Developmental Acquisition of a Rapid Calcium-Regulated Vesicle Supply Allows Sustained High Rates of Exocytosis in Auditory Hair Cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane

Hyperacusis in the Adult Fmr1-KO Mouse Model of Fragile X Syndrome: The Therapeutic Relevance of Cochlear Alterations and BKCa Channels

International audienceHyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs

Kinetics of vesicle release increases with HC maturation.

<p>Cochlear apical HCs were voltage-stepped from holding potential of −90 mV to -10 mV for increasing duration (20 to 3000 ms). To avoid synaptic depression, each pulse of varying duration was separated by a 30 s recovery period. A) Comparative mean ΔC_m responses (RRP+SRP) recorded in P2 (n = 8) and E10 (n = 6) apical HCs. B) RRP depletion, corresponding to the first 100 ms of release, was fitted with a single exponential with τ = 89, 75, 48, and 39 ms at E10 (n = 6), E12 (n = 7), E16 (n = 9) and P2 (n = 8), respectively. (C–D) Comparative ΔC_m responses (RRP + SRP) recorded in P2 or E12 apical HCs using 0.5 (as in A and B) or 2 mM intracellular EGTA (n = 4 and 5, respectively). Contrary to the SRP, note that the RRP (D) is not affected by 2 mM EGTA in both mature and immature HCs.</p

An highly efficient vesicle recruitment allows sustained release in mature HCs.

<p>A)Recording examples of C_m and I_Ca from an E12 apical HC during a train of 100 ms pulses (from -90 mV to -10 mV), each separated by 200 ms. B) Recruitment example evoked in similar conditions in a P2 apical HC. C–D–E) Apical HCs: Comparative mean cumulative ΔC_m changes over number of stimuli in E12 (n = 5) and P2 (n = 4) HCs (C). Comparative mean ΔC_m changes over number of stimuli (D). Contrary to mature P2 HCs, E12 immature HCs cannot sustain constant exocytosis during the train of stimuli. The comparative Ca²⁺ efficiency (fF/pC) calculated after each stimulus is shown in E. (F, G, H) Summary data comparing cumulative ΔC_m changes and Ca²⁺ efficiency at E12 (n = 5), and P2 (n = 4) in basal HCs.</p

Main characteristics of Ca²⁺ dependence of exocytosis in developing chick HCs (LF = low frequency and HF = high frequency).

<p>N indicates power (cooperative) index.</p

Ca²⁺ efficiency of RRP exocytosis increases with maturation.

<p>(A) Examples of I_Ca and ΔC_m recordings following a 100 ms-voltage step from holding potential of -90 mV to -10 mV at E10, E12, E16, and P2 basal HCs. (B) Voltage-dependence of I_Ca and ΔC_m recorded in basal HCs at E10 (n = 6), E12 (n = 9), E16 (n = 9) and P2 (n = 7). I_Ca was measured at its peak value during the voltage pulse shown in A. To limit depression, each consecutive voltage-step was separated by a 30 s recovery period. (C) Synaptic transfer functions relating Q_Ca (charge integral of Ca²⁺ current) and ΔC_m in basal HCs. Data points were fitted using first order power function with ΔC_m = s[I_Ca]<i>^N</i>, where s =  slope factor (Ca²⁺ efficiency; fF/pC) and <i>N</i> =  power index or degree of co-operativity. Values of s and N are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025714#pone-0025714-t001" target="_blank">table 1</a>. (D) Comparative Ca²⁺ efficiency (ΔC_m/Q_Ca) in apical (E10, n = 10; E12, n = 13; E16, n = 11; and P2, n = 9) and basal HCs (n as in B) as a function of developmental age. (E) Comparative cooperative (power) index <i>N</i> from power fit of data in D as a function of age in developing apical and basal HCs.</p

Rate of vesicle replenishment becomes highly sensitive to intracellular EGTA with maturation.

<p>A) Two paired-pulse recordings of I_Ca and ΔC_m in response to two consecutive 100 ms depolarizing steps (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025714#pone-0025714-g003" target="_blank">Fig 3</a>) separated by either 200 ms or 1 s in an E12 apical tall HC using 2 mM intracellular EGTA. (B) Two paired-pulse recordings of I_Ca and ΔC_m as in A but in mature P2 apical HCs. Note the strong depression in ΔC_m after the 2^nd pulse. (C) Comparative RRP recovery after 200 ms inter-pulse using 0.5 or 2 mM intracellular EGTA in E12 (n = 5) and P2 (n = 5) apical HCs. Asterisk indicates statistical significance with p<0.01. (D) Comparative kinetics of RRP recovery using 0.5 or 2 mM intracellular EGTA in mature P2 (n = 5) or immature E12 (n = 5) apical HCs.</p

Synaptic Release Potentiation at Aging Auditory Ribbon Synapses

International audienceAge-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca 2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis

CtBP2 protein (RIBEYE) and otoferlin expression in developing chick HC.

<p>A) Surface preparations of the chick basilar papilla at various developmental stages were labeled with CtBP2 (green) and phalloidin antibodies (blue). Confocal images are averaged over 5–9 Z-stack images of 0.4 µm each and taken from the nucleus area to the bottom of the cell. At right, the graphs indicate the corresponding distribution of the averaged number of ctBP2 spots (ribbons) per HCs from 5 basilar papilla in the low frequency region. Lower bar scale indicates10 µm. B) Confocal images showing otoferlin (red) expression at different developmental stages in the apical low frequency region of chick cochlea (E9, E12 and P2). Note the increasing expression of otoferlin at the HC plasma membrane with development.</p

Kinetics of RRP replesnishment increase during development.

<p>(<b>A</b>) Recording examples of I_Ca and ΔC_m responses during a paired-pulse protocol (two consecutive 100 ms steps from -90 mV to -10 mV separated by 200 ms) at E12 and P2 in apical and basal tall HCs. (<b>B</b>) Comparative paired-pulse recovery (% of first response) at E10 (n = 7), E12 (n = 8), E16 (n = 7) and P2 (n = 4). Recordings were made with an intracellular Ca²⁺ buffer of 0.5 mM EGTA. Note a larger depression in immature pre-hearing HCs E10–E13 as compared to P2 mature HCs. Asterisks indicate a statistical difference with p<0.01. (<b>C</b>) Comparative kinetics of RRP recovery in E12 and E16 apical HCs when varying the inter-pulse time in the paired-pulse protocol. In immature E12 HCs, data points were best fitted with two exponentials with time constants of 800 ms and 6 s (n = 5). In E16 HCs (n = 5), data points were best fitted with a single exponential with time constant of 680 ms. (<b>D</b>) During the paired-pulse protocol, in E12 apical HCs, RRP and I_Ca showed similar depression or slow kinetics of recovery.</p