Chemical identity and connections of medial preoptic area neurons expressing melanin-concentrating hormone during lactation
Introduction
Lactation is an energy-demanding condition characterized by increased circulating levels of oxytocin and prolactin, cessation of the reproductive cycle, induction of maternal behavior and massive food and water consumption (Smith and Grove, 2002). The increase in food intake is an adaptation to the high-energy requirement of milk production and drain (Wade et al., 1996, Barber et al., 1997). In non-lactating animals, the regulation of food intake is orchestrated by a complex net of hypothalamic and extra-hypothalamic connections that have been extensively reported in the literature (Sawchenko, 1998, Elmquist et al., 1999, Elmquist et al., 2005, Niswender et al., 2004, Woods and D’Alessio, 2008).
In lactating females, the pattern of expression of neuropeptides in the arcuate nucleus mirrors that of animals in negative energy balance. Thus, during lactation, the levels of neuropeptide Y (NPY) and agouti-related peptide (AgRP) are increased whereas the levels of proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) are decreased (Smith, 1993, Chen et al., 1999, Smith and Grove, 2002, Xiao et al., 2005). NPY/AgRP and POMC/CART neurons are thought to be the primary sensors for the availability of metabolic cues, including leptin, insulin and oxidable fuels such as glucose and fatty acid (Sawchenko, 1998, Elmquist et al., 1999, Elmquist et al., 2005, Niswender et al., 2004, Woods and D’Alessio, 2008). From the arcuate nucleus, these signals are conveyed to other brain nuclei where neuroendocrine, autonomic and behavioral responses are triggered. For example, NPY/AgRP and POMC/CART neurons in the arcuate nucleus send projections to the paraventricular nucleus of the hypothalamus, to the dorsomedial nucleus of the hypothalamus and to the lateral hypothalamic area (Sawchenko, 1998, Elmquist et al., 1999, Elmquist et al., 2005, Niswender et al., 2004, Woods and D’Alessio, 2008). In the lateral hypothalamic area, NPY/AgRP and POMC/CART terminals are in close apposition to neurons expressing orexin or melanin-concentrating hormone (MCH) (Broberger et al., 1998, Elias et al., 1998, Flier and Maratos-Flier, 1998, Sawchenko, 1998, Elmquist et al., 1999, Elmquist et al., 2005).
The MCH is a neuropeptide that requires post-translational processing of the prohormone preproMCH (ppMCH), which also encodes other putatively neuroactive peptides such as neuropeptide EI (NEI), the neuropeptide glycine–glutamic acid (NGE), the MCH gene overprinted polypeptide (MGOP) and the antisense RNA overlapping MCH gene (AROM) (Nahon et al., 1989, Toumaniantz et al., 1996, Borsu et al., 2000, Bittencourt and Celis, 2008). In male rodents, MCH is expressed in neurons of the LHA and of the incerto-hypothalamic area, and in few cells within the olfactory tubercle and pontine reticular formation (Bittencourt et al., 1992, Sita et al., 2007).
The MCH system has been largely implicated in the control of food intake and energy homeostasis (Qu et al., 1996, Rossi et al., 1997, Flier and Maratos-Flier, 1998). Intracerebroventricular administration of MCH increases feeding (Qu et al., 1996, Rossi et al., 1997, Gomori et al., 2003, Santollo and Eckel, 2008). Mice deficient in MCH are hypophagic and lean and show reduced adipose tissue mass and increased energy expenditure (Shimada et al., 1998).
Previous studies have shown that MCH gene expression and immunoreactivity is induced in neurons of the medial preoptic area (MPO) and anterior aspects of the paraventricular nucleus of the hypothalamus (PVHa) in lactating female rats (Knollema et al., 1992). These findings have been largely neglected, and, therefore, the role played by MCH neurons in the MPO remains unclear. Thus, in the present study, we attempted to determine the chemical identity and possible connections of MPO neurons expressing MCH during lactation.
Section snippets
Animals and treatments
Adult female (70–80 days old) Sprague–Dawley rats were housed two per cage in a light- and temperature-controlled environment (12-h light/12-h dark cycle, 21 ± 2 °C) with food and water available ad libitum. Experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996) and the guidelines established by the University of São Paulo Committee for Ethics and Animal Care in Experimental Research.
Animals were divided into the
Distribution of ppMCH mRNA in the forebrain of lactating females
We initially compared our findings with previous reports. In agreement with Knollema et al. (1992), we found ppMCH mRNA in the MPO and in the PVHa of lactating females (Fig. 1, Fig. 2, Fig. 3). As noted previously (Knollema et al., 1992), we also observed that in the MPO and PVHa, ppMCH mRNA expression increases with the progression of lactation (Fig. 1, Fig. 2, Fig. 3). Thus, in these areas we found low levels of ppMCH mRNA on the 5th day of lactation, moderate expression of ppMCH mRNA on the
Discussion
In the present study we show that MCH neurons in the MPO of lactating rats coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MPO neurons of female rats are densely innervated by terminals that are immunoreactive for neuropeptides related to metabolic control, including AgRP, αMSH and CART. As GABA is an inhibitory neurotransmitter, we predict that these MCH/GABA neurons inhibit their downstream neuronal targets. We further identified these targets and found that MPO neurons
Acknowledgments
We are grateful to Dr. Paul Sawchenko, Wylie Vale and Joan Vaughan for providing the anti-MCH and anti-NEI antisera, as well as the ppMCH cDNA. We would also like to thank Dr. Joel Elmquist and N. Tillakaratne for the gift of the GAD-67 cDNA. In addition, we thank Joelcimar Martins da Silva for her expert technical assistance. Grants from FAPESP (02/11240-8, 03/01096, 04/13849-5, 05/50951-5) supported this work. JCB is a CNPq investigator. CFE is the Distinguished Scholar in Medical Research at
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