Optogenetic activation of medial olivocochlear efferent fibers

TAIS CASTAGNOLA; ELEONORA KATZ; A. BELEN ELGOYHEN; JUAN D. GOUTMAN; CAROLINA WEDEMEYER

Conferencia virtual

Congreso; 44th Annual MidWinter meeting of the Association for Research in Otolaryngology; 2021

During development, inner hair cells (IHCs) in the mammalian cochlea are unresponsive to acoustic stimuli but instead present intrinsic electrical activity, crucial for the normal development of the auditory pathway. During this same period, neurons originating from the medial olivocochlear complex (MOC) transiently innervate the soma of IHCs. This synapse is mediated by acetylcholine (ACh), which activates nicotinic receptors assembled by α9 and α10 subunits and ultimately controls IHC excitability during this period. Even though this is a cholinergic synapse, previous evidence indicates the presence of abundant GABA and presynaptic GABAB receptors. Moreover, the application of GABAB receptors agonists can reduce ACh release. To specifically drive gene expression in MOC efferent fibers, transgenic Chattm2(crc)Lowl/J-Cre (ChAT-cre) and Gad2tm1(cre/ERT2)Zjh/J-Cre (GAD2-cre) mice were mated with a floxed ChR2 line, Ai32. Whole cell patch clamp at the IHC were performed in P9-11 mice while optogenetically stimulated either cholinergic (ChAT-Cre/ChR2) or GABAergic (GAD2-Cre/ChR2) fibers. The expression of Cre in GAD2-cre mice was induced by tamoxifen, injected during P1 to P5 (I.P50µg/g).We showed, for the first time, that cholinergic MOC fibers can be optogenetically activated in ChAT-cre/ChR2 mice. We found that in ChR2 homozygous mice (n=10), Ach release is triggered more robustly than in ChR2 heterozygous mice (n=7). In addition, blocking GABAB receptors with CGP36216 while stimulating ChR2-expressing cholinergic fibers increased the amplitude of the response and decreased synaptic failures, suggesting the presence of GABA together with ACh in the synaptic cleft. Optogenetic experiments were also performed to activate GABAergic fibers in GAD2-cre/ChR2 mice. Preliminary results indicated that activation of ChR2 by light is not sufficient to elicit a measurable cholinergic synaptic response in IHC per se. Additionally, immunohistochemistry techniques were used to characterize cholinergic and GABAergic expression in B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (tdTomato) crossed with ChAT-Cre or GAD2-Cre respectively. Although our functional data in ChAT-Cre/ChR2 mice suggest that, during development, GABA is co-released with ACh from MOC terminals, a more detailed study of the GAD2-Cre /ChR2 mice would be needed to back this conclusion.