Innervation Of Outer Hair Cells Research Articles

353 Eph/Ephrin Signaling Influences Innervation of Outer Hair Cells in Cochlea

OBJECTIVES/GOALS: 48,000,000 people in the U.S. have hearing loss, negatively impacting quality of life and work. Unveiling axon guidance for auditory type II spiral ganglia neurons (SGNs) will aid development of new therapies. I study the role of Eph/Ephrin and planar cell polarity (PCP) signaling during type II SGN turning and outer hair cell (OHC) innervation. METHODS/STUDY POPULATION: This quantitative study was conducted on Efna3 and Vangl2 null mice possessing Neurog1CreERT2 and R26RtdTomato mutations. Spontaneous Cre activity within the Neurogenin1CreERT2 line causes recombination and expression of fluorescent Rosa26 Reporter (R26R)tdTomatoin a restricted number of SGNs, including type IIs. Together, these lines permit SGN sparse labeling. Immunostaining and confocal imaging were used to analyze dsRed in Efna3 and Vangl2 mice and quantify type II SGN turning. In combination, Imaris 3D renderings were used to quantify type II SGN turning, branching, navigation features and temporal effects of EPHRIN-A3-Fc on type IIs via cochlear cultures (a gain-of-function manipulation). For both sexes, 5-6 cochleae per genotype were analyzed and compared by t-test to wildtype (WT) controls. RESULTS/ANTICIPATED RESULTS: Efna3 nulls showed a small rise in type II SGNs incorrectly turning toward the apex at an error rate of 16.0% compared to WTs (n=6; p=0.05). P0 Efna3 nulls had reduced branch number compared to WTs, 4.1 and 7.2, respectively (n=129; p=<0.0001), suggesting EPHRIN-A3 acts as a positive growth cue. In cochlear cultures, EPHRIN-A3-Fc led to type II SGN collapse at E15.5, indicating a repulsive function. However, at P0, EPHRIN-A3-Fc treatment led to type II SGNs with elevated branch numbers compared to Control-Fc treatment: 18.1 and 11.4, respectively (n=116; p=<0.0001). This indicates a positive growth function. At E16.5, EPHRIN-A3 protein immunoreactivity on Deiters’ and pillar cells appears reduced in Vangl2 nulls compared to WT cochleae, suggesting that EPHRIN-A3 acts downstream of PCP signaling. DISCUSSION/SIGNIFICANCE: Results suggest that Eph/Ephrin signaling acts downstream of PCP signaling to mediate type II SGN guidance and EPHRIN-A3 switches its mode of activation. The clinical implications of these findings are that therapeutics targeting EPHRIN-A3 and/or VANGL2 in their given pathways could stimulate new OHC innervation following auditory damage.

486 Molecular Mechanisms of Type II Spiral Ganglion Neuron Development

OBJECTIVES/GOALS: 30,000,000 people in the U.S. have hearing loss, negatively impacting quality of life and work. Understanding auditory axon guidance for spiral ganglia neurons (SGNs) will aid development of new therapies. I study role of Eph/Ephrin signaling in mediating type II SGN turning events, and how planar cell polarity (PCP) signaling modulates this process. METHODS/STUDY POPULATION: This quantitative study was conducted on Efna3 and Vangl2 null mice possessing Neurog1CreERT2 and R26RtdTomato mutations. Spontaneous Cre activity within the Neurogenin 1 CreERT2 line causes recombination and expression of fluorescent Rosa26 Reporter (R26R)tdTomato in a restricted number of SGNs, including type IIs. Together these lines permit SGN sparse labeling. Bulk-labeling was used for Efna3;Vangl2 double knockout (DKO) mutants. Immunostaining and confocal imaging was used to analyze dsRed in Efna3; Vangl2 and NF-200 in DKOs to quantify type II SGN turning. In combination, 3D rendering in Imaris software was used to quantify type II SGN turning, branching and other growth and navigation characteristics. 5-6 cochleae per genotype were analyzed and compared by t-test to wildtype controls. RESULTS/ANTICIPATED RESULTS: EPHRIN-A3 is expressed on the membranes of outer pillar and Deiters’cells of the cochlear epithelium. Efna3 nulls showed a small rise in type II SGNs incorrectly turning toward the apex at an error frequency of 16.9% compared to controls (n=6; p=0.05). Efna3 nulls had reduced branch number/fiber compared to controls, 4.14 and 7.22, respectively (n=129; p DISCUSSION/SIGNIFICANCE: Our results suggest that Eph/Ephrin signaling acts parallel of PCP signaling to mediate type II SGN guidance during development. The clinical implications of these findings are that therapeutics targeting EPHRIN-A3 and/or VANGL2 in this pathway could stimulate new outer hair cell innervation by type II SGNs following auditory damage.

G6PD overexpression protects from oxidative stress and age‐related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL+ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.

Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons.

Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2. We demonstrate using mice of either sex that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration-based deafness therapies.SIGNIFICANCE STATEMENT Planar cell polarity (PCP) signaling has emerged as a complementary mechanism to classical axon guidance in regulating axon track formation, axon outgrowth, and neuronal polarization. The core PCP proteins are also required for auditory circuit assembly, and coordinate hair cell innervation with the tonotopic organization of the cochlea. This is a non-cell-autonomous mechanism that requires the formation of PCP protein complexes between cochlear-supporting cells located along the trajectory of growth cone navigation. These findings are significant because they demonstrate how the fidelity of auditory circuit formation is ensured during development, and provide a mechanism by which PCP proteins may regulate axon outgrowth and guidance in the CNS.

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine‐tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non‐sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience‐independent Ca2+ signals from sensory and non‐sensory cells.

Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.

Identifying the Origin of Effects of Contralateral Noise on Transient Evoked Otoacoustic Emissions in Unanesthetized Mice

Descending neural pathways in the mammalian auditory system are known to modulate the function of the peripheral auditory system. These pathways include the medial olivocochlear (MOC) efferent innervation to outer hair cells (OHCs) and the acoustic reflex pathways mediating middle ear muscle (MEM) contractions. Based on measurements in humans (Marks and Siegel, companion paper), we applied a sensitive method to attempt to differentiate MEM and MOC reflexes using contralateral acoustic stimulation in mice under different levels of anesthesia. Separation of these effects is based on the knowledge that OHC-generated transient evoked otoacoustic emissions (TEOAE) are delayed relative to the stimulus, and that the MOC reflex affects the emission through its innervation of OHC. In contrast, the MEM-mediated changes in middle ear reflectance alter both the stimulus (with a short delay) and the emission. Using this approach, time averages to transient stimuli were evaluated to determine if thresholds for a contralateral effect on the delayed emission, indicating potential MOC activation, could be observed in the absence of a change in the stimulus pressure. This outcome was not observed in the majority of cases. There were also no statistically significant differences between MEM and putative MOC thresholds, and variability was high for both thresholds regardless of anesthesia level. Since the two reflex pathways could not be differentiated on the basis of activation thresholds, it was concluded that the MEM reflex dominates changes in TEOAEs induced by contralateral noise. This result complicates the identification of purely MOC-induced changes on OAEs in mice unless the MEM reflex is inactivated surgically or pharmacologically.

Neuroglobin Expression in the Mammalian Auditory System.

The energy-yielding pathways that provide the large amounts of metabolic energy required by inner ear sensorineural cells are poorly understood. Neuroglobin (Ngb) is a neuron-specific hemoprotein of the globin family, which is suggested to be involved in oxidative energy metabolism. Here, we present quantitative real-time reverse transcription PCR, in situ hybridization, immunohistochemical, and Western blot evidence that neuroglobin is highly expressed in the mouse and rat cochlea. For primary cochlea neurons, Ngb expression is limited to the subpopulation of type I spiral ganglion cells, those which innervate inner hair cells, while the subpopulation of type II spiral ganglion cells which innervate the outer hair cells do not express Ngb. We further investigated Ngb distribution in rat, mouse, and human auditory brainstem centers, and found that the cochlear nuclei and superior olivary complex (SOC) also express considerable amounts of Ngb. Notably, the majority of olivocochlear neurons, those which provide efferent innervation of outer hair cells as identified by neuronal tract tracing, were Ngb-immunoreactive. We also observed that neuroglobin in the SOC frequently co-localized with neuronal nitric oxide synthase, the enzyme responsible for nitric oxide production. Our findings suggest that neuroglobin is well positioned to play an important physiologic role in the oxygen homeostasis of the peripheral and central auditory nervous system, and provides the first evidence that Ngb signal differentiates the central projections of the inner and outer hair cells.

Postnatal maturation of contralateral DPOAE suppression in a precocious animal model (chinchilla) of the human neonate

Conclusion: In the neonatal chinchilla, the degree of contralateral distortion product otoacoustic emission (DPOAE) suppression and the latency and time constants of suppression are immature for 40–60 days. This suggests that olivocochlear efferent innervation of outer hair cells is not fully mature at birth in this animal model, and this may also be the case for human neonates. Objectives: To track postnatal changes in the dynamics of the olivocochlear efferent system in an animal model with cochlear development at birth similar to that in humans. Methods: Real-time measurements of contralateral DPOAE suppression were made in 79 ears of anaesthetized chinchillas, ranging in age from 1 day to 70 days. An adult control group (13 ears) was also tested. DPOAE (2f1-f2; f2 = 4.4 kHz; f2/f1 = 1.22) input/output functions were measured. Dynamics of contralateral broadband noise suppression were measured, including latency and suppression time constants. Results: DPOAE amplitude input/output functions are immature until 20–30 days postnatally. The maturation period for contralateral suppression amplitude is about 30 days. Latency of onset suppression was 40 ms at birth reducing to adult values (23 ms) at 40 days. The DPOAE suppression time constant was about 350 ms at birth and mature (230 ms) at 60 days.

Canal cristae growth and fiber extension to the outer hair cells of the mouse ear require Prox1 activity.

BackgroundThe homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.Methodology/Principal FindingsWe have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.Conclusions/SignificanceThese results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.

Postsynaptic targets of type II auditory nerve fibers in the cochlear nucleus.

Type II auditory nerve fibers, which provide the primary afferent innervation of outer hair cells of the cochlea, project thin fibers centrally and form synapses in the cochlear nucleus. We investigated the postsynaptic targets of these synapses, which are unknown. Using serial-section electron microscopy of fibers labeled with horseradish peroxidase, we examined the border of the granule-cell lamina in mice, an area of type II termination that receives branches having swellings with complex shapes. About 70% of the swellings examined with the electron microscope formed morphological synapses, which is a much higher value than found in previous studies of type II swellings in other parts of the cochlear nucleus. The high percentage of synapses enabled a number of postsynaptic targets to be identified. Most of the targets were small dendrites. Two of these dendrites were traced to their somata of origin, which were cochlear-nucleus "small cells" situated at the border of the granule-cell lamina. These cells did not appear to receive any terminals containing synaptic vesicles that were large and round, indicating a lack of input from type I auditory nerve fibers. Nor did type II swellings or targets participate in the synaptic glomeruli formed by mossy terminals and the dendrites of granule cells. Other type II synapses were axosomatic and their targets were large cells, which were presumed multipolar cells and one cell with characteristics of a globular bushy cell. These large cells almost certainly receive additional input from type I auditory nerve fibers, which provide the afferent innervation of the cochlear inner hair cells. A few type II postsynaptic targets-the two small cells as well as a large dendrite-received synapses that had accompanying postsynaptic bodies, a likely marker for synapses of medial olivocochlear branches. These targets thus probably receive convergent input from type II fibers and medial olivocochlear branches. The diverse nature of the type II targets and the examples of segregated convergence of other inputs illustrates the synaptic complexity of type II input to the cochlear nucleus.

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss.

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.

Reciprocal innervation of outer hair cells in a human infant.

Reciprocal synapses are characterized by the presence of both afferent and efferent types of synaptic specializations between two cells. They have been described at the neural poles of outer hair cells (OHCs) in humans with advanced age and two monkey species. Our objective was to study the innervation of the OHCs and determine if reciprocal synapses were present in a young (8-month-old infant) human subject. We studied the synaptic and cytoplasmic morphology of 162 nerve terminals innervating 29 OHCs using serial section transmission electron microscopy. Seventy-six percent of all OHCs were innervated by terminals with reciprocal synapses. This prevalence increased from the first toward the third row (p < 0.001), and 100% of OHCs in the third row demonstrated at least one reciprocal synapse. The prevalence of terminals with reciprocal synapses was higher in the human infant than in older human subjects and was very similar to what has been reported for the chimpanzee. Reciprocal synapses occur in sufficient numbers to be physiologically significant in primates. The nerve terminals were found to segregate into two groups on the basis of their cytoplasmic morphological characteristics: (1) vesicle-rich/neurofilament-poor (VR/NP) and (2) vesicle-poor/neurofilament-rich (VP/NR). All afferent and reciprocal terminals were of the VP/NR variety. The majority of the efferent terminals originated from VR/NP nerve fibers (classical olivocochlear morphology), but 23.5% of the efferent terminals were VP/NR. The hypothesis that peripheral processes of type II spiral ganglion cells form classical afferent, reciprocal, and a number of purely presynaptic terminals on OHCs is discussed. The presence of different types of synaptic specializations on OHCs formed by nerve fibers of the same type (VP/NR) suggests the existence of reciprocal neuronal circuits between OHCs sharing the dendritic arborization of a type II spiral ganglion cell.

The innervation of outer hair cells: 3D reconstruction from TEM serial sections in the Japanese macaque

Transmission electron micrographs from serial sections were obtained from the neural pole of outer hair cells (OHCs) in the Japanese macaque (Macaca fuscata) and reconstructions of nerve terminals were made using computer software. Data are based on observations of six cells in the basal turn, eight in the middle turn and four in the apex. In general, the number of afferent (type II) terminals on each OHC increased from base to apex, and for a given turn, the numbers appeared unrelated to OHC row. On the other hand, the number of efferent terminals was greater in the middle turn than in other areas, and the number decreased from row 1 to row 3. Reciprocal synapses increased in frequency from the upper basal turn apicalward. The total number of terminals synapsing on an individual OHC increased from base to apex by nearly 100%. Three-dimensional reconstructions showed that nerve fibers terminating on basal and middle turn OHCs ascended directly from sub-OHC regions to synapse on the subnuclear regions of the OHC. In contrast, apical turn fibers ran horizontally at the level of the subnuclear region and the terminals appeared as en passant swellings along a single fiber. Although physiological data are wanting for the macaque, the anatomical findings suggest that functional differences may exist along the length of the cochlea.

Lack of Neurotrophin 3 Causes Losses of Both Classes of Spiral Ganglion Neurons in the Cochlea in a Region-Specific Fashion

Essential functions of neurotrophin 3 (NT-3) in regulating afferent and efferent innervation of the cochlea have been characterized by comparison of normal and NT-3 mutant mice. NT-3 deficiency has striking, region-specific effects, with complete loss of sensory neurons in the basal turn and dramatic but incomplete neuronal loss in the middle and apical turns. The sensory innervation of inner and outer hair cells was reorganized in mutant animals. Instead of a strictly radial pattern of innervation, the axons of remaining sensory neurons projected spirally along the row of inner hair cells to innervate even the most basal inner hair cells. Innervation of outer hair cells was strongly reduced overall and was not detected in the basal turn. The presence of fibers extending to both inner and outer hair cells suggests that subsets of types I and II sensory neurons survive in the absence of NT-3. Likewise, projections of the cochlea to auditory nuclei of the brainstem were attenuated but otherwise present. Equally striking changes in efferent innervation were observed in mutant animals that closely mimicked the abnormal sensory innervation pattern. Despite these impressive innervation deficiencies, the morphology of the organ of Corti and the development of inner and outer hair cells appeared comparatively normal.

Synaptic specializations associated with the outer hair cells of the Japanese macaque

Across species the innervation of outer hair cells (OHCs) shows a remarkable similarity. There are, however, notable differences in fine structure. The present work describes the normal synaptic morphology of OHCs in the Japanese macaque ( Macaca fuscata), as determined by examination of serial sections with transmission electron microscopy. The nerve endings at the base of OHCs were divided primarily into two groups: vesiculated (efferent) and non-vesiculated (afferent). In addition, we found supranuclear efferent nerve endings and reciprocal synapses in all three cochlear turns. We also found presynaptic bodies in OHCs at the afferent synapse, the branching of afferent nerve fibers and axodendritic synapses between afferent and efferent fibers in the outer spiral bundle and just beneath OHCs. In terms of synaptic structure, the data indicate that the Japanese macaque is more similar to that of the human than other species examined to date.

Structural development of sensory cells in the ear

The hair cells of the auditory and balance systems of the inner ear have precise structures and orientations related to function. Hair cells differentiate from a homogenous cell population with the initiation of afferent synaptogenesis and appearance of the apical hair bundle being the first definitive structural sign of hair cell development. The cytoskeletal network then develops and the intercellular membrane junctions become more complex. As auditory function is established in mammalian cochlear hair cells, the lateral membrane acquires certain specializations. Accompanying this there is a change from afferent to efferent innervation of outer hair cells.

Complementary roles of BDNF and NT-3 in vestibular and auditory development.

The physiological role of BDNF and NT-3 in the development of the vestibular and auditory systems was investigated in mice that carry a deleted BDNF and/or NT-3 gene. BDNF was the major survival factor for vestibular ganglion neurons, and NT-3, for spiral ganglion neurons. Lack of BDNF and NT-3 did not affect ingrowth of nerve fibers into the vestibular epithelium, but BDNF mutants failed to maintain afferent and efferent innervation. In the cochlea, BDNF mutants lost type 2 spiral neurons, causing an absence of outer hair cell innervation. NT-3 mutants showed a paucity of afferents and lost 87% of spiral neurons, presumably corresponding to type 1 neurons, which innervate inner hair cells. Double mutants had an additive loss, lacking all vestibular and spiral neurons. These results show that BDNF and NT-3 are crucial for inner ear development and, although largely coexpressed, have distinct and nonoverlapping roles in the vestibular and auditory systems.

A transient afferent innervation of outer hair cells in the postnatal cochlea

In vitro retrograde labeling techniques were used to reconstruct the peripheral terminations of afferent fibers to the outer hair cells in postnatal hamsters. Between postnatal days 4 and 6, both radial and spiral afferent fibers terminated on outer hair cells. The cell bodies which gave rise to such fibers were not distinguishable. These outer radial-fiber terminations may be a transient phenomenon since they were not observed either before or after this period.