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Department of Neuroscience (R.E.C., K.L.G., M.S.S.), Oregon National Primate Research Center, Beaverton, Oregon 97006; and Department of Physiology and Pharmacology (R.E.C., M.S.S.), Oregon Health Science University, Portland, Oregon 97239
Address all correspondence and requests for reprints to: Dr. M. Susan Smith, Oregon National Primate Research Center, Oregon Health Science University, Beaverton, Oregon 97006. E-mail: smithsu{at}ohsu.edu.
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Abstract |
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Introduction |
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Recent physiological and neuroanatomical studies suggest that orexin A may play an important role in control of the hypothalamo-pituitary gonadal axis. For example, it has been shown that central administration of orexin A stimulates LH secretion (11). However, similar to other orexigenic peptides, such as neuropeptide Y (NPY), orexin has been shown to have bimodal effects on LH secretion, with orexin stimulating LH secretion in steroid-primed ovariectomized rats but inhibiting LH secretion in the absence of ovarian steroids (11, 12). It has also been demonstrated that immunoneutralization of orexin A in the brain completely abolishes both LH and PRL surges, but icv administration of orexin leads to a dose-dependent recovery of fasting-suppressed LH surges (13). In addition, tissue explant studies have shown that orexin A stimulates GnRH release from the hypothalamus of both intact male rats and female rats at proestrus (14). Recently a study employing low-dose estrogen replacement in ovariectomized rats, resulting in serum levels similar to those observed in diestrus, demonstrated that low-dose estrogen potentiates the suppressive effects of centrally administered orexin A on LH secretion (15), suggesting that orexin A is inhibitory during diestrus, or low estrogen states, and excitatory during surge conditions. Although orexin is clearly implicated in the regulation of GnRH/LH secretion, the neuroanatomical pathway by which orexin may be mediating its effects in the rat remains to be determined. The first aim of the present study was to investigate whether orexin fibers project to and come in close contact with GnRH neurons and whether GnRH neurons express OX-R1 in the rat.
Similar to orexin, NPY has been found to elicit bimodal effects on LH release, most likely acting through the Y1 (16, 17, 18), Y4 (17, 19, 20, 21), or Y5 (22, 23) receptor subtypes. Recently our laboratory reported that the majority of orexin cell bodies in the LHA expressed Y4-like immunoreactivity (Y4-ir), and some orexin fibers also were positive for Y4-ir (19). Others have shown that activation of Y4 receptors with a Y4 agonist, 1229U91, or a high-affinity ligand for the Y4 receptor, rat pancreatic polypeptide (rPP), stimulates GnRH neurons and in turn LH release in intact male rats (20) and is important in the basal release of LH in the female rat (17). Conversely, when the infertile, leptin-deficient ob/ob mouse is crossed with the Y4 knockout mouse, fertility is partially restored, suggesting the Y4 receptor is involved in suppressing reproductive function (21). Taken together, these data suggest that some of the effects of Y4 activation on LH release may be mediated via orexin. Therefore, the second aim of the present study was to characterize the neuroanatomical relationship between Y4-positive orexin fibers and GnRH neurons.
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Materials and Methods |
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For immunocytochemical studies, animals (n = 4–5, both male and female rats were used in each experiment) were killed under pentobarbital anesthesia by cardiac infusion with ice-cold PBS followed by ice-cold paraformaldehyde in NaPO4 buffer (pH 7.4). The brains were then removed, saturated in 25% sucrose, frozen in cooled isopentane, and stored at -80 C until sectioning on a microtome. A one-in-six series of tissue sections (25 µm) were collected from each animal.
Double- and triple-label immunofluorescence (IF)
All immunofluorescent labeling, as previously reported (18, 22), was performed on floating sections. Double- and triple-label IF for orexin, GnRH, and one of synaptophysin, OX-R1, or Y4 were performed using a cocktail of primary antibodies. The goat polyclonal orexin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1:10,000 (19). The mouse monoclonal GnRH antibody (a kind gift of H. F. Urbanski, Oregon National Primate Research Center, Beaverton, OR) was used at a concentration of 1:500 (22). The rabbit polyclonal synaptophysin antibody (DAKO Corp., Carpinteria, CA) was used at a concentration of 1:200. The rabbit polyclonal OX-R1 antibody (a kind gift of G. Hervieu, SmithKline Beecham Foods International, Harlow, Essex, UK) was used at a concentration of 1:500 (9, 24). The OX-R1 antibody has been extensively characterized and shown to be specific, as evidenced by the agreement in the patterns of receptor mRNA and protein expression (9), specific detection of the receptor in a cell line transfected with OX-R1 (9), and loss of specific staining following preabsorption of the antiserum with the immunogen peptide (24). The rabbit polyclonal Y4 antibody (a kind gift of J. ffrench-Mullen) was used at a 1:10,000 concentration (19). The specificity of this antibody was confirmed by showing similar expression patterns of the mRNA and protein, as determined by Western blot and IF. For each combination of double or triple labeling, all primary antibodies were titrated to establish the maximum dilutions that provided robust signals and minimum nonspecific staining.
First, double labeling for orexin and GnRH was performed. Briefly, floating tissue sections were removed from cryoprotectant and washed in 0.05 M potassium PBS (KPBS). Tissue was incubated in blocking buffer (KPBS + 0.4% Triton X-100 + 2% normal donkey serum) for 20 min to reduce background and then incubated in a cocktail of the orexin and GnRH primary antibodies for 48 h at 4 C. Following washes in KPBS, tissue was incubated for 1 h in a cocktail of fluorescent secondary antibodies. Orexin was visualized with tetramethyl rhodamine isothiocyanate (TRITC) conjugated to a donkey antigoat antibody (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and GnRH was visualized with fluorescein isothiocyanate (FITC) conjugated to a donkey antimouse antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.).
Subsequently double labeling for GnRH and OX-R1 was performed. As in the previous experiment, GnRH was visualized with FITC conjugated to a donkey antimouse antibody (1:200), and OX-R1 was visualized with TRITC conjugated to a donkey antirabbit antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.).
Triple-label experiments for GnRH/orexin/synaptophysin, GnRH/OX-R1/orexin, and GnRH/orexin/Y4 were carried out in a similar manner. In each triple-label experiment, GnRH was visualized with FITC conjugated to a donkey antimouse antibody (1:200). In the first triple-label experiment, orexin was visualized with TRITC conjugated to a donkey antigoat antibody (1:200), and synaptophysin was visualized with Cy5 conjugated to a donkey antirabbit antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.). In the second triple-label experiment, OX-R1 was visualized with TRITC conjugated to a donkey antirabbit antibody (1:200), and orexin was visualized with Cy5 conjugated to a donkey antigoat antibody (1:200). The final triple-label experiment used Cy5 conjugated to a donkey antirabbit antibody (1:200) to visualize Y4 and TRITC conjugated to a donkey antigoat antibody (1:200) to visualize orexin. To enhance the signal for Y4-ir, the BLAST technique of biotinylated tyramide enhancement was used, as reported previously (18, 19). Sections from all experiments were mounted onto subbed slides, cover slipped with glycerol, and sealed.
Confocal laser microscopy
Confocal laser microscopy, as previously described (18, 22), was used to analyze the double- and triple-label IF images for colocalization and close contacts. The TSC SP confocal system (Leica Corp., Germany), consisting of a RBE inverted microscope (Leica Corp., an Ar laser-producing light at 488 nm (for visualization of FITC), a Kr laser-producing light at 568 nm (for visualizing TRITC), and an HeNe laser-producing light at 647 nm (for visualization of Cy5), was used to scan the images. Various objectives (x25, numerical aperture 0.75 and x40, numerical aperture 1.25) were used to scan and capture images. For each experiment, fluorophore signals were checked individually for bleed-through to the apposing detector. All bleed-through was eliminated by adjusting laser intensity and detector window width. To assess colocalization of two signals, a series of continuous optical sections, 0.25 µm or 0.5 µm intervals along the z-axis of the tissue section, were scanned for each fluorescent signal. The signals were obtained for each fluorophore on one series of optical sections and stored separately as a series of 512 x 512 pixel images. The stacks of individual optical slices (0.25 µm or 0.5 µm resolution) were analyzed using the MetaMorph imaging system (Universal Imaging Corp., West Chester, PA) to determine colocalization and contacts. The confocal images are presented as projections of stacks of optical images or as individual slices, as indicated. The brightness and contrast of the images were adjusted in Photoshop to match microscope visualization (Adobe Systems Inc., San Jose, CA).
Data analysis
In the double-label IF studies for GnRH/orexin or GnRH/OX-R1, one series of tissue sections (10–12 sections, 150-µm interval between sections) was used from each animal (n = 4–5 animals). GnRH neurons were analyzed if the entire cell body, including proximal dendritic processes, was visible in the section. For the triple-label IF studies, representative sections from several animals were examined. These studies were performed primarily as a proof of principle, i.e. that GnRH neurons expressing the OX-R1 are contacted by orexin fibers (GnRH/OX-R1/orexin) or orexin contacts observed on GnRH neurons represent potential synaptic contacts (GnRH/orexin/synaptophysin). In the triple-label IF for GnRH/orexin/Y4, there were only a few scattered Y4-positive orexin fibers in the area of GnRH neurons.
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Results |
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, A–C). Sections from four brains were analyzed (more than 500 GnRH neurons), and the results showed that between 75% and 85% of GnRH neurons in each brain were contacted by orexin fibers, and no differences were observed between male and female animals. Using synaptophysin (a protein localized in synaptic vesicles) as a marker of synaptic contacts, orexin fiber butons colocalized with synaptophysin and were associated with GnRH neurons (Fig. 1, D–F). Shown as a stack of images (D) or individual optical slices (0.25 µm resolution, E and F), the spine of an orexin fiber (red) colocalized with synaptophsyin (blue) makes close contact with a GnRH neuron (white represents triple labeling). Additional double-label studies showed that GnRH neurons colocalized with OX-R1-ir (indicated by the yellow color in the low- and high-power examples in Fig. 2, A and B). Consistent with the robust innervation of GnRH neurons by orexin, OX-R1-ir was colocalized in 85 ± 2% of the GnRH neurons examined (485 neurons in five brains). Triple-label IF confirmed that orexin fibers, shown in blue, made contacts onto GnRH neurons expressing OX-R1-ir (Fig. 2, C and D).
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, although there is some OX-R1 expression (red) in this region and orexin fibers do appear to innervate the dorsolateral ME (blue), there is no coexpression of OX-R1 and GnRH (green) and very little interaction between orexin fibers and GnRH nerve terminals.
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depicts three GnRH cell bodies surrounded by orexin fibers. The white arrows indicate two fibers that are Y4/orexin positive. The yellow arrows indicate where orexin-positive fibers were identified coming in close contact with GnRH neurons. Although there were multiple close contacts between orexin fibers and GnRH, as shown previously in Fig. 1
, Y4-positive orexin fibers never came in close contact with GnRH neurons.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). There appears to be no interaction between orexin fibers and GnRH nerve terminals in the ME (Fig. 3). The immunoreactivity of orexin fibers and OX-R1 presented in the present study confirms previous findings demonstrating that orexin A immunoreactive fibers (6), OX-R1 mRNA (7), and immunoreactivity (9) are present in the septal and medial preoptic regions in which GnRH neurons are located. Our data support previously reported findings in the ovine hypothalamus, in which orexin fibers, originating from the LHA, were found in close contact with GnRH neurons (25). However, although the present study found between 75% and 85% of GnRH neurons contacted by orexin fibers and approximately 85% of GnRH neurons expressed OX-R1, the previous study found contacts on only 30% of GnRH neurons. This discrepancy between the two studies may be due to several things, including species differences between the rodent and sheep. If this is the case, it will be important to investigate orexin innervation of GnRH neurons in other species, especially the primate. Another discrepancy may be due to differences in the sensitivity of the antibodies used in the two studies, which were obtained from different sources. The present study shows dense networks of fine immunoreactive fibers (Figs. 1A and 4), but the orexin fibers demonstrated in the ovine hypothalamus (25) were much less dense. Yet another discrepancy might be due to differences in the sensitivity of the confocal microscopy analysis. It is generally accepted that, although confocal microscopy can provide a good approximation of synaptic interactions, the limitations of the technique preclude the degree of certainty obtained using electron microscopy.
Several lines of evidence support the contention that orexin A modulates neuroendocrine function. In addition to a number of studies indicating steroid-dependent effects of central orexin administration on LH release (11, 12, 15), studies using in vitro explants indicate that the stimulatory effects of orexin are at the hypothalamic level (14). It was also reported that orexin A stimulates GnRH release from hypothalamic explants collected from both male rats and female rats in proestrus. These data suggest that the stimulatory effects of orexin on LH release are via direct stimulation of GnRH secretion. Indeed, the present findings provide the functional neuroanatomical pathway by which orexin may stimulate GnRH neurons and subsequent LH release via direct contact with OX-R1 on GnRH cell bodies but not on nerve terminals within the ME. OX-R1 is exclusively coupled to the Gq/11 subtype of G proteins (5, 26), suggesting that orexin action via the OX-R1 on GnRH neurons is excitatory. However, orexin has also been shown to inhibit LH secretion in the absence of gonadal steroids (12), although the mechanism by which gonadal steroids modulate orexin effects on LH secretion remains to be determined. It is possible that the inhibitory actions of orexin on GnRH neurons is indirect, involving the activation of other neurons within the preoptic area that also express OX-R1 (Fig. 2), which in turn may have inhibitory actions on GnRH neurons.
As the name implies, orexin activity is necessary for normal feeding behavior. The central injection of orexin A stimulates food intake in satiated rats (27, 28), whereas neutralization of orexin with an antiorexin antibody dose dependently suppresses spontaneous feeding in fasted rats (29). Furthermore, orexin mRNA is increased with fasting (2). These data suggest that orexin neurons are able to sense the nutritional state of an animal. It has been shown repeatedly that changes in the metabolic status of an animal are tightly linked to changes in reproductive function. Therefore, the ability of orexin to modulate both feeding behavior and reproductive function, along with the present neuroanatomical findings, suggests that the orexin system is in a key position to serve as an integrating signal between energy balance and reproductive function.
In addition, orexin neurons are known to coexpress the long form of the leptin receptor (Ob-Rb) in rat (30) and sheep (25). Leptin, a peripheral metabolic signal released from adipocytes, is important in denoting adiposity and energy balance to the brain. Also, leptin has been shown to play a role in regulating reproductive function. Administration of leptin restores fasting-induced suppression of LH secretion in both sheep and primates (31, 32, 33). However, GnRH neurons do not express Ob-Rb. As previously suggested by similar findings in the ovine hypothalamus (25), direct projections by Ob-Rb-expressing orexin neurons to GnRH neurons may provide one potential neuroanatomical circuit by which leptin regulates GnRH activity.
NPY is another neuropeptide system that may be an important integrating signal for both energy balance/food intake and reproductive function (34). Similar to the effects of orexin, NPY also elicits steroid-dependent effects on LH secretion (35). Orexin neurons in the LHA receive direct synaptic contacts by NPY neurons projecting from the arcuate nucleus and in turn send reciprocal projections to arcuate nucleus NPY neurons (36). Currently, there is evidence implicating the Y1 (16, 17, 18), Y4 (17, 19, 20, 21), and Y5 (22, 23) receptor subtypes in NPY’s modulation of neuroendocrine reproductive function. Of direct relevance, Y4-ir and mRNA are colocalized with orexin neurons in the LHA (19). These data, coupled with the present findings that GnRH neurons express the OX-R1 and are contacted by orexin fibers, demonstrate a unique potential neuronal circuit whereby NPY, acting through the Y4 receptor, can exert its effects on LH release via the modulation of orexin. Because the present study also showed that Y4-positive orexin fibers were never observed in close contact with GnRH neurons, the major actions of the Y4 receptor appear to be through postsynaptic effects on orexin cell bodies within the LHA. Taken together, these data suggest that orexin neurons, by receiving critical input via Y4 and leptin receptors, may provide an important link between the regulation of energy balance and reproductive function.
A number of questions remain concerning the modulatory effects of the Y4-orexin system on GnRH neurons. First, the endogenous ligand for the brain Y4 receptor is not clear. Is it NPY, which has a much lower affinity for the Y4 receptor (37), or other pancreatic polypeptide family ligands? Our recent study characterizing the expression of Y4-ir on orexin neurons also found that administration of rPP, an endogenous ligand of the Y4 receptor, induced robust cFos activation in orexin neurons (19). Furthermore, rPP stimulated a modest increase in food intake and a robust drinking response (19), which is similar to responses by orexin. These data suggest that stimulation of the Y4 receptor by rPP is excitatory to orexin neurons. However, the effects of Y4 on reproductive function have been shown to be both stimulatory (17, 20) and inhibitory (21). Similarly, the effects of orexin have been reported to be both stimulatory and inhibitory to reproductive function (11, 12, 13, 14, 15). Therefore, it is possible the actions of the Y4 receptor on the GnRH/LH axis are mediated via the orexin system.
In summary, these data provide neuroanatomical evidence for a direct link between the orexin system and GnRH neurons. These findings suggest that orexin plays a direct role in the neuroendocrine control of reproduction and may also be uniquely positioned to integrate signals associated with energy balance regulation. Additional studies will be necessary to establish the relative importance of this novel neuronal circuitry in the regulation of reproductive function during normal cyclicity as well as during metabolic changes.
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Footnotes |
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Abbreviations: FITC, Fluorescein isothiocyanate; IF, immunofluorescence; KPBS, potassium PBS; LHA, lateral hypothalamic area; ME, median eminence; NPY, neuropeptide Y; OX-R, orexin receptor; rPP, rat pancreatic polypeptide; TRITC, tetramethyl rhodamine isothiocyanate; Y4-ir, Y4-like immunoreactivity.
Received September 11, 2002.
Accepted for publication January 6, 2003.
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