Degeneration in the ventral cochlear nucleus after severe noise damage in mice.

To study the mechanisms of noise-induced hearing loss and the phantom noise, or tinnitus, often associated with it, we used a mouse model of noise damage designed for reproducible and quantitative structural analyses. We selected the posteroventral cochlear nucleus, which has shown considerable plasticity in past studies, and correlated its changes with the distribution of neurotrophin 3 (NT3). We used volume change, optical density analysis, and microscopic cluster analysis to measure the degeneration after noise exposure. There was a fluctuation pattern in the reorganization of nerve terminals. The data suggest that the source and size of the nerve terminals affect their capacity for regeneration. We hypothesize that the deafferentation of ventral cochlear nucleus is the structural basis of noise-induced tinnitus. In addition, the immunofluorescent data show a possible connection between NT3 and astrocytes. There appears to be a compensatory process in the supporting glial cells during this degeneration. Glia may play a role in the mechanisms of noise-induced hearing loss.

Postnatal development of NT3 and TrkC in mouse ventral cochlear nucleus.

In the developing nervous system, neurotrophin 3 (NT3) and brain-derived neurotrophic factor (BDNF) have been shown to interact with each other and with different parts of a neuron or glia and over considerable distances in time and space. The auditory system provides a useful model for analyzing these events, insofar as it is subdivided into well-defined groups of specific neuronal types that are readily related to each other at each stage of development. Previous work in our laboratory suggested that NT3 and its receptor TrkC in the mouse cochlear nucleus (CN) may be involved in directing neuronal migration and initial targeting of inputs from cochlear nerve axons in the embryo. NT3 is hard to detect soon after birth, but TrkC lingers longer. Here we found NT3 and TrkC around P8 and the peak around P30. Prominent in ventral CN, associated with globular bushy cells and stellate cells, they were localized to different subcellular sites. The TrkC immunostain was cytoplasmic, and that of NT3 was axonal and perisomatic. TrkC may be made by CN neurons, whereas NT3 has a cochlear origin. The temporal pattern of their development and the likelihood of activity-dependent release of NT3 from cochlear axons suggest that it may not be critical in early synaptogenesis; it may provide long-term trophic effects, including stabilization of synapses once established. Activity-related regulation could coordinate the supply of NT3 with inner ear activity. This may require interaction with other neurotrophins, such as BDNF.

Inactivation of fibroblast growth factor receptor signaling in myelinating glial cells results in significant loss of adult spiral ganglion neurons accompanied by age-related hearing impairment.

Hearing loss has been attributed to many factors, including degeneration of sensory neurons in the auditory pathway and demyelination along the cochlear nerve. Fibroblast growth factors (FGFs), which signal through four receptors (Fgfrs), are produced by auditory neurons and play a key role in embryonic development of the cochlea and in neuroprotection against sound-induced injury. However, the role of FGF signaling in the maintenance of normal auditory function in adult and aging mice remains to be elucidated. Furthermore, the contribution of glial cells, which myelinate the cochlear nerves, is poorly understood. To address these questions, we generated transgenic mice in which Fgfr1 and Fgfr2 were specifically inactivated in Schwann cells and oligodendrocytes but not in neurons. Adult mutant mice exhibited late onset of hearing impairment, which progressed markedly with age. The hearing impairment was accompanied by significant loss of myelinated spiral ganglion neurons. The pathology extended into the cochlear nucleus, without apparent loss of myelin or of the deletion-bearing glial cells themselves. This suggests that perturbation of FGF receptor-mediated glial function leads to the attenuation of glial support of neurons, leading to their loss and impairment of auditory functions. Thus, FGF/FGF receptor signaling provides a potentially novel mechanism of maintaining reciprocal interactions between neurons and glia in adult and aging animals. Dysfunction of glial cells and FGF receptor signaling may therefore be implicated in neurodegenerative hearing loss associated with normal aging.

Fine structure of degeneration in the cochlear nucleus of the chinchilla after acoustic overstimulation.

To study plastic changes in the cochlear nucleus after acoustic stimulation, adult chinchillas were exposed once to a 4-kHz octave-band noise at 108 dB SPL for 3 hr. After survival times of 1, 2, 4, 8, and 16 weeks, samples were taken for electron microscopy from a part of the cochlear nucleus, where cochlear nerve fibers degenerated after the noise exposure. Progressive changes in fine structure were characterized as early, intermediate, and late stages of degeneration. Freshly occurring synaptic degeneration appeared in each period from 1-16 weeks. Endings with large round vesicles, putative excitatory synapses of the cochlear nerve, displayed progressive increases in neurofilaments and enlarged synaptic vesicles. Compared to controls, synaptic vesicles seemed fewer, often in small clusters in the interior of endings, and smaller in the synaptic zone. These early changes progressed to mitochondrial disintegration and overt "watery" degeneration. Some surviving endings, however, were shrunken and displaced partially by enlarged spaces in the synaptic complex. Dense-cored vesicles gathered in these endings. In terminals with pleomorphic and flattened vesicles, presumed inhibitory endings, cytological changes appeared within 1 week and persisted for months. The synaptic endings darkened, some vesicles disintegrated, and many smaller flatter vesicles collapsed into heaps. Especially at the presynaptic membrane, vesicles were shriveled, but a few mitochondria were preserved. Without overt signs of synaptic degeneration, some of these cytological changes presumably reflect reduced synaptic activity in the inhibitory endings. These changes may contribute to a continuing process associated with abnormal auditory functions, including hyperacusis and tinnitus.

Fine structure of long-term changes in the cochlear nucleus after acoustic overstimulation: chronic degeneration and new growth of synaptic endings.

The companion study showed that acoustic overstimulation of adult chinchillas, with a noise level sufficient to damage the cochlea, led to cytological changes and degeneration of synaptic endings in the cochlear nucleus within 1-16 weeks. In the present study, the same stimulus was used to study the long-term effects on the fine structure of synaptic endings in the cochlear nucleus. For periods of 6 and 8 months after a single exposure to a damaging noise level, there ensued a chronic, continuing process of neurodegeneration involving excitatory and inhibitory synaptic endings. Electron microscopic observations demonstrated freshly occurring degeneration even as late as 8 months. Degeneration was widespread in the neuropil and included the synapses on the globular bushy cell, which forms part of the main ascending auditory pathway. Neurodegeneration was accompanied by newly formed synaptic endings, which repopulated some of the sites vacated previously by axosomatic endings on globular bushy cells. Many of these synaptic endings must arise from central interneurons. The findings suggest that overstimulation can induce a self-sustaining condition of progressive neurodegeneration accompanied by a new growth of synaptic endings. Noise-induced hearing loss thus may progress as a neurodegenerative disease with the capacity for synaptic reorganization within the cochlear nucleus.

Quantitative study of degeneration and new growth of axons and synaptic endings in the chinchilla cochlear nucleus after acoustic overstimulation.

To determine if acoustic overstimulation altered synaptic connections in the cochlear nucleus, anesthetized adult chinchillas, with one ear protected by a silicone plug, were exposed for 3 hr to a 108-dB octave-band noise, centered at 4 kHz, and allowed to survive for periods up to 32 weeks. This exposure led to cochlear damage in the unprotected ear, mainly in the basal regions of the organ of Corti. The anterior part of the ipsilateral posteroventral cochlear nucleus consistently contained a band of degenerating axons and terminals, in which electron microscopic analysis revealed substantial losses of axons and synaptic terminals with excitatory and inhibitory cytology. The losses were significant after 1 week's survival and progressed for 16-24 weeks after exposure. By 24-32 weeks, a new growth of these structures produced a resurgence in the number of axons and terminals. The net number of excitatory endings fully recovered, but the quantity with inhibitory cytology was only partially recouped. Neuronal somata lost both excitatory and inhibitory endings at first and later recovered a full complement of excitatory but not inhibitory terminals. Dendrites suffered a net loss of both excitatory and inhibitory endings. Excitatory and inhibitory terminals with unidentified postsynaptic targets in the neuropil declined, then increased in number, with excitatory terminals exhibiting a greater recovery. These findings are consistent with a loss and regrowth of synaptic endings and with a reorganization of synaptic connections that favors excitation.

Synaptic nests lack glutamate transporters in the cochlear nucleus of the mouse.

Synaptic nests are closely packed collections of synaptic endings. Nests may be deficient in the glial processes which usually separate terminals in the CNS and which transport much of the glutamate associated with high levels of excitatory activity. We hypothesized that nests might lack glial glutamate transporters, but possibly would conserve neuronal glutamate transporter. Although present throughout the brain, nests are especially numerous in the cochlear nucleus. We performed immunoelectron microscopy by preembedding peroxidase and immunogold methods and by postembedding immunogold to detect expression of the glutamate transporters GLAST, GLT-1, and EAAC1 in the mouse cochlear nucleus. Our results show that the glial transporters, GLAST and GLT-1, are absent in synaptic nests. This deficiency is not compensated by the neuronal transporter EAAC1, which is poorly represented in nests. Outside synaptic nests, all three glutamate transporters are strongly expressed in the cochlear nucleus. Thus, glutamate released outside nests should be quickly bound by transporters and removed from the extracellular glutamate pool. Glutamate released within synaptic nests may persist long enough to permit diffusion to extrajunctional targets in the nest, including presynaptic receptors. Consequently, synaptic nests may play a role in modulation of synaptic activity but also in excitotoxic mechanisms.

Synaptophysin in the cochlear nucleus following acoustic trauma.

Chinchillas are notable for a low-frequency hearing range similar to that of humans and a marked sensitivity to loud noise. A single noise exposure that produces cochlear damage may lead to progressive loss of synaptic endings in the cochlear nucleus, followed by new axonal growth. As an index of synaptic regulation during such changes, we have examined the expression of a synaptic vesicle protein, synaptophysin, in the cochlear nucleus following a damaging acoustic stimulus in adult chinchillas. With one ear protected by a plug, following a 3-h exposure to an octave-band noise of 108 dB sound pressure level, centered at 4 kHz, the unprotected cochlea and the cochlear nuclei exhibited degeneration of hair cells and axons over periods of 7, 14, 30, 90, and 150 days. Axonal degeneration, as revealed by a silver degeneration method, was heavy ipsilateral to the cochlear damage, but sparse degeneration also appeared on the contralateral, unexposed side. Synaptophysin immunostaining underwent a major, bilateral decline in the anteroventral and posteroventral cochlear nuclei, interrupted at intervening periods by transient increases in the numbers of stained structures. A distinction in staining between large perisomatic structures and smaller puncta in the neuropil and between the dorsal and the ventral zones of the ventral cochlear nuclei revealed some variations in the response and degree of recovery of synaptophysin staining. These findings could best be explained by degeneration of synaptic endings followed by new growth of terminals and by regulatory changes in the levels of synaptophysin expression and synaptic vesicle accumulation over time.

Sequential interactions of fibroblast growth factor-2, brain-derived neurotrophic factor, neurotrophin-3, and their receptors define critical periods in the development of cochlear ganglion cells.

We studied the interactions of neurotrophin-3 (NT3) with brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2), and their effects on tyrosine kinase C (TrkC) expression during cochlear ganglion development. Otocysts were explanted from white leghorn chicken embryos at stages when the neuronal precursors normally start to migrate. Cultures were fed with various combinations of NT3, BDNF, and FGF-2. NT3 appeared to have a greater effect on neurite outgrowth than on migration and was enhanced by BDNF. The results from in situ hybridization and immunostaining for TrkC receptor revealed up-regulation of the mRNA and protein by combining NT-3 and BDNF. NT-3 combined with FGF-2 produced down-regulation of receptor. Neutralizing antibody to NT3 had an inhibitory effect on neuronal development, suggesting that endogenous NT3 is normally active during the period examined. The findings suggest an interactive role of NT3 in early neuronal development. The trophic synergism of NT3 and BDNF may result from up-regulation of TrkC. This hypothesis is consistent with immunostaining in the embryonic basilar papilla, which localized TrkC to the initial axonal invasion sites. While the growth factors each produce particular trophic effects, the interactions of these factors define a critical sequence of developmental events based on modulation of receptor expression.

Expression of a voltage-dependent potassium channel protein (Kv3.1) in the embryonic development of the auditory system.

The present study traces the development of a voltage-dependent potassium channel protein (Kv3.1) in the avian homologue of the cochlear nucleus, in the cochleovestibular ganglion, and in the otic epithelium from early developmental stages until near hatching. Immunohistochemistry with antibodies to the carboxy terminus (recognizing the Kv3.1b splice variant) and to the amino terminus (recognizing either form of Kv3.1) was used on Hamburger-Hamilton-staged chicken embryos. There were three periods in the relative levels of immunostaining in these regions. Early (E2-6), when precursor cells proliferate, migrate, and form axons, there was staining when using either antibody. In the middle period (E6-11), marked by hair cell differentiation, dendritic growth, and early synapse formation, staining levels decreased. In the late period (E11-19), when auditory function begins, staining increased rapidly, especially for Kv3.1b. Early Kv3.1 expression occurs in neuronal and hair cell precursors before they differentiate or function. Later, in the otic epithelium, a high level of Kv3.1 in cilia may precede or coincide with the onset of hair cell function. In neurons, some features of its localization correlate with axon outgrowth and synapse formation, others with the onset of neural activity and function.

Developmental expression of a voltage-dependent potassium channel (Kv3.1) in auditory neurons without cochlear input.

Kv3.1, a voltage-dependent potassium channel, has two forms, -a and -b, which differ in expression during development and at the onset of function in the auditory system. To determine whether cochlear nerve input could affect the expression of these two forms, cultures of the developing cochlear nucleus were explanted in the absence of the cochlear nerve at the beginning of cell migration (Hamburger-Hamilton stage 28-30), while neuroblasts continued to migrate onto the culture substrate. After 8, 15, and 22 days in vitro (three survival groups), cultures were immunostained with antibodies recognizing either both forms of Kv3.1 or only the -b form. Only young and newly migrated nerve cells were sampled. In the three survival groups, all nerve cells expressed Kv3.1, among which only 50% or less expressed the -b form. Some of the more differentiated multipolar cells expressed the -b form, but most were labeled with the antibody that recognizes both forms. Thus, in the absence of peripheral input, both forms of Kv3.1 appear at stages very early in development, although not all cells necessarily coexpress both forms. These results agree with other observations in the chick embryo in situ. They are consistent with previous work implicating Kv3.1 in cell migration during early development.

Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation.

To study the effect of FGF in the early development of the sensory neurons of the auditory system, we established a culture preparation of ganglionic neuroblasts engaged in migration and process outgrowth. The presumed anlage of the cochlear ganglion was dissected from E11 otocysts, just as the neuronal precursors were migrating. The cultures were divided into 4 groups and supplemented for 7-9 days with either hrFGF-1 or hrFGF-2 or both or with defined medium only (control group). Measurements of the increase in explant growth, neuroblast migration, and neurite outgrowth were made by time-lapse imaging techniques in living cultures. Either FGF-1 or FGF-2 alone stimulated early migration and outgrowth of the ganglion cells by 5-10x. The effect of combining FGF-1 and FGF-2 was greater than either alone, but less than additive, consistent with a shared receptor. BrdU labeling confirmed that the effect was on migration, not on proliferation. Adding a neutralizing antibody for FGF-2 to the cultures inhibited migration and neurite outgrowth, suggesting an endogenous FGF-2 activity in these functions. Immunocytochemical observations in vitro and in situ with antibodies to FGF-1, FGF-2, or FGF receptor (R1) demonstrated immunopositive staining of the migrating ganglionic neuroblasts, their processes, and growth cones at corresponding stages (E13). Also non-neuronal cells, hair cells, and Schwann cells (in situ) expressed FGF-1 and FGF-2. Evidently both FGF-1 and FGF-2 play important roles in the migration and initial differentiation of cochlear ganglion neurons in the mouse.

Role for basic fibroblast growth factor (FGF-2) in tyrosine kinase (TrkB) expression in the early development and innervation of the auditory receptor: in vitro and in situ studies.

A previous study showed that basic fibroblast growth factor (FGF-2) promotes the effects of brain-derived neurotrophic factor (BDNF) on migration and neurite outgrowth from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage 22 (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes growing into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal outgrowth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor.

Shaw-like potassium currents in the auditory rhombencephalon throughout embryogenesis.

The Shaw subfamily of potassium channel genes, including Kv3.1, are highly expressed within the auditory nuclei of the brainstem, where they have been implicated in the characteristic response properties of particular types of neurons. Potassium currents carried by Kv3.1 are voltage-dependent, have a high activation threshold, are slow to inactivate, and are very sensitive to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). We have investigated the developmental appearance of potassium currents in cell cultures from nucleus magnocellularis and its precursor neuroblasts from the acoustico-vestibular anlage of the chicken. Whole-cell patch recordings revealed that high-threshold, sustained, outward currents were present in 91% of neuroblasts. These currents displayed high sensitivities to TEA and 4-AP. The remaining 9% of neuroblasts exhibited only transient outward currents. Most cells (74%) had both a sustained and an initial transient component of outward current. These current types were observed throughout embryogenesis, beginning with the earliest ages (embryonic day [E]2). During proliferation and migration, and early neuronal differentiation, current levels were low; they incremented gradually during the time when the first synapses occur on dendrites and increased 2- to 3-fold just before hatching, when axosomatic synapses form. These findings suggest that the Shaw subfamily of channels in nucleus magnocellularis may be involved in early neuronal development, as well as in synaptic function later on.

Role in neuronal cell migration for high-threshold potassium currents in the chicken hindbrain.

We have investigated the influence of voltage-dependent, potassium conductances on the migration of embryonic neurons, using a culture preparation taken from the acoustico-vestibular anlage long before the onset of electrical excitability and synaptic function. Whole-cell patch clamp recordings from migrating neuroblasts at Hamburger-Hamilton stage 28 (E 5.5) revealed the exclusive expression of voltage-dependent, high-threshold, outward currents, activating at potentials positive to -20 mV. These currents were completely suppressed by the potassium channel blockers, 1.0 mM tetraethylammonium chloride (TEA) or 1.0 mM 4-aminopyridine (4-AP). In control media, the active migration of individual neuroblasts was recorded at 27 +/- 6 microm per hr. Within minutes after adding either drug to the culture, normal migration completely stopped for several hours. Calcium channel blockers, omega-conotoxin (3 microM) or cadmium chloride (100 microM), slowed, but did not halt, migration, while nickel chloride (100 microM) or N-methyl-D-glucamine (1 mM) had no effect. However, within 8 hr after TEA exposure, migratory activity usually returned. This recovery was associated with the appearance of a previously undetected, low-threshold and 4-AP- sensitive potassium conductance. We suggest that high-threshold, TEA/4-AP-sensitive potassium channels may normally support the migration of these neurons, while their chronic blockade can be compensated by the appearance of novel potassium channels. Potassium currents may act in concert with N-type calcium channels to regulate neuronal migration.

Cochlear nerve projections to the small cell shell of the cochlear nucleus: the neuroanatomy of extremely thin sensory axons.

Labeling cochlear nerve fibers in the inner ear of chinchillas with biotinylated dextran polyamine was used to trace the thin fibers (Type II), which likely innervate outer hair cells. These axons, 0. 1-0.5 microm in diameter, were distinguished from the thicker Type I, fibers innervating inner hair cells, and traced to small-cell clusters in the cochlear nucleus. This study provided two major new insights into the outer hair cell connections in the cochlear nucleus and the potential significance of very thin axons and synaptic nests, which are widespread in the CNS. 1) EM serial reconstructions of labeled and unlabeled material revealed that Type II axons rarely formed synapses with conventional features (vesicles gathered at junctions). Rather, their endings contained arrays of endoplasmic reticulum and small spherical vesicles without junctions. 2) Type II axons projected predominantly to synaptic nests, where they contacted other endings and dendrites of local interneurons (small stellate and mitt cells, but not granule cells). Synaptic nests lacked intrinsic glia and, presumably, their high-affinity amino acid transporters. As functional units, nests and their Type II inputs from outer hair cells may contribute to an analog processing mode, which is slower, more diffuse, longer-lasting, and potentially more plastic than the digital processors addressed by inner hair cells.

Spatial organization of the reciprocal connections between the cat dorsal and anteroventral cochlear nuclei.

We are studying the interconnections between the anteroventral cochlear nucleus (AVCN) and the dorsal cochlear nucleus (DCN). Biotinylated dextran was injected into the DCN, where the best frequency of responses was also recorded. Ventrotubercular neurons in AVCN were labeled, along with cochlear nerve fibers and the axons of cells in DCN. In AVCN, a central band of labeled cochlear nerve axons and large endbulbs was labeled. Bordering this band was a 'fringe' of smaller tuberculoventral axonal endings forming pericellular nests. Most AVCN neurons projecting to DCN were stellate, elongate, or giant cells, located in the posterior division of AVCN, regardless of the DCN injection site. About 75% of the labeled AVCN cells lay within the bands of labeled cochlear nerve fibers. Another 15% were in the outer fringes on either side of these bands, while 10% were outside the bands and the fringes. These findings suggest that most AVCN neurons projecting to the DCN conform to the tonotopic map. A significant portion of the ventrotubercular neurons occupy side-bands in AVCN. Reciprocally, the tuberculoventral tract forms a robust fringe of axonal endings flanking the central bands. The neuronal and axonal bands and side-bands may underlie excitatory and inhibitory signal transformations.

Spatio-temporal diversity in the microenvironments for neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule expression by sensory neurons and their targets during cochleo-vestibular innervation.

Sixteen phases in the microenvironments were defined for the structural development and innervation of the cochleo-vestibular ganglion and its targets. In each phase the cell adhesion molecules, neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule, were expressed differentially by cochleo-vestibular ganglion cells, their precursors, and the target cells on which they synapse. Detected by immunocytochemistry in staged chicken embryos, in the otocyst, neural cell adhesion molecule, but not L1-cell adhesion molecule, was localized to the ganglion and hair cell precursors. Ganglionic precursors, migrating from the otocyst, only weakly expressed neural cell adhesion molecule. Epithelial hair cell precursors, remaining in the otocyst, expressed neural cell adhesion molecule, but not L1-cell adhesion molecule. Post-migratory ganglion cell processes expressed both molecules in all stages. The cell adhesion molecules were most heavily expressed by axons penetrating the otic epithelium and accumulated in large amounts in the basal lamina. In the basilar papilla (cochlea), cell adhesion molecule expression followed the innervation gradient. Neural cell adhesion molecule and L1 were heavily concentrated on axonal endings peripherally and centrally. In the rhombencephalon, primitive epithelial cells expressed neural cell adhesion molecule, but not L1-cell adhesion molecule, except in the floorplate. The neuroblasts and their axons expressed L1-cell adhesion molecule, but not neural cell adhesion molecule, when they began to migrate and form the dorsal commissure. There was a stage-dependent, differential distribution of the cell adhesion molecules in the floorplate. Commissural axons expressed both cell adhesion molecules, but their polysialic acid disappeared within the floorplate at later stages. In conclusion, the cell adhesion molecules are expressed by the same cells at different times and places during their development. They are positioned to play different roles in migration, target penetration, and synapse formation by sensory neurons. A multiphasic model provides a morphological basis for experimental analyses of the molecules critical for the changing roles of the microenvironment in neuronal specification.

Multiple roles of neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1 adhesion molecules during sensory innervation of the otic epithelium in vitro.

To explore the role of cell adhesion molecules in the innervation of the inner ear, antibody perturbation was used on histotypic co-cultures of the ganglionic and epithelial anlagen derived from the otocyst. When unperturbed, these tissues survived and differentiated in this culture system with outgrowth of fasciculated neuronal fibers which expressed neural cell adhesion molecule and L1. The fibers exhibited target choice and penetration, then branching and spreading within the otic epithelium as individual axons. Treatment of the co-cultures, or of the ganglionic anlagen alone, with anti-neural cell adhesion molecule or anti-L1 Fab fragments produced a defasciculation of fibers but did not affect neurite outgrowth. In the co-cultures this defasciculation was accompanied by a small increase in the number of fibers found in inappropriate tissues. However, the antibodies did not prevent fiber entry to the otic epithelium. In contrast, removal of polysialic acid from neural cell adhesion molecule with endoneuraminadase-N, while producing a similar fiber defasciculation, also increased the incidence of fibers entering the epithelium. Nevertheless, once within the target tissue, the individual fibers responded to either Fab or to desialylation by spreading out more rapidly, branching, and growing farther into the epithelium. The findings suggest that fasciculation is not essential for specific sensory fibers to seek out and penetrate the appropriate target, although it may improve their tracking efficiency. Polysialic acid on neural cell adhesion molecule appears to limit initial penetration of the target epithelium. Polysialic acid as well as neural cell adhesion molecule and L1 function are involved in fiber-target interactions that influence the arborization of sensory axons within the otic epithelium.