Elsevier

Brain Research

Volume 928, Issues 1–2, 22 February 2002, Pages 8-17
Brain Research

Research report
Molecular probes of the vestibular nerve: I. Peripheral termination patterns of calretinin, calbindin and peripherin containing fibers

https://doi.org/10.1016/S0006-8993(01)03268-1Get rights and content

Abstract

Vestibular afferents have different physiological properties that can be at least partially correlated with the morphology that the peripheral ending makes with type I and type II hair cells. If the location of the ending in the sensory epithelium is included, the correlations are further improved. It is also known that vestibular afferents can be immunohistochemically stained for a variety of different substances. We have concentrated on three of these markers, calretinin, calbindin and peripherin, because the sources of afferents to the vestibular nuclear complex that contain these substances are restricted, in two cases to the primary afferents. We demonstrate that calretinin is found only in the calyx-only afferents that are located at the apex of the cristae ampullaris and along the striola of the maculae. The area containing stained calyces is equal to or slightly smaller than the central zone of the cristae as defined by the Goldberg group [J. Neurophysiol. 60 (1988) 167]. Calbindin is also found in calyces at the apex of the cristae and along the striola of the otoliths. Examination of adjacent sections of all endorgans indicates that calbindin staining overlaps with calretinin, but is always several hair cells wider on each side. Peripherin also stains fibers in the neuroepithelium. The greatest density of staining is in the peripheral zone of the cristae, i.e. at the base and toward the planum semilunatum. We suggest that these substances are useful markers for specific sets of vestibular afferents.

Introduction

It is well known that the patterns of vestibular primary afferent terminations on type I and/or type II hair cells and their locations in the sensory epithelia can be correlated with the physiological properties of the afferents [1], [15], [17], [33]. It is also known that the primary afferents have different combinations of biochemical markers that can be identified immunohistochemically [2], [5], [10], [11], [13], [14], [19], [32], [34], [38], [40], [44], [47], [49]. The same situation is found in the primary afferents of the somatosensory system. In this system, it has been possible to correlate, to a greater or lesser degree, the immunohistochemical markers with physiological properties. In turn, using immunohistochemical or in situ techniques to detect these phenotypes, it has been possible to examine the organization of primary afferent input to the spinal cord [e.g. [31], [43]], the development of the primary afferents themselves [e.g. [18], [35], [42]] and the response to injury [e.g. 4] and make some implications about the behavioral significance. Here we set the stage for similar studies in the vestibular system.

The central issue is to determine as precisely as possible the groups of primary afferents containing each of several markers. Since different groups of spinal primary afferents contain different transmitters or modulators, such as glutamate, substance P or CGRP, these have been used as markers. In addition, other substances, such as neurofilaments, are also differentially distributed and have been used as markers. While there are at least two reports that some vestibular primary afferents contain substance P [34], [41] it appears that all vestibular primary afferents use glutamate as their principle transmitter. Therefore, we are utilizing markers for substances other than transmitters, specifically two of the calcium-binding proteins and the intermediate filament peripherin. Exactly how the specific properties of any particular substance contribute to the specific phenotype of one group of primary afferents is not clear. However, if they meet certain criteria, these substances are still useful markers.

The first criterion is that the substance is found in only a subset of vestibular afferents. Some substances are ubiquitous in the primary afferents and therefore, are not useful in this context. If a substance is to be a useful marker for examining primary afferent input into the vestibular nuclei, it would be most desirable that the only structures within the vestibular nuclei that contain the marker be primary afferents. It is unlikely that many substances will completely satisfy this criterion, but a marker may still be useful if the sources are restricted. Many of the substances, demonstrated to be present in vestibular afferents, such as parvalbumen [11], [20] and cytochrome oxidase [21], [38], are unsatisfactory for our purposes as they fail one or more of these criteria.

Calretinin, calbindin and peripherin are found in subsets of primary afferents [2], [8], [9], [10], [12], [13], [14], [32], [38], [39], [40], [48]. On the basis of rhizotomies central to the vestibular ganglion, the only appreciable source of calretinin [24] and peripherin [personal observations] fibers in the vestibular nuclei is primary afferents. These substances meet the criteria listed above. Calbindin is found in primary afferents and in cerebellar Purkinje cells [3], [16], [24]. Calbindin in the vestibular nuclei only comes from primary afferents in animals with cerebellectomy [24] or animals with mutations that result in loss of Purkinje cells [3]. Therefore, calbindin is potentially useful.

There are a number of papers describing staining for calbindin in the vestibular periphery or the vestibular ganglion [2], [9], [12], [13], [37], [38], [39], [40], [48], somewhat fewer describing staining for calretinin [10], [13], [14], [38], and only one for peripherin [32]. A variety of species of mammals have been examined. Calretinin and calbindin are typically described as being found in large fibers in the apical regions of the cristae, though they also have been described in hair cells on the slopes of the cristae. The situation is similar for the macula of the utricle. Note that very few reports contain a comparison of the staining patterns for calbindin and calretinin, and none provide a comparison of the patterns for the calcium-binding proteins and peripherin. Our goal is to provide a description of the nature and distribution of the structures containing these three substances in gerbils. A second issue is determining the extent of overlap, if any, in the populations containing these substances. There is evidence suggesting that there is extensive, but incomplete, overlap of calretinin and calbindin. This issue is addressed several ways. In this paper, we examine adjacent sections or double staining of single sections of the sensory epithelia. Presumably because of difficulties removing the saccule intact, there are very few descriptions of the distribution of the immunohistochemical staining patterns in this end organ. Therefore, we include these descriptions. In the companion paper, we present a careful examination of the populations of labeled cells in the vestibular ganglion, the results of two double labeling experiments to examine overlap, and extensive quantitation using dissector based techniques.

Preliminary results have been presented at meetings [22], [23], [26], [27].

Section snippets

Materials and methods

Twenty-six young adult gerbils (Meriones unguiculatus) of both sexes were used in this study. The animals were anesthetized with an overdose of urethane (400 mg/kg, i.p.) and perfused through the left ventricle with warm saline followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). A hole was made in the bone over the lateral or anterior ampulla and the caudal end of the utricle to allow immersion fixation for several hours to overnight.

The ampullae and utricle were removed

Results

Staining the crista with antibodies to calretinin and peripherin reveals distinctly different patterns of staining. Fig. 1 illustrates two, whole-mount cristae that have been stained with antibodies to either calretinin (Fig. 1A) or peripherin (Fig. 1B). The crista in Fig. 1A is slightly tilted and the focal plane is just below the apex. Calretinin staining of the apical ends of calyces appears as circles. It is clear that the staining is confined to the central, apical regions. There is no

Discussion

In gerbils, calretinin antibodies label only calyces and large fibers in both the cristae and maculae. Although Dechesne and co-workers [6], [10] reported some type II hair cell staining in peripheral regions of the cristae, and both types I and II hair cell staining in the utricles of rats, the majority of studies in adult animals emphasize the staining of large fibers and calyces. Calretinin is contained in hair cells during development but is lost by postnatal day 14 in the mouse [6], [51].

Acknowledgements

We thank Cindy Lou Zimmerman for the excellent technical assistance and Dr. Christine Livingston for reading this manuscript. Supported by NIH DC04070 and a grant form the Deafness Research Foundation.

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