Before the onset of sensory function, IHCs drive patterned APs in the auditory nerve that seem to be required for normal wiring of the auditory brain (Walsh & McGee, 1987; Clause et al., 2014). These presensory APs in SGNs are driven by synaptic transmission, evoked by Ca²⁺ spikes in immature IHCs (Fig. 5.2a–c). Calcium spikes are regenerative potentials, similar to Na⁺ APs in neurons. Spike-driven exocytosis in immature IHCs (Kros et al., 1998; Beutner & Moser, 2001) is mediated predominantly by Ca_V1.3 Ca²⁺ channels (Brandt et al., 2003; Marcotti et al., 2003). Although mature IHCs do not spike, they use the same type of voltage-gated Ca²⁺ channels to mediate hearing.

By the onset of hearing, at approximately postnatal day 14 (p14) in mice and rats, reduction in number of Ca_V1.3 channels (Brandt et al., 2003) and upregulation of K⁺ channels disable regenerative Ca²⁺ spikes in IHCs. For example, the large-conductance Ca²⁺– and voltage-activated K⁺ channels (BK channels) carry a hyperpolarizing conductance that ensures a nonspiking, graded response of the mature IHC transmembrane potential (Kros et al., 1998; Oliver et al., 2006). Another developmentally upregulated K⁺ channel, K_V7.4 (KCNQ₄,), defective in human deafness DFNA2 (Kubisch et al., 1999), is partially active when IHCs are at rest, and contributes to setting the IHC resting membrane potential (Oliver et al., 2003). Many aspects of IHC development depend on thyroid hormone signaling (Rüsch et al., 2001; Sendin et al., 2007).

Presensory spiking in IHCs generates bursts of APs in SGNs at p10 in vivo (Fig. 5.2c). These bursts are replaced by mature-looking AP trains in SGNs around p14 (Wong et al., 2013). Experiments in organ of Corti explants have investigated what underlies the temporal pattern of Ca²⁺ spikes in IHCs, but the mechanism is still under debate. Release of ATP onto IHCs from cells in the developmentally transient Kölliker’s organ may be important for hair cell excitation (Tritsch et al., 2007; Tritsch & Bergles, 2010), although patterned activity proceeded in the presence of inhibitors of ionotropic purinoceptors (Sendin et al., 2014). Alternatively, patterned electrical activity may be intrinsic to the IHC but modulated by ATP release (Johnson et al., 2011).

There is general agreement that presensory activity is likely regulated by inhibition of IHCs via the efferent synapses of olivocochlear neurons. Inhibitory cholinergic transmission could periodically interrupt the IHC depolarization resulting from resting mechanotransduction (Walsh & Romand, 1992; Glowatzki & Fuchs, 2000; Sendin et al., 2014), similar to efferent inhibition of mature outer hair cells (Géléoc & Holt, 2003). However, olivocochlear neurons have somas in the brain, and it is unclear how intrinsic activity in their axons is altered in the excised organ of Corti. Whatever the mechanism, dramatic changes in SGN AP trains between p10 and p14 (Fig. 5.2c–f) are concurrent with IHC synaptic maturation.

In mice, SGN fibers reach cells in the differentiating organ of Corti already at birth. The numbers of fibers and synapses in the organ of Corti appear to increase in number during the first postnatal week (Lenoir et al., 1980; Shnerson et al., 1981). Then, presynaptic ribbons and postsynaptic densities (PSDs) decrease in number (Huang et al., 2007, 2012). By p21, IHC–SGN synapses are predominantly mature (Sobkowicz et al., 1982; Grant et al., 2010). This section highlights some structural and functional aspects of synaptic maturation and discusses underlying molecular-anatomical mechanisms.

In the first postnatal week, the IHC Ca²⁺ current and exocytosis increase as they approach their peak sizes. Then, during the second postnatal week, they decline differently as the efficiency with which Ca²⁺ influx triggers exocytosis increases. The number of Ca_V1.3 channels decreases but the smaller Ca²⁺ current of mature IHCs causes comparably large amounts of exocytosis (Beutner & Moser, 2001; Brandt et al., 2005; Zampini et al., 2010). Immunofluorescence microscopy in fixed tissue (Fig. 5.3a–c) as well as Ca²⁺ imaging in live tissue revealed that overall Ca_V1.3 immunoreactivity declined while it accumulated synaptically and the Ca²⁺ influx increased specifically at the ribbon synapses (Wong et al., 2013, 2014). Thus, maturation involved reduction of extrasynaptic Ca²⁺ channels not directly coupled to synaptic vesicle exocytosis. Unlike immature IHCs, Ca²⁺ influx in mature IHCs is largely confined to AZs.

Individual IHC–SGN synapses at p6 displayed several small appositions of AZs and PSDs, only some of them occupied by a presynaptic ribbon. These groups of appositions encircled the perimeter of the bouton contact (Wong et al., 2014). Only after the onset of hearing was a single juxtaposed AZ–PSD complex found per SGN bouton (Fig. 5.3).

As AZs and PSDs decreased in number they increased in size, as shown via electron microscopy (Fig. 5.3e, f) and corroborated with confocal immunohistochemistry using antibodies against Ca_V1.3 Ca²⁺ channels, GluA2/3 glutamate receptors, and the ribbon protein CtBP2 (Fig. 5.3a–c; Wong et al., 2014). The ratio of ribbons to glutamate receptor puncta increased to nearly 1 by p20, indicating that ribbonless AZs disappeared and the 1:1 connection between ribbons and PSDs prevailed for each SGN.

Ribbons are synaptically anchored via the presynaptic protein bassoon (Khimich et al., 2005). In keeping with the notion that Ca²⁺ channels cluster underneath ribbons in the presynaptic density (schematized in Fig. 5.3d, g), bassoon and Ca_V1.3 immunofluorescence closely aligned in elongated stripes at p19 when measured with two-color stimulated emission depletion (STED) microscopy (Wong et al., 2014; Rutherford, 2015). In contrast, before the onset of hearing synaptic Ca_V1.3 channels formed only smaller spot-like clusters.

Two candidate mechanisms for this anatomical refinement are (1) merging—small AZs or PSDs of a synaptic contact coalesce via interactions of scaffold molecules possibly involving transsynaptic regulation and (2) pruning—small AZs and PSDs are selectively eliminated via protein degradation. Bassoon and the similar protein piccolo each inhibit ubiquitin ligase activity (Waites et al., 2013). Their greater abundance might protect the largest of the initially formed AZs.

These structural refinements are accompanied by developmental changes in synaptic function and changes in molecular composition. At p0, rodent IHCs show relatively little Ca²⁺ current or exocytosis. As mentioned previously in this section, this is followed by an increase during week 1, then a decrease in Ca²⁺ current but relatively little decrease in exocytosis during week 2. This increase in efficiency of exocytosis is at least partially due to the positioning of more Ca_V1.3 channels at AZs and fewer Ca_V1.3 channels away from AZs (Fig. 5.3a). Moreover, during the first postnatal week exocytosis seems to employ a different molecular program than later in development. For example, otoferlin, essential for exocytosis in mature IHCs, seems dispensable for presynaptic function at this early stage while the neuronal Ca²⁺ sensor of exocytosis synaptotagmin 2 is temporarily expressed (Beurg et al., 2010; Reisinger et al., 2011). In addition to the increase in efficiency of exocytosis, a change is also observed in the apparent Ca²⁺ dependence of exocytosis when manipulating the Ca²⁺ current by changing the number of open channels (Johnson et al., 2005; Wong et al., 2014).

Two mechanisms have been proposed to contribute to changes in the Ca²⁺ efficiency and apparent Ca²⁺ dependence of exocytosis in IHCs around the onset of hearing: (1) the intrinsic Ca²⁺ dependence of exocytosis changes due to a switch in synaptic protein type and/or (2) tightening of the spatial coupling between Ca²⁺ channels and vesicles at the AZ. A developmental upregulation of synaptotagmin IV has been proposed to underlie the increase in Ca²⁺ efficiency and the linearization of the apparent Ca²⁺ dependence of IHC exocytosis around the onset of hearing (Johnson et al., 2010), which might support hypothesis 1.

The intrinsic Ca²⁺ dependence of exocytosis in mouse IHCs was compared before and after the onset of hearing by measuring the Ca²⁺-dependent rate constant of the fast component of exocytosis, elicited by step changes of [Ca²⁺] in response to intracellular Ca²⁺ uncaging (Wong et al., 2014). The intrinsic Ca²⁺ dependence was found to be similar, which does not support hypothesis 1. In contrast, when changing the Ca²⁺ current by manipulating the number of open channels, a developmental difference was found in the apparent Ca²⁺ dependence (or cooperativity) of exocytosis. The apparent Ca²⁺ cooperativity of exocytosis was supralinear before hearing onset but near linear in mature IHCs, suggesting a transition from Ca²⁺ micro-domain control of exocytosis before the onset of hearing to Ca²⁺ nano-domain control of exocytosis after the onset of hearing. Development of Ca²⁺ nanodomain control of exocytosis upon maturation implies tightening of the spatial coupling between Ca²⁺ influx and exocytosis, which supports hypothesis 2. Indeed, the topography of membrane-proximal vesicles, assumed to form the readily releasable pool, is more ordered around presynaptic densities after the onset of hearing (Fig. 5.3g). For more on the subjects of intrinsic and apparent Ca²⁺ cooperativity as well as Ca²⁺ microdomain and nanodomain control of exocytosis, see Sect. 5.3.3.

Unlike typical L-type Ca²⁺ currents known in other systems to be activated by high-voltage (e.g., in cardiomyocytes of the heart), the L-type Ca²⁺ currents in hair cells of the inner ear activate at relatively hyperpolarized potentials, exhibit fast activation, and undergo slow and mild inactivation (Fuchs et al., 1990; Roberts et al., 1990; Spassova et al., 2001). In mouse cochlea, the pore-forming alpha subunit is Ca_V1.3 (Platzer et al., 2000; Brandt et al., 2003; Dou et al., 2004). Without Ca²⁺ influx through this channel, IHC synaptic exocytosis is abolished (Moser & Beutner, 2000; Brandt et al., 2003) and there is profound deafness in rodents and humans (Zhang et al., 1999; Platzer et al., 2000; Baig et al., 2011).

Hair cells are thusly similar to retinal photoreceptors and bipolar neurons, which also employ L-type Ca²⁺ channels, have synaptic ribbons, and transduce graded receptor potentials for controlling transmitter release (Barnes & Hille, 1989; Heidelberger & Matthews, 1992; Tachibana et al., 1993). They are different from conventional central nervous system (CNS) synapses that use N- and P/Q-type Ca²⁺ channels for transmitter release. The number of channels depends on species, developmental stage, and AZ number which varies by tonotopic location but, on average, the number of Ca²⁺ channels per mature mouse IHC is approximately 1700, with the majority being synaptic (Brandt et al., 2005; Frank et al., 2010; Wong et al., 2014). Evidence from various technical approaches agrees that each AZ of a mature auditory hair cell has on average approximately 100 Ca²⁺ channels in the frog (Roberts et al., 1990; Issa & Hudspeth, 1996; Rodriguez-Contreras & Yamoah, 2001), turtle (Tucker & Fettiplace, 1995), and mouse (Brandt et al., 2005; Zampini et al., 2013).

IHC Ca_V1.3 currents have little Ca²⁺-dependent inactivation (CDI) and activate at relatively negative potentials (Koschak et al., 2001), likely due to the IHC-specific molecular composition of the Ca_V1.3 Ca²⁺ channel complex and specific intracellular modulators of its activity. Ca_Vß₂ was identified to be the predominant ß-subunit of IHCs that co-regulates channel inactivation and enables sufficient numbers of Ca²⁺ channels to accumulate at the AZ (Neef et al., 2009). The Ca_Vα2δ subunit(s) involved in the IHC Ca²⁺ channel remain to be identified. Calmodulin, an obligate mediator of CDI (Lee et al., 2000), is expressed in IHCs, where it regulates CDI of Ca_V1.3 channels (Grant & Fuchs, 2008). However, calmodulin-mediated CDI of Ca_V1.3 channels is antagonized by Ca²⁺ binding proteins (CaBPs), several of which are expressed in IHCs (Yang et al., 2006; Cui et al., 2007). In humans, mutation in the gene coding for CaBP2 results in hearing impairment DFNB93 (Schrauwen et al., 2012).

The list of putative regulators of the IHC Ca_V1.3 Ca²⁺ channel complex is steadily growing and includes bassoon, Rab3-interacting molecule (RIM), RIM-binding protein (Hibino et al., 2002), harmonin, and otoferlin. Of the two described mechanisms of interaction between RIM and Ca²⁺ channels, via RIM–PDZ binding to the proline-rich PDZ interacting motif in the C-terminus of Ca_Vα or via RIM C-terminal C₂ domain binding to Ca_Vß, the Ca_V1.3 Ca²⁺ channel complex seems to employ only the C₂ domain-Ca_Vß binding (Gebhart et al., 2010; Kaeser et al., 2011). Harmonin, a scaffold protein mutated in Usher 1C syndrome (Verpy et al., 2000), is an important organizer of the mechanotransduction machinery in the hair bundle. Harmonin also interacts with Ca_V1.3 via binding of its second PDZ domain to the proline-rich PDZ interacting motif in the Ca_V1.3 C-terminus (Gregory et al., 2011). In this interaction harmonin imposes an inhibition on Ca_V1.3 gating that is relieved by depolarization, thereby contributing to voltage-dependent facilitation of Ca_V1.3. In addition, harmonin appears to facilitate ubiquitination and proteasomal degradation of Ca_V1.3, potentially co-regulating the abundance of Ca²⁺ channels at the presynaptic AZ (Gregory et al., 2011). Finally, proper number and morphology of Ca_V1.3 Ca²⁺ channel clustering have been attributed to the presynaptic scaffold protein bassoon and/or its associated supramolecular ribbon nanomachine (Frank et al., 2010; Jing et al., 2013). IHCs from mice lacking function of bassoon protein had fewer ribbons and less Ca²⁺ channel immunofluorescence at AZs (Fig. 5.4a). Reduction of Ca²⁺ channel immunoreactivity was greatest at the ribbonless AZs. Because the remaining ribbons were more loosely anchored to the AZ than wild-type ribbons (Fig. 5.4b, c), the extent to which functional deficits were due to lack of bassoon alone versus disruption of the entire ribbon complex is unclear.

Presynaptic Ca²⁺ influx has been imaged in living hair cells with confocal microscopy in excised inner ear endorgans. On strong depolarization, spatially confined Ca²⁺ signals rapidly rise and decay with two time constants (Issa & Hudspeth, 1996; Frank et al., 2009), dependent on cytosolic diffusion of free and buffered Ca²⁺ (Roberts, 1993). Among IHC AZs, a marked heterogeneity of Ca²⁺ signal amplitude and voltage of half-maximal activation was observed (Frank et al., 2009). This presynaptic heterogeneity may enable the IHC to decompose sound amongst SGNs having different sensitivities, to encode the entire audible range of sound pressures at any characteristic frequency. For more about synaptic heterogeneity, see Sect. 5.5.

The ensuing Ca²⁺ signal drives rapid exocytosis of the readily releasable pool (RRP) of synaptic vesicles at the AZ, which releases glutamate onto the postsynaptic SGN bouton (Sect. 5.4). The IHC AZ has a molecular composition and structure that enables temporally precise release at high rates over long periods of time, as required for normal hearing (Moser et al., 2006; Matthews & Fuchs, 2010; Rutherford & Pangršič, 2012). The synaptic ribbon tethers synaptic vesicles to its ellipsoid-like surface. Moreover, two rows of vesicles align with the presynaptic membrane density at the base of the ribbon (Frank et al., 2010), some tethered to the plasma membrane. Because of their number and their preferential loss during stimulation, these vesicles are often considered to be the ultrastructural substrate of a finite RRP measured physiologically (Moser & Beutner, 2000; Lenzi et al., 2002). The vesicles immediately surrounding and near ribbons are thought to refill the RRP. Vesicle density can differ between high- and low-frequency hair cells, which may be an important tonotopic specialization (Schnee et al., 2005). After fusion with the plasma membrane, vesicles are regenerated via endocytosis in the perisynaptic space (Neef et al., 2014).

Sound-response properties of single auditory nerve units have been measured with extracellular electrophysiological recording of APs from the central axon of single SGNs in vivo (Kiang, 1965; Taberner & Liberman, 2005). The 1:1 connectivity between IHC AZ and SGN makes these recordings extremely valuable for understanding sound encoding at the IHC afferent synapse but also, more generally for neuroscience, because there is probably no other synaptic connection for which an in vivo readout of a single AZ exists. Computational models have used the acoustic signal as input and the APs of individual SGNs as measured output to describe cochlear filter properties mathematically (Weiss, 1966; Meddis, 2006).

To measure exocytosis of synaptic vesicles, the patch-clamp technique was applied to hair cells in inner ear explants (Parsons et al., 1994). Patch-clamp measurements of presynaptic plasma membrane capacitance allow one to monitor exocytosis and endocytosis because fusion and fission of synaptic vesicle membrane with plasma membrane cause increases and decreases, respectively, in surface area that are proportional to capacitance. Applied to the whole cell, measurements of capacitance changes report the summed activity of all synapses. On average, each AZ in a mouse IHC has RRP of about one dozen vesicles that undergoes exocytosis with a time constant of about 10 ms and is replenished with fast and slow time constants of about 140 ms and 3 s (Moser & Beutner, 2000). For single AZ measurements of exocytosis and synaptic transmission with the patch-clamp technique applied to SGN boutons, see Sect. 5.4.2.

Insights into the molecular composition of transmitter release have been provided along three main avenues of investigation: (1) candidate gene approaches driven by knowledge of conventional synapses (e.g., Safieddine & Wenthold, 1999; Nouvian et al., 2011), (2) genetics of human deafness (e.g., Yasunaga et al., 1999; Ruel et al., 2008), and (3) proteomics (Uthaiah & Hudspeth, 2010; Kantardzhieva et al., 2012; Duncker et al., 2013). The synaptic ribbon is composed primarily of the protein Ribeye (Schmitz et al., 2000), a splice variant of the transcriptional co-repressor CtBP2 that has lysophosphatidylacyl-transferase activity (Schwarz et al., 2011). The presence of ribeye at AZs seem to promote endocytic vesicle regeneration, vesicle tethering/docking/priming, and Ca²⁺-channel clustering in hair cells (Frank et al., 2010; Sheets et al., 2011; Jing et al., 2013; Khimich et al., 2005).

Some components of the presynaptic AZ machinery seem not to be conserved between conventional neuronal synapses and ribbon-type synapses of IHCs, specifically the proteins that mediate Ca²⁺ sensing and lipid membrane fusion. Otoferlin, a multi-C₂-domain ferlin protein specifically expressed in inner ear hair cells is defective in human deafness DFNB9 (Yasunaga et al., 1999) and is currently the best candidate for a vesicular Ca²⁺ sensor. Exocytosis was nearly abolished in otoferlin-deficient IHCs despite the presence of synaptic vesicles at the AZ (Roux et al., 2006). A definitive conclusion on otoferlin as a Ca²⁺ sensor of fusion will require mutagenesis of Ca²⁺ binding sites, biochemical characterization of altered Ca²⁺ binding, and physiological assessment of the Ca²⁺ dependence of IHC exocytosis with the mutant otoferlin. In addition to its putative role as Ca²⁺ sensor, otoferlin seems to facilitate vesicle replenishment (Pangršič et al., 2010).

The core membrane fusion machinery is thought to be conserved at all synapses. In neurons it consists of the soluble NSF attachment protein receptors (SNAREs) synaptobrevin 1 or 2, SNAP25, and syntaxin 1. However, experiments that used neurotoxins and genetic mutations to disable SNARE proteins indicated that IHC exocytosis may operate without neuronal SNARE proteins (Nouvian et al., 2011). Interestingly, otoferlin has been shown to interact with neuronal SNAREs (Roux et al., 2006; Ramakrishnan et al., 2009) but hair cells seem to lack SNARE regulators such as synaptotagmins (Beurg et al., 2010; Reisinger et al., 2011) and complexins (Strenzke et al., 2009; Uthaiah & Hudspeth, 2010). Investigations into the fusion machinery of IHCs are ongoing.

There is an intimate functional relationship and perhaps even direct molecular binding between release-ready vesicles and Ca²⁺ channels in a proximity of 10–30 nm. From the perspective of the Ca²⁺-sensing protein on a given release-ready synaptic vesicle, it seems that only one or very few Ca_V1.3 channels dominate the local [Ca²⁺] (Brandt et al., 2005; Goutman & Glowatzki, 2007; Graydon et al., 2011). In other words, Ca²⁺ control of exocytosis appears to operate in nanodomains. Alternatively, vesicle fusion at a given AZ may be controlled by a Ca²⁺ microdomain (Johnson et al., 2008, 2010; Heil & Neubauer, 2010), in which many Ca²⁺ channels contribute to the local [Ca²⁺] signal acting on individual vesicles.

To test the nanodomain versus microdomain hypotheses, the relative number of Ca_V1.3 channels contributing to exocytosis can be experimentally tested by studying the incremental dependence of RRP exocytosis on Ca²⁺ influx. The apparent Ca²⁺ cooperativity m is obtained by fitting a power function to the relationship between exocytosis and transmembrane Ca²⁺ charge (Q _Ca): exocytosis = A(Q _Ca) ^m , where A is the amplitude of the exocytic response and the exponent m is the apparent cooperativity. Different data points are obtained by manipulating the Ca²⁺ influx, either by changing the number of open channels or by changing the charge through each channel, while depolarizing the IHC for a brief duration to probe the RRP. If m is smaller when manipulating Ca²⁺ influx by changing the number of open Ca²⁺ channels than it is when changing the current through a given channel, then Ca²⁺ nanodomain control of exocytosis is suggested. If m is close to unity then the dependence of RRP exocytosis on Ca²⁺ influx is near linear. This implies little or no cooperativity of Ca²⁺ in its coupling to vesicle fusion and suggests nanodomain stimulus-secretion coupling. In the extreme interpretation of nanodomain, one vesicle undergoes exocytosis for each opening of a Ca²⁺ channel because a sufficient [Ca²⁺] is reached to saturate the sensor. Ca²⁺ from further channels would be insufficient. On the other hand, if comparable estimates of m are obtained for these two types of manipulation of Ca²⁺ influx (changing the number of open Ca²⁺ channels versus changing the current through a given channel), then m should be similar to the intrinsic biochemical Ca²⁺ cooperativity of IHC exocytosis (m = 4; Beutner et al., 2001). This would suggest Ca²⁺ microdomain control (Augustine et al., 1991). In a Ca²⁺ microdomain control of exocytosis, many channels must open with overlapping effects before [Ca²⁺] is high enough to evoke fusion.