Role of nicotinic acetylcholine receptor subunits alpha9 and alpha10 and calcium activated potassium channel SK2 in the development and molecular organization of the olivocochlear synapse.

摘要

The olivocochlear (OC) synapse between the medial efferent fibers originating in the brain stem and cochlear outer hair cells (OHCs) is responsible for modulating the cochlea's response to incoming sound. The OC synapse is primarily cholinergic, and uses calcium permeable excitatory nicotinic acetylcholine receptor (nAChR) composed of alpha9 and alpha10 subunits to activate a hyperpolarizing potassium current in hair cells mediated by SK2, a small conductance calcium activated potassium channel. The functional coupling between the excitatory nAChR and the inhibitory SK2 channel defines the OC-mediated postsynaptic response of OHCs.;Genetic ablation of the alpha9 or alpha10 nAChR subunits, or the SK2 channel results in abnormal development of OC synaptic terminals contacting the OHCs, accompanied by loss of nicotinic activity. Genetic manipulation of the alpha9alpha10 nAChRs and SK2 channels has helped define the role of OC innervation with respect to both OHC electrophysiology and protection against noise-induced trauma. However, null mutation of these genes has also revealed an unexpected developmental role in OC synapse formation and maintenance.;The work described in this thesis reveals that the functional coupling between nAChR and SK2 activity is required for the development and maintenance of OC synapse innervation and structure. Using transgenic mouse models that lack the genes encoding nAChR subunits alpha9 or alpha10, or the SK2 channel, I demonstrate that, while alpha9 and alpha10 nAChR-mediated activity plays an essential role in regulating the spatiotemporal development and stabilization of OC innervation on OHC during early postnatal ages, SK2 channel activity is required for the survival of the OC synapse on the OHCs during late postnatal stages of cochlear development in mice. Loss of SK2-medated OC activity results in degeneration of the OC terminals beginning at approximately 3 weeks of age that is complete by 12 weeks.;In addition, work described here defines a role for alpha9alpha10 nAChRs in the regulation of protein expression related to the OC synapses in a bi-directional manner. Loss of alpha9 and alpha10 nAChR-mediated activity leads to mis-expression of several key synaptic proteins, including trans-synaptic adhesion molecules, presynaptic proteins, and cytoskeletal and CREB pathway proteins. Loss of nAChR activity leads to changes in trans-synaptic adhesion proteins, inducing aberrant retrograde signals to the presynaptic terminals that ultimately impact proteins involved in exocytotic release. Loss of nAChR activity also impacts anterograde signals important for gene transcription. The key component linking both pre- and postsynaptic consequences that follow loss of nAChR activity seems to be altered expression of the trans-synaptic adhesion protein N-Cadherin, which leads to altered post-synaptic CREB signaling, and changes in proteins involved in the presynaptic OC terminal active zone structure/function.;Taken together, these findings reveal an essential role for cochlear hair cell alpha9alpha10 nAChRs and functionally coupled SK2 channels in the regulation of OC innervation and synaptogenesis. In addition, these studies begin to define the molecular composition of the OC synapse which was hitherto unknown. These findings suggest molecular mechanisms by which post-synaptic receptor activity may regulate the formation, maturation and molecular organization of the cholinergic OC synapse in the inner ear.