RESUMEN
Cochlear inner hair cells (IHCs) develop from pre-sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) CaV1.3 channels formed stripe-like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic CaV1.3-channels were selectively reduced, (iv) the intrinsic Ca(2)(+) dependence of fast exocytosis probed by Ca(2)(+) uncaging remained unchanged but (v) the apparent Ca(2)(+) dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca(2)(+) influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca(2)(+) influx through an individual channel dominates the [Ca(2)(+)] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca(2)(+) influx and exocytosis.
Asunto(s)
Calcio/metabolismo , Exocitosis/fisiología , Células Ciliadas Auditivas Internas/fisiología , Modelos Neurológicos , Ganglio Espiral de la Cóclea/fisiología , Sinapsis/fisiología , Animales , Canales de Calcio/metabolismo , Señalización del Calcio , Electrofisiología , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas Internas/citología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mutación , Técnicas de Placa-Clamp , Terminales Presinápticos/ultraestructura , Ganglio Espiral de la Cóclea/citología , Sinapsis/ultraestructuraRESUMEN
EF-hand Ca(2+)-binding proteins are thought to shape the spatiotemporal properties of cellular Ca(2+) signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca(2+) signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca(2+)-dependent inactivation of their Ca(2+) current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca(2+) channels and release sites (effective "coupling distance" of 17 nm). Substitution experiments with synthetic Ca(2+) chelators indicated the presence of endogenous Ca(2+) buffers equivalent to 1 mM synthetic Ca(2+)-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca(2+) buffers regulate presynaptic IHC function for metabolically efficient sound coding.
Asunto(s)
Calbindina 1/metabolismo , Calbindina 2/metabolismo , Señalización del Calcio/fisiología , Exocitosis/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Parvalbúminas/metabolismo , Animales , Calbindina 1/genética , Calbindina 2/genética , Señalización del Calcio/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Exocitosis/efectos de los fármacos , Células Ciliadas Auditivas Internas/citología , Audición/efectos de los fármacos , Audición/fisiología , Ratones , Ratones Noqueados , Parvalbúminas/genética , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
Voltage dependent ion channels can influence signal integration in neurons dramatically. In addition to the classical fast-inactivating Na(+) current that mediates action potentials, many neurons also express persistent sodium current (I(NaP)). Activating at membrane potentials below the threshold for action potentials, this current may amplify excitatory postsynaptic potentials and shape the firing patterns. To determine the qualitative contribution of I(NaP) to the intrinsic firing properties of motoneurons, we eliminated this current by dynamic clamp. As expected, we found that elimination of I(NaP) shifted the rheobase to more positive currents. More interestingly, elimination of I(NaP) increased the steepness of initial frequency-to-current (fI) relation. This suggests that I(NaP) decreases the transient gain and broadens the integration window for short synaptic inputs in spinal motoneurons.