In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Lukasz, Daria; Kindt, Katie S.

doi:10.3791/58794-v

“…After verifying that Nrxn3 is essential for proper synapse organization in both zebrafish and mice, we next assessed the functional impact of fewer synapses. Previous work from our group established assays to assess hair-cell function in living zebrafish (Lukasz and Kindt, 2018). These assays rely on transgenic fish expressing GCaMP6s in hair cells or afferent neurons of the lateral-line system (Zhang et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

“…For functional imaging, 4-6 dpf larvae were anesthetized in 0.04% Tricaine-S (tricaine methanesulfonate, Western chemical, TRS1), pinned to a Sylgard-filled perfusion chamber at the head and tail, and paralyzed by injection of 125 µM α-bungarotoxin (Tocris, 2133) into the heart cavity, as previously described (Lukasz and Kindt, 2018). Larvae were then rinsed three Calcium responses in the afferent process were acquired on a Swept-field confocal system built on a Nikon FN1 upright microscope (Bruker) with a 60x/1.0 NA CFI Fluor waterimmersion objective.…”

Section: Calcium Imaging Of Lateral-line Hair Cells and Afferentsmentioning

confidence: 99%

Presynaptic Nrxn3 is essential for ribbon-synapse assembly in hair cells

Jukic,

Lei,

Cebul

et al. 2024

Preprint

1

0

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Hair cells of the inner ear rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is critical for ribbon-synapse assembly in hair cells. In both mouse and zebrafish models, loss of Nrxn3 results in significantly fewer intact ribbon synapses. In zebrafish we demonstrate that a 60% loss of synapses in nrxn3 mutants dramatically reduces both presynaptic responses in hair cells and postsynaptic responses in afferent neurons. Despite a reduction in synapse function in this model, we find no deficits in the acoustic startle response, a behavior reliant on these synapses. Overall, this work demonstrates that Nrxn3 is a critical and conserved molecule required to assemble ribbon synapses. Understanding how ribbon synapses assemble is a key step towards generating novel therapies to treat forms of age-related and noise-induced hearing loss that occur due to loss of ribbon synapses.

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“…Atoh1 expression is found in IHCs and OHCs during development (Mulvaney & Dabdoub, 2012). As a sensitive green fluorescent protein (GFP)‐based Ca 2+ sensor, GCaMP6s has been used to probe fast Ca 2+ dynamics and low peak Ca 2+ accumulations in neurons (Chen et al., 2013; Lukasz & Kindt, 2018; Shilling‐Scrivo et al., 2021). Compared to other variants, GCaMP6 sensors have similar baseline fluorescence but a higher dynamic range (1.1‐ to 1.6‐fold increase compared to GCaMP5G) and larger signals (>10‐fold compared to GCaMP3) (Chen et al., 2013).…”

Section: Resultsmentioning

confidence: 99%

Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells

Yang,

Murtha,

Climer

et al. 2023

The Journal of Physiology

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Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sensitivity of mammalian hearing. During development, the maturation of OHC afferent connectivity is refined by coordinated spontaneous Ca2+ activity in both sensory and non‐sensory cells. Calcium signalling in neonatal OHCs can be modulated by oncomodulin (OCM, β‐parvalbumin), an EF‐hand calcium‐binding protein. Here, we investigated whether OCM regulates OHC spontaneous Ca2+ activity and afferent connectivity during development. Using a genetically encoded Ca2+ sensor (GCaMP6s) expressed in OHCs in wild‐type (Ocm+/+) and Ocm knockout (Ocm−/−) littermates, we found increased spontaneous Ca2+ activity and upregulation of purinergic receptors in OHCs from Ocm−/− cochlea immediately following birth. The afferent synaptic maturation of OHCs was delayed in the absence of OCM, leading to an increased number of ribbon synapses and afferent fibres on Ocm−/− OHCs before hearing onset. We propose that OCM regulates the spontaneous Ca2+ signalling in the developing cochlea and the maturation of OHC afferent innervation. imageKey points Cochlear outer hair cells (OHCs) exhibit spontaneous Ca2+ activity during a narrow period of neonatal development. OHC afferent maturation and connectivity requires spontaneous Ca2+ activity. Oncomodulin (OCM, β‐parvalbumin), an EF‐hand calcium‐binding protein, modulates Ca2+ signals in immature OHCs. Using transgenic mice that endogenously expressed a Ca2+ sensor, GCaMP6s, we found increased spontaneous Ca2+ activity and upregulated purinergic receptors in Ocm−/− OHCs. The maturation of afferent synapses in Ocm−/− OHCs was also delayed, leading to an upregulation of ribbon synapses and afferent fibres in Ocm−/− OHCs before hearing onset. We propose that OCM plays an important role in modulating Ca2+ activity, expression of Ca2+ channels and afferent innervation in developing OHCs.

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“…Atoh1 expression is found in IHCs and OHCs during development (Mulvaney and Dabdoub, 2012). As a sensitive GFP-based Ca 2+ sensor, GCaMP6s has been used to probe fast Ca 2+ dynamics and low peak Ca 2+ accumulations in neurons (Chen et al, 2013; Lukasz and Kindt, 2018; Shilling-Scrivo et al, 2021). Calcium signaling in the OHCs was investigated using GCaMP6s Ocm wildtype ( Ocm +/+ ) and Ocm knockout ( Ocm -/- ) mice ( Figure 1A-B , see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

Oncomodulin Regulates Spontaneous Calcium Signaling and Maturation of Afferent Innervation in Cochlear Outer Hair Cells

Yang

¹

,

Murtha

²

,

Climer

³

et al. 2023

Preprint

0

View full text Add to dashboard Cite

Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sensitivity of mammalian hearing. During development, the maturation of OHC afferent connectivity is refined by coordinated spontaneous Ca2+activity in both sensory and non-sensory cells. Calcium signaling in neonatal OHCs can be modulated by Oncomodulin (OCM, β-parvalbumin), an EF-hand calcium-binding protein. Here, we investigated whether OCM regulates OHC spontaneous Ca2+activity and afferent connectivity during development. Using a genetically encoded Ca2+sensor (GCaMP6s) expressed in OHCs in wild-type (Ocm+/+) and Ocm knockout (Ocm-/-) littermates, we found increased spontaneous Ca2+activity and upregulation of purinergic receptors in OHCs from GCaMP6s Ocm-/-cochlea immediately following birth. The afferent synaptic maturation of OHCs was delayed in the absence of OCM, leading to an increased number of ribbon synapses and afferent fibers on GCaMP6s Ocm-/-OHCs before hearing onset. We propose that OCM regulates the spontaneous Ca2+signaling in the developing cochlea and the maturation of OHC afferent innervation.

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In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Cited by 5 publications

References 0 publications

Presynaptic Nrxn3 is essential for ribbon-synapse assembly in hair cells

Presynaptic Nrxn3 is essential for ribbon-synapse assembly in hair cells

Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells

Oncomodulin Regulates Spontaneous Calcium Signaling and Maturation of Afferent Innervation in Cochlear Outer Hair Cells

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