Purification and transfection of cochlear Schwann cells.

Schwann cells line nerve fibers in the peripheral nervous system (PNS) and synthesize myelin. In addition, they support neuronal survival, neurite growth and regeneration. In dissociated cultures of postnatal mouse spiral ganglia, regenerating neurites spontaneously associate with Schwann cells. However, the mechanisms and consequences of interactions between cochlear Schwann cells and spiral ganglion neurites have not been examined. Further, the similarities and differences between cochlear Schwann cells and other PNS Schwann cells have not been studied. Experiments to examine these questions will rely on the ability to purify and characterize cochlear Schwann cells. Here we present methods for purifying Schwann cells from postnatal mouse cochleas and for transfecting them with expression plasmids. Dissociated spiral ganglia were plated on poly-D-lysine/laminin in medium containing neurotrophins, leukemia inhibitory factor (LIF), N2 supplement and serum and maintained for 5 days. Cells were harvested with trypsin/EDTA and subjected to an immuno-magnetic purification procedure. After 24 h in vitro, cultures were >85% Schwann cells. Nucleofection of purified Schwann cells with pMax-green fluorescent protein (pMax-GFP) plasmid, or with pEGFP-C-vimentin plasmid returned >45% transfection efficiency. These methods will allow the in-depth characterization of cochlear Schwann cells and an evaluation of their biochemical, functional, and genetic mechanisms that may promote neurite growth from the spiral ganglion.

Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152.

Upon the death of their hair cell synaptic partners, bipolar cochlear spiral ganglion neurons either die or retract their peripheral nerve fibers. Efforts to induce the regrowth of the peripheral neurites have had to rely on limited knowledge of the mechanisms underlying spiral ganglion neurite regeneration and have been restricted by the impracticality of undertaking large numbers of manual analyses of neurite growth responses. Here we have used dissociated cultures of postnatal mouse spiral ganglia to assess the effects of the Rho kinase inhibitor H-1152 on neurite growth and to determine the utility of automated high content analysis for evaluating neurite length from spiral ganglion neurons in vitro. In cultures of postnatal mouse spiral ganglion, greater than 95% of the neurons develop bipolar, monopolar or neurite-free morphologies in ratios dependent on whether the initial medium composition contains leukemia inhibitory factor or bone morphogenetic protein 4. Cultures under both conditions were maintained for 24 h, then exposed for 18 h to H-1152. None of the cultures exposed to H-1152 showed decreased neuronal survival or alterations in the ratios of different neuronal morphologies. However, as measured manually, the population of neurite lengths was increased in the presence of H-1152 in both types of cultures. High content analysis using the Arrayscan VTi imager and Cellomics software confirmed the rank order differences in neurite lengths among culture conditions. These data suggest the presence of an inhibitory regulatory mechanism(s) in the signaling pathway of Rho kinase that slows the growth of spiral ganglion neurites. The automated analysis demonstrates the feasibility of using primary cultures of dissociated mouse spiral ganglion for large scale screens of chemicals, genes or other factors that regulate neurite growth.

Culture conditions determine the prevalence of bipolar and monopolar neurons in cultures of dissociated spiral ganglion.

To gain insight into the mechanisms that control the generation or maintenance of the characteristic bipolar morphology of cochlear spiral ganglion neurons, we have taken advantage of our recently developed procedure for culture of dissociated newborn mouse spiral ganglion. In these cultures, inclusion of the cytokine leukemia inhibitory factor (LIF) in the medium increases neuronal survival and the number of bipolar neurons. Here we tested effects of two other LIF-type cytokines (ciliary neurotrophic factor, CNTF; and human recombinant oncostatin M, hOSM) and of bone morphogenetic protein 4 (BMP4) on survival, morphology and neurite lengths of neurons in cultures of dissociated spiral ganglion. Like LIF, CNTF and hOSM increased neuronal survival and the number of surviving bipolar neurons. BMP4 also increased neuronal survival, but unlike LIF, CNTF and hOSM, increased the number of monopolar neurons and neurons with no neurites. In addition, population histograms demonstrate that the population lengths of the longer and shorter neurites of bipolar neurons were shorter in BMP4 containing cultures than in control or LIF cultures. When LIF and BMP4 were simultaneously added to the cultures, the BMP4 effects predominated. These experiments demonstrate that exposure to different environmental conditions can result in different morphologies in the surviving population of spiral ganglion neurons in culture.

Survival and morphology of auditory neurons in dissociated cultures of newborn mouse spiral ganglion.

We have systematically characterized neuronal survival and growth in cultures derived from newborn/postnatal day 1 mouse cochlea. Dissociated cultures of the cochlear spiral ganglion provide an experimental environment in which to examine molecular mechanisms of survival, development and physiology of auditory neurons. To relate survival to the total number of neurons present in the source tissue, three cochleas from different newborn CD-1 mice were embedded in Araldite resin and serially sectioned at 5 mum thickness. All neurons were counted. To avoid overcounting, each section served as a lookup section for the next, giving 8240+/-423 (S.D.) neurons per ganglion. Cultures maintained in the presence of adjacent non-neural tissue, brain-derived neurotrophic factor, neurotrophin 3, leukemia inhibitory factor (LIF) and 10% fetal bovine serum returned the best overall survival (30%) at 42 h post-plating. Best overall survival required the continuous presence of a serum component(s) larger than 100,000 MW. Plating efficiency (number of neurons that attach to the well after 4 h) was similar in the presence or absence of LIF. Inclusion of LIF maintained 100% survival of plated neurons over 42 h of culture; without LIF, a large fraction of the neurons did not survive. LIF appeared to maintain survival by preferentially preserving a population of bipolar neurons, while having little effect on the number of monopolar neurons. This work provides quantitative measures of survival and morphology of auditory neurons in vitro. The results support the idea that survival of spiral ganglion neurons in vivo may depend on interactions with adjacent, non-neural tissue and raise the possibility that maintenance of bipolar morphology after hair cell damage may require biochemical mechanisms in addition to those induced by neurotrophins.

A temporospatial map of adhesive molecules in the organ of Corti of the mouse cochlea.

In the nervous system, several classes of cell-surface and extracellular matrix molecules have been implicated in processes such as neural growth, fasciculation, pathfinding, target recognition and synaptogenesis, which require cell-to-cell or cell-to-substrate binding. In the developing mouse cochlea, little is known about the types of cell-surface and extracellular matrix molecules existing along the neural growth paths or their possible roles in development. Whole mount and sectioned cochlear tissue were immunolabeled for six different adhesive molecules - neural cell adhesion molecule (NCAM), polysialic acid (PSA), neural cell adhesion molecule L1, E-cadherin, syndecan-1 and tenascin-C. A temporospatial map of adhesive molecule distribution in the basal turns of the mouse cochlea was generated. Distributions of adhesive molecules were compared to each other and to the known progress of neural development in the region. This comparison demonstrated differences in the complements of adhesive molecules between the inner and outer hair cell regions, and variations in the expressions of adhesion molecules among different types of nerve fibers. In addition, developmental changes in the adhesive environment around and beneath the outer hair cells coincided with the known timing of the appearance of morphologically defined efferent synapses. These observations raise the possibility that molecular differences at the cell surface of inner and outer hair cells are one way that ingrowing neurites distinguish different environments to determine their growth routes and synaptic partners in the cochlea. In addition these observations demonstrate the potential for differential signaling of afferent and efferent innervation by altering the microenvironments in which synapses are formed.

Polysialic acid in the cochlea of the developing mouse.

Polysialic acid (PSA), an unusual molecule covalently attached to the neural cell adhesion molecule, NCAM, has been shown to regulate cell-to-cell and cell-to-matrix interactions by interfering with the binding of cell-surface adhesion molecules. We used immunocytochemistry to map the temporospatial distribution of PSA in the mouse cochlea between embryonic day 16 and postnatal day 32 and compared it to the known timetable of neural growth and development. Polysialic acid immunoreactivity develops along a temporospatial gradient beginning in the basal turn and progressing to the apical turn. The expression is transitory on spiral ganglion neurons, intraganglionic bundles, radial bundles, and outer hair cells. Immunoreactivity diminishes progressively from the basal turn to the apical turn. Immunolabeling is maintained to adulthood on fibers in the inner spiral and inner pillar bundles, bundles which have been suggested to sprout continually and grow even in older animals. Inner hair cells are never immunolabeled. The temporo-spatial expression of PSA suggests its involvement in neural growth, whereas its extinction correlates with the time of onset of nerve-receptor interactions.