Microinjection of RFRP-1 in the central nucleus of amygdala decreases food intake in the rat

Abstract

Several members of the RFamide peptide family are known to have role in the regulation of feeding. For example, neuropeptide FF and prolactin-releasing peptide cause anorexigenic, while 26RFa and QRFP result in orexigenic effects in rodents. I.c.v. microinjection of neuropeptide RFRP-1 significantly reduced food and water intake in chicks. However, feeding related effects of RFRP-1 have not been studied in mammals yet. The central part of amygdala (CeA) is essentially involved in the regulation of feeding and body weight. RFRP-1 positive nerve cells were detected in the rat hypothalamus and RFRP-1 immunoreactive fibers were identified in the CeA. RFRP analogs bind with relatively high affinity to the NPFF1 and NPFF2 receptors (NPFF-R). RFRP-1 has potent activity for NPFF1. Significant expression of NPFF1 was detected in the CeA. To evaluate the role of RFRP-1 in feeding regulation rats were microinjected with different doses of RFRP-1 and their food intake were quantified over a 60 min period. Liquid food intake of male Wistar rats was measured after bilateral intraamygdaloid administration of RFRP-1 (25, 50 or 100 ng/side, RFRP-1 dissolved in 0.15 M sterile NaCl/0.4 μl, respectively). The 50 ng dose of RFRP-1 microinjections resulted in significant decrease of food intake. The 25 and 100 ng had no effect. Action of 50 ng (37.8 pmol) RFRP-1 was eliminated by 20 ng (41.4 pmol) RF9 NPFF-R antagonist pretreatment. In open-field test 50 ng RFRP-1 did not modify spontaneous locomotor activity and general behavior of animals did not change. Our results are the first reporting that RFRP-1 injected to the CeA result in a decrease of liquid food consumption. This is a receptor-linked effect because it was eliminated by a NPFF-R selective antagonist.

Introduction

RFRP-1 is a member of the RFamide peptide family. To date, five groups of the RFamide peptides have been documented: NPFF (PQRFa) group, PrRP group, LPXRFamides (RFRPs, GnIH), Kisspeptin group and QFRP (26RFa) group (Ukena and Tsutsui, 2005, Fukusumi et al., 2006, Osugi et al., 2006). Several members of the RFamide family, containing a terminal arginine (R) and amidated phenylalanine (F), affect appetite-associated processes in a wide range of species (Dockray, 2004, Bechtold and Luckman, 2007). Some of these peptides cause anorexigenic or orexigenic effects in rodents (Murase et al., 1996, Lawrence et al., 2002, Chartrel et al., 2003, Bechtold and Luckman, 2006, Takayasu et al., 2006). The mammalian members of the LPXRFamide peptide family are the RFRP-1 and RFRP-3 as well as the RFRP-2. These peptides are derived from a same single precursor protein (Prepro-RFamide-related Peptides). RFRP-2 sequence was absent in rat and mouse preprotein indicating that RFRP-2 has no significant function in these animals (Hinuma et al., 2000). RFRP-3 has emerged as important regulator of reproductive function (Yoshida et al., 2003, Murakami et al., 2008, Pineda et al., 2010a, Pineda et al., 2010b, Smith and Clarke, 2010). I.c.v. administration of RFRP-1 in rats has also been shown to raise circulating levels of prolactin in a concentration-dependent manner and did not affect the secretion of other pituitary peptides (Hinuma et al., 2000). RFRP-1 acts in the hypothalamus to inhibit dopaminergic neuronal activity (Willis et al., 2003). The RFRP gene was expressed in the caudal hypothalamus including the dorsomedial hypothalamus (DMH) and the periventricular nucleus (PerVN) (Fukusumi et al., 2001, Yano et al., 2003). The DMH plays an important role in the control of energy metabolism.

The administration of RFRPs induces c-Fos protein expression in the arcuate nucleus (ARC), which has a key role in the regulation of feeding behavior. I.c.v. administration of RFRP-3 stimulates food intake in male rats and ovariectomized female rats (Yoshida et al., 2003, Murakami et al., 2008). It has been shown, however, that RFRP-1 applied i.c.v. to chicks (Gallus gallus) significantly reduced both food intake and water intake (Newmyer and Cline, 2009).

Administration of RFRPs activates neurons in the locus coeruleus (LC) and nucleus tractus solitarii (NTS), which play important roles in neuroendocrine and behavioral stress responses. Subsequent to stressful stimuli the percentage of RFRP neurons expressing c-Fos protein and the expression of RFRP mRNA in the hypothalamus are increased (Kirby et al., 2009). RFRP fibers are observed in the hypothalamic paraventricular nucleus (PVN) and appear to project directly to cells containing CRH or oxytocin (Qi et al., 2009) in the hypothalamus. Furthermore, i.c.v. administration of RFRP-1 or RFRP-3 induces anxiety-related behavior (Kaewwongse et al., 2011). RFRP-1 plays a role in the processing of pain in mice (Yudin et al., 2006), increases the magnitude and duration of spinal morphine anti-nociception in rats (Jhamandas et al., 2006) and negatively affects adipogenesis in human and mouse cell cultures (Herrera-Herrera and Salazar-Olivo, 2008).

Immunohistological assays identified RFRP-1 immunoreactive cell bodies in the supramammillary nucleus of hypothalamus (suMM), PVN, ventral posteromedial thalamic nucleus (ThVPM), medial preoptic area (MPO) as well as RFRP-1 immunoreactive fibers were detected in the bed nucleus of the stria terminalis (BST), nucleus of the vertical limb of the diagonal band (VDB) and central amygdaloid nucleus (CeA). Several results showed that RFRP-1 and RFRP-3 are produced in the same neurons around the dorsomedial hypothalamus of rats (Fukusumi et al., 2001, Yano et al., 2003, Yano et al., 2004, Yoshida et al., 2003).

Biological responses mediated by RFRPs peptides result from high affinity binding to two NPFF receptors, i.e. NPFF1 receptor and NPFF2 receptor, respectively. The NPFF2 receptors were identified in the amygdala (AMY) (Bonini et al., 2000, Hinuma et al., 2000, Liu et al., 2001, Engstrom et al., 2003) but they were not found in the CeA (Liu et al., 2001). On the other hand, significant expression of NPFF1 receptors were detected in the CeA and the medial, and basolateral amygdaloid nuclei (Liu et al., 2001).

The amygdala plays an important role in feeding and body weight regulation. Molecular biological (Bonini et al., 2000) and immunohistochemical (Liu et al., 2001) investigations have revealed significant expression of NPFF1 receptors in the CeA. Since i.c.v. applied RFRP-1 decreased food intake in chicks (Newmyer and Cline, 2009) and because the rat CeA contains both RFRP-1 fibers and NPFF1 receptors, we hypothesized that RFRP-1 infusion into the CeA of rats may result in decrease of food consumption. In mammals feeding related effects of RFRP-1, however, have not been examined so far. Therefore, our present experiments were designed to examine liquid food intake of male Wistar rats after bilateral RFRP-1 microinjections into the CeA. It was also studied whether application of NPFF receptor antagonist RF-9 can prevent the effect of RFRP-1 in the CeA. In open-field test it was examined whether RFRP-1 microinjections modify spontaneous locomotor activity or induce anxiety-like behavior.