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date: 16 February 2019

The Diversified Form and Function of Cochlear Afferents

Abstract and Keywords

Cochlear afferents differ in form and function. The great majority are type I, large diameter, myelinated neurons that contact a single inner hair cell to transmit acoustic information. Each inner hair cell is presynaptic to a pool of 10–30 type I afferents, among which spontaneous activity and acoustic threshold vary widely. Variation in the number, voltage-gating, and density of L-type calcium channels at each presynaptic active zone (ribbon) may dictate this functional diversity. Despite contacting large numbers of outer hair cells, the scarce, unmyelinated type II afferents are acoustically insensitive, and only weakly depolarized by outer hair cell transmitter release. However, type II afferents respond strongly to adenosine triphosphate released by cochlear tissue damage, providing a biological basis for painful hearing (noxacusis).

Keywords: cochlea, type I afferent, type II afferent, ribbon synapse, inner hair cell, outer hair cell, acoustic threshold, adenosine triphosphate (ATP), noxacusis, hyperacusis

Early neuroanatomists captured the exquisitely patterned innervation of the cochlea (Figure 1) and discussed the implications for functional diversity (Lorente de No, 1937). Certainly this innervation pattern determines the frequency selectivity of each type I cochlear afferent as it matures to contact a single inner hair cell (IHC) along the tonotopic gradient of the cochlea. Additional diversification in activity patterns and acoustic sensitivity correlates with the form and function of each synaptic contact on the IHC. Beyond these quantitative differences among type I afferents, the type II afferents are qualitatively distinct, with extensive arbors that spread hundreds of microns toward the cochlear base, giving off branches to dozens of outer hair cells. These smaller caliber, unmyelinated afferents are poorly, if at all, sensitive to sound, but can be activated strongly by tissue damage. The synaptic determinants of functional diversity among type I afferents and the much greater differences between type I and type II afferents will serve as the focus for this chapter. Details of type I afferent acoustic responses and guidance to the original literature can be found in previous reviews and chapters (e.g., Ruggero, 1992; Young, 2008).

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Figure 1. Drawing of cochlear innervation based on Golgi impregnation in young rat (6–7 days) cochlea. Modified from (Lorente de No, 1937). Type II afferents highlighted in light blue. Type I afferent highlighted in magenta. Type I afferents reduce to a single terminal contact in adult.

Type I Cochlear Afferents

Myelinated, large caliber type I spiral ganglion neurons (SGNs) constitute 95% of cochlear afferents (Spoendlin, 1972). These bipolar neurons have a cell body in the spiral ganglion within Rosenthal’s Canal, with a peripheral process that contacts a single IHC, and a central axon that projects through the modiolus to the cochlear nuclei of the brainstem (Ryugo & Parks, 2003). Decades of research has thoroughly documented the acoustic response properties of type I neurons, and have shown that these can account for the sensitive frequency selectivity of the organism (Young, 2008). Additional features like phase-locking of afferent firing are essential for higher order computations involved in sound localization (Middlebrooks, 2015). Frequency selectivity can be determined by a tuning curve whereby pure tones of different intensity and frequency are used to establish the best (lowest intensity) or characteristic frequency for an individual type I SGN during single unit recording in the VIIIth nerve (Figure 2).

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Figure 2. Constant intensity frequency sweep stimuli were used to determine the selectivity and sensitivity of a single auditory nerve fiber in the guinea pig. Action potentials (vertical ticks) occur at a particular time point, corresponding to a particular frequency during the sweep (on the x-axis). The characteristic frequency (cf) is that which elicits action potentials at the lowest intensity—10 kHz in this example. As intensity rises, action potentials are driven by a wider band of frequencies.

Modified from (Evans, 1972).

Each individual inner hair cell is the sole presynaptic partner of 10 to 30 type I afferents. Thus, this group of afferents will have identical frequency tuning. However, type I afferents differ widely in their acoustic sensitivity and dynamic range (the difference between threshold and saturated sound levels). These differences in acoustic response correlate with their rate of spontaneous activity in the absence of sound (M.C. Liberman, 1978; Sachs & Abbas, 1974; Schmiedt, 1989). Acoustically sensitive type I afferents with a narrow dynamic range have high rates of spontaneous activity—“high-spont” fibers. “Low-spont” type I afferents are less sensitive to sound (i.e., a high threshold) and have a wider, sloping dynamic range (M.C. Liberman, 1978; Sachs & Abbas, 1974; Schmiedt, 1989). Since low- and high-spont afferents are thought to innervate the same inner hair cell (M. C. Liberman, 1982), these differences can be attributed at least in part to properties of the synaptic contacts. These synaptic properties can be probed by studies of spontaneous activity both in vivo and ex vivo.

An important clue is provided by the observation that low frequency tones modulate the spontaneous firing rate both up and down (Johnson, 1980). Firing rate increases during the positive (rarefaction) phase, as expected for hair cell depolarization that increases the open probability of voltage-gated calcium channels driving transmitter release. More revealing is the fact that afferent activity decreases during the negative (compression) phase. This decrease implies that reduced transduction current hyperpolarized the hair cell from a resting potential that permits ongoing calcium channel gating and transmitter release in the absence of sound. This implication was secured by the fundamental discovery that spontaneous activity is dependent on influx through dihydropyridine-sensitive calcium channels (CaV1.3) that drives transmitter release from the presynaptic inner hair cell (Robertson & Paki, 2002; Sueta, Zhang, Sellick, Patuzzi, & Robertson, 2004; S. Y. Zhang, Robertson, Yates, & Everett, 1999). It seems intuitive that higher rates of spontaneous transmitter release might be related to lower thresholds for acoustic stimulation. What synaptic features might underlie differences in spontaneous release rates?

Several studies have described possible structural correlates of these functional differences, reviewed in (M.A. Rutherford, 2016). In pioneering studies of the cat cochlea, it was proposed that larger caliber, mitochrondion-rich afferents contacting the abneural, or pillar side, of inner hair cells corresponded to high-spont, low-threshold afferents. Smaller, mitochondrion-poor afferents terminating on the neural or modiolar surface of the inner hair cell would be low-spont, high-threshold afferents in this scheme (M.C. Liberman, 1980, 1982). Serial section electron microscopy of inner hair cells showed that modiolar ribbons (i.e., low-spont afferents) were two-fold longer in their principal axis (420 nm), than pillar ribbons—200 nm (i.e., high-spont afferents) that tended to be spherical. Antibody labeling to the ribeye protein that constitutes much of the synaptic ribbon dense body was consistent with this pattern, showing that neural (modiolar) immunolabeled ribbons produced a larger diffraction-limited image (presumably reflecting their greater length) than did abneural (pillar) ribbons (L. D. Liberman, Wang, & Liberman, 2011). In contrast, postsynaptic immunolabeling for an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit showed the opposite pattern, with larger AMPA receptor immunopuncta found on the abneural (pillar—“hi-spont”) side, and smaller receptor immunopuncta on the neural (modiolar—“low-spont”) side. These innervation patterns may vary somewhat between species and during development (L. D. Liberman & Liberman, 2016). Rather than a strictly neural/abneural organization, afferent synapses were found preferentially on the flattened, rather than rounded side of inner hair cells in the mouse cochlea (Bullen et al., 2015).

Functional studies of afferent synapses on inner hair cells have confirmed some of these features. Thus, direct recording from pairs of afferents contacting individual inner hair cells showed that spontaneous firing could be similar, or different, but there was zero correlation in spike timing for any afferent pair (Wu, Young, & Glowatzki, 2016). Further, this study showed that the detailed statistics of transmitter release (postsynaptic currents) accounted for the pattern of spontaneous action potentials in the afferent neurons. These findings, together with the tight coupling of synaptic input to the nearby spike initiation zone (M. A. Rutherford, Chapochnikov, & Moser, 2012), cement the hypothesis that the responses of individual type I afferents result directly from the properties of their presynaptic ribbon synapse. It remains to be determined whether postsynaptic heterogeneity or modulation by lateral olivocochlear efferents further affect the diversity of afferent response properties.

The number, size and location of ribbons, calcium channel immunopuncta and calcium microdomains (as observed with low affinity indicator dyes) were thoroughly analyzed in mouse inner hair cells (Frank, Khimich, Neef, & Moser, 2009; Meyer et al., 2009; Ohn et al., 2016). These studies showed that the size of calcium hotspots varied widely within individual inner hair cells. That variability reflected differences both in the voltage-dependence of gating (determined from calcium influx revealed with a fluorescent indicator dye), and in the number of channels (from the size of immunopuncta). The number of calcium channels also correlated with the size of the synaptic ribbon. Neural (modiolar—low- spont synapses) had larger ribbons and stronger calcium signals than those of abneural (pillar—high-spont synapses) signals. These correlations run counter to expectation for determination of spontaneous rate. Rather, these workers suggest that the voltage of half activation is positively shifted for modiolar calcium hotspots (low-spont afferents), requiring stronger depolarization to achieve a given level of calcium influx (as measured by low affinity calcium indicator dyes). A molecular mechanism for differences in voltage gating remains to be determined. Another possible factor is the density of channels at each ribbon. If the interchannel distance is greater on average, this would have an effect like that of stronger calcium buffering that reduces the regional calcium signal (Graydon, Cho, Li, Kachar, & von Gersdorff, 2011; Roberts, 1994). In zebrafish hair cells ribbon size and calcium current magnitude co-vary but correlate negatively with the spontaneous activity of afferents (Sheets et al., 2017)—i.e., larger ribbons with larger calcium currents had lower rates of transmitter release. This study also found that calcium channel density (clustering) was lower at larger ribbons—i.e., greater average interchannel distance. A greater interchannel distance would reduce the probability that flux through stochastically opening calcium channels sums to trigger vesicle fusion, thus lowering the rate of spontaneous activity in the postsynaptic afferent.

Other synaptic parameters have been described that could influence transmission efficacy at hair cell synapses. The large range of synaptic current amplitudes led to the suggestion that the ribbon synapse supported coordinate multivesicular release (Glowatzki & Fuchs, 2002). Spontaneous postsynaptic currents in type I boutons are more or less temporally concise, referred to as monophasic or multiphasic events, with monophasic transmission becoming more prevalent with maturation (Glowatzki & Fuchs, 2002; Grant, Yi, Goutman, & Glowatzki, 2011). The peak postsynaptic depolarization is greater for monophasic transmission, potentially contributing to high-spont, low-acoustic threshold afferent activity and precise temporal coding (M. A. Rutherford et al., 2012). The smaller fraction of synapses that maintain multiphasic transmission in maturity could represent low-spont, high-threshold afferents. An alternative mechanism is that the amplitude and waveform of postsynaptic currents reflects the open time of a fusion pore, so that single vesicles release variable amounts of glutamate over varying time courses (Chapochnikov et al., 2014). Immediate and complete vesicular fusion would produce large, monophasic postsynaptic currents while stuttering opening of the fusion pore would result in multiphasic responses, a variant on the “kiss and run” hypothesis for vesicle cycling (Ceccarelli, Hurlbut, & Mauro, 1972, 1973), reviewed in (J. R. Morgan, Augustine, & Lafer, 2002).

Timing of transmitter release is central to the low frequency phase locking of afferent action potentials. Strikingly, these responses are phase-invariant across a range of stimulus intensities at the characteristic or best frequency of an afferent (Rose, Brugge, Anderson, & Hind, 1967). Phase-constancy also occurs in the timing of postsynaptic events in the type I afferent bouton evoked by cyclical depolarization of the hair cell (Goutman, 2012). Phase constancy results from the opposing effects of increased calcium channel gating to advance transmitter release, with depletion of the readily releasable pool that reduces the probability of release. Together these opposing effects result in constant average timing of release for a range of presynaptic depolarizations. Multivesicular release from ribbons helps to ensure stability in the timing of the afferent response (Wittig & Parsons, 2008). Similar conclusions were reached in a study of phase-locking in afferents of the frog inner ear (G. L. Li, Cho, & von Gersdorff, 2014).

What is clear from the fore-going discussion is that acoustic signaling depends critically on the properties of ribbon synapses between inner hair cells and type I cochlear afferents. Each type I afferent is driven by transmitter release from its private active zone (ribbon), and the release capacities of that ribbon are major determinants of the rate of spontaneous activity, the acoustic sensitivity, the dynamic range and the detailed timing of action potentials. Such synaptic competence is in marked distinction to that found in the cochlea’s other afferents, the type II spiral ganglion neurons that innervate the outer hair cells.

Type II Cochlear Afferents

For more than 50 years, the role of type II afferents in the mammalian cochlea has been a matter of some speculation, but little resolution. Despite beautiful and informative anatomical studies defining their arborization and innervation patterns (Berglund, Benson, & Brown, 1996; Berglund & Brown, 1994; Berglund & Ryugo, 1987; Brown, Berglund, Kiang, & Ryugo, 1988; Perkins & Morest, 1975; Simmons & Liberman, 1988a, 1988b; Smith & Haglan, 1973; Spoendlin, 1972), immunolabeling efforts (e.g., Hafidi, 1998; Hossain, Antic, Yang, Rasband, & Morest, 2005; Huang, Thorne, Housley, & Montgomery, 2007) and intracellular recordings from somata ex vivo (Jagger & Housley, 2003; Reid, Flores-Otero, & Davis, 2004), type II function remains somewhat enigmatic. This is due in large part to their scarcity, small caliber and lack of myelination that make single unit recording in vivo exceedingly difficult. Only three studies have attempted to determine their acoustic sensitivity in guinea pigs (Brown, 1994; Robertson, 1984; Robertson, Sellick, & Patuzzi, 1999), reporting on a total of 28 putative type II neurons (only one confirmed anatomically by fill of the distinctive peripheral arbor), the others based on their conduction velocity and waveform during antidromic stimulation). Of these 28, only one had spontaneous activity and only one responded to intense broadband noise, although type I afferents in these preparations could be driven by sound. Although scarce, these data show that type II afferents are acoustically insensitive, despite being postsynaptic to large numbers of OHCs. This poor acoustic sensitivity had led to suggestions that type II afferents may function as a type of peripheral interneuron, possibly to modulate outer hair cells through putative reciprocal synapses (Thiers, Nadol, & Liberman, 2008). Nonetheless type II afferents do project to the brain.

Tonotopically organized type I afferents project through the VIIIth nerve to the magnocellular regions of the cochlear nucleus (Benson, Berglund, & Brown, 1996; Brown, Berglund, et al., 1988; Fekete, Rouiller, Liberman, & Ryugo, 1984; Y. V. Morgan, Ryugo, & Brown, 1994). Type II afferents from the same region of the spiral ganglion, but innervating outer hair cells about ¼ octave higher in frequency (Brown, 1987), project alongside the type I axons. Type II’s branch through both the ventral and dorsal cochlear nuclei, but morphological synapses have been described primarily in granule cell lamina that border the nuclear cores (Benson & Brown, 1990; Berglund et al., 1996; Berglund & Brown, 1994; Brown, Berglund, et al., 1988). The granule cell regions are thought to serve as integration zones, also receiving projections from polymodal somatosensory areas (Paloff & Usunoff, 1992; Shore & Zhou, 2006; Wright & Ryugo, 1996; Zhan & Ryugo, 2007) and collaterals from the medial olivocochlear (MOC) efferents to the OHCs (Benson & Brown, 1990; Brown, Liberman, Benson, & Ryugo, 1988). Granule cells project at least to the pyramidal neurons of the dorsal cochlear nucleus (Mugnaini, Osen, Dahl, Friedrich, & Korte, 1980) perhaps to integrate information from type II and type I neurons. While type I and II afferents branch in parallel and tonotopically to the dorsal and ventral cochlear nuclei, type II collaterals uniquely innervate small cells in the granule cell lamina that lie between the core nuclei (Benson & Brown, 2004). The similarity of this arrangement to C-fiber innervation of superficial layers of the dorsal horn has been noted (Brown & Ledwith, 1990; Hurd, Hutson, & Morest, 1999). However, while suggesting some differentiation from type I signaling, these central projections do not designate any particular role for type II afferents.

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Figure 3. The intracellular label Biocytin diffused into a type II afferent during intracellular recording, revealing the characteristic dendritic process that spirals toward the cochlear base (to the right). Each type II afferent has one or two terminal arbors from which short branches contact outer hair cells (magnified inset). Intracellular voltage recording shows voltage responses to current steps, including overshooting action potentials. Spontaneous excitatory synaptic potentials are also present (arrowhead).

Modified from (Liu et al., 2015, Weisz et al., 2009).

An alternative strategy has been to make intracellular recordings from type II afferents in excised cochlear tissue from young rats or mice. Somatic recordings in cochlear slices from young rats (postnatal days 7–9) revealed that anatomically-confirmed type II neurons expressed a rapidly-inactivating A-type potassium current and could be depolarized by glutamate or ATP applied to the soma (Jagger & Housley, 2003). Spiral ganglion explants from 6–7 day old mice provided evidence that type II SGNs (identified by peripherin immunolabel) tended to sustained, tonic firing patterns while type I SGNs were phasic (Reid et al., 2004). These differences were more pronounced for SGNs in basal turns of the cochlea.

Tight-seal intracellular recording from the spiral process beneath outer hair cells in apical turns of cochleas excised from young (P5-9) rats (C. Weisz, Glowatzki, & Fuchs, 2009) provided additional information about the excitability and postsynaptic activity of type II afferents. Intracellular labeling revealed the characteristic type II dendritic morphology (Figure 3). Action potentials and small (~4 mV) synaptic potentials were observed in these recordings. At least 10 outer hair cells were synaptically connected to an individual type II afferent (C. J. Weisz, Lehar, Hiel, Glowatzki, & Fuchs, 2012). Maximal stimulation of individual outer hair cells (water jet bundle deflection that caused calcium spikes in these immature cells) released single synaptic vesicles with a 25% probability on average. Dual intracellular recordings from single fibers revealed length constants (the distance that a passive voltage change spreads along a fiber) that ranged from 0.6 to 1.2 mm, encompassing the entire spiral process (C. J. Weisz, Glowatzki, & Fuchs, 2014). These dual recordings also showed that action potentials were initiated near the spiral ganglion, and spread decrementally along the spiral process, consistent with the gradient of sodium channel immunolabeling reported previously (Hossain et al., 2005). Data from the dual recordings were used in compartmental modeling to conclude that maximal stimulation of the entire pool of presynaptic outer hair cells would be required to drive a type II afferent to threshold, consistent with their poor acoustic sensitivity in vivo (Brown, 1994; Robertson, 1984; Robertson et al., 1999).

What determines the efficacy of outer hair cell to type II afferent transmission? It is known that outer hair cells have few ribbons and small voltage-gated calcium currents (Knirsch et al., 2007; Michna et al., 2003) that drive limited vesicular fusion (Beurg et al., 2008). The connectivity of outer hair cells with type II afferents has been examined in the light and electron microscopes (Dunn & Morest, 1975; Simmons & Liberman, 1988b; Siegel & Brownell, 1981; Sobkowicz, Rose, Scott, & Slapnick, 1982; Sobkowicz, Rose, Scott, & Levenick, 1986). The spiral process extends several hundred microns toward the cochlear base, giving off numerous short branches to outer hair cells, predominantly in a single row (Berglund & Ryugo, 1987; Brown, 1987; Echteler, 1992; Fechner, Nadol, Burgess, & Brown, 2001; Ginzberg & Morest, 1983). Type II afferents in the apex of young rat cochleas gave off 6-16 short branches, as revealed with intracellular fills after recording in cochlear explants (Jagger & Housley, 2003). Dendritic contacts with outer hair cells can occur with or without associated presynaptic ribbons (Dunn & Morest, 1975; Gulley & Reese, 1977). This issue was given further attention using a combination of intracellular fiber fills, immunolabeling light microscopy and electron microscopy in the rat cochlea (Martinez-Monedero et al., 2016). Fifteen filled type II afferents had on average 16 (± 1.4 SEM) short branches averaging 10.9 µm (± 1.7 SEM) in length. Each such branch contacted one to three outer hair cells for 23.7 (± 1.5 SEM) outer hair cells per type II fiber. Immunolabeled postsynaptic densities that aligned with immunolabeled presynaptic ribbons (half the total) also were positive for the GluA2 AMPA receptor subunit. “Ribbonless” postsynaptic densities lacked GluA2 immunopuncta. Similar findings were obtained from a “reporter” mouse where only some type II afferents contacts were associated with immunolabeled presynaptic ribbons (Vyas, Wu, Zimmerman, Fuchs, & Glowatzki, 2016). Serial section electron microscopy likewise found that only half the afferent boutons on outer hair cells were associated with presynaptic ribbons. Intracellular recording was used to confirm that postsynaptic currents in type II fibers had biophysical properties and pharmacology consistent with AMPA receptor mediation. Others have reported that only ionotropic kainate receptors (related to but distinct from GluA AMPA receptors) were found at OHC to type II synapses in the adult mice (Fujikawa et al., 2014). Intriguingly, the GluK2 subunit was observed only at those synapses associated with hair cell ribbons.

Outer hair cells release glutamate to activate receptor-gated ion channels with rapid kinetics. These responses are quite similar (allowing for the effect of cable loss on waveform shape) to those occurring in type I afferents below inner hair cells that are mediated by GluA2-containing AMPARs. Ribbon-associated synapses in type II afferents included either the GluA2 (rats) or GluK2 subunit (mice). Taken together the literature drives the conclusion that only some type II afferent boutons are classically postsynaptic. The “ribbonless” contacts may correspond to “silent synapses” as found in the CNS (Faber, Lin, & Korn, 1991), and could provide a substrate for plastic changes.

Molecular Specification of Type II Afferents

Given the marked morphological and functional differences between type I and type II cochlear afferents, one can expect differences in gene expression as well, despite their common origin (Koundakjian, Appler, & Goodrich, 2007). Once validated, the responsible gene promoters then could provide selective expression of reporter proteins, or serve to alter type II afferent activity. So for example, it has been noted that the high expression of neurofilaments in the somata of type II neurons enabled them to be distinguished and quantified using a monoclonal antibody (Berglund & Ryugo, 1986). An early and promising genetic candidate was the protein peripherin, antibodies for which labeled type II, but not type I neurons (Hafidi, 1998). Peripherin immunolabel has been used in several studies as a marker for type II afferents (e.g., Reid et al., 2004), however, the suitability of peripherin as a genetic tool remains uncertain. Not all type II afferents are peripherin-positive (to be discussed), and peripherin may be expressed more widely, especially during development (Hafidi, Despres, & Romand, 1993). Nonetheless, co-labeling with peripherin can be a positive identifier of type II neurons. For example, double-immunolabeling for peripherin showed that the transcription factor Gata3 and a downstream effector Mafb, are maintained in mature type II spiral ganglion neurons (Nishimura, Noda, & Dabdoub, 2017).

Considerable progress has been made in the search for molecular markers of differentiation among dorsal root ganglion afferents of the somatic nervous system (Le Pichon & Chesler, 2014; L. Li et al., 2011; Sun & Dong, 2016). These mouse lines include at least two that show selective expression of reporter proteins in type II, but not type I cochlear afferents. Tyrosine hydroxylase is an enzyme that converts the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), leading to synthesis of dopamine, norepinephrine, and epinephrine and so is a marker of catecholaminergic neurons. Unexpectedly, since there is no evidence to date of catecholaminergic function, a TH-CreER mouse line showed expression of reporter protein (green fluorescent protein or TD-tomato) in type II cochlear afferents (Vyas et al., 2016). There was no overlap with immunolabel for Na/K ATPase, a validated marker of type I afferents and medial olivocochlear efferents (McLean, Smith, Glowatzki, & Pyott, 2009). TH-Cre driven reporter expression and immunolabel of the TH protein itself overlapped throughout the characteristic spiral processes of type II afferents. TH-driven expression also was found in sympathetic nerve fibers within the cochlea and in lateral efferents in 3-4 week old cochleas. Some, but not every, TH-CreER positive type II afferent co-labeled with antibody to peripherin, consistent with the heterogeneous expression of peripherin in type II afferents noted elsewhere (Nishimura et al., 2017).

The Diversified Form and Function of Cochlear AfferentsClick to view larger

Figure 4. Tyrosine hydroxylase and calcitonin gene related peptide alpha drive antiparallel expression gradients in type II spiral ganglion neurons. Spiral ganglion neurons (n = 6) labeled with an antibody to tyrosine hydroxylase (TH) predominated in the cochlear apex. Antibodies to GFP under control of the CGRPα promoter labeled spiral ganglion neurons (n = 8) more heavily in the cochlear base.

Modified from (Wu et al., 2018).

Type II afferents in the cochlear apex express tyrosine hydroxylase more strongly than do those in the cochlear base. This observation was quantified in a study comparing the expression patterns of TH and calcitonin gene related protein (CGRP; Wu, Vyas, Glowatzki, & Fuchs, 2018). CGRP is expressed by a variety of cell types, including small afferents of the dorsal root ganglia where its release increases vasodilation and inflammation to exacerbate pain (Russell, King, Smillie, Kodji, & Brain, 2014). EGFP under control of CGRPα regulatory sequences in a bacterial artificial chromosome was expressed in cochlear efferents, as shown previously by CGRP immunolabel (Kitajiri et al., 1985), and also in a subset of spiral ganglion neurons (Wu et al., 2018). CGRP-positive SGNs had type II-like peripheral arbors, spiraling basally and connecting to outer hair cells. CGRP-dependent reporter expression was completely separate from type I SGNs immunolabeled for Na/K ATPase. As for TH (Vyas et al., 2016), CGRP-positive SGNs overlapped only partially with peripherin immunolabel. Expression patterns of TH and CGRP-positive neurons was quantified by counting labeled somata in 10 equal segments along the entire cochlear spiral. TH expression was consistently higher in apical type II afferents, while CGRP was higher in basal type II afferents (Figure 4). Individual SGNs in middle cochlear regions could be positive for both biomarkers. Thus, as suggested by differences in dendritic arborization (Nayagam, Muniak, & Ryugo, 2011), type II cochlear afferents may be “tonotopically” differentiated, potentially reflecting even further functional sub-specialization.

A Functional Role for Type II Afferents

Since type I cochlear afferents provide acoustic input, what remains for type II afferents to do? Apparently they have something to do with OHCs. In the earliest studies it was suggested that type II afferents might be sensitive to very faint sounds by spatial summation from their numerous presynaptic OHCs (C. Fernandez quoting H. Davis)(Fernandez, 1951; Spoendlin, 1969) a logical but now incorrect assumption. Another idea is that type II afferents are analogous to muscle spindle afferents, providing updates on the mechanical “setpoint” of the cochlea by signaling the activity of OHCs (Berglund & Brown, 1994; Kim, 1984). A peripherin knockout mouse was found to lack sound-evoked suppression of otoacoustic emissions. Type II afferents appeared to be absent from the cochleas of these mice, leading to the suggestion that type II afferents activate the medial olivocochlear efferents that mediate suppression by inhibiting OHCs (Froud et al., 2015). That conclusion was called into question by a subsequent study showing that electrical stimulation of medial olivocochlear efferents also failed to suppress distortion products in these knockout mice, suggesting that peripherin knockout disrupted efferent neurons directly (Maison, Liberman, & Liberman, 2016). More generally, type II afferents are unlikely to be the sole activators of olivocochlear efferents whose acoustic sensitivity is essentially equivalent to that of type I afferents (M. C. Liberman & Brown, 1986; Robertson & Gummer, 1985). Another notion is that type II fibers are a kind of peripheral interneuron, based on a suggestion of reciprocal contacts with OHCs (Nadol, 1983; Thiers et al., 2008) and presynaptic specializations with supporting cells (Fechner et al., 2001). However, even if these peripheral connections are functional (which has not been determined), type II neurons also do project to the cochlear nuclei of the brainstem, distributing branches that largely parallel those of neighboring type I afferents (Berglund & Brown, 1994; Brown, Berglund, et al., 1988) and so they must have central functions as well. More consistent with existing data is the suggestion that type II afferents exist specifically to encode “loudness” (Knirsch et al., 2007).

Type II afferents may serve a still more distinct function, to signal the presence of traumatic, or noxious, levels of sound, as summarized recently (Francis, 2012; K. D. Zhang & Coate, 2017). This hypothesis derives from their morphological similarity to unmyelinated somatic C-fibers, poor acoustic sensitivity (Brown, 1994; Robertson, 1984; Robertson et al., 1999) and weak drive by outer hair cell glutamate release (C. J. Weisz et al., 2012). In support of this hypothesis, when mice with dysfunctional inner hair cell synapses were exposed to traumatizing sound, activity-dependent c-FOS labeling was enhanced in the granule cell domain of the cochlear nucleus (Flores et al., 2015), the specific brainstem destination of type II afferents (Brown, Berglund, et al., 1988). Still more directly, recordings from type II afferents in excised cochlear segments from young rats showed that type II afferents were strongly excited when outer hair cells were ruptured by a mechanical probe (Liu, Glowatzki, & Fuchs, 2015). This response was due in large part to activation of ionotropic (P2X) and metabotropic (P2Y) purinergic receptors in type II afferents. P2Y receptor activation depolarized the type II neuron by the closure of KCNQ-type potassium channels, making the afferent more excitable and lowering the apparent spike threshold (C. Weisz, J.C., 2011). The minutes-long damage response in type II afferents was similar to that of ATP-dependent calcium waves seen in surrounding support cells upon outer hair cell ablation (Gale, Piazza, Ciubotaru, & Mammano, 2004; Lahne & Gale, 2010). Thus, ATP release from supporting cells may drive type II afferent activation, akin to the transient role of Kolliker’s organ cells in spontaneous activity of type I afferents during cochlear maturation (Tritsch, Yi, Gale, Glowatzki, & Bergles, 2007).

The gain of function pathologies tinnitus and hyperacusis arise subsequent to loss of type I afferent input. It is thought that the loss of peripheral input leads to increases in gain at synaptic connections in the central auditory pathway (Auerbach, Rodrigues, & Salvi, 2014). It is not known what, if any contribution type II afferents might make, although their peripheral arbors can persist in damaged cochleas (Ryan, Woolf, & Bone, 1980; Spoendlin, 1971). A guiding analogy can be drawn from the somatic nervous system where the loss of large diameter myelinated afferents (e.g., touch, vibration sensors), but survival of unmyelinated C-fibers, can lead to persistent pain. This was proposed as the “gate theory” (Wall, 1978) whereby rapidly-conducting, large diameter, myelinated somatic afferents normally inhibit the pain pathway. Uncompensated C-fiber input results in chronic pain syndromes. These include hyper-excitability of the peripheral C-fibers as well as changes in central synaptic efficacy (Perl, 1996). In combination with the loss of type I afferent contacts onto inner hair cells (Kujawa & Liberman, 2009; Ruel et al., 2007; Stamataki, Francis, Lehar, May, & Ryugo, 2006), the hyperexcitability of type II afferents could be particularly germane to mechanisms of hyperacusis. Synaptic inputs may increase in number and strength, intrinsic excitability may increase (for example, by down‐regulation of KCNQ channels), and sensitivity to ATP may increase. The cellular and synaptic physiology of damaged cochleas (e.g. Zachary & Fuchs, 2015) will receive increased attention as efforts mount to explore both the loss and gain of function after cochlear trauma.


Work in the author’s laboratory is supported by NIDCD R01DC001508, R01DC016559, R01DC015309, the John E. Bordley Professorship and the David M. Rubenstein Fund for Hearing Research to Johns Hopkins Otolaryngology-HNS. T. Coate and M. Rutherford are thanked for their careful reading of an earlier draft.


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