Influence of feeding status on neuronal activity in the hypothalamus during lipopolysaccharide-induced anorexia in rats

Neuroscience 134 (2005) 933–946

INFLUENCE OF FEEDING STATUS ON NEURONAL ACTIVITY IN THE HYPOTHALAMUS DURING LIPOPOLYSACCHARIDE-INDUCED ANOREXIA IN RATS L. GAUTRON,a R. MINGAM,b A. MORANIS,b C. COMBEb AND S. LAYÉa,b*

A dramatic reduction in food intake is commonly associated with illness during localized and systemic diseases (Hart, 1988; Konsman and Dantzer, 2001). Accordingly, rats injected with lipopolysaccharide (LPS), a gram-negative bacteria-derived endotoxin, develop anorexia (Langhans et al., 1989, 1993; Bret-Dibat et al., 1997; Huang et al., 1999). LPS-induced anorexia is thought to be mediated by a number of pro-inflammatory mediators such as interleukin (IL)-1␤ or prostaglandin E2 (PGE2) acting on the CNS including hypothalamic feeding regulatory nuclei which ultimately control appetite (Uehara et al., 1989; Kent et al., 1994; Layé et al., 2000; Dunn and Swiergiel, 2000; Johnson et al., 2002; Reyes and Sawchenko, 2002). There is evidence indicating that disease-associated anorexia depends on the nutritional status of the organism and pre-inflammation energy stores. In particular, fasting and food restriction reduce IL-1␤- and turpentine-induced anorexia (Mrosovsky et al., 1989; Kent et al., 1994; Lennie et al., 1995; Lennie, 1998; Larson et al., 2002). However, the mechanisms underlying fasting-induced alteration of inflammatory anorexia are poorly understood. Proinflammatory cytokine action in the brain could be in part responsible for this mechanism. Interestingly, fasting and experimental cafeteria diet both increased the constitutive expression of cytokine mRNAs in the normal rat brain (Hansen et al., 1998; Gayle et al., 1999). However, fasting and fasting–refeeding did not modify the induction of mRNA for IL-1, IL-6, tumor necrosis factor-␣ (TNF-␣) and their respective receptors in the rat brain in response to the i.c.v. injection of LPS (Gayle et al., 1999). Even if these results suggest that proinflammatory cytokine-induced anorexia is modulated by fasting, the mechanisms underlying this effect are poorly known. Alternatively, fasting could induce long-lasting neurochemical changes that impact on the brain circuitry recruited by inflammation, hence modulating anorexia. A key factor in the central regulation of food intake is leptin of which plasma levels are reduced upon fasting, thereby altering the transcriptional profile of appetite-related hypothalamic neuropeptides, including neuropeptide Y (NPY), cocaine–amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) (Brady et al., 1990; Thornton et al., 1997; Kristensen et al., 1998; Mizuno et al., 1998; Baskin et al., 1999). In view of these data, we hypothesized that fasting, by decreasing circulating leptin and subsequently changing the NPY/POMC balance, would modulate LPS-induced anorexia and the central response to inflammation in the hypothalamic structures that are involved in the regulation of energy balance. Accordingly,

a Laboratoire des Régulations Neuroendocriniennes, EA 2972, Université Bordeaux I, 33400 Talence, France b UMR Université Bordeaux 2, INRA 1244 FRE CNRS 2723, Laboratoire de Neurobiologie Intégrative, Institut François Magendie, rue Léo Saignat, 33077 Bordeaux, France

Abstract—Fasting attenuates disease-associated anorexia, but the mechanisms underlying this effect are not well understood. In the present study, we investigated the extent to which a 48 h fast alters hypothalamic neuronal activity in response to the anorectic effects of lipopolysaccharide in rats. Male rats were fed ad libitum or fasted, and were injected with i.p. saline or lipopolysaccharide (250 ␮g/kg). Immunohistochemistry for Fos protein was used to visualize neuronal activity in response to lipopolysaccharide within selected hypothalamic feeding regulatory nuclei. Additionally, food intake, body weight, plasma interleukin-1 and leptin levels, and the expression of mRNA for appetite-related neuropeptides (neuropeptide Y, proopiomelanocortin and cocaine–amphetamine-regulated transcript) were measured in a timerelated manner. Our data show that the pattern of lipopolysaccharide-induced Fos expression was similar in most hypothalamic nuclei whatever the feeding status. However, we observed that fasting significantly reduced lipopolysaccharide-induced Fos expression in the paraventricular nucleus, in association with an attenuated lipopolysaccharideinduced anorexia and body weight loss. Moreover, lipopolysaccharide reduced fasting-induced Fos expression in the perifornical area of the lateral hypothalamus. Lipopolysaccharide-induced circulating levels of interleukin-1 were similar across feeding status. Finally, fasting, but not lipopolysaccharide, affected circulating level of leptin and appetiterelated neuropeptides expression in the arcuate nucleus. Together, our data show that fasting modulates lipopolysaccharide-induced anorexia and body weight loss in association with neural changes in specific hypothalamic nuclei. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: leptin, neuropeptides, fasting, cytokines, inflammation, anorexia. *Correspondence to: S. Layé, Laboratoire de Neurobiologie Intégrative, UMR Université Bordeaux 2, INRA 1244, FRE CNRS 2723, Institut François Magendie, rue Léo Saignat, 33077, Bordeaux Cedex, France. Tel: ⫹33-5-57-57-37-20; fax: ⫹33-5-56-98-90-29. E-mail address: [email protected] (S. Layé). Abbreviations: ANOVA, analysis of variance; Arc, arcuate nucleus; CART, cocaine–amphetamine-regulated transcript; CRH, corticotropinreleasing hormone; DAPI, 4=,6-diamidino-2-phenylindole; DMH, dorsomedial hypothalamus; ␤-end, ␤-endorphin; IL, interleukin; LH, lateral hypothalamus; LPS, lipopolysaccharide; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SO, supraoptic nucleus; SSC, standard saline citrate; STAT, signal transducer and activator of transcription; TA, tuberal area; TN, Tris–NaCl.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.03.063

933

934

L. Gautron et al. / Neuroscience 134 (2005) 933–946

we first investigated the neuroanatomical pattern of LPSinduced Fos protein expression as a marker of neuronal activation in the hypothalamus of rats which were fed ad libitum, fasted or fasted–refed. Body weight, food intake, and plasma levels of IL-1␤ and leptin were measured in response to an anorectic dose of LPS. We used signal transducer and activator of transcription 3 (STAT3) translocation and Fos induction in POMC neurons as a marker of leptin and cytokine receptor activation in the hypothalamus (Gautron et al., 2002; Munzberg et al., 2003; Elias et al., 1999). Finally, we compared the expression of the mRNAs for NPY, CART, and POMC in ad libitum or fasted LPS-treated rats.

EXPERIMENTAL PROCEDURES Animals Experiments were performed on 92 adult male Wistar rats (Depré, France), weighing 280 –350 g, that were housed individually under controlled temperature (21⫾2 °C) and lighting conditions (12-h light/dark on from 8:00 p.m.). Animals were acclimatized to the facility and handled daily for 1 week with unrestricted access to water and food. The Animal Care and Use Committee of the University of Bordeaux I approved all protocols.

General experimental design Rats were randomly assigned to two feeding conditions: a) ad libitum: free access to food and water until kill, and b) fasting: food deprivation for 48 h with free access to water. During the 48 h interval, ad libitum animals gained 2.5⫾0.6% of their initial body weight whereas fasted animals lost 8.9⫾0.4%. At the end of this period, rats were given an i.p. injection of sterile saline (0.9% NaCl) or LPS from E. coli (Sigma, USA; lot 0127:B8; 250 ␮g/kg in saline; 0.3 ml/rat). The dose, time course and route of LPS injection were chosen based on previous experiments conducted in our laboratory showing its efficiency on both food intake and Fos expression in the brain (Aubert et al., 1997; Bret-Dibat et al., 1997; Konsman et al., 1999; 2000; Castanon et al., 2001). Injections were given between 7:30 and 8:30 a.m. Five different experiments were carried out with rats submitted to the above-described experimental design.

Experiment 1: Food intake and body weight measurements The first experiment was aimed at studying the impact of LPS on feeding and body weight in ad libitum and fasted rats (n⫽6/group) in a time-related manner. We weighed rats and their food pellets every hour during 6 h after treatment. This interval was chosen on the basis of prior results showing that treatment with LPS produced hypophagia within 1– 6 h of peripheral administration (Aubert et al., 1997; Bret-Dibat et al., 1997; Cross-Mellor et al., 1999; Castanon et al., 2001). Data are presented as mean cumulative food intake and mean body weight changes. For food intake and body weight, a two-way analysis of variance (ANOVA) was carried out (treatment versus time points) in each feeding status. When statistical significance was observed [P⬍0.05] (indicated in text), post hoc analyses were conducted using a Bonferroni test (significance indicated on Fig. 1). Additionally, since food intake cannot be measured in fasted rats, we compared the impact of LPS in ad libitum rats and fasted–refed animals (n⫽4/group). The former group included rats fasted for 48 h with free access to water. At the end of this period, rats were given an i.p. injection of saline or LPS as above-described, and were given free access to food and water. We measured cumulated food intake at 4 h after treatment. LPS effect on food intake of ad libitum and fasted–refed was

calculated as the percent of variation from values of respective saline-treated groups (indicated on Fig. 1B).

Experiment 2: IL-1␤ and leptin assays Ad libitum and fasted rats were anesthetized 2, 4 and 6 h after treatment (n⫽3–7/group) with i.p. overdose of 20% chloral hydrate. Blood was immediately collected by cardiac puncture (1 ml) in EDTA-coated tubes. Blood was centrifuged and plasma stored at ⫺20 °C until assay. Specific ELISAs for IL-1␤ (Safieth-Garabedian et al., 1995) were performed using sheep anti-rat IL-1␤ biotinylated immunoaffinity-purified polyclonal antibody and recombinant cytokine from Dr. Steven Poole (NIBSC, Hertforshire, UK). Revelation used avidin–peroxidase and the chromogen orthophenylene diamine. Optical density was read at 490 nm. The detection limit was 2.44 pg/ml. Leptin levels were measured with a commercial rat leptin radioimmunoassay kit (Linco Research, St. Charles, USA; Cat. RL-83K) according to the manufacturer. The intra- and interassay coefficients of variation ranged between 2.4 – 4.6 and 4.8 –5.7%, respectively, and the limit of sensitivity was 0.5 ng/ml. A two-way ANOVA (feeding status versus treatment) was performed at each time point.

Experiment 3: Fos immunohistochemistry Pilot study. In order to determine a optimal time point of perfusion and LPS-responsive hypothalamic sites, we performed a pilot Fos study in response to LPS in ad libitum and fasted rats (n⫽3/group). The particular dose of LPS used here (250 ␮g/kg) was chosen to ensure that the previously reported effects induced by LPS on behavior would be observed (Aubert et al., 1997; Bret-Dibat et al., 1997). At the time of kill, rats received an i.p. overdose of 20% chloral hydrate, then were perfused transcardially with 50 ml of 0.9% NaCl followed by 200 ml of 4% paraformaldehyde. Brains were removed and post-fixed 2 h in the same fixative at room temperature, submerged in 20% sucrose overnight at 4 °C, then quickly frozen and stored at ⫺80 °C. Frontal sections of the hypothalamus (30 ␮m) were stored at 4 °C in Tris–NaCl buffer (TN) (0.1 M; pH⫽7.4). Free-floating brain sections were incubated in TN containing 0.4% casein, 0.4% Triton X-100 and 0.5% hydrogen peroxide. Brain sections were incubated at 20 °C overnight in TN (0.4% casein, 0.4% Triton X-100) containing polyclonal rabbit anti-Fos antiserum (sc-52; Santa Cruz Biotechnology, USA; 1:1000), followed for 1 h by biotinylated goat anti-rabbit antibodies IgG (Jackson Immunoresearch, USA; 1:2000), and for 1 h by streptavidin/peroxidase complex (Jackson Immunoresearch; 1:2000). Antigen was revealed using the glucose– oxidase–nickel diaminobenzidine method resulting in a dark/blue staining of Fos-positive nucleus. Adjacent sections were counterstained with 4=,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, USA). Sections were mounted on gelatin-coated slides using a Moviol mounting medium, and coverslipped. Stereotaxic coordinates and nomenclatures are based on the Paxinos and Watson (1997) rat brain atlas. Quantitative study. We performed a quantitative evaluation of Fos immunoreactivity in ad libitum and fasted rats at 4 h (peak of Fos) after treatment (n⫽7–10/group). Tissue was prepared as described above and was simultaneously reacted to minimize histochemical variability. Sections were mounted on gelatincoated slides and coverslipped. Fos-positive cells were manually counted on a one in six series of sections (both sides of the brain) using a light microscope (Nikon E400). Fos-based analysis was assessed on hypothalamic nuclei related to feeding as follows: the arcuate nucleus (Arc) (from ⫺2.12 mm to ⫺4.16 mm from bregma; ⬃six sections/rat), the paraventricular nucleus (PVN) (⫺1.80 mm; ⬃two sections/rat), the perifornical area of the lateral hypothalamus (LH) (⫺2.56 mm to ⫺3.60 mm; ⬃six sections/rat), the dorsomedial hypothalamus (DMH) (⫺2.80 mm to ⫺3.60 mm; ⬃five sections/rat), the supraop-

L. Gautron et al. / Neuroscience 134 (2005) 933–946

935

Fig. 1. Graphs illustrating the cumulative food intake in response to i.p. saline or LPS (250 ␮g/kg) of ad libitum rats over 6 h (A), the percentage of feeding reduction occurring 4 h after treatment in ad libitum and fasted–refed rats (B), and the body weight changes of ad libitum and fasted rats (C). Data are expressed as the mean of cumulative food intake (g) ⫾S.E.M. (A, B), and the mean body weight changes (g) from initial weight ⫾S.E.M. (B). Post hoc analysis: * P⬍0.05; ** P⬍0.01 from saline-treated rats of the respective feeding group. The percentages indicated in A= are referring to respective saline-treated groups in each feeding status.

tic nucleus (SO) (⫺1.30 mm to ⫺1.80 mm; ⬃four sections/rat), and additionally the tuberal area (TA) (⫺2.12 mm to ⫺3.30 mm; ⬃five sections/rat). Fos positive cells counting was focused on the mid-rostro caudal part levels of the PVN because it corresponds to the highest Fos expression as compared with more rostral or caudal part of the PVN (data not shown). TA corresponded to the region located between the lateral part of the ventromedial hypothalamus and the LH and including tuber cinereum area and medial tuberal nucleus. Data were presented as the mean number (⫾S.E.M.) of Fos-positive cells per section. A two-way ANOVA was carried out to examine the interaction between LPS and feeding status. When statistical significance was observed [P⬍0.05], post hoc analyses were conducted using a t-test and results indicated on Fig. 6.

Experiment 4: in situ hybridization In situ hybridization for POMC, CART and NPY mRNAs was performed at 4 h after treatment in ad libitum and fasted rats (n⫽4/group). Rats were decapitated and their brains immediately removed, frozen in isopentane, cryosectioned at 14 ␮m generat-

ing frontal sections that were mounted onto gelatin-coated glass slides and stored at ⫺80 °C until processing. POMC, NPY and CART cDNAs were respectively provided by Dr. R. Steiner (University of Washington School of Medicine, Seattle, WA, USA) (Thornton et al., 1997), Dr S. L. Sabol (Laboratory of Biochemical Genetics, Bethesda, USA) (Li et al., 1998), and Dr P. Kristensen (Health Care Discovery, Novo Nordisk, Denmark) (Kristensen et al., 1998). Radioactive riboprobes labeled with 35S-UTP were synthesized according to in vitro transcription procedure as elsewhere described (Cournil et al., 2000). Tissue sections were fixed in 1% paraformaldehyde for 5 min and then rinsed in 0.01 M phosphate buffer saline (2⫻5 min). Next, sections were incubated in 4⫻ standard saline citrate (SSC) followed by 4⫻ SSC containing 0.1 M triethanolamine at pH 8 (7 min), and then 0.1 M triethanolamine and 0.25% acetic anhydride (5 min). All SSC solutions were sterilized and contained diethylpyrocarbonate to prevent RNase contamination. Sections were dehydrated in graded concentrations of ethanol. Radioactive probes were diluted in hybridization buffer (11% dextran sulfate, 20 mM Tris–HCl, 1.2 M EDTA, 0.35 M NaCl, formamide, 1.2⫻ Denhardt) to yield a mixture with

936

L. Gautron et al. / Neuroscience 134 (2005) 933–946

Fig. 2. Plasma levels of IL-1␤ (A) and leptin (B) in ad libitum, fasted or fasted rats at 2, 4 and 6 h after either i.p. saline or LPS (250 ␮g/kg). Data are given as the mean plasma concentration ⫾S.E.M.

106 cpm/50 ␮l. After drying, slides were coverslipped with hybridization mixture (106 cpm/50 ␮l/slide), and then incubated at 55 °C for 17 h in humidified boxes. Sections were digested with RNase A (20 ␮g/ml) at 37 °C for 25 min, rinsed in descending concentrations of SSC (2⫻, 1⫻, 0.5⫻), in 0.1⫻ SSC for 60 min at 65 °C, dehydrated in an ethanol series, air-dried, and exposed to X-ray film (Kodak) for 4 –7 days. Hybridization with sense 35S-labeled riboprobes was carried out in parallel as a control and resulted in no hybridization signal (data not shown). Hybridization signal was measured by computer-assisted densitometry on radioautographic films digitized using a CCD camera (Nikon DXM 1200). For each probe, films were developed and digitized simultaneously and their hybridization signal quantified under standardized conditions using Scion Image software. After image segmentation at a value corresponding just above the mean background value, the mean gray level (arbitrary unit) was measured in a defined selected rectangle enclosing the Arc on

each section. We verified that films background was similar for a given probe. Measurements were performed on four to five sections per rat throughout the medial Arc (from ⫺2.30 to ⫺3.14 mm from Bregma). The mean gray level was calibrated in cpm per mm2 using brain paste standards containing increasing level of 35S-ATP (from 0 to 30 cpm/mm2) as previously described (Cournil et al., 2000). Measurements were presented as the mean of cpm/mm2 (⫾S.E.M.), a value dependent on the amount of mRNA per cell. A two-way ANOVA was carried out (feeding status versus treatment).

Experiment 5: STAT3 and Fos/␤-endorphin (␤-end) immunohistochemistry STAT3 and Fos/␤-end immunohistochemistry were performed at 4 h after treatment in ad libitum and fasted rats (n⫽4/group). To determine whether Fos-positive cells in the Arc were POMC neurons, staining for nuclear Fos was first processed followed by

L. Gautron et al. / Neuroscience 134 (2005) 933–946 cytoplasmic labeling for ␤-end. Fos immunolabeling was performed exactly as described above and the labeled sections were incubated overnight with a rabbit polyclonal anti-␤-end antiserum (1:400; Pape et al., 1996) for 2 h followed by peroxidase-conjugated anti-rabbit antibodies (Jackson Immunoresearch; 1:1000). Diaminobenzidine in presence of hydrogen peroxide was used as a chromogen. We estimated the percentage of Fos-positive cells being simultaneously ␤-end by counting cells harboring a Fospositive black nucleus and a brown cytoplasmic reaction ␤-end immunoreactivity. In order to detect STAT3, sections were processed exactly as for Fos immunohistochemistry but using a polyclonal rabbit anti-STAT3 antiserum (sc-482; Santa Cruz Biotechnology; 1:10,000) and glucose– oxidase–nickel diaminobenzidine methodology. Fos- and STAT3-positive cells were manually counted on a one in six series of sections (both sides of the brain) using a light microscope (Nikon E400). STAT3 immunoreactivity was evaluated in the Arc (from ⫺2.12 mm to ⫺4.16 mm from bregma; ⬃six sections/rat). Data were presented as the mean number (⫾S.E.M.) of STAT3 positive cells per section. A two-way ANOVA was carried out (feeding status versus treatment). Photomicrographic images were captured with a digital camera (Nikon DXM 1200) mounted on a light/fluorescent microscope (Nikon E400) and linked to a computer-based system. Adobe Photoshop was used to produce illustrations and adjust contrast and brightness of the digitized images.

937

al., 1995; Matsunaga et al., 2000), Fos immunoreactivity was induced in cells of PVN of the hypothalamus, periventricular area, SO, DMH, TA, Arc and its retrochiasmatic part after the treatment. As shown in Fig. 3, Fos immunostaining was detectable as soon as 2 h after a LPS injection in the PVN (Fig. 3A), with a peak at 4 h (Fig. 3C). At the 6 h time point, Fos immunoreactivity was still detectable in the PVN (Fig. 3E). PVN was delineated by a DAPI staining performed in adjacent sections (Fig. 3B, D, F). In order to determine whether Fos activation was different in the brain of ad libitum and fasted rats after a LPS treatment, we performed a quantitative study at 4 h post-treatment that corresponds to the peak of expression of Fos (Fig. 3).

RESULTS Effect of feeding status on LPS-induced anorexia, body weight loss and cytokine plasma levels In ad libitum rats, the cumulative food intake was significantly reduced by the LPS treatment (250 ␮g/kg, ip) [P⬍0.01] in a time specific manner [P⬍0.01] with an interaction with the two factors [P⬍0.01] (Fig. 1A). Interestingly, food intake was dependent on feeding status [P⬍0.01] (Fig. 1B). A 4 h LPS treatment significantly reduced food intake in both ad libitum and fasted–refed rats [P⬍0.01], with a trend for interaction [P⬍0.086] (Fig. 1B). Four hours after LPS injection, ad libitum rats ate 7% while fasted– refed ate 25% as compared with their respective control (Fig. 1B). These results suggest that LPS-induced anorexia was stronger in ad libitum rats as compared with fasted rats. LPS treatment significantly decreased body weight of ad libitum rats [P⬍0.01], in a time dependant manner [P⬍0.01], with an interaction of these two factors [P⫽0.0195] (Fig. 1C). In fasted rats, LPS treatment had no significant effect on body weight (Fig. 1C). We further measured IL-1␤ and leptin circulating levels in LPS-treated ad libitum and fasted rats (Fig. 2). There was a significant increase of IL-1␤ circulating levels after a LPS treatment in the plasma of both ad libitum and fasted rats [P⬍0.01] with no significant differences (Fig. 2A). While fasting was accompanied by a dramatic decrease of plasma leptin level [P⬍0.01] (Fig. 2B), LPS did not significantly altered plasma concentration of leptin whatever the time point was [P⫽0.839]. Effect of feeding status on LPS-induced Fos expression in the hypothalamus Rats received LPS (250 ␮g/kg) or vehicle intraperitoneally and were perfused 2, 4 or 6 h later. According to previous studies (Elmquist et al., 1993; Wan et al., 1993; Sagar et

Fig. 3. Photomicrographs of the distribution of Fos immunoreactivity in ad libitum rats 2 (A), 4 (C) and 6 h (E) after i.p. LPS (250 ␮g/kg). Sections were chosen at approximately the same anatomical level to show Fos expression in the PVN. Adjacent sections counterstained with DAPI show the delineation of subnuclei. PaLM, paraventricular nucleus lateral magnocellular part; PaMP, paraventricular nucleus magnocellular posterior part; PaV, paraventricular nucleus ventral part; 3v, third ventricle.

938

L. Gautron et al. / Neuroscience 134 (2005) 933–946

As summarized in Fig. 6, Fos immunoreactivity was low in all the nuclei under study, in the brain of salineinjected rats. Scarce Fos-positive cells were localized in specific hypothalamic areas of saline ad libitum fed rats, namely the DMH, Arc, TA, and LH (Fig. 6B, C, E, F). However, variations were observed across feeding status. In fasted rats, Fos expression was significantly reduced in the Arc (Fig. 6C), but increased in the perifornical area of the LH as compared with ad libitum (Figs. 5A, B; 6F). In fasted–refed rats, the number of Fos-positive cells was enhanced in several hypothalamic areas in comparison to ad libitum and/or fasted group. This was the case in the Arc compared with saline fasted rats (Fig. 6C), in the DMH

(Fig. 6B), and in the PVN (Fig. 6A), the SO (Fig. 6D), and the LH (Fig. 6F). In all LPS-treated rats, the number of Fos-positive cells increased in the Arc (Figs. 4A, D, G; 6C), PVN (Figs. 4B, E, H; 6A), DMH (Figs. 4C, F, I; 6B), SO (Fig. 6D), and the TA (Figs. 5C; 6E). For many structures including the DMH, Arc and SO, there were no statistically significant differences in Fos expression across the feeding status (Fig. 6B–D). Nevertheless, LPS-induced Fos expression was weaker in the PVN of animals that were fasted or fasted–refed (Figs. 4B, E, H; 6A). Moreover, LPS-induced Fos expression in the TA was of lesser magnitude in ad libitum as compared with other feeding status groups (Fig. 6E). Finally, in the LH while in ad libitum rats

Fig. 4. Photomicrographs of the distribution of Fos immunoreactivity in ad libitum, fasted and fasted–refed rats 4 h after i.p. LPS (250 ␮g/kg) (A–I). Sections were chosen at approximately the same anatomical level to show Fos expression in the Arc (A, D, G), the PVN (B, E, F) and the DMH (C, F, I). PaLM, paraventricular nucleus lateral magnocellular part; PaMP, paraventricular nucleus magnocellular posterior part; PaV, paraventricular nucleus ventral part; 3v, third ventricle. Scale bar⫽150 ␮m.

L. Gautron et al. / Neuroscience 134 (2005) 933–946

939

Fig. 5. Photomicrographs of the distribution of Fos immunoreactivity in the LH of saline ad libitum (A), saline fasted (B) and fasted rat 4 h after i.p. LPS (250 ␮g/kg) (C). Sections were chosen at the same anatomical level. f, fornix; scale bar⫽150 ␮m. The line drawing represents approximately the perifornical borderlines.

LPS administration did not produce any statistically significant changes in the number of Fos-positive cells, LPS reduced Fos expression in the perifornical area of the LH in fasted and fasted–refed animals (Figs. 5A–C; 6F). Effects of fasting and LPS on the expression of POMC, CART and NPY mRNAs Fig. 7 shows hybridization signals for POMC, CART and NPY mRNAs in the Arc (Fig. 7A, C, E). There was no statistically significant effect of the feeding status [P⫽0.1992] or LPS treatment [P⫽0.5819] on POMC mRNA expression (Fig. 7B). In contrast, fasting significantly decreased CART mRNA expression [P⬍0.0001], but LPS was without effect [P⫽0.0670] (Fig. 7D). Fasting increased NPY mRNA expression [P⬍0.0001], and LPS had no significant effect [P⫽0.5340] (Fig. 7F). Effects of fasting and LPS on STAT3 immunoreactivity and Fos expression in ␤-end neurons in the Arc STAT3 immunoreactivity in the Arc of both ad libitum and fasted rats treated with saline was similar in terms of number of cells and intensity of staining across feeding status (Fig. 8C). The immunoreactivity was localized in the nucleus and surrounding perinuclear compartment of what appeared to be neuronal cells, based on their size (Fig. 8A). In response to LPS, darkly-stained nuclear STAT3 immunoreactivity appeared in small-sized nuclei (Fig. 8B), and in the same manner in ad libitum and fasted rats (Fig. 8C). Based on their distribution, shape and size, cells displaying LPS-induced STAT3-positive nuclei were likely astrocytes, corroborating our previous results (Gautron et al., 2002). However, since we did not identify the cell type, we cannot totally exclude the activation of STAT3 in neurons. Double immunohistochemistry combining Fos and ␤-end detection (marking POMC neurons) in the Arc, identified a majority of Fos-positive cells in saline ad libitum rats

as POMC neurons (⬃85%) (Fig. 9C). There was no colocalization of Fos and ␤-end in fasted animals (Fig. 9A), and very little (⬃4%) in LPS-treated rats whatever the feeding status (Fig. 9B, D).

DISCUSSION The aim of our study was to investigate the effect of a 48 h fast on the inflammatory response and the hypothalamic neuronal activity in response to the anorectic effects of LPS in rats. We demonstrated for the first time that LPSinduced anorexia and body weight loss were attenuated in 48 h-fasted rats. These effects were accompanied by lower Fos expression in the PVN. Moreover, fasting-induced Fos expression in the LH was inhibited by LPS treatment. Concomitantly, fasting decreased plasma leptin and altered the expression of mRNAs for CART and NPY, but not POMC. In contrast, LPS did not affect these parameters. Taken together, these results suggest that the catabolic response to LPS involves a coordinated mobilization of discrete hypothalamic nuclei, including the activation of the PVN and the inhibition of the LH, which is affected by nutritional status. To determine whether the administration of LPS had an impact on different brain nuclei of ad libitum and fasted rat, we used the expression of Fos immunoreactivity, which has been widely used as a marker of neuronal activation. Fos expression does not imply a direct activation by LPS but results rather from indirect stimulation by endogenously produced cytokines or synaptic inputs. Previous studies performed in our laboratory have shown that the dose of LPS we used (250 ␮g/kg, i.p.) that decreases food intake also induces Fos immunoreactivity and STAT3 activation in the brain (Aubert et al., 1997; Konsman et al., 1999, 2000; Gautron et al., 2002). Studies that have examined the effects of LPS on food intake have used doses ranging from 50 to 1000 ␮g/kg (Langhans et al., 1989; Yirmiya et al., 2001; Cross-Mellor et al., 1999). The 250 ␮g/kg LPS dose and the route of injection (i.p.) we

940

L. Gautron et al. / Neuroscience 134 (2005) 933–946

Fig. 6. Bar graphs summarizing the mean number of Fos-positive cells per section ⫾S.E.M. in different hypothalamic nuclei across experimental groups from rats submitted to different feeding status (ad libitum, gray bars; fasted, white bars; fasted–refed, black bars) 4 h after saline or LPS treatment (A-F). Post hoc: * P⬍0.05 from saline ad libitum; ** P⬍0.01 from saline ad libitum; # P⬍0.05 from saline of each respective feeding status group; ## P⬍0.01 from saline of each respective feeding status group; ⫹ P⬍0.05 from LPS ad libitum.

used induces a reversible sickness symptoms within 24 h (Bret-Dibat et al., 1997) associated with no brain damage (Mouhiate and Pittman, 1998). Fasting and LPS-induced anorexia and weight loss By demonstrating that LPS-induced anorexia and weight loss are attenuated by a 48 h fast, our data both confirm and extend previous results showing that IL-1- or turpentine-induced anorexia is attenuated by fasting or feeding restriction (Mrosovsky et al., 1989; Kent et al., 1994; Len-

nie et al., 1995; Lennie, 1998; Larson et al., 2002). In the case of LPS, McCarthy and colleagues (1986) have previously reported that the i.v. or i.c.v. injections of LPS had no anorectic effects in fasted rats. However, there was no direct comparison of fasted with ad libitum fed rats. In this study, the lack of effect of LPS could have been due to the relative very small dose of LPS that was used (100 ng). Thus, we demonstrate for the first time that a 48 h fast attenuates LPS-induced anorexia and prevents weight loss. The effect of fasting on LPS-induced anorexia was

L. Gautron et al. / Neuroscience 134 (2005) 933–946

941

Fig. 7. Photomicrographs representing the hybridization signal (film macroautoradiographs) of POMC (A), CART (C) and NPY mRNAs (E) in the Arc of saline ad libitum rats. Sections are approximately ⫺2.56 mm from Bregma. Relative quantification of hybridization level (cpm/mm2 ⫾S.E.M.) of POMC (B), CART (D) and NPY mRNAs (F). ANOVA: ## P⬍0.01 from ad libitum of the respective treatment group. Scale bar⫽1 mm.

weak but it is likely that a more prolonged fasting would have totally prevented anorexia. Supporting this view is that turpentine-associated anorexia negatively correlates with the intensity of pre-inflammation body energy restriction (Lennie et al., 1995; Lennie, 1998). A candidate for the reduced anorectic response of fasted rats to LPS is a reduction of pro-inflammatory cytokines in plasma levels and/or in the brain. However, the measurement of plasma IL-1␤ levels revealed a similar LPS-induced increase in fed and fasted animals. Similarly to our results, pre-inflammation food restriction did not influence the stimulatory effect of peripheral turpentine on the release of IL-1␤, IL-6 and IL-6 activity (Lennie et al., 1995, 2001). In addition, centrally administered LPS increased the expression of IL-1␤, IL-6 mRNA and their

receptors in fasted and fed rats (Gayle et al., 1999). Combined, these observations support the hypothesis that LPS-induced anorexia and weight loss vary depending on the feeding status, and this effect perhaps involves central neurochemical changes. Neuronal response to LPS in fed and fasted rats To address the influence of fasting on LPS-induced neurobiological changes, we performed a series of Fos experiments to study the activation of hypothalamic structures involved in the regulation of food intake and energy expenditure. Peripheral administration of LPS activated a specific pattern of hypothalamic nuclei including the PVN, SO, DMH, TA and Arc. It has been shown that fasting and/or

942

L. Gautron et al. / Neuroscience 134 (2005) 933–946

Fig. 8. Photomicrographs of STAT3 immunostaining in the Arc of saline ad libitum (A) and LPS ad libitum (B) rats. Constitutive perinuclear staining (small arrow) is seen in both saline- and LPS-treated rats (A, B), whereas small-sized dark nuclei (arrowhead) appeared after LPS (B). Bar graphs summarizing the mean number of STAT3-positive profiles per section ⫾S.E.M. in the Arc across experimental groups (gray bars, ad libitum; white bars, 48 h fasted). ANOVA: ## P⬍0.01 from saline of each respective feeding status group (C). Scale bar⫽30 ␮m.

feeding restriction attenuates thermogenesis, hypoglycemia and adrenocorticotrope hormone release in LPStreated rats (Shido et al., 1989; Giovambattista et al., 2000). It is not to exclude that modifications in the hypothalamic regulation of sympathetic outflow, feeding and adrenocorticotrope axis may interfere.

Earlier studies demonstrated Fos expression in the same hypothalamic structures at varied time points following peripheral LPS injection (Elmquist et al., 1993; Wan et al., 1993; Sagar et al., 1995). In the present study, the pattern of Fos expression was similar regardless of the time points. The number of Fos-positive cells in several

Fig. 9. Photomicrographs of double-labeled sections combining Fos (black arrows) and ␤-end (white arrowheads) in the Arc of fasted (A, B) or ad libitum (C, D) rats 4 h after either i.p. saline (A, C) or LPS (B, D). Insets in C and D show higher magnification examples of POMC neurons. Scale bar⫽30 ␮m.

L. Gautron et al. / Neuroscience 134 (2005) 933–946

hypothalamic regions was however higher in ad libitum fed rats than in fasted animals 4 h after LPS. Particularly, LPS-induced Fos expression was weaker in the PVN of animals that were fasted or fasted–refed. The activation of PVN neurons may contribute to enhance catabolic activity via their projections toward the pituitary, brainstem and spinal cord, and to the regulation of endocrine and autonomic systems (Sawchenko et al., 2000). Furthermore, lesions of the PVN increase food intake (Gold, 1970; Choi and Dallman, 1999), indicating a pivotal position of the PVN in reducing feeding. Corticotropin-releasing hormone (CRH)-producing cells of the PVN are good candidates to mediate this catabolic effect in view of the distribution pattern of their projections (Swanson and Sawchenko, 1983; Sawchenko et al., 1984). Moreover, the PVN receives both NPY and melanocortin inputs from the Arc (Bell et al., 2000). Interestingly, the anorectic effect of melanocortin agonist is sustained by the stimulation of CRH gene transcription (Lu et al., 2003). Melanocortin activation of CRH expression is mediated by MC4R expressed in a subpopulation of CRH neurons (Lu et al., 2003). Recently, NPY Y1-receptor mRNA has been described to be expressed in MC4-R-positive cells in the PVN, reinforcing the key role of CRH in the regulation of food intake (Kishi et al., 2005). LPS-activated PVN neurons include those expressing CRH, oxytocin and to a lesser extent vasopressin (Rivest and Laflamme, 1995; Sagar et al., 1995; Matsunaga et al., 2000). Moreover, a broad spectrum of neurochemical changes in the PVN is initiated by fasting, among them the expression of neuropeptides such as CRH (Fekete et al., 2000b), but also neurotransmitters receptors, transcription factors and enzymes (Leibowitz, 1988). As a consequence, our observations can be interpreted to suggest that fasting attenuates the catabolic response to LPS by reducing the activity of specific neuronal population in the PVN including CRH neurons. The identity of the exact neuronal population in the PVN that participates in the LPS-induced anorexia remains an important problem that will require further studies to delineate. We also show in the present study that the LH is a brain structure differentially affected by LPS in ad libitum and fasted rats. Fasting-induced Fos expression in the perifornical area of the LH of fasted animals was reduced in response to LPS, indicating an inhibitory effect of LPS on this hypothalamic nucleus. This hypothalamic area functions downstream of NPY- and POMC-signaling to regulate food intake and body weight, but also sleep and arousal (Saper et al., 2002). Neurons stimulated by fasting in the LH presumably express orexigenic peptides such as melanin-concentrating hormone (MCH) and/or orexins (Orx) whose mRNA expression increases following fasting (Qu et al., 1996; Sakurai et al., 1998). LPS-induced cytokines thus seem to activate brain structures involved in catabolic processes and inhibit brain structures implicated in food seeking and reward. The finding that administration of LPS reduces the mRNA expression of the orexigenic peptide MCH in the LH further supports this hypothesis (Sergeyev et al., 2001). The exact circuitry by which the LH

943

is inhibited remains elusive. A direct action of cytokines on LH neurons is possible as IL-1 inhibits the firing rate of glucose-sensitive neurons in the LH (Plata-Salamán et al., 1988). Potential role of leptin and target neuropeptides in the Arc during LPS-induced anorexia Earlier studies reported increased plasma leptin levels in response to LPS (Sarraf et al., 1997; Fink et al., 1998; Faggioni et al., 1999; Mastronardi et al., 2000), making leptin a potential candidate involved in LPS-induced anorexia. Moreover, melanocortin neurons, which are a major target for leptin, have been speculated to be involved in disease-associated anorexia based on neuroanatomical (Sergeyev et al., 2001; Reyes and Sawchenko, 2002), pharmacological (Huang et al., 1999; Wisse et al., 2001), and transgenic mice studies (Marks et al., 2001). In view of these studies, the absence of effect of LPS on plasma leptin concentration, neuropeptides mRNA expression and melanocortin neurons activation in our study is puzzling. However, during the past few years, contradictory data have been accumulated concerning the role of leptin and target neuropeptides in disease-associated anorexia. Accordingly, in a study involving a protocol very similar to ours, LPS (180 ␮g/kg, i.p.) failed to elevate plasma leptin levels in rats 2 h after injection (Giovambattista et al., 2000). Furthermore, leptin-deficient mice were found to be still fully responsive to the anorectic effects of LPS (Faggioni et al., 1997), making the role of leptin in LPS-induced anorexia controversial. These data are corroborated by a very recent report that demonstrate by using obese rat models that an acute injection of LPS mediates anorexia independently of leptin signaling (Lugarini et al., 2005). However, a recent study has evidenced that the immunoneutralization of leptin attenuates LPS-induced anorexia in rats at 8 and 24 h after LPS (Sachot et al., 2004). Therefore, leptin might be involved in the late phase of LPSinduced anorexia. In that case, an examination of leptin level and neuropeptides mRNA should be done over 24 h after the administration of LPS. At first sight it cannot be excluded that microautographic analysis would have facilitated the detection of tiny variation of mRNA expression in response to LPS (Sergeyev et al., 2001). However, other studies failed to find variation of NPY or POMC mRNA expression in the hypothalamus of LPS-treated rodents using RNAse protection assay or in situ hybridization (Turrin et al., 2001; Johnson et al., 2002). Further, Reyes and Sawchenko (2002) surprisingly found that IL-1-induced anorexia was not prevented, but potentiated, by chemical lesion of the Arc or the physical disruption of its efferents. Therefore, hypothalamic POMC neurons are unlikely to be key players in the induction of anorexia in inflammatory conditions. In accordance with this view, we failed to demonstrate activation of Fos in POMC neurons in response to LPS. Our data do not exclude the participation of medullary POMC neurons whose role in food intake was underestimated until a recent report (Fan et al., 2004). This might explain contradictory results between studies that demon-

944

L. Gautron et al. / Neuroscience 134 (2005) 933–946

strated the involvement of melanocortins and their receptors in LPS-induced anorexia and others that did not show the recruitment of hypothalamic POMC neurons. This encourages further studies to analyze the role of medullary POMC neurons in inflammatory stress conditions. The histological analysis of STAT3 nuclear immunoreactivity is a useful and reliable marker for STAT3 activation. Peripheral or central administration of high dose of leptin activates STAT3 translocation in neurons (Hubschle et al., 2001; Munzberg et al., 2003). However, despite the variation of circulating leptin due to feeding status, we could not observe a variation in STAT3 immunoreactivity in the Arc. STAT3 activation is restricted to astrocytes in the brain of LPS-treated rats (Gautron et al., 2002), it is unlikely that STAT3 is activated in POMC neurons. In vivo, astrocytes do not appear directly responsive to leptin (Suzuki et al., 2001). Therefore, it is unlikely that leptin is responsible for STAT3 activation in the brain of LPStreated rats. Other factors that are upregulated by LPS such as IL-6, act in the brain and activate STAT3 translocation in astrocytes (Hubschle et al., 2001; Harre et al., 2003). Fos activation in the arcuate after a LPS treatment was also independent of feeding status and circulating leptin levels. This result reinforced the idea that the recruitment of the Arc 4 h after the LPS treatment is independent of leptin. Alternatively, our findings show that a 48 h fast increased NPY and decreased CART mRNAs in the Arc in accordance with previous findings (Brady et al., 1990; Kristensen et al., 1998; Baskin et al., 1999). We also show that fasted rats did not display any more Fos immunoreactivity in beta endorphin immunoreactive Arc neurons in contrast with fed rats. It has been recently demonstrated that nearly all POMC neurons express beta-endorphin (Cowley et al., 2001). Moreover, by using double immunohistochemistry we found that POMC is strictly co-expressed with beta-endorphin in neurons of the Arc (data not shown). These observations suggest in conjunction with the literature that melanocortin neurons might be actively inhibited when leptin falls (Schwartz et al., 1997; Mizuno et al., 1998; Cowley et al., 2001). All these changes are aimed at the preservation of body energy and favoring food seeking are elicited in the hypothalamus by fasting upon the orchestration of plasma leptin (Ahima, 2000; Saper et al., 2002). NPY and POMC neurons abundantly innervate the PVN (Baker and Herkenham, 1995; Legradi and Lechan, 1998; Fekete et al., 2000a), making them key modulators of the PVN activity. Accordingly, the disruption of arcuato-paraventricular projections changes food intake in rats (Bell et al., 2000). It is very likely that fasting-induced long-term changes in the Arc lead to a differential activity of the PVN, so that the PVN will respond differently to an ulterior LPS challenge. This effect represents a potential substrate for modulating anorexia and body weight loss. For a causal demonstration of this hypothesis, it should be interesting to analyze the impact of leptin administration in animals fasted and treated with LPS on anorexia, body weight loss, appetite-related neuropeptides, and Fos expression at different time points.

CONCLUSION We demonstrated for the first time that fasting attenuates LPS-induced anorexia and prevents weight loss. This effect is due neither to the NPY/POMC balance and the leptin level modification nor to the variation of LPS-induced proinflammatory cytokines production. However, the activation of downstream hypothalamic nuclei such as PVN and LH that are positioned to modulate the catabolic response to LPS is altered in LPS-treated fasted rat. Our findings support the hypothesis that fasting attenuates the catabolic response to LPS via a mechanism involving neural changes in the hypothalamus. Acknowledgments—We gratefully thank Dr. Michèle Guerre-Millo for helping us to perform plasma leptin assay, Pr. Gérard Tramu for having provided us with ␤-end antiserum, Jozina Broussillon and Pierrette Lafon for technical assistance, and Drs. Robert Dantzer and Jan-Pieter Konsman for their helpful comments on the manuscript.

REFERENCES Ahima RS (2000) Leptin and the neuroendocrinology of fasting. Front Horm Res 26:42–56. Aubert A, Kelley KW, Dantzer R (1997) Differential effect of lipopolysaccharide on food hoarding behavior and food consumption in rats. Brain Behav Immunol 11:229 –238. Baker RA, Herkenham M (1995) Arcuate nucleus neurons that project to the hypothalamic paraventricular nucleus: neuropeptidergic identity and consequences of adrenalectomy on mRNA levels in the rat. J Comp Neurol 358:518 –530. Baskin DG, Breininger JF, Schwartz MW (1999) Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 48:828 – 833. Bell ME, Bhatnagar S, Akana SF, Choi S, Dallman MF (2000) Disruption of arcuate/paraventricular nucleus connections changes body energy balance and response to acute stress. J Neurosci 20: 6707– 6713. Brady LS, Smith MA, Gold PW, Herkenham M (1990) Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 52:441– 447. Bret-Dibat JL, Creminon C, Couraud JY, Kelley KW, Dantzer R, Kent S (1997) Systemic capsaicin pretreatment fails to block the decrease in food-motivated behavior induced by lipopolysaccharide and interleukin-1beta. Brain Res Bull 42:443– 449. Castanon N, Bluthe RM, Dantzer R (2001) Chronic treatment with the atypical antidepressant tianeptine attenuates sickness behavior induced by peripheral but not central lipopolysaccharide and interleukin-1beta in the rat. Psychopharmacology 154:50 – 60. Choi S, Dallman MF (1999) Hypothalamic obesity: multiple routes mediated by loss of function in medial cell groups. Endocrinology 140:4081– 4088. Cournil I, Lafon P, Juaneda C, Ciofi P, Fournier MC, Sarrieau A, Tramu G (2000) Glucocorticosteroids up-regulate the expression of cholecystokinin mRNA in the rat paraventricular nucleus. Brain Res 877:412– 423. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, Low MJ (2001) Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411:480 – 484. Cross-Mellor SK, Kent WD, Ossenkopp KP, Kavaliers M (1999) Differential effects of lipopolysaccharide and cholecystokinin on sucrose intake and palatability. Am J Physiol 277:R705–R715.

L. Gautron et al. / Neuroscience 134 (2005) 933–946 Dunn AJ, Swiergiel AH (2000) The role of cyclooxygenases in endotoxin- and interleukin-1-induced hypophagia. Brain Behav Immunol 14:141–152. Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C, Flier JS, Saper CB, Elmquist JK (1999) Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 23:775–786. Elmquist JK, Ackermann MR, Register KB, Rimler RB, Ross LR, Jacobson CD (1993) Induction of Fos-like immunoreactivity in the rat brain following Pasteurella multocida endotoxin administration. Endocrinology 133:3054 –3057. Faggioni R, Fuller J, Moser A, Feingold KR, Grunfeld C (1997) LPSinduced anorexia in leptin-deficient (ob/ob) and leptin receptordeficient (db/db) mice. Am J Physiol 273:R181–R186. Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR, Grunfeld C (1999) Leptin deficiency enhances sensitivity to endotoxin-induced lethality. Am J Physiol 276:R136 –R142. Fan W, Ellacott KL, Halatchev IG, Takahashi K, Yu P, Cone RD (2004) Cholecystokinin-mediated suppression of feeding involves the brainstem melanocortin system. Nat Neurosci 7:335–336. Fekete C, Legradi G, Mihaly E, Huang QH, Tatro JB, Rand WM, Emerson CH, Lechan RM (2000a) alpha-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropinreleasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression. J Neurosci 20:1550 –1558. Fekete C, Legradi G, Mihaly E, Tatro JB, Rand WM, Lechan RM (2000b) alpha-Melanocyte stimulating hormone prevents fastinginduced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Neurosci Lett 289:152–156. Finck BN, Kelley KW, Dantzer R, Johnson RW (1998) In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide. Endocrinology 139: 2278 –2283. Gautron L, Lafon P, Chaigniau M, Tramu G, Laye S (2002) Spatiotemporal analysis of signal transducer and activator of transcription 3 activation in rat brain astrocytes and pituitary following peripheral immune challenge. Neuroscience 112:717–729. Gayle D, Ilyin SE, Plata-Salaman CR (1999) Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus. Am J Physiol 277:R1188 –R1195. Giovambattista A, Chisari AN, Corro L, Gaillard RC, Spinedi E (2000) Metabolic, neuroendocrine and immune functions in basal conditions and during the acute-phase response to endotoxic shock in undernourished rats. Neuroimmunomodulation 7:92–98. Gold RM (1970) Hypothalamic hyperphagia produced by parasagittal knife cuts. Physiol Behav 5:23–25. Hansen MK, Taishi P, Chen Z, Krueger JM (1998) Cafeteria feeding induces interleukin-1beta mRNA expression in rat liver and brain. Am J Physiol 274:R1734 –R1739. Harre EM, Roth J, Gerstberger R, Hubschle T (2003) Interleukin-6 mediates lipopolysaccharide-induced nuclear STAT3 translocation in astrocytes of rat sensory circumventricular organs. Brain Res 980:151–155. Hart BL (1988) Biological basis of the sick animals. Neurosci Biobehav Rev 12:123–137. Huang QH, Hruby VJ, Tatro JB (1999) Role of central melanocortins in endotoxin-induced anorexia. Am J Physiol 276:R864 –R871. Hubschle T, Thom E, Watson A, Roth J, Klaus S, Meyerhof W (2001) Leptin-induced nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei involved in body weight regulation. J Neurosci 21:2413–2424. Johnson PM, Vogt SK, Burney MW, Muglia LJ (2002) COX-2 inhibition attenuates anorexia during systemic inflammation without impairing cytokine production. Am J Physiol Endocrinol Metab 282: E650 –E656.

945

Kent S, Rodriguez F, Kelley KW, Dantzer R (1994) Reduction in food and water intake induced by microinjection of interleukin-1 beta in the ventromedial hypothalamus of the rat. Physiol Behav 56: 1031–1036. Kishi T, Aschkenasi CJ, Choi BJ, Lopez ME, Lee CE, Liu H, Hollenberg AN, Friedman JM, Elmquist JK (2005) Neuropeptide Y Y1 receptor mRNA in rodent brain: distribution and colocalization with melanocortin-4 receptor. J Comp Neurol 482:217–243. Konsman JP, Luheshi GN, Bluthe RM, Dantzer R (2000) The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune signals; a functional anatomical analysis. Eur J Neurosci 12:4434 – 4446. Konsman JP, Kelley K, Dantzer R (1999) Temporal and spatial relationships between lipopolysaccharide-induced expression of Fos, interleukin-1beta and inducible nitric oxide synthase in rat brain. Neuroscience 89:535–548. Konsman JP, Dantzer R (2001) How the immune and nervous systems interact during disease-associated anorexia. Nutrition 17: 664 – 668. Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJL, Hastrup S (1998) Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76. Langhans W, Harlacher R, Scharrer E (1989) Verapamil and indomethacin attenuate endotoxin-induced anorexia. Physiol Behav 46:535–539. Langhans W, Savoldelli D, Weingarten S (1993) Comparison of the feeding responses to bacterial lipopolysaccharide and interleukin-1 beta. Physiol Behav 53:643– 649. Larson SJ, Romanoff RL, Dunn AJ, Glowa JR (2002) Effects of interleukin-1beta on food-maintained behavior in the mouse. Brain Behav Immunol 16:398 – 410. Laye S, Gheusi G, Cremona S, Combe C, Kelley K, Dantzer R, Parnet P (2000) Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression. Am J Physiol Regul Integr Comp Physiol 279:R93–R98. Legradi G, Lechan RM (1998) The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 139:3262–3270. Lennie TA (1998) Relationship of body energy status to inflammationinduced anorexia and weight loss. Physiol Behav 64:475– 481. Lennie TA, McCarthy DO, Keesey RE (1995) Body energy status and the metabolic response to acute inflammation. Am J Physiol 269:R1024 –R1031. Lennie TA, Wortman MD, Seeley RJ (2001) Activity of body energy regulatory pathways in inflammation-induced anorexia. Physiol Behav 73:517–523. Leibowitz SF (1988) Hypothalamic paraventricular nucleus: interaction between alpha 2-noradrenergic system and circulating hormones and nutrients in relation to energy balance. Neurosci Biobehav Rev 12:101–109. Li C, Chen P, Smith MS (1998) The acute suckling stimulus induces expression of neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate nucleus. Endocrinology 139:1645–1652. Lu XY, Barsh GS, Akil H, Watson SJ (2003) Interaction between alpha-melanocyte-stimulating hormone and corticotropin-releasing hormone in the regulation of feeding and hypothalamo-pituitaryadrenal responses. J Neurosci 23:7863–7872. Lugarini F, Hrupka BJ, Schwartz GJ, Plata-Salaman CR, Langhans W (2005) Acute and chronic administration of immunomodulators induces anorexia in Zucker rats. Physiol Behav 84:165–173. Marks DL, Ling N, Cone RD (2001) Role of the central melanocortin system in cachexia. Cancer Res 61:1432–1438. Mastronardi CA, Yu WH, Rettori V, McCann S (2000) Lipopolysaccharide-induced leptin release is not mediated by nitric oxide, but is blocked by dexamethasone. Neuroimmunomodulation 8:91–97.

946

L. Gautron et al. / Neuroscience 134 (2005) 933–946

Matsunaga W, Miyata S, Takamata A, Bun H, Nakashima T, Kiyohara T (2000) LPS-induced Fos expression in oxytocin and vasopressin neurons of the rat hypothalamus. Brain Res 858:9 –18. McCarthy DO, Kluger MJ, Vander AJ (1986) Effect of centrally administered interleukin-1 and endotoxin on food intake of fasted rats. Physiol Behav 36:745–749. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV (1998) Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294 –297. Mouihate A, Pittman QJ (1998) Lipopolysaccharide-induced fever is dissociated from apoptotic cell death in the rat brain. Brain Res 805:95–103. Mrosovsky N, Molony LA, Conn CA, Kluger MJ (1989) Anorexic effects of interleukin 1 in the rat. Am J Physiol 257:R1315–R1321. Munzberg H, Huo L, Nillni EA, Hollenberg AN, Bjorbaek C (2003) Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin. Endocrinology 144:2121–2131. Pape JR, Ciofi P, Tramu G (1996) Suckling-induced Fos-immunoreactivity in subgroups of hypothalamic POMC neurons of the lactating rat: investigation of a role for prolactin. J Neuroendocrinol 8:375–386. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates. San Diego: Academic Press. Plata-Salaman CR, Oomura Y, Kai Y (1988) Tumor necrosis factor and interleukin-1 beta: suppression of food intake by direct action in the central nervous system. Brain Res 448:106 –114. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek R, Kanarek R, Maratos-Flier E (1996) A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380:243–247. Reyes TM, Sawchenko PE (2002) Involvement of the arcuate nucleus of the hypothalamus in interleukin-1-induced anorexia. J Neurosci 22:5091–5099. Rivest S, Laflamme N (1995) Neuronal activity and neuropeptide gene transcription in the brains of immune-challenged rats. J Neuroendocrinol 7:501–525. Sachot C, Poole S, Luheshi GN (2004) Circulating leptin mediates lipopolysaccharide-induced anorexia and fever in rats. J Physiol 561:263–272. Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ (1995) Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 115:1265–1275. Sagar SM, Price KJ, Kasting NW, Sharp FR (1995) Anatomic patterns of Fos immunostaining in rat brain following systemic endotoxin administration. Brain Res Bull 36:381–392. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998) Orexins and orexin receptors: a family of hypothalamic

neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573–585. Saper CB, Chou TC, Elmquist JK (2002) The need to feed: homeostatic and hedonic control of eating. Neuron 36:199 –211. Sarraf P, Frederich RC, Turner EM, Ma G, Jaskowiak NT, Rivet DJ 3rd, Flier JS, Lowell BB, Fraker DL, Alexander HR (1997) Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med 185:171–175. Sawchenko PE, Swanson LW, Vale WW (1984) Corticotropin-releasing factor: co-expression within distinct subsets of oxytocin-, vasopressin-, and neurotensin-immunoreactive neurons in the hypothalamus of the male rat. J Neurosci 4:1118 –1129. Sawchenko PE, Li HY, Ericsson A (2000) Circuits and mechanisms governing hypothalamic responses to stress: a tale of two paradigms. Prog Brain Res 22:61–78. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG (1997) Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46(12):2119 –2123. Sergeyev V, Broberger C, Hokfelt T (2001) Effect of LPS administration on the expression of POMC, NPY, galanin, CART and MCH mRNAs in the rat hypothalamus. Brain Res Mol Brain Res 90: 93–100. Shido O, Nagasaka T, Watanabe T (1989) Blunted febrile response to intravenous endotoxin in starved rats. J Appl Physiol 67:963–969. Suzuki S, Li AJ, Ishisaki A, Hou X, Hasegawa M, Fukumura M, Akaike T, Imamura T (2001) Feeding suppression by fibroblast growth factor-1 is accompanied by selective induction of heat shock protein 27 in hypothalamic astrocytes. Eur J Neurosci 13:2299 –2308. Swanson LW, Sawchenko PE (1983) Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu Rev Neurosci 6:269 –324. Thornton JE, Cheung CC, Clifton DK, Steiner RA (1997) Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology 138:5063–5066. Turrin NP, Gayle D, Ilyin SE, Flynn MC, Langhans W, Schwartz GJ, Plata-Salaman CR (2001) Pro-inflammatory and anti-inflammatory cytokine mRNA induction in the periphery and brain following intraperitoneal administration of bacterial lipopolysaccharide. Brain Res Bull 54:443– 453. Uehara A, Sekiya C, Takasugi Y, Namiki M, Arimura A (1989) Anorexia induced by interleukin 1: involvement of corticotropin-releasing factor. Am J Physiol 257:R613–R617. Wan W, Janz L, Vriend CY, Sorensen CM, Greenberg AH, Nance DM (1993) Differential induction of c-Fos immunoreactivity in hypothalamus and brain stem nuclei following central and peripheral administration of endotoxin. Brain Res Bull 32:581–587. Wisse BE, Frayo RS, Schwartz MW, Cummings DE (2001) Reversal of cancer anorexia by blockade of central melanocortin receptors in rats. Endocrinology 142:3292–3301. Yirmiya R, Pollak Y, Barak O, Avitsur R, Ovadia H, Bette M, Weihe E, Weidenfeld J (2001) Effects of antidepressant drugs on the behavioral and physiological responses to lipopolysaccharide (LPS) in rodents. Neuropsychopharmacology 24:531–544.

(Accepted 30 March 2005)