In the organ of Corti, the afferent axons of Type I and Type II ganglion cells, as well as efferent fibers, are distinguished by their relative diameters, their positions in the tunnel of Corti, their branching patterns, as well as by their innervation targets (; Berglund and Ryugo, 1987
; Romand and Romand, 1987
; Brown and Ledwith, 1990
). In the osseous cochlea, likewise, there are a number of structural features which distinguish these neurons and their processes, e.g., myelination and relative diameters. Thus each class of axon forms an anatomically distinct pathway which can be reliably identified in histological preparations without recourse to markers and double staining.
To begin the search for candidate spike generators, we surveyed the entire cochlea by immunostaining with an antibody which recognizes all known neuronal voltage-gated Na+ channels (Pan Nav; ). High densities of Nav channels were detected at several locations throughout the pathways of the cochlear neurons. For example, in Type I ganglion cells, Nav channels were clustered at the hemi-nodes adjacent to the foramina nervosa (: red) and at all subsequent nodes (: red); these Nav channel clusters were flanked by immunostaining for Caspr (: green), a cell adhesion molecule that is part of the paranodal axoglial junction. The myelinated cell bodies of Type I ganglion cells were surrounded by Caspr immunostaining, but no Nav channel immunoreactivity was seen on the soma. In addition to the patterns described above, Pan Nav immunostaining was also present in the unmyelinated axons of the cochlea (data not shown), including the thinnest processes in the spiral lamina, consistent with Type II ganglion cell afferents. However, Pan Nav staining intensity was sometimes weaker than that seen with the other Nav channel antibodies, since this antibody was very sensitive to the degree of fixation needed to preserve the delicate structures of the cochlea. Therefore, in the remainder of this study we used the subtype specific anti-Nav1.2 and anti-Nav1.6 antibodies, whose epitopes are much less sensitive to fixation, in order to define spike generators in the cochlea.
Figure 2. Pan Nav, Nav1.6, and Caspr in CG cells and fibers. Panel A. Double immunostaining with Pan Nav (Red) and Caspr (Green) antibodies in a horizontal section showing sites of Panels B-E. Scale = 100 μm. Panel B. The first hemi-nodes at the foramina (more ...)
Nav channel clusters in the axons and endings from Type I cochlear ganglion cells.
The bulk of the neural activity coding for sound is produced by the inner hair cells, and this is transmitted at high firing rates by Type I ganglion cells. Therefore, we first examined the spike generator of the Type I ganglion cell in the afferent axons innervating the bases of the inner hair cell bodies (). The first hypothesis we considered was that the location would be in the afferent ending itself or the most peripheral portion of the axon, i.e., the recepto-neural segment.
Within the organ of Corti, all axons are unmyelinated. As seen in horizontal sections parallel to the organ of Corti, the afferent fibers from the Type I ganglion cell innervate the single row of inner hair cells (: IHC), while the distal, preterminal portions of the axons (the recepto-neural segments) penetrate the foramina nervosa (FN), where the first hemi-nodes are located. Nav1.6 was localized in the endings ( and Inset: Arrowhead) and in the short recepto-neural segments of the thin radial afferent axons just beneath each inner hair cell (: Arrow).
At the first hemi-nodes, robust staining for Nav1.6 was detected just within the foramina nervosa of the spiral lamina (Figs. & Inset: *; : * in boxed region). Hemi-nodes were identified by using anti-Caspr, an axonal marker of the paranodal axoglial junction, where myelination begins. Caspr appeared between the Nav1.6 cluster and the beginning of the myelinated fiber layer of the spiral lamina ( Inset: Arrow). Nav1.6 was present in high densities at nodes of Ranvier within the cochlear nerve, in regions both central and peripheral to the ganglion cell bodies (). A similar pattern of nodal staining for Nav1.6 continued in the central nerve root past the Schwann cell-glial junction (not shown). Axons in the spiral lamina also showed the punctuated distribution of Nav1.6 at nodes (: Arrowhead).
In contrast to Nav1.6, immunostaining for Nav1.2 was not detected in the radial afferents, in their endings, or in their axons in the spiral lamina, cochlear ganglion, or cochlear nerve (data not shown). Nav1.2 immunostaining of Type I ganglion cell bodies was not detected. Thus, Nav1.6 appears to be the predominant channel found in processes of Type I ganglion cells. Its distribution is consistent with the location of spike generation in the recepto-neural segment and first hemi-node.
How does the action potential rapidly and reliably traverse the ganglion cell body?
The cochlear ganglion cell is a bipolar neuron, which consists of a large perikaryon with peripheral and central axons. This configuration presents a challenge to the rapid, efficient, and reliable propagation of APs, because the large cell body may act as a current sink due to the dramatic decrease in impedance. This would require a greater ionic current to sustain membrane depolarization and AP propagation. In the case of the Type I ganglion cell, one feature that could compensate for this is the partial myelination of the cell body, including so-called “loose” myelin and sites of close apposition between the neuronal membrane and the loose myelin, first described in the rat by Rosenbluth (1962
) and in the mouse by Romand and Romand (1987
). A more eclectic mechanism, applicable to both Type I and Type II ganglion cells, would be the strategic location of Nav channels at or near the cell body to boost the current density.
When the ganglion cells were immunostained for either Pan Nav or Nav1.6, there was little or no labeling of the neuronal cell bodies (). The peripheral axon extending from the cell body tapers for a short distance before reaching the first node of Ranvier, while the corresponding part of the central process maintains the same diameter. In most cases, we found Nav1.6 at nodes of Ranvier flanking the Type I ganglion cell bodies (). However, there were also rare examples of ganglion cells with Nav1.6 clustered in high densities on the initial segments of the ganglionic axons (: Arrows). In these cases, both the peripheral and central initial segments were well stained, usually for a distance of 10-15 μm (). Since they do not conform to strict criteria for either Type I or Type II ganglion cells (described below), we suggest that these other cells represent varieties of Type I ganglion cells or Type III ganglion cells, as previously described (e.g., Romand and Romand,1987
In the case of the Type I ganglion cell, Caspr was present in a complex arrangement of tangled, web-like processes, which swirled around the surfaces of the ganglion cell bodies in patterns reminiscent of the fine structure of loose myelin, first described in the rat by Rosenbluth (1962
) and in the mouse by Romand and Romand (1987
) (). In contrast, Nav1.2 was not detected at any of the above sites (data not shown).
Navl.6 channel clusters in Type II ganglion cells.
Each Type II ganglion cell typically innervates many outer hair cells spread over a relatively long distance, up to half a cochlear turn (). Their axons are unmyelinated, including the outer spiral fibers, which cross the floor of the tunnel and course in the direction of the basal turn beneath the outer hair cells. Centrally their axons are very thin, often less than 0.5 μm in diameter, and most likely have low firing rates, which could support a modulatory role in central auditory processing. We considered the possibility that Nav subtype expression in the more slowly conducting, unmyelinated Type II ganglion cells might differ from that of the myelinated Type I cells. Furthermore, without loose myelin surrounding the cell bodies of Type II ganglion cells the localization of Nav channels might be different than in the more abundant and myelinated Type I ganglion cells.
In fact, the outer spiral fibers from the Type II ganglion cells were well stained for Nav1.6 beneath the outer hair cells and along their spiral course, where they could be traced through different focal planes (; see supplemental videos V1 and V2 on-line). There was intense labeling of the endings at the bases of the outer hair cells (: Arrows). This staining was continuous throughout the recepto-neural segments of the axons as they joined the outer spiral bundle (: Arrow) and crossed the tunnel floor (: Arrows). The tunnel-crossing fibers from the outer spiral bundle were labeled with Nav1.6, but the axonal diameters (0.5 μm) were much reduced there, compared to the axons beneath the outer hair cells (0.8 - 1.0 μm). In contrast, immunostaining for Nav1.2 was never seen in the Type II afferents, neither on the floor of the tunnel nor in the outer spiral bundle axons or their endings.
Figure 3. Nav1.6 tracks Type II afferent innervation of outer hair cells (OHCs). Panel 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. Panel (more ...)
Type II ganglion cell bodies are not always distinguishable from Type I on the basis of light microscopic morphology. However, by using criteria described for the mouse (Berglund and Ryugo, 1987
), we could identify a number of these cells in the peripheral (lateral) region of the ganglion. Briefly, these cells were distinguished from Type I by an unmyelinated body that was often eccentric with respect to the central and peripheral processes ().Within the cochlear ganglion, Nav1.6 labeling of Type II ganglion cells was restricted to their peripheral and central initial segments (: Arrows). These processes were equal in diameter and maintained the same thickness for a distance of 20 μm from their origins. Usually the central process decreased greatly in diameter after a distance of 20-30 μm. The criteria for this identification were verified in sections stained with an antibody to peripherin, which stained the Type II cell bodies and the outer spiral fibers but not Type I afferents (data not shown). Antibodies against neurofilament-M (Figs. , ) and Kv1.2 (not shown) stained both types of axons throughout the cochlea.
Figure 4. The cochlear efferent innervation uses Nav1.2 but not Nav1.6. Panel 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 OHC (Arrow), where it overlies (more ...)
Sodium channel localization in efferent fibers in the organ of Corti.
The efferent innervation of the organ of Corti is supplied by the olivo-cochlear fibers, which originate in the brain stem (: Efferents). These are not sensory fibers, but they function as a feed-back pathway. Since their initial segments are in the brain, we did not expect to find evidence for a spike generator in the cochlea. The efferent fibers can be distinguished from the afferents at several points in their pathway. The efferents enter the cochlea via the vestibular nerve and take a tangential course in the intraganglionic spiral bundle, whereas the afferents course radially (except in the apical end) into the ganglion and enter the cochlear nerve. In the organ of Corti, many, if not all, of the efferents which supply the inner hair cell region, unlike the afferent fibers, course tangentially and synapse on the afferent fibers beneath the bases of the hair cell bodies. The efferent axons and their endings beneath inner hair cells were prominently stained for neurofilament-M (: Arrowhead, *).
The efferent endings, in contrast to the recepto-neural segments of ganglion cell axons, were not stained for Nav1.6, but were labeled for Nav1.2 (: *). The efferents, in their definitive pathway cross the middle of the tunnel, and their endings, beneath the outer hair cells, contained neurofilament-M (: Arrow) and were clearly labeled for Nav1.2 (: Arrowhead, Arrow) but not for Nav1.6. In the same sections, the Type II afferents, which take a path distinct from the efferents by running on the floor of the tunnel and in the outer spiral bundle, were well stained for Nav1.6 but not Nav1.2 (Double arrows).
Nav channels in a deaf mutant, the quivering mouse.
The structure and molecular composition of nodes of Ranvier and axon initial segments are thought to be stabilized and maintained by ankyrin G in association with beta-IV spectrin (Berghs et al, 2000
; Komada and Soriano, 2002
; Yang et al., 2004
; Lacas-Gervais et al., 2004
). Quivering mice have mutations in beta-IV spectrin and have aberrant clustering of Nav1.6 channels at nodes in the optic nerve (Yang et al., 2004
) as well as hearing deficits (Parkinson et al., 2001
). Previous experiments by us showed that overall levels of Nav channels in the brains of a specific allele of the quivering mouse, the qv3J
mutant, were normal, and that Nav1.2 does not replace Nav1.6 at the disrupted nodes of Ranvier (Yang et al., 2004
); unpublished results). Therefore, we considered the possibility that defective clustering of Nav1.6 channels in the cochlea could contribute to hearing loss in the mutants.
While the light microscopic structure of the cochlea was generally unremarkable, the immunostaining for Nav1.6 revealed major abnormalities. There was little or no staining for Nav1.6 in the mutant cochlear epithelium, in the radial fibers and their endings beneath the inner hair cells, or in the outer spiral fibers and their endings beneath the outer hair cells, although these fibers were stained for neurofilament-M (; compare ). However, there was modest labeling for Nav1.6 of the first hemi-nodes within the spiral lamina (: FN). In the spiral lamina and ganglion, many of the nodes were labeled for Nav1.6, but they were often elongated and disrupted () compared to the wildtype (). Some nodes were unstained for Nav1.6, even though Caspr was clearly labeled (not shown). The abnormal staining for Nav1.6 applies to the myelinated fibers of Type I ganglion cells. We did not attempt to follow the myelinated fibers of the efferents in the intraganglionic spiral bundle. This result contrasts with the sciatic nerve, where the nodes of Ranvier appear to be normal in these mice (Yang et al., 2004
). In the cochlear ganglion, detected clusters of Nav1.6 some distance from the cell body (: Arrows, Arrowheads), presumably at the location of the ganglionic hemi-node, where Caspr is also localized (: Arrowheads). We were unable to identify any initial segments labeled for Nav1.6, although this may reflect the sparse population of Type II ganglion cells. Thus, Nav1.6 was not found in regions without adjacent myelin (i.e., initial segments and the recepto-neural segments), suggesting that Nav channels are no longer stabilized in the qv3J
mice and that the serious disruption in Nav1.6 localization may account for their hearing deficit.
Figure 5. Nav1.6 localization is disrupted in quivering mice. Panel A. In the mutant, the afferent and efferent fibers innervating IHCs and OHCs are present and labeled for NF-M. Panel B. Nav1.6 occurs at the first hemi-nodes near the foramina nervosa (FN), but (more ...)
A model for the generation of APs in the Type II ganglion cell.
We chose to model the Type II ganglion cell in the present study, rather than Type I, because the former, given its greater length and lack of myelination, should present the limiting case for successful AP propagation and thus provide a better opportunity to discover the potential role of sodium channels in these sensory neurons. We defined the outer spiral fiber of the Type II neuron as an axon, consistent with the available light microscopic observations, including length, shape, and branching pattern, as well as immunostaining for neurofilament-M, peripherin, Kv1.2, and Nav channels. This definition is also supported by electron microscopy (e.g., Ginzberg and Morest, 1984
, and many others). To elucidate the possible functional importance of Nav channels in the Type II ganglion cell peripheral afferent axons, we considered the potential effects of adding channels to an otherwise weakly excitable membrane (see Methods).
In , we illustrate the morphology of the model neuron and the location we used for the synaptic input (syn.). In the absence of sodium channels (passive membrane), synaptic potentials of ~40 mV amplitude at the input site caused small (~6 mV) depolarizations of the soma (: Dashed lines and Inset). This result was obtained with an electrotonically compact model neuron, characterized by low Ri (70 Ohm-cm) and high Rm (50 kOhm-cm2
). In the neurocomputational literature, a range for Ri of 150-250 Ohm-cm has been assumed (Anderson et al. 1999
; Archie & Mel, 2000
; Durstewitz et al. 2000
; Rhodes & Llinas, 2001
; Vetter et al. 2001
). As expected from the cable equation, larger values for Ri and smaller values for Rm produced more pronounced attenuation of the hair cell synaptic potential (not shown). In all subsequent modeling experiments, AP propagation was studied under the condition of low Ri and high Rm. This was done to impose a maximum constraint on the significance of actively generated currents for signal propagation.
Figure 6. Modeling action potential (AP) initiation and propagation in Type II ganglion cells. Panel A. The morphological model has 6 regions: (1) central axon (axon_C); (2) central initial segment (ISC); (3) soma; (4) peripheral initial segment (ISP); (5) peripheral (more ...)
The entire neuron, including the cell body, was endowed with a uniform density of sodium and potassium channels (background level of excitability), as specified in the Methods. The densities of the background voltage-gated sodium and potassium conductances were set in such a way that each neuronal compartment triggered a swift AP, whose amplitude overshot zero by at least 20 mV (: Solid red line). However, despite the large amplitude (97.6 mV, measured from base line) and rather long duration (half-width of 1.42 ms), the synaptically evoked AP failed to propagate from the recepto-neural segment (rec_neu) into the soma (: Solid black line). Since the densities of active membrane conductances were uniform throughout the entire neuron, the AP propagation failure was attributed to the sudden increase in diameter at the axon-soma junction point.
It has long been known that a geometrical incongruity can impose a substantial obstacle for propagation of regenerative potentials (Goldstein and Rall 1974
, Parnas et al., 1976
). A traveling AP could perhaps overcome the impedance mismatch between the axon and the soma, if the axon-soma transition were enriched with sodium conductance (Luscher and Larkum, 1998
). However, introducing a ten-fold higher density of sodium channels (hot spot) in the recepto-neural segment (), or in either of the axon initial segments (), was not sufficient to support AP propagation. Only when both central and peripheral axon initial segments (ISC and ISP) were rendered active, would the synaptically evoked APs reliably propagate from the recepto-neural segment into the soma (). Adding or removing sodium channels from the recepto-neural segment did not have a significant effect on the ability of APs to propagate through the axon-soma junction, as long as both axon initial segments were loaded with sodium conductance (not shown). This is not to say that sodium channels in the recepto-neural segment have no role in the processing of the outer hair cell inputs.
The present modeling study revealed two important functions of the recepto-neural hot spot. First, in the absence of this hot spot, the level of synaptic input (gmax) required to generate a nerve impulse in the recepto-neural segment was significantly higher (Suppl. Fig. S1). Second, insertion of sodium channels in the recepto-neural segment dramatically reduced the AP peak latency, measured as a time interval between the onset of the synaptic potential and the peak of the somatic AP (Suppl. Figs. S2A, B). The effect on the somatic AP peak latency was entirely the result of the AP initiation process in the recepto-neural segment, and it was not due to a change in the AP velocity. In fact, the conduction velocity of the evoked AP was almost insensitive to the density of sodium channels in the recepto-neural segment (Suppl. Fig. S2C).
The modeling experiments presented so far establish that, in some cases, a single AP failed to propagate from the initiation site in the outer hair cell layer to the ganglion cell body (), unless both peripheral and central axon initial segments were adequately endowed with sodium channels (). We next examined the range of gNa (sensitive range), in which our model exhibits this particular feature, i.e., a strong dependence on both axonal hot spots. Such a range would serve as a measure of the robustness of the model, the reliability of the results, and the validity of the conclusions laid out in Figures and .
Figure 8. Voltage-gated sodium channels in cochlear nerve cells. Type I cochlear ganglion cells (CG, I) provide rapid transfer of discrete auditory signals from individual inner hair cells (IHC) for precise spatio-temporal processing in the cochlear nucleus (COCH (more ...)
The exploration of the sensitive range was performed with each one of the four different channel mechanisms (gnach
), previously published by Wang et al. (1998
), Traub et al. (2003
), Migliore et al. (2004
), and Hodgkin and Huxley (1952
), respectively. The differences between these channel mechanisms are described in Supplemental Figure S3. The passive parameters, background excitability, and synaptic stimulation were as described for the standard conditions (Methods). In the example shown in , additional Hodgkin-Huxley sodium channels (gnabar_hh
) were inserted in the recepto-neural segment and in both initial segments (ISP and ISC) on top of the sodium channel background level. In this series of experiments, the recepto-neural gnabar_hh
was fixed at 1200 pS/μm2
, while the gnabar_hh
in both initial segments was varied to determine the minimum density of hh sodium channels that would permit propagation of single APs. Membrane potential transients were “recorded” from multiple sites along the recepto-neural segment (rec_neu), peripheral axon (axon_P), soma, and central axon.
Figure 7. Parameter range (sensitive range) for AP propagation. Panel A. The “recording” from the recepto-neural segment (rec_neu = red dot in , 407 μm from soma) is superimposed on the somatic recordings (soma). In the first sweep (more ...)
Using this model, we gradually reduced the gNa in both initial segments concurrently, until the synaptically evoked AP failed to invade the soma (: Heavy line, 928). Adding just 1 pS/μm2 to each initial segment allowed an AP to invade the soma (929). The lower boundary of the sensitive range, therefore, may be defined as the minimum density of sodium conductance (929 pS/μm2), distributed equally in both initial segments, which still supports the propagation of the spike (: Thin line; : Black bars). On the other hand, the upper limit of the sensitive range is the minimum value of gNa, which, when applied to one initial segment alone, would effectively support AP propagation through the axon-soma transition (: Dashed line; : Gray bars). At this relatively high density of sodium conductance, an insertion of new sodium channels in the opposite initial segment had no effect on the outcome of the propagation (not shown). From this point on, the model became insensitive to the distribution of sodium channels between any of the three hot spots. The absolute values for the lower and upper boundaries of the sensitive range are listed in .
Minimal (permissive) gNabar (pS/μm2) required for AP invasion of the cell body based on four different sodium channel mechanisms in three distributions
Three important results emerged from these experiments. First,
regardless of the channel mechanism used, the propagation of an AP in all model neurons was very sensitive to the lack of sodium channels in either central (ISC) or peripheral (ISP) axon initial segment (). Second,
for forward (afferent) propagating APs, the ISP appears to play a slightly greater role than the ISC. In other words, the ISP requires less sodium conductance (Dark gray bars) than the ISC (Light gray bars) to do the same job. This feature was preserved in all model neurons, regardless of the sodium channel mechanism (). Additional evidence for a slightly more effective role of the ISP came from measurements of the amplitude of failing APs in the cell body (: Horizontal line U). During the AP propagation failure, the ensuing peak depolarization of the soma was higher if sodium channels were inserted in the ISP () rather than the ISC (). Third,
multiple-site recordings from the proximal portion of the peripheral axon (: axon_P) revealed that this is the region with the lowest safety margin for AP propagation (). In this region, a severe attenuation of the traveling AP occurred, even when both initial segments were loaded with sodium channels (). But if an AP failed in the region of low safety margin in axon_P, it was reinitiated in the strategically positioned ISP and ISC (: Inset). The characteristic double peak (: Arrow) consists of the failing first spike (Q) and the reflected second spike (R) and has been described in model neurons (Goldstein and Rall, 1974
; Parnas et al., 1976
) and, most importantly, in real neurons (Ramon et al., 1975
; Antic et al., 2000