Introduction

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Acute central administration of corticotropin-releasing factor (CRF) reduces food intake (Britton et al., 1982; Morley and Levine, 1982; Gosnell et al., 1983), induces sympathetic nervous system activation (LeFeuvre et al., 1987; Arase et al., 1988), and activates a stress-like behavioral syndrome in rodents (Britton et al., 1982). The stress-like syndrome is characterized by a response-suppressing as well as a behaviorally "activating" component, which is largely dependent on the novelty of the environment (Britton et al., 1982, 1986a,b; Sutton et al., 1982). The behavioral stress syndrome induced by CRF may be at least partially mediated by its effects on catecholamine release because CRF increases hypothalamic and cortical norepinephrine release as it reduces grooming and locomotor activity and increases perseverative behaviors (Dunn and Berridge, 1987; Matsuzaki et al., 1989; Emoto et al., 1993a; Lavicky and Dunn, 1993). Furthermore, the effects of CRF or stress on food intake, stress-like behaviors, and central norepinephrine release can be reversed with CRF receptor antagonists (Britton et al., 1986b; Krahn et al., 1990; Emoto et al., 1993b; Shimizu et al., 1994), suggesting that CRF-induced anorexia and behavioral stress are specifically mediated by CRF receptors.

There are two primary CRF receptor subtypes, CRF₁ and CRF₂, with at least three splice variants for the CRF₂ subtype (Chen et al., 1993; Perrin et al., 1993; Lovenberg et al., 1995; Kostich et al., 1998). In rodents, CRF₁ receptors have been portrayed as mediating the anxiogenic and "stress-like" behavioral effects of CRF because they are localized in brain areas traditionally associated with emotionality, such as the neocortex, portions of the limbic system (amygdala and hippocampus), and the anterior pituitary (Behan et al., 1996). Furthermore, the CRF₁ knockout mouse shows reduced anxiety and an impaired stress response (Smith et al., 1998; Timpl et al., 1998). The same pattern of effects is observed when antisense to the CRF₁ receptor (Skutella et al., 1998) but not the CRF₂ receptor (Heinrichs et al., 1997) is centrally administered to rodents.

CRF₂ receptors have recently been implicated in the appetite suppressant effects of CRF. Although these receptors do overlap with CRF₁ receptors, in rodents they have an overall distribution pattern that is distinct from CRF₁ and are found in areas associated with the metabolic aspects of sympathetic nervous system activation, such as the ventromedial hypothalamus (Chalmers et al., 1995). Furthermore, the mammalian neuropeptide urocortin (UCN), which is more potent than CRF in activating the CRF₂ receptor (Vaughan et al., 1995), acts to suppress food intake (Spina et al., 1996). Finally, treatment with antisense to the CRF₂ receptor attenuates the effects of CRF on food intake, whereas treatment with the small-molecule CRF₁ antagonist NBI-27914 does not alter the appetite-suppressing effects of CRF (Smagin et al., 1998).

If differential receptor mediation of the "behavioral stress" and appetite suppressant effects of CRF results in separate physiological mechanisms for these CRF properties, then CRF₂ agonists could have potential as antiobesity therapeutics without "stress-like" adverse effects. Conversely, if "behavioral stress" is a necessary physiological component of CRF-induced appetite suppression, then CRF₂ or mixed antagonists may have utility as therapeutics for anxiety-related eating disorders, such as anorexia nervosa. We have explored these ideas in two ways: 1) we have assessed the appetite-suppressant effects of CRF while monitoring behaviors that might be associated with stress, such as locomotor activity and freezing; and 2) we have compared the abilities of a mixed CRF receptor antagonist, a CRF₁-specific antagonist, or a CRF₂-selective antagonist to attenuate CRF-induced appetite suppression (again while monitoring locomotor activity and freezing).

	Materials and Methods

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Subjects were female CD-1 mice (19-22 g; Charles River Breeding Laboratories, Wilmington, MA) that were group-housed (15-16 mice/cage) in the Neurocrine vivarium. The cages used for group housing measured 19 × 10.5 × 8 in. The vivarium was maintained on a 12-h light/dark cycle (7:00 AM-7:00 PM) and a constant temperature of 25-27°C. Mice were food-deprived 16 to 20 h before injection of CRF or UCN. This study was approved by the Institutional Animal Care and Use Committee at Neurocrine Biosciences.

Peptides and Antagonists. All peptides used in this study were synthesized by solid phase methodology on a Beckman 990 peptide synthesizer using t-Boc-protected amino acids and the assembled peptide was deprotected with hydrogen fluoride. The crude peptide product was purified by preparative HPLC. The purity and identity of the final product was ascertained by analytical HPLC and mass spectrometric analysis with an ion-spray ion source.

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View larger version (17K): [in this window] [in a new window]   Fig. 1.   Inhibition plots for UCN (as a comparison standard) and cUTSN (12-41) (106-1011nM) at CRF1 (A) and CRF2 (B) receptors. Inhibition curves for cUTSN (12-41) and NBI-27914 were generated with nonlinear least-squares curve-fitting software (Prizm, Inc.). A range of concentrations of cUTSN (12-41) and NBI-27914 were tested against 0.2nM 125I-Tyro-sauvagine (0.2 nM) for binding to either human CRF1 or CRF2 receptors, which were stably expressed in Chinese hamster ovary cells. The Ki for cUTSN (12-41) at the CRF1 receptor was 9.6 nM and at the CRF2 receptor was 16.1 nM.

Procedures. Peptides were injected into the lateral ventricle with a freehand method where mice were anesthetized with isoflurane for 1.5 to 2 min. Briefly, the injection site was found by visualizing an equilateral triangle between the eyes and the back of the head, with the apex of the triangle as the injection site. Anesthetized mice were injected with a 10-µl Hamilton syringe fitted with a 30-gauge needle. The needle was shortened by a "sleeve" made from peristaltic pump tubing so that the actual injection length was 4 to 4.5 mm. Injection accuracy was verified after completion of each study by a second freehand injection with cresyl violet (1 mg/ml). Success rate (as determined by dye injection) with this method has been 99% in this laboratory.

Statistics. Dose-response relationships were initially tested for statistical significance with one-way ANOVA with Fisher's least-significant difference test used as the post hoc test for means comparisons if the main effects were significant. Half-maximal effective doses were derived from a curve where percentage of maximal effect [(vehicle  highest dose)  (vehicle  dose(x))/(vehicle  highest dose)] × 100] was plotted against the log₁₀ dose. The half-maximal effective dose was defined as the dose that achieved 50% of the maximal effect. Curve-fitting was achieved with a 3-point sigmoid curve-fitting equation (Prizm, Inc., San Diego, CA). In cases where dose-response relationships were assessed over time, a mixed design, repeated measures ANOVA (dose × time) was used for the analysis. Statistically significant interactions were simplified with Fisher's least-significant difference test. Locomotor activity was assessed over the hour-long observation period with a one-way repeated measures ANOVA (dose × time). Photobeam breaks were averaged over 5-min bins, and those averages were used in the repeated measures ANOVA. The relationship between locomotor activity and food intake was assessed by correlating the 1-h food intake against total activity at each time bin, with food intake as the dependent variable. Finally, behavioral observation frequencies were correlated with a Pearson correlation matrix. To determine whether a correlation coefficient was significantly different from zero, a Fisher's r to z transformation was carried out on the correlation.

	Results

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Food Intake. Figure 2 shows that CRF significantly reduced food intake 1 to 6 h after injection (F_5,116 = 19.6; P < .0001), with all doses that were higher than 0.006 nmol significantly reducing food intake compared with the vehicle (P values <.002-.0001) 1 h after treatment. Higher doses were required to suppress feeding 3 and 6 h after injection, with the minimum effective dose at 3 h being 0.06 nmol (P < .009) and the minimum effective dose at 6 h being 0.21 nmol (P < .03). Because CRF appeared to be most potent at the first food-intake measurement (1 h after exposure to food, and 75 min after injection), the half-maximal effective dose (EC₅₀) for CRF was based on data obtained at the 1-h time point. The half-maximal effective dose for CRF-induced suppression of food intake was 23 pmol. cUTSN (12-41) significantly inhibited CRF-induced suppression of food intake in a dose-dependent manner (F_7,109 = 7.3; P < .0001), with all doses significantly attenuating CRF-induced suppression of food intake (P values <.04-.0001), as illustrated in Fig. 3A. When percentage of maximal inhibition of CRF-induced appetite suppression was plotted against log dose cUTSN (12-41), the half-maximal effective dose for inhibition was 1 nmol. Similarly, antisauvagine-30 (1 nmol) significantly attenuated CRF-induced appetite suppression (F_7,158 = 10.7; P < .0001) at all doses (P values <.03-.0001; Fig. 3C). The half-maximal dose for inhibition of CRF-induced appetite suppression was 1.35 nmol. In contrast, NBI-27914 did not significantly attenuate CRF-induced appetite suppression, whether it was given centrally or orally (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   .. Effects of CRF (0.006-0.6 nmol/5 µl; i.c.v.) on cumulative food intake in 16-h food-deprived female CD1 mice 1, 3, and 6 h after injection. *P values <.01-.0001 versus vehicle (n = 10-12 mice/group).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of cUTSN (12-41) (A), NBI-27914 (B), and antisauvagine-30 (C) on CRF (0.04 nmol)-induced suppression of food intake. CRF was coinjected (i.c.v.) with cUTSN (12-41) and NBI-27914 at doses of 0.13 to 10.2 nmol/2.5 µl, and antisauvagine-30 at doses of 0.01 to 10 nmol/2.5 µl. The half-maximal effective dose for inhibition of the CRF effect on food intake was 1 nmol for cUTSN (12-41) and 1.3 nmol for antisauvagine-30. NBI-27914 did not attenuate CRF-induced appetite suppression by 50% at any dose. *P values <.01-.0001 versus CRF (n = 13-15 mice/group). V, vehicle; C, CRF.

Locomotor Activity. Figure 4 shows that CRF significantly altered locomotor activity throughout the 60-min period used to measure its effects on food intake. Repeated measures analysis for CRF revealed a significant effect of treatment (CRF; F_5,95 = 4.6; P < .0008). CRF appeared to reduce the initial exploratory activity observed in vehicle-treated mice at all doses. This "damping" of exploratory activity began to disappear after 30 min, as suggested by a significant interaction between time and treatment for CRF (F_55,1045 = 3.7; P < .0001). Figure 5A shows that the general CRF antagonist cUTSN(12-41) significantly attenuated these effects of CRF on locomotor activity (F_7,109 = 11.2; P < .000). As for CRF alone, these effects began to subside after 30 min, as suggested by a significant interaction effect (F_77,1199 = 14.8; P < .0001). Similarly, antisauvagine-30 significantly attenuated the CRF-induced reduction in activity (F_7,1837 = 7.9; P < .0001), although the minimal effective dose was higher (1 nmol) than it was for cUTSN (12-41) (0.1 nmol; Fig. 5C). NBI-27914 did not significantly attenuate the effects of CRF on locomotor activity at any dose (Fig. 5B).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of CRF on total locomotor activity during food intake measurements. Total locomotor activity is expressed as a function of time (in 5-min bins) and CRF dose (0.006-0.63 nmol/5 µl). Activity is expressed as average number of photocell beam breaks/5 min bin/group.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of cUTSN (12-41) (A), NBI-27914 (B), and antisauvagine-30 (C) on CRF (0.04 nmol)-induced suppression of exploratory locomotor activity. In all groups except vehicle, CRF was coinjected (i.c.v.) with cUTSN (12-41) and NBI-27914 at doses of 0.13 to 10.2 nmol/2.5 µl, and antisauvagine-30 at doses of 0.01 to 10 nmol/2.5 µl. n = 13-15 mice/group. V, vehicle; C, CRF.

Snapshot Observations. Observations of freezing behavior, averaged over the entire 60-min period, were significantly increased in CRF-treated (F_4,26 = 5.2; P < .003) mice. When locomotor activity, frequency of freezing, piloerection, and overall food intake in CRF-treated mice were compared in a correlation matrix, overall food intake correlated positively with locomotor activity (r = 0.35; P < .0001) and negatively with piloerection (r = 0.3; P < .0001) and freezing (r = 0.36; P < .0001). Piloerection and freezing correlated positively with each other (r = 0.31; P < .0001) and negatively with locomotor activity (r values = 0.32, 0.28; P values <.0001). Furthermore, when food intake was regressed against total and ambulatory locomotor activity, freezing observations, and piloerection observations in a multiple regression analysis, all of the above-mentioned variables predicted food intake (overall r = 0.87; P < .0001). There were 300 total observations for each variable.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Comparison of CRF receptor antagonists' effects on total 60-min food intake (A), freezing observations averaged over 1 h (B), and piloerection observations averaged over 1 h (C). cUTSN (12-41) (1 nmol), antisauvagine-30 (1 nmol), or NBI-27914 (30 nmol) were coinjected with CRF (0.04 nmol). NBI-27914 (20-40 mg/kg p.o.) also was administered by oral gavage 30 min before CRF injection (0.04 nmol/5 µl). #P values <.0001 versus vehicle; *P values <.007-.0001 versus CRF.

	Discussion

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CRF dramatically reduced food intake in CD1 mice, with a half-maximal effective dose of 23 pmol, suggesting that CRF is at least as effective in inhibiting food intake in mice as it is in rats (Britton et al., 1982; Gosnell et al., 1983; Spina et al., 1996; Smagin et al., 1998). Furthermore, both the mixed antagonist cUTSN (12-41) and the CRF₂-selective antagonist antisauvagine-30 dose dependently reversed the effects of CRF on food intake. In contrast, the CRF₁-specific antagonist NBI-27914 had no effect on CRF-induced suppression of food intake. Thus, our pharmacological data support the initial hypothesis that the appetite-suppressant effects of CRF are mediated by the CRF₂ receptor, which was based on a comparison of the effects of CRF₁ and CRF₂ antisense administration on CRF-induced appetite suppression (Smagin et al., 1998).

In addition to suppressing food intake, CRF reduced exploratory locomotor activity (total and ambulatory) in a dose-dependent manner. These "immobilizing" effects of CRF on the exploration that normally occurs in a novel environment have been observed previously, although in an open field arena (Britton et al., 1982; Conti et al., 1994; Moreau et al., 1997). Interestingly, the dose of CRF that inhibited exploratory locomotor activity by 50% was similar to the ED₅₀ dose derived for the effects of CRF on food intake (63 pmol). Consistent with the above-mentioned findings, CRF-induced suppression of exploratory locomotor activity correlated significantly with the overall reduction in food intake induced by CRF.

The CRF-induced reduction in exploratory locomotor activity that accompanied appetite suppression was significantly attenuated by cUTSN (12-41) with a half-maximal dose, which was similar to that calculated for inhibition of CRF-induced appetite suppression (1 nmol). As with CRF-induced appetite suppression, NBI-27914 did not significantly attenuate CRF-induced immobility at any dose, whereas antisauvagine-30 attenuated the CRF effects on exploratory activity. Antisauvagine-30, however, was less potent than cUTSN (12-41) in its effects on CRF-induced exploratory activity suppression, which is somewhat surprising because of its very potent effects on all of the other measures. Whether this reflects anything about the role of CRF₂ receptors in CRF-induced behavioral suppression is unclear. Overall, the locomotor activity data suggest that 1) CRF-induced appetite suppression is directly related to the effects of these peptides on immobility, and 2) the CRF₁ receptor does not appear to be primarily involved in the reduction of locomotor activity that accompanies the effects of CRF on food intake.

Snapshot observation data also suggested that CRF-induced appetite suppression was a function of its nonappetitive behavioral effects. The number of freezing behavior observations in CRF-treated mice correlated in a statistically significant, negative direction with total food intake and locomotor activity. This is consistent with the idea that in a novel environment, CRF induced a generalized immobility, which directly competed with eating behavior. Furthermore, total food intake correlated in a negative direction with the number of piloerection observations. Piloerection, in turn, correlated positively with freezing behavior. When total and ambulatory locomotor activity, freezing, and piloerection were all regressed against total food intake as the dependent variable, all of these variables reliably predicted food intake in mice.

Again, the CRF₂-selective antagonist antisauvagine-30 had effects very similar to the mixed antagonist cUTSN (12-41) in that both significantly attenuated most of these CRF-induced behaviors, whereas the specific CRF₁ antagonist NBI-27914 failed to attenuate any of them. Oral doses of NBI-27914 actually appeared to exaggerate CRF-induced freezing and piloerection. Because central administration of this CRF₁-selective antagonist did not have such an effect, it is possible that the effects of orally administered NBI-27914 on CRF-induced freezing and piloerection are a consequence of some nonspecific mechanism related to the relatively high systemic doses used. Collectively, the snapshot and locomotor activity data suggest that 1) the suppression of food intake induced by exogenous CRF is a function of the effects that CRF has on competing behaviors, and 2) that the CRF₁ receptor does not appear primarily involved in any of the above-mentioned CRF-induced behaviors that accompany suppression of food intake.

Although the finding that NBI-27914 did not attenuate CRF-induced suppression of food intake was certainly in line with previous observations (Smagin et al., 1998), it was somewhat surprising that NBI-27914 did not effect the general behavioral suppression we observed after injection with CRF. Previous work has shown that several CRF₁ antagonists attenuate stress or CRF-induced anxiogenic behavior in mice and rats, with the light-dark conflict and plus maze as measures of anxiogenic behavior (Okuyama et al., 1999) and performance in the shock-escape task or fear-potentiated startle as measures of "stress-like" behavior (Schulz et al., 1996; Mansbach et al., 1997). All of the above-mentioned paradigms, however, involved stressors or centrally administered CRF as pretreatments, followed by, or concurrent with tests in environments that were also psychological stressors (Okuyama et al., 1999). Thus, in all of the cases mentioned above, CRF₁ antagonists have attenuated anxiogenic behaviors that were enhanced by a previous aversive experience or by moderate-to-high doses of CRF (0.2 nmol) immediately before anxiety testing. In contrast, the reduction in exploratory activity, freezing, and piloerection that accompanied CRF-induced suppression of food intake was an immediate response to even low doses of CRF (0.02 nmol) where no previous experience with stress was required. Perhaps CRF₁ receptors mediate responses to severe forms of stress or fear (where large amounts of endogenous CRF are released), and CRF₂ receptors mediate physiological responses to mild or moderate stress and excitation.

Indeed, several very recent articles have suggested that the CRF₂ receptor could play a role in the effects of CRF on certain forms of anxiety and stress. Liebsch et al. (1999) have shown that pretreatment with CRF₁ antisense attenuated social defeat-induced, anxiety-related behavior, but failed to attenuate the increased immobility observed in the forced swim task. Furthermore, CRF₂ antisense increased the immobility normally observed in the forced swim task, rather than having no effect, which might be expected if the CRF₂ receptor was not involved in stress-like behaviors (Liebsch et al., 1999). Another group (Radulovic et al., 1999) has shown that antisauvagine-30 attenuated the anxiogenic effects of a CRF injection into the CRF₂-dense lateral septum (Chalmers et al., 1995).

Therefore, either 1) NBI-27914 simply did not reach central CRF₁ receptors and the effects of antisauvagine-30 were mediated by its low-affinity binding to CRF₁ receptors, or 2) CRF₂ receptors mediate the entire anorexic syndrome induced by exogenous CRF. Resolution of these issues, of course, depends on the availability of a more selective, nonpeptide CRF₂ antagonist than antisauvagine-30.

In summary, the effects of CRF on food intake and/or appetite are significantly correlated with reduced exploration in addition to freezing and piloerection, suggesting that CRF may suppress food intake by activating behaviors that compete with eating. Furthermore, both the mixed CRF antagonist cUTSN (12-41) and the CRF₂-selective antagonist antisauvagine-30 attenuated CRF-induced appetite suppression as well as the behaviors associated with CRF-induced appetite suppression. In contrast, the CRF₁-specific antagonist NBI-27914 did not attenuate either CRF-induced appetite suppression or the stress-like behaviors that were associated with CRF-induced anorexia. Collectively, these data argue that the effects of CRF on food intake are associated with a behavioral stress-like syndrome, and that the CRF₂ receptor may play a more extensive role in the central effects of exogenous CRF than previously believed. These data also may suggest that CRF₂ antagonists could have therapeutic utility in disorders where anorexia is accompanied by a neuropsychiatric component, such as anorexia nervosa (Herzog et al., 1996; Pollice et al., 1997).