Research paperLocalization of kainate receptors in inner and outer hair cell synapses
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.
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.
References (71)
- et al.
Immunocytochemical localization of neurofilament subunits in the spiral ganglion of normal and neomycin-treated guinea pigs
Hear. Res.
(1989) - et al.
Kainate receptors differentially regulate release at two parallel fiber synapses
Neuron
(2002) - et al.
The efferent medial olivocochlear-hair cell synapse
J. Physiol.
(2012) - et al.
Synaptic profiles during neurite extension, refinement and retraction in the developing cochlea
Neural Dev.
(2012) Functional role of the olivo-cochlear bundle: a motor unit control system in the mammalian cochlea
Hear. Res.
(1989)- et al.
Kainate receptors in health and disease
Neuron
(2013) Morphological differences among radial afferent fibers in the cat cochlea: an electron-microscopic study of serial sections
Hear. Res.
(1980)- et al.
Physiology and anatomy of single olivocochlear neurons in the cat
Hear. Res.
(1986) - et al.
GRM7 variants associated with age-related hearing loss based on auditory perception
Hear. Res.
(2012) - et al.
Organization of NMDA receptors at extrasynaptic locations
Neuroscience
(2010)
Off bipolar cells express distinct types of dendritic glutamate receptors in the mouse retina
Neuroscience
Habenula “cholinergic” neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes
Neuron
Horseradish peroxidase injection of physiologically characterized afferent and efferent neurones in the guinea pig spiral ganglion
Hear. Res.
The continuing search for outer hair cell afferents in the guinea pig spiral ganglion
Hear. Res.
Immunocytochemical localization of neurofilament protein subunits in the spiral ganglion of the adult rat
Brain Res.
Parasynaptic signalling by fast neurotransmitters: the cerebellar cortex
Neuroscience
Neurofilament localization and phosphorylation in the developing inner ear of the rat
Hear. Res.
Modulation of hair cell efferents
Hear. Res.
Homeostatic control of synaptic transmission by distinct glutamate receptors
Neuron
Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca2+-calmodulin at mossy fiber-CA3 synapses
J. Neurochem.
Lac z Histochemistry and immunohistochemistry reveal ephrin-B ligand expression in the inner ear
J. Histochem. Cytochem.
Antidromic responses of single units from the spiral ganglion
J. Neurophysiol.
Kainate receptors
Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice
Development
Distribution of kainate receptor subunits at hippocampal mossy fiber synapses
J. Neurosci.
Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea
Synapse
Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells
J. Neurocytol.
Neurotransmitters and neuromodulators of the mammalian cochlea
Physiol. Rev.
Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons
Eur. J. Neurosci.
GRM7 variants confer susceptibility to age-related hearing impairment
Hum. Mol. Genet.
Distribution of the glutamate/aspartate transporter GLAST in relation to the afferent synapses of outer hair cells in the guinea pig cochlea
J. Assoc. Res. Otolaryngol.
Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus
J. Neurosci.
Transmitter release at the hair cell ribbon synapse
Nat. Neurosci.
Localization of PDZD7 to the stereocilia ankle-link associates this scaffolding protein with the Usher syndrome protein network
J. Neurosci.
Localization of kainate receptors to the presynaptic active zone of the rod photoreceptor in primate retina
Vis. Neurosci.
Cited by (26)
Diverse identities and sites of action of cochlear neurotransmitters
2022, Hearing ResearchCitation Excerpt :Post-synaptic KARs containing GluK5 and 2 have been reported at the adult IHC to type I afferent synapse, and GluK2 is also reported pre-synaptically. Immature IHCs also label for GluK5, which disappears in adulthood (Fujikawa et al., 2014). No specific functional role for kainate receptors has been confirmed at the IHC-type I SGN synapse, but it is suggested that the pre-synaptic receptors could modulate neurotransmitter release, and that the post-synaptic receptors likely regulate neuronal excitability (Fujikawa et al., 2014).
2.15 - Anatomy and Development of the Inner Ear
2020, The Senses: A Comprehensive Reference: Volume 1-7, Second EditionThe use of animal models to study cell transplantation in neuropathic hearing loss
2019, Hearing ResearchPre- and postsynaptic ionotropic glutamate receptors in the auditory system of mammals
2018, Hearing Researchα-Actinin Anchors PSD-95 at Postsynaptic Sites
2018, NeuronRecent advances in the development and function of type II spiral ganglion neurons in the mammalian inner ear
2017, Seminars in Cell and Developmental BiologyCitation Excerpt :These morphological data support the conventional wisdom that, on a per-cell basis, OHCs play a more limited role in transducing acoustic signals compared to IHCs. In addition to these morphological variations, the composition of glutamate receptors may also be different between postsynaptic type I and type II afferents [33,66]. Kainate-type glutamate receptors GluK2 and GluK5 are expressed in both type I and type II SGNs [66].