A, TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) secretion during neonatal development. Diagram shows BDNF secretion, expressed as percentage of control secretions at P0 (black bars) and P20 (grey bars) from rat utricles at low temperature (LT) and control (C) conditions, under an electrical stimulation protocol in absence (S) or presence of TTX (STTX) in the bathing solution. Each column represents the mean of three experiments (±s.d.). Statistically significant differences are indicated by asterisks: * P < 0.05. B, developmental evolution of Nav1.2 and BDNF immunoreactivities. Laser confocal microscopy micrographs of double immunofluorescence staining using antibodies directed against Nav1.2 Na+ channel subunit (a–c) or BDNF (d–f), (red labelling) and neurofilament (green labelling) at three developmental stages P0-3, P10 and P20. Ba, at early stages P0-P3, most of the neonatal hair cell (asterisks) membranes are immunostained with anti-Nav1.2 Na+ channel antibodies. In addition, a strong level of staining within the apical part of the epithelium involving both hair cells and supporting cells is clearly identified (upper arrows). Some basal membranes of supporting cells (lower arrow) are also labelled. Bb, at P10, Nav1.2 immunostaining is detected in few sensory cells (asterisk), while it is intensely recovered within the apical region and the basal intracellular compartment of supporting cells (upper and lower arrows). Bc, in the mature utricular sensory epithelium (P20), Nav1.2 immunoreactivity is mainly restricted to the basal intracellular compartments of supporting cells (arrows). Sensory cells are devoid of immunostaining except for isolated cells (asterisk) remaining labelled. Bd, the cytoplasm of hair cells (asterisks) and the middle part of supporting cells (arrow) display a strong BDNF immunostaining at neonatal stages. Be and f, from P10 stage, BDNF immunoreactivity is no longer detected in sensory cells and appears as restricted to the apex of supporting cells (upper arrows). Neurofilament co-staining (green labelling) shows the epithelium innervation. Calyceal endings of afferent fibres innervating type I sensory cells are clearly identified. Scale bar = 10 µm.