We have employed both in vitro patch clamp recordings of hair cell synaptic vesicle fusion and in vivo single unit recording of cochlear nerve activity to study, at the same synapse, the time course, control, and physiological significance of readily releasable pool dynamics. Exocytosis of the readily releasable pool was fast, saturating in less than 50 ms, and recovery was also rapid, regaining 95% of its initial amplitude following a 200-ms period of repolarization. Longer depolarizations (greater than 250 ms) yielded a second, slower kinetic component of exocytosis. Both the second component of exocytosis and recovery of the readily releasable pool were blocked by the slow calcium buffer, EGTA. Sound-evoked afferent synaptic activity adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. Comparison of readily releasable pool amplitude, capture distances of calcium buffers, and number of vesicles tethered to the synaptic ribbon suggested that readily releasable pool dynamics reflect the depletion of release-ready vesicles tethered to the synaptic ribbon and the reloading of the ribbon with vesicles from the cytoplasm. Thus, we submit that rapid recovery of the cochlear hair cell afferent fiber synapse from short-term adaptation depends on the timely replenishment of the synaptic ribbon with vesicles from a cytoplasmic pool. This apparent rapid reloading of the synaptic ribbon with vesicles underscores important functional differences between synaptic ribbons in the auditory and visual systems.

Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, 2-hydroxypropyl-β-cyclodextrin (HPβCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPβCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPβCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology.

Tyrosine receptor kinase B (TrkB)-mediated signaling modulates synaptic structure and strength in hippocampal and other neurons, but the underlying mechanisms are poorly understood. Full-length and truncated TrkB are diffusely distributed throughout the dendrites and soma of rat hippocampal neurons grown in vitro. Manipulation of TrkB-mediated signaling resulted in dramatic changes in the number and synaptic localization of postsynaptic NMDA receptor (NMDAR) and GABA A receptor (GABA A R) clusters. BDNF treatment resulted in an increase in the number of NMDAR and GABA A R clusters and increased the proportion of clusters apposed to presynaptic terminals. Downregulation of TrkB signaling resulted in a decrease in receptor cluster number and synaptic localization. Examination of the time course of the effects of BDNF on receptor clusters showed that the increase in GABA A R clusters preceded the increase in NMDAR clusters by at least 12 hr. Moreover, the TrkB-mediated effects on NMDAR clusters were dependent on GABA A R activation. Although TTX, APV, and CNQX treatment had no effect, blockade of GABA A Rs with bicuculline abolished the BDNF-mediated increase in NMDAR cluster number and synaptic localization. In contrast, application of exogenous GABA prevented the decrease in NMDAR clusters induced by BDNF scavenging. Together, these results suggest that TrkB-mediated signaling modulates the clustering of postsynaptic GABA A Rs and that receptor activity is required for a subsequent upregulation of NMDAR clusters. Therefore, TrkB-mediated effects on postsynaptic neurotransmitter clusters may be part of a mechanism that balances inhibitory and excitatory synaptic transmission in developing neural circuits.

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