Differential expression of ryanodine receptor in the developing rat cochlea

Ryanodine receptors (RyRs) are one of the intracellular calcium channels involved in regulation of intracellular free calcium concentration ([Ca²⁺]i). The immunolocalization of RyRs was investigated in the developing rat cochlea at different postnatal days (PND). The change of [Ca²⁺]i in isolated outer hair cells (OHCs) was determined. Morphological results showed low expression of RyRs in the Kolliker’s organ from the PND 5 group. RyR expression in inner hair cells (IHCs) increased as the rats aged, and was mature after PND 14. RyRs in OHCs were expressed near the synaptic area of afferent and efferent nerves. RyRs in supporting cells were expressed widely and strongly. The application of ACh, ryanodine + ACh, and thapsigargin + ACh could induce a significant increase in [Ca²⁺]i in OHCs in the presence of extracellular calcium. This increase of [Ca²⁺]i induced by ACh was caused by not only the calcium influx through surface calcium channels, but also the calciuminduced calcium release (CICR) from intracellular RyR-sensitive calcium stores. Morphological and Ca imaging results suggested that RyRs expression is related to cochlear maturity, and may play an important role in its function.Key words: ryanodine receptor, development of cochlea, Ca²⁺, calcium-induced calcium release.Changes in intracellular Ca²⁺ concentration ([Ca²⁺]i) play an important role in cellular communication. The intracellular Ca²⁺ concentration is mainly mediated by two pathways: calcium entry via membrane calcium channels and calcium release from intracellular stores. The latter is mediated by 1, 4, 5-inositol triphosphate receptors (IP3R) and ryanodine receptors (RyRs). Mechanism of calcium release induced by RyRs has been believed to be calcium-induced calcium release (CICR) (Chakraborti et al., 2007). In the mammalian auditory system, calcium released from intracellular calcium stores in inner hair cells (IHCs), outer hair cells (OHCs), Deiters’ cells, and basal cells of stria vascularis plays an important role in the regulation of auditory transduction and electrochemical equilibrium of the cochlea (Bobbin, 2002; Marcotti et al., 2004). Neural transmitters, such as glutamate, ATP, acetylcholine (ACh), and substance P, can activate calcium signaling in spiral ganglia neurons, and then regulate the excitability of auditory neurons (Skinner et al., 2003). It has been reported that RyRs are widely expressed in the IHCs, OHCs, supporting cells in the organ of Corti’s, and in spiral ganglia neurons (SGNs) (Lioudyno et al., 2004; Morton-Jones et al., 2006). In the OHCs, the RyRs are mainly expressed under the reticular lamina, where BK channels are also strongly expressed (Beurg et al., 2005). Other studies also revealed that RyRs expressed in the synaptic area at the bottom of OHCs and the adjacent Deiters’ cells called as synaptoplasmic cistern (Lioudyno et al., 2004; Morton-Jones et al., 2006). Lioudyno et al., 2004; Morton-Jones et al., 2006).Three subtypes of RyRs have been identified expressed in the cochlea. RyR1 was observed in the synapses of OHCs using an SP6-RNA probe. RyR2 was mainly expressed in Corti’s organ and the cytoplasm of IHCs. RyR3 was found in the axons of SGNs located in the inner spiral plexus and the terminal of outer spiral fiber innervating OHCs (Lioudyno et al., 2004; Morton-Jones et al., 2006). These distinct expression patterns suggest that RyRs mediated calcium signaling plays important roles in auditory function.

Table 1

Location of RyR expression in previous references.

Reference	Localization of RyR expression
Lioudyno et al., 2004	OHCs and Deiters’ cells (RyR1) Synaptic pole of OHCs
Morton-Jones et al., 2006	Adult rat cochlea (three types of RyR) SGN cell bodies (RyR)
Beurg et al., 2005	Deiters’ cells, IHCs and lateral membrane side and basal synaptic areas of OHCs (RyR1)
Skinner et al., 2003	Reticular laminae and lateral cell wall of the OHCs and vicinity of the SGNs’ membrane (RyRs)

Open in a separate windowRyR-mediated Ca²⁺ release may be involved in auditory neurotransmission, sound transduction, cochlear electrochemical homeostasis, and development (Morton-Jones et al., 2006). However, the relationship between calcium-induced calcium release (CICR) mediated by RyRs and the development of auditory function is unclear. CICR has been proposed to be evoked by calcium release from intracellular RyR-sensitive stores (Fabiato, 1983). On the other hand, there were fewer studies on the development of the inner ear in rats. Development of the rat auditory system started from the embryonic period and extended to the postnatal period. The developmental stage of the primary auditory afferent pathway and the auditory central in the brainstem was nearly mature at PND 14 (Auestad et al., 2003; Lee et al., 2007). However, whether the RyR expression promotes the auditory system maturate in the development of rat is still unclear. In this study, the distribution and expression of RyRs in the developing rat cochlea was investigated by use of immunofluorescent stain and confocal microscopy. Calcium imaging identified that RyR-mediated Ca²⁺ release may play an important role in the cochlear maturity.