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Am J Physiol Regul Integr Comp Physiol 285: R1037-R1045, 2003; doi:10.1152/ajpregu.00383.2002
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Peptides that Regulate Food Intake

Cholecystokinin and D-fenfluramine inhibit food intake in oxytocin-deficient mice

Rose C. Mantella,1 Linda Rinaman,2 Regis R. Vollmer,1 and Janet A. Amico1,3

Departments of 1Pharmaceutical Sciences, 3Medicine, and 2Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Submitted 25 June 2002 ; accepted in final form 2 May 2003

ABSTRACT

Results from previous studies indicate that oxytocin (OT)-containing neural pathways are activated in laboratory rats after systemic administration of CCK or D-fenfluramine and that centrally released OT may participate in the anorexigenic effects of these treatments. To explore the relationship between feeding behavior and OT function, the effects of CCK and D-fenfluramine on feeding and central c-Fos expression were compared in wild-type (OT+/+) and OT-deficient mice (OT-/-) of C57BL/6 background. Male OT+/+ and OT-/- mice were administered saline or CCK (1, 3, or 10 µg/kg ip) after overnight food deprivation. Saline-treated OT+/+ and OT-/- mice consumed equivalent amounts of food after an overnight fast. CCK inhibited deprivation-induced food intake in a dose-dependent manner to a similar extent in both genotypes. CCK treatment also induced similar hindbrain and forebrain patterns of increased c-Fos expression in mice of both genotypes. After treatment with D-fenfluramine (10 mg/kg ip), both OT+/+ and OT-/- mice consumed significantly less food than untreated controls, with no difference between genotypes. We conclude that OT signaling pathways are unnecessary for the anorexigenic effects of systemically administered CCK and D-fenfluramine in C57BL/6 mice.

c-Fos; hypothalamus; dorsal vagal complex; anorexia

OXYTOCIN (OT), a nine-amino acid peptide, is synthesized within magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) as well as magnocellular neurons of the supraoptic nucleus (SON) of the hypothalamus (41). Magnocellular OT neurons that project to the posterior lobe of the pituitary gland are the source of systemically secreted OT, and parvocellular OT neurons that project to extrapituitary sites are the source of centrally released OT (6). Peripherally secreted OT elicits milk ejection during lactation and promotes uterine contractility at parturition (12). Central OT is believed to influence reproductive function (4, 31), maternal behavior (25), anxiety (39), and to inhibit consumption of food (34) and sodium chloride solution (NaCl) in the laboratory rat (32).

OT levels in the plasma of rats increase after administration of certain anorexigens (35). For example, systemic administration of anorexigenic doses of CCK produced plasma concentrations of OT that were proportional to the inhibition of food intake (16, 35). Subsequently, central, but not systemic, administration of synthetic OT was found to inhibit food intake in a dose-dependent manner (5, 21). Thus increased plasma levels of OT were thought to serve as a marker indicative of central OT release, and central OT signaling pathways were thought to mediate inhibitory effects on food intake (35). Support for this idea came from findings that the anorexigenic effect of CCK could be blunted by central pretreatment with an OT receptor antagonist (22).

Brain regions that are activated in rats after CCK treatment have been mapped on the basis of neuronal expression of c-Fos, a protein product of the early response gene c-fos (10). CCK treatment increased c-Fos expression in both magnocellular and parvocellular OT neurons of the PVN and in magnocellular OT neurons of the SON (23, 26). Interestingly, similar activation of OT neurons was observed in rats after treatment with the appetite suppressant D-fenfluramine (13, 19), a serotonin releaser and reuptake inhibitor (9). The observations suggest that central OT pathways may also mediate the anorexigenic effects of D-fenfluramine.

Mice deficient in OT (40) provide a rodent model in which to study the proposed inhibitory effects of central OT on ingestive behaviors. Recently, we reported that OT-deficient (OT-/-) mice manifest a robust ingestion of NaCl solution after overnight fluid deprivation (2). When presented with a choice of water vs. 0.5 M NaCl, OT-/- mice ingested greater amounts of NaCl solution than OT+/+ cohorts (2). Because centrally released OT is believed to inhibit salt appetite in the laboratory rodent, presumably the absence of OT predisposes these mice to enhanced ingestion of NaCl-containing solutions.

In this study, we investigated food intake in OT-deficient mice. We hypothesized that OT-/- mice might consume more food after an overnight fast and might be less sensitive to the anorexigenic effects of systemically administered CCK or D-fenfluramine compared with wild-type (OT+/+) mice due to the absence of central OT signaling pathways thought to inhibit food intake. We measured food intake in OT-/- and OT+/+ mice under baseline conditions and after an overnight fast in the presence or absence of systemically administered CCK or in nonfasted mice after systemic administration of D-fenfluramine. We also evaluated c-Fos expression in specific subregions of the hindbrain and forebrain in mice of both genotypes after systemic CCK treatment.

MATERIALS AND METHODS

Animals

Male OT+/+ and OT-/- mice (6-8 mo of age, of C57BL/6 background) of the F4 and F5 generation were used in this study. Dr. W. Scott Young III (National Institute of Mental Health, Bethesda, MD) generated the OT-/- mice (40), and the breeding pairs were purchased from Jackson Laboratories, Bar Harbor, ME. Animals were bred in temperature-controlled, viral-free quarters of the University of Pittsburgh Animal Facility and housed in standard suspended boxes in groups of up to four per cage under a 12:12-h light/dark cycle (lights on at 0700). Food pellets and water were provided ad libitum. The growth rates of OT+/+ and OT-/- mice in our colony were monitored for 24 mo and did not differ (unpublished data).

Mice used for this study were housed individually for the week before and during each experimental protocol. The genotype of each mouse was determined using DNA extracted from a tail sample and prepared for PCR using methods previously described (2, 40). The studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh and conform to the "Guiding Principles for Research Involving Animals and Human Beings" (1).

Experimental Procedure

Experiment 1A: the effect of CCK on deprivation-induced food intake. Before experiments using CCK (Bachem, King of Prussia, PA), mice were acclimated to a nutritionally balanced liquid diet (BioServ, Frenchtown, NJ). Liquid diet was provided in graduated feeding tubes calibrated in 0.1-ml increments, which allowed for an accurate measure of food intake over a short period. Cumulative 24-h food intake was monitored for 4 days in OT+/+ (n = 8) and OT-/- (n = 8) mice.

A within-subjects crossover design was employed whereby each animal served as its own control. Animals that failed to acclimate to the liquid diet were removed from the study. OT+/+ (n = 7) and OT-/- (n = 8) mice were randomly assigned to one of two groups. The first group received an intraperitoneal injection (10 µl/kg body wt) of 0.9% saline vehicle in the first experimental test and then received an intraperitoneal injection of a similar volume of CCK (1, 3, or 10 µg/kg dissolved in 0.9% saline) 1 wk later in the second experimental test. A 100 µg/g dose of CCK was administered, but this dose resulted in immobilization of the mice for the entire 60-min observation period. The doses of CCK used for this study have been reported to decrease food intake and to increase plasma OT concentrations in laboratory rats (35). The second group of animals received the same treatments in reverse (CCK in the first test and saline in the second).

Liquid diet was removed the evening before each experimental test and reintroduced the following morning (18 h later). The effect of acute systemic CCK on food intake is short lived (3, 35); therefore, liquid diet was reintroduced immediately after intraperitoneal injection of CCK or saline vehicle. Subsequent liquid diet intake was recorded every 5 min for 30 or 60 min.

Experiment 1B: CCK-induced c-Fos expression. After an overnight fast (1600-0800) mice were injected intraperitoneally with 3 µg/kg CCK (n = 4 of each genotype), 10 µg/kg CCK (n = 3 OT+/+), or saline vehicle (n = 3 of each genotype). Food was not reintroduced. One hour after CCK or vehicle injection, mice were anesthetized by intraperitoneal injection of pentobarbital sodium and then perfused transcardially with 0.15 M NaCl followed by fixative (0.1 M sodium phosphate buffer containing 4% paraformaldehyde, 1.4% lysine, and 0.2% sodium metaperiodate) modified from McLean and Nakane (17). Fixed brains were removed from the skull, postfixed at 4°C for 10-18 h, and then cryoprotected for 24-72 h by immersion in aqueous 25% sucrose solution (4°C) before sectioning. Coronal 40-µm-thick sections were cut using a freezing-stage microtome, and tissue was stored at -20°C in cryoprotectant (38) until further processing.

Before immunocytochemical procedures, sections were removed from cryopreservant, rinsed in PBS, treated for 30 min in 1% sodium borohydride (Sigma, St. Louis, MO), and rinsed again in PBS. Primary and secondary antisera were diluted in PBS containing 0.3% Triton X-100 and 1% normal donkey serum. Rinses were performed in multiple changes of PBS over 30 min. The c-Fos antiserum used in this study (provided by Drs. Philip Larson and Jens Mikkelsen, Planum Institute, Denmark) was generated in rabbits immunized with amino acids 4-17 of synthetic c-Fos protein. The specificity of this antiserum has been verified (28). Tissue sections were incubated for 48 h at 4°C in rabbit anti-c-Fos (1:50,000), rinsed, and then incubated in biotinylated donkey anti-rabbit IgG (1:600; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. After rinsing, sections were processed using the Vectastain Elite avidin-biotin immunoperoxidase method (Vector Laboratories, Burlington, CA). A solution of diaminobenzadine (DAB), nickel sulfate, and H2O2 was used to generate blue-black nuclear c-Fos immunolabeling.

In two OT-/- and OT+/+ mice administered 3 µg/kg CCK, a second set of tissue sections was processed for dual immunoperoxidase localization of c-Fos plus either tyrosine hydroxylase (TH) for brain stem sections (1:40,000; mouse monoclonal antiserum, Chemicon, Temecula, CA), OT (rabbit anti-OT, 1:20,000; Peninsula Laboratories, Belmont, CA), or AVP (1:20,000; rabbit polyclonal antiserum, Peninsula, Belmont, CA) for forebrain sections. In three OT+/+ mice administered 10 µg/kg CCK, tissue sections were processed for dual immunoperoxidase localization of c-Fos plus OT. For this purpose, nuclear c-Fos immunolabeling was generated using a nickel-enhanced DAB reaction as described above, whereas cytoplasmic TH, AVP, or OT immunolabeling was subsequently generated using a nonenhanced DAB reaction to create brown immunoprecipitate. Immunolabeled tissue sections were mounted onto Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), cleared in graded alcohols and xylene, and placed under a coverslip using Histomount (VWR, Bridgeport, NJ).

The specificities of the c-Fos, TH, AVP, and OT antisera were verified by preabsorbing each antiserum at its working dilution with a 10 mg/ml solution of the appropriate synthetic immunogen. In each case, forebrain and brain stem immunoperoxidase labeling was abolished, providing evidence for antisera specificity.

General patterns of treatment-induced c-Fos expression were evaluated qualitatively by microscopic inspection of immunolabeled brainstem and forebrain sections. Particular attention was paid to the hypothalamus, amygdala, bed nucleus of the stria terminalis, and parabrachial complex. In addition, a quantitative analysis of c-Fos expression was conducted in the hindbrain through the rostrocaudal level of the area postrema (AP) and in the dorsal vagal complex (DVC), which includes the nucleus of the solitary tract (NST) and the dorsal motor nucleus of the vagus (DMV). For this purpose, c-Fos-positive neurons in each case were counted in three to four hindbrain tissue sections (at 160-µm intervals). Cells were considered c-Fos-positive when their nucleus contained blue-black nuclear immunoreactivity, regardless of intensity. The number of c-Fos-positive neurons within the NST, AP, and DMV was determined in each CCK- or vehicle-injected mouse. Potential differences in the number of c-Fos-positive neurons were tested for statistical significance by using three-way ANOVA, with mouse phenotype (OT+/+ or OT-/-), experimental treatment group (control or CCK), and brain stem region (AP, NST, DMV) as independent variables. When F values indicated significant overall main effects of treatment group and brain stem region on c-Fos activation values (see RESULTS), the ANOVA was followed up with planned t comparisons. Results were considered statistically significant when P < 0.05.

Experiment 2: the effect of D-fenfluramine on food intake. Before experiments using D-fenfluramine (Sigma, St. Louis, MO), mice were acclimated for 1 wk to ad libitum access to powdered rodent chow (ProLab RMH 3000 5P00, LabDiet/Purina). Specially designed feeders permitted an accurate measurement of food intake. To construct the feeders, a beaker (2 cm in diameter x 5.5-cm high), filled with pulverized food, was fitted to the center of a bowl (7 cm in diameter x 4-cm high). Wire mesh was placed on top of the open space between the outside of the beaker and the inside of the bowl. The mice were able to access the food in the beaker, and spilled food was collected in the outer bowl. Feeders were tared and food was weighed at the beginning and end of each 24 h. Fresh food was provided daily, and cumulative 24-h food intake was recorded daily. On the first test day (baseline condition), food intake of untreated mice was recorded every 30 min for 12 h, beginning at 1900. One week later, mice were injected intraperitoneally with 10 mg/kg D-fenfluramine dissolved in 0.9% saline (10 µl/g body wt) at 1900, and food intake was again recorded every 30 min for 12 h. The amount of food consumed over 12 h after D-fenfluramine treatment was compared with the amount of food consumed over 12 h during the prior baseline condition.

The dose of D-fenfluramine used in this study was based on a preliminary dose-response experiment. Twelve-hour feeding responses to D-fenfluramine were determined in OT+/+ and OT-/- mice (n = 3 of each genotype). Mice were tested at 2- to 3-wk intervals with 3 and 10 mg/kg ip D-fenfluramine. These doses have been reported to decrease food intake in nonfasted laboratory mice (30).

Statistics

Values are expressed as group means ± SE. Group data were analyzed by repeated-measures ANOVA. When the overall F ratio was significant, pairwise comparisons were made with the Bonferroni/Dunn post hoc comparison. Differences were considered significant when P < 0.05.

RESULTS

Experiment 1A: Effect of CCK on Deprivation-Induced Food Intake

During acclimation, mice of each genotype consumed equivalent volumes of liquid diet. On the third day of acclimation, neither cumulative 24-h food intake (OT+/+, 21.0 ± 1.4 ml vs. OT-/-, 18.4 ± 1.6 ml, P = 0.99) nor body weights (OT+/+, 28.0 ± 0.8 g vs. OT-/-, 28.7 ± 2.1 g, P = 0.73) differed between genotypes.

Thirty-minute food intake after overnight (18 h) food deprivation was not significantly different in saline-treated OT+/+ and OT-/- mice. In both genotypes, systemic administration of CCK decreased deprivation-induced food intake in a dose-dependent manner (Fig. 1). Administration of 1 µg/kg CCK significantly inhibited food intake (ANOVA, F1,12, P = 0.004) to a similar degree in both genotypes (ANOVA, F1,12, P = 0.20). The 3 µg/kg dose of CCK also inhibited food intake (ANOVA F1,26, P < 0.0001) to a similar degree in both genotypes (ANOVA, F1,12, P = 0.55). The 10 µg/kg dose of CCK significantly inhibited food intake (ANOVA, F1,12, P < 0.0001), but this dose resulted in immobilization of the mice for the first 20 min of observation. Therefore, we extended the observation time to 60 min for this group of animals. However, we did not observe genotypic differences in food intake after this CCK dose, even with the extended observation period (ANOVA, F1,12, P = 0.28).



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  		Fig. 1. Cumulative food consumption of wild-type and oxytocin (OT)deficient (OT+/+ and OT-/-, respectively) mice treated with saline or CCK after overnight food deprivation. Volume of liquid diet consumed was recorded every 5 min for 30 or 60 min after injection and reintroduction of food. Animals treated with 1 µg/kg (A; P = 0.004), 3 µg/kg (B; P < 0.001), or 10 µg/kg CCK (C; P < 0.001) consumed less food than those treated with saline. However, no differences were observed between genotypes after administration of saline (P > 0.05) or CCK at 1 µg/kg (P = 0.20), 3 µg/kg (P = 0.55), or 10 µg/kg (P = 0.28).

 

Experiment 1B: CCK-Induced c-Fos Expression

Intraperitoneal injection of 3 µg/kg CCK activated a small number of OT-positive neurons in the hypothalamus of OT+/+ mice (data not shown) and the 10 µg/kg dose of CCK robustly activated c-Fos in OT-positive magno- and parvocellular neurons of the paraventricular (PVN) and magnocellular neurons of the supraoptic nuclei (SON) of the hypothalamus of OT+/+ mice (Fig. 2). After an overnight fast and intraperitoneal saline injection, c-Fos-immunoreactive cells were sparse in the DVC and limbic forebrain of both OT+/+ and OT-/- mice (Fig. 3). In addition, intraperitoneal injection of 3 µg/kg CCK led to activation of c-Fos expression in the DVC (Fig. 3), parabrachial nucleus, PVN (Fig. 4), SON, the bed nucleus of the stria terminalis, and the central nucleus of the amygdala in mice of both genotypes. DVC neurons activated by CCK included cells in the vicinity of OT-immunopositive fibers (Fig. 3). In both OT+/+ and OT-/- mice, neurons activated by CCK included TH-positive neurons in the NST (Fig. 3); however, AVP-positive neurons in the PVN were not affected (Fig. 4). Three-way ANOVA of c-Fos activation values in the NST, AP, and DMV failed to reveal any genotype-related differences in neural activation, although significant main effects of treatment group (CCK vs. saline) and brain stem region (NST, DMV, AP) were found. As shown in Figs. 3 and 5, CCK treatment significantly increased c-Fos expression in the AP and NST, but not in the DMV, relative to c-Fos expression in control (saline treated) mice. These experimental treatment- and region-related effects were similar in OT-/- and OT+/+ mice. Quantitative analysis of c-Fos expression in the NST, AP, and DMV failed to reveal any genotype-related differences in CCK-induced neural activation (Fig. 5). Qualitative analysis of labeling in other regions of the brain stem and forebrain also failed to reveal any genotype-related differences in the distribution or density of CCK-induced c-Fos expression.



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  		Fig. 2. Color photomicrographs illustrating c-Fos immunostaining (blue-black nuclei) in the dorsal vagal complex (A; DVC) paraventricular nucleus (B; PVN), and supraoptic nucleus (SON) of the hypothalamus (C)ofOT+/+ mice treated with 10 µg/kg CCK. Tissue sections are double-labeled for c-Fos and OT (brown cells). Arrows, cells that are double labeled for c-Fos and OT. DMV, dorsal motor nucleus of the vagus; tr, tractus solitaris.

 


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  		Fig. 3. Color photomicrographs illustrating c-Fos immunostaining (blue-black nuclei) in the DVC of OT-/- and OT+/+ mice. A-D: hindbrain tissue sections are double labeled for c-Fos and tyrosine hydroxylase (TH; brown cells). Few c-Fos-positive neurons are present within the nucleus of the solitary tract (NST) in control (con; saline injected) OT-/- or OT +/+ mice (A and B). Conversely, robust c-Fos expression is observed within the NST of OT-/- and OT+/+ mice after CCK treatment (3 µg/kg; C and D). TH neurons are among those activated to express c-Fos in mice of both genotypes after CCK. Scale bar in A (100 µm) applies to all panels.

 


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  		Fig. 4. Color photomicrographs illustrating c-Fos immunostaining (blue-black nuclei) in the PVN in OT-/- and OT+/+ mice. A-D: tissue sections are double labeled for c-Fos and AVP (brown cells). Few c-Fos-positive neurons are present within the PVN of control (saline injected) OT-/- or OT+/+ mice (A and B). Conversely, robust c-Fos expression is observed within the PVN of OT-/- and OT+/+ mice after CCK treatment (3 µg/kg; C and D). Few AVP neurons are activated to express c-Fos in mice of either phenotype after CCK treatment. Scale bar in A (100 µm) applies to all panels.

 


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  		Fig. 5. Bar graphs depicting the mean number of c-Fos-positive neurons present within the area postrema (AP), NST, and DMV in OT-/- and OT+/+ mice after intraperitoneal injection of CCK (3 µg/kg) or saline (control). CCK treatment significantly increased c-Fos expression within the AP and NST in mice of both genotypes (P < 0.001 for both regions, in both phenotype groups), whereas CCK did not significantly activate DMV neurons (P > 0.05 in both phenotypes). There were no significant genotype-related differences in the number of c-Fos-positive neurons present within the AP, NST, or DMV (P > 0.05 for each comparison).

 

Experiment 2: Effect of D-fenfluramine on Food Intake

Cumulative 24-h food intake was similar in OT+/+ and OT-/- mice (4.18 ± 0.45 vs. 4.11 ± 0.35 g, P = 0.91) on the fourth day of acclimation to powdered diet. Mice treated with 10, but not 3 mg/kg, of D-fenfluramine consumed less food than untreated controls during 12 h of observation. The inhibitory effects of 10 mg/kg of D-fenfluramine on feeding were observed in both OT+/+ (P < 0.0001) and OT-/- (P = 0.03) mice. The magnitude of drug-induced anorexia was not different between the two genotypes (ANOVA, F1,10, P = 0.39; Fig. 6).



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  		Fig. 6. Cumulative food consumption of OT+/+ and OT-/- mice treated with 10 mg/kg D-fenfluramine. Amount of diet consumed was recorded every 30 min for 12 h after D-fenfluramine injection. Compared with untreated control animals, D-fenfluramine decreased food intake in both OT+/+, P < 0.0001, and OT-/- mice, P = 0.027. However, food intake was not different between D-fenfluramine-treated OT+/+ and OT-/- mice, P = 0.39.

 

DISCUSSION

In the present study, we investigated whether a congenital absence of OT in mice alters their feeding response to two anorexigenic agents. Because CCK and D-fenfluramine activate OT systems when administered at doses that suppress food intake (30, 35), we hypothesized that mice deficient in OT might be less responsive to the anorexigenic effects of these agents. However, this hypothesis was not supported. Food intake was decreased similarly by each agent in OT+/+ and OT-/- mice. We conclude that OT is unnecessary for the anorexigenic effects of systemically administered CCK or D-fenfluramine in C57BL/6 mice.

Increased plasma OT after administration of CCK in fasted rats has been interpreted as a marker for concomitant activation of central OT signaling pathways (35), which serve as the functional mediator of feeding inhibition. Central (5, 21), but not systemic (5), administration of OT or an OT agonist results in a dose-dependent inhibition of food intake in fasted rats. The OT-deficient mouse does not synthesize or release OT either centrally or peripherally and, therefore, provides an ideal model to assess the role of endogenous OT signaling in food intake. Our present findings indicate that the amount of food consumed under normal conditions and after an overnight fast is not affected by a congenital lack of OT, arguing against a critical role for this peptide in either the short- or long-term modulation of food intake in mice under physiological conditions.

In rats, plasma concentrations of OT are directly correlated with the magnitude by which food intake is suppressed by various anorexigenic treatments (16, 35). Additionally, pretreatment of rats centrally with an OT receptor antagonist attenuates (but does not eliminate) the anorexigenic effects of CCK (22). Administration of 10 µg/kg of CCK robustly activated OT neurons in the PVN and SON of OT+/+ mice.

Therefore, we predicted that the inhibitory effects of this dose of CCK on food intake would be greater in OT+/+ than OT-/- mice if OT were necessary for the anorexigenic effects of CCK in mice. However, all three doses of CCK used in this study inhibited deprivation-induced feeding to a similar extent in mice of both genotypes. Despite activation of OT neurons, we did not observe behavioral differences in food intake between genotypes. Species-related differences in neuroendocrine responses to CCK treatment have been reported. For example, systemically administered CCK increases plasma OT levels in rats (35), but not in monkeys (36) or humans (18). Although we have not measured plasma OT in response to CCK administration, magnocellular OT-positive hypothalamic neurons, which give rise to peripheral OT release, express c-Fos in wild-type C57BL/6 mice after CCK treatment.

Prior studies in rat indicate that systemically administered CCK binds to CCK-A receptors on gastrointestinal vagal afferents, thereby stimulating gastric and duodenal sensory input to the DVC (14, 20, 29, 42). The resulting viscerosensory signal subsequently is relayed from the DVC to other brain stem and forebrain regions, including the hypothalamus. Of particular relevance to the present study is evidence that hypothalamic neurons activated in rats after CCK treatment include OT neurons that project reciprocally to the DVC (23). This descending, OT-containing projection pathway has been implicated indirectly in contributing to the anorexigenic effects of CCK (23, 37) by activating NST inhibitory interneurons, which, in turn, inhibit gastric vagal motor neurons. Microinjection of OT into the DVC mimics the inhibitory effects of PVN stimulation on vagally mediated gastric motility (15), whereas administration of an OT antagonist into the DVC produces an abrupt increase in baseline gastric motility (7). Parvocellular OT neurons in the PVN have been proposed to provide tonic inhibitory control over vagally mediated gastric motility, such that activation of this descending, OT-containing pathway contributes to CCK-induced anorexia by enhancing local NST-mediated inhibition of gastric vagal motor outflow. Thus we hypothesized that CCK treatment would activate fewer DVC neurons (in particular, fewer NST neurons) in OT-/- mice compared with OT+/+ mice. Instead, we found that both the distribution and number of DVC neurons activated to express c-Fos after CCK treatment were similar in OT-/- and OT+/+ mice. Therefore, the absence of OT signaling pathways does not alter the ability of systemic CCK treatment to activate DVC neurons in mice.

In addition, we found that TH-positive neurons in the NST (i.e., catecholaminergic cells) were activated in mice of both genotypes after CCK treatment, consistent with previous findings in rat (27, 29). AVP-positive hypothalamic neurons were not activated after CCK treatment in either OT-/- or OT+/+ mice, consistent with evidence in rats that CCK activates OT but not AVP-containing neurons (37).

In this study, we also administered the appetite suppressant D-fenfluramine, which induces satiety by augmenting serotonin release and inhibiting its reuptake (8). Like CCK, D-fenfluramine activates OT neurons of the PVN (13, 33). Activation of central OT signaling pathways may partially underlie the appetite suppressant effect of D-fenfluramine in the rat (e.g., see Ref. 13), although this idea has not been directly tested (for example, by blocking central OT receptors prior to D-fenfluramine administration). A complete dose-response curve was not performed because of information provided in the literature reporting that 3 and 10 mg/kg D-fenfluramine inhibited food intake ~30 and 100%, respectively, in C57BL/6J mice (30). Therefore, both of these doses were used for this study. In the present study, D-fenfluramine decreased food intake to the same degree in mice of both genotypes. The ability of D-fenfluramine to reduce food intake may be mediated by other regulators of food intake. For instance, the melanocortin system enhances the anorectic efficacy of D-fenfluramine (11). Therefore, we conclude that neural signaling pathways other than those that use OT are sufficient to support D-fenfluramine-induced anorexia in mice.

Perspectives

Despite the absence of central OT pathways in OT-/- mice, baseline or deprivation-induced food intake was similar in OT+/+ and OT-/- mice. In summary, our findings indicate that OT plays no critical role in either short- or long-term controls of feeding in C57BL/6 mice, nor in the anorexigenic effects of two dissimilar pharmacological agents (i.e., CCK and D-fenfluramine). The C57BL/6 mouse may have modifying genes that compensate for the deletion of the OT gene, a phenomenon that has been reported in other gene deletion models for some strains of mice (24). Deletion of the OT gene in more than one strain of mouse may help to resolve this possibility. Additional experiments will be necessary to determine whether centrally administered OT inhibits food intake in C57BL/6 mice as it does in rats and whether mice from different background strains might exhibit differential responsiveness to the feeding-suppressive effects of central OT.

DISCLOSURES

Financial support for this work was provided, in part, by National Institutes of Health Grants HD-37268 (to J. A. Amico) and MH-01208 (to L. Rinaman).

ACKNOWLEDGMENTS

The authors acknowledge the technical assistance of Dr. H.-M. Cai, A. Czerveny, J. T. Johnson, J. R. Karam, and Dr. X. Li in the performance of these studies.

FOOTNOTES  

Address for reprint requests and other correspondence: R. C. Mantella, Dept. of Pharmaceutical Sciences, Univ. of Pittsburgh, 904 Salk Hall, Pittsburgh, PA 15261 (E-mail rcmst22{at}pitt.edu).

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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