Membrane electrical activity also provides a strong success government both in vivo and in vitro. While sgn survival may be increased by direct electrical stimulation via an implanted electrode after hair cell loss sgn survival is promoted by depolarization, accomplished by raising extracellular K, in vitro. Survival reactions as a result of membrane electrical activity need Ca2 influx natural compound library through L type voltage gated calcium channels and subsequent activation of at least three separate calcium dependent protein kinases: cyclic AMP dependent protein kinase, and Ca2 /calmodulin dependent kinases II and IV. However, excessive Ca2 trend is dangerous and contributes to SGN death. These Ca2 dependent signaling pathways control many areas of neuronal function aside from survival, including synaptic maintenance and direction, growth and plasticity. The consequences of membrane electrical activity and i on SGN neurite growth have not been thoroughly investigated Metastasis but it is clear that at least one Ca2 dependent signal, CaMKII, can be a powerful negative regulator of neurite growth. Given the relevance of SGN axon growth to cochlear implant technology together with the potential of hair cell regeneration, we have investigated the effects of membrane depolarization on SGN neurite extension in vitro. Increasing levels of e contributes to a dosedependent decrease in SGN neurite lengths, even at levels which promote SGN emergency. These effects on neurite outgrowth result from reduced expansion of existing processes together with development of neurite processes. This inhibition of neurite growth by depolarization requires numerous types voltage gated calcium channels and activation of calpain, a protease. II. STRATEGIES Spiral ganglion cultures Dissociated spiral ganglion cultures were prepared as previously described. Briefly, ganglia were plated on polyornithine/laminin covered 8 pan HDAC inhibitor well culture chambers, dissociated with trypsin, dissected from postnatal day 5 rat pups, and maintained in high glucose Dulbeccos Modified Eagles Medium with N2 product and fresh insulin in a humidified incubator with 6. Five minutes CO2. Three hr after plating the cultures were placed in experimental or get a grip on conditions, preserved for 48 hr to permit for neurite growth, and then fixed for immunofluorescence. We typically obtained 1000 SGNs/well in cultures maintained in NT3 equivalent to 2000 SGNs/cochlea, just like the plating efficiency of other ways of culturing rat SGNs. Cultures were depolarized with elevated extracellular K 30 mM or 80 mM or maintained in control nondepolarizing 5 mM K method, as previously described. Some cultures were depolarized in the presence of the next VGCC inhibitors singly or in combination: the N type channel blocker conotoxin GVIA, the M type channel blocker verapamil, and the P/Q type channel blocker agatoxin. Gene transfer into SGNs Another culture procedure was employed for gene transfer into SGNs.