Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling

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Abstract

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs.

Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis.

Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

Introduction

In the auditory system, like as in other sensory systems, characterization of distinct cell types is one prerequisite for a functional analysis of afferent and efferent pathways. The auditory system differs from other sensory systems in the complexity of its subcortical nuclear network. Four major processing levels are interposed between the primary afferents (auditory nerve fibers) and the thalamorecipient laminae of the primary cortical field (review: Webster, 1992): The cochlear nucleus (CN) is the first relay station of the central auditory pathway. The auditory nerve fibers innervate three major divisions within this nuclear complex, the anteroventral, the posteroventral, and the dorsal cochlear nucleus (AVCN, PVCN, DCN). Several distinct principal cell types, for example spherical and globular bushy cells, stellate cells and octopus cells were firstly described for cat (Osen, 1969, Brawer et al., 1974), guinea pig (Hackney et al., 1990), rabbit (Disterhoft et al., 1980), chinchilla (Morest et al., 1990), and rat (Wouterlood et al., 1984). The distribution of these different cell types and the specific afferent innervation patterns within each of CN divisions led to an even more refined parcellation of CN divisions (Irvine, 1986, Webster, 1992, Morest, 1993). However, most of these earlier investigations of cell types were mainly based on Nissl staining, silver impregnations, and the application of various tract tracer techniques. Recently, staining by components of extracellular matrix was used to identify cell types and to mark locations of some specific cell types in the CN (Lurie et al., 1997, Cant and Benson, 2006). In the present study, antibodies directed against intracellular calcium-binding proteins (CaBPs) were used to describe principal cell types and their distribution in the CN of two different mammals in addition to preceding morphological studies.

CaBPs, which selectively bind calcium with high affinity, are characterized by a common structural motif—the EF-hand (Persechini et al., 1989, Heizmann and Braun, 1992, Ikura, 1996, Niki et al., 1996, Lewit-Bentley and Réty, 2000). Among such EF-hand CaBPs, parvalbumin (PV), calbindin-D28k (CB) and calretinin (CR) are known to be specific markers of neurons (Celio and Heizmann, 1981, Heizmann and Hunziker, 1991, Heizmann and Braun, 1995). These proteins are widely dispersed throughout the cytosol of the neurons up to its thinnest processes. Thus, antibodies against these CaBPs are excellent tools to visualize the neuronal architecture and to provide almost Golgi-like staining (for reviews see Baimbridge et al., 1992, Andressen et al., 1993, Seto-Ohshima, 1994). The labeling can even help to reveal specific connectivities between afferents and their target neurons (Baimbridge et al., 1992, Kevetter and Leonard, 1997).

To date, the organization of the auditory brainstem nuclei has been analyzed by differential expression patterns for PV, CB, and CR only in eutherians: e.g. in guinea pig (Caicedo et al., 1996), bat (Zettel et al., 1991, Vater and Braun, 1994), rat (Friauf, 1993, Friauf, 1994, Lohmann and Friauf, 1996, Por et al., 2005), chinchilla (Kelley et al., 1992, Frisina et al., 1995), gerbil (Braun and Piepenstock, 1993, Korada and Schwartz, 2000), human (Bazwinsky et al., 2003) and rhesus monkey (Bazwinsky et al., 2005). A common result of these studies on the above listed mammals was a widespread PV-immunoreactivity in morphologically distinct cell types, while CB- and CR-immunolabeling is restricted to specific cell subpopulations and axonal terminals.

Hypotheses about the functional significance of CaBP distributions vary greatly. It is generally thought that CaBPs are involved in converting calcium signals into intracellular responses (Berggard et al., 2002) thereby effecting a wide variety of cell activities, such as cytoskeleton organization, cell motility and differentiation, cell-cycle regulation, and transport of calcium into intracellular compartments (Heizmann and Braun, 1995, Niki et al., 1996, Billing-Marczak and Kuznicki, 1999).

The main goal of our study was to provide additional knowledge about the mammalian CN principal cell types by use of PV, CB and CR as neuronal markers in two different mammalian species, an eutherian species the Mongolian gerbil (Meriones unguiculatus) and for the first time to investigate a metatherian species, the gray short-tailed opossum (Monodelphis domestica). It would be of interest to know whether similar auditory brainstem organization and principal cell types are present in Meriones and Monodelphis, species belonging to two mammalian groups, which have been evolved successfully and independently since Cretaceous (review: Aitkin, 1995). Structural species differences and varying pattern of neuronal labeling may be related to adaptive differences in auditory function.

Immunofluorescence labeling was applied to reveal the three CaBPs PV, CB and CR, whereby always two of the three markers were visualized in varying combinations. This enabled an allocation of specific CaBPs to distinct cell types and, at the same time, allowed for the detection of CaBP co-expression in one and the same neuron. We also examined whether each individual of a particular cell type expresses the same CaBP. Therefore, in each section all neurons were visualized by the combined application of two neuronal markers, neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5). Both markers label neurons in the central and peripheral nervous system as well as neuroendocrine cells (Schmechel et al., 1978, Thompson et al., 1983, Thompson and Day, 1988).

NSE is a brain-specific acidic isoenzyme of the glycolytic enzyme enolase (reviewed by Marangos and Schmechel, 1987). NSE immunohistochemistry does not label glia, endothelial cells, and other non-neuronal structures. The labeling is confined to the neuronal cytoplasm, revealing the soma, dendrites, and axons; the nucleus is not marked. Also the second neuronal marker, PGP 9.5 has been characterized as a cytoplasmatic enzyme, ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., 1989), which constitutes 1–2% of the proteins in the brain.

The methods mentioned above enabled a detailed examination of the distribution of CB-, PV- and CR-containing cell types and terminals in Meriones and Monodelphis cochlear nucleus with emphasis on principal cell types of each CN subregion.

Section snippets

Tissue preparation

The care and use of animals in this study were approved by the Saxonian District Government, Germany, according to the animal care law. The experiments were conducted in accordance with international standards on animal welfare as well as being compliant with the European Communities Council Directive of 24 November 1986 and National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four adult Mongolian gerbils (M. unguiculatus; 35–53 g; 1–3 months) and four gray

Positive control for CaBP-immunolabeling

Immunohistochemical CaBP-labeling of neurons in the cerebellum of Meriones und Monodelphis was used to evaluate the quality of the labeling of CN neurons. Neurons of the cerebellum were chosen as positive control cells because of their well-known CaPB-expression, which has been described for a number of mammals. Purkinje cells show a strong PV- and CB-immunoreactivity, as previously reported (Rogers et al., 1990, Heizmann and Braun, 1990; review: Andressen et al., 1993). Calretinin is strongly

Discussion

In the present study we compared for the first time principal cell types of the CN of an eutherian species (M. unguiculatus) and those of a metatherian species (M. domestica) using various combinations of double labeling for PV, CR and CB and the combined staining of CaBPs and pan-neuronal markers. In mammalian phylogeny, the Metatheria and the Eutheria are sister groups, which have evolved separately from an ancestor common to both and are very successfully adapted to their environments

Summary

The present study revealed similarities between Monodelphis and Meriones, which indicate a convergence between metatherian and eutherian mammals. However, we were also able to report on functionally based, species-specific differences, e.g. spherical bushy cells.

Comparison of the distribution pattern of CaPBs in different cell types in mammalian species revealed a more ubiquitous occurrence of PV, but at the same time a more cell-specific and complementary expression pattern of CB and CR in

Acknowledgements

The study was supported by DFG RU 390 17-15-1 (SPP) and DFG GRK 250/1-96. The authors are thankful to Dr. Luminita Göbbel for improving an earlier version of the manuscript. The authors would also like to thank Dr. Mühlbauer and Mrs. Mühlbauer for careful proof-reading the English of the manuscript. We thank Dr. R. Kretzschmar for providing the Golgi stained material for cell identification.

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