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Published in final edited form as: Hear Res. 2015 Sep 25;330(0 0):18–25. doi: 10.1016/j.heares.2015.09.007

Synaptic Studies Inform the Functional Diversity of Cochlear Afferents

PA Fuchs 1, E Glowatzki 1
PMCID: PMC4674337  NIHMSID: NIHMS726481  PMID: 26403507

Abstract

Type I and type II cochlear afferents differ markedly in number, morphology and innervation pattern. The predominant type I afferents transmit the elemental features of acoustic information to the central nervous system. Excitation of these large diameter myelinated neurons occurs at a single ribbon synapse of a single inner hair cell. This solitary transmission point depends on efficient vesicular release that can produce large, rapid, suprathreshold excitatory postsynaptic potentials. In contrast, the many fewer, thinner, unmyelinated type II afferents cross the tunnel of Corti, turning basally for hundreds of microns to form contacts with ten or more outer hair cells. Although each type II afferent is postsynaptic to many outer hair cells, transmission from each occurs by the infrequent release of single vesicles, producing receptor potentials of only a few millivolts. Analysis of membrane properties and the site of spike initiation suggest that the type II afferent could be activated only if all its presynaptic outer hair cells were maximally stimulated. Thus, the details of synaptic transfer inform the functional distinctions between type I and type II afferents. High efficiency transmission across the inner hair cell’s ribbon synapse supports detailed analyses of the acoustic world. The much sparser transfer from outer hair cells to type II afferents implies that these could respond only to the loudest, sustained sounds, consistent with previous reports from in vivo recordings. However, type II afferents could be excited additionally by ATP released during acoustic stress of cochlear tissues.

Introduction

Vertebrate sensory hair cells are canonical ‘short receptors’ having neither axons nor action potentials, but relying instead on transmitter release driven directly by the receptor potential that alters voltage-gated calcium influx. As in retinal photoreceptors and bipolar cells, transmitter release from hair cells occurs at ribbon synapses where vesicles are tethered to a dense body, and where voltage-gated calcium channels cluster in the plasma membrane (Nouvian et al., 2006; Matthews and Fuchs, 2010). Thus, ribbon synapse function will strongly influence information transfer from the inner ear to the CNS – particularly in the case of type I cochlear afferents whose single dendrite receives synaptic input from a single ribbon in one inner hair cell. This unusually solitary connection becomes still more intriguing given that activity patterns are thought to differ among the group of type I afferents contacting a single inner hair cell (Merchan-Perez and Liberman, 1996). Beyond the inner hair cell to type I afferent synapse however, still greater anatomical diversity is found in the cochlea (type II afferents) and vestibular end-organs (calyx, bouton, dimorphic afferents) whose functional diversity is only beginning to be revealed. Given the daunting complexity of vestibular synapses, this presentation will focus on a comparison of the structure and function of type I and type II cochlear afferents, with particular attention to details of transmitter release from their respective presynaptic hair cells.

Type I and type II afferents have distinctly different peripheral arbors (Fig. 1). The predominant type I afferents (90–95% of the total) are larger diameter, myelinated and extend a single unbranched dendrite to the inner hair cell row. The minority type II afferents are thinner and unmyelinated, with a peripheral process that crosses the tunnel of Corti then turns to extend hundreds of microns toward the cochlear base (Berglund and Ryugo, 1987). Each type II afferent gives off short branches to multiple outer hair cells, usually within one row (Brown, 1987; Jagger and Housley, 2003).

Figure 1.

Figure 1

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Typical morphology of type I and type II afferents as shown by HRP fills in the mouse cochlea. (From Berglund and Ryugo, 1987, with permission).

Type I afferents transmit fundamental aspects of acoustic stimuli including timing, intensity and frequency composition (Kiang et al., 1967; Sachs and Abbas, 1974; Sachs and Young, 1979; Young and Sachs, 1979; Young, 2008). Afferent frequency selectivity results from the specific innervation of single inner hair cells along the tonotopic axis of the cochlea; while the coding of timing and intensity requires transformation from the acoustic receptor potential to a related pattern of transmitter release onto the afferent dendrite (Fuchs, 2005; Moser et al., 2006). Multi-vesicular release from inner hair cell ribbon synapses may facilitate this transformation (Glowatzki and Fuchs, 2002; Wittig and Parsons, 2008; Goutman, 2012; Rutherford et al., 2012). The small caliber and scarcity of type II afferents severely limits single-unit recording, with only a small number reported, all insensitive to sound (Robertson, 1984; Brown, 1994; Robertson et al., 1999). Somatic recordings from excised tissue, or dissociated ganglion neurons have revealed some features of excitability (Jagger and Housley, 2003; Reid et al., 2004). More recent intracellular recordings from the spiral process have measured excitability and synaptic inputs, and support the notion that type II afferents could be activated only by the loudest sounds (Weisz et al., 2009; Weisz et al., 2012; Weisz et al., 2014).

Capacitance measurements from hair cells and postsynaptic recordings from afferent fibers have been used to document transmitter release from both inner and outer hair cells. Substantially more information exists for the inner hair cell to type I afferent synapse, using capacitance recording to detail developmental changes (Beutner and Moser, 2001; Johnson et al., 2009) and differences along the tonotopic axis (Johnson et al., 2008), as well as the influence of various genetic manipulations, e.g., knockout of bassoon (Khimich et al., 2005) or synaptotagmins (Beurg et al., 2010; Johnson et al., 2010; Reisinger et al., 2011). From such measurements it is possible to estimate the size of the readily-releasable pool (RRP) of vesicles (400–500 in gerbil IHCs (Johnson et al., 2009) corresponding to 10–14 vesicles per ribbon; likewise 280 in mouse IHCs, corresponding to ~11 per each of 25 ribbons (Beutner and Moser, 2001). Immuno-label shows many fewer ribbons in most outer hair cells, consistent with their smaller total RRP (135–143 in gerbil OHCs – (Johnson et al., 2009)) and small voltage-gated calcium currents (Knirsch et al., 2007). However, the calcium current on a per ribbon basis seems to be about the same as for inner hair cells, as well as the number of vesicles in the RRP for each ribbon, 13 in OHCs of gerbil. Outer hair cells from newborn rats (P2–3) have many more ribbons, larger voltage-gated calcium currents and a far larger RRP = 1200, and these values all fall with maturation (Knirsch et al., 2007). These issues also have been examined by direct postsynaptic measurement of release probability – see below.

Type I afferents

Intracellular recording from type I afferent contacts on inner hair cells provides a privileged view of ribbon function with unmatched signal-to-noise and temporal resolution (Glowatzki and Fuchs, 2002). This vantage point has confirmed that glutamate released from the hair cell activates the AMPA class of postsynaptic receptors (Fig. 2A), and first revealed the wide (20-fold) range of amplitudes of stochastically-released postsynaptic currents (i.e., constant presynaptic membrane potential) (Fig. 2B). ‘Monophasic’ responses varied in amplitude but were characterized by uninterrupted rising and falling phases. Irregularly-shaped ‘multiphasic’ responses were generally smaller and seemingly composed of subunits. These and other observations suggested that the ribbon synapse can execute spontaneous multi-vesicular release, as suggested previously on the basis of sharp electrode recordings in the guinea pig cochlea (Siegel, 1992). How this might be accomplished remains uncertain, although the microstructure of the ribbon constrains calcium entering through nearby voltage-gated channels, creating locally-high clouds that could trigger release of already docked vesicles (Roberts, 1994; Graydon et al., 2011). The relatively large conductance of hair cell calcium channels (Zampini et al., 2014) could promote this process. Compound fusion of vesicles prior to release also has been proposed on the basis of ultrastructural studies (Matthews and Sterling, 2008). In the context of classical vesicular release hypotheses, multi-vesicular release would correspond to variations in quantum content (number of vesicles), whereas compound fusion would yield a variable quantal size. Variable quantal size also is the basis of a recent uniquantal proposal (Chapochnikov et al., 2014). In this model, a chattering fusion pore releases variable amounts of glutamate from single fused vesicles. This in combination with an unsaturated pool of postsynaptic receptors can explain many features of release from inner hair cell ribbons, although perhaps not that from all hair cells. A wide range of postsynaptic current amplitudes also is found at hair cell afferent synapses in frog and turtle (Keen and Hudspeth, 2006; Li et al., 2009; Schnee et al., 2013). In both of these species, in contrast to the IHC afferent synapse, many ribbon synapses release onto a single afferent fiber. Current hypotheses regarding the underlying mechanisms include multi-vesicular release and ‘complex’ postsynaptic currents produced by temporal summation of overlapping release from multiple ribbons.

Figure 2.

Figure 2

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Stochastic postsynaptic currents in type I afferent (hair cell at rest or subject to potassium depolarization in excised apical turn of young rat cochlea). A. Rapidly gating inward currents were sensitive to cyclothiazide, a specific modulator of AMPA-type glutamate receptors. B. Spontaneous postsynaptic currents varied widely in amplitude within each recording. (Reproduced from Figures 2 and 3, Glowatzki and Fuchs, 2002, with permission)

What all observations and models have in common is that the rate of transmitter release from hair cell ribbons is strongly calcium-dependent. This dependence takes the form of an increased rate of vesicular fusion events as cytoplasmic calcium increases. Paired pre- and postsynaptic recordings established this fact for frog saccular hair cells (Keen and Hudspeth, 2006; Li et al., 2009) and at the ribbon synapse of inner hair cells (Goutman and Glowatzki, 2007). Progressive depolarization of mammalian inner hair cells increased the frequency but not mean amplitude of postsynaptic currents. This same conclusion was reached using different levels of cytoplasmic buffer in the hair cell to alter release frequency (Fig. 3). Paired recordings also provided a mechanism for adaptation of afferent activity. Postsynaptic currents declined to a lower steady-state during sustained depolarization of the presynaptic hair cell. This decline was shown to be due to decreased event frequency; presumably from depletion of the readily-releasable vesicular pool (the presynaptic calcium current was virtually constant). The rate of decline (initial time constant ~ 7 ms) was similar to that reported for adaptation of afferent firing rates to high frequency tone bursts (Taberner and Liberman, 2005), supporting the hypothesis that central signaling depends fundamentally on transmission at the ribbon synapse.

Figure 3.

Figure 3

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Pair recordings from inner hair cell and type I afferent dendritic bouton (young rat cochlea). Transmitter release was evoked under different conditions of calcium buffering. An increase of intracellular EGTA concentration, or change from EGTA to BAPTA greatly reduced the frequency, but not average size of evoked synaptic currents. (From Figure 4, Goutman and Glowatzki 2007, copyright © by the National Academy of Sciences)

The main effect of presynaptic depolarization (calcium level in the hair cell) is to alter the rate of release events. This process is subject to depression (depletion) and voltage-dependent facilitation (Goutman and Glowatzki, 2011) that together set the release probability for any particular presynaptic membrane potential, as seen also for frog hair cell transmission (Cho et al., 2011). As a consequence, the peak timing of release remains constant at different release rates (Goutman, 2012), supporting the phase-constancy with intensity of afferent firing patterns seen for acoustic stimulation at the best frequency (Rose et al., 1967). A similar observation has been reported for transmitter release from hair cells of the frog amphibian papilla (Li et al., 2014).

The majority of these recordings were carried out on cochlear tissue excised from neonatal animals (before postnatal day 14), during a time when capacitance measurements indicate that release is immature (Beutner and Moser, 2001; Johnson et al., 2009). Postsynaptic recordings from older animals (>P19) revealed a shift toward larger, faster, monophasic events with relatively fewer multiphasic events (Grant et al., 2010). In older fibers the synaptic amplitudes could be normally distributed about a mean of several hundred pA (Fig. 4) – as though the mature pattern of release was for monophasic, multivesicular events, perhaps related to the observed higher efficiency coupling of calcium influx to capacitance increase (Beutner and Moser, 2001; Johnson et al., 2009). Nonetheless, some older synapses retained significant numbers of multiphasic events of lower amplitude, leading to the suggestion that these may serve low-spontaneous rate afferent fibers, with many more sub-threshold synaptic potentials.

Figure 4.

Figure 4

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Postsynaptic currents in type I afferents of the mature rat cochlea. Cumulative fraction plots (A, D) and histograms (B, C, E, and F) illustrate the amplitude distribution for EPSCs in type I afferents. These tend to cluster around much larger mean amplitudes than in younger tissue. Note also that small events still occur in some recordings from mature cochlear tissue (from Grant et al., 2010, Figure 7, with permission).

Type II afferents

The same technique developed for type I afferents was adapted to make tight-seal, intracellular recordings from the peripheral processes of type II afferents as they spiral beneath the rows of outer hair cells in apical cochlear explants from 5–9 day-old rats (Weisz et al., 2009). These revealed typical, fast, ionotropic synaptic excitation by glutamate release from outer hair cells (Figure 5A). Sequential stimulation of individual outer hair cells showed that ten or more were presynaptic to a given type II afferent (Figure 5B). The strength of transmission from each hair cell was low, appearing to consist of single vesicles released once for every four maximal presynaptic stimuli (Weisz et al., 2012). Similarly uniquantal release seems to occur as well at the type I vestibular hair cell to calyx afferent synapse (Rennie and Streeter, 2006; Dulon et al., 2009; Eatock and Songer, 2011; Sadeghi et al., 2014).

Figure 5.

Figure 5

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Synaptic inputs from outer hair cells (OHCs) to type II afferents. A. Examples of excitatory synaptic currents (EPSCs) evoked by waterjet stimulation of OHC. Waterjet pulse begins at time 0 and elicits regenerative depolarizations (calcium spikes) from OHCs whose half-amplitude duration extends from 7–40 ms (as determined in separate recordings). B. Mapping synaptically-coupled OHCs. Red circles are OHCs that produced EPSCs when stimulated, blue were silent. Black circles indicate position of type II recording pipette. Amplitude histograms of EPSCs from near (@ 30 μm) and far (@109 μm) OHCs had similar distributions and little amplitude variation, indicating single events (vesicles), and no cable loss over that distance (single exemplar – cell F - of amplitude histograms summarized in Figs. 6 and 7, Weisz et al., 2012).

Release was evoked onto type II afferents with a strong mechanical stimulus (waterjet puff) that reliably elicited calcium action potentials from these immature outer hair cells. Since this same stimulus was used in earlier studies on young inner hair cells (Beutner and Moser, 2001; Glowatzki and Fuchs, 2002), it is possible to compare the efficacy of transmission from young outer and inner hair cells stimulated to produce action potentials. One measure of synaptic strength is the quantum content, or average number of vesicles released for each presynaptic action potential, e.g., 200–300 at the mammalian neuromuscular junction (Boyd and Martin, 1956). Work on young inner hair cells suggested that each action potential caused the release of 40–50 vesicles at a single synaptic contact (Beutner et al., 2001; Glowatzki and Fuchs, 2002), in marked contrast to the very much lower probability of release from each outer hair cell onto type II afferents (0.25 vesicles per action potential). These comparisons inspire two types of questions: what might be the structural bases for these significant functional differences; and, what are the larger implications for cochlear signaling?

Many laboratories have described the presynaptic structures of outer hair cells, among them (Siegel and Brownell, 1981; Nadol, 1983; Simmons and Liberman, 1988; Hashimoto and Takasaka, 1989; Weisz et al., 2012). Immuno-labeled ribbons are relatively scarce, from 2–5 per outer hair cell, depending on species, development and cochlear position, see for example (Fujikawa et al., 2014) compared to the 20 or so found in the typical inner hair cell. Assuming that vesicular release from outer hair cells occurs at ribbons, as from inner hair cells, one might expect substantial differences in ribbon size, or the number and disposition of vesicles to account for the observed differences in transmitter release. However, a direct comparison in the apical turn of the young (P9) rat cochlea (equivalent to the locus of postsynaptic recordings) showed no difference in ribbon size and only a two to three-fold difference in vesicle numbers (Weisz et al., 2012). Outer hair cell ribbons were slightly taller and thinner (but with the same volume), with approximately half as many tethered and docked vesicles as found on inner hair cell ribbons. However, in addition to being fewer in number, vesicles at outer hair cell ribbons also appear to be more heterogeneous in size and disposition than those of inner hair cells (Fig. 6) as observed in studies of the developing and cultured mouse cochlea (Sobkowicz et al., 1982).

Figure 6.

Figure 6

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Ribbons and vesicles of inner hair cells (IHC) and outer hair cells (OHCs) from the apical turn of a P9 rat cochlea (Lehar, Hiel and Fuchs, unpublished).

All together however, it is unclear how these relatively minor distinctions could explain the major differences in release efficiency between these types of hair cells. Both inner and outer hair cells can express otoferlin and synaptotagmin IV which are implicated in the linear calcium dependence of voltage-dependent capacitance measurements (Beurg et al., 2008; Dulon et al., 2009; Johnson et al., 2009; Johnson et al., 2010), but further characterization of component proteins may reveal differing molecular mechanisms. For example, the vesicular glutamate transporter VGlut3 appears to be absent from outer hair cells, but is required for transmission from inner hair cells (Akil et al., 2012). Another possibility is suggested by the observation that clusters of intra-membrane particles in freeze fracture EM – thought to include voltage-gated calcium channels (Roberts et al., 1990)) are about 4-fold smaller under ribbons of outer hair cells compared to those of inner hair cells in the guinea pig cochlea (Saito, 1990). Clusters of postsynaptic intramembranous particles occur in type II afferents at a density of 3000/μm2 (Saito and Hama, 1984). While comparable numbers aren’t available for the type I afferent, if these represent glutamate receptors one might expect larger such aggregations as another element of more effective transmission. The better-organized ribbons of most other hair cells are thought to ensure precise and reliable timing of release (Wittig and Parsons, 2008; Buran et al., 2010) to encode the information content of sound. By comparison, the outer hair cell to type II afferent connection seems poorly-designed for such coding.

Implications for function

Recognizing the limitations of room-temperature recordings from immature, excised cochlear tissue, nonetheless it is worth considering the implications of these results. Transmission from outer hair cells to type II afferents is extremely weak by comparison to that between inner hair cells and type I afferents in identical experimental conditions. Based on measurements of length constant using two intracellular electrodes, a compartmental model suggests that at least 6 outer hair cells (out of a pool of 20–25) would have to release a vesicle to bring the type II afferent to action potential threshold (Weisz et al., 2014). These estimates are based on maximal stimulation of the outer hair cell (calcium action potentials) – so presumably corresponding to maximal acoustic stimulation. If type I and II afferents project to similar regions of the cochlear nuclei (as HRP fills suggest - (Brown et al., 1988) it is worth considering what combination of frequency and intensity might co-activate type II and I afferents. The synaptic input zone of type II fibers lies about 750 μm basal from the tunnel crossing (presumably where neighboring type I fibers reside). Thus, type II afferents will be co-activated by sounds loud enough for maximal basilar membrane vibration basal to the CF for a given type I afferent. This will occur as increasingly louder sounds result in asymmetric basal-ward extension of the traveling wave on the basilar membrane (Von Békésy, 1960).

Beyond acoustic stimulation, type II afferents respond vigorously to ATP (Fig. 7). It has been shown that damage to hair cells triggers a calcium-driven release of ATP among supporting cells (Gale et al., 2004), providing a potential source of type II excitation during acoustic trauma. Thus, type II afferents may be analogous to somatic C-fibers, i.e., nociceptors. Both are smaller diameter, unmyelinated, with wide-spread peripheral arbors that are sensitive to ATP. It has been shown that type II central terminals form synaptic contacts in the small cell cap and/or granule cell domain of the cochlear nuclei (Brown et al., 1988; Berglund and Brown, 1994; Benson and Brown, 2004) but the onward connections remain to be determined. Are there connections to central pain pathways?

Figure 7.

Figure 7

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ATP stimulates type II afferents. A. ATP triggers inward current in voltage-clamp, and B. large depolarization from the resting potential (from Weisz et al., 2009, Figure 3, with permission).

Summary

Type I and type II cochlear afferents differ in morphology, innervation pattern and synaptic physiology, implying significantly different roles in cochlear function. It is well-established that type I afferents encode the information content of sound, and that efficacious transmitter release from inner hair cell ribbon synapse enables that information transfer. The function of type II afferents remains unresolved, but weak synaptic transmission from outer hair cells reinforces the conclusion that information coding must be limited. An alternative hypothesis is that type II afferents are analogous to nociceptive C-fibers in skin, being small diameter, unmyelinated sensors with very high thresholds. That analogy is further strengthened by the sensitivity of type II fibers to ATP, a recognized noxious stimulant (Burnstock, 2013) released by supporting cells upon hair cell damage (Gale et al., 2004).

Highlights.

  • Hair cell to afferent synapses differ markedly in efficacy.

  • Single inner hair cell ribbons activate each type I afferent.

  • Multiple presynaptic outer hair cells only weakly excite each type II afferent.

  • Type II afferents respond strongly to ATP, a noxious stimulant.

Acknowledgments

With thanks to J. Goutman, E. Yi, L. Grant, C.J. Weisz, C. Liu, R. Martinez-Monodero, P. Vyas, J-J. Wu, H. Hiel and M. Lehar who contributed to work discussed here. Supported by NIDCD R01 DC011741 (PAF and EG), R01 DC001508 (PAF), NIDCD R01 DC006476, R01 DC012957 (EG) and NIDCD P30 DC005211 to the Center for Hearing and Balance at Johns Hopkins University School of Medicine.

Footnotes

A keynote lecture delivered to the Inner Ear Biology Workshop in Kyoto, Japan, November 4th, 2014.

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