Co-release of GABA and ACh from medial olivocochlear neurons as a fine regulatory mechanism of cochlear efferent inhibition.

During development, inner hair cells (IHCs) in the mammalian cochlea are unresponsive to acoustic stimuli but instead exhibit spontaneous activity. During this same period, neurons originating from the medial olivocochlear (MOC) complex transiently innervate IHCs, regulating their firing pattern which is crucial for the correct development of the auditory pathway. Although the MOC-IHC is a cholinergic synapse, previous evidence indicates the widespread presence of gamma-aminobutyric acid (GABA) signaling markers, including presynaptic GABAB receptors (GABABR). In this study, we explore the source of GABA by optogenetically activating either cholinergic or GABAergic fibers. The optogenetic stimulation of MOC terminals from GAD;ChR2-eYFP and ChAT;ChR2-eYFP mice (of either sex) evoked synaptic currents in IHCs, which were blocked by α-bungarotoxin. This suggests that GABAergic fibers release acetylcholine (ACh) and activate α9α10 nicotinic acetylcholine receptors (nAChRs). Additionally, MOC cholinergic fibers release not only ACh but also GABA, as the effect of GABA on ACh response amplitude was prevented by applying a GABABR blocker. Using optical neurotransmitter detection and calcium imaging techniques, we examined the extent of GABAergic modulation at the single synapse level. Our findings suggest heterogeneity in GABA modulation, as only 15 out of 31 recorded synaptic sites were modulated by applying the GABABR specific antagonist, CGP 35348 (100-200 µM). In conclusion, we provide evidence indicating that GABA and ACh are co-released from at least a subset of MOC terminals. In this circuit, GABA functions as a negative feedback mechanism, locally regulating the extent of cholinergic inhibition at certain efferent-IHC synapses during an immature stage.Significance statement Before hearing onset, the medial olivocochlear (MOC) efferent system of the mammalian cochlea regulates the pattern of IHC spontaneous firing rate through the activation of α9α10 nAChRs. However, GABA is also known to have a modulatory role at the MOC-IHC synapse. Our results suggest that GABA is co-released from at least a subset of MOC terminals, working as a precise regulatory mechanism for ACh release. Furthermore, we demonstrate that not all synaptic contacts within a single IHC are equally modulated by GABA.