Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Abstract

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and transmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

Diversity matters — extending sound intensity coding by inner hair cells via heterogeneous synapses

“…This was not amenable to the dual‐color imaging of Ca 2+ signals and glutamate release (Özçete & Moser, 2021), which limits the reliability of the approach and calls for validation by more resolved recordings. Future studies should also capture other key synaptic parameters such as SV tethering and docking, ideally at different functional stages (Chakrabarti et al , 2018, 2022), for which differences among AZs have not yet been studied in detail. Most importantly, the field will need to work on relating synaptic heterogeneity and neural firing diversity, which is far from trivial given the methodological differences of e.g.…”

“…Electron tomography allows the definition of SV subpools based on their tethering and docking status (Fernández‐Busnadiego et al , 2010; Chakrabarti et al , 2018). Comparative electron tomography of pillar and modiolar synapses, ideally following optogenetic stimulation and high‐pressure freezing (Chakrabarti et al , 2022), is expected to majorly advance our understanding of the nanoanatomy and nanophysiology of the IHC synapse. Since the postsynaptic density is typically well accessible in these samples, the presynaptic parameters and the size of the postsynaptic density (PSD) can be simultaneously approached, but typically not fully captured given the limit of typical 200 kV microscopes to a sample thickness of 250 nm or less.…”

“…(C–E) Optogenetic approach to mapping the synaptic position of functionally distinct SGNs on IHCs (C): ChR2‐H134R‐mediated IHC photocurrents lead to robust photodepolarization (D) and ensuing glutamate release revealed by recordings of light‐evoked EPSCs from postsynaptic boutons of SGNs (E). This way, IHCs can be stimulated in a less invasive way than using patch clamp (Adapted from Chakrabarti et al , 2022).…”

Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing

“…Such docked SVs could account for the size of the rapid RRP in auditory ribbon synapses of the adult bullfrog, which is resistant to Ca 2+ buffers like EGTA and BAPTA (Graydon et al, 2011). In murine IHCs, the number of docked SVs is rather limited under rest (Chakrabarti et al, 2018(Chakrabarti et al, , 2022Kroll et al, 2020) and strikingly increases with stimulation (Chakrabarti et al, 2022). In the Pclo gt/gt mutants, we observed a general reduction of the MP-SV pool, which might be the result of a larger proportion of small-sized ribbons.…”

“…For retinal ribbon synapses, a gradient of SVs has been observed depending on the activity state, with a depletion at the ribbon stage upon darkness (Jackman et al, 2009). At wild-type ribbon synapses in IHCs, such gradients or activity-induced depletion of SVs cannot be observed, neither at the membrane nor at the ribbon (Chakrabarti et al, 2018(Chakrabarti et al, , 2022. To our surprise, the observed structural alterations were not accompanied by functional changes on the whole IHC level: Ca 2+ currents and exocytosis of Pclo gt/gt IHCs were comparable to the controls.…”