D.K. Morest

Neuronal and transneuronal degeneration of auditory axons in the brainstem after cochlear lesions in the chinchilla: Cochleotopic and non-cochleotopic patterns

Terminal axonal degeneration in the brain following cochlear lesions was studied with the Nauta-Rasmussen method. Losses of hair cells and myelinated cochlear fibers were assessed. The cochleotopic map projected, from apex to base, on the ventral-to-dorsal axes of the cochlear nuclei. The cochleotopic correspondence was better for loss of cochlear nerve fibers and inner hair cells, than for outer hair cells. Cochlear fibers were traced to all parts of the cochlear nucleus, including the small-cell shell, also to cell-group Y and the flocculus. Terminal axonal degeneration in nuclei of the superior olivary complex, lateral lemniscus, and inferior colliculus was interpreted as transynaptic, since degenerated axons could not be traced to these locations from the cochlear nerve or trapezoid body. Moreover, biotinylated dextran amine injection in the basal turn of scala media of a normal cochlea labeled cochlear nerve fibers projecting to the high-frequency regions of the cochlear nuclei and to the flocculus, but not to more central auditory nuclei. This is the first detailed account of transynaptic degeneration in the ascending auditory pathway resulting from cochlear damage in an adult mammal. These findings are consistent with a dystrophic process depending on hair-cell loss and/or direct damage to cochlear nerve fibers.

New Growth of Axons in the Cochlear Nucleus of Adult Chinchillas after Acoustic Trauma

This study determined the effect of acoustic overstimulation of the adult cochlea on axons in the cochlear nucleus. Chinchillas were exposed to an octave-band noise centered at 4 kHz at 108 dB sound pressure level for 1.75 h. One chinchilla was never exposed to the noise, and several others had one ear protected by an ear plug or prior removal of the malleus and incus. Exposure of unprotected ears caused loss of inner and outer hair cells and myelinated nerve fibers, mostly in the basal half of the cochlea. Cochlear nerve fiber degeneration, ipsilateral to the exposed ears, was traced to regions of the cochlear nucleus representing the damaged parts of the cochlea. In silver impregnations of a deafferented zone in the posteroventral cochlear nucleus, the concentration of axons decreased by 43% after 1 month and by 54% after 2 months. However, by 8 months, the concentration of thinner axons, with diameters of less than 0.46 microm, increased by 46-90% over that at 2 months. The concentration of axons with larger diameters did not change. Between 2 and 8 months small axonal endings appeared next to neuronal cell bodies. This later increase of thinner axons and endings is consistent with a reactive growth of new axons of relatively small diameter. The emergence of small perisomatic boutons suggests that the new axons formed synaptic endings, which might contribute to an abnormal reorganization of the central auditory system and to the pathological changes that accompany acoustic overstimulation.

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 weeks 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.

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 5–10×. 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. J. Neurosci. Res. 62:40–55, 2000.

LONG-TERM DEGENERATION IN THE COCHLEAR NERVE AND COCHLEAR NUCLEUS OF THE ADULT CHINCHILLA FOLLOWING ACOUSTIC OVERSTIMULATION

Adult chinchillas were exposed once to an octave‐band noise, centered at 4 kHz, and allowed to survive for 16 days or for 1, 2, 4, and 8 months. Axonal degeneration was mapped in the cochlear nucleus, using the Nauta‐Rasmussen silver method, and related to hair cell damage and to loss of myelinated nerve fibers in the osseous spiral lamina of the cochlea. Axonal degeneration in the dorsal cochlear nucleus had already reached a peak by 16 days and disappeared after 1 month. Meanwhile, myelinated nerve fiber degeneration in the cochlea extended basally, followed 2 weeks to 2 months later by spread of axonal degeneration into the corresponding high‐frequency region of the ventral cochlear nucleus. Axonal degeneration occurred early in the low‐frequency region of the ventral cochlear nucleus, followed 2–4 weeks later by spread of myelinated fiber degeneration into more apical regions of the cochlea. New degeneration of axons in the cochlear nerve and in the ventral cochlear nucleus continued to occur for up to 8 months after stimulation. These findings imply that plastic changes in the central auditory pathways could play a role in the long‐term effects of cochlear damage and acoustic overstimulation, possibly leading to a chronic neurodegenerative condition in the ear and in the brain. Microsc. Res. Tech. 41:205–216, 1998.

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.

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.

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.

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.

Basic fibroblast growth factor affects neuronal migration and differentiation in normotypic cell cultures from the cochleovestibular ganglion of the chick embryo.

To study the role of basic fibroblast growth factor (FGF-2) in the development of sensory neurons, the cochleovestibular ganglion of the chicken embryo provides a well-characterized structure. This permits use of morphological markers in a cell culture preparation comparable to the normal embryo (normocytic). Otocysts were explanted from white leghorn embryos at Hamburger-Hamilton Stages 14-16, when ganglion cell precursors normally start migrating from the otic epithelium. The cultures were supplemented with either fetal bovine serum or human recombinant FGF-2 (in defined medium or serum) for 2 or 5 days. FGF-2 increased explant growth, neuroblast migration, and neurite outgrowth 2- to 10-fold in the first 2 days. Neuronal morphology appeared within 2-3 days with FGF-2 but required at least 4-5 days with serum. FGF-2 in defined medium stimulated early migration and differentiation, but without serum led to degeneration after 5 days. In serum, growth was later and slower but continued for at least 3 weeks. When explants were cultured in serum with a neutralizing antibody to FGF-2, but no FGF added, neuroblast migration and elongation were decreased by 2- to 4-fold, compared to serum alone. Immunocytochemistry demonstrated FGF receptor sites on the migrating ganglionic neuroblasts, on their processes and growth cones, and in the incipient ganglion and otic epithelium at Stages 15-17, both in the embryo and in vitro. The findings suggest that FGF-2 stimulates early migration and differentiation of ganglion cells by activating the receptors of neuroblasts or their precursors in the embryonic otocyst. However, other factors must sustain their later development.