Mark A. Rutherford

Bassoon and the synaptic ribbon organize Ca2+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca²+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca²+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca²+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca²+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca²+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca²+ channels and vesicles, and (2) promote vesicle replenishment.

Spike Encoding of Neurotransmitter Release Timing by Spiral Ganglion Neurons of the Cochlea

Mammalian cochlear spiral ganglion neurons (SGNs) encode sound with microsecond precision. Spike triggering relies upon input from a single ribbon-type active zone of a presynaptic inner hair cell (IHC). Using patch-clamp recordings of rat SGN postsynaptic boutons innervating the modiolar face of IHCs from the cochlear apex, at room temperature, we studied how spike generation contributes to spike timing relative to synaptic input. SGNs were phasic, firing a single short-latency spike for sustained currents of sufficient onset slope. Almost every EPSP elicited a spike, but latency (300–1500 μs) varied with EPSP size and kinetics. When current-clamp stimuli approximated the mean physiological EPSC (≈300 pA), several times larger than threshold current (rheobase, ≈50 pA), spikes were triggered rapidly (latency, ≈500 μs) and precisely (SD, <50 μs). This demonstrated the significance of strong synaptic input. However, increasing EPSC size beyond the physiological mean resulted in less-potent reduction of latency and jitter. Differences in EPSC charge and SGN baseline potential influenced spike timing less as EPSC onset slope and peak amplitude increased. Moreover, the effect of baseline potential on relative threshold was small due to compensatory shift of absolute threshold potential. Experimental first-spike latencies in response to a broad range of stimuli were predicted by a two-compartment exponential integrate-and-fire model, with latency prediction error of <100 μs. In conclusion, the close anatomical coupling between a strong synapse and spike generator along with the phasic firing property lock SGN spikes to IHC exocytosis timing to generate the auditory temporal code with high fidelity.

Disruption of the Presynaptic Cytomatrix Protein Bassoon Degrades Ribbon Anchorage, Multiquantal Release, and Sound Encoding at the Hair Cell Afferent Synapse

Inner hair cells (IHCs) of the cochlea use ribbon synapses to transmit auditory information faithfully to spiral ganglion neurons (SGNs). In the present study, we used genetic disruption of the presynaptic scaffold protein bassoon in mice to manipulate the morphology and function of the IHC synapse. Although partial-deletion mutants lacking functional bassoon (BsnΔEx4/5) had a near-complete loss of ribbons from the synapses (up to 88% ribbonless synapses), gene-trap mutants (Bsngt) showed weak residual expression of bassoon and 56% ribbonless synapses, whereas the remaining 44% had a loosely anchored ribbon. Patch-clamp recordings and synaptic CaV1.3 immunolabeling indicated a larger number of Ca2+ channels for Bsngt IHCs compared with BsnΔEx4/5 IHCs and for Bsngt ribbon-occupied versus Bsngt ribbonless synapses. An intermediate phenotype of Bsngt IHCs was also found by membrane capacitance measurements for sustained exocytosis, but not for the size of the readily releasable vesicle pool. The frequency and amplitude of EPSCs were reduced in BsnΔEx4/5 mouse SGNs, whereas their postsynaptic AMPA receptor clusters were largely unaltered. Sound coding in SGN, assessed by recordings of single auditory nerve fibers and their population responses in vivo, was similarly affected in Bsngt and BsnΔEx4/5 mice. Both genotypes showed impaired sound onset coding and reduced evoked and spontaneous spike rates. In summary, reduced bassoon expression or complete lack of full-length bassoon impaired sound encoding to a similar extent, which is consistent with the comparable reduction of the readily releasable vesicle pool. This suggests that the remaining loosely anchored ribbons in Bsngt IHCs were functionally inadequate or that ribbon independent mechanisms dominated the coding deficit.

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis.

Cochlear inner hair cells (IHCs) develop from pre‐sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) CaV1.3 channels formed stripe‐like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic CaV1.3‐channels were selectively reduced, (iv) the intrinsic Ca2+ dependence of fast exocytosis probed by Ca2+ uncaging remained unchanged but (v) the apparent Ca2+ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca2+ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca2+ influx through an individual channel dominates the [Ca2+] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca2+ influx and exocytosis.

Concurrent Maturation of Inner Hair Cell Synaptic Ca2+ Influx and Auditory Nerve Spontaneous Activity around Hearing Onset in Mice

Hearing over a wide range of sound intensities is thought to require complementary coding by functionally diverse spiral ganglion neurons (SGNs), each changing activity only over a subrange. The foundations of SGN diversity are not well understood but likely include differences among their inputs: the presynaptic active zones (AZs) of inner hair cells (IHCs). Here we studied one candidate mechanism for causing SGN diversity—heterogeneity of Ca2+ influx among the AZs of IHCs—during postnatal development of the mouse cochlea. Ca2+ imaging revealed a change from regenerative to graded synaptic Ca2+ signaling after the onset of hearing, when in vivo SGN spike timing changed from patterned to Poissonian. Furthermore, we detected the concurrent emergence of stronger synaptic Ca2+ signals in IHCs and higher spontaneous spike rates in SGNs. The strengthening of Ca2+ signaling at a subset of AZs primarily reflected a gain of Ca2+ channels. We hypothesize that the number of Ca2+ channels at each IHC AZ critically determines the firing properties of its corresponding SGN and propose that AZ heterogeneity enables IHCs to decompose auditory information into functionally diverse SGNs.

Spikes and Membrane Potential Oscillations in Hair Cells Generate Periodic Afferent Activity in the Frog Sacculus

To look for membrane potential oscillations that may contribute to sensory coding or amplification in the ear, we made whole-cell and perforated-patch recordings from hair cells and postsynaptic afferent neurites in the explanted frog sacculus, with mechanoelectrical transduction (MET) blocked. Small depolarizing holding currents, which may serve to replace the in vivo resting MET current, evoked all-or-none calcium spikes (39–75 mV amplitude) in 37% of hair cells tested, and continuous membrane potential oscillations (14–28 mV; 15–130 Hz) in an additional 14% of cells. Spiking hair cells were on average taller and thinner than nonspiking hair cells, and had smaller outward currents through delayed rectifier channels (IKV) and noninactivating calcium-activated potassium channels (IBK,steady), and larger inward rectifier currents (IK1). Some spiking hair cells fired only a brief train at the onset of a current step, but others could sustain repetitive firing (3–70 Hz). Partial blockade of IBK changed the amplitude and frequency of oscillations and spikes, and converted some nonspiking cells into spiking cells. Oscillatory hair cells preferentially amplified sinusoidal stimuli at frequencies near their natural oscillation frequency. Postsynaptic recordings revealed regularly timed bursts of EPSPs in some afferent neurites. EPSP bursts were able to trigger afferent spikes, which may be initiated at the sodium channel cluster located adjacent to the afferent axons most peripheral myelin segment. These results show that some frog saccular hair cells can generate spontaneous rhythmic activity that may drive periodic background activity in afferent axons.

Hair cells use active zones with different voltage dependence of Ca2+ influx to decompose sounds into complementary neural codes

Significance We hear sounds varying in intensity over six orders of magnitude using spiral ganglion neurons (SGNs), each of which changes its firing rates over only a fraction of this range. Somehow, the SGNs with different dynamic ranges collectively encode the full range of sound levels represented in the receptor potential of the inner hair cell (IHC) in the mammalian cochlea. Our study, combining subcellular imaging, mouse genetics, and auditory systems physiology, offers a unifying synaptic hypothesis for wide dynamic range sound encoding in the spiral ganglion. We propose that IHCs, from one receptor potential but via presynaptic active zones that vary in the voltage dependence of Ca2+ influx, generate complementary codes on sound pressure level in functionally distinct SGNs. For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca2+ influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca2+ influx ranged over ∼20 mV. Ca2+ influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca2+ channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca2+ influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca2+ influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca2+ influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca2+ channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.

Molecular anatomy and physiology of exocytosis in sensory hair cells

Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca(2+)-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular-anatomical specializations that underlie the unique synaptic physiology of hair cells.

Maturation of NaV and KV Channel Topographies in the Auditory Nerve Spike Initiator before and after Developmental Onset of Hearing Function

Auditory nerve excitation and thus hearing depend on spike-generating ion channels and their placement along the axons of auditory nerve fibers (ANFs). The developmental expression patterns and native axonal locations of voltage-gated ion channels in ANFs are unknown. Therefore, we examined the development of heminodes and nodes of Ranvier in the peripheral axons of type I ANFs in the rat cochlea with immunohistochemistry and confocal microscopy. Nodal structures presumably supporting presensory spiking formed between postnatal days 5 (P5) and P7, including Ankyrin-G, NaV1.6, and Caspr. These immature nodal structures lacked low-voltage-activated KV1.1 which was not enriched at juxtaparanodes until approximately P13, concurrent with the developmental onset of acoustic hearing function. Anatomical alignment of ANF spike-initiating heminodes relative to excitatory input from inner hair cell (IHC) ribbon synapses continued until approximately P30. High-voltage-activated KV3.1b and KV2.2 were expressed in mutually exclusive domains: KV3.1b was strictly localized to nodes and heminodes, whereas KV2.2 expression began at the juxtaparanodes and continued centrally along the first internode. At spike-initiating heminodes in the distal osseous spiral lamina, NaV1.1 partly overlapped NaV1.6 and ankyrin-G. ANFs displayed KV7.2 and KV7.3 at heminodes, nodes, internodes, and the unmyelinated synaptic terminal segments beneath IHCs in the organ of Corti. In response to sound, spikes are initiated at the heminode, which is tightly coupled to the IHC ribbon synapse ∼20–40 μm away. These results show that maturation of nodal alignment and ion channel content may underlie postnatal improvements of ANF excitability and discharge synchrony. SIGNIFICANCE STATEMENT Acoustic and electrical hearing depends on rapid, reliable, and precise spike generation in auditory nerve fibers. A limitation of current models and therapies is a lack of information on the identities and topographies of underlying ion channels. We report the developmental profile of the auditory nerve spike generator with a focus on NaV1.1, NaV1.6, KV1.1, KV2.2, KV3.1b, KV7.2, and KV7.3 in relation to the scaffold ankyrin-G. Molecular anatomy of the spike generator matures in the weeks after developmental onset of hearing function. Subcellular positioning of voltage-gated ion channels will enable multicompartmental modeling of auditory nerve responses elicited by afferent chemical neurotransmission from hair cells and modulated by efferent neurotransmitters or evoked by extracellular field stimulation from a cochlear implant.

Resolving the structure of inner ear ribbon synapses with STED microscopy

Synapses are diverse in form and function; however, the mechanisms underlying this diversity are poorly understood. To illuminate structure/function relationships, robust analysis of molecular composition and morphology is needed. The molecular‐anatomical components of synapses—vesicles, clusters of voltage‐gated ion channels in presynaptic densities, arrays of transmitter receptors in postsynaptic densities—are only tens to hundreds of nanometers in size. Measuring the topographies of synaptic proteins requires nanoscale resolution of their molecularly specific labels. Super‐resolution light microscopy has emerged to meet this need. Achieving 50 nm resolution in thick tissue, we employed stimulated emission depletion (STED) microscopy to image the functionally and molecularly unique ribbon‐type synapses in the inner ear that connect mechano‐sensory inner hair cells to cochlear nerve fibers. Synaptic ribbons, bassoon protein, voltage‐gated Ca2+ channels, and glutamate receptors are inhomogeneous in their spatial distributions within synapses; the protein clusters assume variations of shapes typical for each protein specifically at cochlear afferent synapses. Heterogeneity of substructure among these synapses may contribute to functional differences among auditory nerve fibers. The morphology of synaptic voltage‐gated Ca2+ channels matures over development in a way that depends upon bassoon protein, which aggregates in similar form. Functional properties of synaptic transmission appear to depend on voltage‐gated Ca2+ channel cluster morphology and position relative to synaptic vesicles. Super‐resolution light microscopy is a group of techniques that complement electron microscopy and conventional light microscopy. Although technical hurdles remain, we are beginning to resolve the details of molecular nanoanatomy that relate mechanistically to synaptic function. Synapse, 69:242–255, 2015. ©2015 Wiley Periodicals, Inc.