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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Feb 3, 2012.
Published in final edited form as:
PMCID: PMC3155688
NIHMSID: NIHMS315875

Inflammation-induced lethargy is mediated by suppression of orexin neuron activity

Abstract

In response to illness, animals subvert normal homeostasis and divert their energy utilization to fight infection. An important and unexplored feature of this response is the suppression of physical activity and foraging behavior in the setting of negative energy balance. Inflammatory signaling in the hypothalamus mediates the febrile and anorectic responses to disease, but the mechanism by which locomotor activity (LMA) is suppressed has not been described. Lateral hypothalamic (LHA) orexin (Ox) neurons link energy status with LMA, and deficiencies in Ox signaling lead to hypoactivity and hypophagia. In the present work, we examine the effect of endotoxin-induced inflammation on Ox neuron biology and LMA in rats. Our results demonstrate a vital role for diminished Ox signaling in mediating inflammation-induced lethargy. This work defines a specific population of inflammation-sensitive, arousal-associated Ox neurons and identifies a proximal neural target for inflammatory signaling to Ox neurons, while eliminating several others.

Keywords: orexin, lethargy, inflammation, hypothalamus, cachexia, arousal

INTRODUCTION

The maintenance of energy homeostasis is crucial to ensure long-term survival. Normally, energy intake and expenditure are precisely controlled, stabilizing body weight over an animal’s lifetime. Because infectious illness is an acute threat to life, systems have evolved that sacrifice energy homeostasis in order to provide the greatest chance of survival. Indeed, all of the behaviors and metabolic changes typical of sickness, including fever, anorexia, and lethargy, are linked directly to a highly organized strategy of reassigning biological and physiological priorities to fight infection (Dantzer, 2001).

During disease locally produced inflammatory signals alter the activity of hypothalamic homeostatic systems. For example, fever is caused by prostaglandin E2-mediated activation of cold-sensitive neurons in the preoptic area (Gordon and Heath, 1980), while inflammatory cytokines regulate pro-opiomelanocortin (POMC-) and agouti-related peptide (AgRP-) expressing neurons in the arcuate nucleus (Scarlett et al., 2007; Scarlett et al., 2008) leading to anorexia (Grossberg et al., 2010) and increased thermogenesis (Arruda et al., 2010). Sickness behavior is characterized by the suppression of normal locomotor responses to circadian, metabolic, and emotional stimuli, thereby minimizing energy expenditure. These locomotor deficits are not improved by restoring food intake in immune challenged animals (Marks et al., 2001), suggesting that locomotor activity (LMA) is independently regulated during disease. The inflammation-sensitive population of neurons that mediates the lethargic response to sickness has not yet been identified, however.

One set of neurons implicated in the allostatic control of arousal and physical activity is the Ox (hypocretin)-expressing neurons of the perifornical LHA (Trivedi et al., 1998). Ox neurons are activated during waking, stress, exposure to novel environments, and undernutrition (Sakurai, 2007). Ox neurons are also responsive to circulating signals of energy status and mediate the increase in foraging behavior exhibited by food-restricted (FR) animals (Yamanaka et al., 2003; Mieda et al., 2004; Sakurai, 2007). Ox neurons are linked to other motivated behaviors as well, including drug-seeking and sexual behavior (Harris and Aston-Jones, 2006). Taken together, this work demonstrates that orexins serve as a crucial intermediate manipulating arousal in response to metabolic needs, stress, and reward-oriented stimuli.

Several lines of evidence suggest that Ox neurons mediate inflammation-induced lethargy. First, Ox knockout mice exhibit marked hypophagia, hypolocomotion, hypersomnolence, and an inability to adapt to restricted feeding, a phenotype resembling sickness behavior (Chemelli et al., 1999; Mieda et al., 2004). Second, a localized reduction in cFos immunoreactivity (IR) in the LHA has been observed following inflammatory insults, indicating reduced neuron activity in this region (Becskei et al., 2008; Gaykema and Goehler, 2009). Third, LHA glucose-sensitive neurons have reduced electrical activity in the presence of interleukin-1 beta (IL-1β) or tumor necrosis factor-alpha (TNF-α) (Plata-Salaman et al., 1988). In the present work we use lipopolysaccharide (LPS)- and tumor-induced models of inflammation to investigate whether suppression of Ox signaling mediates the lethargic response to sickness. We also evaluate the proximal neural input by which inflammation inhibits Ox neurons and investigate the connection between the central melanocortin and orexin systems.

MATERIALS AND METHODS

Animals and surgical procedures

Male Sprague Dawley (250–450 g; Charles River Laboratories) or F344/NTacfBR rats (200–250 g; Taconic Farms) and Nts-cre/GFP mice (Leinninger et al., 2011) were maintained on a normal 12 h light/dark period with lights on 0600 –1800 h; corresponding to ZT 0–12 at 22–24°C with ad libitum access to food (Purina rodent diet 5001; Purina Mills) and water, unless otherwise noted. Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Oregon Health and Science University or the University of Michigan. The generation of the Nts-cre/GFP mice is detailed by (Leinninger et al., 2011).

Locomotor Activity and Temperature

Voluntary home cage LMA and brown adipose tissue (BAT) temperature were measured using implantable telemetric transponders (MiniMitter). Animals were anesthetized using 2% isoflurane, a small midline incision was made just anterior to the BAT, and transponders were implanted beneath the BAT, with care taken to leave innervation unperturbed. For rats receiving icv injections, transponders were implanted during the lateral ventricle cannulation surgery. Rats were individually housed and allowed to acclimate for at least 5 days before temperature and net movement in x-, y-, and z-axes was recorded in 1 min intervals (Vital View, MiniMitter).

Implantation of lateral ventricle cannulae

Sprague Dawley or F344/NTacfBR rats were anesthetized with 2% isoflurane and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). A sterile guide cannula with obturator stylet (Plastics One) was stereotaxically implanted into the lateral ventricle. The coordinates used were: 1.0 mm posterior to bregma, 1.5 mm lateral to midline, and 4.1 mm below the surface of the skull (Paxinos and Watson, 1998). The cannula was fixed in place with dental cement. The animals were individually housed after surgery for a minimum of 1 wk, and were handled daily for at least 3 d before each treatment.

Dark phase LPS injections

For evening experiments, on the day of the experiment at ZT 10.5, individually housed animals received ip injections of LPS [250 µg/kg (Sigma-Aldrich)] dissolved in 0.5% low-endotoxin BSA (Equitech-Bio) in 0.9% saline or 0.5% BSA in 0.9% saline alone, and were placed in their home cage without food. Behavioral studies were conducted with a crossover design, with activity and temperature monitored throughout the evening. The following morning at ZT 23.5, rats were anesthetized with 2% isoflurane and CSF was collected by percutaneous cisterna magna puncture (Desarnaud et al., 2004). Protease inhibitor cocktail (Roche Diagnostics) was added to the CSF before snap freezing on dry ice and storage at −80°C. Following 5 d of recovery treatment groups were switched and the experiment was repeated. For histology experiments, rats or mice were sacrificed at ZT 13.5 and processed for IHC as described below. Experiments were performed on serial days to minimize variation in time-of-sacrifice between animals.

Central OxA replacement experiments

For the acute OxA replacement experiments, male Sprague Dawley rats were implanted with lateral ventricle cannulae and telemetric transponders. After 5 d of recovery and handling, LPS was administered at ZT 10.5 and food was removed, as described above, followed by icv injection of OxA (1 µg; California Peptide Research) dissolved in aCSF or aCSF alone. In the lethargy prevention experiment (1), icv injections were conducted at ZT 11.5; in the lethargy reversal experiment (2), icv injections were performed at ZT 15. Following injection, rats were returned to their cages and activity and temperature were measured. For the subchronic Ox replacement experiment (3), rats were implanted with Alzet icv brain infusion kits connected to 3-day osmotic minipumps [model 1003D, 1 µl/h (Durect)] on the day before the experiment using the above coordinates for lateral ventricle cannulation. Pumps were filled with OxA dissolved in aCSF (3 µg/µl) or aCSF. During pump assembly and priming, the catheters connecting the minipumps to the cannulae were filled with saline with a small bubble to separate the saline from the drug. These catheters were measured such that the drug began delivery into the brain approximately 1–2 h before onset of the dark phase on the experimental day. During minipump implantation, the distance from the bubble to the cannula was confirmed to ensure appropriate treatment start time. On the day of the experiment at ZT 10.5, rats were injected ip with LPS or veh, as described above, except that food was weighed and returned to the cage. Activity, temperature, and food intake were measured during the subsequent 24 h period.

Central anti-inflammatory treatments

Sprague Dawley rats were implanted with lateral ventricle cannulae and subcutaneous telemetric transponders and allowed to recover for 5 d before the beginning of these experiments. Peripheral LPS treatments were performed as described above with slight modifications. In the central IL-1 blockade experiment, rats were co-treated with icv IL-1ra (4 µg) dissolved in aCSF + 0.5% BSA v. vehicle and ip LPS (250 µg/kg) v. vehicle injection at ZT 10.5 (1630 h). Food was weighed and returned to the cages as a positive control for central drug action. Food intake, body weight, temperature, and activity were recorded. Following 5 d of recovery, rats were crossed-over such that all animals received a different ip and icv treatment and the experiment was repeated. For nonspecific, systemic PG blockade, rats were pretreated with ip indomethacin (indo; 4 mg/kg) dissolved in DMSO v. vehicle at ZT 10 (1600 h). Rats were subsequently treated with ip LPS (250 µg/kg) v. vehicle injection at ZT 10.5 (1630 h). For central COX-2 inhibition, rats were co-treated with icv NS-398 (5 µg) dissolved in 50% DMSO/50% 0.01 M PBS v. vehicle and ip LPS (250 µg/kg) v. vehicle injection at ZT 10.5 (1630 h). In both PG blockade experiments, rats had ad libitum access to food and water and data were collected as described in the IL-1ra experiment. Body temperature was monitored as a positive control for PG inhibition.

Central opioid blockade

Sprague Dawley rats were implanted with lateral ventricle cannulae and subcutaneous telemetric transponders and allowed to recover for 5 d before the beginning of the experiment. Peripheral LPS treatments were performed as described above with slight modifications. Rats were co-treated with icv naltrexone (NTX; 5 µg) dissolved in normal saline v. vehicle and ip LPS (250 µg/kg) v. vehicle injection at ZT 10.5 (1630 h). Food intake, body weight, temperature, and activity were recorded. Following 5 d of recovery, rats were crossed-over such that all animals received a different ip and icv treatment and the experiment was repeated.

RNA preparation and qRT-PCR in FR rats

Male Sprague Dawley rats were allowed ad libitum access to food or FR for 8 d as described in main text. On experiment day, rats were injected ip with LPS (250 µg/kg) v. vehicle at 0730 h (ZT 1.5). One hour later (ZT 2.5) the animals were deeply anesthetized using sodium pentobarbital (65 mg/kg), the thoracic cavity was opened and blood was collected by cardiac puncture of the right ventricle. Blood was stored on ice until the end of the experiment, then plasma was isolated and frozen at −80°C. Rats were then perfused with RNase-free DEPC 0.01M PBS + heparin sodium (15,000 U/L) to flush the vasculature in the hypothalamus. Hypothalamic blocks with median eminence attached were isolated, preserved in RNAlater solution (Ambion, Inc., Austin, TX), and stored at 4°C overnight. RNA was extracted the next day and used for qRT-PCR analysis as described previously (Scarlett et al., 2008). cDNA was synthesized and RT-PCR reactions were run using prevalidated TaqMan master mix and primer-probes (Applied Biosystems, Foster City, CA). Statistical analysis using 2-way ANOVA followed by post hoc analysis using Bonferroni’s corrected t test performed on dCt values. Raw Ct values from 18S endogenous controls were compared between groups to validate observed changes in target genes.

Plasma IL-6 ELISA

Plasma was collected as described above. IL-6 ELISA (R&D Systems, Minneapolis, MN) was performed according to manufacturer instruction. All samples were run at 1:2 dilution as well as 1:200 dilution to ensure concentration was within the dynamic range of the assay.

In situ hybridization histochemistry

Fresh frozen brains were collected and processed for in situ hybridization as previously described (Scarlett et al., 2007) with the following modifications. Hypothalamic coronal sections (30 µm) were collected in a 1:6 series from the diagonal band of Broca (bregma 0.50 mm) caudally through the mammillary bodies (bregma −5.00 mm). Antisense 33P-labeled rat preproorexin (Hcrt) riboprobe (corresponding to bases 18–420 of rat Hcrt; GenBank accession no. NM_ 013179) (0.1 pmol/ml) was denatured, dissolved in hybridization buffer along with tRNA (1.7 mg/ ml), and applied to slides. Controls used to establish the specificity of the Hcrt riboprobe included slides incubated with an equivalent concentration of radiolabeled sense Hcrt riboprobe or radiolabeled antisense probe in the presence of excess (1000X) unlabeled antisense probe. Slides were covered with glass coverslips, placed in a humid chamber, and incubated overnight at 55°C. The following day, slides were treated with RNase A and washed under conditions of increasing stringency. Slides were dipped in 100% ethanol, air dried, and then dipped in NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Slides were developed 6 d later and coverslipped. Blinded counts of the number of silver grain clusters (corresponding to radiolabeled Hcrt mRNA) in each hypothalamic nucleus (user-defined) as well as the number of silver grains in each cell were made using Grains 2.0.b software (University of Washington, Seattle, WA).

Double-label in situ hybridization was used for simultaneous visualization of Hcrt mRNA with IL-1RI, TNF-R, LIF-R, IκBα, MC3-R and MC4-R in the rat brain (n=3) was performed as previously reported (Scarlett et al., 2007), with slight modifications. Tissue was prepared as described above. Antisense digoxigenin-labeled rat Hcrt riboprobe and antisense 33P-labeled IL-1RI (bases 207–930 of rat Il1r1; GenBank accession no. M95578) (0.2 pmol/ml), IκBα (bases 112–922 of rat Nfbia; GenBank accession no. NM_001105720) (0.1pmol/ml) TNF-R (bases 63–1129 of rat Tnfrsf1a; GenBank accession no. NM_013091) (0.2 pmol/ml), LIF-R (bases 785–1645 of rat Lifr; GenBank accession no. NM_031048) (0.2 pmol/ml), MC3-R (bases 90–486 of rat Mc3r; GenBank accession no. NM_001025270) (0.3 pmol/ml), or MC4-R (bases 557–1006 of rat Mc4r; GenBank accession no. NM_013099) (0.3 pmol/ml). Co-expression of radiolabeled and digoxigenin-labeled mRNA was assessed using criteria previously described (Cunningham et al., 2002). Signal-to-background ratios for individual cells were calculated; an individual cell was considered to be double-labeled if it had a signal-to-background ratio of 2.5 or more. For each animal, the amount of double-labeling was calculated as a percentage of the total number of Hcrt-expressing cells and then averaged across animals to produce mean ± SEM.

Perfusion and IHC

For histology experiments, rats or mice were deeply anesthetized using sodium pentobarbital (65 mg/kg), and sacrificed by transcardial perfusion fixation with 150 mL (rats) or 15 mL (mice) ice cold 0.01 M PBS + heparin sodium (15,000 U/L) followed by 200 mL (rats) or 25 mL (mice) 4% paraformadehyde (PFA) in 0.01 M PBS. Brains were post-fixed in 4% PFA overnight at 4°C and cryoprotected in 20% sucrose for 24 h at 4°C before being stored at −80°C until used for IHC. Free-floating sections were cut at 30 µm from perfused brains using a sliding microtome (Leica SM2000R; Leica Microsystems, Bannockburn, IL). Three (rats) or four (mice) sets of sections were generated from the hypothalamus of each brain. Hypothalamic sections were collected from the division of the optic chiasm (bregma −2.0 mm, rats; bregma −1.0 mm, mice) caudally through the mammillary bodies (bregma −5.00 mm, rats; bregma −3.0 mm, mice). The sections were incubated for 1 h at room temperature in blocking reagent (5% normal donkey serum in 0.01 M PBS and 0.1% Triton X-100). After the initial blocking step, the sections were incubated in rabbit anti-c-Fos antibody (PC38; EMD Biosciences, Inc., San Diego, CA) diluted 1:50,000, rabbit anti-cFos antibody (SC-52; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:25,000, and goat anti-OxA (SC-8070; Santa Cruz Biotechnology) diluted 1:1,500 in blocking reagent for 72 h at 4°C, followed by incubation in donkey anti-rabbit Alexa 594 (1:500; Invitrogen) and donkey anti-goat Alexa 488 (1:500; Invitrogen) for 2 h at room temperature. Triple-IF histochemistry was performed as described above with the following modifications. Nes-cre/GFP sections were incubated in blocking reagent as described above, then washed and incubated in the anti-cFos and anti-OxA antisera as well as chicken anti-GFP (Abcam, Cambridge, MA) diluted 1:1000 for 72 h @ 4°C. Sections were then incubated in donkey anti-rabbit Alexa 594, donkey anti-chicken-FITC (Jackson Immunoresearch, West Grove, PA), and donkey anti-goat-DyLight 649 (Jackson Immunoresearch) for 2 h at room temperature. Between each stage, the sections were washed thoroughly with 0.01 M PBS. Incubating the sections in the absence of primary antisera was used to ensure specificity of the secondary antibodies. Sections were mounted onto gelatin-coated slides, coverslipped using Vectashield mounting media (Vector Laboratories, Burlingame, CA), and viewed under a fluorescence microscope (Leica 4000 DM; Leica Microsystems, Bannockburn, IL, or Zeiss LSM710; Carl Zeiss, Inc.).

Peripheral LPS and central IL-1β injections in food-entrained rats

Sprague Dawley rats were implanted with lateral ventricle cannulae and subcutaneous telemetric transponders and allowed to recover for 5 d before the beginning of food entrainment. FR rats were individually housed, handled, and allowed access to food from ZT 4–6 each day for 8 days. Food intake, body weight, activity, and temperature were recorded daily. On day 9 rats were injected ip with LPS (250 µg/kg) or veh (LPS experiments) or injected icv with IL-1β [100 ng; (R&D Systems)] dissolved in aCSF + 0.5% BSA or veh (IL-1β experiments) at ZT 1.5. In the behavioral studies, all animals then received icv injections of OxA (1 µg) or aCSF at ZT 3. Food was placed in the cages from ZT 4–6 and food intake, body weight and activity were recorded. FAA was measured during the 60 min preceding food presentation. For histology studies, rats were sacrificed at ZT 3.5 by transcardial perfusion fixation as described below. Experiments were performed on serial days to minimize variation in time of treatment and sacrifice between animals.

Tumor implantation

Male F344/NTacfBR rats were individually housed, and divided into four age and weight-matched groups: sham-operated controls treated with aCSF; tumor-bearing rats treated with aCSF; tumor-bearing rats treated with AgRP; and sham-operated rats that were pair fed with the tumor-bearing/aCSF group. Seven days prior to tumor implantation, animals had lateral ventricle cannulae and biotelemetry devices implanted in a single surgery. On d 0, the animals were anesthetized with 2% isoflurane, and fresh tumor tissue (0.2–0.3 g) from a rat donor was implanted subcutaneous into the flank of the rat as previously described (Scarlett et al., 2008). The tumor is a methylcholanthrene-induced sarcoma that does not metastasize and induces anorexia, cachexia, and lethargy (Smith et al., 1993). Beginning on d 10 after surgery, rats received 5 µl icv injections of aCSF or AgRP (1 nmol) daily at ZT 10. On d 13 after tumor implantation, tumor growth had fallen within the predetermined endpoints of the study, according to the Oregon Health & Science University Institutional Animal Care and Use Committee Policy on Tumor Burden, and the animals were killed. The brains were immediately removed and frozen on dry ice. Brains were stored at −80°C until in situ hybridization analysis.

OxA radioimmunoassay (RIA)

OxA concentration was measured in rat CSF samples, collected as described above, using a commercially available RIA kit (Phoenix Pharmaceuticals). RIA was performed in duplicate on 25 µl CSF samples according to manufacturer instructions. The lowest detectable level was 22.47 pg/mL. The interassay variability (assessed by replicate analysis of a 250 pg/mL standard) was 0.31%.

Image processing and statistical analysis

Confocal photomicrographs between matched brain sections were taken using a Zeiss LSM710 (Carl Zeiss, Inc.) under identical microscope conditions. Images were merged with NIH ImageJ software. Photomicrographs were only collected to show representative images. Data were graphed and analyzed using GraphPad Prism 5. Comparisons between two groups at a single time point were performed using two-tailed Student’s t test. Comparisons between three or more groups used a one-way ANOVA with post-hoc Bonferonni corrected t test. All comparisons involving two treatments or multiple time points used two-way ANOVA followed by post-hoc Bonferonni corrected t test. Differences between groups were considered significant when p < 0.05.

RESULTS

LPS-induced inhibition of home cage LMA is associated with suppressed dark phase Ox signaling

Diminished voluntary home cage LMA is a hallmark behavior associated with both sickness and insufficient Ox signaling. To investigate a possible connection, rats were injected with LPS (250 µg/kg, n = 7) or vehicle (veh, n = 10) 90 min prior to lights off (Zeitgeber Time [ZT] 10.5). We observed nearly complete inhibition of the normal dark phase LMA in LPS-treated animals (Figure 1A). This experiment was repeated with the addition of CSF collection either at lights off or just before lights on, at the zenith of CSF Ox concentration (Desarnaud et al., 2004). We measured a significant decrease in CSF OxA concentration in LPS-treated animals (n = 6) compared to veh (n = 5) at ZT 0, suggesting reduced Ox release throughout the dark phase in these animals (LPS, 887 ± 78 pg/mL; veh, 1720 ± 130 pg/mL; p < 0.001), though no significant change was observed at the onset of the dark phase, during the nadir of OxA concentration (Figure 1B). This is unlikely a transcriptional effect, as no change in Ox mRNA expression was observed 1 h or 8 h after LPS administration (mRNA quantity relative to vehicle; LPS 1 h, 0.98 ± 0.1-fold; LPS 8 h, 0.97 ± 0.1-fold; n.s.).

Figure 1
LPS-induced inhibition of home cage LMA is associated with reduced dark phase accumulation of OxA in the CSF

In nocturnal rodents, the activity of Ox neurons increases during the dark phase, coincident with increased Ox release and increased wakefulness and activity (Estabrooke et al., 2001; MartÌnez et al., 2002). We examined whether LPS administration just before onset of the dark phase suppresses the vespertine induction of cFos IR in Ox neurons. LPS-treated rats (n=4) exhibited significantly reduced cFos IR in Ox neurons compared to control (n=3; p < 0.01) (Figures 2A–H). Because anatomically distinct populations of Ox neurons may have different functions (Harris and Aston-Jones, 2006), colocalization was quantified in dorsomedial nucleus (DMH), perifornical area (PFA), and lateral LHA subpopulations (anatomy described in Figure 2H). 2-way ANOVA showed a significant effect of treatment (F(1,15) = 25.35; p < 0.001) and anatomic location (F(2,15) = 87.18; p < 0.001). No change in cFos IR was observed in DMH Ox neurons, however post hoc analysis by Bonferroni t test demonstrated a significant decrease in cFos IR in both PFA (p < 0.01) and LHA (p < 0.05) Ox neurons (Figures 2A–H). No effect of rostrocaudal location on Ox neuron cFos IR was observed. The number of Ox IR neurons counted did not differ between treatment groups (Total, Veh – 1075 ± 159 cells, LPS – 1123 ± 90.1 cells; DMH, Veh – 196.3 ± 48.7 cells, LPS – 189.8 ± 23.6 cells; PFA, Veh – 492.5 ± 38.9 cells, LPS – 546.5 ± 58.9 cells; LHA, Veh – 386.3 ± 97.6 cells, LPS – 386.8 ± 28.3 cells).

Figure 2
LPS treatment blocks the vespertise rise in cFos IR in Ox neurons

Central (icv) OxA replacement prevents the onset of LPS-induced lethargy

To test the hypothesis that a specific inhibition of Ox signaling underlies the reduced LMA exhibited by LPS-treated rats, we undertook a series of icv OxA replacement studies. We found that central OxA administration influences LMA and arousal for approximately 90 min after injection (data not shown), in accordance with previous studies (Hagan et al., 1999). We also found that a 1 µg bolus icv injection of OxA had no significant effect on LMA in healthy rats (data not shown). Because the duration of Ox action is relatively short compared to the behavioral effects of LPS, we tested the effect of Ox replacement in three paradigms: (1) acute LPS-induced lethargy prevention, (2) acute dark phase lethargy reversal, and (3) subchronic (overnight) OxA infusion. In the acute lethargy prevention experiment (1), rats treated with ip LPS or veh at ZT 10.5 were administered icv OxA or aCSF at ZT 11.5, 30 min before onset of the dark period (n = 6/group). While OxA had no effect on LMA in veh-treated rats, OxA restored LMA in LPS-treated rats to near-control levels during the subsequent 90 min (Figures 3A and 3B). Following this period, LPS/OxA rats reduced their activity to the level of LPS/aCSF rats for the remainder of the dark phase.

Figure 3
Central bolus OxA replacement prevents and reverses LPS-induced lethargy

In the lethargy reversal experiment (2), rats were treated with LPS v. veh and subsequently centrally injected with OxA (veh/OxA n=4; LPS/OxA n=6) v. aCSF (veh/aCSF n=3; LPS/aCSF n=3) at ZT 15, 3 h after the onset of the dark phase. We found that LPS inhibited LMA compared to veh and that this was reversed by OxA administration during the 90 min following treatment (Figures 3C and 3D). Post hoc analysis demonstrated significant differences between veh/aCSF and LPS/aCSF groups (p < 0.001) as well as between LPS/OxA and LPS/aCSF groups (p < 0.05).

In the subchronic OxA administration experiment (3), rats were implanted with icv minipumps to deliver OxA (3 µg/h) or aCSF throughout the dark phase, with ad-libitum access to food. This dose elicits no acute or long term effects on body weight, food intake, brown adipose temperature, plasma corticosterone, or fat depot mass in rats (Haynes et al., 1999). Rats were treated with LPS or vehicle at ZT 10.5, as described above, yielding 3 groups – veh/aCSF (n=10), LPS/aCSF (n=7), and LPS/OxA (n=7). Though both LPS-treated groups moved significantly less than vehicle-treated controls, LPS/OxA-treated rats showed an amelioration of this effect throughout the dark phase (Figures 4A and 4B; see Movie 1). Post hoc analysis of dark phase and total 24 h LMA demonstrated a significant effect of OxA treatment on LPS-treated rats (p < 0.05). OxA treatment reinstated normal crepuscular LMA peaks, which are suppressed in the LPS/aCSF rats. OxA treatment had no effect on either food intake or body weight in LPS-treated rats (Figures 4C and 4D), suggesting that the increase in LMA was not due to increased feeding behavior.

Figure 4
Continuous icv OxA infusion ameliorates LPS-induced decreases in LMA

LPS blocks FAA and Ox neuron activity in food-entrained rats

24 h cyclic FR paradigms induce entrainment of biological rhythms to adapt to the period of food availability. This behavior is marked by an increase in LMA in the period immediately preceding food availability [food-anticipatory activity (FAA)] that is dependent on Ox signaling but independent of SCN master clocks, which remain phase locked to the light/dark cycle (Wakamatsu et al., 2001; Mieda et al., 2004). Therefore, we undertook several studies investigating the effect of inflammation on FAA and the concomitant increase in Ox neuron activity. We first evaluated whether ip LPS could inhibit FAA, and if central OxA administration could restore this behavior. As the animals entrained to FR, LMA maxima shifted from two crepuscular peaks to a single activity peak during the hour preceding feeding (Figure 5A). FR rats were injected ip with LPS or vehicle at ZT 1.5 and subsequently treated icv with OxA or aCSF at ZT 3 (n=7–9/group). We found that LPS potently inhibited FAA compared to vehicle treatment, but that OxA treatment restored activity during this period to control levels. OxA treatment had no significant effect on veh-treated rats (Figures 5B and 5C). LPS inhibited food intake, and this effect was not reversed following OxA treatment (Figure 5D). To determine whether Ox neurons are inhibited in this paradigm, the above experiment was repeated without icv injection and rats were sacrificed at ZT 3.5, during the peak of FAA. As a control for the effects of FR, a group of ad-libitum fed, veh-treated rats were sacrificed at the same time (n=4/group). We found that FR significantly increased cFos IR in Ox neurons compared to ad-libitum fed animals, but that LPS treatment significantly reduced the percentage of total Ox neurons exhibiting cFos IR compared to vehicle (F(2,27) = 72.37; p < 0.001) (Figures 5E and 5F). When counted by anatomic location, we found that FR specifically activated Ox neurons in the DMH and PFA, but not in the LHA. The LPS-induced suppression of Ox neuron activity was localized to the PFA regions of neurons (p < 0.001), while no difference was observed between veh- or LPS-treated FR rats in the DMH or LHA populations. No difference in the total number of Ox IR neurons was observed between groups (Total, FR/Veh – 1164 ± 59.9 cells, FR/LPS – 1093 ± 123 cells, Ad-lib - 1173 ± 147 cells; DMH, FR/Veh – 201.8 ± 42.8 cells, FR/LPS – 191.8 ± 35.1 cells, Ad-lib – 210.5 ± 13.1 cells; PFA, FR/Veh – 515.0 ± 70.5 cells, FR/LPS – 469.8 ± 52.5 cells, Ad-lib – 514.5 ± 68.7 cells; LHA, FR/Veh – 447.0 ± 35.4 cells, FR/LPS – 431.0 ± 68.6 cells, Ad-lib – 448.3 ± 87.3 cells). Previous studies demonstrated that calorie restriction dampens cytokine responses to pro-inflammatory stimuli (Lord et al., 1998). Two hours following LPS treatment, we found that the induction of serum IL-6 and hypothalamic inflammatory markers (IL-1β, IL-6, SOCS-3, IκBα) remained intact in FR rats (Table 1).

Figure 5
Central OxA administration restores FAA in FR rats treated with ip LPS or icv IL-1β
Table 1
FR rats exhibit normal inflammatory response to LPS

Central IL-1β blocks FAA and Ox neuron activity in food-entrained rats

To test whether central inflammation in the absence of peripheral inflammation is sufficient to suppress Ox neuron activity and FAA, we measured LMA and food intake in FR rats following icv administration of the inflammatory cytokine, IL-1β. Rats were then treated with central OxA or vehicle as described above (n=5/group). IL-1β treatment potently blocked FAA, and this effect was ameliorated by OxA administration with no effect on food intake (Figures 5G–5J). Two-way ANOVA showed a significant effect of both treatment (F(1,21) = 21.31; p < 0.001) and anatomic location (F(2,21) = 35.72; p < 0.001) on the immunohistochemical (IHC) colocalization of cFos and OxA as well as an interaction between these variables (F(2,21) = 3.886; p < 0.05) (Figure 5J). Post hoc analysis revealed that FR induced cFos IR in DMH and PFA Ox neurons, while IL-1β specifically reduced cFos IR in PFA Ox neurons, with no significant effect on either DMH or LHA populations (PFA, p < 0.001). No significant differences between total Ox IR neurons were observed between treatment groups (Total, FR/Veh – 1158 ± 68.1 cells, FR/IL-1β – 1091 ± 151 cells, Ad-lib - 1190 ± 175 cells; DMH, FR/Veh – 200.0 ± 37.3 cells, FR/ IL-1β – 191.3 ± 43.0 cells, Ad-lib – 217.0 ± 2.0 cells; PFA, FR/Veh – 524.6 ± 75.0 cells, FR/ IL-1β – 452.0 ± 47.4 cells, Ad-lib – 505.3 ± 81.1 cells; LHA, FR/Veh – 433.8 ± 42.5 cells, FR/ IL-1β – 447.7 ± 73.5 cells, Ad-lib – 467.7 ± 95.8 cells). These data demonstrate that central inflammation is sufficient to suppress FAA in an Ox-dependent manner and suggest that central and peripheral inflammatory challenges share a common pathway for suppressing PFA Ox neuron activity.

Ox neurons are not direct targets for inflammatory signaling

Because inflammatory cytokines directly modulate the activity of hypothalamic melanocortin and thermoregulatory neurons, we tested whether Ox neurons also respond directly to cytokines. Using dual-label in situ hybridization, we did not observe colocalization of IL-1RI, LIF-R, or TNF-R mRNA with preproorexin (Ox) mRNA (Figures 6A, 6C, and 6D). Further, we assessed whether Ox neurons receive direct inflammatory input by measuring the expression of IκBα, which is induced in response to activation of the inflammatory NF-κB signaling pathway. Though we observed IκBα expression in the LHA of LPS-treated rats, IκBα did not colocalize with Ox, indicating that Ox neurons do not directly respond to pro-inflammatory signals (Figure 6B).

Figure 6
Ox neurons are not direct targets for inflammatory signaling

Because icv IL-1β is sufficient to suppress FAA in FR rats, we tested whether central IL-1 signaling is necessary for the induction of lethargy by LPS. Although LPS suppressed vespertine LMA compared to vehicle (F(1,18) = 32.64; p < 0.001), co-administration of icv IL-1 receptor antagonist (IL-1ra; 4 µg) with ip LPS at ZT 10.5 (n=5–6/group) had no effect on vespertine (ZT 12–14) LMA in LPS- or veh-treated rats (veh/veh, 2435 ± 170 counts; veh/LPS, 840.0 ± 210 counts; IL-1ra/veh, 1899 ± 253 counts; IL-1ra/LPS, 1027 ± 211 counts; F(1,18) = 2.804; p = 0.11), despite ameliorating LPS-induced anorexia (veh/veh, 25.6 ± 2.4 g; veh/LPS, 8.78 ± 1.9 g; IL-1ra/veh, 22.4 ± 2.5 g; IL-1ra/LPS, 16.6 ± 2.5 g; F(1,18) = 5.397; p < 0.05). We observed that IL-1ra partially restored LMA in LPS-treated rats approximately 8 h following treatment, suggesting a role for IL-1 signaling in propagating the acute behavioral response to LPS.

Several studies have further implicated that prostaglandin (PG) signaling is an essential step in the induction of sickness behavior (Crestani et al., 1991; Serrats et al., 2010). Systemic (ip) pretreatment with the nonselective cyclooxygenase (COX) −1/2 inhibitor indomethacin (4 mg/kg) (n=5–6/group) at ZT 10 potently blocked LPS-induced pyresis (peak Δ T; veh/veh, 1.88 ± 0.2 °C; indo/veh, 1.97 ± 0.2 °C; veh/LPS, 4.13 ± 0.5 °C; indo/LPS, 1.94 ± 0.1 °C; 2-way ANOVA, interaction F(1,18) = 16.76; p < 0.001), but no significant effect on LMA was observed (veh/veh, 2474 ± 321 counts; indo/veh, 2617 ± 406 counts; veh/LPS, 1439 ± 241 counts; indo/LPS, 1793 ± 228 counts). Statistical analysis by 2-way ANOVA demonstrated a significant effect of LPS treatment on LMA (F(1,18) = 8.278; p < 0.05), but no effect of indomethacin and no interaction between treatments. Central pretreatment with the COX-2 specific NS-398 (5 µg) also suppressed the febrile response to LPS, but, like indomethacin, had no significant effect on LMA (data not shown). Together, these data demonstrate that neither central IL-1 signaling nor PG signaling is required to produce lethargy in response to LPS.

LPS-induced lethargy is not melanocortin dependent

These results indicate that the LPS-induced inhibitory input onto Ox neurons is mediated by a proximal, inflammation-sensitive neuron population. We tested whether inflammation-induced activation of central melanocortin signaling could relay this information by assessing the expression of melanocortin receptors on Ox neurons using dual-label in situ hybridization. We found no coexpression of either the type 4 (MC4R) or type 3 (MC3R) melanocortin receptors in cells expressing Ox (Figures 7A and 7B).

Figure 7
LPS-induced lethargy is not melanocortin-dependent

We tested the potential role of increased melanocortinergic tone on the suppression of LMA by administering the non-specific MC3R/MC4R antagonist SHU-9119 (1 nmol) in animals treated with LPS v. veh (n=4–6/group). Food was removed from the cages to control for feeding-induced suppression of LMA. Pharmacologic blockade of melanocortin receptors in LPS-treated animals did not increase LMA, and SHU treatment alone suppressed LMA to levels approximating those of LPS (Figures 7C and 7D). We also tested the effects of melanocortin blockade on LMA and food intake in a tumor-bearing rat model of chronic inflammation. Tumor-bearing or sham-implanted rats were centrally treated with the endogenous MC3R/MC4R antagonist, AgRP (1 nmol) or aCSF during the final 4 d of the experiment. The sham animals were either fed ad-libitum or pair-fed to tumor-bearing animals and were treated with aCSF, yielding 4 groups (Sham/ad-lib, Sham/pair-fed, Tumor/aCSF, Tumor/AgRP; n=3/group). Nightly administration of AgRP did not ameliorate lethargy in tumor bearing rats, despite restoring food intake (Figures 7E and 7F). Again, melanocortin antagonism suppressed LMA in healthy (sham-operated) rats.

Arcuate POMC neurons release both alpha-melanocyte stimulating hormone (α-MSH), which binds MC3R & MC4R, and β-endorphin, which binds the opioid receptors. Opioids inhibit Ox neuron activity via the mu-opioid receptor (µOR), which is expressed by Ox neurons (Georgescu et al., 2003; Li and van den Pol, 2008). Blockade of central opioid signaling by icv pretreatment with the non-selective OR antagonist naltrexone (10 µg) had no effect on vespertine LMA (veh/veh, 2011 ± 130 counts; NTX/veh, 2342 ± 395 counts; veh/LPS, 703 ± 151 counts; NTX/LPS, 809 ± 164 counts) or overnight food intake in LPS- or veh-treated rats (n=6/group).

LHA neurotensin (Nts) neurons are activated during LPS-induced inflammation

LHA Nts-expressing neurons, many of which also express the long form of the leptin receptor (LepRb), provide an inhibitory input on Ox neurons when activated by leptin (Leinninger et al., 2011). Because leptin is closely structurally related to class I helical cytokines (Zhang et al., 1997) and shares intracellular signaling pathways with IL-6 family cytokines (Banks et al., 2000), we tested whether these neurons are also activated during inflammatory challenge. We injected male Nts-cre/GFP mice with LPS or veh (n=4/group) at ZT 10.5 and looked at cFos IR in Nts- and Ox-IR neurons at ZT 12.5. Because many cells in multiple nuclei express Nts, we only counted those cells that overlap with the Ox neuron distribution, where the Nts-LepRb+ cells are located. We found a significant increase in the total number of Nts neurons with cFos-IR as well as the percentage of total Nts neurons co-labeled with cFos in LPS-treated rats, but no difference in total Nts neurons per section between groups (Figures 8A–8H). This corresponded with a significant decrease in cFos IR in Ox neurons following LPS treatment (Figures 8F, 8G, and 8I). A small percentage of GFP-IR cells were also labeled for Ox, (< 1%) and cFos IR was not counted in these cells. This finding is consistent with the hypothesis that LHA Nts neurons are activated during inflammatory challenge and may contribute to the inhibition of Ox neuron activity.

Figure 8
LPS treatment induces cFos IR in LHA NTS neurons while suppressing vespertine activation of Ox neurons in NTS-cre/GFP mice

Tumor-bearing rats exhibit decreased LMA and fewer medial Ox-expressing neurons

Behavioral inhibition has also been described in cancer patients and tumor-bearing animals (Dantzer, 2001; Ryan et al., 2007). We used a syngenic methylcholanthrene-induced sarcoma model to evaluate the role of Ox signaling in the inhibition of volitional activity in tumor-bearing rats. Tumor-bearing animals (n=12) exhibited a steady decline in total daily LMA during days 10–13, when tumor growth is maximal, compared to sham (n=6). On the final day of the experiment (d 13 after implantation), tumor-bearing rats showed significantly less dark phase activity compared to their own baseline activity (average of days 3–5 post-implantation)(Figure 9A) and exhibit a significant decrease in 24 h LMA compared to sham-operated controls (Figure 9B). In situ hybridization showed a significant decrease in the number of Ox-expressing cells in tumor-bearing animals, specifically in the PFA Ox neuron distribution, but not in the DMH or LHA. No difference in grains/cell was observed (Figures 9C–9F). These data indicate that suppression of Ox signaling may play a role in chronic disease, as well, and support the anatomic localization of inflammation-sensitive Ox neurons to the perifornical hypothalamus.

Figure 9
Tumor-induced inflammation inhibits home cage LMA and reduces Ox mRNA expression in the perifornical hypothalamus

DISCUSSION

Despite the ubiquitous suppression of physical activity and arousal in response to illness, the neural mediators of this response are unknown. We now demonstrate that inflammation-induced lethargy is mediated by suppression of normal Ox signaling. We show that crepuscular and FR-associated increases in Ox neuron activity and OxA release are suppressed by inflammatory challenge. Despite robust movement suppression, icv OxA replacement restored LMA in LPS-treated rats in both circadian- and FR-conditions of increased locomotion. This effect is not due to a nonspecific effect of pharmacological OxA administration, as the same dose of icv OxA did not increase LMA in veh-treated rats. Suppression of Ox neuron activity is unlikely mediated by direct inflammatory or melanocortin signaling, but we show here evidence that Nts-expressing GABAergic interneurons may play a key role in mediating Ox neuron inhibition.

Previous studies demonstrate that the medial (DMH/PFA) and lateral (LHA) Ox neuron populations exhibit distinct effects on behavior (Harris and Aston-Jones, 2006). Our cFos IR data support an anatomically based functional dichotomy for Ox neurons, as we observed increases in cFos IR in DMH and PFA, but not LHA Ox neurons during both vespertine and food anticipatory peaks in LMA. We also observed a specific suppression of cFos IR in PFA Ox neurons, more than DMH, in response to LPS or central IL-1β. This finding separates the medial arousal-associated Ox neuron population into inflammation-sensitive (PFA) and inflammation-insensitive (DMH) populations and indicates that the activity of the PFA Ox neuron population is necessary for normal LMA responses to both circadian and non-circadian cues. It is important to note that our division of the Ox neuron distribution into these three anatomic zones is artificial. While the majority of inflammation-sensitive Ox neurons lie in the perifornical hypothalamus, our anatomic zones only approximate the actual division between inflammation-sensitive and –insensitive Ox neurons. Tract tracing has identified differences in innvervation between medial (DMH/PFA) and lateral (LHA) Ox neurons that are consistent with previously reported roles for medial Ox neurons in mediating arousal and lateral Ox neurons influencing reward and feeding (Yoshida et al., 2006). Similar examination of afferents to and projections from DMH and PFA subpopulations may define the circuits underlying arousal modulation and inflammation sensitivity.

Initially, we hypothesized that inflammatory cytokines inhibit Ox neurons by directly binding to these neurons. We found no evidence for the expression of cytokine receptors or the expression of NF-κB response genes following LPS treatment in Ox neurons. Previous reports describe a prominent role for PG signaling in pyresis (Engblom et al., 2003), anorexia (Lugarini et al., 2002), cFos induction (Dallaporta et al., 2007), and HPA activation (Serrats et al., 2010). In accordance with Serrats et al. (2010), we found no effect of COX inhibition on LMA, indicating that LPS-induced lethargy is PG-independent. However, we demonstrated that central IL-1β is sufficient, but not necessary to suppress LMA and Ox neuron activity. Blocking nerve conduction through the dorsal vagal complex (DVC) prevents ip LPS-induced social withdrawal and cFos IR in multiple nuclei (Marvel et al., 2004), identifying the brainstem as a crucial mediator of sickness behavior. Given that the DVC and central IL-1β can each independently mediate lethargy, we postulate that a population of inflammation-sensitive neurons that receive afferent input from the DVC integrates these two inflammatory signals and relays this signal to Ox neurons.

Recently, Leinninger et al. reported that leptin activates GABAergic Nts neurons in the LHA, which then inhibit Ox neuron activity (2011). That LHA Nts neurons can respond to leptin indicates that, like arcuate nucleus POMC neurons, these neurons may be cytokine-sensitive (Scarlett et al., 2007; Grossberg et al., 2010). The LPS-induced decrease in cFos IR in Ox neurons was associated with an increase in cFos IR in LHA Nts neurons, indicating that these interneurons play a common role in the allostatic suppression of LMA by both overnutrition and inflammation. The relationship between increased perifornical Nts neuron activation and decreased Ox neuron activation may not be causative. Furthermore, Nts neurons may inhibit Ox neurons via release of GABA, as with the leptin-responsive neurons. Alternatively the peptide neurotransmitter neurotensin may mediate this effect. Modulation of Ox neuron activity by LPS and leptin both alter the behavioral response to amphetamine (Fishkin and Winslow, 1997; Nakamura et al., 2000; Leinninger et al., 2011). This suggests exploitation of similar circuitry and supports a role for VTA dopamine signaling in mediating the LMA effects of LPS. However, the effects of leptin and LPS are not completely congruent. In contrast to leptin, LPS does not acutely induce Ox mRNA expression. This may be explained by recruitment of unique, but overlapping Ox-regulating pathways by leptin and LPS. Further anatomic and functional studies are needed to elucidate the role of LHA Nts neurons in mediating LPS-induced suppression of Ox neurons.

Restoration of LMA in LPS-treated rats by icv OxA did not influence food intake, despite the well-documented orexigenic properties of OxA. After icv injection, LPS/OxA-treated animals exhibit normal home cage exploratory behavior, including investigation of their food hopper, but, unlike veh-treated rats, these animals seldom consume food (Movie 1). Therefore, the increase in LMA induced by OxA replacement was not simply an increase in feeding behavior, but rather represented a more active animal in lieu of depressed feeding. We previously reported that inflammation induces anorexia by increasing central melanocortin signaling, and that melanocortin antagonism restores feeding (Marks et al., 2001). We found no evidence that Ox neurons receive direct input from melanocortinergic neurons, though we observed MC4R expression in non-Ox neurons throughout the LHA. Melanocortins may, via MC4R, regulate the activity of LHA neurons that influence the Ox system, such as Nts neurons. Specific activation of AgRP neurons using designer receptors exclusively activated by designer drugs (DREADD) potently induces foraging behavior in the absence of food (Krashes et al., 2011). However, AgRP treatment failed to restore LMA in LPS-treated rats suggesting that either an alternative neurotransmitter released by AgRP neurons (e.g. NPY, GABA) coordinates feeding and LMA, or that non-synaptically delivered AgRP acts ectopically to suppress LMA. Thus, in the setting of increased melanocortin tone following LPS administration, OxA provides an insufficient orexigenic stimulus to induce food intake. Coversely, blockade of MC4R has no effect on Ox-dependent suppression of LMA. This marks the first time these two features of sickness behavior have been mechanistically separated.

Though the present work focuses primarily on mechanisms of acute illness-induced lethargy, our TB rat studies suggest that Ox plays a role in the lethargy of chronic disease, as well. TB rats exhibited a decrease in the number of PFA Ox mRNA-expressing neurons, indicating either specific degeneration of these neurons or transcriptional repression of Ox synthesis in these cells. Either phenomenon, if responsible for cancer-associated lethargy, should be sensitive to Ox replacement. That the same neurons appear to be affected by both acute and chronic inflammatory insult indicates a similar mechanism underlies both phenomena. The effective restoration of LMA throughout the dark phase by subchronic OxA infusion in LPS-treated rats demonstrates that Ox replacement is a viable therapeutic avenue for sickness-induced lethargy. Although allostatic suppression of LMA and food intake by inflammation is an important, and reversible, part of the response to acute infection, chronic dysregulation of metabolism leads to wasting, debilitation, and death. We propose that Ox agonists would be useful in chronically ill patients to improve quality of life and independent living.

Footnotes

The authors have no conflicts of interest to report.

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