Developmental changes in the voltage-gated Ca2+ channels (VGCC) that mediate acetylcholine (ACh) release at the transient efferent-inner hair cell synapse.

GRACIELA KEARNEY; JAVIER ZORRILLA DE SAN MARTÍN; CAROLINA WEDEMEYER; ANA B ELGOYHEN; ELEONORA KATZ

San Diego, California

Congreso; 37th Annual Midwinter meeting of the Association for Research in Otolaryngology; 2014

Association for Research in Otolaryngology

Since birth until the onset of hearing (postnatal day (P) 12 in mice), inner hair cells (IHCs) are innervated by medial olivocochlear fibers. At P9-11, ACh release is supported by both P/Q and N-type VGCCs and negatively regulated by L-type VGCC, coupled to BK channel activation. We previously reported that at P5-7, P/Q- but not N-type VGCC partially support ACh release and that blocking BK channel increases the quantum content (m) (Zorrilla de San Martín et al., ARO Abstracts 2012). Our goal is now to determine which other type/s of VGCCs mediate ACh release at this (P5-7) and earlier stages (P3) and whether BK channels and L-type VGCCs are functionally coupled. Postsynaptic responses were monitored in whole-cell voltage-clamped IHCs while electrically stimulating the efferent fibers in P3 and P5-7 mouse cochleas. At P5-7, SNX-482, the R-type VGCC antagonist, significantly reduced m (control: 0.84±0.01; 500 nM SNX: 0.37±0.05, n=2; p0.50 and m = 1.10±0.42 control; 2.67±0.80 Bay-K; 2.23±0.16 Bay-K+Ibtx; n=3; p >0.2). Moreover, nifedipine had no effect on m after blocking both P/Q-type VGCC and BK channels with 200 nM ω-AgaIVA and 100 nM Ibtx, respectively (m = 0.91±0.25 ω-Aga+Ibtx and 1.08±0.29 for ω-Aga+Ibtx+Nife; p>0.5, n=3). Besides, Bay-K had no effect on m after blocking BK channels (control: 0.95, Ibtx: 2.7, Ibtx+Bay-K: 2.8). Altogether, these results show that L-type VGCCs do not support but negatively modulate release by activating BK channels. Preliminary experiments show that at P3, ω-AgaIVA, does not affect m (control: 054±0.06; ω-AgaIVA 0.62±0.25; n=2; p>0.5), suggesting that P/Q-type VGCCs do not support release at this stage. At P3, Bay-K significantly enhanced m by 382±120%; n=2), suggesting that L-type VGCCs might be also be modulating release at this stage. We conclude that at P5-7, both P/Q and R-type VGGC support ACh release and that Ca2+ entry through L-type VGCCs, negatively modulate this process by activating BK channels. Supported by grants from University of Buenos Aires to EK, ABE; CONICET and ANPCYT to ABE, EK, NIH to PAF, ABE.