- Email: [email protected]
Melanin concentrating hormone (MCH): A novel neural pathway for regulation of GnRH neurons
Brain Research 1041 (2005) 117 – 124 www.elsevier.com/locate/brainres
Research report
Melanin concentrating hormone (MCH): A novel neural pathway for regulation of GnRH neurons Patricia S. Williamson-Hughesa, Kevin L. Grovea, M. Susan Smitha,b,T a
Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA b Department of Physiology and Pharmacology, Oregon Health and Science University, Beaverton, OR 97006, USA Accepted 6 November 2004 Available online 23 March 2005
Abstract The link between the state of energy balance and reproductive function is well known. Thus, signals denoting negative energy balance and the accompanying hyperphagic drive are likely to be factors in the suppression of gonadotropin releasing hormone (GnRH) activity. We have previously found that appetite-regulating systems, such as neuropeptide Y (NPY) in the arcuate nucleus (ARH) and orexin in the lateral hypothalamic area (LHA), send fiber projections that come in close apposition with GnRH neurons. Furthermore, the appropriate receptors, NPY Y5 and OR-1, respectively, are coexpressed on GnRH neurons, providing neuroanatomical evidence for a direct link between the NPY and orexin systems and GnRH neurons. Therefore, these orexigenic neuropeptide systems are potential candidates that convey information about energy balance to GnRH neurons. The current studies focused on melanin concentrating hormone (MCH), another orexigenic neuropeptide system located in the LHA that is sensitive to energy balance. The results showed that MCH fiber projections came in close apposition with approximately 85–90% of GnRH cell bodies throughout the preoptic area and anterior hypothalamic area in the rat. In addition, the MCH receptor (MCHR1) was coexpressed on about 50–55% of GnRH neurons. These findings present evidence for a possible direct neuroanatomical pathway by which MCH may play a role in the regulation of GnRH neuronal function. Thus, MCH is another potential signal that may serve to integrate energy balance and reproductive function. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autocrine regulation Topic: Hypothalamic–pituitary–gonadal regulation Keywords: GnRH; MCH; MCHR1; Immunofluorescence; Confocal microscopy; Neuroanatomy
1. Introduction It is well established that food intake and energy balance can have profound effects on reproductive function, as conditions involving an energy deficit, such as fasting or exercise-induced amenorrhea, are marked by suppressed fertility [25,37]. The notion of regulation of reproductive function by energy homeostasis through integration of appetite-mediating neuronal systems of the hypothalamus
T Corresponding author. Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA. Fax: +1 503 690 5569. E-mail address: [email protected] (M.S. Smith). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.066
with GnRH neuronal activity has a firm foundation [26,36]. Many neuropeptide systems involved in appetite regulation, such as NPY, agouti-related protein (AGRP), pro-opiomelanocortin (POMC), orexin, and MCH, have also been shown to participate in the regulation of reproductive function (see reviews, Refs. [18,33]). These same systems also express leptin receptors [10,13]. Leptin, a peripheral signal of adiposity, is an important satiety factor [20] and a modulator of reproductive function [37]. Therefore, leptin can serve as a peripheral signal that transmits information about the status of energy balance to these neuropeptide systems that have been linked to the regulation of reproductive function. Our laboratory and others have shown that hypothalamic orexigenic neuropeptide systems send direct projections to
118
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
GnRH neurons [33]. Fiber projections from ARH NPY neurons come in close apposition with GnRH neurons [21,41]. Furthermore, GnRH neurons coexpress the NPY Y5 receptor subtype [4], whereas the NPY Y1 receptor subtype is expressed on presynaptic terminals that come in close contact with GnRH neurons [21]. Studies from our group have also shown that orexin neurons in the lateral hypothalamic area (LHA) send direct projections to GnRH neurons, which also coexpress the OX-1 receptor [5]. Similar connections between orexin fibers and GnRH neurons have also been reported in the sheep [17]. These studies provide neuroanatomical evidence for direct links between appetite-regulating neuropeptide systems, such as NPY and orexin, and GnRH neurons, and establish a framework by which information about energy balance can be directly transmitted to GnRH neurons. The present studies focus on MCH, a potent orexigenic agent produced by neurons located in the LHA and zona incerta, as another potential integrating signal between energy status and reproductive function [38]. Previous studies have shown that MCH levels in the LHA are increased in response to fasting and negative energy balance [19,28]. There are also several studies showing that MCH can affect LH secretion. MCH administration into the third ventricle inhibits LH secretion in the presence of low levels of estrogen [40], the hormone milieu that would be present during states of negative energy balance and suppression of cyclic reproductive function [33]. In contrast, others have shown that MCH stimulates LH release under conditions of high levels of estrogen that can induce an LH surge [9,15,23,24]. Both MCH fibers and the MCHR1 receptor subtype are located in areas of the brain that contain GnRH neurons [2,30], although whether MCH has direct effects on GnRH neuronal activity has not been established. The goal of this study was to investigate whether MCH fibers send direct projections to GnRH neurons and whether GnRH neurons express MCHR1 in the rat.
2. Materials and methods 2.1. Experimental animals Virgin cycling female rats (Sprague–Dawley, Simenson, Gilroy, CA) were maintained under a 12:12 light–dark cycle (lights on at 0700 h) and constant temperature (23 F 2 8C). Food and water were provided ad libitum. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee. The stage of the estrous cycle was determined by vaginal smear, and animals were sacrificed during diestrus under pentobarbital anesthesia by intracardiac infusion with ice-cold saline followed by ice-cold 4% paraformaldehyde (pH 7.4). Following a 20-min perfusion, brains were removed and placed in 4%
paraformaldehyde for 24 h, then in 25% sucrose for 3 days. The brains were frozen on crushed dry ice and stored at 80 8C until sectioned on a sliding microtome at a thickness of 25 Am into a 1:6 series of tissue sections. Sections were stored in cryoprotectant until immunohistochemistry (IHC) or in situ hybridization (ISH) was performed. 2.2. Experiment 1: Double and triple label IHC for MCH, GnRH, and synaptophysin IHC was performed as previously reported [4,5] on a 1:3 series of floating sections. Double or triple label IHC for MCH, GnRH, or synaptophysin was performed using a cocktail of primary antibodies. The rabbit polyclonal MCH antibody (Phoenix Pharmaceuticals, Belmont, CA) was used at a concentration of 1:1000, which produced a pattern of MCH cell labeling in the LHA and zona incerta that was identical to that reported previously [2,11,39]. The mouse monoclonal GnRH antibody (a kind gift of Dr. Henryk Urbanski, Oregon National Primate Research Center, Beaverton, OR) was used at a concentration of 1:2000 [5]. The goat polyclonal synaptophysin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1:1000. For each combination of double or triple label IHC, all primary antibodies were titrated to establish the maximum dilutions that provided robust signals and minimal nonspecific staining. The experimental design and IHC protocols for these studies were similar to those used to establish orexin fiber contacts on GnRH neurons [5]. First, double-label IHC was used to determine if MCH fibers come in close apposition with GnRH neurons (n = 3). The tissue was incubated in a cocktail of MCH and GnRH primary antibodies for 48 h at 4 8C. Following washes in 0.05 M potassium phosphate-buffered saline (KPBS), the tissue was incubated for 1 h in a cocktail of fluorescent secondary antibodies: MCH was visualized with tetramethyl rhodamine isothiocyanate (TRITC) conjugated to a donkey anti-rabbit antibody and GnRH was visualized using fluorescein isothiocyanate (FITC) conjugated to a donkey anti-mouse antibody. All secondary antibodies were used at 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA). In the second IHC study, synaptophysin was used to confirm the presence of synaptic contacts. Triple-label IHC for MCH, GnRH, and synaptophysin was performed in a similar manner as described above. MCH was visualized with TRITC conjugated to a donkey anti-rabbit antibody, GnRH was visualized using FITC conjugated to a donkey anti-mouse antibody, and synaptophysin was visualized with Cy5 conjugated to a donkey anti-goat antibody. All secondary antibodies were used at 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections from all experiments were mounted onto subbed slides, cover slipped with glycerol, and sealed.
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
2.3. Experiment 2: ISH for MCHR1 and IHC for GnRH The brains (n = 4) were sectioned at 25 Am using a sliding microtome under RNAse-free conditions and stored in RNAse-free cryoprotectant. ISH was performed on a 1:3 series of floating sections. The cRNA probe [19] to detect MCHR1 mRNA (a kind gift of Eleftheria Maratos-Flier, Joslin Diabetes Center, Boston, MA) was transcribed from a 800-bp cDNA with 100% of the UTP labeled with 33P. The ISH protocol has been described previously [8,19]. Incubation with the antisense RNA probe (5 107 cpm/ml) was for 17 h at 55 8C, followed by a series of washes in SSC at increased stringency. The sections were then subjected to the standard IHC protocol, described above for GnRH, except that GnRH was visualized with 3,3V-diaminobenzidine (DAB). Sections were first exposed to radiographic film (3 days, 4 8C) to confirm the MCHR1 signal, and then the slides were dipped in NTB-2 radiographic emulsion (Eastman Kodak) and stored at 4 8C for 15 days. Control experiments were performed using a labeled sense probe in the ISH protocol, which resulted in a complete loss of specific labeling. Standard bright field and dark field microscopies were simultaneously applied to visualize the location of GnRH neurons and silver grains for MCHR1 mRNA. GnRH cell bodies were examined throughout the preoptic area and anterior hypothalamus. Silver grain expression over individual GnRH cells was qualitatively assessed, and only cells with clear silver grain clusters over the soma were counted as positively labeled with MCHR1 mRNA. This conservative approach may have underestimated the number of MCHR1-positive GnRH neurons. Because of the qualitative nature of this method and the small number of animals used, the data are expressed as percent MCHR1positive GnRH neurons of the total number of GnRH neurons examined. 2.4. Confocal microscopic analysis The detection of MCH close appositions with GnRH neurons and colocalization with synaptophysin were conducted as previously described using confocal microscopy [4,5,21]. The TSC SP confocal system (Leica Corp., Germany) consisted of a RBE inverted microscope, with an Ar laser for producing light at 488 nm, a Kr laser for producing light at 568 nm, and a HeNe laser for producing light at 647 nm. Various objectives were used to scan and capture images (25, numerical aperture 0.75; 40, numerical aperture 1.25) for analysis throughout the entire rostral to caudal extent of GnRH neurons. Optical sectioning was used to assess colocalization of two signals in series captured at 0.25- to 0.5-Am intervals along the z-axis of the tissue section. Optical sections were taken in 1024 1024 pixel images and analyzed using the MetaMorph imaging system (Universal Imaging, West Chester, PA). GnRH neurons were analyzed for apposition of MCH
119
fibers only if the entire cell body was visible in the section. Because of the qualitative nature of this methodology and the small number of animals used, the data are presented as percent GnRH neurons contacted of the total number of GnRH neurons examined. Confocal images are presented as projections of stacks of optical sections or as individual optical sections as indicated. The brightness and contrast of the images were adjusted in Photoshop to match the microscope visualization (Adobe Systems, San Jose, CA).
3. Results An abundance of MCH fibers was observed throughout the preoptic area and anterior hypothalamus, as previously reported [2,11,34]. Double- and triple-label IHF showed that MCH fibers made close appositions on GnRH neurons (Figs. 1A and B). Sections from 3 brains were analyzed by confocal microscopy (584 total GnRH neurons) and the results showed that approximately 85– 90% of GnRH neurons were contacted by MCH fibers (Figs. 1A and B), and the variability among the 3 animals was not large. To provide additional confirmation of the close apposition of MCH fibers with GnRH neurons, a smaller number of representative sections were stained for synaptophysin (a protein localized in synaptic vesicles), as a marker of synaptic contacts. MCH fiber buttons colocalized with synaptophysin and were associated with GnRH neurons (Fig. 1C). Shown as a single optical slice (Figs. 1C1 to C4, 0.5 Am thick), the MCH terminal can be seen coming in close apposition with the GnRH neuron. MCH innervation was also investigated in the region of GnRH nerve terminals in the median eminence. The results showed that there was considerable overlap between the two types of fibers in the external zone of the median eminence, as shown by the yellow color representing close associations between MCH and GnRH fibers (Figs. 1D–E). MCHR1 mRNA expression was observed throughout the brain, including the cortex, nucleus accumbens, bed nucleus of the stria terminalis, anterior hypothalamic area, and preoptic area, as previously reported (Figs. 2 and 3A; [16,30]). Additional double label IHC and ISH showed that MCHR1 was colocalized with GnRH neurons (Figs. 3B and C). GnRH neurons labeled by IHC-DAB staining were easily identified (Fig. 3B, preoptic area). Analysis of sections from 4 rat brains (378 total cells examined) revealed that about 50–55% of GnRH neurons expressed the MCHR1 (Fig. 3C). The variability among the animals was not large. The percentage of cells expressing the MCHR increased from rostral to caudal, as only about 30% of GnRH neurons in the area of the Diagonal Band of Broca expressed MCHR1, while about 70% of the GnRH neurons in the more caudal sections of the anterior hypothalamic area expressed MCHR1. These results were
120
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
in relative agreement with the amount of MCH fibers, which were less abundant in the area of the Diagonal Band of Broca and increasingly abundant in close proximity to the LHA.
4. Discussion This report is the first to provide evidence for a possible direct functional neuroanatomical pathway between the
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
121
Fig. 2. MCHR1 mRNA expression throughout the brain. Dark field photomicrographs of silver grains representing MCHR1 mRNA in the cortex (CTX), anterior hypothalamus (AHA), nucleus accumbens (AcB), and bed nucleus of the stria terminalis (BST). Scale bar: 500 Am.
MCH system and GnRH neurons in the rat. The experiments showed that the connections between the MCH system and GnRH neurons are very robust, as about 85–90% of GnRH neurons were contacted by MCH fibers and about 50–55% of GnRH neurons had detectable levels of MCHR1 mRNA (Figs. 1–3). Due to the conservative approach for counting positive cells, it is likely that this is an underestimate of the number of GnRH cells that coexpress the MCHR1. The results of the present study confirm previous findings demonstrating MCH fibers and MCHR1 mRNA in the areas where GnRH neurons are located [2,30]. In addition, MCH fibers and MCHR1 receptor mRNA were abundant in other preoptic areas known to project to and influence GnRH neuronal activity. Thus, MCH may have both direct and indirect actions on GnRH neurons. This may also reflect
the dual inhibitory/stimulatory actions of MCH on GnRH/ LH release. Taken together, these results suggest that the MCH system is in a key position to serve as an integrating signal between energy balance and reproductive function. It is well established that MCH is a regulator of food intake; MCH expression is increased in response to fasting and injections of MCH into the hypothalamus increase food intake [38]. Furthermore, disruption of the MCH gene results in a hypophagic animal [32]. MCH has complex interrelationships with a number of neuronal systems that are involved in the regulation of food intake and energy balance, such as orexin and NPY [3]. For example, orexin and MCH neurons have many connections within the LHA [3]. MCH neurons in the LHA receive direct synaptic contacts by NPY neurons projecting from the ARH and in
Fig. 1. MCH fibers come in close apposition with GnRH neurons. (A) Confocal micrograph representing a 20-Am section showing GnRH cell bodies (green) in the preoptic area surrounded by MCH fibers (red). White arrowheads indicate close appositions (represented by the yellow color) of MCH fibers on GnRH neurons. (B) Higher magnification of confocal micrograph (20-Am-thick stack of optical sections). White arrowheads indicate close appositions (represented by the yellow color) of MCH fibers (red) on GnRH neurons (green). (C) Confocal micrograph of triple label immunofluorescence for MCH (red), GnRH (green), and synaptophysin (blue). The micrograph represents a 10-Am-thick stack of optical sections (40 optical sections at 0.25-Am thickness). White arrowheads indicate close appositions between MCH fibers and GnRH neurons. (C1 to C4) Single optical sections (0.5 Am thick) at intervals indicated in the upper right corner and showing the MCH terminal coming in close apposition with the GnRH neuron. Colocalization between MCH and synaptophysin is shown as white. (D) Low magnification confocal micrograph showing the overlap of MCH (red) and GnRH (green) fibers in the median eminence. Close appositions (within 0.5 Am) between MCH and GnRH fibers are represented by the yellow color. (E) Higher magnification confocal micrograph (5 Am thick) of MCH and GnRH fibers in the median eminence. Scale bars: A, 100 Am; B, 25 Am; C, 10 Am; D, 100 Am; E, 25 Am.
122
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
Fig. 3. MCHR1 mRNA expression in GnRH neurons. (A) Low power photomicrograph showing silver grain clusters in the lateral preoptic area (LPOA). (B–C) Double label ISH and IHC for GnRH and MCHR1 mRNA. (B) Bright field image of GnRH neurons stained with DAB. (C) Dark field image showing MCHR1 silver grains. Arrowheads indicate examples of GnRH neurons that express MCHR1 mRNA. Scale bars: A, 100 Am; B–C, 20 Am.
turn send reciprocal projections to ARH NPY neurons [3,11]. Antagonism of the NPY Y-1 receptor diminishes the MCH-induced feeding response [6]. Therefore, there appears to be complex relationships among the MCH, orexin, and NPY orexigenic systems that would serve to integrate the feeding response to negative energy balance. Since these same orexigenic systems have also been shown to make direct contacts with GnRH neurons (Figs. 1 and 2; [5,21]), their interrelationships would also provide coordinated information about the status of energy balance to the reproductive system. There is considerable evidence supporting the notion that MCH might modulate reproductive function. MCH has been shown to stimulate GnRH release from median eminence explants under conditions of high estrogen [9]. The ability of MCH to have direct effects on the median eminence is consistent with the extensive overlap and close associations of MCH and GnRH fibers shown in this study (Fig. 1). The effects of MCH on GnRH/LH secretion appear to be bimodal, that is, stimulatory in the presence of high levels of estrogen [9,15,23,24] and inhibitory in the presence of low levels of estrogen [40], as has been reported for other neuropeptides, such as NPY and orexin. Thus, under conditions of negative energy balance, MCH levels in the
LHA would be increased and levels of estrogen would be low. Therefore, MCH could play a role in the suppression of GnRH/LH secretion [42]. This notion is supported by the observation that animals lacking the MCH gene are still capable of reproducing, even though they are hypophagic and have low levels of leptin, reflecting the state of negative energy balance [32]. The present findings provide a neuroanatomical framework by which MCH may affect GnRH, either by direct effects through MCHR1s on GnRH cell bodies or by direct contacts on GnRH fibers and terminals in the median eminence. Since this study only investigated the expression of MCHR1 mRNA, we can only hypothesize that there might be MCHR1 expression on GnRH terminals in the median eminence. However, the lack of a suitable antibody makes this impossible to confirm at this time. The regularity of close associations between MCH and GnRH fibers in the median eminence does support this hypothesis. MCHR1 has been shown to be coupled to both Gi and Gq subtypes of G proteins, suggesting the capability to be both inhibitory and stimulatory [27]. At this time, it is not known how GnRH neurons might be affected by the direct actions of MCH or the mechanisms by which the levels of estrogen alter these actions. In addition, MCH neurons are known to coexpress the long form of the leptin receptor [Ob-Rb] in rat (see review, Ref. [29]). Leptin is released from adipocytes and denotes adiposity and energy balance to the brain [12,14]. The low levels of leptin during states of negative energy balance [33,37] have been shown to play a role in the suppression of GnRH neuronal activity [1,7,22]. Administration of leptin can restore fasting-induced suppression of LH secretion [25] and can greatly improve reproductive and neuroendocrine function in hypothalamic amenorrhea resulting from strenuous exercise or low weight [42]. Since GnRH neurons do not express Ob-Rb, direct projections by Ob-Rb-expressing MCH neurons to GnRH neurons may provide one potential neuroanatomical circuit by which leptin regulates GnRH activity, as has been suggested for orexin neurons [5]. The effects of leptin on MCH neurons may also be indirect through actions on ARH/NPY neurons [10], which make direct connections with MCH neurons [3,11]. There is considerable evidence that MCH expression is downregulated by leptin, and injections of leptin block MCH-induced feeding in the rat [38]. In contrast, MCH expression is elevated in ob/ob mice, which are obese and lack the leptin protein, and in obese Zucker rats, which lack leptin signaling [31,35]. Thus, MCH neurons appear to be very sensitive to peripheral signals of energy balance, such as leptin. During states of negative energy balance, the low levels of leptin would result in increased MCH expression, which may be an important signal to suppress GnRH neuronal activity. Conversely, administration of leptin would suppress MCH expression and decrease the inhibitory effect of MCH on GnRH neurons, thus restoring LH secretion.
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
In summary, these results provide neuroanatomical evidence that the MCH system may be directly linked to GnRH neurons, suggesting that MCH may play a role in integrating the status of energy balance with regulation of reproductive function. At this time, it is unclear how important this novel neurocircuit is to the regulation of reproductive function during normal conditions as well as during metabolic challenges. Acknowledgments This work was supported by NIH grants HD-14643, RR00163, and HD18185. References [1] C.L. Adam, Z.A. Archer, D.W. Miller, Leptin actions on the reproductive neuroendocrine axis in sheep, Reproduction 61 (2002) 283 – 297. [2] J.C. Bittencourt, C.F. Elias, Melanin-concentrating hormone and neuropeptide EI projections from the lateral hypothalamic area and zona incerta to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers, Brain Res. 805 (1998) 1 – 19. [3] C. Broberger, L. De Lecea, J.G. Sutcliffe, T. Hokfelt, Hypocretin/ orexin- and melanin-concentrating hormone-expressing cells from distinct populations in the rodent lateral hypothalamus: relationship to the Neuropeptide Y and agouti-gene-related protein systems, J. Comp. Neurol. 402 (1998) 460 – 474. [4] R.E. Campbell, J.M.H. ffrench-Mullen, M.A. Cowley, M.S. Smith, K.L. Grove, Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype, Neuroendocrinology 74 (2001) 106 – 119. [5] R.E. Campbell, K.L. Grove, M.S. Smith, Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers, Endocrinology 144 (2003) 1542 – 1548. [6] C.L. Chaffer, M.J. Morris, The feeding response to melaninconcentrating hormone is attenuated by antagonism of the NPYY1receptor in the rat, Endocrinology 143 (2002) 191 – 197. [7] F.F. Chehab, J. Qiu, K. Mounzih, A. Ewart-Toland, S. Ogus, Leptin and reproduction, Nutr. Rev. 60 (2002) S39 – S46. [8] P.L. Chen, M.S. Smith, Melanocortin 4 receptor-mediated hyperphagia and activation of neuropeptide Y (NPY) expression in the dorsomedial hypothalamus during lactation, J. Neurosci. 24 (2004) 5091 – 5100. [9] S.R. Chiocchio, M.G. Gallardo, P. Louzan, V. Gutnisky, J.H. Tramezzani, Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels, Biol. Reprod. 64 (2001) 1466 – 1472. [10] M.A. Cowley, J.L. Smart, M. Rubinstein, M.G. Cerdan, S. Diano, T.L. Horvath, R.D. Cone, M.J. Low, Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus, Nature 411 (2001) 480 – 484. [11] C.F. Elias, C.B. Saper, E. Maratos-Flier, N.A. Tritos, C. Lee, J. Kelly, J.B. Tatro, G.E. Hoffman, M.M. Ollmann, G.S. Barsh, T. Sakurai, M. Yanagisawa, J.K. Elmquist, Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area, J. Comp. Neurol. 402 (1998) 442 – 459. [12] J.S. Flier, Obesity wars: molecular progress confronts an expanding epidemic, Cell 116 (2004) 337 – 350.
123
[13] J.M. Friedman, Leptin, leptin receptors, and the control of body weight, Nutr. Rev. 56 (1998) S38 – S46. [14] J.M. Friedman, The function of leptin in nutrition; weight, and physiology, Nutr. Rev. 60 (2002) S1 – S14. [15] M.I. Gonzalez, B.I. Baker, C.A. Wilson, Stimulatory effect of melanin concentrating hormone on luteinizing hormone release, Neuroendocrinology 66 (1997) 254 – 262. [16] G.J. Hervieu, J.E. Cluderay, D. Harrison, J. Meakin, P. Maycox, S. Nasir, R.A. Leslie, The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat, Eur. J. Neurosci. 12 (2000) 1194 – 1216. [17] J. Iqbal, S. Pompolo, T. Sakurai, I.J. Clarke, Evidence that orexincontaining neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus, J. Neuroendocrinol. 13 (2001) 1033 – 1041. [18] S.P. Kalra, W.R. Crowley, Neuropeptide Y: a novel neuroendocrine peptide in the control of pituitary hormone secretion, and its relation to luteinizing hormone, Front. Neuroendocrinol. 13 (1992) 1 – 46 (Review). [19] E.G. Kokkotou, N.A. Tritos, J.W. Mastaitis, L. Slieker, E. MaratosFlier, Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain, Endocrinology 142 (2001) 680 – 686. [20] S.J. Konturek, J.W. Konturek, T. Pawlik, T. Brzozowski, Brain–gut axis and its role in the control of food intake, J. Physiol. Pharmacol. 55 (2004) 137 – 154. [21] C. Li, P. Chen, M.S. Smith, Morphological evidence for direct interaction between arcuate nucleus neuropeptide Y (NPY) neurons and gonadotropin releasing hormone (GnRH) neurons and the possible involvement of NPY Y1 receptors, Endocrinology 140 (1999) 5382 – 5390. [22] C.S. Mantzoros, Role of leptin in reproduction, Ann. N. Y. Acad. Sci. 900 (2000) 174 – 183. [23] J.F. Murray, R.A.H. Adan, R. Walker, B.I. Baker, A.J. Thody, W.A.J. Nijenhuis, J. Yukitake, C.A. Wilson, Melanin-concentrating hormone, melanocortin receptors and regulation of luteinizing hormone release, J. Neuroendocrinol. 12 (2000) 217 – 223. [24] J.F. Murray, J.G. Mercer, R.A. Adan, J.J. Datta, C. Aldairy, K.M. Moar, B.I. Baker, M.J. Stock, C.A. Wilson, The effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone, J. Neuroendocrinol. 12 (2000) 1133 – 1139. [25] S. Nagatani, P. Guthikonda, R.C. Thompson, H. Tsukamura, K.I. Maeda, D.L. Foster, Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting, Neuroendocrinology 67 (1998) 370 – 376. [26] S. Ohkura, T. Tanaka, S. Nagatani, D.C. Bucholtz, H. Tsukamura, K.I. Maeda, D.L. Foster, Central, but not peripheral, glucosesensing mechanisms mediate glucoprivic suppression of pulsatile luteinizing hormone secretion in the sheep, Endocrinology 141 (2000) 4472 – 4480. [27] P. Pissios, D.J. Trombly, I. Tzameli, E. Maratos-Flier, Melaninconcentrating hormone receptor 1 activates extracellular signalregulated kinase and synergizes with G(s)-coupled pathways, Endocrinology 144 (2003) 3514 – 3523. [28] D. Qu, D.S. Ludwig, S. Gammeltoft, M. Piper, M.A. Pelleymounter, M.J. Cullen, W.F. Mathes, R. Przypek, R. Kanarek, E. Maratos-Flier, A role for melanin-concentrating hormone in the central regulation of feeding behaviour, Nature 380 (1996) 243 – 247. [29] A. Sahu, Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance, Front. Neuroendocrinol. 24 (2003) 225 – 253. [30] Y. Saito, M. Cheng, R.M. Leslie, O. Civelli, Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain, J. Comp. Neurol. 435 (2001) 26 – 40. [31] G. Segal-Liberman, R.L. Bradley, E. Kokkotou, M. Carlson, D.J. Trombly, X. Wang, S. Bates, M.G. Myers Jr., E. Maratos-Flier,
124
[32]
[33]
[34]
[35]
[36]
[37]
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124 Melanin concentrating hormone is a critical mediator of the leptindeficient phenotype, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10085 – 10090. M. Shimada, N.A. Tritos, B.B. Lowell, J.S. Flier, E. Maratos-Flier, Mice lacking melanin-concentrating hormone are hypophagic and lean, Nature 396 (1998) 670 – 674. M.S. Smith, K.L. Grove, Integration of the regulation of reproductive function and energy balance: lactation as a model, Front. Neuroendocrinol. 23 (2002) 225 – 256. T.L. Steininger, T.S. Kilduff, M. Behan, R.M. Benca, C.F. Landry, Comparison of hypocretin/orexin and melanin-concentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain, J. Chem. Neuroanat. 27 (2004) 165 – 181. A. Stricker-Krongrad, T. Dimitrov, B. Beck, Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats, Mol. Brain Res. 92 (2001) 43 – 48. S.D. Sullivan, S.M. Moenter, Gamma-aminobutyric acid neurons integrate and rapidly transmit permissive and inhibitory metabolic cues to gonadotropin-releasing hormone neurons, Endocrinology 145 (2004) 1194 – 1202. F.S.L. Thong, T.E. Graham, Leptin and reproduction: is it a critical
[38]
[39]
[40]
[41]
[42]
link between adipose tissue, nutrition and reproduction? Can. J. Appl. Physiol. 24 (1999) 317 – 336. N.A. Tritos, E. Maratos-Flier, Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone, Neuropeptides 33 (1999) 339 – 349. N.A. Tritos, J.W. Mastaitis, E. Kokkotou, E. Maratos-Flier, Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus, Brain Res. 895 (2001) 160 – 166. H. Tsukamura, R.C. Thompson, S. Tsukahara, S. Ohkura, F. Maekawa, R. Moriyama, Y. Niwa, D.L. Foster, K.I. Maeda, Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat, J. Neuroendocrinol. 12 (2000) 529 – 534. G.F. Turi, Z. Liposits, S.M. Moenter, C. Fekete, E. Hrabovszky, Origin of neuropeptide Y-containing afferents to gonadotropinreleasing hormone neurons in male mice, Endocrinology 144 (2003) 4967 – 4974. C.K. Welt, J.L. Chan, J. Bullen, R. Murphy, P. Smith, A.M. DePaoli, A. Karalis, C.S. Mantzoros, Recombinant human leptin in women with hypothalamic amenorrhea, N. Engl. J. Med. 351 (2004) 987 – 997.
Research report
Melanin concentrating hormone (MCH): A novel neural pathway for regulation of GnRH neurons Patricia S. Williamson-Hughesa, Kevin L. Grovea, M. Susan Smitha,b,T a
Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA b Department of Physiology and Pharmacology, Oregon Health and Science University, Beaverton, OR 97006, USA Accepted 6 November 2004 Available online 23 March 2005
Abstract The link between the state of energy balance and reproductive function is well known. Thus, signals denoting negative energy balance and the accompanying hyperphagic drive are likely to be factors in the suppression of gonadotropin releasing hormone (GnRH) activity. We have previously found that appetite-regulating systems, such as neuropeptide Y (NPY) in the arcuate nucleus (ARH) and orexin in the lateral hypothalamic area (LHA), send fiber projections that come in close apposition with GnRH neurons. Furthermore, the appropriate receptors, NPY Y5 and OR-1, respectively, are coexpressed on GnRH neurons, providing neuroanatomical evidence for a direct link between the NPY and orexin systems and GnRH neurons. Therefore, these orexigenic neuropeptide systems are potential candidates that convey information about energy balance to GnRH neurons. The current studies focused on melanin concentrating hormone (MCH), another orexigenic neuropeptide system located in the LHA that is sensitive to energy balance. The results showed that MCH fiber projections came in close apposition with approximately 85–90% of GnRH cell bodies throughout the preoptic area and anterior hypothalamic area in the rat. In addition, the MCH receptor (MCHR1) was coexpressed on about 50–55% of GnRH neurons. These findings present evidence for a possible direct neuroanatomical pathway by which MCH may play a role in the regulation of GnRH neuronal function. Thus, MCH is another potential signal that may serve to integrate energy balance and reproductive function. D 2005 Elsevier B.V. All rights reserved. Theme: Endocrine and autocrine regulation Topic: Hypothalamic–pituitary–gonadal regulation Keywords: GnRH; MCH; MCHR1; Immunofluorescence; Confocal microscopy; Neuroanatomy
1. Introduction It is well established that food intake and energy balance can have profound effects on reproductive function, as conditions involving an energy deficit, such as fasting or exercise-induced amenorrhea, are marked by suppressed fertility [25,37]. The notion of regulation of reproductive function by energy homeostasis through integration of appetite-mediating neuronal systems of the hypothalamus
T Corresponding author. Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA. Fax: +1 503 690 5569. E-mail address: [email protected] (M.S. Smith). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.066
with GnRH neuronal activity has a firm foundation [26,36]. Many neuropeptide systems involved in appetite regulation, such as NPY, agouti-related protein (AGRP), pro-opiomelanocortin (POMC), orexin, and MCH, have also been shown to participate in the regulation of reproductive function (see reviews, Refs. [18,33]). These same systems also express leptin receptors [10,13]. Leptin, a peripheral signal of adiposity, is an important satiety factor [20] and a modulator of reproductive function [37]. Therefore, leptin can serve as a peripheral signal that transmits information about the status of energy balance to these neuropeptide systems that have been linked to the regulation of reproductive function. Our laboratory and others have shown that hypothalamic orexigenic neuropeptide systems send direct projections to
118
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
GnRH neurons [33]. Fiber projections from ARH NPY neurons come in close apposition with GnRH neurons [21,41]. Furthermore, GnRH neurons coexpress the NPY Y5 receptor subtype [4], whereas the NPY Y1 receptor subtype is expressed on presynaptic terminals that come in close contact with GnRH neurons [21]. Studies from our group have also shown that orexin neurons in the lateral hypothalamic area (LHA) send direct projections to GnRH neurons, which also coexpress the OX-1 receptor [5]. Similar connections between orexin fibers and GnRH neurons have also been reported in the sheep [17]. These studies provide neuroanatomical evidence for direct links between appetite-regulating neuropeptide systems, such as NPY and orexin, and GnRH neurons, and establish a framework by which information about energy balance can be directly transmitted to GnRH neurons. The present studies focus on MCH, a potent orexigenic agent produced by neurons located in the LHA and zona incerta, as another potential integrating signal between energy status and reproductive function [38]. Previous studies have shown that MCH levels in the LHA are increased in response to fasting and negative energy balance [19,28]. There are also several studies showing that MCH can affect LH secretion. MCH administration into the third ventricle inhibits LH secretion in the presence of low levels of estrogen [40], the hormone milieu that would be present during states of negative energy balance and suppression of cyclic reproductive function [33]. In contrast, others have shown that MCH stimulates LH release under conditions of high levels of estrogen that can induce an LH surge [9,15,23,24]. Both MCH fibers and the MCHR1 receptor subtype are located in areas of the brain that contain GnRH neurons [2,30], although whether MCH has direct effects on GnRH neuronal activity has not been established. The goal of this study was to investigate whether MCH fibers send direct projections to GnRH neurons and whether GnRH neurons express MCHR1 in the rat.
2. Materials and methods 2.1. Experimental animals Virgin cycling female rats (Sprague–Dawley, Simenson, Gilroy, CA) were maintained under a 12:12 light–dark cycle (lights on at 0700 h) and constant temperature (23 F 2 8C). Food and water were provided ad libitum. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee. The stage of the estrous cycle was determined by vaginal smear, and animals were sacrificed during diestrus under pentobarbital anesthesia by intracardiac infusion with ice-cold saline followed by ice-cold 4% paraformaldehyde (pH 7.4). Following a 20-min perfusion, brains were removed and placed in 4%
paraformaldehyde for 24 h, then in 25% sucrose for 3 days. The brains were frozen on crushed dry ice and stored at 80 8C until sectioned on a sliding microtome at a thickness of 25 Am into a 1:6 series of tissue sections. Sections were stored in cryoprotectant until immunohistochemistry (IHC) or in situ hybridization (ISH) was performed. 2.2. Experiment 1: Double and triple label IHC for MCH, GnRH, and synaptophysin IHC was performed as previously reported [4,5] on a 1:3 series of floating sections. Double or triple label IHC for MCH, GnRH, or synaptophysin was performed using a cocktail of primary antibodies. The rabbit polyclonal MCH antibody (Phoenix Pharmaceuticals, Belmont, CA) was used at a concentration of 1:1000, which produced a pattern of MCH cell labeling in the LHA and zona incerta that was identical to that reported previously [2,11,39]. The mouse monoclonal GnRH antibody (a kind gift of Dr. Henryk Urbanski, Oregon National Primate Research Center, Beaverton, OR) was used at a concentration of 1:2000 [5]. The goat polyclonal synaptophysin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1:1000. For each combination of double or triple label IHC, all primary antibodies were titrated to establish the maximum dilutions that provided robust signals and minimal nonspecific staining. The experimental design and IHC protocols for these studies were similar to those used to establish orexin fiber contacts on GnRH neurons [5]. First, double-label IHC was used to determine if MCH fibers come in close apposition with GnRH neurons (n = 3). The tissue was incubated in a cocktail of MCH and GnRH primary antibodies for 48 h at 4 8C. Following washes in 0.05 M potassium phosphate-buffered saline (KPBS), the tissue was incubated for 1 h in a cocktail of fluorescent secondary antibodies: MCH was visualized with tetramethyl rhodamine isothiocyanate (TRITC) conjugated to a donkey anti-rabbit antibody and GnRH was visualized using fluorescein isothiocyanate (FITC) conjugated to a donkey anti-mouse antibody. All secondary antibodies were used at 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA). In the second IHC study, synaptophysin was used to confirm the presence of synaptic contacts. Triple-label IHC for MCH, GnRH, and synaptophysin was performed in a similar manner as described above. MCH was visualized with TRITC conjugated to a donkey anti-rabbit antibody, GnRH was visualized using FITC conjugated to a donkey anti-mouse antibody, and synaptophysin was visualized with Cy5 conjugated to a donkey anti-goat antibody. All secondary antibodies were used at 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections from all experiments were mounted onto subbed slides, cover slipped with glycerol, and sealed.
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
2.3. Experiment 2: ISH for MCHR1 and IHC for GnRH The brains (n = 4) were sectioned at 25 Am using a sliding microtome under RNAse-free conditions and stored in RNAse-free cryoprotectant. ISH was performed on a 1:3 series of floating sections. The cRNA probe [19] to detect MCHR1 mRNA (a kind gift of Eleftheria Maratos-Flier, Joslin Diabetes Center, Boston, MA) was transcribed from a 800-bp cDNA with 100% of the UTP labeled with 33P. The ISH protocol has been described previously [8,19]. Incubation with the antisense RNA probe (5 107 cpm/ml) was for 17 h at 55 8C, followed by a series of washes in SSC at increased stringency. The sections were then subjected to the standard IHC protocol, described above for GnRH, except that GnRH was visualized with 3,3V-diaminobenzidine (DAB). Sections were first exposed to radiographic film (3 days, 4 8C) to confirm the MCHR1 signal, and then the slides were dipped in NTB-2 radiographic emulsion (Eastman Kodak) and stored at 4 8C for 15 days. Control experiments were performed using a labeled sense probe in the ISH protocol, which resulted in a complete loss of specific labeling. Standard bright field and dark field microscopies were simultaneously applied to visualize the location of GnRH neurons and silver grains for MCHR1 mRNA. GnRH cell bodies were examined throughout the preoptic area and anterior hypothalamus. Silver grain expression over individual GnRH cells was qualitatively assessed, and only cells with clear silver grain clusters over the soma were counted as positively labeled with MCHR1 mRNA. This conservative approach may have underestimated the number of MCHR1-positive GnRH neurons. Because of the qualitative nature of this method and the small number of animals used, the data are expressed as percent MCHR1positive GnRH neurons of the total number of GnRH neurons examined. 2.4. Confocal microscopic analysis The detection of MCH close appositions with GnRH neurons and colocalization with synaptophysin were conducted as previously described using confocal microscopy [4,5,21]. The TSC SP confocal system (Leica Corp., Germany) consisted of a RBE inverted microscope, with an Ar laser for producing light at 488 nm, a Kr laser for producing light at 568 nm, and a HeNe laser for producing light at 647 nm. Various objectives were used to scan and capture images (25, numerical aperture 0.75; 40, numerical aperture 1.25) for analysis throughout the entire rostral to caudal extent of GnRH neurons. Optical sectioning was used to assess colocalization of two signals in series captured at 0.25- to 0.5-Am intervals along the z-axis of the tissue section. Optical sections were taken in 1024 1024 pixel images and analyzed using the MetaMorph imaging system (Universal Imaging, West Chester, PA). GnRH neurons were analyzed for apposition of MCH
119
fibers only if the entire cell body was visible in the section. Because of the qualitative nature of this methodology and the small number of animals used, the data are presented as percent GnRH neurons contacted of the total number of GnRH neurons examined. Confocal images are presented as projections of stacks of optical sections or as individual optical sections as indicated. The brightness and contrast of the images were adjusted in Photoshop to match the microscope visualization (Adobe Systems, San Jose, CA).
3. Results An abundance of MCH fibers was observed throughout the preoptic area and anterior hypothalamus, as previously reported [2,11,34]. Double- and triple-label IHF showed that MCH fibers made close appositions on GnRH neurons (Figs. 1A and B). Sections from 3 brains were analyzed by confocal microscopy (584 total GnRH neurons) and the results showed that approximately 85– 90% of GnRH neurons were contacted by MCH fibers (Figs. 1A and B), and the variability among the 3 animals was not large. To provide additional confirmation of the close apposition of MCH fibers with GnRH neurons, a smaller number of representative sections were stained for synaptophysin (a protein localized in synaptic vesicles), as a marker of synaptic contacts. MCH fiber buttons colocalized with synaptophysin and were associated with GnRH neurons (Fig. 1C). Shown as a single optical slice (Figs. 1C1 to C4, 0.5 Am thick), the MCH terminal can be seen coming in close apposition with the GnRH neuron. MCH innervation was also investigated in the region of GnRH nerve terminals in the median eminence. The results showed that there was considerable overlap between the two types of fibers in the external zone of the median eminence, as shown by the yellow color representing close associations between MCH and GnRH fibers (Figs. 1D–E). MCHR1 mRNA expression was observed throughout the brain, including the cortex, nucleus accumbens, bed nucleus of the stria terminalis, anterior hypothalamic area, and preoptic area, as previously reported (Figs. 2 and 3A; [16,30]). Additional double label IHC and ISH showed that MCHR1 was colocalized with GnRH neurons (Figs. 3B and C). GnRH neurons labeled by IHC-DAB staining were easily identified (Fig. 3B, preoptic area). Analysis of sections from 4 rat brains (378 total cells examined) revealed that about 50–55% of GnRH neurons expressed the MCHR1 (Fig. 3C). The variability among the animals was not large. The percentage of cells expressing the MCHR increased from rostral to caudal, as only about 30% of GnRH neurons in the area of the Diagonal Band of Broca expressed MCHR1, while about 70% of the GnRH neurons in the more caudal sections of the anterior hypothalamic area expressed MCHR1. These results were
120
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
in relative agreement with the amount of MCH fibers, which were less abundant in the area of the Diagonal Band of Broca and increasingly abundant in close proximity to the LHA.
4. Discussion This report is the first to provide evidence for a possible direct functional neuroanatomical pathway between the
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
121
Fig. 2. MCHR1 mRNA expression throughout the brain. Dark field photomicrographs of silver grains representing MCHR1 mRNA in the cortex (CTX), anterior hypothalamus (AHA), nucleus accumbens (AcB), and bed nucleus of the stria terminalis (BST). Scale bar: 500 Am.
MCH system and GnRH neurons in the rat. The experiments showed that the connections between the MCH system and GnRH neurons are very robust, as about 85–90% of GnRH neurons were contacted by MCH fibers and about 50–55% of GnRH neurons had detectable levels of MCHR1 mRNA (Figs. 1–3). Due to the conservative approach for counting positive cells, it is likely that this is an underestimate of the number of GnRH cells that coexpress the MCHR1. The results of the present study confirm previous findings demonstrating MCH fibers and MCHR1 mRNA in the areas where GnRH neurons are located [2,30]. In addition, MCH fibers and MCHR1 receptor mRNA were abundant in other preoptic areas known to project to and influence GnRH neuronal activity. Thus, MCH may have both direct and indirect actions on GnRH neurons. This may also reflect
the dual inhibitory/stimulatory actions of MCH on GnRH/ LH release. Taken together, these results suggest that the MCH system is in a key position to serve as an integrating signal between energy balance and reproductive function. It is well established that MCH is a regulator of food intake; MCH expression is increased in response to fasting and injections of MCH into the hypothalamus increase food intake [38]. Furthermore, disruption of the MCH gene results in a hypophagic animal [32]. MCH has complex interrelationships with a number of neuronal systems that are involved in the regulation of food intake and energy balance, such as orexin and NPY [3]. For example, orexin and MCH neurons have many connections within the LHA [3]. MCH neurons in the LHA receive direct synaptic contacts by NPY neurons projecting from the ARH and in
Fig. 1. MCH fibers come in close apposition with GnRH neurons. (A) Confocal micrograph representing a 20-Am section showing GnRH cell bodies (green) in the preoptic area surrounded by MCH fibers (red). White arrowheads indicate close appositions (represented by the yellow color) of MCH fibers on GnRH neurons. (B) Higher magnification of confocal micrograph (20-Am-thick stack of optical sections). White arrowheads indicate close appositions (represented by the yellow color) of MCH fibers (red) on GnRH neurons (green). (C) Confocal micrograph of triple label immunofluorescence for MCH (red), GnRH (green), and synaptophysin (blue). The micrograph represents a 10-Am-thick stack of optical sections (40 optical sections at 0.25-Am thickness). White arrowheads indicate close appositions between MCH fibers and GnRH neurons. (C1 to C4) Single optical sections (0.5 Am thick) at intervals indicated in the upper right corner and showing the MCH terminal coming in close apposition with the GnRH neuron. Colocalization between MCH and synaptophysin is shown as white. (D) Low magnification confocal micrograph showing the overlap of MCH (red) and GnRH (green) fibers in the median eminence. Close appositions (within 0.5 Am) between MCH and GnRH fibers are represented by the yellow color. (E) Higher magnification confocal micrograph (5 Am thick) of MCH and GnRH fibers in the median eminence. Scale bars: A, 100 Am; B, 25 Am; C, 10 Am; D, 100 Am; E, 25 Am.
122
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
Fig. 3. MCHR1 mRNA expression in GnRH neurons. (A) Low power photomicrograph showing silver grain clusters in the lateral preoptic area (LPOA). (B–C) Double label ISH and IHC for GnRH and MCHR1 mRNA. (B) Bright field image of GnRH neurons stained with DAB. (C) Dark field image showing MCHR1 silver grains. Arrowheads indicate examples of GnRH neurons that express MCHR1 mRNA. Scale bars: A, 100 Am; B–C, 20 Am.
turn send reciprocal projections to ARH NPY neurons [3,11]. Antagonism of the NPY Y-1 receptor diminishes the MCH-induced feeding response [6]. Therefore, there appears to be complex relationships among the MCH, orexin, and NPY orexigenic systems that would serve to integrate the feeding response to negative energy balance. Since these same orexigenic systems have also been shown to make direct contacts with GnRH neurons (Figs. 1 and 2; [5,21]), their interrelationships would also provide coordinated information about the status of energy balance to the reproductive system. There is considerable evidence supporting the notion that MCH might modulate reproductive function. MCH has been shown to stimulate GnRH release from median eminence explants under conditions of high estrogen [9]. The ability of MCH to have direct effects on the median eminence is consistent with the extensive overlap and close associations of MCH and GnRH fibers shown in this study (Fig. 1). The effects of MCH on GnRH/LH secretion appear to be bimodal, that is, stimulatory in the presence of high levels of estrogen [9,15,23,24] and inhibitory in the presence of low levels of estrogen [40], as has been reported for other neuropeptides, such as NPY and orexin. Thus, under conditions of negative energy balance, MCH levels in the
LHA would be increased and levels of estrogen would be low. Therefore, MCH could play a role in the suppression of GnRH/LH secretion [42]. This notion is supported by the observation that animals lacking the MCH gene are still capable of reproducing, even though they are hypophagic and have low levels of leptin, reflecting the state of negative energy balance [32]. The present findings provide a neuroanatomical framework by which MCH may affect GnRH, either by direct effects through MCHR1s on GnRH cell bodies or by direct contacts on GnRH fibers and terminals in the median eminence. Since this study only investigated the expression of MCHR1 mRNA, we can only hypothesize that there might be MCHR1 expression on GnRH terminals in the median eminence. However, the lack of a suitable antibody makes this impossible to confirm at this time. The regularity of close associations between MCH and GnRH fibers in the median eminence does support this hypothesis. MCHR1 has been shown to be coupled to both Gi and Gq subtypes of G proteins, suggesting the capability to be both inhibitory and stimulatory [27]. At this time, it is not known how GnRH neurons might be affected by the direct actions of MCH or the mechanisms by which the levels of estrogen alter these actions. In addition, MCH neurons are known to coexpress the long form of the leptin receptor [Ob-Rb] in rat (see review, Ref. [29]). Leptin is released from adipocytes and denotes adiposity and energy balance to the brain [12,14]. The low levels of leptin during states of negative energy balance [33,37] have been shown to play a role in the suppression of GnRH neuronal activity [1,7,22]. Administration of leptin can restore fasting-induced suppression of LH secretion [25] and can greatly improve reproductive and neuroendocrine function in hypothalamic amenorrhea resulting from strenuous exercise or low weight [42]. Since GnRH neurons do not express Ob-Rb, direct projections by Ob-Rb-expressing MCH neurons to GnRH neurons may provide one potential neuroanatomical circuit by which leptin regulates GnRH activity, as has been suggested for orexin neurons [5]. The effects of leptin on MCH neurons may also be indirect through actions on ARH/NPY neurons [10], which make direct connections with MCH neurons [3,11]. There is considerable evidence that MCH expression is downregulated by leptin, and injections of leptin block MCH-induced feeding in the rat [38]. In contrast, MCH expression is elevated in ob/ob mice, which are obese and lack the leptin protein, and in obese Zucker rats, which lack leptin signaling [31,35]. Thus, MCH neurons appear to be very sensitive to peripheral signals of energy balance, such as leptin. During states of negative energy balance, the low levels of leptin would result in increased MCH expression, which may be an important signal to suppress GnRH neuronal activity. Conversely, administration of leptin would suppress MCH expression and decrease the inhibitory effect of MCH on GnRH neurons, thus restoring LH secretion.
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124
In summary, these results provide neuroanatomical evidence that the MCH system may be directly linked to GnRH neurons, suggesting that MCH may play a role in integrating the status of energy balance with regulation of reproductive function. At this time, it is unclear how important this novel neurocircuit is to the regulation of reproductive function during normal conditions as well as during metabolic challenges. Acknowledgments This work was supported by NIH grants HD-14643, RR00163, and HD18185. References [1] C.L. Adam, Z.A. Archer, D.W. Miller, Leptin actions on the reproductive neuroendocrine axis in sheep, Reproduction 61 (2002) 283 – 297. [2] J.C. Bittencourt, C.F. Elias, Melanin-concentrating hormone and neuropeptide EI projections from the lateral hypothalamic area and zona incerta to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers, Brain Res. 805 (1998) 1 – 19. [3] C. Broberger, L. De Lecea, J.G. Sutcliffe, T. Hokfelt, Hypocretin/ orexin- and melanin-concentrating hormone-expressing cells from distinct populations in the rodent lateral hypothalamus: relationship to the Neuropeptide Y and agouti-gene-related protein systems, J. Comp. Neurol. 402 (1998) 460 – 474. [4] R.E. Campbell, J.M.H. ffrench-Mullen, M.A. Cowley, M.S. Smith, K.L. Grove, Hypothalamic circuitry of neuropeptide Y regulation of neuroendocrine function and food intake via the Y5 receptor subtype, Neuroendocrinology 74 (2001) 106 – 119. [5] R.E. Campbell, K.L. Grove, M.S. Smith, Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers, Endocrinology 144 (2003) 1542 – 1548. [6] C.L. Chaffer, M.J. Morris, The feeding response to melaninconcentrating hormone is attenuated by antagonism of the NPYY1receptor in the rat, Endocrinology 143 (2002) 191 – 197. [7] F.F. Chehab, J. Qiu, K. Mounzih, A. Ewart-Toland, S. Ogus, Leptin and reproduction, Nutr. Rev. 60 (2002) S39 – S46. [8] P.L. Chen, M.S. Smith, Melanocortin 4 receptor-mediated hyperphagia and activation of neuropeptide Y (NPY) expression in the dorsomedial hypothalamus during lactation, J. Neurosci. 24 (2004) 5091 – 5100. [9] S.R. Chiocchio, M.G. Gallardo, P. Louzan, V. Gutnisky, J.H. Tramezzani, Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels, Biol. Reprod. 64 (2001) 1466 – 1472. [10] M.A. Cowley, J.L. Smart, M. Rubinstein, M.G. Cerdan, S. Diano, T.L. Horvath, R.D. Cone, M.J. Low, Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus, Nature 411 (2001) 480 – 484. [11] C.F. Elias, C.B. Saper, E. Maratos-Flier, N.A. Tritos, C. Lee, J. Kelly, J.B. Tatro, G.E. Hoffman, M.M. Ollmann, G.S. Barsh, T. Sakurai, M. Yanagisawa, J.K. Elmquist, Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area, J. Comp. Neurol. 402 (1998) 442 – 459. [12] J.S. Flier, Obesity wars: molecular progress confronts an expanding epidemic, Cell 116 (2004) 337 – 350.
123
[13] J.M. Friedman, Leptin, leptin receptors, and the control of body weight, Nutr. Rev. 56 (1998) S38 – S46. [14] J.M. Friedman, The function of leptin in nutrition; weight, and physiology, Nutr. Rev. 60 (2002) S1 – S14. [15] M.I. Gonzalez, B.I. Baker, C.A. Wilson, Stimulatory effect of melanin concentrating hormone on luteinizing hormone release, Neuroendocrinology 66 (1997) 254 – 262. [16] G.J. Hervieu, J.E. Cluderay, D. Harrison, J. Meakin, P. Maycox, S. Nasir, R.A. Leslie, The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat, Eur. J. Neurosci. 12 (2000) 1194 – 1216. [17] J. Iqbal, S. Pompolo, T. Sakurai, I.J. Clarke, Evidence that orexincontaining neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus, J. Neuroendocrinol. 13 (2001) 1033 – 1041. [18] S.P. Kalra, W.R. Crowley, Neuropeptide Y: a novel neuroendocrine peptide in the control of pituitary hormone secretion, and its relation to luteinizing hormone, Front. Neuroendocrinol. 13 (1992) 1 – 46 (Review). [19] E.G. Kokkotou, N.A. Tritos, J.W. Mastaitis, L. Slieker, E. MaratosFlier, Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain, Endocrinology 142 (2001) 680 – 686. [20] S.J. Konturek, J.W. Konturek, T. Pawlik, T. Brzozowski, Brain–gut axis and its role in the control of food intake, J. Physiol. Pharmacol. 55 (2004) 137 – 154. [21] C. Li, P. Chen, M.S. Smith, Morphological evidence for direct interaction between arcuate nucleus neuropeptide Y (NPY) neurons and gonadotropin releasing hormone (GnRH) neurons and the possible involvement of NPY Y1 receptors, Endocrinology 140 (1999) 5382 – 5390. [22] C.S. Mantzoros, Role of leptin in reproduction, Ann. N. Y. Acad. Sci. 900 (2000) 174 – 183. [23] J.F. Murray, R.A.H. Adan, R. Walker, B.I. Baker, A.J. Thody, W.A.J. Nijenhuis, J. Yukitake, C.A. Wilson, Melanin-concentrating hormone, melanocortin receptors and regulation of luteinizing hormone release, J. Neuroendocrinol. 12 (2000) 217 – 223. [24] J.F. Murray, J.G. Mercer, R.A. Adan, J.J. Datta, C. Aldairy, K.M. Moar, B.I. Baker, M.J. Stock, C.A. Wilson, The effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone, J. Neuroendocrinol. 12 (2000) 1133 – 1139. [25] S. Nagatani, P. Guthikonda, R.C. Thompson, H. Tsukamura, K.I. Maeda, D.L. Foster, Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting, Neuroendocrinology 67 (1998) 370 – 376. [26] S. Ohkura, T. Tanaka, S. Nagatani, D.C. Bucholtz, H. Tsukamura, K.I. Maeda, D.L. Foster, Central, but not peripheral, glucosesensing mechanisms mediate glucoprivic suppression of pulsatile luteinizing hormone secretion in the sheep, Endocrinology 141 (2000) 4472 – 4480. [27] P. Pissios, D.J. Trombly, I. Tzameli, E. Maratos-Flier, Melaninconcentrating hormone receptor 1 activates extracellular signalregulated kinase and synergizes with G(s)-coupled pathways, Endocrinology 144 (2003) 3514 – 3523. [28] D. Qu, D.S. Ludwig, S. Gammeltoft, M. Piper, M.A. Pelleymounter, M.J. Cullen, W.F. Mathes, R. Przypek, R. Kanarek, E. Maratos-Flier, A role for melanin-concentrating hormone in the central regulation of feeding behaviour, Nature 380 (1996) 243 – 247. [29] A. Sahu, Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance, Front. Neuroendocrinol. 24 (2003) 225 – 253. [30] Y. Saito, M. Cheng, R.M. Leslie, O. Civelli, Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain, J. Comp. Neurol. 435 (2001) 26 – 40. [31] G. Segal-Liberman, R.L. Bradley, E. Kokkotou, M. Carlson, D.J. Trombly, X. Wang, S. Bates, M.G. Myers Jr., E. Maratos-Flier,
124
[32]
[33]
[34]
[35]
[36]
[37]
P.S. Williamson-Hughes et al. / Brain Research 1041 (2005) 117–124 Melanin concentrating hormone is a critical mediator of the leptindeficient phenotype, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 10085 – 10090. M. Shimada, N.A. Tritos, B.B. Lowell, J.S. Flier, E. Maratos-Flier, Mice lacking melanin-concentrating hormone are hypophagic and lean, Nature 396 (1998) 670 – 674. M.S. Smith, K.L. Grove, Integration of the regulation of reproductive function and energy balance: lactation as a model, Front. Neuroendocrinol. 23 (2002) 225 – 256. T.L. Steininger, T.S. Kilduff, M. Behan, R.M. Benca, C.F. Landry, Comparison of hypocretin/orexin and melanin-concentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain, J. Chem. Neuroanat. 27 (2004) 165 – 181. A. Stricker-Krongrad, T. Dimitrov, B. Beck, Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats, Mol. Brain Res. 92 (2001) 43 – 48. S.D. Sullivan, S.M. Moenter, Gamma-aminobutyric acid neurons integrate and rapidly transmit permissive and inhibitory metabolic cues to gonadotropin-releasing hormone neurons, Endocrinology 145 (2004) 1194 – 1202. F.S.L. Thong, T.E. Graham, Leptin and reproduction: is it a critical
[38]
[39]
[40]
[41]
[42]
link between adipose tissue, nutrition and reproduction? Can. J. Appl. Physiol. 24 (1999) 317 – 336. N.A. Tritos, E. Maratos-Flier, Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone, Neuropeptides 33 (1999) 339 – 349. N.A. Tritos, J.W. Mastaitis, E. Kokkotou, E. Maratos-Flier, Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus, Brain Res. 895 (2001) 160 – 166. H. Tsukamura, R.C. Thompson, S. Tsukahara, S. Ohkura, F. Maekawa, R. Moriyama, Y. Niwa, D.L. Foster, K.I. Maeda, Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat, J. Neuroendocrinol. 12 (2000) 529 – 534. G.F. Turi, Z. Liposits, S.M. Moenter, C. Fekete, E. Hrabovszky, Origin of neuropeptide Y-containing afferents to gonadotropinreleasing hormone neurons in male mice, Endocrinology 144 (2003) 4967 – 4974. C.K. Welt, J.L. Chan, J. Bullen, R. Murphy, P. Smith, A.M. DePaoli, A. Karalis, C.S. Mantzoros, Recombinant human leptin in women with hypothalamic amenorrhea, N. Engl. J. Med. 351 (2004) 987 – 997.