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

Abstract

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.

Micropatterned ECM substrates reveal complementary contribution of low and high affinity ligands to neurite outgrowth

“…A dose-dependent effect of these ECM proteins has also been found in studies using micropatterned substrates. When PLL is used as background substrate (through precoating), neurite preference for LN is observed only at rather high LN concentrations, whereas in case of direct competition between LN and FN, neurites prefer LN, independently of the concentration of LN [Gundersen 1987;Evans et al, 2007]. In this study, we also noticed such dose-dependent effects (data not shown).…”

“…Several molecular substrate micropatterning techniques, such as lithography and lCP, have been developed as a means to investigate cellular processes such as proliferation, apoptosis, morphogenesis, differentiation and neurite outgrowth [Gundersen, 1987;Clark et al, 1993;Gomez and Letourneau, 1994;Kuhn et al, 1995;Gomez et al, 1996;Tai and Buettner, 1998;Renaudin et al, 1999;Wheeler et al, 1999;Corey and Feldman, 2003;Adams et al, 2005;Turney and Bridgman, 2005;Thery et al, 2006;Evans et al, 2007;Fink et al, 2007]. This study illustrates the efficacy and versatility of micropatterned substrates to analyze the molecular interactions underlying the point-to-point navigation of individual growth cones.…”

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

“…Regrowth of SGN peripheral axons toward the stimulating electrodes might allow reduced currents, more refined stimulation paradigms, and improved functional outcomes (Rubinstein, 2004). Thus, there is growing interest in therapeutic strategies to induce regrowth of SGN peripheral processes towards a stimulating electrode (Bianchi and Raz, 2004, Roehm and Hansen, 2005, Wittig et al, 2005, Evans et al, 2007. To the extent that chronic depolarization mimics electrical stimulation in vivo (Hegarty et al, 1997, Miller et al, 2003, inhibition of neurite outgrowth by depolarization suggests an additional consideration in the development of such strategies.…”

Orientation of spiral ganglion neurite extension in electrical fields of charge-balanced biphasic pulses and direct current in vitro

“…neurites among the dissociated SGN cultures. There were often Schwann cells and fibroblasts surrounding the neurites (data not shown), which could not only be obstacles to the migration of a growth cone, but could also act as guidance cues to neurites (Evans et al, 2007). Furthermore, the digesting procedure for the preparation of dissociated cultures may destroy membranous proteins, which are essential to neurite guidance.…”