Elsevier

Brain Research

Volume 846, Issue 2, 6 November 1999, Pages 196-203
Brain Research

Research report
Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea

https://doi.org/10.1016/S0006-8993(99)02034-XGet rights and content

Abstract

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca2+ ions ([Ca2+]i) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca2+]i. The ACh concentration-response curve indicated an apparent dissociation constant (Kd) of 8 μM and a Hill coefficient of 1.0. Removing extracellular free Ca2+ did not suppress the ACh-induced Ca2+ responses suggesting an intracellular Ca2+-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca2+ response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca2+ responses similar to those observed with ACh, with an apparent Kd of 10 μM and a Hill coefficient of 1.0. The cholinergic-induced Ca2+ response was reversibly blocked by muscarinic antagonists with the following order of potency, atropine>4-DAMP>methoctramine>pirenzepine. Nicotine (10 to 100 μM) did not evoke Ca2+ responses and the nicotinic antagonist curare (10 μM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.

Introduction

Acetylcholine (ACh) has long been recognized as one of the main neurotransmitter of the olivocochlear efferent system, which regulates mechanical and electrical activity of the mammalian auditory organ 4, 16. This olivocochlear efferent system is subdivided into a medial olivocochlear efferent system (MOC) and a lateral olivocochlear efferent system (LOC) 30, 35.

The MOC is formed by efferent fibers, originating from neuronal cell bodies located in the medial superior olive, which project in the cochlea predominantly to the outer hair cells (OHC) and form large axosomatic synapses at their base. The cholinergic synapse of the MOC has been extensively studied at the post-synaptic level in isolated OHC. ACh has been shown to hyperpolarize OHC via the activation of ligand-gated receptors (nAChRs) permeable to calcium 3, 19. These nAChRs are presumably composed of nicotinic α9 subunits [10]. The calcium influx flowing through these nAChRs activates co-localized SK-type Ca2+-activated K+ channels, known to be expressed in OHC [9], producing a means for a fast hair cell hyperpolarization 3, 15. A similar fast inhibitory cholinergic synapse has been described in hair cells of the chick cochlea [14].

On the other hand, the LOC originates essentially in the ipsilateral superior olive and projects fibers below the inner hair cells (IHC). These fibers form synapses “en passant” with the radial auditory afferent dendrites of the myelinated bipolar neurons of the cochlear spiral ganglion. These myelinated neurons innervating IHC are called type I and constitute about 95% of the total neurons contained in the spiral ganglion of the cochlea. These type I neurons convey the acoustic-evoked electrical signals issued from IHC to the brain. The precise role of the efferent synapses contacting the afferent endings of type I neurons below IHC remains essentially unknown. Immunocytochemical and enzymatic studies have suggested that ACh could act as a neurotransmitter in these lateral olivocochlear endings on IHC afferent dendrites 11, 12. Furthermore, a study by Felix and Ehrenberger [13]suggested that ACh has here an excitatory action on afferent type I neurons. They showed that microiontophoresis of ACh in vivo, at the subsynaptic area below IHC, produced an increase in subsynaptic spiking activity of the afferent nerve fibers.

Contrary to the rather well characterized OHC efferent synapse, we still do not know which types of cholinergic receptors are involved in IHC afferent neurons. Indeed, recent studies using RT-PCR and riboprobes in situ hybridization have suggested that both muscarinic 6, 32and nicotinic receptor mRNA 27, 28are expressed by spiral ganglion neurons. However, there is no direct evidence to date of functional cholinergic receptors, either muscarinic or nicotinic, in spiral ganglion neurons of the mammalian cochlea.

The purpose of our study was to determine, by using indo-1 spectrofluorometry, whether external application of ACh triggers Ca2+ signals in isolated cochlear neurons. Specifically, we studied the pharmacology of the ACh-evoked Ca2+ responses and determined whether it involved Ca2+ flux via nicotinic receptors as in OHC [3]or Ca2+ mobilization by activation of G-protein-linked ACh receptors (metabotropic). Our results demonstrated for the first time the presence of functional muscarinic receptors mediating Ca2+ mobilization in cochlear spiral ganglion neurons.

Section snippets

Cochlear neurons isolation

Newborn rats (3 to 30 days after birth) were deeply anesthetized with an i.m. injection of 0.1 to 0.2 ml of a solution consisting of 1 vol. of 50 mg/ml ketamine (Ketalar, Parke-Davis Courbevoie France) and 1 vol. of 2% xylazine (Rompun; Bayer, Leverkusen, Germany). The animals were then decapitated and cochleae were extracted and placed in Dulbecco's phosphate-buffered saline (DPBS from Sigma containing (in g/l): CaCl2·2H20, 0.133; MgCl2·6H20, 0.1; KCl, 0.2; KH2PO4, 0.2; NaCl, 8.0; Na2HPO4,

ACh raises intracellular Ca2+

The resting intracellular Ca2+ level ([Ca2+]i) of rat spiral ganglion neurons isolated at different stage of post-natal development from 3 to 30 days were determined to be 114±54 nM (n=193). There was no significant difference in resting [Ca2+]i between neurons obtained from rats of different ages. This value was comparable to the resting [Ca2+]i previously measured in isolated cochlear hair cells 7, 8. A pressure-puff application of 50 μM ACh evoked a rapid and transient increase in [Ca2+]i (

Discussion

Our study demonstrated for the first time the presence of functional cholinergic receptors in mammalian afferent cochlear neurons. The data showed that the neurotransmitter ACh mobilized intracellular Ca2+ through the activation of muscarinic receptors (mAChRs). This process is generally known to involve stimulation of phosphoinositide-specific phospholipase C (PLC) and the concomitant formation of inositol 1,4,5-triphosphate (InsP3) which binds to specific receptor-operated Ca2+ channels on

Acknowledgements

The authors wish to thank the “Fondation Pour La Recherche Médicale” (Paris France) and The Conseil Régional d'Aquitaine for grant support to our laboratory.

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    • Survival and stimulation of neurite outgrowth in a serum-free culture of spiral ganglion neurons from adult mice

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      The pioneering work on culturing dissociated spiral ganglion neurons was carried out more than a decade ago with embryonic rats and chicken (Lefebvre et al., 1990a; Yamaguchi and Ohmori, 1990). Various protocols have since been established for culturing spiral ganglion neurons from a range of species and ages, such as embryonic mice (Rabejac et al., 1994; Vazquez et al., 1994); neonatal mice (Kita et al., 2005; Lin et al., 1998; Mo and Davis, 1997; Whitlon et al., 2006), rats (Dazert et al., 1998; Hegarty et al., 1997; Lefebvre et al., 1990b; Malgrange et al., 1996; Marzella et al., 1997; Ripoll and Rebillard, 1997; Rome et al., 1999; Zheng et al., 1995), and gerbils (Lin, 1997); and adult rats (Lefebvre et al., 1991), guinea pigs (Anderson et al., 2006; Rask-Andersen et al., 2005), and humans (Rask-Andersen et al., 2005). Sensory epithelia have also been cultured from the cochlea of adult guinea pigs (Zhao, 2001); most experiments with spiral ganglion neurons, however, have been conducted with neonatal samples, possibly because adult neurons are in general more difficult to culture (Banker and Goslin, 1998).

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