Elsevier

Progress in Neurobiology

Volume 54, Issue 2, February 1998, Pages 127-148
Progress in Neurobiology

Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear

https://doi.org/10.1016/S0301-0082(97)00054-3Get rights and content

Abstract

The synapses between sensory cells in the inner ear and the afferent dendrites of ganglion cells are well suited to investigations of fundamental mechanisms of fast synaptic signalling. The presynaptic elements can be isolated for electrophysiological and functional studies while the synapses can be easily recognized in the electron microscope due to their distinct morphological features. This allows for a broader range of correlative functional and structural analyses than can be applied to synapses in the central nervous system (CNS). As in most fast excitatory synapses in the CNS the transmitter in the afferent hair cell synapses appears to be glutamate or a closely related compound. Recent studies have revealed many of the key molecular players at this type of synapse and how they are spatially and functionally coupled. By use of high resolution immunogold cytochemistry it has been shown that AMPA glutamate receptors are specifically expressed in the postsynaptic specialization of afferent hair cell synapses (except at those established by outer hair cells in the organ of Corti) and that their density varies as a function of the distance from the release sites (demonstrated for the afferent contacts of inner hair cells). The glutamate transporter GLAST is localized in supporting cell membranes and concentrated in those membrane domains that face the synaptic regions. Glutamine synthetase and phosphate-activated glutaminase—which are responsible for the interconversion of glutamate and glutamine—are selectively localized in non-neuronal and neuronal elements, respectively. Taken together with quantitative immunogold data on the cellular compartmentation of glutamate and glutamine the above findings suggest that the sensory epithelia in the inner ear sustain a cycling of glutamate carbon skeletons. In this process, the supporting cells may carry out functions analogous to those of glial cells in the CNS. Functional and morphological analyses of the presynaptic membrane indicate that l-type Ca2+-channels and Ca2+-activated K+-channels are colocalized and clustered at the active zone. Influx through the l-type channels triggers synaptic release and their close spatial association with Ca2+-activated K+-channels appears to be critical for frequency tuning. The focal expression of different Ca2+-channels combined with a high intracellular buffering capacity permits several Ca2+-signalling pathways to operate in parallel without undue interference. The molecular organization of the afferent hair cell synapsesreflects the functional demand for speed and precision and attests to the ability of the pre- andpostsynaptic elements to target and anchor key proteins at specific membrane domains.

Introduction

Glutamate is now well established as a major excitatory transmitter in the central nervous system (Fonnum, 1984; Ottersen et al., 1994; Stone, 1995). Evidence is mounting that glutamate also mediates fast signalling in the first synapse in the auditory and vestibular pathways, i.e. the contact between the hair cells and afferent dendrites of spiral and vestibular ganglion cells (Klinke, 1986; Eybalin, 1993; Puel, 1995). It is important to unravel the organization and function of this type of synapse, not only because of its central position in auditory and vestibular physiology, but also because it occurs outside the CNS and hence in a cellular microenvironment that differs considerably from that of central glutamatergic synapses. Thus by comparing the organization of hair cell synapses with that of central synapses one may obtain insight into what constitutes the essential building blocks of a functioning glutamate synapse. Another reason for focusing on the afferent hair cells synapses is their presumed involvement in auditory pathology related to noise trauma, presbycusis, and some forms of peripheral tinnitus (Puel, 1995).

The afferent hair cell synapses in the inner ear are not uniform (Friedmann and Ballantyne, 1984; Dallos et al., 1996). Their physiological and morphological characteristics depend on the identity of the presynaptic element, and the activation threshold and spontaneous discharge rate may vary even among afferent dendrites apposed to a single sensory cell (Merchan-Perez and Liberman, 1996). Superimposed on this are substantial species differences. However, the afferent hair cell synapses share certain fundamental features that justify their being treated as one group. They represent the site where a graded receptor potential, induced by the mechanical deflection of the sensory hair bundle, is transformed into a classical all or none response in the form of action potentials in the postsynaptic ganglion cell processes (Koyano and Ohmori, 1996). Further, a general property of afferent hair cell synapses is that they show a high rate of spontaneous release. This release is modulated during mechanotransduction and requires a rapid recycling of vesicles (Parsons et al., 1994). The characteristic physiological properties of the hair cell synapses are associated with, and probably dependent on, a number of specializations, the most notable of which is a presynaptic dense body to which the synaptic vesicles are tethered. The molecular basis for vesicle anchoring and guidance differs from that in central synapses and does not seem to involve synapsin (Favre et al., 1986; Mandell et al., 1990; Zidanic and Fuchs, 1996).

Certain aspects of afferent synaptic transmission have been investigated more thoroughly in the organ of Corti than in the vestibular epithelium while for other aspects the converse is true. Hence, an advantage of considering the different categories of afferent hair cell synapses in the same review is that it helps establish a more coherent picture of the afferent synapse than would have been possible had the different synapses been treated on an individual basis. But this strategy calls for a word of caution. Notably, generalizations across synapses may obscure bona fide biological differences, and discrepancies between data sets dismissed as being due to methodological factors may in fact represent synaptic heterogeneities. These caveats should be borne in mind when conclusions are drawn from data based on different methodological approaches, species, and synapse types.

The scope of the present review is limited to the afferent synapses. Emphasis will be placed on recent studies that have revealed how key molecules in glutamatergic transmission (receptors, ion channels, transporters, and enzymes responsible for transmitter synthesis and degradation) are arranged to form a functional synapse in the mammalian inner ear. The extensive literature that is available on the efferent innervation of the sensory epithelia will not be dealt with here. The reader is referred to excellent reviews of Eybalin (1993)and Puel (1995).

Section snippets

Morphological specializations of afferent hair cell synapses

The ultrastructural characteristics of the afferent hair cell synapses bear some resemblance to those of other synapses in which quantal transmitter release is triggered by graded membrane potentials (von Gersdorff and Matthews, 1994; Parsons et al., 1994; Rao-Mirotznik et al., 1995). The most conspicuous features are found presynaptically where there is a dense body, a halo of vesicles, and a thickening of the presynaptic membrane (Gleisner et al., 1973; Hama and Saito, 1977; Jacobs and

Glutamate in cochlear hair cells

The two major excitatory amino acid transmitter candidates in the CNS are glutamate and aspartate. Early microdissection analyses in rat and guinea-pig by Godfrey et al. (1976), Godfrey et al. (1986)revealed an enrichment of both of these amino acids in the hair cell region of the organ of Corti. Altschuler et al. (1989)subsequently demonstrated, at the light microscopic level, that hair cells were more strongly immunoreactive for glutamate than were the supporting cells. These results were

Compartmentation of glutamine: introduction of the glutamate– glutamine cycle

Glutamine is a major precursor and breakdown product of glutamate. Quantitative immunogold analyses of the vestibular epithelium (Usami and Ottersen, 1995) indicated that the supporting cells were consistently enriched with this amino acid compared to the hair cells (Fig. 3). The organ of Corti showed a more complex pattern of immunoreactivity inasmuch as the level of glutamine immunoreactivity was high in those types of supporting cells that are situated adjacent to the hair cells (such as

Glutamate transporters, glutamine synthetase, and phosphate-activated glutaminase at the hair cell synapse

The finding of a high glutamine level and a low glutamate–glutamine ratio in supporting cells, a feature also characteristic of glial cells in the CNS, raised the question whether the two cell types carry out analogous functions in regard to glutamate-glutamine cycling. If so, one would predict that the supporting cells are equipped with one or several glutamate transporters, as well as with GS.

Release of glutamate

Based on the immunohistochemical data reviewed above one can conclude that hair cells contain glutamate and that they are associated with a machinery for clearance of glutamate from the extracellular space. Both are essential requirements for glutamatergic transmission but it also needs to be shown that the hair cells are actually capable of glutamate release under conditions mimicking synaptic activity. Only recently has the latter requirement been met, as will be discussed below.

General comments on receptor targeting

The release data referred to above cannot be accepted as compelling evidence in favour of glutamate signalling at the afferent hair cell synapses unless it is demonstrated that the ganglion cells express glutamate receptors, and that these receptors are inserted in the postsynaptic membrane within a physiologically relevant distance from the release site.

The importance of the latter requirement can hardly be overemphasized. The recent observation of Landsend et al. (1997)is a case in point.

Organization of ion channels at the afferent hair cell synapses

The graded membrane potential induced by the deflection of the hair bundle is coupled to transmitter release through the activation of dihydropyridine-sensitive (l-type), voltage-dependent calcium channels (Fuchs et al., 1990; Roberts et al., 1990; Art et al., 1995; review: Fuchs, 1996). This differs from the situation in the CNS where glutamate release is believed to be effected primarily by opening of dihydropyridine-insensitive calcium channels (Tsien et al., 1988; Dunlap et al., 1995). The l

Conclusions

The afferent hair cell synapse now emerges as one of the best characterized glutamatergic synapses in the nervous system. Key molecules involved in the release, action, removal, and metabolism of the transmitter have been localized at high resolution and further studies will undoubtedly clarify how the physiological properties of the synapse correlate with its molecular organization. Important aims of future research will be to unravel the molecular mechanisms underlying the presumed

Acknowledgements

Studies conducted in the authors' laboratories were supported by EU Biomed grant BMH4-96-0851, the Norwegian Research Council, and Sasakawa's foundation. The data on PAG were obtained in collaboration with I. A. Torgner, B. Roberg and E. Kvamme. I. A. Torgner provided the PAG antibody. We would like to thank K. Osen and C. Hackney for critical reading of the manuscript.

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