Elsevier

Hearing Research

Volume 314, August 2014, Pages 20-32
Hearing Research

Research paper
Localization of kainate receptors in inner and outer hair cell synapses

https://doi.org/10.1016/j.heares.2014.05.001Get rights and content

Highlights

  • Adult IHC afferent synapses have postsynaptic KARs with GluK2&5 + presynaptic GluK2.

  • Adult OHC afferent terminal GluRs are KARs containing GluK2 and GluK5.

  • At P8, OHC afferent terminals also have GluK1 and GluK3.

  • Adult OHC efferent terminal synapses have KARs with GluK1, GluK2, and GluK5.

Abstract

Glutamate plays a role in hair cell afferent transmission, but the receptors that mediate neurotransmission between outer hair cells (OHCs) and type II ganglion neurons are not well defined. A previous study using in situ hybridization showed that several kainate-type glutamate receptor (KAR) subunits are expressed in cochlear ganglion neurons. To determine whether KARs are expressed in hair cell synapses, we performed X-gal staining on mice expressing lacZ driven by the GluK5 promoter, and immunolabeling of glutamate receptors in whole-mount mammalian cochleae. X-gal staining revealed GluK5 expression in both type I and type II ganglion neurons and OHCs in adults. OHCs showed X-gal reactivity throughout maturation from postnatal day 4 (P4) to 1.5 months. Immunoreactivity for GluK5 in IHC afferent synapses appeared to be postsynaptic, similar to GluA2 (GluR2; AMPA-type glutamate receptor (AMPAR) subunit), while GluK2 may be on both sides of the synapses. In OHC afferent synapses, immunoreactivity for GluK2 and GluK5 was found, although GluK2 was only in those synapses bearing ribbons. GluA2 was not detected in adult OHC afferent synapses. Interestingly, GluK1, GluK2 and GluK5 were also detected in OHC efferent synapses, forming several active zones in each synaptic area. At P8, GluA2 and all KAR subunits except GluK4 were detected in OHC afferent synapses in the apical turn, and GluA2, GluK1, GluK3 decreased dramatically in the basal turn. These results indicate that AMPARs and KARs (GluK2/GluK5) are localized to IHC afferent synapses, while only KARs (GluK2/GluK5) are localized to OHC afferent synapses in adults. Glutamate spillover near OHCs may act on KARs in OHC efferent terminals to modulate transmission of acoustic information and OHC electromotility.

Graphical abstract

Drawing illustrates the distribution of kainate receptor subunits, GluK1,2,4,5, and the AMPA receptor subunit, GluA2, at synapses on inner (IHC) and outer (OHC) hair cells in the adult rat, as described in this study. At IHC afferent (a) synapses, GluA2, GluK2 and GluK5 are postsynaptic and GluK2 is presynaptic. OHC afferent synapses bearing ribbons (usually one or two; colored green in drawing) have GluK2 and GluK5, while those lacking ribbons have small amounts of GluK5 only; efferent (e) synapses have GluK1, GluK2, and GluK5.

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Introduction

Hearing requires both the transduction of sounds into electrical impulses by cochlear hair cells and the relaying and processing of that information along a cascade of neurons (nerve cells) in the brain via chemical synapses between them. Defects in any of these processes can lead to hearing loss. The first synapses in this sequence link the sound-transducing hair cells with the processes of the primary neurons, called spiral ganglion cells, which relay information to the brainstem. Synaptic transmission between the hair cell and the ganglion cell processes depends on release of a chemical neurotransmitter, mainly glutamate, and its binding to neurotransmitter receptor molecules.

About 95% of auditory afferent neurons are type I ganglion neurons (Spoendlin, 1969). Each type I neuron sends a peripheral projection to an inner hair cell (IHC), forming a ribbon synapse (Liberman, 1980, Liberman, 1982, Moser et al., 2006, Safieddine et al., 2012), where the presynaptic neurotransmitter glutamate and postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-isoxazolepropionic acid)-type glutamate receptors (AMPARs) mediate excitatory transmission (Matsubara et al., 1996, Ruel et al., 1999, Glowatzki and Fuchs, 2002). Multivesicular release at this synapse achieves frequent excitatory postsynaptic currents (EPSCs) in type I afferent neurons and causes continuous and rapid transmission of acoustic information. Type II neurons constitute the remaining 5 percent of afferent neurons. Each type II neuron forms a thin, unmyelinated dendrite contacting many outer hair cells (OHCs). Each OHC has 3 to 15 afferent terminals (Liberman et al., 1990) and some of them do not bear a ribbon synapse (Hashimoto and Kimura, 1988, Liberman et al., 1990, Huang et al., 2012). OHC/type II afferent transmission is also glutamatergic, but EPSCs in type II neurons are much smaller in frequency and amplitude, and significantly slower in kinetics compared to averaged EPSCs recorded in type I neurons (Weisz et al., 2009), so that summated stimulation is required to produce an action potential in type II afferent neurons (Weisz et al., 2012). The specific receptor involved remains unknown, because there is no immunoreactivity for AMPARs in type II afferent nerve terminals (Matsubara et al., 1996, Liberman et al., 2011).

OHCs also are innervated by myelinated fibers from the medial olivocochlear (MOC) efferent projections, forming several synapses at the base (Liberman and Brown, 1986). The MOC system is cholinergic and suppresses the electromotile response of OHCs (Wersinger and Fuchs, 2011; Elgoyhen and Katz, 2012), providing a feedback system to optimize cochlear amplification (LePage, 1989). Acetylcholine (ACh) is the only well-defined neurotransmitter in OHC efferent synapses, but additional molecules are proposed to be involved in the transmission as neuromodulators, including gamma amino butyric acid (GABA). It is suggested that GABAergic signaling contributes to the long-term maintenance of hair cells or normal OHC electromotility (Maison et al., 2006, Maison et al., 2009). Furthermore, a recent study shows that released GABA acts on presynaptic GABAB receptors expressed in OHC efferent terminals to downregulate the release of ACh (Wedemeyer et al., 2013).

Ionotropic glutamate receptors (GluRs) include three major families; NMDA-type, AMPA-type and kainate-type receptors (KARs). GluRs mediate neurotransmission at excitatory synapses, but neuronal KAR-mediated EPSCs have distinct physiological feature of smaller amplitude and slower deactivation kinetics, compared to AMPA-mediated EPSCs and are shaped by auxiliary KAR subunits, such as tolloid-like 1 (NETO1) and NETO2 proteins (Lerma and Marques, 2013). In addition, presynaptic KARs can act as neuromodulators of synapse transmission to control transmitter release in a bidirectional manner; frequency-dependent facilitation and depression of transmitter release via presynaptic KARs have been demonstrated at mossy fiber synapses in the hippocampus (Lerma, 2003) and at parallel fiber synapses in the cerebellar cortex (Delaney and Jahr, 2002). Moreover, postsynaptic KARs are thought to regulate neuronal excitability both through slowly deactivated ionotropic KARs and through G protein-coupled metabotropic KARs (Lerma and Marques, 2013).

A previous study using in situ hybridization showed that several KAR subunits (GluK1, GluK2, GluK4, GluK5) are expressed in cochlear ganglion neurons (Niedzielski and Wenthold, 1995). Another study reported that KARs are expressed in IHC afferent synapses and suggested that KARs contribute to hair cell acoustic transmission, based on physiological data using a GluK1-specific antagonist (Peppi et al., 2012). These findings suggest that KARs are involved in normal cochlear function for synaptic transmission or modulation. In order to determine whether KARs are expressed in synapses of IHCs and OHCs, we performed immunolabeling of all the subtypes of KARs in the adult mammalian cochlea. We also performed auditory testing on mice that lacked GluK5. We found that KARs (GluK2/GluK5) are the main postsynaptic GluRs in OHC afferent synapses and that the expression pattern of KARs shows developmental changes. Interestingly, KARs are also expressed in OHC efferent terminals. Moreover, we detected both pre- and postsynaptic KARs in IHC afferent synapses.

Section snippets

Animals

GluK5 knockout mice (GluK5 KO; strain B6.129P2-Grik5tm1Dgen/J), was obtained from the Jackson Laboratories (generated by Deltagen). This strain has been backcrossed to C57BL/6 mice and has a targeted deletion inside the GluK5 (Grik5) gene (from base 1936 to base 2006) that is replaced with a reporter gene, lacZ. We confirmed that X-gal staining in brain sections of the mice (data not shown) is consistent with previous studies of GluK5 cellular distribution in the brain (Darstein et al., 2003).

GluK5 is expressed in OHCs, cochlear ganglion cells and vestibular hair cells in adult

We performed X-gal staining on GluK5 KO mice to determine the cellular distribution of GluK5 in the cochlea and vestibule. GluK5 expression in cochlear hair cells in whole-mount cochleae showed developmental changes (Fig. 1A–C). Both IHCs (arrow) and OHCs (arrowhead) showed lacZ reactivity with equal intensity at P8 (Fig. 1A); but IHCs showed reduced reactivity at P14 (Fig. 1B) and reactivity was lost completely at 1.5 months old (Fig. 1C). On the other hand, OHCs maintained reactivity

Discussion

In this study (Supplementary Fig. S5), we found that: 1) adult IHC afferent synapses have postsynaptic KARs containing GluK2 and GluK5 (in addition to AMPARs as described previously) and presynaptic KARs with GluK2. 2) The adult OHC afferent terminal GluRs are KARs containing GluK2 and GluK5. 3) At P8, OHC afferent terminals also have GluK1 and GluK3. 4) OHC efferent terminal synapses have KARs with GluK1, GluK2, and GluK5.

Acknowledgments

This work was supported by the Intramural Research Program of the NIDCD at the National Institutes of Health. We also thank Dr. Stephan Brenowitz for reviewing the manuscript, and Dr. Kai Chang for advice related to the GluK5 mutant mouse.

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