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, 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. Keywords auditory neuron; axon growth; Ca 2+ /calmodulin dependent kinase II

Spiral ganglion neurons (SGNs) extend processes that interact with Schwann cells (SCs) and with oligodendrocytes (OLs) and astrocytes (ACs). We investigated the ability of these glial cells to support SGN neurite growth. In the presence of cultured ACs, OLs and SCs, SGN neurites tended to follow SCs and OLs and cross-over ACs. Most neurites initially followed the type of glial cell on which the neuronal cell body was found. To determine the influence of homogeneous populations of glia on neurite growth, SG explants were plated on cultured SCs, ACs or OLs. The number of neurites/explant extending onto SCs (463.89±16.25) was significantly greater than the number extending onto ACs (111.38±38.73) or OLs (6.75±2.21), indicating that populations of central glia inhibit SGN neurite growth. Treatment with cell-permeant cpt-cAMP or forskolin (FSK) each significantly increased the number of neurites on OLs (133.54±25.59 and 292.25±83.57, respectively). cpt-cAMP and FSK each also increased the number of neurites on ACs (213.19± 36.06 and 208.64±59.25, respectively), however the difference was not significant compared with control. The neurites on ACs and OLs failed to grow radially in a well-fasciculated pattern as on SCs. In explants plated on the borders of cultured OL-SC or AC-SC groups, more neurites extended onto SCs compared with OLs and ACs. Conditioned media from OL or AC cultures did not reduce neurite length, implying that the inhibition of neurite growth by central glia is not due to soluble factors. Taken together, these results demonstrate that homogeneous populations of central glia inhibit SGN neurite growth.

Objective-To determine the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts.Methods-VS xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor, trastuzumab, or saline for 2 weeks. Animals also received BrdU injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging (MRI) over the treatment period. Cell death was analyzed by terminal dUTP nick end labeling (TUNEL).Results-Tumors could be distinguished with T2 weighted MRI sequences. Trastuzumab significantly reduced the proliferation of VS cells compared to control (p<0.01) as determined by BrdU uptake. Control tumors demonstrated slight growth over the 12 week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p<0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percent of TUNEL-positive VS cells (p<0.01).Conclusions-In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis type 2.

Spiral ganglion neurons (SGNs) provide afferent innervation to the cochlea and rely on contact with hair cells (HCs) for their survival. Following deafferentation due to hair cell loss, SGNs gradually die. In a rat culture model, we explored the ability of prosurvival members of the Bcl-2 family of proteins to support the survival and neurite outgrowth of SGNs. We found that overexpression of either Bcl-2 or Bcl-xL significantly increases SGN survival in the absence of neurotrophic factors, establishing that the Bcl-2 pathway is sufficient for SGN cell survival and that SGN deprived of trophic support die by an apoptotic mechanism. However, in contrast to observations in central neurons and PC12 cells where Bcl-2 appears to promote neurite growth, both Bcl-2 and Bcl-xL overexpression dramatically inhibit neurite outgrowth in SGNs. This inhibition of neurite growth by Bcl-2 occurs in nearly all SGNs even in the presence of multiple neurotrophic factors implying that Bcl-2 directly inhibits neurite growth rather than simply rescuing a subpopulation of neurons incapable of extending neurites without additional stimuli. Thus, although overexpression of prosurvival members of the Bcl-2 family prevents SGN loss following trophic factor deprivation, the inhibition of neurite growth by these molecules may limit their efficacy for support of auditory nerve maintenance or regeneration following hair cell loss.