Localization and neuronal response of RFamide related peptides in the rat central nervous system

doi:10.1016/S0006-8993(03)02877-4

Brain Research

Volume 982, Issue 2, 29 August 2003, Pages 156-167

https://doi.org/10.1016/S0006-8993(03)02877-4 Get rights and content

Abstract

RFamide related peptides (RFRP)-1 and RFRP-3 are neuropeptides derived from the same preproprotein. We have examined the distribution of RFRP-1 and RFRP-3 immunoreactivities (irs) in the rat central nervous system using specific antibodies. Neuronal cell bodies containing both RFRP-1 and RFRP-3 were detected within the caudal portion of the hypothalamus, the periventricular nucleus (PerVN), and the portion around or above the ventromedial nucleus of the hypothalamus. Both immunohistochemical and in situ hybridization analyses showed that neurons containing RFRP immunoreactivity and mRNA were distinct from those of neuropeptide FF, which contains the same structure at the C-terminus, Pro–Glu–Arg–Phe-NH₂, as RFRP-3. Fibers containing both RFRP-1 and RFRP-3 were widely distributed in the brain: the lateral septal nucleus in the telencephalon, the paraventricular thalamic nucleus, various hypothalamic nuclei, the periaqueductal gray in the midbrain, the parabrachial nucleus in the pons, and the nucleus tractus solitarius (NTS) in the medulla oblongata. Only RFRP-1-ir was detected within the posterior gray horn in the spinal cord. Only RFRP-3-ir was detected in several thalamic nuclei and the spinal cord, especially at the posterior intermediate sulcus and within the anterior gray horn. Intracerebroventricular administration of RFRPs induced c-Fos expression in the anterior portion of the NTS, locus coeruleus, the nucleus of incertus, supraoptic nucleus, PerVN and the arcuate nucleus of the hypothalamus. These results show that RFRP-1 and RFRP-3 are widely distributed in the rat central nervous system and might be involved in various functions such as the neuroendocrine system or pain modulation.

Introduction

RFamides are peptides with an arginine–phenylalanine-amide structure at the carboxy-terminal, which have various bioactivities. Although they have been found abundantly in invertebrates [24], [27], [29], only a few RFamide peptides have been identified in mammals.

Neuropeptide FF (NPFF) and neuropeptide AF (NPAF), which are derived from the same gene, modulate the action of morphine [20], [23], [31], cardiovascular regulation [25], and the increase in somatostatin secretion from the pancreas [4]. The other RFamide peptide, prolactin releasing peptide (PrRP), has various functions in the hypothalamic endocrine system [10], [16], [17], [18], [28].

We searched for novel RFamides on the basis of information in the human genome database. We identified a novel gene that encodes at least three different neuropeptides with an Arg–Phe-NH₂ structure at their C-terminal, termed RFamide related peptides (RFRP-1, -2, -3), whereas the genes of rodents would generate only RFRP-1 and RFRP-3 [7]. In cells transfected with orphan receptor OT7T022, synthetic RFRP-1 and RFRP-3 bound the receptor and decreased the intracellular cAMP level via Gi/Go [5], [7]. RFRP mRNA and RFRP-1 immunoreactivity (ir) were detected within the neuronal cell bodies in the caudal portion of the hypothalamus: the PerVN and the portion around or above VMN. On the other hand, its receptor, rOT7T022, mRNA was expressed in the lateral portion of the PVN and the PerVN in the hypothalamus [7]. Some neurons with rOT7T022 in the PerVN simultaneously expressed tyrosine hydroxylase, a key enzyme of catecholaminergic neurons [2]. Intracerebroventricular (i.c.v.) administration of RFRP-1 induced an increase in plasma prolactin in a dose-dependent manner [7]. On the other hand, an enzyme immunoassay revealed that significant quantities of RFRP-1 and RFRP-3 were detected in various areas of the brain stem in addition to the hypothalamus [5], [32]. This finding suggested that RFRPs are involved not only in the secretion of prolactin but also in the function of other neuroendocrine or neurotransmission. A recent study [15] revealed that i.c.v. administration of RFRP-1 as well as NPFF impaired the action of morphine. These results suggested that RFRP and NPFF might modulate the action of morphine in the brain.

Previous studies have argued that NPFF immunoreactive cell bodies were located between the DMN and the VMN of the hypothalamus [13], [21], [30]. This area is very close to the portion in which RFRP mRNA-positive signals and RFRP-1-ir were detected [7]. In addition, NPFF contains the same chemical structure as the C-terminal of RFRP-3: Pro–Glu–Arg–Phe-NH₂. In this study, we examined the distribution of RFRP-1 and RFRP-3 immunoreactive neurons and fibers in the rat central nervous system by immunohistochemistry using specific antibodies. We also examined whether RFRP-ir was colocalized with NPFF-ir in the same neuronal cell bodies. Moreover, to investigate the possible action of RFRP-1 or RFRP-3, we detected the c-Fos expression after i.c.v. administration of RFRP-1 or RFRP-3 in the rat central nervous system.

Section snippets

Animals

Male Wistar rats (8–9 weeks old) were purchased from Charles River Japan (Yokohama, Japan). They were provided with food and water ad libitum, and kept under conditions of controlled lighting (07:00–19:00 h lights on) and temperature (22 °C) until use for experiments. All experiments in this study were carried out following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The Committee of Animal Research in Kyoto Prefectural University of Medicine also

RFRP immunoreactive cell bodies in the rat brain

To determine whether RFRP-1-ir and RFRP-3-ir coexisted in the same perikarya, we examined double-labeling immunohistochemistry. The results showed that RFRP-1 immunoreactive neurons were completely coincident with RFRP-3 immunoreactive neurons within the caudal PerVN and the portion around and above the VMN (Fig. 1a–c). In addition, under these fluorescent immunohistochemical conditions, RFRP-1-ir in the cell bodies was relatively weak but that in the neuronal fibers around the cell bodies was

Discussion

In the present study we elucidated the distribution of RFRP-1 and RFRP-3 immunoreactive neuronal perikarya and nerve fibers in the rat central nervous system (CNS). They were broadly distributed in the CNS, particularly in the hypothalamus and the lower brain stem. In general, RFRP-1-ir and RFRP-3-ir were detected in the same neuronal perikarya and nerve fibers. However, we also found subtle differences in the distribution between RFRP-1 and RFRP-3 immunoreactive nerve fibers. For instance,

Acknowledgements

Grant sponsor: the Ministry of Education, Science, Sports and Culture, Japan.

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¹: Takahiko Yano and Norio Iijima contributed equally to this work.

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Research reportLocalization and neuronal response of RFamide related peptides in the rat central nervous system

Abstract

Introduction

Section snippets

Animals

RFRP immunoreactive cell bodies in the rat brain

Discussion

Acknowledgements

Neuroscience

Neuropeptides

Biochim. Biophys. Acta

J. Chem. Neuroanat.

Brain Res.

J. Biol. Chem.

Neurosci. Lett.

Neurosci. Lett.

Brain Res.

Prog. Neurobiol.

FEBS Lett.

Neuropeptides

Brain Res.

Biochem. Biophys. Acta

Distribution of tyrosine-hydroxylase-immunoreactive neurons in the hypothalamus of rats

J. Comp. Neurol.

Stress selectively increases fos protein in dopamine neurons innervating the prefrontal cortex

Cereb. Cortex

Research report
Localization and neuronal response of RFamide related peptides in the rat central nervous system