Calcium channels that support acetylcholine release at the transient olivocochlear-inner hair

JAVIER ZORRILLA DE SAN MARTÍN; CAROLINA WEDEMEYER; PAUL A. FUCHS; ANA BELÉN ELGOYHEN; ELEONORA KATZ

Phoenix, Arizona, USA

Congreso; 31st Midwinter Meeting of the Association for Research in Otolaryngology; 2008

Before the onset of hearing, inner hair cells (IHC) of the mammalian cochlea are transiently innervated by medial olivocohlear efferent fibers. This synapse is cholinergic, inhibitory and mediated by the á9á10 nicotinic cholinergic receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. á9á10 nicotinic cholinergic receptor. During postnatal development, IHC undergo dramatic changes in cholinergic sensitivity, pattern of innervation and expression of key postsynaptic proteins. In developing synapses, synaptic modifications likely take place concurrently in both postsynaptic cells and presynaptic terminals. In mammals, fast synaptic transmission at both central and peripheral synapses is mediated by multiple types of voltage-gated Ca2+ channels (VGCCs), including N-type (Cav2.2), P/Q (Cav2.1) type and R-type (Cav2.3). So far, nothing is known concerning the VGCCs that support ACh release at the efferent-IHC synapse. Therefore, in this work we evaluated the type/s of Ca2+ channels coupled to ACh release at this synapse before the onset of hearing. We used the acutely isolated cochlear preparation from mice and evaluated the effects of different Ca2+ channel blockers on the quantal content (m) of transmitter release and on the amplitude of spontaneous synaptic currents. Postsynaptic cholinergic currents in IHCs, voltage-clamped at -90 mV, were evoked by electrically stimulating the efferent fibers. Both ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse. ù-conotoxin-GVIA (300 nM), an N-type VGCC blocker, and ù-Agatoxin-IVA (200 nM), a P/Q-type VGCC blocker, significantly reduced m (49 ± 17%, n=5 and 52 ± 8%, n=4, respectively). When both toxins were applied together, ACh release was reduced by 96% (n=1). Neither of the toxins affected the amplitude of spontaneous synaptic currents (n=2-3), indicating that their site of action is only presynaptic. Our results suggest that both N and P/Q-type VGCCs support ACh release at the transient efferent-IHC synapse.