Changes in Hypothalamic Corticotropin-Releasing Hormone, Neuropeptide Y, and Proopiomelanocortin Gene Expression during Chronic Rapid Eye Movement Sle
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《内分泌学杂志》
Laboratory of Physiology (M.K.), Richard N. Dixon Science Research Building Department of Biology, Morgan State University, Baltimore, Maryland 21251
Department of Anatomy and Neurobiology (W.W.L., G.E.H.), University of Maryland School of Medicine, Baltimore, Maryland 21201
Abstract
Chronic rapid eye movement (paradoxical) sleep deprivation (REM-SD) of rats leads to two conspicuous pathologies: hyperphagia coincident with body weight loss, prompted by elevated metabolism. Our goals were to test the hypotheses that 1) as a stressor, REM-SD would increase CRH gene expression in the hypothalamus and that 2) to account for hyperphagia, hypothalamic gene expression of the orexigen neuropeptide Y (NPY) would increase, but expression of the anorexigen proopiomelanocortin (POMC) would decrease. Enforcement of REM-SD of adult male rats for 20 d with the platform (flowerpot) method led to progressive hyperphagia, increasing to approximately 300% of baseline; body weight steadily declined by approximately 25%. Consistent with changes in food intake patterns, NPY expression rapidly increased in the hypothalamic arcuate nucleus by d 5 of REM-SD, peaking at d 20; by contrast, POMC expression decreased progressively during REM-SD. CRH expression was increased by d 5, both in mRNA and ability to detect neuronal perikaryal staining in paraventricular nucleus with immunocytochemistry, and it remained elevated thereafter with modest declines. Taken together, these data indicate that changes in hypothalamic neuropeptides regulating food intake are altered in a manner consistent with the hyperphagia seen with REM-SD. Changes in CRH, although indicative of REM-SD as a stressor, suggest that the anorexigenic actions of CRH are ineffective (or disabled). Furthermore, changes in NPY and POMC agree with current models of food intake behavior, but they are opposite to their acute effects on peripheral energy metabolism and thermogenesis.
Introduction
SLEEP DEPRIVATION (SD) is a remarkable stressor. Unlike commonly used stress modalities in rats such as immobilization, adaptation to SD is not possible because, without exception, unremitting SD is fatal (1, 2). As a chronic stressor, SD is pervasive and thus differs from most long-term stress paradigms, which are intermittent. SD is also different for the reason that within days, rats exhibit a progressively developing hyperphagia while body weight continuously decreases (3, 4, 5, 6, 7). Chronic SD therefore results in a state of negative energy balance with increased energy expenditure (1, 2, 8) and elevated resting metabolic rate (9).
In addition to hyperphagia and loss of body weight, SD brings about a unique cluster of pathologies that causes significant morbidity (reviewed in Refs.6 and 10). Physically, rats appear increasingly debilitated and wasted with development of skin lesions (6, 10). Integrity of the immune system becomes compromised (11, 12). Hormonally, there is a generalized depression of the endocrine system with declines in levels of anabolic hormones (13), thyroid hormones (3, 8, 14), sex steroids (15, 16), and leptin (9, 13, 17). In addition, peripheral sympathetic tone is increased (8, 15, 18), and the hypothalamic-pituitary-adrenal (HPA) axis is activated, with elevated hypothalamic CRH (this study), increased circulating ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), and adrenal hypertrophy (3, 7, 20). Others, however, find no change in the HPA axis during SD (6, 10).
In humans, sleep loss is multifactorial, presenting with myriad other syndromes that lead to morbidity and generalized stress. For instance, critically ill patients in intensive care units are subjected to severe disruption in sleep, caused by preexisting illness, medications, the heightened amount of care given, or other factors (24, 25). Insomnia, which is often secondary to serious diseases, is predictive of psychological disorders and poorer quality of physical health (26). Sleep-disordered breathing is a major risk factor for cerebrovascular disease (27), and patients with obstructive sleep apnea have higher incidences of cardiovascular disease (28) and the metabolic syndrome (29). Studies also show that sleep-deprived humans, similar to rats, have elevated metabolic rate (30), increased sympathetic tone and cortisol (31), compromised immune function (24), and increased food intake concomitant with elevated serum ghrelin but decreased leptin (31).
Our interest in SD hyperphagia was prompted by the general observation that with most stressors, food intake is typically blunted. One such example is the potent psychological stress brought on by immobilization (32, 33, 34). Clearly, the food intake response is opposite to that of sleep-deprived rats, despite the latter showing clear symptoms of stress (e.g. elevated corticosterone) (7, 15, 16, 19, 20, 21, 22, 23). A noteworthy feature of SD-associated hyperphagia is that because body weight always declines, the amount of food consumed is insufficient to sustain elevated metabolism. Not surprisingly, necropsies of chronically sleep-deprived rats reveal virtually no remaining abdominal depots of white adipose tissue (1, 2), and stomachs are typically engorged with food (M. Koban, unpublished observations); additionally, hepatic and skeletal muscle glycogen levels are depleted within the first few days of SD (35).
Food intake behavior is governed by a complex array of central neurotransmitters, hypothalamic and brain stem neuropeptides, and peripheral peptides. For example, within the hypothalamic arcuate nucleus (Arc), neuropeptide Y (NPY) is the most powerful orexigen known; its less potent counterpart, also in the Arc, is the anorexigenic neuropeptide, -MSH, a cleavage product of proopiomelanocortin (POMC) (36, 37). For this study, our goal was to chronically deprive rats of rapid eye movement (REM) sleep to test the hypothesis that hypothalamic NPY and POMC gene expression would be significantly increased and decreased, respectively, to explain the hyperphagic response. Furthermore, because there are elevations in plasma ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), we reasoned that CRH would be up-regulated within the hypothalamic paraventricular nucleus (PVN) to activate the HPA axis.
Materials and Methods
Animals
Male Sprague-Dawley rats (4–5 months of age; Harlan, Indianapolis, IN) were housed individually in standard shoebox plastic cages with ad libitum chow (TekLad, Madison, WI) and water; ambient temperature averaged 24 C and photoperiod was a 12-h light, 12-h dark cycle with lights on at 0900 h. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Morgan State University, and they comply with National Institutes of Health guidelines.
For 2 wk, rats were accustomed to routine handling and the novel environment of the SD tanks (see below) by placement onto the platforms for approximately 1 h each day. Body weights were obtained and 24-h food consumption was estimated by weighing leftover chow. These data were gathered daily from 0900–1000 h, during 1 wk of baseline and up to 20 d of experiments. Food intake was normalized as grams per day per kilogram body weight taken to the 0.67 power to compensate for differences in metabolic rate as a function of body mass (38). No corrections were made for scattered crumbs because they were continually washed out.
SD paradigm
The SD paradigm employed in this study is known as the platform (i.e. flowerpot) method. A rat resides on a small circular platform (e.g. inverted flowerpot) surrounded by water. Rats have easy access to food and water (standard water bottle); they can groom themselves, engage in limited exploratory behavior, and rest by lying down. When a rat lapses into REM (paradoxical) sleep, muscle atonia causes it to make facial contact with or to fall into the water, abruptly awakening it to continuously enforce the paradigm. The platform method is therefore selective for significantly abolishing REM sleep. Electroencephalography studies have validated its effectiveness, although slow-wave sleep can also be disrupted (39, 40, 41, 42).
Two large Plexiglas SD tanks are each divided into five separate compartments of 30 x 30 x 40 cm. Each compartment has a 10-cm high column onto which a 10-cm diameter Plexiglas platform is attached. For most REM-SD studies of approximately 96 h duration, smaller platforms (6–7 cm) are typically used (3, 4, 5, 7, 15, 16, 19, 20, 21, 22, 23, 43); however, beyond this time point, we have noted that rats become completely exhausted. Ten-centimeter platforms thereby provide more space and mobility and minimize confinement as a possible restraint-like stressor. For this study, rats were sleep-deprived for 5 (n = 6), 10 (n = 6), or 20 (n = 5) d. Six rats in their home cages served as controls.
Inlet and outlet ports on opposite ends allow continuous water flow to carry away waste and debris and floods each chamber to 1 cm below the platform surface. Rats increasingly experience water immersions after approximately 2 wk so water temperature was adjusted to approximately 30 C to lessen cooling effects.
Tissue preparation
On the final day of REM-SD, all the animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, ip), administered heparin (100 U) directly into the heart, and perfused transcardially with saline containing 2% sodium nitrite followed by 2.5% acrolein in buffered 4% paraformaldehyde (44). The brains were removed and sunk in 30% sucrose solution, frozen, and sectioned at 25 μm on a freezing sliding microtome into 1-in-12 series. The sections were collected in cryoprotectant antifreeze solution (45). Sections were stored at –20 C until they were processed for immunocytochemical analysis of CRH, -MSH, ACTH1–25, and NPY or for in situ hybridization (ISH) analysis of mRNAs for CRH, NPY, and POMC.
Immunohistochemistry
The immunocytochemical method used for staining NPY, POMC derivatives (-MSH or ACTH1–25), and CRH immunoreactivity has been published previously (44, 46). Briefly, the sections were removed from the cryoprotectant, combined to form a 1-in-6 series, rinsed in potassium PBS (KPBS) at 0.05 M (pH 7.4), treated with 1% sodium borohydride (Sigma Chemical Co., St. Louis, MO) for 10 min, rinsed, and then incubated with rabbit anti-NPY (1:70,000) (Sigma), rabbit anti-CRH (1:50,000) (gift from Dr. John Olschwaka, University of Rochester), rabbit anti-ACTH (1:200,000) (AC-6; gift from Dr. Roger Guillemin, Salk Institute), or rabbit anti--MSH (1:30,000) (Immunonuclear, Stillwater MN; catalog no. 12288, lot 8410018) in KPBS with 0.4% Triton X-100 for 48 h at 4 C. After rinsing, the tissue was incubated for 1 h at room temperature in biotinylated goat antirabbit IgG (heavy and light chains; Vector Laboratories, Burlingame, CA) at a concentration of 1:600 in KPBS with 0.4% Triton X-100, rinsed, and of incubated 1 h in avidin-biotin complex solution at room temperature (ABC Elite Kit; Vector Laboratories) (4.5 μl of A and B/ml KPBS with 0.4% Triton X-100 incubation mixture). After rinsing in KPBS and then in 0.175 M sodium acetate (NaOAc) solution, the antibody-peroxidase complex was visualized with nickel sulfate (25 mg/ml), 3,3'-diaminobenzidine HCl (Ni-DAB; 0.2 mg/ml), and H2O2 (0.83 μl of a 3% solution/ml) in 0.175 M NaOAc. Staining times were approximately 15–20 min. The tissue was transferred to NaOAc to stop the reaction, rinsed in KPBS, placed into 0.9% saline, and mounted onto gelatin subbed slides. The slides were dried overnight, dehydrated through alcohols, cleared, and coverslipped.
ISH: probe preparation
NPY.
Briefly, the NPY cDNA construct (gift from Dr. A. Sahu, University of Pittsburgh) contains an insert that included most (residues 1–511) of the sequenced cDNA of rat brain NPY ligated into the EcoRI site of the Bluescribe M13 vector (47). For the antisense NPY riboprobe, the plasmid was linearized with FspI and transcribed with T3 RNA polymerase to yield a probe of 511 bp of the rat NPY cDNA. For the sense-strand mRNA, the DNA was linearized with SmaI and transcribed with T7 RNA polymerase.
POMC.
The POMC cDNA construct (gift of Dr. S. J. Watson, University of Michigan) contains 666 bp ligated into pGEM 4Z. For antisense POMC riboprobes, the plasmid was linearized with EcoRI and transcribed with T7 RNA polymerase to yield a probe complementary to nucleotides 89–755 of the rat POMC DNA. For sense strands, the DNA was linearized with HindIII and transcribed with Sp6 RNA polymerase.
CRH.
The CRH cDNA construct (gift of Dr. Paul Sawchenko, Salk Institute) contains 1197 bp ligated into Bluescript SK. For antisense CRH riboprobes, the plasmid was linearized with HindIII and transcribed with T7 polymerase to yield a probe complementary to nucleotides 1–1997 of the CRH cDNA. For sense-strand cRNA, the DNA was linearized with SmaI and transcribed with T3 polymerase.
For all probes, the in vitro transcription reaction consisted of 50 μl mixture containing 2 mM biotin-CTP (Life Technologies, Gaithersburg, MD), 3 μg linearized CRH, NPY, or POMC, 5 mM dithiothreitol, 40 U RNAsin ribonuclease inhibitor (Promega, Madison, WI), 40 U T7 RNA polymerase (Life Technologies), 0.5 mM CTP, and 1 mM each ATP, GTP, and UTP. The transcription reaction was developed at 37 C for 90 min and stopped by the addition of 1 μl of EDTA.
ISH: hybridization and detection
The general strategy for the ISH reactions involved hybridization with a biotinylated riboprobe, followed by detection of the biotin using a slight modification of the avidin-biotin complex (ABC) method initially described by Berghorn et al. (46) that lowered the concentration of the chromogen-peroxide solution as described below. The method reveals clusters of specific mRNAs within the cells, and these are easily quantified by counting the RNA clusters in each cell (46) or determining the OD of each cell.
Day 1 (RNase free).
Freely floating 25-μm tissue sections from two 1-in-12 tissue wells combined to form a 1-in-6 series of sections encompassing the entirety of the hypothalamic Arc for NPY and POMC, and the PVN for CRH, were removed from the cryoprotectant antifreeze, rinsed in KPBS made with 0.1% diethylpyrocarbonate-treated water (DEPC H2O), and then incubated in 1% sodium borohydride/KPBS and DEPC H2O for 15 min to remove residual aldehydes derived from the paraformaldehyde-acrolein fixation. Sections were then rinsed repeatedly to remove the sodium borohydride solution, and the ISH procedure was initiated. The tissue was rinsed in 0.1 M triethanolamine buffer (pH 8.0) twice and then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine buffer for 10 min at room temperature. Next, the tissue was rinsed in 2x SSC for 10 min at room temperature. Sections were prehybridized for 2 h at 37 C by using hybridization buffer [50% deionized formamide, 10% dextran sulfate, 1x Denhardt’s solution, 300 mM NaCl, 8 mM Tris (pH 8.0), 0.8 mM EDTA, and 15% DEPC H2O] containing heat-denatured torula yeast RNA (2 mg/ml) (Ambion, Austin, TX). Sections were rinsed with 2x SSC for 10 min. After these rinses, the sections were hybridized with biotinylated NPY, biotinylated POMC, or biotinylated CRH riboprobes (final probe concentration was 600 ng/kb/ml). Probes and torula yeast RNA (0.5 mg/ml) were denatured by heating to 85–90 C for 3 min, put on ice for 5 min, mixed with hybridization buffer, and placed into polystyrene microbeakers containing the sections. The hybridization was continued overnight at 37 C.
Day 2.
Tissue was rinsed with 4x SSC for 30 min, rinsed once with RNase buffer [10 mM Tris (pH 8.0), 500 mM NaCl, 0.75 mM EDTA) heated to 37 C, followed by a 30-min incubation of RNase (20 μg/ml) in RNase buffer at 37 C. Sections were rinsed with RNase buffer and then incubated in RNase buffer for 30 min at 37 C. After 40 min of rinsing in 2x SSC and one rinse with 0.1x SSC (heated to 42 C), sections were incubated in 0.1x SSC for 60 min at 42 C. Tissue was rinsed once with 0.1x SSC at room temperature followed by 1 h of rinsing with KPBS. Sections were then incubated in goat antibiotin (Vector Laboratories) at a concentration of 1:100,000 in KPBS with 0.4% Triton X-100 at 4 C for 48 h.
Day 4.
Sections were rinsed in KPBS and then immersed with rabbit antigoat secondary antibody (Vector Laboratories) at 1:600 in KPBS with 0.4% Triton X-100 at room temperature for 1 h. The tissue was then rinsed and placed into avidin-biotin complex solution (ABC Elite Kit; Vector Laboratories) for 1 h at room temperature. Sections were rinsed in KPBS, followed by rinses with 0.175 M NaOAc. The biotinylated probes were visualized using nickel sulfate (12.5 mg/ml), 3,3-diaminobenzidine HCl (Ni-DAB; 0.1 mg/ml), and H2O2 (0.42 μl of a 3% solution/ml of final mixture in 0.175 M NaOAc). This solution is one half the concentration of Ni-DAB that normally is used for ABC immunocytochemistry to keep background staining to a minimum. Staining proceeded for approximately 15–20 min. The reaction was stopped by rinsing the tissue with the NaOAc solution. After rinsing in KPBS, sections were placed into saline and mounted onto gelatin subbed slides. After drying overnight, the sections were dehydrated in graded ethanol solutions, cleared in Histoclear, and coverslipped with Histomount. mRNA was visualized as small clusters within the cell cytoplasm. At the electron microscopic level, these clusters accurately represent the ribosomal clusters in the cytoplasm.
Data analysis
Assessment of changes in ACTH, -MSH, and NPY immunoreactivity were qualitative and based mainly on changes in the ability to detect cells and their intensity. For NPY in particular, the fact that the entire Arc contained abundant axon profiles obscured the ability to count weakly labeled neuron perikarya in control animals, and it is for this reason that descriptions of immunocytochemical data for NPY and POMC products were mainly qualitative.
For CRH, with the antibody we used, without colchicine pretreatment, the detection of neurons immunoreactive for the peptide serves as a useful tool for reflecting levels of protein expression. To analyze the CRH peptide, images from each side of the PVN that encompassed all the CRH cells were captured using a x10 objective and a Nikon Optiphot-equipped Edge microscope (Edge Corp., Santa Monica, CA) linked to a Retiga (EX) digital camera (Biovision Technologies, Exton, PA). Analysis was performed on a Macintosh G4 and used Signal Analytic’s IP Spectrum software (Vienna, VA). The borders of the PVN at the level of the medial parvocellular division were outlined as the region of interest. All detectible cells were segmented, and the area occupied by CRH immunoreactive cells for each nucleus determined. Use of this approach avoided problems of overlapping and partial cells. By setting a minimum area for the structures analyzed, the thin fibers that coursed from the cells were not included in the analysis unless they were in direct contact with an immunoreactive soma. Because the most prominent fibers were outside the borders of the portion of the PVN assayed, those few axons did not greatly influence the measures taken. The analysis also included determination of the perikaryal staining intensity. This measure was accomplished by the determination of the mean gray level of the selected cells minus the background selected from a region adjacent to the PVN where no CRH cells or fibers were located.
With the method we used for ISH of CRH, POMC, and NPY mRNA, clusters of RNA were distributed within the cytoplasm of the cells, and this feature enables the level of expression to be reliably quantified based on comparisons with 35S-labeled riboprobes (46) by RNA cluster image analyses and with solution hybridization analyses (48). Electron microscopic examination of these clusters validates their interpretation as ribosomal clusters (49) and reinforces their utility for quantitative assessments. The advantage to using this approach in freely floating fixed sections is that the same sections can be used for ISH analysis and immunocytochemistry. A pilot study determined that use of each cells’ OD level was equally able to detect accurate differences in the level of expression and, when clusters were numerous, avoided problems of multiple grains that came into contact with each other being counted as single (large) grains. Multiple images were captured with a x100 objective on each side of the parvocellular division of the PVN for CRH and on each side of the Arc for NPY or POMC, to encompass the entirety of the region in which cells were located. A stack of images in the Z plane at 0.2-μm intervals was captured for each field and flattened to visualize the total mRNA clusters present in each cell. The background value of the surrounding tissue (devoid of clusters) was subtracted from each cell, and the OD (mean gray level minus background value) of the cell cytoplasm bearing mRNA clusters was determined for each cell. Borders of the cell’s cytoplasm were determined by the limit of the presence of scattered mRNA clusters. Values were averaged for all the cells detected in each animal. Data are expressed as mean OD ± SEM.
Statistical analysis
Baseline food intake and body weight were averaged (± SEM) and assigned a value of 100%. Changes in these quantities during REM-SD are shown as percent change from baseline. Nonparametric Kruskal-Wallis (KW) testing was used for analysis of food intake and body weight, followed by Dunn’s posttest. For analysis of expression of the peptides, ANOVA with post hoc analysis using Hsu’s test were performed. The relationship between the levels of POMC or NPY expression and days of REM-SD was determined with Spearman nonparametric correlation analysis. Overall, a value of P < 0.05 was interpreted as a significant difference.
Results
Our data show that long-term REM-SD resulted in stereotypical changes in food consumption and body weight (Fig. 1). Within 5 d of REM-SD, there was a significant increase in food consumption that was maintained over the time course of the experiment (Fig. 1A) (KW = 66.454; P < 0.0001) and peaking at about 300% of baseline. Accompanying this hyperphagia was a progressive decrease in body weight so that by d 15, rats had lost almost 25% of their mass (Fig. 1B) (KW = 151.87; P < 0.0001). Some of the data for d 15–20 were lost, accounting for the gaps.
CRH
Staining of CRH peptide in the PVN was affected by REM-SD. CRH neurons normally synthesize the peptide and transport it rapidly from the neurons. Consequently, CRH neuronal staining in home cage control animals was often not easily detected or was weak (Fig. 2A). Five and 10 d of REM-SD produced striking increases in the detection of CRH within PVN perikarya (Fig. 2, C and E). As a percentage of the parvocellular PVN, there was more than a 10-fold increase in the area occupied by CRH immunoreactive cells by 5 d of REM-SD, and this increase was maintained for up to 20 d (Fig. 3A). In addition, within the neurons detected, the intensity of CRH immunoreactivity increased over the first 10 d (Fig. 2, C and E), but then at 20 d, it became more variable with rats showing signs of advanced morbidity, which is characteristic of chronic SD (6, 10).
The mRNA data reflected that seen for peptide expression. Home cage controls showed extremely low levels of CRH mRNA within the parvocellular neurons with only scattered mRNA clusters noted (Fig. 2B), yielding low OD for the cells. REM-SD increased the level of expression of CRH mRNA within the parvocellular PVN neurons (Fig. 2, D and F). By d 5, CRH mRNA expression increased by nearly 70% over control values (Fig. 3C) (P < 0.01), and by d 10, although still maintaining higher levels than controls (P < 0.05), CRH mRNA declined somewhat, just as had been noted for the CRH peptide. After 20 d of REM-SD, CRH mRNA levels became more erratic.
NPY
Expression of NPY mRNA was low in home cage controls and significantly increased over the course of REM-SD. At baseline, the clusters of mRNA in the Arc were easily detected (Fig. 4, A and E) but were of low density. Striking progressive increases for mRNA per cell (Figs. 4, B–D and F–H), reflected in the changes in NPY OD (Fig. 5), were noted with REM-SD. The mRNA expression doubled by d 5 (P = 0.0404) and then reached a 4-fold peak after d 20 (P < 0.0003 compared with control animals) (Fig. 4). Regression analysis indicated that the levels of mRNA increased with length of REM-SD (Spearman = 0.5769; P = 0.0040).
NPY immunoreactivity also showed changes, although these were not easily quantified. As REM-SD was enforced, the detection of NPY neurons in the Arc became more apparent and by d 10–20, NPY neurons were quite easily distinguished from the abundant axon profiles found in the nucleus (Fig. 6).
POMC
In home cage controls, scattered neurons expressing the products of the POMC gene (ACTH1–24 and -MSH; Fig. 7, A and C) were located in the Arc and the ventral hypothalamic areas lateral to the Arc. Staining of either ACTH1–24 or -MSH suggested reduced levels of expression in that after long-term REM-SD, fewer perikarya with either of the POMC-related products were detected compared with controls (Fig. 7, B and D). As with the other peptides examined, POMC mRNA levels changed in a fashion consistent with observed immunocytochemical data. A gradual reduction in POMC mRNA was noted over the course of REM-SD (Fig. 8). Initially, mRNA levels were high and then declined in a linear fashion with REM-SD (Fig. 9) (Spearman = –0.5769; P = 0.0067); by d 20, POMC mRNA levels were significantly lower than the controls and had fallen to 56% of control values (P = 0.0109).
Discussion
There are three findings of significance in this study of REM-SD of rats for up to 20 d. First, we show that neurons of the hypothalamic Arc progressively and robustly increase NPY gene expression over the time course of the experiment. Second, gene expression of POMC in the Arc decreased with length of REM-SD. The third point of interest is that CRH gene expression in neurons of the PVN was up-regulated early in the REM-SD regimen and remained elevated.
As noted in the Introduction, REM-SD represents a potent and chronic stressor that gives rise to a number of pathologies. Toward the end of our experiment, rats were showing signs of advanced morbidity (see also Refs.6 and 10), which likely accounts for more variable gene expression of CRH by d 20 compared with earlier time points. Peptide expression, based on the ability to retain CRH within the PVN neurons, did not return to baseline in the d 20 group, indicating that feedback mechanisms for down-regulating CRH while attempting to restore equilibrium to the CRH cells were only partially effective. Studies by Lopez et al. (50) showed that repeated unpredictable stress to rats results in persistent up-regulation of CRH because of loss of glucocorticoid receptor function in the hippocampus. It is possible that similar mechanisms in REM-SD promote CRH expression in the face of elevated corticosteroid levels. Another possibility is that upon prolonged REM-SD, responsiveness of the HPA axis is abnormal because of loss of cooperative effects of vasopressin on the corticotrophs (51) and that some animals experience falls in corticosteroid levels as death nears. Additional study of the central nervous system regulation of the HPA is needed to resolve this issue.
The reciprocal changes in hypothalamic NPY and POMC are consistent with SD-associated hyperphagia and the rapidly developing state of negative energy balance. Moreover, the marked decline in circulating leptin that occurs within a few days of total- or REM-SD (9, 13, 17) is in accord with increased food consumption. Taken together, the patterns of change in hypothalamic NPY and POMC and serum leptin agree with current models of the regulation of food intake (37, 52). Interestingly, when human volunteers are sleep-deprived for 88 h, rapid and sustained changes in the diurnal pattern of circulating leptin occur (53). The authors relate their findings to the so-called night-eating syndrome (54), and similar changes in eating patterns may partially explain total- or REM-SD hyperphagia in rats.
A difficulty develops, however, when our results are evaluated in the context of the stress response and with the present understanding of the circuitry and hierarchy of hypothalamic neuropeptides involved in feeding and energy metabolism. The issues are complex, and compelling questions arise.
One issue is concerned with the stress response and food intake. There is an abundant body of literature showing that stress blunts appetite. For instance, psychological stressors such as immobilization (32, 34) or social defeat (55) and physical stress caused by lipopolysaccharide administration (56) or electric footshock (57) all result in diminished food intake. Evidence points to increased hypothalamic CRH to be responsible for the anorexia (58, 59). Rats undergoing REM-SD also increase CRH gene expression (this study) and have elevated serum ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), all of which are well-accepted indicators of activation of the stress response. What is not clear, however, is despite there being a strong central CRH signal that otherwise would be expected to suppress appetite, sleep-deprived rats unfailingly exhibit hyperphagia. This suggests that during REM-SD, the anorexic effect of CRH may be inhibited or overridden by pathways that sense and try to correct the energy deficit of this stressor.
A second issue involves energy metabolism and hyperphagia during total- or REM-SD. The sleep-deprived rat progressively increases energy expenditure and metabolic rate (1, 2, 8, 9), and it makes sense that food intake increases many-fold to fuel the process. In the present study, although we provide evidence that one of the mechanisms sustaining rapidly developing hyperphagia is increased hypothalamic NPY gene expression, there are perplexing results stemming from intracerebroventricular (icv) or PVN administration of NPY. Prominently, acute NPY results in dose-dependent decreases in several indicators of energy metabolism, including sympathetic nerve activity to brown adipose tissue (BAT) (60, 61), binding of GDP to BAT mitochondria (62, 63), UCP-1 mRNA expression (64), and oxygen consumption (63). Under certain physiologically relevant circumstances, persistently increased hypothalamic NPY can be elicited, concomitant with development of a state of negative energy balance. For example, chronically up-regulated hypothalamic NPY occurs with energetically costly conditions such as lactation (65), food restriction or deprivation (66, 67, 68), and cold exposure and acclimation (69). Complementary to acutely applied NPY studies (60, 61, 62, 63, 64), lactation leads to decreased regulatory thermogenesis (70) with down-regulation of uncoupling protein gene expression (71) and decreased peripheral norepinephrine turnover (72). Of significant interest is that cold acclimation brings about hyperphagia in rats (73, 74, 75) with elevated NPY in the hypothalamic Arc (69), but in sharp contrast to acute NPY studies, there is increased sympathetic activity (76) and circulating thyroid hormones (77, 78), and most importantly, thermogenic capacity is markedly elevated, together with increased resting metabolic rate (77, 78, 79). Whereas studies of acute NPY administration and of lactation suggest that up-regulated hypothalamic NPY should dial down thermogenesis during REM-SD, instead, it becomes elevated. This scenario is not readily explained by our current understanding of the neurocircuitry governing food intake and energy metabolism, and unraveling this problem poses a challenging puzzle.
The flip side of this matter involves POMC, the counterpart to NPY in the hypothalamic Arc. As with NPY, POMC has an important role in energy metabolism and food intake. Physiological conditions that demand increased food intake, such as during lactation, stem from increased NPY and decreased POMC (80). Our finding of a decline in Arc POMC with time of REM-SD further explains the hyperphagic response, but similar to elevated NPY, the low POMC levels fail to account for increased REM- or total-SD-associated metabolism. That is, if POMC, its cleavage end-product, -MSH, or various agonists or antagonists are administered icv, there is evidence showing that the melanocortin system stimulates energy metabolism (81, 82). Although the pattern of POMC gene expression is entirely in agreement with food intake behavior during chronic REM-SD, it is opposite to what occurs metabolically in the periphery.
CRH also has a role in the regulation of energy metabolism because when the peptide is applied centrally, there is an increase in BAT thermogenesis (83, 84, 85). What this means is that CRH stimulation of pathways emerging from the PVN, which raise the level of sympathetic activity to increase BAT thermogenesis (86), remains intact. Undoubtedly, it is likely that there are complex (and for the moment, perplexing) interactions between CRH, NPY, and POMC neurons in the hypothalamus and other regions such as the brain stem, that, when clarified, will explain their opposing actions on food intake and energy metabolism.
As the circuitry governing sleep/wakefulness and interactions with energy metabolism unravels, additional players enter the scene that could explain the metabolic sequellae observed after REM-SD. For example, a mechanism involving orexin A and orexin B is certainly likely. Studies on orexins have mainly focused on their role in sleep/arousal (87), but central administration of orexin A increases metabolic rate in anesthetized rats (88, 89), although some of its effect on thermogenesis may be attributed to stimulation of spontaneous physical activity (90). Orexin fibers innervate the intermediolateral cell columns in the spinal cord (i.e. the preganglionic sympathetic fibers) (91), and stimulation of these neurons is believed to be one site where orexin regulates metabolism. Indeed, administration of orexin increases excitability of sympathetic preganglionic neurons in vivo and in vitro (92, 93), and it results in increased sympathetic tone and cardiovascular function (94). Moreover, upon awakening, the activity of orexin neurons increases (95), and quite interestingly, REM-SD for 96 h produces a near doubling of orexin levels in the cisternal cerebrospinal fluid of rats (96). At this point, it is premature to conclude that orexin is responsible for enhancing sympathetic tone to increase metabolic rate in total- or REM-SD. Nevertheless, a broader view of the literature shows all ranges of effect with orexins. As an example, icv-administered orexin A was recently shown to decrease sympathetic nerve activity to BAT (whereas orexin B increased it) (97), an effect not in agreement with the increased metabolic rate with icv- or Arc-administered orexin A (88, 89). With the knowledge that orexin neurons project widely in the central nervous system (98), it is now critical to determine whether selective projections of the orexin system play a major role in metabolic stimulation during REM-SD. Although we have only begun to explore the possible changes in the orexin system after REM-SD in our model, preliminary data suggest that it is up-regulated.
In summary and conclusion, we provide evidence that the stereotypical hyperphagia of chronically sleep-deprived rats is mediated, at least in part, by up-regulation of NPY and down-regulation of POMC in the hypothalamic Arc. That REM-SD constitutes a stressor is demonstrated by increased gene expression of CRH within the PVN. Although these changes are in agreement with current models of food intake regulation, there remain thought-provoking disconnects between the role of these neuropeptides in food intake, the stress response, and peripheral thermogenesis.
Acknowledgments
We thank Kevin L. Swinson for his valuable technical assistance.
Footnotes
This study was funded by National Institutes of Health Grants 2-SO6-GM-51971-04 and G12-RR-17581-02 (to M.K.) and NS 43788 (to G.E.H.).
First Published Online October 6, 2005
Abbreviations: Arc, Arcuate nucleus; BAT, brown adipose tissue; DAB, 3,3'-diaminobenzidine; DEPC, diethylpyrocarbonate; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular; ISH, in situ hybridization; KPBS, potassium PBS; KW, Kruskal-Wallis; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; REM, rapid eye movement; SD, sleep deprivation.
Accepted for publication September 23, 2005.
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Department of Anatomy and Neurobiology (W.W.L., G.E.H.), University of Maryland School of Medicine, Baltimore, Maryland 21201
Abstract
Chronic rapid eye movement (paradoxical) sleep deprivation (REM-SD) of rats leads to two conspicuous pathologies: hyperphagia coincident with body weight loss, prompted by elevated metabolism. Our goals were to test the hypotheses that 1) as a stressor, REM-SD would increase CRH gene expression in the hypothalamus and that 2) to account for hyperphagia, hypothalamic gene expression of the orexigen neuropeptide Y (NPY) would increase, but expression of the anorexigen proopiomelanocortin (POMC) would decrease. Enforcement of REM-SD of adult male rats for 20 d with the platform (flowerpot) method led to progressive hyperphagia, increasing to approximately 300% of baseline; body weight steadily declined by approximately 25%. Consistent with changes in food intake patterns, NPY expression rapidly increased in the hypothalamic arcuate nucleus by d 5 of REM-SD, peaking at d 20; by contrast, POMC expression decreased progressively during REM-SD. CRH expression was increased by d 5, both in mRNA and ability to detect neuronal perikaryal staining in paraventricular nucleus with immunocytochemistry, and it remained elevated thereafter with modest declines. Taken together, these data indicate that changes in hypothalamic neuropeptides regulating food intake are altered in a manner consistent with the hyperphagia seen with REM-SD. Changes in CRH, although indicative of REM-SD as a stressor, suggest that the anorexigenic actions of CRH are ineffective (or disabled). Furthermore, changes in NPY and POMC agree with current models of food intake behavior, but they are opposite to their acute effects on peripheral energy metabolism and thermogenesis.
Introduction
SLEEP DEPRIVATION (SD) is a remarkable stressor. Unlike commonly used stress modalities in rats such as immobilization, adaptation to SD is not possible because, without exception, unremitting SD is fatal (1, 2). As a chronic stressor, SD is pervasive and thus differs from most long-term stress paradigms, which are intermittent. SD is also different for the reason that within days, rats exhibit a progressively developing hyperphagia while body weight continuously decreases (3, 4, 5, 6, 7). Chronic SD therefore results in a state of negative energy balance with increased energy expenditure (1, 2, 8) and elevated resting metabolic rate (9).
In addition to hyperphagia and loss of body weight, SD brings about a unique cluster of pathologies that causes significant morbidity (reviewed in Refs.6 and 10). Physically, rats appear increasingly debilitated and wasted with development of skin lesions (6, 10). Integrity of the immune system becomes compromised (11, 12). Hormonally, there is a generalized depression of the endocrine system with declines in levels of anabolic hormones (13), thyroid hormones (3, 8, 14), sex steroids (15, 16), and leptin (9, 13, 17). In addition, peripheral sympathetic tone is increased (8, 15, 18), and the hypothalamic-pituitary-adrenal (HPA) axis is activated, with elevated hypothalamic CRH (this study), increased circulating ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), and adrenal hypertrophy (3, 7, 20). Others, however, find no change in the HPA axis during SD (6, 10).
In humans, sleep loss is multifactorial, presenting with myriad other syndromes that lead to morbidity and generalized stress. For instance, critically ill patients in intensive care units are subjected to severe disruption in sleep, caused by preexisting illness, medications, the heightened amount of care given, or other factors (24, 25). Insomnia, which is often secondary to serious diseases, is predictive of psychological disorders and poorer quality of physical health (26). Sleep-disordered breathing is a major risk factor for cerebrovascular disease (27), and patients with obstructive sleep apnea have higher incidences of cardiovascular disease (28) and the metabolic syndrome (29). Studies also show that sleep-deprived humans, similar to rats, have elevated metabolic rate (30), increased sympathetic tone and cortisol (31), compromised immune function (24), and increased food intake concomitant with elevated serum ghrelin but decreased leptin (31).
Our interest in SD hyperphagia was prompted by the general observation that with most stressors, food intake is typically blunted. One such example is the potent psychological stress brought on by immobilization (32, 33, 34). Clearly, the food intake response is opposite to that of sleep-deprived rats, despite the latter showing clear symptoms of stress (e.g. elevated corticosterone) (7, 15, 16, 19, 20, 21, 22, 23). A noteworthy feature of SD-associated hyperphagia is that because body weight always declines, the amount of food consumed is insufficient to sustain elevated metabolism. Not surprisingly, necropsies of chronically sleep-deprived rats reveal virtually no remaining abdominal depots of white adipose tissue (1, 2), and stomachs are typically engorged with food (M. Koban, unpublished observations); additionally, hepatic and skeletal muscle glycogen levels are depleted within the first few days of SD (35).
Food intake behavior is governed by a complex array of central neurotransmitters, hypothalamic and brain stem neuropeptides, and peripheral peptides. For example, within the hypothalamic arcuate nucleus (Arc), neuropeptide Y (NPY) is the most powerful orexigen known; its less potent counterpart, also in the Arc, is the anorexigenic neuropeptide, -MSH, a cleavage product of proopiomelanocortin (POMC) (36, 37). For this study, our goal was to chronically deprive rats of rapid eye movement (REM) sleep to test the hypothesis that hypothalamic NPY and POMC gene expression would be significantly increased and decreased, respectively, to explain the hyperphagic response. Furthermore, because there are elevations in plasma ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), we reasoned that CRH would be up-regulated within the hypothalamic paraventricular nucleus (PVN) to activate the HPA axis.
Materials and Methods
Animals
Male Sprague-Dawley rats (4–5 months of age; Harlan, Indianapolis, IN) were housed individually in standard shoebox plastic cages with ad libitum chow (TekLad, Madison, WI) and water; ambient temperature averaged 24 C and photoperiod was a 12-h light, 12-h dark cycle with lights on at 0900 h. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Morgan State University, and they comply with National Institutes of Health guidelines.
For 2 wk, rats were accustomed to routine handling and the novel environment of the SD tanks (see below) by placement onto the platforms for approximately 1 h each day. Body weights were obtained and 24-h food consumption was estimated by weighing leftover chow. These data were gathered daily from 0900–1000 h, during 1 wk of baseline and up to 20 d of experiments. Food intake was normalized as grams per day per kilogram body weight taken to the 0.67 power to compensate for differences in metabolic rate as a function of body mass (38). No corrections were made for scattered crumbs because they were continually washed out.
SD paradigm
The SD paradigm employed in this study is known as the platform (i.e. flowerpot) method. A rat resides on a small circular platform (e.g. inverted flowerpot) surrounded by water. Rats have easy access to food and water (standard water bottle); they can groom themselves, engage in limited exploratory behavior, and rest by lying down. When a rat lapses into REM (paradoxical) sleep, muscle atonia causes it to make facial contact with or to fall into the water, abruptly awakening it to continuously enforce the paradigm. The platform method is therefore selective for significantly abolishing REM sleep. Electroencephalography studies have validated its effectiveness, although slow-wave sleep can also be disrupted (39, 40, 41, 42).
Two large Plexiglas SD tanks are each divided into five separate compartments of 30 x 30 x 40 cm. Each compartment has a 10-cm high column onto which a 10-cm diameter Plexiglas platform is attached. For most REM-SD studies of approximately 96 h duration, smaller platforms (6–7 cm) are typically used (3, 4, 5, 7, 15, 16, 19, 20, 21, 22, 23, 43); however, beyond this time point, we have noted that rats become completely exhausted. Ten-centimeter platforms thereby provide more space and mobility and minimize confinement as a possible restraint-like stressor. For this study, rats were sleep-deprived for 5 (n = 6), 10 (n = 6), or 20 (n = 5) d. Six rats in their home cages served as controls.
Inlet and outlet ports on opposite ends allow continuous water flow to carry away waste and debris and floods each chamber to 1 cm below the platform surface. Rats increasingly experience water immersions after approximately 2 wk so water temperature was adjusted to approximately 30 C to lessen cooling effects.
Tissue preparation
On the final day of REM-SD, all the animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, ip), administered heparin (100 U) directly into the heart, and perfused transcardially with saline containing 2% sodium nitrite followed by 2.5% acrolein in buffered 4% paraformaldehyde (44). The brains were removed and sunk in 30% sucrose solution, frozen, and sectioned at 25 μm on a freezing sliding microtome into 1-in-12 series. The sections were collected in cryoprotectant antifreeze solution (45). Sections were stored at –20 C until they were processed for immunocytochemical analysis of CRH, -MSH, ACTH1–25, and NPY or for in situ hybridization (ISH) analysis of mRNAs for CRH, NPY, and POMC.
Immunohistochemistry
The immunocytochemical method used for staining NPY, POMC derivatives (-MSH or ACTH1–25), and CRH immunoreactivity has been published previously (44, 46). Briefly, the sections were removed from the cryoprotectant, combined to form a 1-in-6 series, rinsed in potassium PBS (KPBS) at 0.05 M (pH 7.4), treated with 1% sodium borohydride (Sigma Chemical Co., St. Louis, MO) for 10 min, rinsed, and then incubated with rabbit anti-NPY (1:70,000) (Sigma), rabbit anti-CRH (1:50,000) (gift from Dr. John Olschwaka, University of Rochester), rabbit anti-ACTH (1:200,000) (AC-6; gift from Dr. Roger Guillemin, Salk Institute), or rabbit anti--MSH (1:30,000) (Immunonuclear, Stillwater MN; catalog no. 12288, lot 8410018) in KPBS with 0.4% Triton X-100 for 48 h at 4 C. After rinsing, the tissue was incubated for 1 h at room temperature in biotinylated goat antirabbit IgG (heavy and light chains; Vector Laboratories, Burlingame, CA) at a concentration of 1:600 in KPBS with 0.4% Triton X-100, rinsed, and of incubated 1 h in avidin-biotin complex solution at room temperature (ABC Elite Kit; Vector Laboratories) (4.5 μl of A and B/ml KPBS with 0.4% Triton X-100 incubation mixture). After rinsing in KPBS and then in 0.175 M sodium acetate (NaOAc) solution, the antibody-peroxidase complex was visualized with nickel sulfate (25 mg/ml), 3,3'-diaminobenzidine HCl (Ni-DAB; 0.2 mg/ml), and H2O2 (0.83 μl of a 3% solution/ml) in 0.175 M NaOAc. Staining times were approximately 15–20 min. The tissue was transferred to NaOAc to stop the reaction, rinsed in KPBS, placed into 0.9% saline, and mounted onto gelatin subbed slides. The slides were dried overnight, dehydrated through alcohols, cleared, and coverslipped.
ISH: probe preparation
NPY.
Briefly, the NPY cDNA construct (gift from Dr. A. Sahu, University of Pittsburgh) contains an insert that included most (residues 1–511) of the sequenced cDNA of rat brain NPY ligated into the EcoRI site of the Bluescribe M13 vector (47). For the antisense NPY riboprobe, the plasmid was linearized with FspI and transcribed with T3 RNA polymerase to yield a probe of 511 bp of the rat NPY cDNA. For the sense-strand mRNA, the DNA was linearized with SmaI and transcribed with T7 RNA polymerase.
POMC.
The POMC cDNA construct (gift of Dr. S. J. Watson, University of Michigan) contains 666 bp ligated into pGEM 4Z. For antisense POMC riboprobes, the plasmid was linearized with EcoRI and transcribed with T7 RNA polymerase to yield a probe complementary to nucleotides 89–755 of the rat POMC DNA. For sense strands, the DNA was linearized with HindIII and transcribed with Sp6 RNA polymerase.
CRH.
The CRH cDNA construct (gift of Dr. Paul Sawchenko, Salk Institute) contains 1197 bp ligated into Bluescript SK. For antisense CRH riboprobes, the plasmid was linearized with HindIII and transcribed with T7 polymerase to yield a probe complementary to nucleotides 1–1997 of the CRH cDNA. For sense-strand cRNA, the DNA was linearized with SmaI and transcribed with T3 polymerase.
For all probes, the in vitro transcription reaction consisted of 50 μl mixture containing 2 mM biotin-CTP (Life Technologies, Gaithersburg, MD), 3 μg linearized CRH, NPY, or POMC, 5 mM dithiothreitol, 40 U RNAsin ribonuclease inhibitor (Promega, Madison, WI), 40 U T7 RNA polymerase (Life Technologies), 0.5 mM CTP, and 1 mM each ATP, GTP, and UTP. The transcription reaction was developed at 37 C for 90 min and stopped by the addition of 1 μl of EDTA.
ISH: hybridization and detection
The general strategy for the ISH reactions involved hybridization with a biotinylated riboprobe, followed by detection of the biotin using a slight modification of the avidin-biotin complex (ABC) method initially described by Berghorn et al. (46) that lowered the concentration of the chromogen-peroxide solution as described below. The method reveals clusters of specific mRNAs within the cells, and these are easily quantified by counting the RNA clusters in each cell (46) or determining the OD of each cell.
Day 1 (RNase free).
Freely floating 25-μm tissue sections from two 1-in-12 tissue wells combined to form a 1-in-6 series of sections encompassing the entirety of the hypothalamic Arc for NPY and POMC, and the PVN for CRH, were removed from the cryoprotectant antifreeze, rinsed in KPBS made with 0.1% diethylpyrocarbonate-treated water (DEPC H2O), and then incubated in 1% sodium borohydride/KPBS and DEPC H2O for 15 min to remove residual aldehydes derived from the paraformaldehyde-acrolein fixation. Sections were then rinsed repeatedly to remove the sodium borohydride solution, and the ISH procedure was initiated. The tissue was rinsed in 0.1 M triethanolamine buffer (pH 8.0) twice and then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine buffer for 10 min at room temperature. Next, the tissue was rinsed in 2x SSC for 10 min at room temperature. Sections were prehybridized for 2 h at 37 C by using hybridization buffer [50% deionized formamide, 10% dextran sulfate, 1x Denhardt’s solution, 300 mM NaCl, 8 mM Tris (pH 8.0), 0.8 mM EDTA, and 15% DEPC H2O] containing heat-denatured torula yeast RNA (2 mg/ml) (Ambion, Austin, TX). Sections were rinsed with 2x SSC for 10 min. After these rinses, the sections were hybridized with biotinylated NPY, biotinylated POMC, or biotinylated CRH riboprobes (final probe concentration was 600 ng/kb/ml). Probes and torula yeast RNA (0.5 mg/ml) were denatured by heating to 85–90 C for 3 min, put on ice for 5 min, mixed with hybridization buffer, and placed into polystyrene microbeakers containing the sections. The hybridization was continued overnight at 37 C.
Day 2.
Tissue was rinsed with 4x SSC for 30 min, rinsed once with RNase buffer [10 mM Tris (pH 8.0), 500 mM NaCl, 0.75 mM EDTA) heated to 37 C, followed by a 30-min incubation of RNase (20 μg/ml) in RNase buffer at 37 C. Sections were rinsed with RNase buffer and then incubated in RNase buffer for 30 min at 37 C. After 40 min of rinsing in 2x SSC and one rinse with 0.1x SSC (heated to 42 C), sections were incubated in 0.1x SSC for 60 min at 42 C. Tissue was rinsed once with 0.1x SSC at room temperature followed by 1 h of rinsing with KPBS. Sections were then incubated in goat antibiotin (Vector Laboratories) at a concentration of 1:100,000 in KPBS with 0.4% Triton X-100 at 4 C for 48 h.
Day 4.
Sections were rinsed in KPBS and then immersed with rabbit antigoat secondary antibody (Vector Laboratories) at 1:600 in KPBS with 0.4% Triton X-100 at room temperature for 1 h. The tissue was then rinsed and placed into avidin-biotin complex solution (ABC Elite Kit; Vector Laboratories) for 1 h at room temperature. Sections were rinsed in KPBS, followed by rinses with 0.175 M NaOAc. The biotinylated probes were visualized using nickel sulfate (12.5 mg/ml), 3,3-diaminobenzidine HCl (Ni-DAB; 0.1 mg/ml), and H2O2 (0.42 μl of a 3% solution/ml of final mixture in 0.175 M NaOAc). This solution is one half the concentration of Ni-DAB that normally is used for ABC immunocytochemistry to keep background staining to a minimum. Staining proceeded for approximately 15–20 min. The reaction was stopped by rinsing the tissue with the NaOAc solution. After rinsing in KPBS, sections were placed into saline and mounted onto gelatin subbed slides. After drying overnight, the sections were dehydrated in graded ethanol solutions, cleared in Histoclear, and coverslipped with Histomount. mRNA was visualized as small clusters within the cell cytoplasm. At the electron microscopic level, these clusters accurately represent the ribosomal clusters in the cytoplasm.
Data analysis
Assessment of changes in ACTH, -MSH, and NPY immunoreactivity were qualitative and based mainly on changes in the ability to detect cells and their intensity. For NPY in particular, the fact that the entire Arc contained abundant axon profiles obscured the ability to count weakly labeled neuron perikarya in control animals, and it is for this reason that descriptions of immunocytochemical data for NPY and POMC products were mainly qualitative.
For CRH, with the antibody we used, without colchicine pretreatment, the detection of neurons immunoreactive for the peptide serves as a useful tool for reflecting levels of protein expression. To analyze the CRH peptide, images from each side of the PVN that encompassed all the CRH cells were captured using a x10 objective and a Nikon Optiphot-equipped Edge microscope (Edge Corp., Santa Monica, CA) linked to a Retiga (EX) digital camera (Biovision Technologies, Exton, PA). Analysis was performed on a Macintosh G4 and used Signal Analytic’s IP Spectrum software (Vienna, VA). The borders of the PVN at the level of the medial parvocellular division were outlined as the region of interest. All detectible cells were segmented, and the area occupied by CRH immunoreactive cells for each nucleus determined. Use of this approach avoided problems of overlapping and partial cells. By setting a minimum area for the structures analyzed, the thin fibers that coursed from the cells were not included in the analysis unless they were in direct contact with an immunoreactive soma. Because the most prominent fibers were outside the borders of the portion of the PVN assayed, those few axons did not greatly influence the measures taken. The analysis also included determination of the perikaryal staining intensity. This measure was accomplished by the determination of the mean gray level of the selected cells minus the background selected from a region adjacent to the PVN where no CRH cells or fibers were located.
With the method we used for ISH of CRH, POMC, and NPY mRNA, clusters of RNA were distributed within the cytoplasm of the cells, and this feature enables the level of expression to be reliably quantified based on comparisons with 35S-labeled riboprobes (46) by RNA cluster image analyses and with solution hybridization analyses (48). Electron microscopic examination of these clusters validates their interpretation as ribosomal clusters (49) and reinforces their utility for quantitative assessments. The advantage to using this approach in freely floating fixed sections is that the same sections can be used for ISH analysis and immunocytochemistry. A pilot study determined that use of each cells’ OD level was equally able to detect accurate differences in the level of expression and, when clusters were numerous, avoided problems of multiple grains that came into contact with each other being counted as single (large) grains. Multiple images were captured with a x100 objective on each side of the parvocellular division of the PVN for CRH and on each side of the Arc for NPY or POMC, to encompass the entirety of the region in which cells were located. A stack of images in the Z plane at 0.2-μm intervals was captured for each field and flattened to visualize the total mRNA clusters present in each cell. The background value of the surrounding tissue (devoid of clusters) was subtracted from each cell, and the OD (mean gray level minus background value) of the cell cytoplasm bearing mRNA clusters was determined for each cell. Borders of the cell’s cytoplasm were determined by the limit of the presence of scattered mRNA clusters. Values were averaged for all the cells detected in each animal. Data are expressed as mean OD ± SEM.
Statistical analysis
Baseline food intake and body weight were averaged (± SEM) and assigned a value of 100%. Changes in these quantities during REM-SD are shown as percent change from baseline. Nonparametric Kruskal-Wallis (KW) testing was used for analysis of food intake and body weight, followed by Dunn’s posttest. For analysis of expression of the peptides, ANOVA with post hoc analysis using Hsu’s test were performed. The relationship between the levels of POMC or NPY expression and days of REM-SD was determined with Spearman nonparametric correlation analysis. Overall, a value of P < 0.05 was interpreted as a significant difference.
Results
Our data show that long-term REM-SD resulted in stereotypical changes in food consumption and body weight (Fig. 1). Within 5 d of REM-SD, there was a significant increase in food consumption that was maintained over the time course of the experiment (Fig. 1A) (KW = 66.454; P < 0.0001) and peaking at about 300% of baseline. Accompanying this hyperphagia was a progressive decrease in body weight so that by d 15, rats had lost almost 25% of their mass (Fig. 1B) (KW = 151.87; P < 0.0001). Some of the data for d 15–20 were lost, accounting for the gaps.
CRH
Staining of CRH peptide in the PVN was affected by REM-SD. CRH neurons normally synthesize the peptide and transport it rapidly from the neurons. Consequently, CRH neuronal staining in home cage control animals was often not easily detected or was weak (Fig. 2A). Five and 10 d of REM-SD produced striking increases in the detection of CRH within PVN perikarya (Fig. 2, C and E). As a percentage of the parvocellular PVN, there was more than a 10-fold increase in the area occupied by CRH immunoreactive cells by 5 d of REM-SD, and this increase was maintained for up to 20 d (Fig. 3A). In addition, within the neurons detected, the intensity of CRH immunoreactivity increased over the first 10 d (Fig. 2, C and E), but then at 20 d, it became more variable with rats showing signs of advanced morbidity, which is characteristic of chronic SD (6, 10).
The mRNA data reflected that seen for peptide expression. Home cage controls showed extremely low levels of CRH mRNA within the parvocellular neurons with only scattered mRNA clusters noted (Fig. 2B), yielding low OD for the cells. REM-SD increased the level of expression of CRH mRNA within the parvocellular PVN neurons (Fig. 2, D and F). By d 5, CRH mRNA expression increased by nearly 70% over control values (Fig. 3C) (P < 0.01), and by d 10, although still maintaining higher levels than controls (P < 0.05), CRH mRNA declined somewhat, just as had been noted for the CRH peptide. After 20 d of REM-SD, CRH mRNA levels became more erratic.
NPY
Expression of NPY mRNA was low in home cage controls and significantly increased over the course of REM-SD. At baseline, the clusters of mRNA in the Arc were easily detected (Fig. 4, A and E) but were of low density. Striking progressive increases for mRNA per cell (Figs. 4, B–D and F–H), reflected in the changes in NPY OD (Fig. 5), were noted with REM-SD. The mRNA expression doubled by d 5 (P = 0.0404) and then reached a 4-fold peak after d 20 (P < 0.0003 compared with control animals) (Fig. 4). Regression analysis indicated that the levels of mRNA increased with length of REM-SD (Spearman = 0.5769; P = 0.0040).
NPY immunoreactivity also showed changes, although these were not easily quantified. As REM-SD was enforced, the detection of NPY neurons in the Arc became more apparent and by d 10–20, NPY neurons were quite easily distinguished from the abundant axon profiles found in the nucleus (Fig. 6).
POMC
In home cage controls, scattered neurons expressing the products of the POMC gene (ACTH1–24 and -MSH; Fig. 7, A and C) were located in the Arc and the ventral hypothalamic areas lateral to the Arc. Staining of either ACTH1–24 or -MSH suggested reduced levels of expression in that after long-term REM-SD, fewer perikarya with either of the POMC-related products were detected compared with controls (Fig. 7, B and D). As with the other peptides examined, POMC mRNA levels changed in a fashion consistent with observed immunocytochemical data. A gradual reduction in POMC mRNA was noted over the course of REM-SD (Fig. 8). Initially, mRNA levels were high and then declined in a linear fashion with REM-SD (Fig. 9) (Spearman = –0.5769; P = 0.0067); by d 20, POMC mRNA levels were significantly lower than the controls and had fallen to 56% of control values (P = 0.0109).
Discussion
There are three findings of significance in this study of REM-SD of rats for up to 20 d. First, we show that neurons of the hypothalamic Arc progressively and robustly increase NPY gene expression over the time course of the experiment. Second, gene expression of POMC in the Arc decreased with length of REM-SD. The third point of interest is that CRH gene expression in neurons of the PVN was up-regulated early in the REM-SD regimen and remained elevated.
As noted in the Introduction, REM-SD represents a potent and chronic stressor that gives rise to a number of pathologies. Toward the end of our experiment, rats were showing signs of advanced morbidity (see also Refs.6 and 10), which likely accounts for more variable gene expression of CRH by d 20 compared with earlier time points. Peptide expression, based on the ability to retain CRH within the PVN neurons, did not return to baseline in the d 20 group, indicating that feedback mechanisms for down-regulating CRH while attempting to restore equilibrium to the CRH cells were only partially effective. Studies by Lopez et al. (50) showed that repeated unpredictable stress to rats results in persistent up-regulation of CRH because of loss of glucocorticoid receptor function in the hippocampus. It is possible that similar mechanisms in REM-SD promote CRH expression in the face of elevated corticosteroid levels. Another possibility is that upon prolonged REM-SD, responsiveness of the HPA axis is abnormal because of loss of cooperative effects of vasopressin on the corticotrophs (51) and that some animals experience falls in corticosteroid levels as death nears. Additional study of the central nervous system regulation of the HPA is needed to resolve this issue.
The reciprocal changes in hypothalamic NPY and POMC are consistent with SD-associated hyperphagia and the rapidly developing state of negative energy balance. Moreover, the marked decline in circulating leptin that occurs within a few days of total- or REM-SD (9, 13, 17) is in accord with increased food consumption. Taken together, the patterns of change in hypothalamic NPY and POMC and serum leptin agree with current models of the regulation of food intake (37, 52). Interestingly, when human volunteers are sleep-deprived for 88 h, rapid and sustained changes in the diurnal pattern of circulating leptin occur (53). The authors relate their findings to the so-called night-eating syndrome (54), and similar changes in eating patterns may partially explain total- or REM-SD hyperphagia in rats.
A difficulty develops, however, when our results are evaluated in the context of the stress response and with the present understanding of the circuitry and hierarchy of hypothalamic neuropeptides involved in feeding and energy metabolism. The issues are complex, and compelling questions arise.
One issue is concerned with the stress response and food intake. There is an abundant body of literature showing that stress blunts appetite. For instance, psychological stressors such as immobilization (32, 34) or social defeat (55) and physical stress caused by lipopolysaccharide administration (56) or electric footshock (57) all result in diminished food intake. Evidence points to increased hypothalamic CRH to be responsible for the anorexia (58, 59). Rats undergoing REM-SD also increase CRH gene expression (this study) and have elevated serum ACTH and corticosterone (7, 15, 16, 19, 20, 21, 22, 23), all of which are well-accepted indicators of activation of the stress response. What is not clear, however, is despite there being a strong central CRH signal that otherwise would be expected to suppress appetite, sleep-deprived rats unfailingly exhibit hyperphagia. This suggests that during REM-SD, the anorexic effect of CRH may be inhibited or overridden by pathways that sense and try to correct the energy deficit of this stressor.
A second issue involves energy metabolism and hyperphagia during total- or REM-SD. The sleep-deprived rat progressively increases energy expenditure and metabolic rate (1, 2, 8, 9), and it makes sense that food intake increases many-fold to fuel the process. In the present study, although we provide evidence that one of the mechanisms sustaining rapidly developing hyperphagia is increased hypothalamic NPY gene expression, there are perplexing results stemming from intracerebroventricular (icv) or PVN administration of NPY. Prominently, acute NPY results in dose-dependent decreases in several indicators of energy metabolism, including sympathetic nerve activity to brown adipose tissue (BAT) (60, 61), binding of GDP to BAT mitochondria (62, 63), UCP-1 mRNA expression (64), and oxygen consumption (63). Under certain physiologically relevant circumstances, persistently increased hypothalamic NPY can be elicited, concomitant with development of a state of negative energy balance. For example, chronically up-regulated hypothalamic NPY occurs with energetically costly conditions such as lactation (65), food restriction or deprivation (66, 67, 68), and cold exposure and acclimation (69). Complementary to acutely applied NPY studies (60, 61, 62, 63, 64), lactation leads to decreased regulatory thermogenesis (70) with down-regulation of uncoupling protein gene expression (71) and decreased peripheral norepinephrine turnover (72). Of significant interest is that cold acclimation brings about hyperphagia in rats (73, 74, 75) with elevated NPY in the hypothalamic Arc (69), but in sharp contrast to acute NPY studies, there is increased sympathetic activity (76) and circulating thyroid hormones (77, 78), and most importantly, thermogenic capacity is markedly elevated, together with increased resting metabolic rate (77, 78, 79). Whereas studies of acute NPY administration and of lactation suggest that up-regulated hypothalamic NPY should dial down thermogenesis during REM-SD, instead, it becomes elevated. This scenario is not readily explained by our current understanding of the neurocircuitry governing food intake and energy metabolism, and unraveling this problem poses a challenging puzzle.
The flip side of this matter involves POMC, the counterpart to NPY in the hypothalamic Arc. As with NPY, POMC has an important role in energy metabolism and food intake. Physiological conditions that demand increased food intake, such as during lactation, stem from increased NPY and decreased POMC (80). Our finding of a decline in Arc POMC with time of REM-SD further explains the hyperphagic response, but similar to elevated NPY, the low POMC levels fail to account for increased REM- or total-SD-associated metabolism. That is, if POMC, its cleavage end-product, -MSH, or various agonists or antagonists are administered icv, there is evidence showing that the melanocortin system stimulates energy metabolism (81, 82). Although the pattern of POMC gene expression is entirely in agreement with food intake behavior during chronic REM-SD, it is opposite to what occurs metabolically in the periphery.
CRH also has a role in the regulation of energy metabolism because when the peptide is applied centrally, there is an increase in BAT thermogenesis (83, 84, 85). What this means is that CRH stimulation of pathways emerging from the PVN, which raise the level of sympathetic activity to increase BAT thermogenesis (86), remains intact. Undoubtedly, it is likely that there are complex (and for the moment, perplexing) interactions between CRH, NPY, and POMC neurons in the hypothalamus and other regions such as the brain stem, that, when clarified, will explain their opposing actions on food intake and energy metabolism.
As the circuitry governing sleep/wakefulness and interactions with energy metabolism unravels, additional players enter the scene that could explain the metabolic sequellae observed after REM-SD. For example, a mechanism involving orexin A and orexin B is certainly likely. Studies on orexins have mainly focused on their role in sleep/arousal (87), but central administration of orexin A increases metabolic rate in anesthetized rats (88, 89), although some of its effect on thermogenesis may be attributed to stimulation of spontaneous physical activity (90). Orexin fibers innervate the intermediolateral cell columns in the spinal cord (i.e. the preganglionic sympathetic fibers) (91), and stimulation of these neurons is believed to be one site where orexin regulates metabolism. Indeed, administration of orexin increases excitability of sympathetic preganglionic neurons in vivo and in vitro (92, 93), and it results in increased sympathetic tone and cardiovascular function (94). Moreover, upon awakening, the activity of orexin neurons increases (95), and quite interestingly, REM-SD for 96 h produces a near doubling of orexin levels in the cisternal cerebrospinal fluid of rats (96). At this point, it is premature to conclude that orexin is responsible for enhancing sympathetic tone to increase metabolic rate in total- or REM-SD. Nevertheless, a broader view of the literature shows all ranges of effect with orexins. As an example, icv-administered orexin A was recently shown to decrease sympathetic nerve activity to BAT (whereas orexin B increased it) (97), an effect not in agreement with the increased metabolic rate with icv- or Arc-administered orexin A (88, 89). With the knowledge that orexin neurons project widely in the central nervous system (98), it is now critical to determine whether selective projections of the orexin system play a major role in metabolic stimulation during REM-SD. Although we have only begun to explore the possible changes in the orexin system after REM-SD in our model, preliminary data suggest that it is up-regulated.
In summary and conclusion, we provide evidence that the stereotypical hyperphagia of chronically sleep-deprived rats is mediated, at least in part, by up-regulation of NPY and down-regulation of POMC in the hypothalamic Arc. That REM-SD constitutes a stressor is demonstrated by increased gene expression of CRH within the PVN. Although these changes are in agreement with current models of food intake regulation, there remain thought-provoking disconnects between the role of these neuropeptides in food intake, the stress response, and peripheral thermogenesis.
Acknowledgments
We thank Kevin L. Swinson for his valuable technical assistance.
Footnotes
This study was funded by National Institutes of Health Grants 2-SO6-GM-51971-04 and G12-RR-17581-02 (to M.K.) and NS 43788 (to G.E.H.).
First Published Online October 6, 2005
Abbreviations: Arc, Arcuate nucleus; BAT, brown adipose tissue; DAB, 3,3'-diaminobenzidine; DEPC, diethylpyrocarbonate; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular; ISH, in situ hybridization; KPBS, potassium PBS; KW, Kruskal-Wallis; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; REM, rapid eye movement; SD, sleep deprivation.
Accepted for publication September 23, 2005.
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