Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle

Abstract

Spontaneous calcium transients are present during early postnatal development in the mouse retina and cochlea, and play an important role in maturation of the sensory organs and neural circuits in the central nervous system (CNS). It is not known whether similar calcium transients occur during postnatal development in the vestibular sensory organs. Here we demonstrate spontaneous intracellular calcium transients in sensory hair cells (HCs) and supporting cells (SCs) in the murine utricular macula during the first two postnatal weeks. Calcium transients were monitored using a genetically encoded calcium indicator, GCaMP5G (G5), at 100 ms-frame −1 in excised utricle sensory epithelia, including HCs, SCs, and neurons. The reporter line expressed G5 and tdTomato (tdT) in a Gad2-Cre dependent manner within a subset of utricular HCs, SCs and neurons. Kinetics of the G5 reporter limited temporal resolution to calcium events lasting longer than 200 ms. Spontaneous calcium transients lasting 1-2 s were observed in the expressing population of HCs at birth and slower spontaneous transients lasting 10-30 s appeared in SCs by P3. Beginning at P5, calcium transients could be modulated by application of the efferent neurotransmitter acetylcholine (ACh). In mature mice, calcium transients in the utricular macula occurred spontaneously, had a duration 1-2 s, and could be modulated by the exogenous application of acetylcholine (ACh) or muscarine. Long-lasting calcium transients evoked by ACh in mature mice were blocked by atropine, consistent with previous reports describing the role of muscarinic receptors expressed in calyx bearing afferents in efferent control of vestibular sensation. Large spontaneous and ACh evoked transients were reversibly blocked by the inositol trisphosphate receptor (IP 3 R) antagonist aminoethoxydiphenyl borate (2-APB). Results demonstrate long-lasting calcium transients are present in the utricular macula during the first postnatal week, and that responses to ACh mature over this same time period.

ATP and ACh Evoked Calcium Transients in the Neonatal Mouse Cochlear and Vestibular Sensory Epithelia

“…Spontaneous and ACh evoked Ca 2+ transients in utricular hair cells and supporting cells have been reported previously during the first postnatal week, but ATP evoked transients were not observed (Holman et al, 2019(Holman et al, , 2020. Here, we report the presence of long-lasting Ca 2+ transients evoked by 100 µM ATP (Figure 5).…”

“…Semi-intact vestibular preparations or apical and mid-turn sections of the cochlea from Gad2-Cre; PC-G5-tdT mice of either sex were studied from acutely dissected bony labyrinths in 4°C extracellular buffer [EB (mM): 5.8 KCl, 155 NaCl, 0.9 MgCl 2 , 1.3 CaCl 2 , 0.7 NaHPO 4 , 5,6 glucose, 10 HEPES, 1 Na pyruvate, pH 7.4; osmolality ∼308 mmol kg –1 ] ( Holman et al, 2019 ). Dissected tissue from postnatal days 4–7 (P4-P7; day of birth is P0) were transferred to a recording chamber and immobilized with a nylon harp (Warner Instrument, RC-22C), and continuously perfused with EB at room temperature (21–23°C).…”

“…Previous studies have shown in the cochlea that binding of extracellular ATP to G-protein coupled P2Y2 and P2Y4 receptors, expressed on the endolymphatic surface of the developing sensory epithelium, activates phospholipase-C dependent generation of IP 3 ( Gale et al, 2004 ; Beltramello et al, 2005 ; Piazza et al, 2007 ; Ceriani et al, 2019 ). Additionally, muscarinic ACh receptors in the vestibular epithelium triggers IP 3 dependent CICR in specific vestibular hair cells ( Holman et al, 2019 , 2020 ). In some cases, gap junctions between adjacent support cells can lead to relatively slow propagation of intercellular Ca 2+ waves.…”

Synaptic mitochondria regulate hair-cell synapse size and function

“…In addition to evoked presynaptic- and mito-Ca 2+ signals in hair cells, we also observed instances of spontaneous presynaptic- and mito-Ca 2+ signals (Example, Figure 4A–A’’’, Video 2). Numerous studies have demonstrated that mammalian hair cells have spontaneous presynaptic-Ca 2+ influx during development (Eckrich et al, 2018; Holman et al, 2019; Marcotti et al, 2003; Tritsch et al, 2007; Tritsch et al, 2010). Therefore, we predicted that similar to mammals, spontaneous presynaptic-Ca 2+ uptake may be a feature of development.…”

Developmental GAD2 Expression Reveals Progenitor-like Cells with Calcium Waves in Mammalian Crista Ampullaris

“…We examined vestibular neuroepithelium from the first filial generation of GAD2-IRES-Cre crossed with a dual reporter GCaMP5G-tdTomato line ( Holman et al., 2019 ). In this study, GAD2-tdTomato (GAD2-tdT) cells were observed in the EC and specific zones within vertical cristae .…”