Spiral Ganglion Neurite Research Articles

Spiral Ganglion Outgrowth and Hearing Development in p75NTR-Deficient Mice

To explore the role of nerve growth factor receptor p75^NTR during the terminal neuronal development of the mammalian cochlea the onset of hearing and the in vitro response of spiral ganglion neurites to neurotrophin 3 (NT-3), which is known to play a critical role during neonatal inner ear development, were investigated in p75^NTR-deficient mice (p75^NTR–/–). Auditory-evoked brain stem response recordings from p75^NTR–/– and wild-type (WT) littermates were measured from postnatal days (PD) 8 to 23. Additionally, spiral ganglion explants from p75^NTR–/– and WT animals were dissected and cultured in an organotypic tissue culture system. In both groups, spiral ganglion neurite outgrowth was analyzed with and without NT-3 supplementation. No significant differences in the onset of hearing of mutant mice compared to the WT mice were detected, and both groups showed a similar development of hearing until PD 23. After stimulation with NT-3, neurite outgrowth was enhanced in both p75^NTR–/– and WT mice. However, neurites from p75^NTR–/– spiral ganglion explants were longer in both culture conditions. Moreover, NT-3 did not significantly enhance neurite number in p75^NTR–/–, as it did in WT mice. P75^NTR has a remarkable influence on spiral ganglion neurite growth behavior. However, p75^NTR does not seem to be essential for the development of basic hearing function in the first 3 postnatal weeks.

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca 2+ or treatment with a combination of L-type, N-type, and P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 μM), we demonstrate that depolarization activates calpains. Calpeptin (15 μM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization.

Hearing development and spiral ganglion neurite growth in VASP deficient mice

Vasodilator-stimulated phosphoprotein (VASP) has been found to be involved in intracellular signalling pathways and to play an important role in the actin associated organization and formation of the cytoskeleton. Since differential VASP expression was noted in inner ear tissues, the present study was performed to investigate the hearing development in VASP deficient mice. Hearing development in VASP−/− mice and wild type animals was investigated by auditory brain stem (ABR) measurements. In addition, inner ear tissues of wild type animals were tested for VASP expression using PCR, Western blot analysis, in situ hybridisation, and immunohistochemistry. To compare spiral ganglion (SG) neurite growth, SG explants from VASP−/− and wild type mice were analyzed under cell culture conditions. The electroacoustical results of the present study indicate that VASP deficient mice present with a later onset of hearing during postnatal development compared to wild type animals. Transient VASP expression was detected in neonatal SG of wild type mice. Tissue culture experiments with SG explants from VASP−/− animals revealed significant alterations in SG neurite extension compared to wild types. The present findings suggest a role for VASP during neonatal development of the mammalian cochlea and allow speculation on a possible delayed innervation of cochlear hair cells due to changes in SG neurite growth in VASP-deficient mice. Temporary VASP deficits in the neonatal inner ear may be compensated by related proteins like MENA leading to a delayed but complete development of hearing function in VASP−/− animals.

Laminin and fibronectin modulate inner ear spiral ganglion neurite outgrowth in an in vitro alternate choice assay

Extracellular matrix (ECM) molecules have been shown to function as cues for neurite guidance in various populations of neurons. Here we show that laminin (LN) and fibronectin (FN) presented in stripe micro-patterns can provide guidance cues to neonatal (P5) inner ear spiral ganglion (SG) neurites. The response to both ECM molecules was dose-dependent. In a LN versus poly-L-lysine (PLL) assay, neurites were more often observed on PLL at low coating concentrations (5 and 10 microg/mL), while they were more often on LN at a high concentration (80 microg/mL). In a FN versus PLL assay, neurites were more often on PLL than on FN stripes at high coating concentrations (40 and 80 microg/mL). In a direct competition between LN and FN, neurites were observed on LN significantly more often than on FN at both 10 and 40 microg/mL. The data suggest a preference by SG neurites for LN at high concentrations, as well as avoidance of both LN at low and FN at high concentrations. The results also support a potential model for neurite guidance in the developing inner ear in vivo. LN, in the SG and osseus spiral lamina may promote SG dendrite growth toward the organ of Corti. Within the organ of Corti, lower concentrations of LN may slow neurite growth, with FN beneath each row of hair cells providing a stop or avoidance signal. This could allow growth cone filopodia increased time to sample their cellular targets, or direct the fibers upward toward the hair cells.

Environmental Micropatterning for the Study of Spiral Ganglion Neurite Guidance

The projection of neuronal processes is guided by a variety of soluble and insoluble factors, which are sensed by a fiber’s growth cone. It is the differential distribution of such guidance cues that determine the direction in which neurites grow. The growth cone senses these cues on a fine scale, using extensible filopodia that range from a few to tens of µm in length. In order to study the effects of guidance cues on spiral ganglion (SG) neurites, we have used methods for distributing both soluble and insoluble cues on a scale appropriate for sensing by growth filopodia. The scale of these methods are at the micro, rather than nano, level to match the sensing range of the growth cone. Microfluidics and transfected cells were used to spatially localize tropic factors within the fluid environment of extending neurites. Micropatterning was used to present neurites with stripes of insoluble factors. The results indicate that differentially distributed permissive, repulsive and stop signals can control the projection of SG neurites. Implications for future micropatterning studies, for SG development and for the growth of deafferented SG dendrites toward a cochlear implant are discussed.

EphA4 provides repulsive signals to developing cochlear ganglion neurites mediated through ephrin-B2 and -B3.

The ephrins and Eph receptors make up two large families of bi-directional signaling molecules that are known to play a role in the development of the nervous system. Recently, expression of EphA4 in the developing cochlea was shown, with strong expression in cells lining the osseous spiral lamina (OSL) through which afferent dendrites must pass to reach the organ of Corti (OC). It was also demonstrated that ephrin-B2 and -B3, both of which are known to interact with EphA4, are expressed by spiral ganglion (SG) neurons. To investigate the functional role of EphA4 in the development of inner ear neurons, neonatal rat SG explants were cultured for 72 hours on uniformly coated surfaces or near stripes of EphA4/IgG-Fc-chimera. Control explants were cultured on or near IgG-Fc and EphA1/IgG-Fc-chimera. To assess the roles of ephrin-B2 and -B3 in EphA4 signaling, SG explants were cultured with or without anti-ephrin-B2 and/or -B3 blocking antibodies. Growth patterns of SG neurites at the border of EphA4 receptor stripes showed repulsion, characterized by turning, stopping and/or reversal. In the case of IgG-Fc and EphA1, the neurites grew straight onto the stripes. Treatment with either anti-ephrin-B2 or -B3 blocking antibodies significantly reduced the repulsive effect of an EphA4 stripe. Moreover, when both antibodies were used together, neurites crossed onto EphA4 stripes with no evidence of repulsion. The results suggest that EphA4 provides repulsive signals to SG neurites in the developing cochlea, and that ephrin-B2 and -B3 together mediate this response.

Branching of spiral ganglion neurites is induced by focal application of fibroblast growth factor-1.

During the terminal innervation of the developing organ of Corti, fibroblast growth factor-1 (FGF-1) messenger RNA has been shown to be transiently expressed in the sensory epithelium of the rat, suggesting that this growth factor may contribute to developmental processes such as innervation and synaptogenesis of the inner and outer hair cells. In a previous study it has been demonstrated that exogenous FGF-1 supports rat spiral ganglion neurite extension in vitro, whereas a secreted form of FGF-1 produced by transfected fibrocytes induces neurite branching and targeting. Response of spiral ganglion neurites to FGF-1-coupled beads was compared with the response to noncoupled control beads. Effects of multiple focal sources of FGF-1 to outgrowing spiral ganglion neurites were investigated on explants from postnatal day 4 rat spiral ganglion samples that were cultured in the presence of FGF-1 covalently coupled to polybead microspheres. After fixation and immunocytochemical labeling of the explants the growth patterns of the extending neuronal processes were evaluated. When spiral ganglion neurites were observed near clusters of FGF-1 beads, they formed a plexus-like network characterized by significantly higher branching in the vicinity of the beads. However, fibers did not appear to terminate on the beads. Plexus-like formations were not seen at a distance from FGF-1 coupled beads or in the vicinity of control beads lacking FGF-1 on their surface. The results of the study indicate that spiral ganglion neurites branch in response to focal sources of FGF-1, suggesting an important role of this growth factor for hair cell innervation during the terminal development of the sensory epithelium.

Clostridium difficile Toxin B, an Inhibitor of the Small GTPases Rho, Rac and Cdc42, Influences Spiral Ganglion Neurite Outgrowth

Objective--Neurotrophins and extracellular matrix (ECM) molecules are involved in neurite guidance during the development of spiral ganglion (SG) neurons. Several intracellular signaling molecules can be activated by ECMs and neurotrophins via their cognate receptors. In other systems these include the Rho small GTPases, which influence reorganization of the actin cytoskeleton that is required for axon growth. The aim of this study was to determine whether neurotrophin-3 (NT-3)-mediated SG neurite outgrowth on laminin-1 (LN) is dependent on the activation of the small GTPases Rho/Rac/Cdc42. Material and methods--SG explants from postnatal Day 4 rats were cultured on LN with and without NT-3 and increasing concentrations of Clostridium difficile Toxin B, an inhibitor of Rho GTPases. After fixation and immunocytochemical labeling, neurite growth was evaluated. Results--Treatment with C. difficile Toxin B without NT-3 lead to a dose-dependent decrease in the length and number of processes on LN. In contrast, C. difficile Toxin B had no significant effect on NT-3-mediated stimulation of neurite growth on LN, in terms of either number or length. Conclusion--The results suggest that the Rho GTPases (Rho, Rac and Cdc42) are not involved in the pathways linking NT-3 signals to neurite outgrowth, but appear to be involved in LN signaling in these neurons. However, NT-3 can override or bypass LN signaling to promote neurite extension.

Interaction of spiral ganglion neuron processes with alloplastic materials in vitro

The cochlear implant (CI) involves the introduction of alloplastic materials into the cochlea. While current implants interact with cochlear neurons at a distance, direct interactions between spiral ganglion (SG) neurites and implants could be fostered by appropriate treatment with neurotrophic factors. The interactions of fibroblasts and osteoblasts with alloplastic materials have been well studied in vitro and in vivo. However, interactions of inner ear neurons with such alloplastic materials have yet to be described. To investigate survival and growth behavior of SG neurons on different materials, SG explants from post-natal day 5 rat SG were cultured for 72 h in the presence of neurotrophin-3 (10 ng/ml) on titanium, gold, stainless steel, platinum, silicone and plastic surfaces that had been coated with laminin and poly- L-lysine. Neurite outgrowth was investigated after immunohistological staining for neurofilament, by image analysis to determine neurite extension and directional changes. Neurite morphology and adhesion to the alloplastic material were also evaluated by scanning electron microscopy (SEM). On titanium, SG neurites reached the highest extent of outgrowth, with an average length of 662 μm and a mean of 31 neurites per explant, compared to 568 μm and 21 neurites on gold, 574 μm and 24 neurites on stainless steel, 509 μm and 16 neurites on platinum, 281 μm and 12 neurites on silicone and 483 μm and 31 neurites on plastic. SEM revealed details of adhesion of neurites and interaction with non-neuronal cells. The results of this study indicate that the growth of SG neurons in vitro is strongly influenced by alloplastic materials, with titanium exhibiting the highest degree of biocompatibility with respect to neurite extension. The knowledge of neurite interaction with different alloplastic materials is of clinical interest, as development in CI technology leads to closer contact of implanted electrodes with surviving inner ear neurons.

Histomorphometric assessment of spiral ganglion neurite outgrowth in vitro

During the last years, various groups of growth factors have been identified to influence spiral ganglion cell survival and neurite extension in the mammalian cochlea. To evaluate and compare the effects of different growth factors, a precise histomorphometrical analysis of neurite outgrowth patterns has to be applied. The here presented technique is compared to already published methods, that only approximately estimate the neurite length. A software has been developed to analyse digitalised scans of spiral ganglion cells and to measure the length of the neurites. Therefore, the neurites are being separated in any given number of straight lines. The totals of these lines can then be added like a polygon. This polygon method was compared to a semi-quantitative procedure in which the neurite length was determined by concentric circles that were crossed by the neurites in a certain distance. The accuracy of both methods was analysed. Both methods were performed in 20 specimen of neonatal rat spiral ganglion cells after in vitro stimulation with neurotrophic factors. The semi-quantitative method has shown to involve a systematic error between +/- 10 to 15 %. The polygon method, on the contrary, has a systematic error of around +/- 1 %, which admits much more accurate measurement of spiral ganglion neurite outgrowth. With the described polygon method, spiral ganglion neurite growth patterns in cell culture studies can be characterised more precisely and, thus, helps to better differentiate the action domain of neurotrophic factors.

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase influences neurotrophin-induced spiral ganglion neurite growth

Phosphatidylinositol 3-kinase (PI3K) is considered to be an important enzyme in cell signaling, mediating certain aspects of neurotrophin signals from the cell surface receptor to the nucleus. The participation of PI3K in the mediation of neurotrophin-induced effects in the spiralganglion (SG) of neonatal rats was investigated in vitro. SG explants were stimulated with neurotrophin (NT)-3 and treated with Wortmannin, a specific inhibitor of PI3K. After fixation and immunhistochemical staining, the growth of the SG neurites was evaluated. Stimulation with NT-3 lead to significant increases in number and length of neurites, when compared to non-stimulated controls. Treatment of NT-3 stimulated SG explants resulted in a dose-dependent reduction of both parameters, whereas the neurite growth of non-stimulated control explants was not significantly influenced by the incubation with Wortmannin. The results demonstrate that neurotrophin-induced neurite growth from SG explants can be modulated with the PI3K inhibitor Wortmannin and indicate that PI3K is a key enzyme in the mediation of NT-3 effects in cochlear neurons. These observations together with results of previous studies suggest that the activation of PI3K as well as Ras and MEK are essential for neurite growth in cochlear neurons. Further knowledge of cell signaling mechanisms influencing SG neurite growth could lead to new therapeutical strategies for the treatment of inner ear diseases.

The effects of laminin-1 on spiral ganglion neurons are dependent on the MEK/ERK signaling pathway and are partially independent of Ras

Laminin-1 (LN) is expressed along the route of neural growth from spiral ganglion (SG) neurons towards the developing organ of Corti, and has been shown to enhance neurite outgrowth from SG neurons in vitro. Signal transduction pathways linking LN signaling at the cell membrane to the cell nucleus can involve a variety of signaling molecules. Data from other systems suggest the potential involvement of the small G protein Ras, and the mitogen-activated protein kinases (MAPKs) Erk and/or p38. To assess these possibilities, the length and number of processes extending from SG explants cultured on LN-coated surfaces were evaluated after treatment with the Ras inhibitor FTI-277, the p38 inhibitor SB203580 and MAPK kinase (MEK) inhibitor U0126, which operates immediately upstream of the Erk MAPK. Treatment with the Ras inhibitor at levels known to inhibit the H- and N-Ras isoforms had no effect, while FTI-277 levels known to inhibit K-Ras reduced only neurite length. Suppression of MEK resulted in a decrease of both parameters, while incubation with the p38 inhibitor had no effect. The results of this study suggest that MEK plays a central role in LN signaling in SG neurites. While K-Ras signaling may participate in MEK-dependent increases in neurite length, the MEK-dependent increase in neurite number appears to be activated by a different intracellular pathway.

Focal delivery of fibroblast growth factor-1 by transfected cells induces spiral ganglion neurite targeting in vitro.

Sensory cells in the cochlea of the rat transiently express acidic fibroblast growth factor (FGF-1) during the developmental period of terminal innervation in the sensory epithelium. To explore the potential role of FGF-1 in terminal innervation events, the response of cochlear ganglion neurons to FGF-1 was evaluated in culture. Explants from the spiral ganglion of postnatal day 5 rats were cultured in the presence of exogenous FGF-1, with or without heparin. FGF-1 in the culture medium produced a dose-dependent increase in the number and length of neurites produced by spiral ganglion neurons, a response that was enhanced by heparin. To assess the effects of FGF-1 produced by a focal, cellular source, additional explants were cocultured with 3T3 cell transfectants that secrete FGF-1. Neurites that came into contact with FGF-1 secreting cells branched, formed bouton-like terminal swellings on the surface of the transfectants, and stopped extending. The results suggest that FGF-1 may stimulate neurite extension into the sensory epithelium of the cochlea and that focal production of FGF-1 may contribute to the formation of contacts on sensory cells by developing neurites.