Effect of the anorectic fatty acid synthase inhibitor C75 on neuronal activity in the hypothalamus and brainstem

Abstract

Intraperitoneal (i.p.) injection of C75, a fatty acid synthase inhibitor, causes a rapid (≤2-h) and persistent (to at least 24-h) ≈95% decrease in food intake. The persistent effect seems to be due to inhibition of the fasting-induced up-regulation of expression of hypothalamic orexigenic neuropeptides neuropeptide Y and agouti-related protein and down-regulation of expression of anorexigenic neuropeptides pro-opiomelanocortin/α-melanocyte-stimulating hormone and cocaine-amphetamine-related transcript. The effect of C75 on neuronal activity in the hypothalamus and brainstem was assessed by c-Fos expression. Consistent with its effect on neuropeptide expression, C75 blocked fasting-induced c-Fos expression in the arcuate nucleus (Arc), lateral hypothalamic area (LHA), and paraventricular nucleus (PVN) 10–24 h after i.p. injection. However, i.p. C75 induced a rapid (≤2-h) c-Fos expression in the nucleus of the solitary tract (NTS) and area postrema of the brainstem but not in the Arc or LHA. Intracerebroventricular administration of C75 rapidly induced c-Fos expression in the Arc, PVN, and NTS, supporting a central role of C75 in the regulation of food intake. Thus, suppression of food intake by C75 administered i.p. seems to be mediated in two phases, a rapid initial phase via the NTS/area postrema of the brainstem and a delayed phase via the Arc, LHA, and PVN of the hypothalamus. The delayed effect of C75 on the Arc, LHA, and PVN correlates well with its ability to interfere with the fasting-induced effects on the expression of key orexigenic (neuropeptide Y and agouti-related protein) and anorexigenic (pro-opiomelanocortin/α-melanocyte-stimulating hormone and cocaine-amphetamine-related transcript) messages in the hypothalamus.

Peripheral neural and hormonal signals that indicate global energy status are monitored in several regions of the brain, notably the hypothalamus and brainstem (1, 2). Within the hypothalamus, three interconnected regions, i.e., the arcuate nucleus (Arc), paraventricular nucleus (PVN), and lateral hypothalamic area (LHA), play important roles in regulating food intake and energy expenditure (1, 2). The Arc produces both orexigenic and anorexigenic neuropeptides that are sent to the LHA and PVN via dense projections (1, 2). The PVN is a heterogeneous nucleus richly supplied by axons projecting from the Arc, LHA, and brainstem and thus is the site of convergence of multiple neural inputs that affect food intake (1, 2). The LHA was viewed classically as a “hunger center,” because stimulation of this area of the hypothalamus increases food intake, whereas its destruction attenuates feeding and causes weight loss (1). The regulation of food intake also involves extrahypothalamic regions including the nucleus of the solitary tract (NTS) and the closely related area postrema (AP) in the brainstem (1, 2). Satiety information emanating from the gastrointestinal tract such as cholecystokinin generated during the course of a meal is primarily conveyed to the NTS/AP by means of afferent fibers of the vagus nerve and afferents passing into the spinal cord from the upper gastrointestinal tract (1–7). The NTS sends projections to the PVN in the hypothalamus and is considered to be the first central relay nucleus in the process of termination of individual meals (1, 6, 8).

Recent studies (9–13) showed that i.p. injection of fatty acid synthase (FAS) inhibitors, i.e., cerulenin and C75, dramatically reduces food intake and body weight in both lean mice and ob/ob mice. In mice these effects do not result from visceral illness or malaise, because C75 administered i.p. at a dosage that resulted in >90% suppression of 24-h food intake did not induce conditioned taste aversion (12). The FAS inhibitors seem to act centrally, because when administered at much lower levels by intracerebroventricular (i.c.v.) injection, they cause marked reductions in food intake and body weight (9, 13). The anorectic effect of FAS inhibitors is elicited, at least in part, at the level of hypothalamus. Thus, C75 blocked the normal fasting-induced up-regulation of the hypothalamic orexigenic neuropeptide mRNAs for neuropeptide Y (NPY) and agouti-related protein (AgRP). Conversely, C75 blocked the normal fasting-induced down-regulation of the anorexigenic neuropeptide mRNAs for pro-opiomelanocortin and cocaine-amphetamine-related transcript in the hypothalamus (9, 10, 12).

The present study was undertaken to identify the regions within the hypothalamus and the brainstem that are activated or suppressed by the FAS inhibitor C75 by using immunohistochemistry to assess c-Fos expression, a well established marker for neuronal activation (14). The evidence presented indicates that the long-term/persistent (24-h postinjection) effect of C75 is largely on the hypothalamus, whereas the short-term (1- to 2-h) effect seems to be mediated through the brainstem.

Materials and Methods

Handling of Mice and Administration of C75.

Animal experiments were conducted in accordance with guidelines of the Johns Hopkins University School of Medicine Institutional Animal Care and Use Committee. Male BALB/c mice (20–25 g) from Charles River Breeding Laboratories were acclimated for 1 week to a 12-h light (1:30 a.m.–1:30 p.m.)/12-h dark (1:30 p.m.–1:30 a.m.) cycle at 22°C and fed standard laboratory chow ad libitum unless otherwise noted. Mice were either fed ad libitum or fasted for the time indicated.

C75 was administered either by i.p. (i.p.; 30 mg/kg of body weight) or i.c.v. (10 μg in 3 μl of RPMI medium 1640) injection. One, 11.5, and 24 h after i.p. injection, cumulative food intake was measured, mice were killed, brains were sectioned, and slices were subjected to immunohistochemical staining for c-Fos. All i.p. injections were given 1 h before the start of the dark cycle. For i.c.v. injection, mice were anesthetized with metofane and given 3 μl of RPMI medium 1640 (control) or C75 in RPMI medium 1640 into the lateral ventricle with a calibrated 10-μl Hamilton syringe.

Transcardial Perfusion, Immunohistochemical Staining, and Data Analysis.

Mice were anesthetized deeply by inhalation of halothane and perfused transcardially with PBS, pH 7.4, followed by ice-cold 4% formaldehyde in PBS, pH 7.4. Brains were removed, postfixed in the same fixative overnight, and cryoprotected in 30% sucrose in PBS, pH 7.4. Brains were frozen in OCT compound with powdered dry ice and sectioned into 30-μm coronal sections with a sliding microtome.

Diaminobenzidine immunohistochemistry was performed as described (8). Tissue sections were rinsed briefly with PBS, followed by a blocking step with 10% heat-inactivated goat serum (GIBCO) and 0.3% Triton X-100 in PBS [10% normal goat serum/Triton (NGST)] for 1 h at room temperature, and then incubated for 48 h in rabbit primary antiserum (c-fos, Ab-5, polyclonal Ab, 1:40,000 in 1% NGST; Oncogene, Cambridge, MA) at 4°C with gentle agitation. Sections were washed three times each for 10 min in 1% NGST, incubated in biotinylated secondary Ab (1:200 diluted in 1% NGST, Vectastain ABC kit, Vector Laboratories) for 1 h, washed for three times each for 10 min in 0.3% Triton X-100 in PBS (PBST), and incubated with avidin-biotinylated peroxidase complex (1:100 in PBST, Vectastain ABC kit, Vector Laboratories) solution for 1 h. Sections then were washed three times for 10 min each in 0.2 M phosphate buffer, pH 7.4, and transferred to diaminobenzidine (peroxidase substrate kit, Vector Laboratories) solution for development. Sections then were washed three times each for 10 min in 0.1 M phosphate buffer, pH 7.4, dried overnight, mounted with p-xylenebis(pyridinium)bromide mounting medium (Electron Microscopy Sciences, Fort Washington, PA), and coverslipped.

Sections were analyzed qualitatively with an Olympus light microscope, and Fos-immunoreactivity (ir) in appropriate regions was quantified. An observer unaware of treatment groups counted the stained cells by using a scale grid and a ×10 (for LHA) or ×20 (for all other areas, i.e., Arc, PVN, and NTS/AP) objective. The position of the counting grid within each brain region was delineated through adjacent landmarks. For each region, two to three adjacent sections were counted, and for each section both sides of the brain were counted. The average number of stained cells on both sides was used to represent the number of c-Fos-ir-positive cells on that section, and animals from each treatment group were compared by using the mean number on all sections counted in that region. Photomicrographs were produced by capturing images under a ×20 or ×10 objective with a digital camera and processed on an Apple Macintosh computer using IP-LAB software.

Differences between groups in food intake, body weight, and c-Fos-ir in response to drug injection were evaluated with Student's t tests. In all cases, P < 0.05 (two-tailed) indicated statistical significance.

Primary Culture of Hypothalamic Neurons and Immunocytochemistry.

The methods for primary culture of hypothalamic neurons were based on previously described protocols with minor modifications (15–17). Briefly, hypothalamic tissues were harvested from 21-day-old Wistar rats (Charles River Breeding Laboratories) and digested with papain (Worthington). Hypothalamic neurons were purified with a Nycoprep (Nycomed, Oslo) gradient step as described (15). Cells then were plated onto chambered glass slides coated with laminin (Becton Dickinson Labware) and poly-D-lysine (Sigma) and cultured in B27/Neurobasal A medium (GIBCO) supplemented with nerve growth factor (Roche Molecular Biochemicals) under 10% carbon dioxide at 37°C. On day 5 postplating, cell monolayers were subjected to standard immunocytochemical treatment. Briefly, medium was removed, and cultures were rinsed with PBS (pH7.4). Then, cells were treated with cold methanol at −20°C for 20 min, and after rehydration cells were blocked with 4% BSA in PBS and incubated with three pairs of Abs diluted in 4% BSA overnight at 4°C: (i) rabbit anti-FAS polyclonal Ab (1:500, gift of Stuart Smith, Children's Hospital, Oakland Research Institute, Oakland, CA) and mouse anti-Tau-1 monoclonal Ab (1:1,000, Chemicon); (ii) rabbit anti-acetyl-CoA carboxylase (ACC) α subtype polyclonal Ab (1:200, gift of Lee Witters, Department of Medicine and Biochemistry, Dartmouth Medical School, Hanover, NH) and mouse anti-Tau-1 monoclonal Ab; and (iii) mouse anti-ACC β subtype (1:200, gift of Lee Witters) and rabbit anti-microtubule-associated protein subtype 2 (MAP-2) polyclonal Ab (Chemicon). After primary Ab incubation, the cells were rinsed once with PBS and then incubated with Alexa-fluor 546-labeled Fab′ fragment of goat anti-rabbit IgG (Molecular Probes) together with an Alexa-fluor 488-labeled Fab′ fragment of goat anti-mouse IgG (Molecular Probes) diluted in 4% BSA. Then, the cells were rinsed with PBS, mounted with ProLong antifade reagents (Molecular Probes), and coverslipped. The fluorescent images were visualized under a ×63/oil objective of a Carl Zeiss fluorescence microscope, and pictures were captured with a digital camera. The colocalized images were generated and analyzed by using IP-LAB software on an Apple Macintosh computer.

Results

Previously we showed that during the 24-h period after a single i.p. injection of C75 (30 mg/kg body weight), food intake of mice was blocked, producing a virtual fasted state. It was discovered that the fasting-induced changes (induced by the inhibitor) in expression of key hypothalamic orexigenic and anorexigenic neuropeptide mRNAs were blocked. Thus, C75 prevented fasting-induced up-regulation of NPY and AgRP mRNAs and down-regulation of pro-opiomelanocortin and cocaine-amphetamine-related transcript mRNAs. In the present study we sought to determine whether C75 activates (or suppresses) neurons in specific regions of the hypothalamus as assessed by immunohistochemical staining of c-Fos. It is known that expression of c-Fos is a rapid event indicative of neuronal activation in the hypothalamus in response to various stimuli including fasting (14, 18–20).

Effect of i.p. Injection of C75 on Food Intake, Body Weight, and Expression of c-Fos in the Hypothalamus and Brainstem 24 h After Injection.

Intraperitoneal administration of C75 at 30 mg/kg body weight inhibited food intake of mice by ≥95% within 2 h after i.p. injection (Fig. 1A). This inhibition was persistent, lasting for at least 24 h. By 24 h of C75-induced “fasting,” body weight decreased by 12%, which was comparable with the body-weight loss of mice fasted for 24 h (Fig. 1B).

Figure 1.

Effect of C75 on food intake and body weight. BALB/c mice (n = 9) were either fasted (n = 3) or treated with vehicle (n = 3, control) or C75 (n = 3, 30 mg/kg body weight) by i.p. injection (1 h before “lights off”) and fed ad libitum. (A) After injection, food consumption of vehicle-treated (●) or C75-treated (■) mice was measured 1, 4, 8, 10.5, and 23 h after lights off. (B) Percent body-weight (BW) change of control (ctrl), fasted (F), and C75-treated mice at 24 h postinjection was determined. Values are means ± SEM. *, P < 0.01 vs. control group.

Consistent with previous reports (14, 19), fasting markedly increased neuronal activity in the Arc, LHA, and PVN as evidenced by c-Fos immunostaining of hypothalamic sections (Fig. 2 A–C). This change in c-Fos expression was most likely due to activation of NPY/AgRP neurons that promote food intake. Consistent with known effects of C75 on the expression of hypothalamic orexigenic and anorexigenic neuropeptide mRNAs (9, 10, 12), C75 prevented the fasting-induced increase of c-Fos expression in these regions of the hypothalamus. Quantification of the data shows that the largest effects on c-Fos expression were on the Arc and LHA, where c-Fos staining was decreased by 70% and 83%, respectively (Fig. 2 D and E). The basis for the lower, although significant, suppressive effect of C75 on c-Fos expression in the PVN (Fig. 2 C and F) is discussed below. In all cases the effects of C75 were statistically significant.

Figure 2.

c-Fos expression at different levels of the hypothalamus in fed, fasted, and C-75-treated mice and the reversal of Fos-ir 24 h after i.p. injection of C75 (30 mg/kg body weight). (A) Arc (×20 objective). (B) LHA (×10 objective). (Bb1–Bb3) LHA (×20 objective). (C) PVN (×20 objective). 3V, third ventricle; ME, median eminence. (D–F) The bar graphs quantify c-Fos staining (n = 3–6). AL, ad libitum; F, fasted; C75, C75-treated. Values are the mean ± SEM. Differences between treatment groups in each region were assessed by Student's t test. *, P < 0.01 vs. ad libitum; +, P < 0.01 vs. fasted; ++, P < 0.01 vs. ad libitum; **, P < 0.05 vs. fasted.

After a 24-h fast there was no significant change in c-Fos expression in the NTS or AP of the brainstem; however, C75 caused a substantial increase in both of these regions (Fig. 3). Axons from the NTS project to the PVN, thus when activated the NTS is known to activate the PVN. This fact may account for the inability of C75 to lower the fasting-induced increase of c-Fos expression in the PVN to the same extent as in the Arc and LHA (Fig. 2 C and F).

Figure 3.

Effect of fasting and C75 treatment on c-Fos expression in the AP and NTS of the caudal brainstem. (A) Ad libitum (n = 4). (B) Fasted for 24 h (n = 4). (C) C75-treated (30 mg/kg, n = 4). (Magnification, ×20.) cc, central canal. (D) Bar graphs of quantified results. *, P < 0.01 vs. ad libitum or fasted. AL, ad libitum; F, fasted; C75, C75-treated.

Because food intake was blocked rapidly (≤2 h) by C75 administered i.p. (Fig. 1A), the short-term effect of C75 on c-Fos expression in the same regions of the hypothalamus was also investigated. It was anticipated that because the level of c-Fos expression in fed mice (Fig. 2, ad lib) was already low, C75 would not cause significant further lowering. This finding was found to be the case for the Arc and LHA (Fig. 4 E and F). Unexpectedly, however, C75 provoked a marked (P < 0.01) increase in c-Fos expression in the PVN 1 h postinjection (Fig. 4 A, B, and G). This finding suggested that the C75-induced activation of neurons of the PVN might have resulted from activation of other brain centers known to be involved in the regulation of food intake, e.g., the NTS and AP of the brainstem.

Figure 4.

Effect of C75 on c-Fos expression in the PVN of the hypothalamus and in the AP and NTS of the caudal brainstem 1 h after i.p. injection. BALB/c mice received i.p. injections of vehicle (A and C) or 30 mg/kg C75 (B and D). The photomicrographs are at ×20 (n = 4). 3V, third ventricle; cc, central canal. (E–H) Bar graphs of quantified results. *, P < 0.01 vs. control (ctrl).

Rapid Effect of i.p. Administration of C75 on the Expression of c-Fos in the Brainstem.

Experiments were conducted to determine whether the rapid activation of neurons in the PVN, caused by i.p. C75, correlated with activation of c-Fos expression in the brainstem. After an i.p. injection of vehicle, c-Fos staining in the brainstem was undetectable (Fig. 4 C and H). However, 1 h after i.p. administration of C75, intense staining occurred in both the NTS and AP areas after i.p. administration of C75 (Fig. 4 D and H). The rapid activation of neurons in the PVN is consistent with the activation of neurons in these centers, because the NTS sends projections to the PVN (1, 6–8) and is known to be activated by feeding (21) and anorexigenic agents such as leptin (8).

Effect of i.c.v. Administration of C75 on c-Fos Expression in the Hypothalamus and Brainstem.

To explore further the short-term effects of C75 on feeding centers of the hypothalamus and brainstem, C75 was administered by i.c.v. injection, and 2 h later expression of c-Fos in these areas was investigated. Food intake during this 2-h period was reduced by ≈80% in mice treated with C75 (results not shown). As shown in Fig. 5 A–D, C75 strongly activated c-Fos expression in the Arc and PVN compared with mock-injected controls. C75 had no significant effect on c-Fos expression in the LHA (results not shown). In the NTS/AP area, there was a marked increase of c-Fos expression in the NTS in the caudal brainstem (Fig. 5 E, F, and I). In these regions very strong c-Fos staining was induced rapidly after i.p. administration of C75 (Fig. 4).

Figure 5.

Effect of C75 administered by i.c.v. injection on c-Fos expression in the Arc and PVN of the hypothalamus and in the AP and NTS of the caudal brainstem. c-fos staining was performed 2 h after i.c.v. injection of C75 (10 μg in 3 μl) or vehicle (3 μl). The photomicrographs are at ×20 (n = 3). 3V, third ventricle; ME, median eminence; cc, central canal. (G–I) The bar graphs show quantified results. *, P < 0.01 vs. control (ctrl).

Distribution of FAS, ACC-α, and ACC-β in Primary Cultured Neurons.

Previous studies (12) demonstrated that the target of C75, i.e., FAS, was localized in mouse PVN and Arc of the hypothalamus. The enzymes that generate (ACC-α and -β) malonyl-CoA, substrate of FAS, were also shown to be expressed in the Arc (12). To elucidate the cellular distribution pattern of these enzymes in hypothalamic neurons in primary culture, immunocytochemistry was performed on dissociated hypothalamic neuronal cultures with Abs against FAS, ACC-α, and ACC-β. On day 5 postplating, there is a 2- to 2.5-fold increase of release of NPY into the culture medium within 30 min after stimulation of 50 mM potassium chloride (unpublished results), which showed that the cultured neurons at this stage were in a viable and physiological state. Neurons were identified by the use of Abs against MAP-2 and Tau, two molecular markers for central neurons (22). FAS-ir and ACC-α-ir were found in Tau-1-positive neurons and have a diffuse distribution mainly in the cytoplasm (Fig. 6 A–F). ACC-β-ir was also present in the MAP-2-positive neurons, giving a punctuate appearance in the cytoplasm characteristic of mitochondrial localization (Fig. 6 G and H). These distribution patterns verified that the enzymes responsible for the metabolism of malonyl-CoA, the substrate of FAS, are localized in the hypothalamic neurons, which suggests that the molecular mechanism of the observed c-fos response in the hypothalamus under C75 treatment is at least partly due to the signaling through an increase of malonyl-CoA resulting from a direct inhibition of FAS by C75.

Figure 6.

Immunocytochemical localization of FAS, ACC-α, and ACC-β in dissociated primary hypothalamic neuronal culture. (A and D) Neuronal marker Tau is shown in green fluorescence. Diffuse staining/distribution of FAS-ir (B) and ACC-α-ir (E) in the cytoplasm (red fluorescence) is shown. (C and F) Colocalization/merge (yellow overlay) of Tau and FAS-ir or Tau and ACC-α-ir, respectively. (G) Neuronal marker MAP-2 exhibits red fluorescence in a representative neuron. (H) Punctuate distribution of ACC-β-ir in the same neuron.

Discussion

This investigation identifies specific regions of the hypothalamus in which fasting-induced neuronal activity is suppressed by C75, a potent inhibitor of FAS. These findings are consistent with the pattern of inhibition caused by C75 of the fasting-induced changes in the expression of hypothalamic neuropeptides (9, 10, 12). Previously we found that 24 h after a single i.p. injection, C75 prevented fasting-induced up-regulation of hypothalamic NPY and AgRP and down-regulation of pro-opiomelanocortin and cocaine-amphetamine-related transcript mRNAs in the hypothalamus. In the present investigation, we assessed the long-term (≤24-h) and short-term (≤2-h) effects of the inhibitor on neuronal activity in key centers of the hypothalamus (and brainstem) known to be involved in the regulation of food intake. After a 24-h fast neuronal activity as indicated by c-Fos expression was increased markedly in the Arc, LHA, and PVN (summarized in Fig. 7 A–C). These high levels of neuronal activity reflect the state of hunger. Similar to its effect in blocking fasting-induced changes in the expression of the orexigenic and anorexigenic neuropeptides, C75 prevented the fasting-induced increase of expression of c-Fos in Arc, PVN, and LHA (Fig. 7 A–C). It is likely that the major c-Fos-expressing neurons activated by fasting were the “orexigenic” NPY/AgRP neurons, and presumably it is these neurons that are targeted by C75. These findings indicate that the “longer-term” effects of C75, i.e., those elicited 11.5–24 h after i.p. injection, are accounted for primarily by effects on the hypothalamus.

Figure 7.

Kinetics of expression of c-Fos in the Arc, LHA, and PVN of the hypothalamus and in the AP and NTS of the caudal brainstem after initiation of fasting or i.p. injection of C75 (30 mg/kg). c-Fos expression was quantified from the results shown in Figs. 2–4. (A–C) Results are expressed as the ratio of fasted/ad libitum (ad lib) or C75-treated/ad libitum. (D and E) The number of c-Fos-positive cells in ad libitum-fed, fasted, or C75-treated mice is shown.

The presence of the key enzymes responsible for the metabolism of malonyl-CoA, the substrate of FAS, in the hypothalamic neurons provides a solid basis for the c-fos response by C75 treatment. Recently we showed that C75 effectively blocks the 24-h fasting-induced decrease of hypothalamic malonyl-CoA concentration (Z. Hu and M.D.L., unpublished results), which correlates well with the pattern of prevention of fasting-induced expression of c-fos in hypothalamic nuclei by C75. This finding suggests that the reversal of fasting-induced c-fos expression by C75 is most likely due to signaling caused by an increased malonyl-CoA concentration that results from the inhibition of FAS by C75.

Because the activation of Arc neurons, as assessed by c-Fos expression, is not sufficiently rapid (does not occur ≤1 h after i.p. C75 treatment; Fig. 7A), to account for rapid inhibition of food intake (i.e., within 1 h after i.p. C75 treatment), the Arc does not seem to be involved in the “short-term” mechanism by which food intake is inhibited. Rather, the short-term mechanism seems to involve activation of NTS and AP neurons of the caudal brainstem, which is known to be involved in the inhibition of food intake (1–7), because these regions rapidly (within 1 h) express c-Fos in response to C75 (Fig. 7 D and F). This explanation is consistent with the rapid initial (≤1-h) activation of c-Fos expression in the PVN by C75 (Fig. 7C). It should be noted that the PVN receives dense projections from the NTS of the brainstem.

There is a precedent for the involvement of the brainstem in appetite control. Thus, the anorexigenic peptide cholecystokinin, which acutely terminates food intake, acts on vagal afferents to activate c-Fos expression rapidly in both the NTS/AP and PVN. Three possible modes of action for the anorectic effect of C75 come to mind: (i) systemic C75 may act directly on neurons in the NTS/AP; (ii) C75 may stimulate vagal afferent terminals directly in a manner analogous to cholecystokinin (3, 4); or (iii) C75 may stimulate vagal afferents to the brainstem through visceral irritation/illness or malaise. The latter is not likely because at an i.p. dosage that suppresses 24-h food intake by ≥90%, C75 does not induce a conditioned taste aversion in mice (12). The possibility that C75 may function through direct interaction with the NTS and AP also has a precedent. Leptin receptors are located within the NTS and leptin rapidly (≤2 h after systemic infusion) activates c-Fos expression in both the PVN and NTS in the rat (8, 20, 23). The AP is a circumventricular organ where the blood–brain barrier is deficient; thus, this area has ready access to the blood-borne agents. The very intense c-Fos staining of these regions persists to 11.5 and 24 h (Fig. 7 D and E), suggesting that this signaling pathway may be activated continuously and contributes to the suppression of food intake.

The PVN of the hypothalamus is a heterogeneous nucleus and receives inputs from both the Arc and NTS. Thus, either of these inputs can activate the PVN. What we observe in the PVN 24 h after treatment with C75 is the net result (Figs. 2 C and F and 6C), i.e., a compromise between its interaction with the Arc of the hypothalamus and the NTS of the brainstem.

Abbreviations

Arc

arcuate nucleus

PVN

paraventricular nucleus

LHA

lateral hypothalamic area

NTS

nucleus of the solitary tract

AP

area postrema

FAS

fatty acid synthase

NPY

neuropeptide Y

AgRP

agouti-related protein

i.c.v.

intracerebroventricular administration

ir

immunoreactivity

ACC

acetyl-CoA carboxylase

MAP-2

microtubule-associated protein subtype 2