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

Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years

Subjectivity in a context of environmental change: opening new dialogues in mental health research

In a period of unstable experimentation with challenges of globalization of associated risks, and disenchantment with ‘enduring injustice’, we bring forward a consideration of subjectivity to the study of environmental change and mental health. We begin by identifying how mainstream climate change and mental health studies are unable to explain the emergent and co-evolutionary pathways of agency. As a means of freeing these studies of their objective dimensions of linear-causation, we argue in favour of a re-positioning of subjectivity within an appreciation of recognition conflicts and beyond the over-deterministic interpretations of power centres—state, market or religion. We draw on one example of scientific research that was conducted in a region undergoing strong environmental, social and cultural changes, in the state of São Paulo/Brazil, with the aim to open mental health research to new dialogues, to which we contribute with the notion of the ‘pluriversal subject’

Chick cochlear hair cell exocytosis mediated by dihydropyridine-sensitive calcium channels

A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells).Tall hair cell depolarization resulted in changes in cell membrane capacitance (ΔCm) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (ICa) and ΔCm were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around −10 mV.Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed ICa and blocked ΔCm. Conversely, the DHP agonist Bay K 8644 increased both ICa and ΔCm amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels

Direct measurement of single-channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca2+ ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage-gated Ca2+ channels in bullfrog saccular hair cells by means of perforated and cell-attached patch-clamp techniques. Whole-cell Ca2+ current records provided hints that hair cells express L-type as well as dihydropyridine-insensitive Ca2+ currents.Single Ca2+ channel records confirmed the presence of L-type channels, and a distinct Ca2+ channel, which has sensitivity towards ω-conotoxin GVIA. Despite its sensitivity towards ω-conotoxin GVIA, the non-L-type channel cannot necessarily be considered as an N-type channel because of its distinct voltage-dependent gating properties.Using 65 mm Ca2+ as the charge carrier, the L-type channels were recruited at about –40 mV and showed a single-channel conductance of 13 pS. Under similar recording conditions, the non-L-type channels were activated at ∼–60 mV and had a single-channel conductance of ∼16 pS.The non-L-type channel exhibited at least two fast open time constants (τo = 0.2 and 5 ms). In contrast, the L-type channels showed long openings (τo =∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τo = 0.3 and 10 ms).The number of functional channels observed in patches of similar sizes suggests that Ca2+ channels are expressed singly, in low-density clusters (2–15 channels) and in high-density clusters (20–80 channels). Co-localization of the two channel subtypes was observed in patches containing low-density clusters, but was rare in patches containing high-density clusters.Finally, we confirmed the existence of two distinct Ca2+ channel subtypes by using immunoblot and immunohistochemical techniques

Tonotopic Distribution of Short-Term Adaptation Properties in the Cochlear Nerve of Normal and Acoustically Overexposed Chicks

Cochlear nerve adaptation is thought to result, at least partially, from the depletion of neurotransmitter stores in hair cells. Recently, neurotransmitter vesicle pools have been identified in chick tall hair cells that might play a role in adaptation. In order to understand better the relationship between adaptation and neurotransmitter release dynamics, short-term adaptation was characterized by using peristimulus time histograms of single-unit activity in the chick cochlear nerve. The adaptation function resulting from 100-ms pure tone stimuli presented at the characteristic frequency, +20 dB relative to threshold, was well described as a single exponential decay process with an average time constant of 18.6 ± 0.8 ms (mean ± SEM). The number of spikes contributed by the adapting part of the response increased tonotopically for characteristic frequencies up to ∼0.8 kHz. Comparison of the adaptation data with known physiological and anatomical hair cell properties suggests that depletion of the readily releasable pool is the basis of short-term adaptation in the chick. With this idea in mind, short-term adaptation was used as a proxy for assessing tall hair cell synaptic function following intense acoustic stimulation. After 48 h of exposure to an intense pure tone, the time constant of short-term adaptation was unaltered, whereas the number of spikes in the adapting component was increased at characteristic frequencies at and above the exposure frequency. These data suggest that the rate of readily releasable pool emptying is unaltered, but the neurotransmitter content of the pool is increased, by exposure to intense sound. The results imply that an increase in readily releasable pool size might be a compensatory mechanism ensuring the strength of the hair cell afferent synapse in the face of ongoing acoustic stress