Purinergic Modulation of Acetylcholine Release through P2Y and A1 Receptors at the Efferent-Inner Hair Cell Synapse in the Developing Inner Ear

FACUNDO ALVAREZ HEDUAN; ELEONORA KATZ; JUAN GOUTMAN; ANA BELÉN ELGOYHEN

Baltimore, MD

Congreso; Annual 38th Midwinter Meeting of the Association for Research in Otolaryngology; 2015

Association for Research in Otolaryngology

Before the onset of hearing (postnatal day (P) 12 in mice) inner hair cells (IHCs) are transiently innervated by medial olivocochlear (MOC) efferent fibers. Acetylcholine released by these fibers activates alpha9alpha10 nicotinic receptors coupled to SK2 calcium-activated potassium channels, leading to inhibitory post synaptic currents (IPSCs). During this developmental period, IHCs fire spontaneous sensory-independent action potentials that are required for normal development of the auditory pathway. Recent studies suggest that this spontaneous activity is driven and/or modulated by ATP released from cochlear supporting cells and that ACh released from efferent fibers would also contribute to this regulation. The main goal of this study is to investigate the role of ATP and their products of hydrolysis such as adenosine in modulating the MOC-IHC synapse. IHC from Balb/c mice at P9-11 were recorded in the wholecell patch-clamp mode while applying different agonists and antagonists of purinergic receptors. Neurotransmitter release ARO Abstracts 15 Volume 38, 2015 was evoked by electrical stimulation of the efferent axons. 100 double pulse (interval: 25 ms) stimuli protocols were applied at a frequency of 1 Hz. The quantal content (m) was estimated as the ratio between the mean amplitude of evoked synaptic currents and the mean amplitude of spontaneous synaptic currents. ATP reversibly decreased m in a concentration dependent manner compared to control (1 μM: 15±7 %, 10 μM: 52±14 %; 50 μM: 57±11 %, 100 μM: 51±6 %). Suramin, a non-specific P2 antagonist, abolished the effect of ATP. PPADS, an antagonist with a preferential effect on P2X receptors, and TNP-ATP, a specific P2X antagonist, did not modify ATP-induced inhibition. Furthermore, alpha,beta-MeATP, a specific P2X agonist, had no effect on m. Both non-hydrolyzable ATP analog ATPγS, and the specific P2Y agonist 2-MeSADP, mimicked the effect of ATP (39±9 % and 58±7 % compared to control, respectively). Similar effects of ATP and ATPγS were observed during trains of stimuli. Adenosine decreased m in a reversible and concentration dependent manner (1 μM: 42±8 %, 10 μM: 70±5 %, 100 μM: 72±4 %). CGS15943, a specific P1 receptor antagonist, abolished the effect of adenosine, while NECA, a specific P1 agonist, decreased m (10 μM: 74±0.5 %). Our results show that both ATP and adenosine inhibit ACh release at the MOC-IHC synapse through the activation of P2Y and A1 receptors, respectively. These results suggest that ATP may have multiple roles in the developing cochlea.