Immunocytochemical detection of the neurokinin B receptor (NK3) on melanin-concentrating hormone (MCH) neurons in rat brain

Abstract

The presence of the neurokinin B receptor (NK3 receptor) in the rat lateral hypothalamus and the zona incerta was previously reported. The aim of the present study was to define its cellular localization in these areas. Investigations, coupling immunocytochemical and in situ hybridization techniques, focussed on two neuron populations: the melanin-concentrating hormone (MCH) neurons and a population of neurons recognized by an ovine prolactin antiserum (PRL-ir neurons). While PRL-ir neurons did not exhibit NK3 immunoreactivity, 57%±6% of MCH neurons were strongly stained by the NK3 antiserum. These results suggest that neurokinin B is involved in the regulation of MCH neuron activity via the NK3 receptor; they provide new bases for further investigations on MCH role in the control of food and water intake.

Introduction

Tachykinins constitute a family of structurally related peptides involved in the regulation of many biological processes. The major members of this family are substance P, neurokinin A (NKA) and neurokinin B (NKB). Their actions as neurotransmitters/neuromodulators are mediated by three distinct receptors termed NK1, NK2 and NK3 (review in Patacchini and Maggi, 1995). These receptors belong to the superfamily of G protein-coupled receptors with seven transmembrane domains (Nakanishi, 1991). Their regional distribution has been extensively investigated by radioligand binding and autoradiography (see Ding et al., 1996). In the brain, NK2 receptor is poorly represented if not absent (Whitty et al., 1995); NK1 and NK3 are both present; their mRNA and binding sites are distributed in several regions including the hypothalamus where they are particularly abundant (Tsuchida et al., 1990; Humpel and Saria, 1993). NK3 shows greater abundance than NK1 but has a more restricted distribution; recently its presence in neurons located in the dorsal and lateral hypothalamic areas as well as in the zona incerta of the rat was demonstrated by using in situ hybridization and immunocytochemical techniques (Ding et al., 1996). Since we are particularly interested in two neuron populations exclusively located in these areas, the melanin-concentrating hormone (MCH) neurons (Fellmann et al., 1987; Risold et al., 1992; Deray et al., 1994) and a population of cells containing a peptide related to prolactin (PRL-ir neurons) (Griffond et al., 1994, Griffond et al., 1995; Grillon et al., 1996), we tested their immunoreactivity (ir) to a specific NK3 antibody by double immunocytochemical labelling or by coupling immunocytochemistry and in situ hybridization. Indeed the roles of these populations are still poorly understood and their connections with other neuron systems remain unknown. Some data suggest that the PRL-ir neurons could be involved in food intake (Bahjaoui-Bouhaddi et al., 1994) and water homeostasis (Grillon et al., 1996) regulation. Concerning MCH, many studies dealing with its functions in mammals have been published suggesting that MCH acts as a neurotransmitter/neuromodulator involved in the control of goal-oriented behaviours or general arousal (for review, see Nahon, 1994). Very recent data pointed out a role in the control of food intake and feeding behaviour (Presse et al., 1996; Qu et al., 1996) as well as in the regulation of fluid homeostasis (Parkes, 1996). While investigations on molecular and physiological aspects are very numerous, evidence on hormonal or neuronal factors regulating MCH synthesis and secretion is still lacking. Corticosteroids are thought to positively regulate the expression of the MCH gene and the secretion of the peptide (Parkes and Vale, 1992; Presse et al., 1992); however, no glucocorticoid receptor-producing neurons have been previously detected in the lateral hypothalamus, suggesting that these hormones do not act directly on MCH neurons. It was also shown that glutamate induced a stimulation of MCH release from hypothalamic neurons in primary culture, and that this effect was mediated by NMDA receptors (Compagnone et al., 1993). Identifying receptors carried by MCH and PRL-ir neurons could provide a substrate for further physiological studies and for a better understanding of their regulatory pathways. In this prospect, we report here that MCH but not PRL-ir neurons possess NK3 receptor.