A CALCIUM IMAGING APPROACH TO INVESTIGATE TYPE II COCHLEAR AFFERENT ACTIVITY AFTER TRAUMA

Abstract

Auditory information is sent to the central nervous system from the cochlea through type I and type II cochlear afferents. While the function of the type I afferents is relatively well understood, definitive proof for the role of type II afferents lags far behind. Limited recordings from type II afferents suggest they are activated by near maximal outer hair cell stimulation, ATP, and by rupture of nearby outer hair cells. Taken together, these lines of evidence suggest that type II afferents sense damaging levels of sound. Recordings of type II afferents have been confined to the low-frequency regions of excised organs of Corti in pre-hearing animals due to the intractable nature of these methods. THCreER:GCaMP6f, DrD2Cre:GCaMP6f, and Tac1Cre:GCaMP6f mice were used to overcome some of these limitations by introducing a genetically encoded fluorescent calcium indicator in type II afferents spanning the range of the organ of Corti. In concordance with direct recordings, most type II afferents are inactive without external stimuli but become more and more responsive after depolarization with increasing levels of external potassium. Moreover, pre-hearing type II afferents in both the apex and the base reliably experience calcium transients following hair cell ablation, recapitulating previous electrophysiological recordings. Additionally, both indirect and direct methods of measuring epithelial cell activity suggest that these cell types also strongly respond to ablation. The combination of calcium imaging and innovative dissection techniques allowed for reliable recording of type II afferent activity in response to ablation in mature and noise-exposed animals. While the response is changed and less likely to occur, mature type II afferents can still experience calcium transients to focal ablation. Type II afferents in noise-exposed animals appear to become sensitized both through an increase in the number of presynaptic ribbons and by shifting their response to focal ablation. Overall, calcium imaging is capable of capturing type II afferent responses to damage even in mature and noise-exposed animals, further supporting their role as putative auditory nociceptors.