Cochleovestibular nerve development is integrated with migratory neural crest cells.

The cochleovestibular (CV) nerve, which connects the inner ear to the brain, is the nerve that enables the senses of hearing and balance. The aim of this study was to document the morphological development of the mouse CV nerve with respect to the two embryonic cells types that produce it, specifically, the otic vesicle-derived progenitors that give rise to neurons, and the neural crest cell (NCC) progenitors that give rise to glia. Otic tissues of mouse embryos carrying NCC lineage reporter transgenes were whole mount immunostained to identify neurons and NCC. Serial optical sections were collected by confocal microscopy and were compiled to render the three dimensional (3D) structure of the developing CV nerve. Spatial organization of the NCC and developing neurons suggest that neuronal and glial populations of the CV nerve develop in tandem from early stages of nerve formation. NCC form a sheath surrounding the CV ganglia and central axons. NCC are also closely associated with neurites projecting peripherally during formation of the vestibular and cochlear nerves. Physical ablation of NCC in chick embryos demonstrates that survival or regeneration of even a few individual NCC from ectopic positions in the hindbrain results in central projection of axons precisely following ectopic pathways made by regenerating NCC.

Developmental changes in the protective effect of exogenous brain-derived neurotrophic factor and neurotrophin-3 against ototoxic drugs in cultured rat vestibular ganglion neurons.

We examine developmental changes in the responsiveness of rat vestibular ganglion neurons (VGNs) to two neurotrophic factors (NTFs), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and investigate the protective effects of these NTFs against ototoxic drugs during postnatal development in dissociated cultures. VGNs were obtained from rats on postnatal days (P) 1, 3, 7 and 14. BDNF facilitated neuronal survival as well as neurite sprouting of VGNs obtained from younger rats (P1 and P3), whereas these effects were not observed in older rats (P7 and P14). BDNF was also effective in facilitating neurite extension in VGNs at each of the postnatal ages. NT-3 also facilitated neuronal survival and neurite extension of VGNs from younger rats but these effects were significantly smaller than those of BDNF (p < 0.05). The protective effects of BDNF and NT-3 against ototoxic drugs, gentamicin and cisplatin, were also age-dependent: they were effective for neuronal survival, neurite sprouting and neurite extension in VGNs from younger rats, whereas these effects tended to disappear in VGNs from older rats. Analysis of the changes in the expression of the receptors of NTFs revealed that expression of TrkB and TrkC proteins and their mRNA did not change during the developmental period, whereas expression of p75(NTR) protein was down-regulated together with that of p75(NTR) mRNA during the developmental period. Developmental changes in the responsiveness to exogenous NTFs in VGNs, which is not caused by the changes of their receptors but probably caused by changes in the intracellular signaling pathways, should be taken into consideration in the prevention of neuronal degeneration caused by ototoxic drugs.

Excitatory actions of GABA in developing chick vestibular afferents: effects on resting electrical activity.

The aim of this study was to characterize the effect of γ-aminobutyric acid (GABA) in the resting multiunit activity of the vestibular afferents during development using the isolated inner ear of embryonic and postnatal chickens (E15-E21 and P5). GABA (10(-3) to 10(-5) M; n = 133) and muscimol (10(-3) M) elicited an increase in the frequency of the basal discharge of the vestibular afferents. We found that GABA action was dose-dependent and inversely related to animal age. Thus, the largest effect was observed in embryonic ages such as E15 and E17 and decreases in E21 and P5. The GABAA receptor antagonists, bicuculline (10(-5) M; n = 10) and picrotoxin (10(-4) M; n = 10), significantly decreased the excitatory action of GABA and muscimol (10(-3) M). Additionally, CNQX 10(-6) M, MCPG 10(-5) M and 7ClKyn 10(-5) M (n = 5) were co-applied by bath substitution (n = 5). Both the basal discharge and the GABA action significantly decreased in these experimental conditions. The chloride channel blocker 9-AC 0.5 mM produced an important reduction in the effect of GABA 10(-3) (n = 5) and 10(-4) M (n = 5). Thus, our results suggest an excitatory role of GABA in the resting activity of the vestibular afferents that can be explained by changes in the gradient of concentration of Cl(-) during development. We show for the first time that the magnitude of this GABA effect decreases at later stages of embryonic and early postnatal development. Taking into account the results with glutamatergic antagonists, we conclude that GABA has a presynaptic action but is not the neurotransmitter in the vestibular afferent synapses, although it could act as a facilitator of the spontaneous activity and may regulate glutamate release.

Development of spontaneous activity and response properties of primary lagenar neurons in the chick.

Here, we report for the first time developmental changes in spontaneous activity and in response properties of single nerve fibers from the macular chick lagena. Such aspects are important in order to get insight into the functional role of the lagena which remains undetermined. For this purpose, we used intracellular and extracellular single-unit recording techniques in an isolated inner ear preparation from the chicken at ages E15 and P1. At E15, afferent fibers displayed a low irregular spontaneous discharge rate (41 +/- 14 spikes/s, CV = 1.17 +/- 0.1), which was replaced by regular high frequency spontaneous activity at P1 (CV = 0.48 +/- 0.8, 89 +/- 27 spikes/s). During the developmental period including E15, the percentage of silent neurons was 60% while that of P1 was 40%. The synaptic activity was higher at E15 than at P1. The action potential waveform generated at E15 had small amplitude and derivative depolarization, and consequently, a large duration in correlation with respect to action potential waveform at P1 (respectively: 53 +/- 2 vs. 65 +/- 3 mV, 60 +/- 11 vs. 109 +/- 20 mV/ms, 3.6 +/- 0.4 vs. 1.1 +/- 0.12 ms). In addition, we recognized two response dynamics to the injection of current steps: phasic, or rapidly adapting neurons and tonic, or slowly adapting neurons. Our results indicate similar developmental processes for the lagena as described for the vestibular system in other species, in agreement with the known morphological characteristics of this otholitic end organ. The presence of more than one subtype of afferent neuron also correlates with previous reports on vestibular afferents with analogous electrophysiological properties, strongly suggesting the vestibular nature of the lagena.

Differentiation of the facio-vestibulocochlear ganglionic complex in human embryos of developmental stages 13-15.

A study was made on 18 embryos of developmental stages 13-15 (5(th) week). Serial sections made in horizontal, frontal, and sagittal planes were stained with routine histological methods and some of them were treated with silver. In embryos of stage 13, the otic vesicle is at the rhombomere 5, and close to the vesicle is the facial-vestibulocochlear ganglionic complex in which the geniculate, vestibular, and cochlear ganglion may be discerned. These ganglia are well demarcated in embryos of stage 14. In the last investigated stage (15(th)) the nerve fibres of the ganglia reach the common afferent tract.

EphB2 guides axons at the midline and is necessary for normal vestibular function.

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

The duration of neurotrophic factor independence in early sensory neurons is matched to the time course of target field innervation.

To investigate how the onset of neurotrophic factor dependence in neurons is coordinated with the arrival of their axons in the target field, we have studied the survival of four populations of cranial sensory neurons whose axons reach their common central target field, the hindbrain, at different times. We show that neurons whose axons reach the hindbrain first survive for a short time in culture before responding to brain-derived neurotrophic factor (BDNF). Neurons whose axons reach the hindbrain later survive longer before responding to BDNF. These differences in survival, which arise prior to gangliogenesis, may play a role in coordinating trophic interactions for cranial sensory neurons.

Differential expression of Eph receptors and ephrins in the cochlear ganglion and eighth cranial nerve of the chick embryo.

The cochleovestibular ganglion of the chick differentiates at early embryonic stages as VIIIth nerve axons enter the brainstem. The tonotopic organization of the auditory portion of the VIIIth nerve can be discerned at the time axons initially reach their brainstem targets. The mechanisms underlying this early organization are not known. Eph receptor tyrosine kinases and their ligands, the ephrins, have a demonstrated role in guiding axons to topographically appropriate locations in other areas of the nervous system. In order to begin to test whether Eph proteins have a similar role in the auditory system, we investigated the tonotopic expression of several Eph receptors and ephrins in the VIIIth nerve during embryonic ages corresponding to the initial innervation of the auditory brainstem. Expression patterns of EphA4, EphB2, EphB5, ephrin-A2, and ephrin-B1 were evaluated immunohistochemically at embryonic days 4 through 10. Protein expression was observed in the cochlear ganglion and VIIIth nerve axons at these ages. EphB5, ephrin-A2, and ephrin-B1 were expressed throughout the nerve. EphA4 and EphB2 had complementary expression patterns within the nerve, with EphA4 expression higher in the dorsolateral part of the nerve and EphB2 expression higher in the ventromedial part of the nerve. These regions may correspond to auditory and vestibular components, respectively. Moreover, EphA4 expression was higher toward the low-frequency region of both the centrally and peripherally projecting branches of cochlear ganglion cells. Regional variation of Eph protein expression may influence the target selection and topography of developing VIIIth nerve projections.

Dlx gene expression during chick inner ear development.

Members of the Dlx gene family play essential roles in the development of the zebrafish and mouse inner ear, but little is known regarding Dlx genes and avian inner ear development. We have examined the inner ear expression patterns of Dlx1, Dlx2, Dlx3, Dlx5, and Dlx6 during the first 7 days of chicken embryonic development. Dlx1 and Dlx2 expression was seen only in nonneuronal cells of the cochleovestibular ganglion and nerves from stage 21 to stage 32. Dlx3 marks the otic placode beginning at stage 9 and becomes limited to epithelium adjacent to the hindbrain as invagination of the placode begins. Dlx3 expression then resolves to the dorsal otocyst and gradually becomes limited to the endolymphatic sac by stage 30. Dlx5 and Dlx6 expression in the developing inner ear is first seen at stages 12 and 13, respectively, in the rim of the otic pit, before spreading throughout the dorsal otocyst. As morphogenesis proceeds, Dlx5 and Dlx6 expression is seen throughout the forming semicircular canals and endolymphatic structures. During later stages, both genes are seen to mark the distal surface of the forming canals and display expression complementary to that of BMP4 in the vestibular sensory regions. Dlx5 expression is also seen in the lagena macula and the cochlear and vestibular nerves by stage 30. These findings suggest important roles for Dlx genes in the vestibular and neural development of the avian inner ear.

Differential expression of microtubule associated protein MAP-2 in developing cochleovestibular neurons and its modulation by neurotrophin-3.

Microtubule associated proteins (MAPs) are essential cytoskeletal proteins in developing neurons. The present study was undertaken to analyze the expression of MAP2 and its isoforms (a,b,c) during the embryonal and early post-hatching development of chicken cochleovestibular ganglion (CVG) neurons. Moreover, we have investigated MAP2 expression in primary cultures of CVG neurons, and whether it is regulated by neurotrophin-3 (NT3). The expression of MAP2 immunoreactivity (IR) was studied using both Western blot and immunohistochemistry on tissue sections and primary cultures. In vivo MAP2c was expressed from incubation day 4 (E4) to E10, and MAP2b was found in all embryonal stages studied and at post-hatching day 10 (P10), whereas MAP2a was restricted to the post-hatching periods. The cellular localization of IR was in the neuronal perikarya and their peripheral processes (dendrites) but not in axons. Primary cultures matched the in vivo pattern of MAP2 expression, and IR was localized in neuronal cell bodies and the initial segment of the neuronal processes. Exogenous NT3 regulated the expression of MAP2 isoforms in a dose dependent manner. At the survival dose of 0.5 ng/ml NT3, the main MAP2 expression was MAP2c. Conversely, at the neuritogenic dose of 5 ng/ml NT3 increased MAP2b and MAP2a expression, but not MAP2c. The present results demonstrate that MAP2 isoforms are developmentally regulated, thus suggesting that each isoform is specifically involved in CVG neuron maturation. Furthermore, we provide evidence of MAP2 regulation in culture by the neurotrophic factor NT3.

Cell proliferation during early development of the chick embryo otic anlage: quantitative comparison of migratory and nonmigratory regions of the otic epithelium.

During development of the otic anlage, a certain proportion of epithelial cells migrate toward the mesenchymal compartment to form part of the acoustic-vestibular ganglion. The migrating cells are observed only in the zone of the otic anlage that will make contact with the acoustic-vestibular ganglion (so-called ganglion zone). In Hamburger and Hamilton's stages 13 to 16, the number of epithelial cells that migrate is relatively low, but it becomes steadily higher from stage 17 on. In the otic anlage of chick embryos, between developmental stages 9 and 21 (48 to 94 hours of incubation), mitotic index, apical or basal localization within the epithelium of dividing cells, and orientation of the mitotic spindles were analyzed. These features in the ganglion zone were compared with observations in the rest of the otic epithelium, where migratory processes do not take place. In stages 13 to 15, when few epithelial cells are migrating, the mitotic index (MI) in the ganglion zone of the otic anlage is similar to that in nonmigratory regions. In more advanced stages, however, when cell migration becomes accelerated, the MI in the migratory zone of the otic wall is significantly higher than that in the rest of the otic epithelium. This suggests an intimate relationship between the migration of otic epithelial cells and a high rate of cell proliferation, the possible nature of which is discussed. Although the majority of mitoses in the otic anlage are located at the apical surface of the epithelium, from stage 13 onward, a few dividing cells are seen in the basal third of the epithelium. Furthermore, these basal mitoses appear exclusively in the migratory zone of the otic anlage, thus suggesting a possible relationship between epithelial cell migration and basal mitosis. During the developmental period prior to stage 18, no significant differences in mitotic spindle orientation are noted between migratory and nonmigratory zones of the otic anlage. In contrast, in stages of maximal otic epithelial cell migration (stages 19 to 21), the frequency of mitoses with the spindle axis oriented radially is significantly higher in the migratory zone. These findings point toward a close correlation between increased frequency of radial mitotic spindle orientation and intense cell migration, although the exact nature of this relationship is as yet unknown.

Studies on cell migration and axon guidance in the developing distal auditory system of the mouse.

The events that take place along the potential route of distal auditory axons (future vestibular component) prior to and during their outgrowth were examined morphologically using timed mouse embryos. During embryonic (E) day 9.5 a discrete zone of cell death appears in the rostrolateral wall of the otic cup. Necrosis is accompanied by outward migration of epitheloid cells from the same region of the otic wall. Temporally and spatially correlated with these two events is the widening of extracellular spaces between otic neuroepithelial cells and the breakdown of basement membrane. During E 10.5 migrating epitheloid cells condense to form a funnel-shaped configuration. This cellular "funnel" begins narrowly at the dorsorostrolateral wall of the otocyst and broadens as it reaches the auditory ganglion. During E 11.5 through E 12.5, "pioneer" distal auditory axons take a circuitous route and ascend from the auditory ganglion to enter the otocyst. Axons extend toward the otocyst moving along cells of the "funnel," maintaining an orientation similar to that of the cells that compose it. Axon growth cones enter the otocyst at sites devoid of basement membrane and invade the wall of the otocyst moving tangentially along radially arranged cells that bridge the otocyst and the "funnel." These observations demonstrate that a preformed, funnel-shaped tissue exists along the future route of the auditory fibers. We suggest that the "funnel" may influence the growth and directionality of distal auditory axons as they extend from the auditory ganglion to the wall of the otocyst. At the otic wall, the transition provided by "bridge" epitheloid cells, together with the absence of basement membrane at specific sites of the otic wall, provide the auditory axons with a route into the otocyst.

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.

Pathway specificity of reticulospinal and vestibulospinal projections in the 11-day chicken embryo.

The organization of the axonal pathways of reticulospinal and vestibulospinal projections in the 11-day chicken embryo was ascertained through retrograde tracing experiments. An in vitro preparation of the brainstem and cervical spinal cord facilitated precisely localized tracer applications. Single- and double-labelling experiments involving high cervical injections of tracers in combination with selective lesions defined the specific pathways by which different brainstem neurons project to the spinal cord. Coherent, and in many cases distinct, groups of reticulospinal and vestibulospinal neurons could thus be identified on the basis of their position and projection pathway. The organization of these groups and their projections in the 11-day chicken embryo is similar to that in avian and other vertebrate adults and therefore serves as a reference point for studies of pathfinding by bulbospinal axons during early development.

Sensory organ generation in the chicken inner ear: contributions of bone morphogenetic protein 4, serrate1, and lunatic fringe.

The chicken inner ear is a remarkably complex structure consisting of eight morphologically distinct sensory organs. Unraveling how these sensory organs are specified during development is key to understanding how such a complex structure is generated. Previously, we have shown that each sensory organ in the chicken inner ear arises independently in the rudimentary otocyst based on Bone morphogenetic protein 4 (Bmp4) expression. Here, we compare the expression of Bmp4 with two other putative sensory organ markers, Lunatic Fringe (L-fng) and chicken Serrate1 (Ser1), both of which are components of the Notch signaling pathway. L-fng and Ser1 expression domains were asymmetrically distributed in the otic cup. At this early stage, expression of L-fng is similar to Delta1 (Dl1), in an anteroventral domain apparently corresponding to the neurogenic region, while Ser1 is expressed at both the anterior and posterior poles. By the otocyst stage, the expression of both L-fng and Ser1 largely coincided in the medial region. All presumptive sensory organs, as identified by Bmp4 expression, arose within the broad L-fng- and Ser1-positive domain, indicating the existence of a sensory-competent region in the rudimentary otocyst. In addition, there is a qualitative difference in the levels of expression between L-fng and Ser1 such that L-fng expression was stronger in the ventral anterior, whereas Ser1 was stronger in the dorsal posterior region of this broad domain. This early difference in expression may presage the differences among sensory organs as they arise from this sensory competent zone.

Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in culture.

The effect of the availability of synaptic targets on neuronal survival was tested by explanting the cochleovestibular ganglion from embryonic day 3-1/2 chick embryos and maintaining it in the presence or absence of appropriate synaptic target tissues for 14 days in culture. The targets were the inner ear, peripherally, and the myelencephalon, centrally. Light and electron microscopic observations showed that the ganglion cells in the explants with targets present had generally achieved a degree of differentiation comparable to that of their counterparts in embryonic day 14 embryos. The variety of cell types seen in the normal embryonic day 14 ganglia was also evident in vitro. In ganglia explanted without peripheral or central targets, few neurons survived. Ganglia explanted with either peripheral or central target intact showed considerably better survival than those explanted without any target. Ganglia explanted with only the peripheral target (the inner ear) survived equally as well as those with both central and peripheral targets. Ganglia cultured with the central target (myelencephalon) did not survive as well as those with peripheral targets. The effect of the peripheral target on the ganglion was less clear-cut when ganglia were first dissected from their targets and then recombined in culture. However, the results of such experiments in which nerve fascicles were traced in serial sections from ganglia to target areas, suggest that the actual innervation of target cells, as well as proximity of ganglia to target tissues, could influence neuronal survival. Establishment of innervation appeared to be selective, in that the closest available target area was not always the one contacted by the ganglionic fibers. The present findings are consistent with a role of neuron-target cell interactions in supporting neuronal survival in the cochleovestibular ganglion of the chick embryo. Both the central and the peripheral targets are implicated in trophic interactions with the sensory neurons.

Two distinct phases and mechanisms of axonal growth shown by primary vestibular fibres in the brain, demonstrated by parvalbumin immunohistochemistry.

Antibodies to parvalbumin label intensely a small number of non-overlapping fibre systems in embryonic rat brain. All are in hindbrain--the oculomotor and trochlear motor fibres, the acoustic and vestibular fibres of the VIIIth nerve, and an unidentified group of fibres which ascend under the dorsal surface in caudal medulla. Of these, the vestibular fibres are the first to acquire parvalbumin immunoreactivity, and we have used this property to follow the growth of their axons in the brain. This occurs in two phases. In the first, occurring at embryonic days 12-14, the axons grow in small groups or fascicles under the pial surface to their most distant terminal zones rostrally in the cerebellum and caudally in the descending vestibular nuclei. This growth is directed towards the two sites where germinal neuroepithelium is expanding over the medullary velum in forming the cerebellum and lateral recess of the IVth ventricle. In a second stage, commencing at E15, individual collaterals branch from these fascicles to arborize amongst their presumptive synaptic targets (cells of the vestibular nuclei and vestibulocerebellum) located in the sub-ventricular and ventricular layers. In this phase the axons follow a radial route, at right angles to their original subpial course, possibly by growing along radial glial processes. The target cells then migrate to their final position with the vestibular axons maintaining contact with them. The vestibular fibres are the first axons to enter the cerebellum, but from E15 onwards their fascicles are joined by increasing numbers of non-vestibular fibres following the same course. These other axons, and the movement of cells to form the deep cerebellar nuclei, separate the fascicles of vestibular fibres so that their course into the cerebellum becomes very diffuse. Thus this single set of axons grow, not only in two distinct phases, but also follow distinctly different substrates for growth in each. Furthermore, they then appear to act as pioneer fibres guiding the entry or egress of later-developing axons to or from the cerebellum.

Neuroepithelial 'compartments' and the specification of vestibular projections.

The implication that there exist coherent vestibulo-ocular neuron pools with specific functions may provide new insight into how conjugate eye movements are synthesized within the vestibulo-ocular reflex. The systematic relationship between pool position and synergistic principle terminations, the 'hodological mosaic' suggests, moreover, a determinate groundplan established by developmental mechanisms operative at early stages in the hindbrain neuroepithelium. From such a groundplan, evolutionary and use-dependent modifications could mold connectivity patterns functionally appropriate for each species and individual. How the expression of developmentally regulatory genes contributes to establishing the mosaic organization of the vestibular system is the current focus of our research.

The developmental segregation of posterior crista and saccular vestibular fibers in mice: a carbocyanine tracer study using confocal microscopy.

The developmental segregation of gravistatic input mediated by saccular fibers and of angular acceleration input mediated by posterior crista (PC) fibers was analyzed for the first time in a developing mammal using carbocyanine dye tracing in fixed tissue. The data reveal a more extensive projection of either endorgan in 7-day-old mice (P7) than has previously been reported in adult mammals. While we confirm and extend many previous findings, we also describe a novel segregation of saccular and posterior crista fibers in the anterior half of the medial vestibular nucleus (Mv) not reported before. Our developmental analysis shows a progressive segregation of posterior crista and saccular fibers to their respective discrete projection areas between embryonic day 15 (E15) and birth (P0). Retention of overlap in young adult animals appears to reflect the early embryonic overlap found in most areas. The vestibular projection does not show a topological projection as has been described in many other sensory systems. We propose that the unique projection features of the vestibular endorgans may relate to the transformation of vestibular signals into a motor output in the three neuron reflex arc of the VOR, of which the primary vestibular projection constitutes the first leg.

Developmental regulation of a neurite-promoting factor influencing statoacoustic neurons.

The present study investigated a target-derived, neurite-promoting factor (NPF) released by the developing chick otocyst and its effects on statoacoustic ganglia (SAG). SAG explants cultured in the absence of otocysts produced little neurite outgrowth at all stages of development examined (E4-E13). However, extensive neurite outgrowth was seen when E4-E6 SAG were cultured in the presence of otocysts of the same age. The amount of neurite outgrowth observed in cocultures steadily decreased at later developmental stages. E7-E9 cocultures produced less outgrowth and E10-E13 cocultures produced the least outgrowth compared to E4-E6 cocultures. Additionally, otocysts from older stages were unable to promote outgrowth of E4 SAG. Thus, the level of the factor released by the otocysts declined during development. In contrast, neurite outgrowth was promoted when E10-E15 SAG were cocultured in the presence of younger stage otocysts. Our data indicate that the release of NPF from chick otocysts decreased from E6 to E13, although the ability of SAG neurons to respond to the NPF was maintained throughout development.