Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue

Ceriani, Federico; Ciubotaru, Catalin D.; Bortolozzi, Mario; Mammano, Fabio

doi:10.1007/978-1-4939-3615-1_13

Cited by 12 publications

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“…The intracellular Ca 2+ level in OHCs increased rapidly but decayed with a relatively long fluorescence decay time constant (~300 ms; Ceriani et al, 2016). Calcium signals are evident in OHCs.…”

Section: Resultsmentioning

confidence: 95%

“…E Simultaneous cell-attached patch clamp recording(top) and Ca 2+ imaging (bottom) obtained from a P1 OHC from wild-type mouse at room temperature. The intracellular Ca 2+ level in OHCs increased rapidly but decayed with a relatively long fluorescence decay time constant (~300 ms;Ceriani et al, 2016). F Percentage of apical and basal OHCs showing spontaneous Ca 2+ signals at near body temperature and as a function of postnatal age.…”

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Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

et al. 2019

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Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine‐tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non‐sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience‐independent Ca2+ signals from sensory and non‐sensory cells.

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Section: Resultsmentioning

confidence: 95%

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confidence: 99%

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

et al. 2019

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“…Ca 2+ signals were recorded using a custom-built spinning disk confocal microscope (Ceriani et al, 2016a). Fluorescence excitation was produced by light emitted from a 470 nm LED (M470L2, Thorlabs) filtered through a BP460–480 filter (Olympus), and directed onto the sample through a 515 DCXR dichromatic mirror (Chroma Technology).…”

Section: Methodsmentioning

confidence: 99%

Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea

et al. 2016

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Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients.SIGNIFICANCE STATEMENT The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information.

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“…( E ) Simultaneous cell-attached patch clamp recording (top) and Ca 2+ imaging (bottom) obtained from a P1 OHC from wild-type mice at room temperature. The intracellular Ca 2+ level in OHCs increased rapidly but decayed with a relatively long fluorescence decay time constant (~300 ms) ( Ceriani et al, 2016 ). ( F ) Percentage of apical and basal OHCs showing spontaneous Ca 2+ spikes at near body temperature and as a function of postnatal age.…”

Section: Resultsmentioning

confidence: 99%

Spontaneous and coordinated Ca²⁺activity of cochlear sensory and non-sensory cells drives the maturation of OHC afferent innervation

Ceriani

Hendry

Jeng

et al. 2018

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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 3 adult cochlea (Guinan, 1996). However, OHCs are also innervated by Type II afferent fibres 49 that, unlike the Type I fibres contacting IHCs, which encode sound timing, intensity and 62We found that during a narrow, critical period of postnatal development, OHCs fire 63 spontaneous Ca 2+ spikes immediately preceding their functional maturation (~P7-P8 (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 4 Results 70The functional development of OHCs was studied primarily in the apical third of the Figure 1C, Movie 1) and room temperature (~20°C: Figure 1D (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.)The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 5 stopping a couple of days later ( Figure 1F). This correlated with a decrease in the maximum 95 Ca 2+ -related changes in fluorescence intensity (ΔF/F 0 ) for Ca 2+ measured from active OHCs 96( Figure `1G), which could be due to a progressive reduction of AP bursts with age. 97Calcium transients were abolished in Ca 2+ free solution (Figure 2A, Figure 3C, Movie 5). We quantified the average correlation 119All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OHCs ( Figure 3D). However, the r s was independent of the amplitude (ΔF/F 0 ) of the Ca 2+ 127 signal ( Figure 3E) measured as a pixel average over the entire spread of the Ca 2+ wave. 128Therefore, the coordination of the electrical activity between nearby OHCs was dependent on 129 the lateral spread, but not the amplitude, of the Ca 2+ waves. 130We then sought to identify how spontaneous Ca 2+ activity from the non-sensory cells of the easier to see that the Ca 2+ waves originating in the GER were able to travel to the LER and 140propagate through Deiters' cells ( Figure 4D; Supplementary Figure 1B). In order to test 141whether Deiters' cells mediate signal transfer from the GER to the OHCs, we analysed Ca 2+ 142 signals after removal of Deiters' cells ( Figure 5C), which was performed using gentle suction 143 via a small pipette (~3-4 µm in diameter). This procedure is widely used to gain access to the 144 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OHCs ( Figure 5A,B, Movie 6). We found that Ca 2+ elevation in OHCs associated ...

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Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue

Cited by 12 publications

References 10 publications

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea

Spontaneous and coordinated Ca²⁺activity of cochlear sensory and non-sensory cells drives the maturation of OHC afferent innervation

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Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue

Cited by 12 publications

References 10 publications

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea

Spontaneous and coordinated Ca2+activity of cochlear sensory and non-sensory cells drives the maturation of OHC afferent innervation

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Spontaneous and coordinated Ca²⁺activity of cochlear sensory and non-sensory cells drives the maturation of OHC afferent innervation