Presynaptic voltage-dependent Ca2+ channels involved in synaptic transmission at the mouse medial olivocochlear-outer hair cell synapse at the onset of hearing

LUCAS VATIINO; ANA BELÉN ELGOYHEN; ELEONORA KATZ

Baltimore, MD

Congreso; 40th Midwinter Meeting of the Association for Research in Otolaryngology; 2017

Association for Research in Otolaryngology

Medial olivocochlear (MOC) fibers begin to innervate the outer hair cells (OHCs), the final targets of this efferent system, at postnatal day (P)6. From birth to hearing onset (P12 in altricial rodents) MOC fibers also innervate the inner hair cells (IHCs). At P9-11, ACh release at the transient MOC-IHC synapse is supported by P/Q- and N-type voltage-gated Ca2+ channels (VGCC). At this age, Ca2+ flowing in through L-type VGCC activates large conductance Ca2+-activated potassium (BK) channels exerting a negative control on release likely by curtailing the duration of the terminal action potential (Zorrilla de San Martín et al., 2010). At earlier more immature postnatal ages (P5), however, N-type VGCC are not involved and release is mediated by P/Q- and R-type VGCCs. Interestingly, at this earlier developmental stage, L-type VGCCs have a dual role: they activate BK channels but also support release indicating less compartmentalization of presynaptic molecules involved in synaptic transmission (Kearney et al., ARO Abstracts 2014). So far, nothing is known about the presynaptic ion channels involved in transmitter release at the MOC-OHC synapse. Using isolated mouse cochleas at P11-13 we show by whole-cell voltage-clamp recordings in OHCs while stimulating the efferent axons that the quantum content of transmitter release (m) is very low (around 0.3-0.4) and mediated by both P/Q (200 nM ω-agatoxin VIA blocked ~74% of the control response n = 4, p < 0.03) and R-type VGCC (500 nM SNX482 blocked ~60% of the control response n = 7; p < 0.01). N-type VGCC, however, do not participate in evoked release at this synapse (500 nM ω-conotoxin IVA, had no significant effect on m; n = 7). In addition, we show that BK channels are functionally expressed at the MOC-OHC synapse as their block by iberiotoxin, a specific BK antagonist, significantly increased release (200 nM Ibtx caused a 188% increase in m; n = 6; p < 0.002). We also show that both the L-type VGCC antagonist nifedipine (3 μM) and the agonist BayK (10 μM) significantly increased m (154%, p < 0.02 and 216%, p < 0.05, respectively). Preliminary occlusion experiments with iberiotoxin and the dihydropiridines also indicate that Ca2+ flowing in through L-type VGCC is both activating BK channels and partially supporting ACh release. This resembles the MOC-IHC synapse at early immature stages (P5) and suggests that the MOC-OHCs synapse is functionally immature at the onset of hearing.Support:UBA&ANPCyT:EK,ABE; NIH: PAF,ABE.