair cell synapse.
Keywordshair cell afferent efferent hearing auditory nerve
Processing of sound in the cochlea involves both afferent and efferent innervation. The majority of afferent fibers, the myelinated type I fibers, make glutamatergic synapses with the inner hair cells (IHCs) and allow fast and precise transmission to the brain (Fuchs et al. 2003). Unmyelinated type II fibers make en passant synapses onto the outer hair cells (OHCs; Spoendlin 1973) and have a yet unknown role. Efferent feedback to the cochlea is provided by olivocochlear fibers originating in the brainstem. Activation of myelinated medial efferent terminals contacting OHCs suppresses cochlear function (Guinan 1996). Activation of the unmyelinated lateral efferent fibers that form axodendritic synapses onto type I afferent fibers causes either slow suppression or enhancement of afferent activity (Groff and Liberman 2003).
Mechanisms to maintain hyperpolarized membrane potentials and restore intracellular concentration gradients are essential for excitable cells and vary depending on their particular physiology (Blanco 2005). The Na,K-ATPase (NKA) is a membrane-bound protein that uses energy from the hydrolysis of adenosine triphosphate (ATP) to extrude three sodium ions for every two potassium ions taken into the cell. Neurons with high firing rates especially depend upon the NKA to sustain activity, since the NKA maintains concentration gradients and also hyperpolarizes the membrane. The functional NKA is comprised of two α subunits, which contain the residues necessary for ATP hydrolysis and ion transport and two β subunits. The diversity of α (1–4), β (1–3), and also FXYD (1–7) subunits (Geering 2005) and the highly regulated tissue-specific and developmental expression patterns are no doubt responsible for tailoring the NKA transport properties to specific cellular demands. In this study, we investigated the neuronal distribution of the NKA α subunit (NKAα) in the cochlea to determine its contribution to setting and modulating activity patterns in the various neuronal cell types.
Because inhibitors of the NKAα are known to reduce the endocochlear potential (Konishi and Mendelsohn 1970), previous research investigating the localization of the NKAα in the cochlea has focused on its expression in structures, like the stria vascularis and spiral ligament, responsible for maintaining the endocochlear potential (Wangemann 2006). Nonetheless, earlier work also reported expression of the NKAα in the neuronal elements of the mammalian cochlea using a variety of techniques, including enzyme cytochemistry (Kerr et al. 1982), in situ hybridization (Ryan and Watts 1991), and immunohistochemistry (Schulte and Adams 1989; Iwano et al. 1990; McGuirt and Schulte 1994; Schulte and Steel 1994; ten Cate et al. 1994; Nakazawa et al. 1995; Zuo et al. 1995; Erichsen et al. 1996; Peters et al. 2001; Weber et al. 2001).
Using immunofluorescence in whole-mount preparations of the organ of Corti and spiral ganglion, we performed a variety of double labeling experiments with antibodies against the NKAα and markers identifying particular subsets of neurons or supporting cells within the cochlea. Our results unambiguously identify the cell types expressing particular isoforms of the NKAα and, importantly, suggest novel roles for the NKAα in regulating neuronal activity in the cochlea.
Primary antibodies used in the study
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Peptide derived from rat neurofilament 200 sequence
Peptide derived from conserved sequence of the sodium channel
Generously provided by Dr. James Trimmer, University of California Davis
Santa Cruz Biotechnology
Peptide derived from rat NKAα2 sequence
Generously provided by Dr. Thomas Pressley, Texas Tech University
Developmental Studies Hybridoma Bank
Specificity of NKAα antibodies
The NKAα antibodies used in this study were chosen because their specificity has been well characterized previously. Specificity of the antibodies for the NKAα3 has been verified by the vendor using Western blot analysis of rat brain extracts (sc-16052) or canine skeletal muscle extracts (MA3-915). Specificity of the antibodies for the NKAα1 has been verified by the vendor using Western blot analysis of rat brain microsomes (C464.6) and by Arystarkhova and Sweadner (1996) using Western blot analysis of membrane preparations from rat kidney, brainstem, and adult and neonatal heart (α6F). Specificity of the antibodies for the NKAα2 has been verified by Pressley (1992) using Western blot analysis of rat brain microsomes (anti-HERED) and by the vendor using immunohistochemistry to show membrane localization in rat retinal cells.
Fluorescence images were acquired using an Olympus Fluoview FV1000 confocal microscope with a 60× Olympus PlanoApo oil immersion lens (N. A. 1.42) under the control of the Olympus Fluoview FV1000 version 1.6a software. z-Stacks (30 to 90 optical sections) and were collected at 0.3 to 0.5 μm. Except for subpanels D–I in Figure 7, which are micrographs of single optical sections, all images are presented as z-projections through the entire optical stack.
The NKAα3 is expressed in the spiral ganglion and the organ of Corti
To confirm the specificity of the monoclonal antibody against NKAα3, double labeling experiments with a monoclonal and polyclonal anti-NKAα3 were performed (Fig. 1C–K). High magnification micrographs show colocalized immunoreactivity for both the monoclonal (red) and polyclonal (green) anti-NKAα3 for the spiral ganglion somata (Fig. 1C–E) and fibers within the inner (Fig. 1F–H) and outer (Fig. 1I–K) hair cell region. Additionally, both antibodies show immunoreactivity restricted to the cell membranes, consistent with the transmembrane localization of the NKAα. The patterns of immunoreactivity for both the monoclonal and polyclonal anti-NKAα3 suggest that both antibodies are specifically recognizing the NKAα3 in the rat organ of Corti and spiral ganglion. To unambiguously identify the structures expressing NKAα3, we undertook a series of double labeling experiments with antibodies against NKAα3 and known markers of the specific neuronal cell types in the cochlea.
NKAα3 is present in type I but not in type II spiral ganglion somata
NKAα3 is present in type I afferent terminals contacting the IHCs
Previous work has shown that voltage-gated sodium channels in type I afferent fibers are localized at the heminode just before the first site of myelination and at subsequent unmyelinated nodes (Hossain et al. 2005). To determine if the region of greatest NKAα3 immunoreactivity corresponded to the initial unmyelinated segment of the type I afferent terminals, we double-labeled cochlear turns with a polyclonal anti-NKAα3 (red) and a monoclonal antibody known to recognize all neuronal voltage-gated sodium channels, including those expressed in the cochlea (green; K58/35; Rasband et al. 1999). Indeed, NKAα3 immunoreactivity appears greatest in the initial unmyelinated segment of the type I afferent fibers and does not overlap with the site of sodium channel enrichment in the first heminode (Fig. 3G). Although others have reported expression of the NKAα3 in the myelinated segments (Nakazawa et al. 1995) and nodes of Ranvier (Schulte and Adams 1989) of the afferent fibers, our experiments showed NKAα3 immunoreactivity that was most intense in the initial segments of the afferent fibers directly contacting the IHCs and not enriched in the subsequent unmyelinated nodes.
NKAα3 is not present in lateral olivocochlear efferents that contact the IHC afferent terminals
NKAα3 is present in medial olivocochlear efferents that contact the OHCs
NKAα3 is absent in type II afferent fibers that contact the OHCs
NKAα1 is present in supporting cells surrounding the IHCs
The aim of this study was to characterize the neuronal expression of NKAα in the organ of Corti and spiral ganglion. Compared to earlier work, our study utilized double immunofluorescent labeling and higher resolution micrographs of whole-mount preparations of the rat cochlea to unambiguously assign NKAα immunoreactivity to specific subtypes and subcellular structures of neurons. We identified NKAα3 in the somata and postsynaptic afferent terminals of type I SGNs that contact the IHCs and in the presynaptic medial efferent terminals that contact the OHCs. No NKAα3 immunoreactivity was detected in type II SGNs that contact the OHCs or in the lateral efferent terminals that contact the type I afferent terminals. Thus, NKAα3 expression appears restricted to the myelinated nerve fibers of the cochlea. Such somatic, axonal, and dendritic expression of NKAα3 has been reported in a variety of preparations (reviewed in Dobretsov and Stimers 2005). Thus, there does not seem to be a particular subcellular compartment to which NKAα3 expression is restricted in either the central or peripheral nervous system.
Our findings are consistent with earlier research using immunocytochemistry that identified the NKAα3 in the SGNs and cochlear nerve in the gerbil (Schulte and Adams 1989; McGuirt and Schulte 1994; Nakazawa et al. 1995), rat (ten Cate et al. 1994; Peters et al. 2001), and mouse (Erichsen et al. 1996). NKAα immunoreactivity was localized to the nerve terminals underlying the IHCs and OHCs in the human cochlea (Weber et al. 2001). Ultrastructural analysis attributed NKAα immunoreactivity in the terminals underlying the IHCs to the type I afferent and not lateral efferent terminals in the gerbil (Nakazawa et al. 1995). Additional work has shown that NKAα immunoreactivity in the terminals underlying the IHCs and OHCs is due to expression of the NKAα3 in both the gerbil (Schulte and Adams 1989; McGuirt and Schulte 1994) and rat (ten Cate et al. 1994).
We also tested for the presence of the other NKAα isoforms in the organ of Corti and spiral ganglion. We found NKAα1 in supporting cells surrounding the IHCs (see discussion below). We found no NKAα1 immunoreactivity in the afferent terminals. Previous studies have reported weak to moderate NKAα1 immunoreactivity in the SGNs and cochlear nerve of the rat (ten Cate et al. 1994; Peters et al. 2001; Delprat et al. 2007) and mouse (Erichsen et al. 1996), and one study reports expression of the NKAα in the afferent terminals underlying the OHCs in the gerbil (Nakazawa et al. 1995). Although we did not find NKAα1 immunoreactivity in either type I or II afferent terminals, we did see NKAα1 labeling in the spiral ganglion (data not shown) but were unable to identify unequivocally the NKAα1-expressing cells as either the SGNs or ensheathing satellite cells. In the region of the spiral ganglion, NKAα1 immunoreactivity in the gerbil cochlea (McGuirt and Schulte 1994; Schulte and Steel 1994) and NKAα immunoreactivity in the human cochlea (Weber et al. 2001) were attributed to the satellite cells. We were unable to detect NKAα2 immunoreactivity in the organ of Corti or spiral ganglion with either of two antibodies used (data not shown). While some groups have reported exclusive expression of the NKAα2 in the supralimbal fibrocytes (McGuirt and Schulte 1994; Schulte and Steel 1994; Peters et al. 2001), others have reported additional weak or transient NKAα2 immunoreactivity in the cochlear nerve (ten Cate et al. 1994; Erichsen et al. 1996).
Our results suggest that the myelinated nerve fibers of the organ of Corti express only NKAα3. Although NKAα1 is often considered to be ubiquitously expressed, the exclusive expression of NKAα3 has been observed in a variety of neurons (McGrail et al. 1991) including those that maintain tonic and/or rapid rates of firing, such as the afferent and efferent nerve fibers innervating the muscle stretch receptor (Dobretsov et al. 2003), neurons of the rat medial geniculate nucleus in auditory thalamus (Senatorov et al. 1997), as well as the calyx of Held afferent terminals in the medial nucleus of the trapezoid body in auditory brainstem (Kim et al. 2007). The selective expression of NKAα3 provides unique insight into the physiological demands on the myelinated fibers in the cochlea. The NKAα3 is distinguished from the NKAα1 by its relatively low affinity for intracellular sodium (Jewell and Lingrel 1991; Munzer et al. 1994; Zahler et al. 1997; Crambert et al. 2000), high affinity for ATP (Jewell and Lingrel 1991), and lack of inhibition at hyperpolarized potentials (Balshaw et al. 2000; Crambert et al. 2000). Thus, NKAα3 may be especially suited for the myelinated fiber types of the cochlea that sustain high rates of activity that would be expected to raise intracellular sodium concentrations, deplete ATP, and maintain hyperpolarized membrane potentials. In fact, the NKAα1 isoform, which has a much steeper voltage dependence of its transport rate compared to the NKAα3 isoform, may respond too quickly to action potential depolarization and prevent sustained firing (Dobretsov and Stimers 2005).
In testing for other NKAα isoforms in the organ of Corti, we found NKAα1 immunoreactivity in the glial-like supporting cells surrounding the IHCs, including the inner phalangeal cells. Previous ultrastructural work reported NKAα1 immunoreactivity of the inner phalangeal and border cells in rat (Iwano et al. 1990). These supporting cells express the glutamate transporter GLAST and are thought to remove glutamate from the extracellular space that has been released from the IHC (Furness and Lehre 1997; Hakuba et al. 2000; Furness and Lawton 2003; Glowatzki et al. 2006). The expression of NKAα1 in these supporting cells is not unexpected: glutamate transporters rely on sodium gradients to transport glutamate (Danbolt 2001). The NKAα inhibitors ouabain and vanadate have been shown to block the clearance of extracellular glutamate in cultured rat cortical astrocytes, suggesting that glutamate transporters are dependent on NKAα activity (Abe and Saito 2000). Our experiments suggest that GLAST-mediated glutamate uptake by the supporting cells adjacent to IHCs may be dependent upon the activity of NKAα1.
Our knowledge about the functional involvement of the NKAα in hearing has come from experiments involving inhibitors of the NKAα and also from experiments using mice with genetic deletions of the NKAα. Perilymphatic perfusion of ouabain, an inhibitor of the NKAα, causes a rapid reduction of the endocochlear potential (Konishi and Mendelsohn 1970; Kuijpers and Bonting 1970; Kusakari et al. 1978), and researchers have identified NKAα1 in the strial marginal cells and NKAα2 in the lateral wall fibrocytes (McGuirt and Schulte 1994; Schulte and Steel 1994; reviewed in Wangemann 2006). The contribution of the NKAα1 and NKAα2 in maintaining the endocochlear potential is supported by the characterization of mice carrying deletions of these subunits. Although homozygous deletion of either NKAα1 or NKAα2 is lethal at birth, heterozygous deletions are viable (Moseley et al. 2007). Despite normal cochlear morphology, NKAα1+/− and NKAα2+/− mice show reductions in the endocochlear potential and progressive, age-dependent hearing loss (Diaz et al. 2007). A possible neuronal contribution to hearing loss in these mice has not been excluded.
The contribution of the NKAα3 to hearing is less obvious. Homozygous deletion of NKAα3 is lethal; however, heterozygous deletion is viable (Moseley et al. 2007). The hearing of NKAα3+/− mice has not been reported. More recent studies investigating the perfusion of ouabain directly through the round window of the gerbil cochlea provide evidence for an additional neuronal role of the NKAα in the cochlea. Specifically, these experiments show threshold increases or complete abolishment of the compound action potential without a reduction of the endocochlear potential (Schmiedt et al. 2002; Wang et al. 2006). Moreover, chronic exposure to ouabain is associated with degeneration of type I but not type II SGNs in the gerbil cochlea (Lang et al. 2005). These findings are consistent with our observation of the NKAα3 in type I but not type II spiral ganglion afferent terminals. Especially in rodents, the NKAα3 isoform is much more sensitive to inhibition by ouabain than the NKAα1 isoform and slightly more sensitive than the NKAα2 isoform (O’Brien et al. 1994). Thus, specific doses of ouabain could be expected to inhibit the NKAα3 isoform and reduce or abolish the compound action potential without necessarily affecting the NKAα1-and NKAα2-dependent endocochlear potential.
The expression pattern of the NKAα in the organ of Corti and spiral ganglion suggests that both the NKAα1 and NKAα3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-IHC synapse. Thus, in addition to maintaining the endocochlear potential, the NKAα may contribute directly to the functioning of these synapses in the inner ear. Future investigation of the particular β (1–3) and FXYD (1–7) subunits coexpressed with the NKAα1 and NKAα3 will identify the additional molecular players involved in tailoring the NKAα transport properties at each of these synapses. Ultimately, clarifying the molecular components and neuronal contribution of the NKA in the cochlea will suggest ways in which its activity may be altered by exogenous inhibitors as a possible means to treat hearing loss (Aperia 2007) and also, very excitingly, by naturally occurring, endogenously expressed inhibitors (Nesher et al. 2007; Schoner and Scheiner-Bobis 2007) that may contribute to both normal and pathological hearing states.
We would like to thank Mr. D. Mark Gay and Dr. Richard Dillaman at the University of North Carolina at Wilmington for excellent assistance with confocal microscopy, Dr. Paul A. Fuchs at the Johns Hopkins University for scientific advice and comments on the manuscript, Dr. Richard W. Aldrich at the University of Texas at Austin for supporting the preliminary experiments performed in his laboratory then at Stanford University, and Dr. Kathleen J. Sweadner at the Massachusetts General Hospital and the Harvard Medical School for advice and recommendations regarding antibodies against the Na,K-ATPase. The α6F monoclonal antibody developed by Dr. Douglas Fambrough was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The monoclonal antibody anti-HERED was generously provided by Dr. Thomas Pressley at the Texas Tech University. The monoclonal antibody K58/23 was generously provided by Dr. James Trimmer at the University of California at Davis. This work was supported by National Institute of Deafness and Communication Disorders grants (DC00276 to Dr. Paul Fuchs and DC006476 to E. G.), a research award from the National Organization for Hearing Research (to S. J. P), a new investigator research award from the American Academy of Audiology (to S. J. P.) and a research grant from the Deafness Research Foundation (to S. J. P.).
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