Where is the spike generator of the cochlear nerve? Voltage-gated sodium channels in the mouse cochlea

Abstract

The origin of the action potential in the cochlea has been a long-standing puzzle. Because voltage-dependent Na+ (Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the type I and type II ganglion cells. In most type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.

Figure 1.

The auditory pathway. A, Low-magnification photomicrograph showing the relationships of the organ of Corti (OC), spiral lamina (SL), cochlear ganglion (CG), cochlear nerve (CN), and cochlear nucleus (COCH NUC) at the level of the basal coil of the cochlea. The arrowhead indicates the Schwann cell- oligodendroglial border. SPT, Spinal trigeminal nucleus. B, Scheme of cochlear innervation. Afferent fibers from myelinated type I ganglion cells innervate individual inner hair cells (IHC) in a single row in OC. Afferents from unmyelinated type II ganglion cells cross the tunnel of Corti (T) to innervate groups of outer hair cells (OHC), which are arranged in three rows. Both types of ganglion cells project to the cochlear nucleus. The efferent fibers arise in the brainstem and project to hair cells or their afferent endings in a complicated pattern, which is simplified for clarity in the diagram. All fibers enter and leave the OC through the FN. C, Horizontal section showing three rows of OHCs, the phalangeal processes (PP) covering the tunnel, and one row of IHCs. D, In a plane below the IHCs, Nav1.6 is in the afferent endings (arrowhead) beneath the unlabeled IHC bases. Also stained are the afferent radial fibers (arrow) leading through the FN to their first heminodes (^*), which are intensely labeled. On the outer side of the tunnel, at the bottom of the field, in a plane beneath the OHCs, the afferent fibers of the outer spiral bundle are intensely stained as they run longitudinally. Inset, In another plane of focus, there is intense labeling of afferent IHC endings (arrowhead) and the first heminodes (^*). Scale bars: A, 0.2 mm; C, 25 μm; D (for D, inset), 10 μm.

Figure 2.

Pan Nav, Nav1.6, and Caspr in cochlear ganglion (CG) cells and fibers. A, Double immunostaining with Pan Nav (red) and Caspr (green) antibodies in a horizontal section showing sites from B-E. Scale bar, 100 μm. B, The first heminodes at the foramina nervosa stain for Pan Nav; the paranodes stain for Caspr. C, In the spiral lamina, Pan Nav at the nodes of Ranvier and Caspr at the paranodes are consistently labeled. D, E, In the CG, the nodes of Ranvier are consistently labeled for Pan Nav, including those (arrows) that flank type I ganglion cell bodies, whereas the loose myelin around the cell bodies (^*) and paranodes are stained for Caspr (arrowheads, bracket). In E, a type I ganglion cell body out of the plane of focus is outlined to show the relationship of the central (right arrow) and peripheral (left arrow) nodes, which are stained for Pan Nav. Scale bars: B-E, 10 μm. F, Cross section from the basal turn showing the bony spiral lamina (SL), CG, and central root of the cochlear nerve (arrowhead). Scale bar, 50 μm. G, Polygonal field indicated in F, showing the tunnel (T), outer hair cells (OHC), and SL, labeled for NF-M and Nav1.6. The first heminode (box in SL) is heavily labeled for Nav1.6 and less visibly for NF-M, whereas the NF-M-labeled myelinated nerve fibers runout of the field centrally toward the CG. Nodes of Ranvier are positive for Nav1.6 (arrowhead). Insets (two focal planes), heminodes (^*) and nodes of Ranvier (arrowhead) are labeled for Nav1.6 (red). Caspr (green) is labeled at the paranode of the heminodes and flanking nodes (arrow). Scale bar, 10 μm. H, In a higher-power field (from F) of the CG and nerve root, Nav1.6 staining is present in the axons at nodes (arrow head), bordered at the paranodes by Caspr. Nonspecific immunoreactivity (red) appears in the bony capsule around CG; this is commonly seen in cochlear immunohistology. Scale bar, 25 μm. I, Example of a type I GC from the preceding panel. The cell body (^*) and central paranode (arrowhead) are outlined by immunostained Caspr up to the first node of Ranvier (arrow), which is stained for Nav1.6. J, Both central and peripheral initial segments of a type II cell are immunostained for Nav1.6 in heavy clusters (arrows), which increase progressively away from the cell body and stop sharply at a distal point. K, L, Higher magnification of type I CG cells, surrounded by a Caspr-stained lacework, presumably associated with myelin or loose myelin. M, N, Initial axon segments of putative type III CG cells (arrows). In both the central and peripheral initial segments, Nav1.6 colocalizes with neurofilament-M (M). Scale bars: I-N, 10 μm.

Figure 3.

Nav1.6 tracks type II afferent innervation of outer hair cells (OHCs). A′, In a horizontal plane, just below the OHCs, the afferent endings are labeled (arrows) in row 1. Also stained are preterminal portions of outer spiral fibers. A″, The same location in a deeper plane shows an immunopositive afferent curving down to enter the outer spiral bundle (arrows). A‴, Still deeper beneath the OHCs are four afferent fibers in the outer spiral bundle (arrow). B, In a plane below the preceding (section from Fig. 1 D), the afferent outer spiral fibers are labeled along their entire lengths beneath the three rows of OHCs (1, 2, 3). C, Immunopositive outer spiral fiber (arrows) descends and crosses the tunnel floor (T). DC, Deiters cells; OP, outer pillar cell body; IP, inner pillar cell body. Scale bars, 10 μm.

Figure 4.

The cochlear efferent innervation uses Nav1.2 but not Nav1.6. A, An efferent fiber (arrowhead) and its ending (^*) are labeled for NF-M beneath an IHC. Other efferent fibers cross the tunnel (T): one ends beneath an outer hair cell (OHC; arrow), in which it overlies an afferent ending labeled for Nav1.6 (yellow). Afferent fiber heminodes in the spiral lamina (SL) and afferent type II fibers beneath OHCs (two arrows) are colabeled with Nav1.6 and NF-M. B, Some of the inner spiral bundle efferent endings beneath the inner hair cells (IHC; ^*), the tunnel crossing efferents (arrowhead), and their endings (arrow) beneath row 1 of the OHCs are labeled for Nav1.2. Outer spiral fiber type II afferents (two arrows) are labeled for Nav1.6 beneath the OHCs and at the heminodes in the SL. Scale bars, 10 μm.

Figure 5.

Nav1.6 localization is disrupted in quivering mice. A, In the mutant, the afferent and efferent fibers innervating inner hair cells (IHC) and outer hair cells (OHC) are present and labeled for NF-M. T, Tunnel. B, Nav1.6 occurs at the first heminodes near the FN but not in the afferent fibers innervating hair cells. C, In a mutant, a type I cochlear ganglion (CG) cell is labeled for NF-M (arrows). A small cluster of Nav1.6 can be detected on the central axon (arrowhead), presumably at the first flanking node of Ranvier. D, In a mutant, a type I CG cell (^*) initial segment is bordered by Caspr staining at the first flanking node of Ranvier on the peripheral axon (arrowhead). E, In the spiral lamina (SL) of a wild-type mouse, the peripheral processes are stained for Nav1.6 at nodes of Ranvier (arrowhead). F, In the corresponding locations from a mutant, Nav1.6 localization is disrupted, so that some nodes appear to be larger (arrowheads) compared with wild type. Scale bars, 10 μm.

Figure 6.

Modeling AP initiation and propagation in type II ganglion cells. A, The morphological model has six regions: (1) central axon (axon_C); (2) ISC; (3) soma; (4) ISP; (5) peripheral axon (axon_P); and (6) recepto-neural segment with six endings on hair cells (syn.). The gray horizontal line marks the boundary between axon_P and the recepto-neural segment, just proximal to the organ of Corti and the FN. The red dot marks the recording site in the recepto-neural segment (rec_neu.). Inset, Perisomatic region showing ISC and ISP (red). B, Under standard conditions (see Materials and Methods), a synaptically evoked AP in the recepto-neural region (solid red line) fails to invade the soma (solid black line). In the next sweep (dashed lines), sodium channels are removed from the entire neuron, and the same synaptic stimulation is repeated. Inset, The same sweep (dashed lines) shown on a slower time scale. C, Same as in B, except the entire length of the recepto-neural segment, from FN to the distal tip, is a hot spot, i.e., loaded with a uniform density of the Hodgkin-Huxley sodium conductance (g_{nabar_hh} = 1200 pS/μm²) on top of the global background sodium channel density (g_{nabar_hh} = 120 pS/μm²). The AP fails to invade the soma. C-F, Insets, A red sleeve indicates the hot spots in the corresponding experiments. D, E, When a second hot spot is inserted in the ISC (D) or ISP (E), an AP generated in the recepto-neural segment (red trace) fails to invade the soma (black trace). The horizontal line π marks the amplitude of the peak somatic depolarization. F, With all three of the hot spots loaded (g_{nabar_hh} = 1200 pS/μm²), the AP successfully invades the soma (black trace).

Figure 7.

Parameter range (sensitive range) for AP propagation. A, The “recording” from the recepto-neural segment (rec_neu is red dot in Fig. 6 A, 407 μm from soma) is superimposed on the somatic recordings (soma). In the first sweep (heavy line), when g_{nabar_hh} in each initial segment (ISP and ISC) is set to 928 pS/μm², an AP fails to invade the soma. In the second sweep (thin line), adding 1 pS/μm² to each initial segment allows an AP to invade the soma, making 929 pS/μm² the lower limit of the sensitive range. B, At higher densities (g_{nabar_hh} = 1200 pS/μm²) distributed equally in both initial segments, AP amplitude and peak latency of the somatic spike improve (solid lines). A sign of the failing axonal AP appears as a hump on the rising phase of the spike. The second sweep (dashed line) is an example in which channels are inserted in ISC alone at the minimal density (g_{nabar_hh} = 1853 pS/μm²) that permits invasion of the soma (upper limit of sensitive range). C, Simulations with four different channel mechanisms (nach, naf, naxn, and nahh) show similar sensitive ranges. Bars mark the normalized permissive channel density in the ISP (dark gray) or ISC (light gray) alone. Gray values are normalized with respect to Na⁺ conductance, distributed equally between both initial segments (black bars). Absolute densities (in picosiemens per square micrometer) are in Table 2. D, With g_{nabar_hh} at 929 pS/μm² in both initial segments (ISP, ISC), an AP barely invades the central axon. Multisite recordings reveal the transients in different neuronal compartments. Black triangles mark the recording sites on a schematic of a type II ganglion cell. Numbers are micrometers from cell body. Q marks the peak of the failing AP (hump potential). R marks the peak of the reinitiated (reflected) AP. E, Same as D, with g_{nabar_hh} at 1200 pS/μm². Despite high channel density, a hump potential (arrow) persists in the region of low safety margin for AP propagation in the proximal part of the peripheral axon.

Figure 8.

Voltage-gated sodium channels in cochlear nerve (CN) cells. Type I cochlear ganglion cells (CG, I) provide rapid transfer of discrete auditory signals from individual inner hair cells (IHC) for precise spatiotemporal processing in the cochlear nucleus (COCH NUC) by the shortest route. To support this divergent processing at a high level of temporal precision, the afferent fibers from IHCs have Nav1.6 channels available for voltage generators on their terminals and axons in the organ of Corti, at the first heminodes just central to the FN, at subsequent nodes of Ranvier and nodes flanking the cell bodies (I). Type II ganglion cells (CG, II) provide slower processing for many outer hair cells (OHC) spread out over a longer distance. To support this convergent mode of processing in a temporally secure relationship to IHC activity, the afferent fibers from the OHC have Nav1.6 channels over their entire course in the organ of Corti. Both initial segments at the CGII cell body express Nav1.6, but it is uncertain what specific sodium channels occur at low density on the rest of the axon. The efferents use Nav1.2 in the organ of Corti. Caspr is expressed at the paranodes of type I fibers and in the myelinated layers around the CGI cell bodies. SL, Spiral lamina; T, tunnel.