Neurotrophic factors modulate hair cells and their potassium currents in chick otocyst explants

Abstract

Neurotrophins, retinoids and their receptors are present in the sensory epithelia of the inner ear during development. We show that these factors modulate the proliferation of hair cells and their K+-currents when the embryonic day 3 (ED 3) presumptive inner ear (i.e. otocyst) is maintained in organ culture. All trans-retinoic acid (RA) increases hair cell differentiation and enhances the acquisition of outward currents, including a delayed rectifier and a fast activating, transient type, voltage-gated potassium current. In contrast, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decrease ionic current activity, and the addition of RA with the neurotrophins enhances this inhibitory response in an age-dependent manner. We measured the total number of cells per explant over time to determine precisely when and how these factors inhibit explant growth. We found that high concentrations of BDNF and NT-3 administered together, and low concentrations of both neurotrophins combined and administered with RA suppress otocyst cell numbers after 24 h in vitro. This suppressive response is induced by RA and NT-3, not by RA and BDNF. The suppressive or inhibitory influence of NT-3 and RA is the result of NT-3 binding to the low affinity receptor, p75NTR, not the result of RA increasing mRNA levels for the high affinity receptor, trkC. However, trk may act with p75NTR, as disruption of trk signalling alleviates the inhibitory response induced by NT-3 and RA. Our data suggest that various combinations and/or concentration gradients of these factors can differentially regulate inner ear development and hair cell excitability.

A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances

“…The effects of all-trans retinoic acid show that our in-vitro model recapitulates responses observed in primary tissue from the vertebrate inner ear (Kelley et al, 1993;Raz & Kelley, 1999;Sokolowski et al, 1999). We show that retinoic acid upregulates expression of myosin VIIa and enhances expression of potassium conductances similar to those observed in native hair cells.…”

“…I K,late was expressed only in cells cultured in serum and was not activated by retinoic acid. Despite this, retinoic acid enhanced current density as predicted from studies on its effects on developing chick inner ear in vitro, where it enhanced hair cell differentiation and the expression of voltage-gated potassium currents (Sokolowski et al, 1999). These effects are probably indirect as we found no evidence for activation of specific ion channel subunits in VOT-E36.…”

“…It has been tested as a potential agent for protection (Ahn et al, 2005) and regeneration of mammalian hair cells (Lefebvre et al, 1993;Kopke et al, 2001) although the results regarding regeneration have been controversial (Chardin & Romand, 1995;Romand & Chardin, 1999). There are several reports of the specific effects of adding retinoic acid to hair cell progenitors in organotypic culture, including suppression of cell division and the early onset of cell differentiation (Represa et al, 1990;Kelley et al, 1993), enhanced expression of myosin VIIa and the transcription factor Brn3c (Raz & Kelley, 1999), and enhanced expression of voltage-gated potassium conductances (Sokolowski et al, 1999).…”

Role of nerve growth factor in modulation of gastric afferent neurons in the rat

“…Recently, Winston et al (37) reported an upregualtion of VR-1 mRNA in dorsal root ganglion neurons cultured in the presence of NGF. Others have reported changes in the expression of both ligand-and voltage-gated ion channels (8,34), which will also affect the functional properties of afferent neurons and alter their firing patterns.…”

Protein–protein interactions of a Kvβ subunit in the cochlea

“…Once the cochlea was removed as described above, the tegmentum vasculosum was cut away with iridectomy scissors. As described previously (Sokolowski et al,1999), the cochlea was placed in a low‐calcium saline containing protease XXIV (0.09 mg/ml) (Sigma, St. Louis, MO), and the tectorial membrane was removed with forceps after 2–4 min. The cochlea was rinsed in normal divalent saline and tissue was fixed and embedded as described previously (Sokolowski and Cunningham,1996).…”