Calcium- and otoferlin-dependent exocytosis by immature outer hair cells.
Immature cochlear outer hair cells (OHCs) make transient synaptic contacts (ribbon synapses) with type I afferent nerve fibers, but direct evidence of synaptic vesicle exocytosis is still missing. We thus investigated calcium-dependent exocytosis in murine OHCs at postnatal day 2 (P2)-P3, a developmental stage when calcium current maximum amplitude was the highest. By using time-resolved patch-clamp capacitance measurements, we show that voltage step activation of L-type calcium channels triggers fast membrane capacitance increase. Capacitance increase displayed two kinetic components, which are likely to reflect two functionally distinct pools of synaptic vesicles, a readily releasable pool (RRP; tau = 79 ms) and a slowly releasable pool (tau = 870 ms). The RRP size and maximal release rate were estimated at approximately 1200 vesicles and approximately 15,000 vesicles/s, respectively. In addition, we found a linear relationship between capacitance increase and calcium influx, like in mature inner hair cells (IHCs). These results give strong support to the existence of efficient calcium-dependent neurotransmitter release in immature OHCs. Moreover, we show that immature OHCs, just like immature IHCs, are able to produce regenerative calcium-dependent action potentials that could trigger synaptic exocytosis in vivo. Finally, the evoked membrane capacitance increases were abolished in P2-P3 OHCs from mutant Otof-/- mice defective for otoferlin, despite normal calcium currents. We conclude that otoferlin, the putative major calcium sensor at IHC ribbon synapses, is essential to synaptic exocytosis in immature OHCs too.
Beurg
Maryline
M
Equipe Neurophysiologie de la Synapse Auditive, Inserm U587 et Université Victor Segalen, Institut des Neurosciences de Bordeaux, Centre Hospitalier Universitaire Pellegrin, 33076 Bordeaux, France.
Safieddine
Saaid
S
Roux
Isabelle
I
Bouleau
Yohan
Y
Petit
Christine
C
Dulon
Didier
D
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D018072:Hair Cells, Auditory, Outer / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis / Q000502:physiology*; D051379:Mice; D008817:Mice, Mutant Strains; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology / Q000502:physiology*
D008565:Membrane Proteins; C516233:otoferlin protein, mouse; D002118:Calcium