Electrophysiological correlates of progressive sensorineural pathology in carboplatin-treated chinchillas

Carboplatin produces progressive damage to auditory nerve fibers, spiral ganglion neurons (SGNs) and inner hair cells (IHC) in the chinchilla cochlea but leaves outer hair cells intact. Within 1 h after injection, many afferent terminals beneath IHCs and myelin lamellae surrounding SGN processes are vacuolated. One day after injection, approximately half of the nerve fibers are missing. IHCs are intact at 2 days, but 20-30% are missing at 3 days. We studied the electrophysiological correlates of this progressive morphological damage by recording cochlear microphonics (CM), distortion product otoacoustic emissions (DPOAE), summating potentials (SP), compound action potentials (CAP) and midbrain evoked potentials (IC-EVP) before and 1 h, 12 h, 1 days, 3 days, 5 days, 7 days and 14 days after carboplatin injection (75 mg/kg IP) in four chinchillas. CM and DPOAEs tended to be unchanged or enhanced. CAP and SP showed little change until Day 3, when amplitudes were reduced in all animals and CAP thresholds were elevated by 9 dB; amplitudes declined further between Days 3 and 5 but not thereafter. IC-EVP amplitudes decreased on Days 3 or 5 but thresholds were relatively unchanged. All animals showed some recovery of IC-EVP between Days 7 and 14, including one with 70% enhancement on Day 14. The results indicate that threshold and amplitude measures fail to detect peripheral pathology until some relatively high threshold level of damage has been exceeded. This has important implications for monitoring peripheral damage and interpreting electrophysiological test results in animals and humans.     

Deafferentation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-week-delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate- and delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers.     

Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.     

Schwann cells revert to non-myelinating phenotypes in the deafened rat cochlea

Loss of sensory hair cells within the cochlea results in a permanent sensorineural hearing loss and initiates the gradual degeneration of spiral ganglion neurons (SGNs) - the primary afferent neurons of the cochlea. While these neurons are normally myelinated via Schwann cells, loss of myelin occurs as a precursor to neural degeneration. However, the relationship between demyelination and the status of Schwann cells in deafness is not well understood. We used a marker of peripheral myelin (myelin protein zero; P0) and a marker of Schwann cells (S100) to determine the temporal sequence of myelin and Schwann cell loss as a function of duration of deafness. Rat pups were systemically deafened for periods ranging from 2 weeks to greater than 6 months by co-administration of frusemide and gentamicin. Cochleae were cryosectioned and quantitative immunohistochemistry used to determine the extent of P0 and S100 labelling within the peripheral processes, SGN soma and their central processes within the modiolus. SGN density was also determined for each cochlear turn. P0 labelling decreased throughout the cochlea with increasing duration of deafness. The reduction in P0 labelling occurred at a faster rate than the SGN loss. In contrast, S100 labelling was not significantly reduced compared with age-matched controls in any cochlear region until 6 months post-deafening. These results suggest that Schwann cells may revert to non-myelinating phenotypes in response to deafness and exhibit greater survival traits than SGNs. The potential clinical significance of these findings for cochlear implants is discussed.     

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.     

Developmental segregation in the efferent projections to auditory hair cells in the gerbil

The auditory receptor epithelium of mammals receives efferent innervation from neurons within and surrounding the superior olivary complex of the brainstem (Warr [1975] J. Comp. Neurol. 161:159-181). Disruption of this pathway during early postnatal life, when olivocochlear axons are forming their final connections with auditory hair cells and nerve fibers, can lead to profound and permanent hearing impairments (Walsh et al. [1998] J. Neurosci. 18:3859-3869). Identification of the possible causes for this deterioration in auditory function requires a better understanding of the normal developmental interactions that occur between efferent axons and their target cells within the cochlea. To provide such information, we labeled developing efferent fibers at a constant location within the gerbil cochlea by using the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI). The terminal arbors of these neurons were then reconstructed by using digital confocal microscopy. By postnatal day (P) 2, the efferent arbors associated with inner hair cells (IHCs) and outer hair cells (OHCs) displayed distinctly different morphologies closely resembling those described for adult animals (Brown [1987] J. Comp. Neurol. 260:605-619). Unlike their mature counterparts, however, P2 efferent axons frequently branched to contact both types of auditory hair cells. Unexpectedly, between P4 and P6, both IHC and OHC efferent axons produced additional branches that crossed the tunnel of Corti to invade the OHC zone. By P8, all of these supernumerary connections were eliminated, yielding completely segregated efferent pathways to IHCs and OHCs.     

Chronic neurotrophin delivery promotes ectopic neurite growth from the spiral ganglion of deafened cochleae without compromising the spatial selectivity of cochlear implants

Cochlear implants restore hearing cues in the severe–profoundly deaf by electrically stimulating spiral ganglion neurons (SGNs). However, SGNs degenerate following loss of cochlear hair cells, due at least in part to a reduction in the endogenous neurotrophin (NT) supply, normally provided by hair cells and supporting cells of the organ of Corti. Delivering exogenous NTs to the cochlea can rescue SGNs from degeneration and can also promote the ectopic growth of SGN neurites. This resprouting may disrupt the cochleotopic organization upon which cochlear implants rely to impart pitch cues. Using retrograde labeling and confocal imaging of SGNs, we determined the extent of neurite growth following 28 days of exogenous NT treatment in deafened guinea pigs with and without chronic electrical stimulation (ES). On completion of this treatment, we measured the spread of neural activation to intracochlear ES by recording neural responses across the cochleotopically organized inferior colliculus using multichannel recording techniques. Although NT treatment significantly increased both the length and the lateral extent of growth of neurites along the cochlea compared with deafened controls, these anatomical changes did not affect the spread of neural activation when examined immediately after 28 days of NT treatment. NT treatment did, however, result in lower excitation thresholds compared with deafened controls. These data support the application of NTs for improved clinical outcomes for cochlear implant patients. J. Comp. Neurol. 521:2818–2832, 2013. © 2013 Wiley Periodicals, Inc.     

Distribution of the crossed olivocochlear bundle in the chinchilla's cochlea

The efferent crossed olivocochlear bundle (COCB) was transected in the brain stem of the chinchilla, and the animals sacrificed 7 to 96 days later. Electron microscopy revealed that all the large efferent nerve endings on outer hair cells in the basalmost 2 mm (round window region) of the cochlea had degenerated, 87.5% in the remainder of the first turn, 70% in the second turn and 43% in the third turn. Only a few degenerating nerve fibers were seen in the medial spiral tract (inner spiral and tunnel bundles) of the experimental animals. Nerve fibers were counted in the medial spiral tracts of the cochleas of control animals as well as in those animals whose COCB had been transected. There were considerable individual variations in the fiber numbers, and statistical analysis showed no significant difference between the numbers of nerve fibers in normal and experimental animals. The cochlear microphonics (CM) and nerve action potentials (AP) of acute animals were assessed before and after COCB section. The CM and AP of the chronic experimental animals were compared with responses from normals. Overall, no changes in a physiological response of the anesthetized chinchilla could be attributed to complete section of the COCB.     

Combining Cell-Based Therapies and Neural Prostheses to Promote Neural Survival

Cochlear implants provide partial restoration of hearing for profoundly deaf patients by electrically stimulating spiral ganglion neurons (SGNs); however, these neurons gradually degenerate following the onset of deafness. Although the exogenous application of neurotrophins (NTs) can prevent SGN loss, current techniques to administer NTs for long periods of time have limited clinical applicability. We have used encapsulated choroid plexus cells (NTCells; Living Cell Technologies, Auckland, New Zealand) to provide NTs in a clinically viable manner that can be combined with a cochlear implant. Neonatal cats were deafened and unilaterally implanted with NTCells and a cochlear implant. Animals received chronic electrical stimulation (ES) alone, NTs alone, or combined NTs and ES (ES + NT) for a period of as much as 8 months. The opposite ear served as a deafened unimplanted control. Chronic ES alone did not result in increased survival of SGNs or their peripheral processes. NT treatment alone resulted in greater SGN survival restricted to the upper basal cochlear region and an increased density of SGN peripheral processes. Importantly, chronic ES in combination with NTs provided significant SGN survival throughout a wider extent of the cochlea, in addition to an increased peripheral process density. Re-sprouting peripheral processes were observed in the scala media and scala tympani, raising the possibility of direct contact between peripheral processes and a cochlear implant electrode array. We conclude that cell-based therapy is clinically viable and effective in promoting SGN survival for extended durations of cochlear implant use. These findings have important implications for the safe delivery of therapeutic drugs to the cochlea.     

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: Evidence for a waiting period

Reconstructions of the efferent innervation of the hamster (Mesocricetus auratus) cochlea were done during postnatal development. Efferent neurons were labeled via injections of biocytin and horseradish peroxidase into the crossed olivocochlear (OC) bundles using an in vitro brainstem technique. Such injections retrogradely labeled cell bodies in ventral periolivary regions of the superior olive consistent with their being medial OC neurons. Anterogradely labeled axons were traced to the cochlea, where they terminated on or below inner hair cells (IHCs) prior to postnatal day 5 (P5). After P5, labeled axons terminated on IHCs and outer hair cells (OHCs) and after P10, the majority of labeled axons terminated on the OHCs. In the electron microscope, small labeled terminals containing densely packed synaptic vesicles were found both adjacent to IHCs (axosomatic) as well as apposed to afferent and efferent fibers below IHCs prior to P5. By P10, large labeled terminals were axosomatic to OHCs and no longer found on IHCs. Consistent with previous reports, these data suggest that medial OC axons form part of an early primary innervation on and below IHCs before terminating on OHCs. This raises the possibility that OC neurons demonstrate a period of waiting below an intermediate target similar to that described in the development of thalamocortical projections. © 1996 Wiley-Liss, Inc.     

Calpain inhibitors protect auditory sensory cells from hypoxia and neurotrophin-withdrawal induced apoptosis

Inhibitors of calpain have been shown to protect nerve growth factor (NGF)-deprived ciliary ganglion neurons and hypoxic cortical neurons. Calpains have been identified in the cochlea and are active during ischemic injury. Since apoptosis can be initiated by loss of neurotrophic support, hypoxia, and ototoxins (e.g., cisplatin, CDDP), the role of calpain inhibitors under these conditions was examined in auditory hair cells and neurons. Dissociated spiral ganglion neuron (SGN) cell cultures and organ of Corti explants from P3 rats were used to test the efficacy of calpain inhibitors as otoprotective molecules. Our results indicate that calpain inhibitor I, calpain inhibitor II, and leupeptin all provided significant protection of SGNs against neurotrophin-withdrawal and hypoxia-induced apoptosis. The increase in neuronal survival ranged from 2.16 to 2.31 times greater than in untreated neurotrophin-withdrawn SGN cell cultures. BOC-Asp(Ome)-Fluoromethyl Ketone (B-D-FMK), a general caspase inhibitor, increased neuronal survival 2.16 times more. Neuronal survival rates were from 1.88 to 2.27 times greater than in untreated, hypoxic neurons and hair cell survival rates were from 1.98 to 2.03 times greater than untreated, hypoxic organ of Corti explants. However, protection of auditory hair cells and neurons from CDDP-induced damage (10 and 6 μg/ml, respectively) was limited with any of these calpain inhibitors. Apoptotic pathways initiated by neurotrophin-deprivation and ototoxic stress (e.g., CDDP) have been shown to be different. Our results agree with this finding, with neurotrophin-withdrawal and hypoxia, but not CDDP damage-induced apoptosis being calpain-dependent.     

Synaptophysin in the developing cochlea

The immunoreactivity to SY38 (anti-synaptophysin antibody) was investigated in rat and guinea-pig cochleas during development. In rat pups SY38 reactivity first appeared in the inner spiral bundle (below inner hair cells) at postnatal day 3. Later on (days 10 and 15) the basal pole of outer hair cells (OHCs) was also reactive. In fetal guinea-pigs, the inner spiral bundle was reactive on day 45 of gestation, while the reactivity occurred below OHCs on day 62 of gestation. A preliminary electron microscopic finding (from a guinea-pig 62 days of gestation) indicated that SY38 immunoreactivity is localized within varicosities of efferent (olivo-cochlear) endings. Synaptophysin is thus present in the cochlea at the level where the two efferent systems terminate. Moreover, the occurrence of SY38 immunoreactivity, first at the ISB then at the OHC levels, is in accordance with the observation that the maturation of lateral efferents preceeds that of medial efferents.     

Unmyelinated auditory type I spiral ganglion neurons in congenic Ly5.1 mice

With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics, including 1) large aggregates of unmyelinated SGNs in the apical and middle turns, 2) symmetrical junction‐like contacts between the unmyelinated neurons, 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters, 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells, 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies compared with control mice with similar ABR threshold, and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the “human‐like” feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties. J. Comp. Neurol. 518:3254–3271, 2010. © 2010 Wiley‐Liss, Inc.     

Current concepts in cochlear ribbon synapse formation

In mammals, hair cells and spiral ganglion neurons (SGNs) in the cochlea together are sophisticated “sensorineural” structures that transduce auditory information from the outside world into the brain. Hair cells and SGNs are joined by glutamatergic ribbon‐type synapses composed of a molecular machinery rivaling in complexity the mechanoelectric transduction components found at the apical side of the hair cell. The cochlear hair cell ribbon synapse has received much attention lately because of recent and important findings related to its damage (sometimes termed “synaptopathy”) as a result of noise overexposure. During development, ribbon synapses between type I SGNs and inner hair cells form in the time window between birth and hearing onset and is a process coordinated with type I SGN myelination, spontaneous activity, synaptic pruning, and innervation by efferents. In this review, we highlight new findings regarding the diversity of type I SGNs and inner hair cell synapses, and the molecular mechanisms of selective hair cell targeting. Also discussed are cell adhesion molecules and protein constituents of the ribbon synapse, and how these factors participate in ribbon synapse formation. We also note interesting new insights into the morphological development of type II SGNs, and the potential for cochlear macrophages as important players in protecting SGNs. We also address recent studies demonstrating that the structural and physiological profiles of the type I SGNs do not reach full maturity until weeks after hearing onset, suggesting a protracted development that is likely modulated by activity.     

Prosurvival and proapoptotic intracellular signaling in rat spiral ganglion neurons in vivo after the loss of hair cells

Neurons depend on afferent input for survival. Rats were given daily kanamycin injections from P8 to P16 to destroy hair cells, the sole afferent input to spiral ganglion neurons (SGNs). Most SGNs die over an approximately 14-week period after deafferentation. During this period, the SGN population is heterogeneous. At any given time, some SGNs exhibit apoptotic markers--TUNEL and cytochrome c loss--whereas others appear nonapoptotic. We asked whether differences among SGNs in intracellular signaling relevant to apoptotic regulation could account for this heterogeneity. cAMP response element binding protein (CREB) phosphorylation, which reflects neurotrophic signaling, is reduced in many SGNs at P16, P23, and P32, when SGNs begin to die. In particular, nearly all apoptotic SGNs exhibit reduced phospho-CREB, implying that apoptosis is due to insufficient neurotrophic support. However, >32% of SGNs maintain high phospho-CREB levels, implying access to neurotrophic support. By P60, when approximately 50% of the SGNs have died, phospho-CREB levels in surviving neurons are not reduced, and SGN death is no longer correlated with reduced phospho-CREB. Activity in the proapoptotic Jun N-terminal kinase (JNK)-Jun signaling pathway is elevated in SGNs during the cell death period. This too is heterogeneous: <42% of the SGNs exhibited high phospho-Jun levels, but nearly all SGNs undergoing apoptosis exhibited elevated phospho-Jun. Thus, heterogeneity among SGNs in prosurvival and proapoptotic signaling is correlated with apoptosis. SGN death following deafferentation has an early phase in which apoptosis is correlated with reduced phospho-CREB and a later phase in which it is not. Proapoptotic JNK-Jun signaling is tightly correlated with SGN apoptosis.     

Ouabain-Induced Cochlear Degeneration in Rat

Ouabain, a potent inhibitor of the Na+/K+-ATPase pump, selectively destroys spiral ganglion neurons (SGNs) in gerbils and mice, whereas in guinea pigs it preferentially damages cochlear hair cells. To elucidate the effects of ouabain on the rat inner ear, a species widely used in research, 5 μl of 1 or 10 mM ouabain was applied to the round window membrane. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses (ABR) were used to identify functional deficits in hair cells and neurons, respectively, and histological techniques were used to characterize cochlear pathologies. High-frequency ABR thresholds were elevated after treatment with 1 mM ouabain, whereas DPOAEs remained normal. In contrast, 10 mM ouabain increased ABR thresholds and reduced DPOAE amplitudes. Consistent with the physiological changes, 1 mM ouabain only damaged the SGNs and auditory nerve fibers in the basal turn of the cochlea whereas 10 mM ouabain destroyed both SGNs and cochlear hair cells; damage was greatest near the base and decreased toward the apex. The nuclei of degenerating SGNs and hair cells were condensed and fragmented and many cells were TUNEL-positive, morphological features of apoptotic cell death. Thus, ouabain-induced cochlear degeneration in rats is apoptotic and concentration dependent; low concentrations preferentially damage SGNs in the base of the cochlea, producing an animal model of partial auditory neuropathy, whereas high concentrations damage both hair cells and SGNs with damage decreasing from the base toward the apex.     

Semaphorin 3A induces acute changes in membrane excitability in spiral ganglion neurons in vitro

The development and survival of spiral ganglion neurons (SGNs) are dependent on multiple trophic factors as well as membrane electrical activity. Semaphorins (Sema) constitute a family of membrane‐associated and secreted proteins that have garnered significant attention as a potential SGN “navigator” during cochlea development. Previous studies using mutant mice demonstrated that Sema3A plays a role in the SGN pathfinding. The mechanisms, however, by which Sema3A shapes SGNs firing behavior are not known. In these studies, we found that Sema3A plays a novel role in regulating SGN resting membrane potential and excitability. Using dissociated SGN from pre‐hearing (P3–P5) and post‐hearing mice (P12–P15), we recorded membrane potentials using whole‐cell patch clamp recording techniques in apical and basal SGN populations. Recombinant Sema3A was applied to examine the effects on intrinsic membrane properties and action potentials evoked by current injections. Apical and basal SGNs from newborn mice treated with recombinant Sema3A (100 ng/ml) displayed a higher resting membrane potential, higher threshold, decreased amplitude, and prolonged latency and duration of spikes. Although a similar phenomenon was observed in SGNs from post‐hearing mice, the resting membrane potential was essentially indistinguishable before and after Sema3A exposure. Sema3A‐mediated changes in membrane excitability were associated with a significant decrease in K⁺ and Ca²⁺ currents. Sema3A acts through linopirdine‐sensitive K⁺ channels in apical, but not in the basal SGNs. Therefore, Sema3A induces differential effects in SGN membrane excitability that are dependent on age and location, and constitutes an additional early and novel effect of Sema3A SGNs in vitro.     

Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5-9 d postnatal) rat cochlea. A "high potassium puffer" was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02-0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A "receptive field" map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 μm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1-11). EPSCs evoked from presynaptic OHCs separated by >100 μm did not differ in amplitude or waveform, implying that the type II fiber's length constant exceeded the length of the synaptic input zone. Together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.     

Hair cell innervation by spiral ganglion neurons in the mouse

Horseradish peroxidase (HRP) was injected extracellularly into the auditory nerve of adult mice so that the enzyme could infuse individual spiral ganglion neurons. Forty-two well-stained neurons were reconstructed through serial sections from their cell bodies to peripheral terminations in the organ of Corti with the aid of a light microscope and drawing tube. No neuron was observed to innervate both inner and outer hair cells (IHCs and OHCs). Previous observations from neonatal mammals that reported that IHCs and OHCs were innervated by the same neuron are thus presumed to describe a transient developmental phenomenon. Two populations of spiral ganglion neurons were determined on the basis of the differences in receptor innervation. The type I neurons innervated exclusively IHCs by way of thick (1-2 microns) radial fibers, whereas the type II neurons innervated only OHCs by way of thin (approximately 0.5 micron) outer spiral fibers. Certain features of the peripheral process in the vicinity of the cell body were highly correlated with fiber type. This pattern of separate innervation of IHCs and OHCs by type I and type II neurons, respectively, may represent the general plan of afferent organization for the adult mammalian cochlea.