Postnatal development of orexin/hypocretin in rats

doi:10.1016/S0169-328X(00)00080-2

Molecular Brain Research

Volume 78, Issues 1–2, 31 May 2000, Pages 108-119

https://doi.org/10.1016/S0169-328X(00)00080-2 Get rights and content

Abstract

We examined developmental changes of orexins/hypocretins and their receptors (OX1R and OX2R) in the rat hypothalamus from postnatal day 0 to 10 weeks, using in situ hybridization histochemistry for the prepro-orexin, OX1R and OX2R mRNAs and immunohistochemistry for orexin-A and orexin-B. The prepro-orexin mRNA was weakly detected in the lateral hypothalamic area (LHA) from days 0 to 15. Orexin-A- and -B-like immunopositive cells and fibers were not detected from days 0 to 10, but they were observed after day 15. The prepro-orexin mRNA in the LHA markedly increased between days 15 and 20. The OX1R mRNA was detected in the ventromedial hypothalamic area (VMH) at day 0. The OX2R mRNA was not detected in the paraventricular nucleus (PVN) at days 0 and 1, but weakly observed on day 5. The OX1R mRNA in the VMH and OX2R mRNA in the PVN gradually increased throughout the postnatal period. Next, we examined the effects of milk deprivation and intraperitoneal (i.p.) administration of leptin on the hypothalamic prepro-orexin mRNA in pups. Although 24-h milk deprivation did not affect the level of the prepro-orexin mRNA at days 5 and 10, i.p. administration of leptin from days 0 to 3 caused a significant increase in the prepro-orexin mRNA on days 5 and 10. These results suggest that the development of orexins may be associated with developmental changes such as increase of leptin, weaning, feeding and sleep/wakefulness states.

Introduction

Orexigenic peptides in the hypothalamus such as neuropeptide Y (NPY) and galanin are involved in regulating not only food intake in adult animals but also milk and water intake in preweanling animals [15], [19]. Food, water and maternal deprivation causes a significant increase of the preproNPY mRNA in the arcuate nucleus of neonatal rats [15]. Neonatal overfeeding and hyperinsulinism causes an increased number of galanin-positive neurons in the paraventricular nucleus (PVN) [19].

Recently discovered orexigenic peptides, orexin-A and -B are identical to hypocretin-1 and -2 [5], [23]. De Lecea et al., using Northern blot analysis, demonstrated that the hypocretin mRNA was not detected in the hypothalamus at embryonic ages and postnatal days 1 and 5 [5]. They also showed that hypocretin mRNA was increased dramatically after the third postnatal week [5]. However, little is known, about developmental changes of orexins/hypocretins in the hypothalamus during the neonatal period.

Orexins/hypocretins producing neurons are exclusively distributed in the lateral hypothalamic area (LHA), the posterior hypothalamic area and the perifornical nucleus in rats [5], [23]. Orexins have been discovered by ligands of the orphan receptors (orexin type 1 receptor, OX1R; and orexin type 2 receptor, OX2R) [23]. OX1R and OX2R are widely distributed in the rat brain, and there are some different distributions in the regional sites of the rat brain [24]. In particular, in the hypothalamus, OX1R mRNA is most abundant in the ventromedial hypothalamic area (VMH) and OX2R mRNA is most plentiful in the PVN [24].

In the present study, in order to investigate the development of orexins/hypocretins in the postnatal rat hypothalamus, we examined the expression of the prepro-orexin gene in the rat hypothalamus and the expression of the OX1R gene in the VMH and the OX2R gene in the PVN during postnatal day 0 to 10 weeks, using in situ hybridization histochemistry. We also examined the developmental changes of immunoreactivity for orexin-A and orexin-B in the postnatal rat hypothalamus.

Leptin, which is the product of the ob gene, may be involved in the postnatal development of the neuroendocrine axis [1], [6]. In mice, postnatal leptin surge in plasma was observed on day 10 and decreased to adult level after weaning [1]. Leptin has a major effect on the neuroendocrine response to food deprivation in adult mice [2]. Thus, we examined the effects of milk deprivation and systemic administration of leptin during the neonatal period on the expression of the prepro-orexin gene in the hypothalamus, using in situ hybridization histochemistry.

Section snippets

Animals

Sprague–Dawley rats were used in all experiments. Timed pregnant Sprague–Dawley rats were obtained from Seac Yoshitomi (Fukuoka, Japan). They were housed in plastic cages in an air-conditioned room (23–25°C) under a 12-h light (07:00–19:00 h)/12-h dark (19:00–07:00 h) cycle with free access to food and water. All procedures were in accordance with the Japanese Physiological Society’s guidelines for animal care.

Development

The rats on postnatal days 0, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 10 weeks were used

Postnatal developmental change of the prepro-orexin gene expression

The prepro-orexin gene expression was observed in the LHA, the posterior hypothalamic area and the perifornical nucleus in rats from postnatal day 0 to 10 weeks (Fig. 1). There were no significant signals in any other hypothalamic region in rats from postnatal day 0 to 10 weeks. The expression of the prepro-orexin gene was weakly detected at postnatal day 0 and increased gradually from postnatal days 0 to 15 (neonatal and infantile periods) (Fig. 1, Fig. 2). Between postnatal days 15 and 20

Discussion

In the present study, in situ hybridization histochemistry revealed that the prepro-orexin mRNA exhibited in the rat hypothalamus from postnatal days 0 to 15 and markedly increased between days 15 and 20.

Northern blot analysis showed that hypocretin mRNA was not detected in the rat hypothalamus at embryonic ages and postnatal days 1 and 5 but increased dramatically after the third postnatal week [5]. As in situ hybridization histochemistry may be more sensitive to detect mRNA than Northern blot

Acknowledgements

We thank Drs. M. Nakazato and S. Matsukura (Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki, Japan) for helpful discussions. This study was supported by Grant-in-Aid for Scientific Research no. 10470019, 10218210 for H.Y., no. 11670078 for Y.U. from the Ministry of Education, Science, Sports and Culture, Japan, the research grant from the Ministry of Health and Welfare and the Salt Science Research Foundation.

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¹: Present address: Clinical Laboratory, Kyunin Co., Kitakyushu 806-0046, Japan.

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Research reportPostnatal development of orexin/hypocretin in rats

Abstract

Introduction

Section snippets

Animals

Development

Postnatal developmental change of the prepro-orexin gene expression

Discussion

Acknowledgements

J. Biol. Chem.

Cell

Brain Res.

J. Nutr.

Cell

FEBS Lett.

Brain Res. Bull.

Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding: implication for energy homeostasis and neuroendocrine function

J. Clin. Invest.

Role of leptin in the neuroendocrine response to fasting

Nature

Regulation of neuronal and glial proteins by leptin: implication for brain development

Endocrinology

Narcolepsy in orexin knockout mice: molecular genetics sleep regulation

Cell

The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity

Proc. Natl. Acad. Sci. USA

Developmental changes in obese gene expression and circulating leptin peptide concentrations

Biochem. Biophys. Res. Commun.

Research report
Postnatal development of orexin/hypocretin in rats