Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs

doi:10.1016/S0006-8993(98)01345-6

Brain Research

Volume 819, Issues 1–2, 20 February 1999, Pages 58-64

https://doi.org/10.1016/S0006-8993(98)01345-6 Get rights and content

Abstract

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1–4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

Introduction

Glutamate is the most likely neurotransmitter between hair cells and primary afferents in the inner ear (reviews; Refs. 8, 16, 18). This amino acid is concentrated in hair cells in the vestibular end organs and organ of Corti 5, 21, 22, and glutamate receptor subunits are expressed by vestibular as well as spiral ganglion cells 9, 15, 20. A recent high resolution immunogold analysis of the organ of Corti demonstrated AMPA GluR2/3 and GluR4 immunoreactivities at the postsynaptic specialization of afferent synapses established by inner hair cells, suggesting that AMPA receptor subunits may be involved in the afferent transmission [14]. The absence of detectable AMPA receptor immunoreactivity in the synaptic region of the outer hair cells pointed to considerable differences between inner and outer hair cells in regard to glutamatergic signalling [14]. The vestibular end organs, which are phylogenetically related to the organ of Corti, are also endowed with two types of sensory hair cell (types I and II), and differences in synaptic responses have been reported between chaliceal nerve endings surrounding type I cells and bouton type endings contacting type II cells [26]. This raises the question whether glutamate receptors are differentially expressed in these two types of synapse. To address this issue, we investigated the localization of AMPA receptors in the crista ampullaris by means of a postembedding immunogold procedure. A second aim was to resolve whether AMPA receptors also occur in the less well known but morphologically distinct synapse between type II hair cells and the outer face of the nerve chalices 7, 10, 19.

Section snippets

Tissue preparation

The inner ear was fixed by the perilymphatic perfusion method 1, 25. Four Wistar rats (250–300 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and 1 ml of fixative (see below) was injected into the oval window during a period of 20–30 s. Subsequently, the animals were perfused intracardially with 2% dextran (mw 70,000) in 0.1 M phosphate buffer (PB, pH 7.4, 4°C, 15 s), followed by the fixative that was used for perilymphatic perfusion (room temperature, 50

The synapses of type II cells with bouton type endings

Each type II hair cell is contacted by several afferent bouton type endings which usually display one synapse each. This type of synapse was immunolabelled for GluR4 (Fig. 1A) as well as GluR2/3 (Fig. 1B,C). Gold particles occurred throughout the postsynaptic specialization. Very few gold particles were observed in extrasynaptic membranes.

The synapses of type I cells with nerve chalices

Each type I hair cell makes several small synaptic contacts with the inner face of its afferent chalice. These contacts were associated with gold particles

Discussion

The present results revealed that AMPA receptor subunits are expressed at the postsynaptic specialization of afferent dendrites, in nerve chalices as well as in bouton type endings. This is in line with a transmitter role of glutamate in both types of synapse 5, 9, 13, 15, 18, 20, 21.

Acknowledgements

We would like to thank Dr. R.J. Wenthold for his generous gift of antibodies, B. Riber, K.M. Gujord, and S. Komatsu for technical assistance. Support is gratefully acknowledged from the Ministry of Education of Japan, the Norwegian Research Council, J.E. Isberg's Fund, and the EU Biomed programme.

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The tetrapeptide FGLM-NH₂ derived from substance P (SP) can be used to treat corneal disorders when combined with SSSR, which is a tetrapeptide derived from insulin-like growth factor-1 (IGF-1). We examined the influence of FGLM-NH₂ plus SSSR when locally applied to the unilateral inner ear of guinea pigs with vestibular disorder induced by AMPA. A total of 18 Hartley white guinea pigs were assigned to groups receiving either FGLM-NH₂ plus SSSR, artificial perilymph, or no treatment at all. A hole was drilled adjacent to the round window, with AMPA then infused into the hole in order to induce the vestibular disorder. Thereafter, FGLM-NH₂ plus SSSR or artificial perilymph was delivered via an osmotic pump that was inserted into the hole. Sinusoidal rotation tests were used for observing spontaneous nystagmus and for measurements of the vestibulo-ocular reflexes (VOR). Two animals from each group were immunohistochemically examined at 24 h after the treatment. Spontaneous nystagmus decreased immediately after FGLM-NH₂ plus SSSR infusion. The recovery of the VOR gains was statistically faster than that seen in the control group at 3 and 7 days after treatment. Immunohistochemical examination revealed that many synaptic ribbons, which are markers of the synapse, were stained in the FGLM-NH₂ plus SSSR group compared with the untreated group. Topical application of FGLM-NH₂ plus SSSR accelerates functional recovery from AMPA-induced vestibular disorders by facilitating synaptic regeneration in guinea pigs.
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Glutamate transmission from vestibular end organs to central vestibular nuclear complex (VNC) plays important role in transferring sensory information about head position and movements. Three isoforms of vesicular glutamate transporters (VGLUTs) have been considered so far the most specific markers for glutamatergic neurons/cells. In this study, VGLUT1 and VGLUT2 were immunohistochemically localized to axon terminals in VNC and somata of vestibular primary afferents in association with their central and peripheral axon endings, and VGLUT1 and VGLUT3 were co-localized to hair cells of otolith maculae and cristae ampullaris. VGLUT1 and VGLUT2 defined three subsets of Scarpa's neurons (vestibular ganglionic neurons): those co-expressing VGLUT1 and VGLUT2 or expressing only VGLUT2, and those expressing neither. In addition, many neurons located in all vestibular subnuclei were observed to contain hybridized signals for VGLUT2 mRNA and a few VNC neurons, mostly scattered in medial vestibular nucleus (MVe), displayed VGLUT1 mRNA labelling. Following unilateral ganglionectomy, asymmetries of VGLUT1-immunoreactivity (ir) and VGLUT2-ir occurred between two VNCs, indicating that the VNC terminals containing VGLUT1 and/or VGLUT2 are partly of peripheral origin. The present data indicate that the constituent cells/neurons along the vestibular pathway selectively apply VGLUT isoforms to transport glutamate into synaptic vesicles for glutamate transmission.
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These data are consistent with PICK1 interacting with GluR2 in presynaptic vesicles. The presence of GluR2 in presynaptic terminals, which we show for the first time in intact, mature, hippocampal synapses, and which has been reported in several other synapses (Matsubara et al., 1996, 1999; Satake et al., 2000; Schenk et al., 2003; Pittaluga et al., 2006; Chavez et al., 2006; Feligioni et al., 2006) suggests that GluR2 is involved in the modulation of transmitter release. In previous studies from our laboratory, we used a mixture of antibodies that recognized all AMPAR subunits rather than antibodies directed specifically to GluR2 (Takumi et al., 1999).
AMPA receptors have been identified in different populations of presynaptic terminals and found to be involved in the modulation of neurotransmitter release. The mechanisms that govern the expression of presynaptic AMPA receptors are not known. One possibility is that pre- and postsynaptic AMPA receptors are regulated according to the same principles. To address this hypothesis we investigated whether protein interacting with C kinase 1 (PICK1), known to interact with AMPA receptors postsynaptically, also is expressed presynaptically, together with AMPA receptors. Subfractionation and high-resolution immunogold analyses of the rat hippocampus revealed that GluR2 and PICK1 are enriched postsynaptically, but also in presynaptic membrane compartments, including the active zone and vesicular membranes. PICK1 and GluR2 are associated with the same vesicles, which are immunopositive also for synaptophysin and vesicle-associated membrane protein 2. Based on what is known about the function of PICK1 postsynaptically, the present data suggest that PICK1 is involved in the regulation of presynaptic AMPA receptor trafficking and in determining the size of the AMPA receptor pool that modulates presynaptic glutamate release.
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Chicken (Gallus gallus) brains were used to investigate the typology and the immunolabel pattern for the subunits composing the AMPA-type glutamate receptors (GluR) of hindbrain neurons of the dorsal (dND) and ventral nuclei (vND) of the Deiter's vestibular complex (CD), which is the avian correspondent of the lateral vestibular nucleus (LVN) of mammals. Our results revealed that neurons of both divisions were poor in GluR1. The vND, the GluR2/3+ and GluR4+ label presented no area or neuronal size preference, although most neurons were around 75%. The dND neurons expressing GluR2/3 are primarily around 85%, medium to large-sized 85%, and predominantly 60% located in the medial portion of the rostral pole and in the lateral portion of the caudal pole. The majority of dND neurons containing GluR4 are also around 75%, larger (70% are large and giant), exhibiting a distribution that seems to be complementary to that of GluR2/3+ neurons. This distinct arrangement indicates functional differences into and between the DC nuclei, also signaling that such variation could be attributed to the diverse nature of the subunit composition of the GluRs. Discussion addresses the morphological and functional correlation of the avian DC with the LVN of mammals in addition to the high morphological correspondence, To include these data into the modern comparative approach we propose to adopt a similar nomenclature for the avian divisions dND and vND that could be referred as dLVN and vLVN.
Edaravone protects the vestibular periphery from free radical-induced toxicity in response to perilymphatic application of (±)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid
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Intracochlear infusion of (±)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) was performed with a syringe pump in guinea pigs, and peripheral vestibular dysfunction was induced. Animals were administered edaravone systemically or topically. In the systemic application group, animals were administered edaravone once a day for 7 days after AMPA infusion. In the topical application group, edaravone-soaked gelfoam was placed on the round window membrane just after, 12 h after or 24 h after AMPA infusion. Spontaneous nystagmus was observed after AMPA infusion. Immunohistochemistry for 4-hydroxy-2-nonenal (4-HNE), a marker of free radical-induced lipid peroxidation, was performed 24 h after AMPA infusion. In addition, caloric tests were performed to evaluate vestibular function 1 week after AMPA infusion. Animals in both groups showed decreased spontaneous nystagmus, but results were not significant. Animals treated topically with edaravone within 12 h of AMPA infusion showed normal morphology of the ampullar sensory epithelia of the lateral semicircular canals and showed a good response to the caloric tests. 4-HNE immunoreactivity in the sensory epithelia was very low in these animals. In contrast, untreated animals and animals treated with edaravone systemically or topically 24 h after AMPA infusion showed morphologic hair cell damage, reduced caloric response and remarkable 4-HNE immunoreactivity in the sensory epithelia. These results indicate that topical application of edaravone within 12 h after damage protects the vestibular periphery from free radical-induced toxicity in response to intracochlear infusion of AMPA.

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Research reportImmunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs

Abstract

Introduction

Section snippets

Tissue preparation

The synapses of type II cells with bouton type endings

The synapses of type I cells with nerve chalices

Discussion

Acknowledgements

Micron

Neuron

Brain Res.

Hearing Res.

Prog. Neurobiol.

Prog. Brain Res.

Brain Res.

J. Biol. Chem.

The vestibular nerve of the chinchilla: II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals

J. Neurophysiol.

Glutamate-like immunoreactivity in the peripheral vestibular system of mammals

Hearing Res.

Glutamate receptors on type I vestibular hair cells of guinea-pig

Eur. J. Neurosci.

Neurotransmitters and neuromodulators of the mammalian cochlea

Physiol. Rev.

Research report
Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs