NKCC1 in Neonatal Cochlear Support Cells Reloads Ions Necessary for Cochlear Spontaneous Activity

In the auditory system, the spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from inner supporting cells (ISCs). This ATP release sets off a cascade, activating purinergic autoreceptors, opening of Ca 2+ -activated Cl- channel TMEM16A, Cl- efflux and osmotic cell shrinkage. Then, the shrunken ISCs efficiently regain their original volume, suggesting the existence of mechanisms for refilling Cl- and K + , priming them for subsequent activity. This study explores the potential involvement of NKCCs (Na+ -K+ -Cl- cotransporters) and KCCs (K+ -Cl- cotransporters) in ISC spontaneous activity, considering their capability to transport both Cl- and K+ ions across the cell membrane. Employing a combination of immunohistochemistry, pharmacological interventions, and shRNA experiment, we unveiled the pivotal role of NKCC1 in cochlear spontaneous activity. Immunohistochemistry revealed robust NKCC1 expression in ISCs, persisting until the 2nd postnatal week. Intriguingly, we observed a developmental shift in NKCC1 expression from ISCs to synaptophysin-positive efferent terminals at postnatal day 18, hinting at its potential involvement in modulating synaptic transmission during the post-hearing period. Experiments using bumetanide, a well-known NKCC inhibitor, supported the functional significance of NKCC1 in ISC spontaneous activity. Bumetanide significantly reduced the frequency of spontaneous extracellular potentials (sEP) and spontaneous optical changes (sOCs) in ISCs. NKCC1-shRNA experiments conducted in cultured cochlear tissues further supported these findings, demonstrating a substantial decrease in event frequency and area. Taken together, we revealed the role of NKCC1 in shaping the ISC spontaneous activity that govern auditory pathway development.

Distribution of Kv3 Subunits in Cochlear Afferent and Efferent Nerve Fibers Implies Distinct Role in Auditory Processing

Kv3family K + channels, by ensuring speedy repolarization of action potential, enable rapid and high frequency neuronal firing and high precision temporal coding of auditory information in various auditory synapses in the brain. Expression of different Kv3 subtypes within the auditory end organ has been reported. Yet, their precise role at the hair cell synaptic transmission has not been fully elucidated. Using immunolabeling and confocal microscopy we examined the expression pattern of different Kv3 family K + channel subunits in the nerve fibers innervating the cochlear hair cells.Kv3.1b was found in NKA-positive type 1 afferent fibers, exhibiting high signal intensity at the cell body, the unmyelinated dendritic segment, first heminode and nodes of Ranvier.Kv3.3 signal was detected in the cell body and the unmyelinated dendritic segment of NKA-positive type 1 afferent fibers but not in peripherin-positive type 2 afferent. Kv3.4 was found in ChAT-positive LOC and MOC efferent fibers as well as peripherin-positive type 2 afferent fibers. Such segregated expression pattern implies that each Kv3 subunits participate in different auditory tasks, for example, Kv3.1b and Kv3.3 in ascending signaling while Kv3.4 in feedback upon loud noise exposure.