Chemoarchitecture of the primate dorsal raphe nucleus

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Abstract

The dorsal raphe nucleus (DR) harbours the largest single collection of serotonin (5-HT)-containing neurons in the brain but also comprises other types of chemospecific neurons. The aim of the present study was to characterise morphologically and immunohistochemically the DR in the squirrel monkey (Saimiri sciureus). The morphology of the DR 5-HT-immunoreactive (ir) neurons was analysed and their distribution compared to that of neurons displaying immunoreactivity for either tyrosine hydroxylase (TH), γ-aminobutyric acid (GABA), substance P (SP), calbindin-D28k (CB), calretinin (CR) or parvalbumin (PV). The 5-HT-ir neurons were distributed in a highly heterogeneous manner throughout the rostrocaudal extent of the DR. The morphology and density of the 5-HT neurons were found to vary significantly in the major subdivisions of the primate DR, that is, the median, ventral, dorsal, ventrolateral, lateral and caudal subnuclei. Numerous SP-, GABA- and PV-ir neurons occurred in all six subnuclei of the DR. The distribution of SP-ir neurons was largely in register with that of 5-HT-ir neurons. Neurons expressing the other neuronal markers (TH, CB, CR) were not present in all six DR subnuclei and their distribution was either complementary to, or in register with, that of 5-HT-ir neurons. The median subnucleus was unique because it contained all the different types of chemospecific neurons. This study has revealed that the primate DR is chemically highly heterogeneous, a finding that may explain the multifarious influence that this nucleus exerts upon various forebrain structures.

Introduction

The dorsal raphe nucleus (DR) is the largest single collection of neurons containing serotonin (5-hydroxytryptamine, 5-HT) in the entire brain (Dahlström and Fuxe, 1964, Parent et al., 1981, Steinbusch, 1981, Steinbusch et al., 1981, Descarries et al., 1982, Felten and Sladek, 1983, Azmitia and Gannon, 1986, Baker et al., 1990, Törk and Hornung, 1990, Jacobs and Azmitia, 1992). Serotonin is thought to be involved in a wide variety of complex physiological and behavioral processes, such as autonomic functions, vigilance states, mood, affect, learning and memory (see Jacobs and Azmitia, 1992, Wang and Nakai, 1994for reviews). The fact that the DR is a major source of serotoninergic innervation to the cerebral cortex, basal ganglia, hypothalamus and thalamus (Steinbusch and Nieuwenhuys, 1983, Jacobs and Azmitia, 1992), where many of these functions are integrated, makes this relatively small brainstem nucleus particularly attractive.

Neurons of the DR produce a wide variety of transmitter-related molecules other than 5-HT, including dopamine (Ochi and Shimizu, 1978), glutamate (Clements et al., 1991), γ-aminobutyric acid (GABA) (Nanopoulos et al., 1982, Belin et al., 1983), substance P (SP) (Chan-Palay et al., 1978, Hökfelt et al., 1978, Ljungdahl et al., 1978) and enkephalin (Hökfelt et al., 1977, Uhl et al., 1979, Glazer et al., 1981, Moss et al., 1981). These various molecules are produced either by distinct neurons in the DR or are coexpressed with other chemical markers in different combinations.

The present immunohistochemical study was undertaken to analyse the morphological organisation of the serotoninergic neurons in the DR of the squirrel monkey and to correlate this arrangement with the distribution of various other neurochemical markers. As examples of neurons utilizing classical small molecule transmitters other than serotonin, we looked at the distribution of tyrosine hydroxylase (TH) and GABA, as markers of dopaminergic and GABAergic neurons, respectively. As for neurons producing neuroactive peptides, we analysed the distribution of the tachykinin SP. Finally, we studied the cellular localization of three calcium-binding proteins of the ‘EF hand’ family, namely calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV), because very little is known about these proteins, which could play a major role in the functioning of DR neurons in both normal and pathological conditions.

This investigation is the first part of an ongoing research project on the anatomical and functional organisation of the DR in primates. The normative data reported here will serve as a framework for future investigations, which will involve double-immunostaining analysis of the coexpression of various neuronal markers in single DR neurons, as well as tract-tracing studies of the efferent projections of each of the DR subnuclei in the squirrel monkey.

Section snippets

Experimental procedures

The data reported here were collected from nine adult squirrel monkeys (Saimiri sciureus), weighing 800–1000 g, that had previously been used for tract-tracing experiments with biocytin or biotin dextran amine. These small microiontophoretic injections were aimed at the striatum and did not involve the brainstem. In one case, colchicine (500 μg dissolved in 50 μl distilled water) was stereotaxically injected in the lateral ventricles under deep anaesthesia with a 100 μl Hamilton microsyringe,

Results

The morphological characteristics and patterns of distribution of the various types of chemospecific neurons in the DR were remarkably similar in the nine monkeys examined in the present study. Three animals showed some slight variations that were largely due to differences in the plane of section. The distribution of the various neuronal types was virtually identical in the other six monkeys. Thus, the overall mappings shown below (Fig. 1Fig. 2Fig. 3) can be considered as highly representative

Discussion

The present study has provided evidence for the existence of various types of chemospecific neurons, other than serotoninergic neurons, in the DR of primates. Neurons immunoreactive for either CB, CR, or TH were preferentially located in sectors of the DR devoid of 5-HT-ir neurons, whereas neurons immunostained for SP, PV and GABA were distributed in regions populated by 5-HT-ir neurons. The significance of these findings will now be discussed in the light of previous data obtained with a

Acknowledgements

The authors express their gratitude to Carole Emond and Lisette Bertrand for technical assistance. This research was supported by grant MT-5781 from the Medical Research Council of Canada to A. Parent. A. Charara was holding a Studentship from the FCAR.

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