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Fasting Activates the Nonhuman Primate Hypocretin (Orexin) System and Its Postsynaptic Targets

Departments of Obstetrics and Gynecology (S.D., T.L.H.) and Neurobiology (B.H., T.L.H.), Yale University School of Medicine, New Haven, Connecticut, 06520; Division of Neuroscience (H.F.U.), Oregon Regional Primate Research Center, Beaverton, Oregon, 97006; and Department of Anatomy and Histology (P.S.), Szent Istvan University, School of Veterinary Medicine, Budapest, Hungary, 1071

Address all correspondence and requests for reprints to: Tamas L. Horvath, Department of Obstetrics and Gynecology, Yale Medical School, 333 Cedar Street, FMB 339, New Haven, Connecticut 06520. E-mail: tamas.horvath{at}yale.edu.

Abstract

In rodents, hypocretin (HCRT, also called orexin) influences a variety of endocrine, autonomic, and metabolic functions. The present study was undertaken to determine whether the HCRT-producing circuit is involved in the hypothalamic regulation of homeostasis in primates as well. We studied female monkeys (Cercopithecus aethiops) that were either fed or fasted for 24 h. Immunocytochemistry revealed HCRT-producing perikarya exclusively in the lateral hypothalamus-perifornical region and dorsomedial hypothalamus of the monkey brain. HCRT axons and axon terminals were present in different parts of the hypothalamus and adjacent forebrain and thalamic nuclei. The 24-h fast resulted in an approximately 50% decline in circulating leptin levels and significantly elevated c-fos expression in the perifornical region; in the dorsomedial, ventromedial, and arcuate nuclei; and in the medial preoptic area. In the dorsomedial nucleus and perifornical region of fasted monkeys, three times more HCRT-neurons expressed nuclear c-fos than those of the normally fed controls. Neurons in different parts of the hypothalamus and basal forebrain that expressed c-fos, but did not contain HCRT, were targets of HCRT-immunopositive boutons establishing asymmetric synapses. In the arcuate nucleus, subsets of these HCRT-targeted c-fos-expressing cells contained neuropeptide Y. The present study provides the first experimental evidence to implicate HCRT in the hypothalamic regulation of homeostasis in primates. The fact that these lateral hypothalamic cells have leptin receptors and can be activated by a metabolic challenge and that they innervate diverse brain regions indicates that the HCRT system may be a key integrator of environmental cues in their regulation of diverse brain activity.

ENERGY HOMEOSTASIS IS regulated by discrete central nervous system circuits that, in predominantly hypothalamic areas, are sensitive to peripheral metabolic signals (1). A great part of our current understanding of human physiology and disorders of the hypothalamus is inferred from rodent models, whereas little is known about the primate hypothalamic circuits that control homeostasis. In particular, it is not clear whether the same peptidergic circuits known to be involved in the hypothalamic regulation of rodent homeostasis have similar functions in the primate brain.

Hypocretin (HCRT, also called orexin), a peptide produced exclusively in the hypothalamus (2, 3, 4), has emerged as an important regulator of autonomic, endocrine, and metabolic functions of rodents (3, 5, 6). HCRT cells of rats and monkeys were found to express leptin receptor immunoreactivity and to provide synaptic input to distinct hypothalamic and brainstem circuits known to participate in the central regulation of endocrine, metabolic, and autonomic systems (4, 7). Although the determination of synaptic interactions between HCRT and other hypothalamic systems provided the map via which signals may travel within the hypothalamus, that does not demonstrate whether that signaling modality is involved in homeostatic regulation. For that determination, the cytochemical identification of c-fos as an indicator of cell activity under different metabolic conditions may be used.

After metabolic challenge, distinct populations of hypothalamic neurons begin to express c-fos in rats (8) as well as in monkeys (9). Using this paradigm, the current study aimed to determine whether, in the monkey hypothalamus, HCRT cells and their postsynaptic targets are activated by a metabolic challenge (fasting).

Materials and Methods

Animals
Adult (3.5–4.0 kg) female African green monkeys (Cercopithecus aethiops; n = 6) were used. All monkeys were previously ovariectomized for use in an unrelated experiment; three monkeys were killed after fasting (food withdrawal for 24 h), whereas the other three were killed without fasting. The primate tissue was collected under protocols approved by the Yale Animal Care and Use Committee. After receiving a lethal dose of anesthetic, monkeys were killed by a transcardial perfusion of 500 ml heparinized saline (0.9%) followed by 2000 ml of fixative consisting of 4% paraformaldehyde, 15% saturated picric acid, and 0.08% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The mediobasal hypothalamus was dissected out and postfixed for an additional 1.5 h in glutaraldehyde-free fixative. Tissue blocks were washed and stored in 0.1 M PB at 4 C.

Light and electron microscopic double immunostaining
Light microscopic double immunostaining for HCRT and c-fos was carried out according to our previously published protocol. Sections were incubated with one of the primary antisera (rabbit anti-HCRT, 1:2000) and processed with the avidin-biotin-peroxidase technique. The immunoreaction was visualized with a modified version of the nickel-diaminobenzidine (DAB) reaction (15 mg DAB, 0.12 mg glucose oxidase, 12 mg ammonium chloride, 600 µl 0.05 M nickel ammonium sulfate, and 600 µl 10% ß-D-glucose in 30 ml PB), resulting in a dark-blue reaction product. After several rinses in PB, the sections were further incubated in sheep anti-c-fos (1:2000; Cambridge Research Biochemicals Inc., Wilmington, DE) for 24 h at 4 C and processed with the peroxidase-anti-peroxidase technique. The tissue-bound peroxidase was visualized with a DAB reaction (15 mg DAB and 165 µl 0.3% H₂O₂ in 30 ml PB), resulting in a light-brown reaction product. After visualization of tissue antigens, some sections were processed for electron microscopy (1% OsO₄ in PB for 30 min., dehydrated through increasing ethanol concentrations using 1% uranyl acetate in the 70% ethanol for 30 min) and flat-embedded in araldite between liquid release-coated slides (Electron Microscopy Sciences, Fort Washington, PA).

The specificity of the HCRT antisera has been thoroughly tested by us and others in previous studies (2, 4).

Analysis of c-fos expressing cells in single- and double-labeled (c-fos and HCRT) material
To estimate the induction of c-fos expression in the primate hypothalamus after 24 h of fasting, matching sections from the hypothalami of control and fasted monkeys were coincubated in the same vials for either single immunostaining for c-fos or for double immunostaining of c-fos and HCRT (see above). To ensure that matching sections were coprocessed, sections were selected using the comparative assessment of the location of the following anatomical landmarks: optic chiasm or tract, shape of the third ventricle, anterior commissure, fornix, mammilo-thalamic tract, and median eminence. One group of sections (either the control or the experimental) was marked by placing a notch on one side of the tissue block before vibratome sectioning. The analyzer was blind to the code, which was revealed after the collection of the counts. When the immunostaining was completed (three sets of coincubations from six monkeys—three control and three experimental), matching sections were placed on slides, coverslipped, and analyzed using light microscopy. The number of c-fos-immunolabeled cells, those that were labeled for HCRT, and those that were labeled for both antigens were noted in several nuclei of the hypothalamus (see below). Only those cells on the surface of the section were counted. Guided by the stereotaxic atlas of the African Green monkey (10), the following hypothalamic areas were analyzed: medial preoptic area (MPOA), parvicellular and magnocellular paraventricular nucleus, arcuate (infundibular) nucleus (ARC), dorsomedial hypothalamic nucleus, ventromedial nucleus, and the lateral hypothalamus perifornical region. After determining the mean values, the Student’s t test was used for comparison. Significance was concluded at P < 0.05.

Light microscopic triple immunostaining
Because HCRT is not present in neuronal perikarya of the ARC, c-fos is only expressed in cell nuclei, and neuropeptide Y (NPY) is expressed in the cytoplasm of neurons, we triple-labeled hypothalamic sections using our triple-labeling protocol (4). Sections were first incubated for 24 h at room temperature with a mixture of the HCRT and c-fos antisera and processed with the avidin-biotin-peroxidase, and the tissue-bound peroxidase was visualized by a nickel-diaminobenzidine reaction, resulting in a dark-blue to black color. Subsequently, sections were further immunostained for NPY using sheep anti-NPY (Auspep Pty Ltd., Perkwille, Australia) and the peroxidase antiperoxidase method, and the tissue-bound peroxidase was visualized by a DAB reaction to give a light-brown reaction product.

Leptin measurements
All of the blood samples were collected in the morning, and the serum was stored frozen. Samples were collected before and after animals were fasted. Leptin was measured using primate leptin RIA kit (Linco Research, Inc., St. Charles, MO). The minimum detectable concentration at 95% binding was 0.7 ng/ ml, and the interassay coefficient of variation was 6%; all of the serum samples were assayed together in a single RIA. A Student’s t test with two-tail probabilities was used for statistical comparisons of leptin values between the two groups. Significance was concluded at P < 0.05.

Results

Circulating leptin levels in fasted and normally fed control African green monkeys
The circulating leptin level values did not differ between the fasted and control groups at 0 h (6.7 ± 0.9 vs. 6.5 ± 0.7 ng/ml; P > 0.05), but they were significantly lower in the fasted animals after the 24-h fast (2.5 ± 0.5 vs. 6.3 ± 0.8 ng/ml; P < 0.05).

HCRT in the hypothalamus.
The overall pattern of HCRT immunolabeling in monkey corresponded to our earlier description (4, 7). HCRT-immunoreactive perikarya were present exclusively in the lateral hypothalamus, perifornical region, and, to a lesser extent, in the dorsomedial hypothalamus, whereas their projections were abundant in different hypothalamic nuclei. We detected no obvious differences between the HCRT immunoreactivity in the hypothalamus of control vs. fasted animals.

c-fos.
In accordance with a recent report (9), c-fos immunoreactivity was detected in different parts of the hypothalamus of both fasted and normally fed control monkeys. In control animals, a moderate number of c-fos-labeled nuclei were homogenously distributed in the MPOA; periventricular regions; paraventricular nucleus (both parvo- and magnocellular regions); anterior hypothalamus; suprachiasmatic, arcuate, dorsomedial, and ventromedial nuclei; lateral hypothalamus; and perifornical region. The number of c-fos-expressing cells in individual hypothalamic subnuclei of fasted monkeys was distinctly different from the control values (Fig. 1, A–C). In fasted animals, a significantly higher number of c-fos-expressing cells was detected in the MPOA (235 ± 22 vs. 489 ± 35*; *, P < 0.05), lateral hypothalamus-perifornical (LH-PF) region (343 ± 17 vs. 889 ± 41*; *, P < 0.05; Fig. 1, A and B), and arcuate (205 ± 34 vs. 341 ± 42*; *, P < 0.05) and dorsomedial nuclei (276 ± 41 vs. 452 ± 26*; *, P < 0.05).

View larger version (82K): [in this window] [in a new window]   FIG. 1. Light micrographs of the monkey perifornical region (pf) after immunostaining for c-fos taken from control (A) and fasted animals (B). Bar scale, 100 µm. f, Fornix. C, Bar graph showing the number of c-fos-immunoreactive nuclei in different parts of the hypothalamus in control (white bars) and fasted (black bars) animals.

c-fos expression in HCRT cells
c-fos immunoreactivity was detected in cell nuclei of HCRT-immunoreactive cells of both control and 24-h-fasted animals (Fig. 2, A–D). There was no difference in the mean number of HCRT-labeled cells in control and fasted animals (502 ± 24 vs. 535 ± 33, P > 0.05; Fig. 2E). However, the percentage of HCRT cells expressing nuclear c-fos was robustly and significantly elevated in the fasted animals when compared with control values (P < 0.05). In the control LH-PF, 135 ± 35 HCRT cells exhibited nuclear c-fos labeling, which represented 26.8% of the total HCRT cells counted. In contrast, in fasted animals, 418 ± 38 HCRT cells exhibited c-fos immunoreactivity, which represented 78.1% of the total HCRT cells counted (Fig. 2). The number of c-fos-expressing neurons that contained HCRT in control monkeys (135 ± 35) represented approximately 75% of all the cells that were c-fos immunopositive (182 ± 29) in the LH-PF region. This ratio remains similar in fasted animals, in which 418 + 38 of the total c-fos-immunolabaled nuclei (562 ± 42) were in HCRT-immunoreactive perikarya (Fig. 2F).

View larger version (77K): [in this window] [in a new window]   FIG. 2. Fasting-induced c-fos expression was associated exclusively with cell nuclei (A and C). In the perifornical region, many nuclear c-fos-labeled cells (red nuclei in B and D) were also immunopositive for HCRT. Bar scales (in A and C), 10 µm (for A and B) and 100 µm (for C and D). E, Bar graph showing c-fos-immunoreactive HCRT neurons (white bars) and total HCRT neurons (black bars) counted in control and fasted animals. F, Bar graph showing c-fos-immunoreactive HCRT neurons (white bars) and total c-fos-immunolabeled neurons (black bars) counted in control and fasted animals.

View larger version (173K): [in this window] [in a new window]   FIG. 3. In all regions of the monkey hypothalamus in which c-fos induction was observed after fasting in cell nuclei, HCRT boutons were observed in close apposition to these activated cells (A), and electron microscopy showed (B and C) that these contacts were synapses (arrows in C point to membrane specializations). Bar scales, 10 µm (for A) and 1 µm (for B and C).

View larger version (134K): [in this window] [in a new window]   FIG. 4. A subset of fasting-activated, HCRT-targeted cells were NPY cells in the ARC. Bar scale, 10 µm.

The present study provides the first suggestive evidence that the HCRT system of nonhuman primates is involved in the hypothalamic signal transduction associated with metabolic alterations. The fact that short-term food withdrawal underlies robust activation of transcriptional mechanisms in the primate lateral hypothalamic HCRT system, together with the widespread distribution of HCRT projections in homeostatic centers and beyond, argues for a coordinating role for HCRT in autonomic, endocrine, and metabolic processes. This assertion is in line with evidence that is available for the role of HCRT in the modulation of food intake, blood pressure, and arousal (3, 5, 6, 11, 12).

In a previous study (4), the monkey ARC was found to receive a robust HCRT innervation, with HCRT axons heavily innervating NPY cells (4). Whether this ARC NPY system may participate in the modulation of HCRT on food intake in primates, as suggested in the rat (13, 14), remains to be tested. The present observation of HCRT input to fasting-activated NPY cells in the African green monkey lends support to this possibility. Nevertheless, it is also conceivable that the activation of HCRT neurons by fasting occurs secondary to the activation of the ARC. However, ARC NPY cells are most likely protected by the blood-brain barrier, and the direct projection from the ARC to the lateral hypothalamus is not robust in the nonhuman primate (4). It may also be plausible that, because of the HCRT system is heavily involved with the regulation of sleep/wake cycles, it is disturbance of sleep during fasting that affects HCRT neuronal function. Alternatively, should metabolic signals affect HCRT cells during fasting, sleep disturbances may be the consequence HCRT neuronal activation.

Leptin receptor immunoreactivity has been detected in the primate lateral hypothalamic HCRT cells (4), and leptin is suggested to cross the blood-brain barrier (15). Thus, the observed induction of c-fos gene expression in the HCRT cells may be a direct consequence of the diminishing leptin levels we observed after the short-term fasting. However, fasting paradigms, particularly when used on primates, do not allow for the distinction of the effect of particular hormones, because they underlie complex responses of a variety of endocrine mechanisms. For example, in the rodent, evidence is available to indicate that circulating insulin and/or glucose levels have a regulatory influence on hypothalamic HCRT expression (16). Future studies will determine the extent of contribution of each of the involved humoral and metabolic signals in the activation of primate HCRT systems.

Interestingly, in the hypothalamic nuclei (including the ARC, LH-PF, dorsomedial nucleus, and MPOA), in which short-term fasting induced c-fos expression the most robustly, activated cells received multiple HCRT inputs. In rodents, HCRT exerts excitatory postsynaptic currents (17), and intracerebroventricular administration of HCRT induces c-fos expression in a manner not dissimilar to what we describe here in the nonhuman primate (18, 19). Because all the synaptic membrane specializations between HCRT axons and c-fos-expressing perikarya in the primate hypothalamus were asymmetrical, stimulatory synapses, the activation of hypothalamic circuitry by metabolic challenge may be mediated, in part, by the HCRT system.

Acknowledgments

We thank Anthony van den Pol for providing the HCRT antisera.

Footnotes

This work was supported by NIH Grant RR-14451. T.L.H. was an Albert Szent-Gyorgyi Fellow.

Abbreviations: ARC, Arcuate nucleus; DAB, diaminobenzidine; HCRT, hypocretin; LH-PF, lateral hypothalamus-perifornical; MPOA, medial preoptic area; NPY, neuropeptide Y; PB, phosphate buffer.

Received March 3, 2003.

Accepted for publication June 24, 2003.

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