synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

Abstract

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.

Author Summary

Ribbon synapses are found in the ear and eye and facilitate the transmission of sensory information to the brain. In hair cells of the ear, the molecules required for ribbon function have not been fully explored. Zebrafish are ideal for investigating molecular components of these specialized synapses because of the ability to study ribbon function using genetic, cellular, and physiological methods. Here, we explore the role of the lipid phosphatase Synaptojanin at the hair cell synapse. Synaptojanin has been previously implicated in synaptic vesicle recycling in conventional synapses, and we also find that the number of synaptic vesicles are reduced in mutant synaptojanin hair cells. Mutant synaptojanin larvae have obvious equilibrium defects, and our electrophysiological recordings revealed that synaptic transmission from hair cells to neurons projecting to the brain is impaired in terms of both rate and accuracy. When stimulated at high frequency or for prolonged periods, mutant synaptojanin hair cells release vesicles out of phase with mechanical stimuli, thus compromising the transfer of sensory information to the brain.