Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells.

Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking.

A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse.

Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.

Dye Tracking Following Posterior Semicircular Canal or Round Window Membrane Injections Suggests a Role for the Cochlea Aqueduct in Modulating Distribution.

The inner ear houses the sensory epithelium responsible for vestibular and auditory function. The sensory epithelia are driven by pressure and vibration of the fluid filled structures in which they are embedded so that understanding the homeostatic mechanisms regulating fluid dynamics within these structures is critical to understanding function at the systems level. Additionally, there is a growing need for drug delivery to the inner ear for preventive and restorative treatments to the pathologies associated with hearing and balance dysfunction. We compare drug delivery to neonatal and adult inner ear by injection into the posterior semicircular canal (PSCC) or through the round window membrane (RWM). PSCC injections produced higher levels of dye delivery within the cochlea than did RWM injections. Neonatal PSCC injections produced a gradient in dye distribution; however, adult distributions were relatively uniform. RWM injections resulted in an early base to apex gradient that became more uniform over time, post injection. RWM injections lead to higher levels of dye distributions in the brain, likely demonstrating that injections can traverse the cochlea aqueduct. We hypothesize the relative position of the cochlear aqueduct between injection site and cochlea is instrumental in dictating dye distribution within the cochlea. Dye distribution is further compounded by the ability of some chemicals to cross inner ear membranes accessing the blood supply as demonstrated by the rapid distribution of gentamicin-conjugated Texas red (GTTR) throughout the body. These data allow for a direct evaluation of injection mode and age to compare strengths and weaknesses of the two approaches.

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse.

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon's contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.

Calcium-dependent synaptic vesicle trafficking underlies indefatigable release at the hair cell afferent fiber synapse.

Sensory hair cell ribbon synapses respond to graded stimulation in a linear, indefatigable manner, requiring that vesicle trafficking to synapses be rapid and nonrate-limiting. Real-time monitoring of vesicle fusion identified two release components. The first was saturable with both release rate and magnitude varying linearly with Ca(2+), however the magnitude was too small to account for sustained afferent firing rates. A second superlinear release component required recruitment, in a Ca(2+)-dependent manner, of vesicles not in the immediate vicinity of the synapse. The superlinear component had a constant rate with its onset varying with Ca(2+) load. High-speed Ca(2+) imaging revealed a nonlinear increase in internal Ca(2+) correlating with the superlinear capacitance change, implicating release of stored Ca(2+) in driving vesicle recruitment. These data, supported by a mass action model, suggest sustained release at hair cell afferent fiber synapse is dictated by Ca(2+)-dependent vesicle recruitment from a reserve pool.

Tracking vesicle fusion from hair cell ribbon synapses using a high frequency, dual sine wave stimulus paradigm.

Recent experiments describe a technique for tracking membrane capacitance during depolarizations where membrane conductance is varying. This is a major advance over traditional technologies that can only monitor capacitance when conductance is constant because it gives direct information regarding release kinetics from single stimulations. Presented here is additional data supporting the use of this technology with multiple conductances being active including BK-Ca-activated potassium channels, SK Ca-activated potassium conductances and also the rapidly activating sodium conductance. It goes further to illustrate the ability to monitor rapid capacitative changes. And finally, it points out the need to evaluate single step responses because of the use-dependent movement of vesicles.