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

KEARNEY G; ZORRILA DE SAN MARTIN J; WEDEMEYER C; ELGOYHEN AB; KATZ E

Congreso; 36th Midwinter Meeting, Association for Research in Otolaryngology; 2014

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 Mart¨ªn 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¡À0.01; 500 nM SNX: 0.37¡À0.05, n=2; p<0.05).  Surprisingly, both the L-type VGCC antagonist (Nifedipine) and the agonist (Bay-K) enhanced m (%increment = 161¡À21, 3 mM Nife; 294¡À26, 10 mM Bay-K). This suggested that at this earlier stage, Ca2+ entry through L-type VGCC might be both supporting release and activating BK channels. However, occlusion experiments showed that Ibtx had no effect on m after incubation with L-type VGCC modulators (m = 0.56¡À0.18 control; 1.64¡À0.86 Nife; 1.85¡À1.02 Nife+Ibtx; n=2, p>0.50 and m = 1.10¡À0.42 control; 2.67¡À0.80 Bay-K; 2.23¡À0.16 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 ¦Ø-AgaIVA and 100 nM Ibtx, respectively (m = 0.91¡À0.25 ¦Ø-Aga+Ibtx and 1.08¡À0.29 for ¦Ø-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, ¦Ø-AgaIVA, does not affect m (control: 054¡À0.06; ¦Ø-AgaIVA 0.62¡À0.25; 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¡À120%; 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.