Elsevier

Brain Research

Volume 1039, Issues 1–2, 28 March 2005, Pages 63-74
Brain Research

Research report
Presence and distribution of three calcium binding proteins in projection neurons of the adult rat cochlear nucleus

https://doi.org/10.1016/j.brainres.2005.01.057Get rights and content

Abstract

The presence and distribution of three cytoplasmic calcium binding proteins, calbindin, calretinin, and parvalbumin, have been investigated in the projection neurons of the cochlear nucleus complex in adult rats by using immunohistochemistry in free-floating slices. Identification of the individual cell types was carried out on the basis of their intranuclear localization, morphological characteristics, and (in the cases of pyramidal and bushy neurons) by retrograde labeling with rhodamine-dextran. The most important findings were confirmed by using confocal microscopy. The data obtained in these experiments are the first to demonstrate the presence of parvalbumin in pyramidal neurons and globular and spherical bushy cells of rat cochlear nucleus, whereas octopus and giant cells did not show positivity for parvalbumin. Calretinin was not present in either Purkinje-like cells or giant neurons. According to the double immunolabeling co-localization experiments, the pyramidal neurons, Purkinje-like cells, globular bushy cells, and octopus cells express two different calcium binding proteins in their cytoplasm (although in different combinations) whereas giant cells and spherical bushy cells contain solely calbindin and parvalbumin, respectively. The presence of calretinin in globular bushy cells provides a tool for distinguishing them from spherical bushy cells. The immunolabeling of the fibers and axonal endings of the acoustic nerve in the ventral part of the cochlear nucleus indicated that these structures are also parvalbumin positive. It is concluded that the heterogenous cell composition of the cochlear nucleus is accompanied by a rather complex expression pattern of the cytoplasmic calcium binding proteins.

Introduction

Like other mammalian cells, neurons posses a broad variety of proteins that transiently bind Ca2+. These calcium binding proteins can be classified on the basis of their molecular structure, localization, Ca2+ binding properties, etc. [1], [24], [37]. From a functional point of view, however, the most intriguing question is their involvement in the modulation of cellular activity. Some calcium binding proteins exert well-defined regulatory actions (e.g., calmodulin, calpains), whereas others have not been hitherto related directly to specific cellular functions. Nevertheless, even these proteins may affect various Ca2+-dependent regulatory processes as they are able to influence the extent and duration of activity-related intracellular [Ca2+] changes [1], [37].

Cytoplasmic Ca2+ plays a rather complex role in neurons. On one hand, nerve cells posses several functions that are triggered or modulated by intracellular [Ca2+] transients but, on the other hand, unusually high or long-lasting [Ca2+]i elevations seem to be involved in the genesis of numerous neuronal disorders [20], [27], [41]. It is not surprising, therefore, that nerve cells express a considerable amount of Ca2+ buffer proteins, apparently as a defense tool against Ca2+ overload and the consequent Ca2+-induced cytotoxicity. The most common calcium buffers of the neurons are parvalbumin (PA), calbindin-D28K (CB), and calretinin (CR) [1], [6], [15], all of them belonging to the EF-hand family. While PA has been described in other cell types (e.g., in skeletal muscle fibers), CB and CR seem to be exclusively present in the nervous system.

There were several efforts to describe the distribution of the aforementioned proteins in the central nervous system (CNS) of different species using various techniques [1], [15]. These studies revealed similarities between the expression patterns of some neuron populations indicating common developmental origin and/or functional relationship [1]. An especially strong correlation has been established between the synaptic refinement and the expression levels of the Ca2+ buffer proteins in the course of ontogenesis in different parts of the CNS [15], [21]. In fact, the development of the hearing function appears to be a good model for analyzing the postnatal maturation of neuronal networks, as the newborns of several species lack hearing and it takes several days or weeks until the auditory function fully develops. It is also known that impairment of the peripheral elements of the auditory apparatus leads to altered morphology and/or function of the central auditory neurons, including modifications of calcium binding protein expression [3], [29], [44]. Data obtained from various species agree that CB, CR, and PA are present at all parts of the auditory pathway although their expression levels show considerable cell-to-cell, developmental, and interspecies variability (see [2], [19], [23], [40], [42] and further references cited in these papers).

Previously, we have described the membrane properties of two projection neurons (bushy and pyramidal cells) of the rat cochlear nucleus (CN) [7], [11], [30], [31]. As the functional properties of these cells might be strongly influenced by intracellular modulatory mechanisms (including [Ca2+]i changes), the characterization of the Ca2+ homeostasis has also been attempted. The results of these experiments on pyramidal cells [12], [13], [35] necessitated, however, a better characterization of their cytoplasmic Ca2+ buffer capacity. Moreover, to be able to compare the Ca2+ handling of the different types of neurons, such data from other CN cells are also required. In order to gain a more complete understanding of the presence and distribution of the three main Ca2+ buffer proteins in the rat CN, double immunolabeling experiments using both fluorescence microscopy and confocal microscopy were carried out in adult, fully matured animals. Some of the results presented here are in good accordance with earlier findings. Moreover, our results also provide new information about the co-expression of the calcium binding proteins in the primary projection neurons of the CN.

Section snippets

Preparation of cochlear nucleus

Free-floating sections were prepared from 30-day-old Wistar rats following the steps of an earlier detailed description [36] (the protocol was authorized by the Ethical Committee of the University of Debrecen). Briefly, after decapitation of the animal, the brain was prepared, the meninges and the major blood vessels were removed, then the brain was cut in half by making a sagittal section in the midline. When retrograde tracing was performed (see below), the last step was omitted. The

Calcium binding proteins in the DCN

The best-characterized projection neuron of the DCN is the pyramidal (or fusiform) cell. The axons of this cell population join the dorsal acoustic stria and run to the contralateral inferior colliculus. Consequently, the back-tracing of the pyramidal cells was performed via cuts made in the area of the dorsal acoustic stria in a distance which allowed the tracer to reach the ipsilateral neurons in the given incubation time. Fig. 1A shows the result of such an experiment, where it can be seen

Discussion

The aim of the present experiments was to describe the presence and distribution of the three most common cytoplasmic calcium binding proteins in projection neurons of the CN of adult rats. Earlier data about the occurrence of these proteins in this nucleus were collected from different species or from a given species at different ages. Thus, it seemed desirable to conduct a survey using the same technique in the same species with fully matured auditory pathway. On the basis of our earlier

Acknowledgments

This work was supported by grants from the Hungarian Science Foundation (OTKA, T046067, 13B0-0/19/TS01) and from the Wellcome Trust (075243/Z/04/Z). The authors are indebted to Mrs. C. Matesz, Drs. P. Szentesi and P. Szűcs for methodological help and discussions as well as to Mrs. I. Varga for technical assistance.

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