No abstract

SUMMARYRibbon synapses of the ear, eye and pineal gland contain a unique protein component: Ribeye. Ribeye consists of a novel aggregation domain spliced to the transcription factor CtBP2 and is one of the most abundant proteins in synaptic ribbon bodies. Although the importance of Ribeye for the function and physical integrity of ribbon synapses has been shown, a specific role in synaptogenesis has not been described. Here, we have modulated Ribeye expression in zebrafish hair cells and have examined the role of Ribeye in synapse development. Knockdown of ribeye resulted in fewer stimulus-evoked action potentials from afferent neurons and loss of presynaptic Ca V 1.3a calcium channel clusters in hair cells. Additionally, afferent innervation of hair cells was reduced in ribeye morphants, and the reduction was correlated with depletion of Ribeye punctae. By contrast, transgenic overexpression of Ribeye resulted in Ca V 1.3a channels colocalized with ectopic aggregates of Ribeye protein.Overexpression of Ribeye, however, was not sufficient to create ectopic synapses. These findings reveal two distinct functions of Ribeye in ribbon synapse formation -clustering Ca V 1.3a channels at the presynapse and stabilizing contacts with afferent neurons -and suggest that Ribeye plays an organizing role in synaptogenesis.

Many auditory, vestibular, and lateral-line afferent neurons display spontaneous action potentials. This spontaneous spiking is thought to result from hair-cell glutamate release in the absence of stimuli. Spontaneous release at hair-cell resting potentials presumably results from Ca V 1.3 L-type calcium channel activity. Here, using intact zebrafish larvae, we recorded robust spontaneous spiking from lateralline afferent neurons in the absence of external stimuli. Consistent with the above assumptions, spiking was absent in mutants that lacked either Vesicular glutamate transporter 3 (Vglut3) or Ca V 1.3. We then tested the hypothesis that spontaneous spiking resulted from sustained Ca V 1.3 activity due to depolarizing currents that are active at rest. Mechanotransduction currents (I MET ) provide a depolarizing influence to the resting potential. However, following block of I MET , spontaneous spiking persisted and was characterized by longer interspike intervals and increased periods of inactivity. These results suggest that an additional depolarizing influence maintains the resting potential within the activation range of Ca V 1.3. To test whether the hyperpolarization-activated cation current, I h participates in setting the resting potential, we applied I h antagonists. Both ZD7288 and DK-AH 269 reduced spontaneous activity. Finally, concomitant block of I MET and I h essentially abolished spontaneous activity, ostensibly by hyperpolarization outside of the activation range for Ca V 1.3. Together, our data support a mechanism for spontaneous spiking that results from Ca 2ϩ -dependent neurotransmitter release at hair-cell resting potentials that are maintained within the activation range of Ca V 1.3 channels through active I MET and I h .

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.