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 (IP3R) 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.
Highlights
During development, immature vestibular sensory epithelia include hair cells (HCs), supporting cells (SCs), and undifferentiated precursor cells
A maximum intensity projection (MIP) from confocal images taken through seventymicrons of the utricle and saccule (1 μm per slice) shows tdT expressing cells primarily in the extrastriolar region of the utricle and saccule in Gad2-G5-tdT mice at postnatal day 1 (P1) (Figure 1A; Olympus FV100, 40XW obj)
In mature mice from this transgenic cross, HCs with strong tdT expression are primarily extrastriolar type 1, which are known to be contacted by calyx endings of dimorphic afferent neurons (Eatock et al, 1998; Goldberg, 2012)
Summary
Immature vestibular sensory epithelia include hair cells (HCs), supporting cells (SCs), and undifferentiated precursor cells. The maturation of action potential discharge rate, regularity of discharge inter-spike intervals, and sensitivity to physiological stimulation follows a similar time course as hair cells and synapses reaching mature properties near postnatal day 10 (Curthoys, 1983; Desmadryl and Sans, 1990). During this period, spontaneous bursts of action potentials occur in vestibular ganglion neurons in the absence of gravito-inertial stimulation, thereby providing inputs to vestibular pathways in the central nervous system (CNS). How spontaneous vestibular afferent nerve activity is generated, if it is modulated during postnatal development by efferent action, and what role it might play in establishing functional vestibular physiology are as yet unknown
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