Role of fibroblast growth factor 8 in neurite outgrowth from spiral ganglion neurons in vitro.

Many neurons degenerate after injuries resulting from overstimulation, drugs, genetic mutations, and aging. Although several growth factors and neurotrophins delay degeneration and promote regrowth of neural processes, the role of fibroblast growth factor 8 (FGF8) in mammalian spiral ganglion neurons (SGN) neurite outgrowth has not been examined. This study develops and uses SGN cell cultures suitable for experimental analysis, it investigates whether FGF8a and FGF8b isoforms affect the neurite outgrowth from SGN cultured in vitro. We found that both FGF8a and FGF8b promoted the outgrowth of neurites from cultured SGN. This response is mediated by FGF receptors and involves the activation of IκBα-mediated NFκB signaling pathway. These findings suggest that, besides its morphogenetic role during development, FGF8 may have trophic functions in the adult which are relevant to regeneration.

Cisplatin-induced ototoxicity: effect of intratympanic dexamethasone injections.

HYPOTHESIS: Intratympanic (IT) application of dexamethasone will reduce ototoxicity associated with systemic cisplatin therapy. BACKGROUND: Cisplatin is a common chemotherapeutic drug often dose-limited by ototoxicity attributed to the formation of reactive oxygen and nitrogen species damaging critical inner ear structures. Steroids have been shown to reduce formation of reactive oxygen species and thus may reduce ototoxicity. In the present pilot study, we test this hypothesis by IT administration of dexamethasone in a novel murine model of cisplatin ototoxicity. METHODS: Click- and pure-tone-evoked auditory brainstem responses (ABRs) in young CBA/J mice were measured. The first phase consisted of a dosing study to identify the optimal cisplatin dose for ototoxicity. In the next phase, ABR thresholds were measured in cisplatin-treated mice after 5 days of IT injection of 24 mg/ml of dexamethasone in 1 ear and normal saline in the opposite ear to serve as controls. RESULTS: Intraperitoneal injection of 14 mg/kg of cisplatin induces significant hearing loss (click-evoked ABR threshold elevation = 12 +/- 7 dB, mu +/- standard error of the mean) with acceptable mortality (20%). The ears that received IT dexamethasone in cisplatin-treated mice had minimal ABR threshold shifts with the click, 8 and 16 kHz of stimuli. There was no significant difference between IT dexamethasone and IT saline ears at 32 kHz. CONCLUSION: IT dexamethasone protected the mouse ear against cisplatin-induced ototoxicity in a frequency-dependent manner. The present results suggest that IT dexamethasone may be a safe, simple, and effective intervention that minimizes cisplatin ototoxicity without interfering with the chemotherapeutic actions of cisplatin.

Interactive roles of fibroblast growth factor 2 and neurotrophin 3 in the sequence of migration, process outgrowth, and axonal differentiation of mouse cochlear ganglion cells.

A growth factor may have different actions depending on developmental stage. We investigated this phenomenon in the interactions of fibroblast growth factor 2 (FGF2) and neurotrophins on cochlear ganglion (CG) development. The portions of the otocyst fated to form the CG and cochlear epithelium were cocultured at embryonic day 11 (E11). Cultures were divided into groups fed with defined medium, with or without FGF2 and neurotrophin supplements, alone or in combination, for 7 days. We measured the number of migrating neuroblasts and distances migrated, neurite outgrowth, and axonlike processes. We used immunohistochemistry to locate neurotrophin 3 (NT3) and its high-affinity receptor (TrkC) in the auditory system, along with FGF2 and its R1 receptor, at comparable developmental stages in vitro and in situ from E11 until birth (P1) in the precursors of hair cells, support cells, and CG cells. Potential sites for interaction were localized to the nucleus, perikaryal cytoplasm, and cell surfaces, including processes and growth cones. Time-lapse imaging and quantitative measures support the hypothesis that FGF2 alone or combined with neurotrophins promotes migration and neurite outgrowth. Synergism or antagonism between NT3 and other factors suggest interactions at the receptor level. Formation of axons, endings, and synaptic vesicle protein 2 were increased by interactions of NT3 and FGF2. Similar experiments with a mutant overexpressor for FGF2 suggest that endogenous FGF2 supports migration and neurite outgrowth of CG neuroblasts as well as proliferation, leading to accelerated development. The findings suggest interactive and sequential roles for FGF2 and NT3.

Plasticity of synaptic endings in the cochlear nucleus following noise-induced hearing loss is facilitated in the adult FGF2 overexpressor mouse.

In adult mammals a single exposure to loud noise can damage cochlear hair cells and initiate subsequent episodes of degeneration of axonal endings in the cochlear nucleus (CN). Possible mechanisms are loss of trophic support and/or excitotoxicity. Fibroblast growth factor 2 (FGF2), important for development, might be involved in either mechanism. To test this hypothesis, we noise-exposed FGF2 overexpressor mice and observed the effects on synaptic endings by immunolabelling for SV2, a synaptic vesicle protein, at 1, 2, 4, and 8 weeks after noise exposure. SV2 staining was observed in two major locations; perisomatic, representing axo-somatic terminals, and neuropil, representing axo-dendritic terminals. The wildtype (WT) lost both perisomatic and neuropil clusters with an intervening period of modest recovery for the perisomatic. In contrast, in the overexpressor, the perisomatic clusters remained unchanged after intervening periods of increase. The neuropil clusters underwent a period of initial decline, followed by a transient recovery and ultimate decline. Changes in SV2 immunostaining correlated with changes in vesicular glutamate and GABA transporters at synapses and, in the overexpressor, with staining changes for FGF2 and FGF receptor 1. These molecules may contribute to the synaptic reorganization after noise damage; they may protect and/or aid recovery of synapses after overstimulation.

Site-specific interactions of neurotrophin-3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus.

Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes.

Development of synapses and expression of a voltage-gated potassium channel in chick embryonic auditory nuclei.

The potassium channel protein, Kv3.1, is abundantly expressed in the chick auditory pathway. Its b-isoform is found in nucleus magnocellularis, which receives the cochlear input, both before and after the establishment of synaptic connections. It is also present in cell cultures in the absence of any peripheral input. However, the expression of this isoform in the embryo has been shown to increase with development. Here, we address the question of the correlation between maturation of synapses in the auditory pathway and the pattern of expression of the b-isoform in a series of embryos prepared for immunohistochemistry at Hamburger-Hamilton stages equivalent to E10, E12, E14, and E17. We show here that this subunit translocates from the perinuclear cytoplasm to the cell membrane domain in nucleus magnocellularis at the time that cochlear nerve endings emerge as endbulbs of Held (E17). In nucleus laminaris, by this time, while abundant Kv3.1b occurs in the perinuclear cytoplasm, a translocation to the cell membrane domain has not yet occurred, and the mature peri-synaptic localization is delayed to a later stage. This difference suggests a hierarchy in the developmental expression of Kv3.1. An unexpected finding is the expression of the a-isoform of Kv3.1 in astrocytes, especially those which surround the developing nuclei and their connecting fibers. We also report here for the first time the presence of Kv3.1b in the initial segments of axons at the times when they begin to form. Our observations suggest that the Kv3.1 channel protein is regulated through mechanisms linked to the development of synaptic activity.

Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction.

Analyses of the effect of ryanodine in vertebrate brain slices have led to the conclusion that presynaptic ryanodine receptors (RYRs) may have several functions in synaptic release, including causing large-amplitude miniature postsynaptic currents (mPSCs) by promoting concerted multivesicular release. However, the role of RYRs in synaptic release is controversial. To better understand the role of RYRs in synaptic release, we analyzed the effect of RYR mutation on mPSCs and evoked postsynaptic currents (ePSCs) at the Caenorhabditis elegans neuromuscular junction (NMJ). Amplitudes of mPSCs varied greatly at the C. elegans NMJ. Loss-of-function mutations of the RYR gene unc-68 (uncoordinated 68) essentially abolished large-amplitude mPSCs. The amplitude of ePSCs was also greatly suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells, suggesting that RYRs acted presynaptically. A combination of removing extracellular Ca2+ and UNC-68 function eliminated mPSCs, suggesting that influx and RYR-mediated release are likely the exclusive sources of Ca2+ for synaptic release. Large-amplitude mPSCs did not appear to be caused by multivesicular release, as has been suggested to occur at vertebrate central synapses, because the rise time of mPSCs was constant regardless of the amplitude but distinctive from that of ePSCs, and because large-amplitude mPSCs persisted under conditions that inhibit synchronized synaptic release, including elimination of extracellular Ca2+, and mutations of syntaxin and SNAP25 (soluble N-ethylmaleimide-sensitive factor attachment protein 25). These observations suggest that RYRs are essential to normal quantal size and are potential regulators of quantal size.

Where is the spike generator of the cochlear nerve? Voltage-gated sodium channels in the mouse cochlea.

The origin of the action potential in the cochlea has been a long-standing puzzle. Because voltage-dependent Na+ (Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the type I and type II ganglion cells. In most type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.

Organization of inhibitory feed-forward synapses from the dorsal to the ventral cochlear nucleus in the cat: a quantitative analysis of endings by vesicle morphology.

The main ascending, excitatory pathway from the cochlea undergoes synaptic interruption in the dorsal and ventral cochlear nuclei. The dorsal cochlear nucleus also forms a feed-forward circuit, which receives cochlear input and projects to the ventral cochlear nucleus by a tuberculo-ventral tract. This circuit may provide an inhibitory fringe (side bands) surrounding the center bands of the main ascending pathway. Biotinylated dextran injections into the dorsal cochlear nucleus anterogradely labeled the tuberculo-ventral tract and its endings in the anteroventral cochlear nucleus but also retrogradely filled cochlear nerve fibers and their terminals in the same regions. To distinguish tuberculo-ventral from cochlear nerve terminals, we used electron microscopy of the immunolabeled endings. Images were digitized and filter-enhanced, and the sizes and shapes of synaptic vesicles were used to construct quantitative profiles of the terminal types. The cochlear nerve endbulbs mapped to the same iso-frequency band of the injection site (main band). Flanking the main band were smaller labeled endings. About 45% of labeled terminals were pleomorphic and equally represented in the main band and side bands. Therefore, if there is an inhibitory fringe in the main projection pathway, it was not selective for tuberculo-ventral tract endings. Surprisingly, an excitatory category of round vesicles of intermediate size was labeled in the main band but not in the side bands. These intermediate endings may balance the feed-forward inhibition from the tuberculo-ventral tract. The quantitative method devised for classification of ending types by their vesicle profiles should be a generally useful tool for analysis.

Regeneration of the inner ear as a model of neural plasticity.

The publication of a paper entitled "Direct transdifferentiation gives rise to the earliest new hair cells in regenerating avian auditory epithelium" in the Journal of Neuroscience Research offers the opportunity to call attention to a well-developed line of research on the auditory receptor of birds, which should be of interest to students of regeneration and plasticity of the mature nervous system in higher vertebrates, including mammals. Although hair cell proliferation normally stops before hatching, destruction of the auditory receptors of the chicken may be followed by complete regeneration of hair cells. Most of the new hair cells arise from a new wave of proliferation, but Roberson et al. show that about one-third of the new hair cells are formed without undergoing cell division and thus may differentiate from so-called supporting cells or cells with an "intermediate morphology." This finding suggests some models for regeneration of this neuroepithelium, including the possibility that mature supporting cells could transform directly into hair cells. The present Mini-Review discusses some of the models for neural regeneration that future studies might address in the light of our current knowledge and the new report. The possibility is raised that transitional forms of hair cell and supporting cell precursors may reside in the inner ear in a quiescent state until stimulated by damage.

BetaIV spectrins are essential for membrane stability and the molecular organization of nodes of Ranvier.

High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on betaIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv(3J)"quivering" mice, whose betaIV spectrins have a truncated proline-rich "specific" domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv(3J) mice, which lack betaIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv(3J) nodes, some with detectable betaIV spectrins, are less affected. In contrast, a larger truncation of betaIV spectrins in qv(4J) mice, deleting the SD, PH, and ankyrinG binding domains, causes betaIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt betaIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.

Precursors of neurons, neuroglia, and ependymal cells in the CNS: what are they? Where are they from? How do they get where they are going?

Neurons, neuroglia (astrocytes and oligodendrocytes), and ependymal cells are three distinct categories of neural cells in the central nervous system. In the mature brain and spinal cord, the classical histological criteria define these cells by their microscopic structure very well. During development, the precursors for all of these cells reside within the epithelium of the neural plate and its successor, the neural tube. These precursor cells are the undifferentiated, primitive neuroepithelium of the classical literature. As the cerebral vesicles enlarge and their walls thicken, the primitive neuroepithelial cells elongate, maintaining a radial orientation until they migrate. Although many, but not all, of these cells span the extent of the ventricular wall, they are the precursors of neurons, neuroglia, and ependymal cells. Thus, it is useful to retain their classical designation as primitive neuroepithelial cells and to treat them as neural precursor cells. Neural precursor cells are neither neuroglia nor neurons. It is not appropriate to call them radial glial cells anymore than it is to call them radial neuronal cells. The term "radial glia" has long been used to describe the mature, elongated astrocytes, represented by Bergmann cells in the cerebellum and Müller cells in the retina. Inevitably, during development, transitional forms between neural precursor cells and the neurons, neuroglia, and ependymal cells will occur. Such transitional cells are known as neuroblasts, glioblasts, or ependymoblasts, even though they may be postmitotic. Alternative terms are "immature neurons," "immature neuroglia," and "immature ependymal cells." The migration of many neural precursor cells is accomplished by translocation rather than free cellular locomotion. There is both direct and indirect evidence to document the translocation of the nuclear/perikaryal/somal complex through the leading process of primitive neuroepithelial cells. This is conspicuous in the neocortex, where the discrete radial arrangement of pyramidal cells may result from translocation of neuroblasts, while their leading processes still contact the pial surface. Migration by translocation occurs throughout the CNS. GLIA 43:6-18, 2003.

Intracellular fibroblast growth factor produces effects different from those of extracellular application on development of avian cochleovestibular ganglion cells in vitro.

In an avian coculture system, the neuronal precursors of the cochleovestibular ganglion typically migrated from the otocyst and differentiated in response to soluble fibroblast growth factor (FGF-2), which had free access to FGF receptors on the cell surface. Free FGF-2 switched cells from a proliferation mode to migration, accompanied by increases in process outgrowth, fasciculation, and polysialic acid expression. Microsphere-bound FGF-2 had some of the same effects, but in addition it increased proliferation and decreased fasciculation and polysialic acid. As shown by immunohistochemistry, FGF-2 that was bound to latex microspheres depleted the FGF surface receptor protein, which localized with the microspheres in the cytoplasm and nucleus. For microsphere-bound FGF-2, the surface receptor-mediated responses to FGF-2 appear to be limited and the door opened to another venue of intracellular events or an intracrine mechanism.

Fibroblast growth factors (FGFs) in the cochlear nucleus of the adult mouse following acoustic overstimulation.

To see if fibroblast growth factors (FGFs) might function in the central changes following auditory overstimulation we tracked immunostaining in the cochlear nucleus of adult mice with monoclonal antibodies to FGFs (FGF-1, FGF-2) and FGF receptor. After exposure nearly all outer hair cells died, while inner hair cell and fiber loss were restricted to a region midway along the cochlear spiral. FGFs staining in the cochlear nucleus appeared in hypertrophied astrocytes in the regions of nerve fiber degeneration only. For normal-sized astrocytes there was an increase in the number stained and the intensity of staining across all frequency domains, but not in neurons. The increases were modest at 3-7 days, pronounced at 14 days, modest again by 30 days, and back to control levels by 60 days. FGF receptor staining of neurons occurred equally in all mice, exposed or not. The findings suggest that astrocytes play a role in the central responses to acoustic overstimulation and cochlear damage, involving FGFs, possibly regulating the activity of intrinsic neurons or signaling axonal growth. Not limited to regions of cochlear nerve fiber and inner hair cell loss, the changes in FGFs may represent a reaction to outer hair cell damage which spreads broadly across the central pathways.