Effects of m-cpp and mesulergine on dietary choices in deprived rats: Possible mechanisms of their action

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1 Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Effects of m-cpp and mesulergine on dietary choices in deprived rats: Possible mechanisms of their action Spyridon Antonatos, Panagiota Galanopoulou Department of Experimental Pharmacology, Medical School, University of Athens, 75, M. Asias, str, Athens 11527, Goudi, Greece Accepted 26 August 2005 Available online 20 October 2005 Abstract Although it has been well established that compounds that stimulate 5-HT 2C and/or 5-HT 1B receptors induce hypophagia by promoting satiety process, the relative role of these receptor subtypes in dietary choices remains to be fully determined. m-cpp is considered a useful probe of 5-HT 2C receptor function in vivo and its administration reduces food intake and appetite in humans and rats. Conversely, the nonselective 5-HT 2C receptor antagonist mesulergine elicits feeding in rats. Food intake and dietary choices were measured in a fooddeprivation experimental protocol employing male Wistar rats. Animals were given access for a 4-h period to a pair of isocaloric diets. These two diets were enriched in protein or carbohydrate proportions, respectively, but fat content was held constant. The mixed 5-HT 2C/1B receptor agonist, m-cpp, led to a dose-dependent hypophagia, due to substantial reduction in carbohydrate consumption while protein intake was spared (0.62, 1.25 and 2.50 mg/kg i.p., respectively). The non-selective 5-HT 2C receptor antagonist and also D 2 agonist, mesulergine, on its own produced a significant dose-dependent increase in both protein and carbohydrate diets (1.0 and 3.0 mg/kg i.p., respectively). Combined treatment with m-cpp, at its maximum effective dose, and mesulergine dose-dependently reversed m-cpp-induced hypophagia, during the 4-h test period. In order to clarify the effects of mesulergine on dietary choices since it is simultaneously a dopamine agonist besides its antiserotonergic properties, the D 2 agonist apomorphine was also used. Apomorphine caused a dose-dependent increase in protein intake while carbohydrate and total food intake remained nearly unchanged (0.5 and 1.0 mg/kg i.p., respectively). It is concluded that the mesulergine-induced hyperphagic response on both diets is the expression of a dual mode of action, due to its 5-HT 2C antagonist activity together with D 2 agonist properties. The results further indicate that the activation of hypothalamic 5-HT 2C receptors may be involved in both protein sparing and carbohydrate suppressing effects of 5-HT (m-cpp-like effect), whereas an important role in increase of protein consumption seems to have the dopaminergic system probably through D 2 receptors (apomorphine-like and mesulergine-like effects, respectively). D 2005 Elsevier Inc. All rights reserved. Keywords: Apomorphine; Carbohydrate enriched diet; Dietary choices; Dopamine (DA) receptors; D 2 ; Food deprivation schedule; 5-Hydroxytryptamine (5-HT) receptors; 5-HT 2C ; Isocaloric diets; m-cpp; Mesulergine; Protein enriched diet 1. Introduction Abbreviations: CED, Carbohydrate enriched diet; DA, Dopamine; DMN, Dorsomedial nucleus of hypothalamus; 5-HT, 5-hydroxytryptamine (serotonin); LHA, Lateral area of hypothalamus; m-cpp, 1-(3-chlorophenyl)piperazine; PED, Protein enriched diet; PVN, Paraventricular nucleus of hypothalamus; SB , N-(1-methyl-5-indonyl)-NV-(3-pyridyl) urea hydrochloride; SB , 6-chloro-5-methyl-1-[2(2-methylpyridyl-3-oxy)-pyrid-5-yl carbamoyl] indoline; TFI, Total food intake; VMN, Ventromedial nucleus of hypothalamus. Corresponding author. Tel.: , ; fax: address: sorfeas@otenet.gr (S. Antonatos). The serotonergic system has an inhibitory role in appetite behaviour and has been the main target for drug development against obesity (Blundell and Halford, 1998; Brownell and Fairburn, 2002; Halford and Blundell, 2000; Leibowitz, 1991; Simansky, 1996, 1998). Furthermore, there is a close relationship between satiety, dietary choices and serotonergic activities (Blundell, 1984, 1991; Curzon, 1990; Halford et al., 2000; Leibowitz, 1990). Clinical and preclinical studies have shown that administration of compounds which enhance serotonergic neurotransmission (i.e., fenfluramine, fluoxetine and sertraline) induce hypophagia in both animals and humans (De Vry and /$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi: /j.pnpbp

2 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Schreiber, 2000; Garattini et al., 1992; Hewitt et al., 2002; Kitchener and Dourish, 1994; Sargent et al., 1997; Simansky and Vaidya, 1990). Conversely, administration of serotonergic receptor antagonists reducing serotonin (5-HT) activity may increase food intake (Curzon et al., 1997; De Vry and Schreiber, 2000; Dourish et al., 1989; Fletcher, 1988). Cloning and radioligand techniques have allowed the subdivision of serotonin (5-HT, 5-hydroxytryptamine) receptors into 7 distinct families, with each of 14 receptor subtypes that are recognized at present having its own properties (Hoyer et al., 2002). A variety of approaches have suggested that concurrent activation of 5-HT 2C and 5-HT 1B receptors located on hypothalamic sites leads to full expression of serotonergic satiety (De Vry et al., 2000, 2003; Simansky, 1996, 1998). Importantly, a confluence of evidence supports that 5-HT 2C receptors contribute substantially to the serotonergic suppression of feeding (Giorgetti and Tecott, 2004). Receptor binding studies and functional models in rats have demonstrated that m-cpp has approximately 10-fold selectivity for the 5-HT 2C over the 5-HT 1B receptor subtype (Kennett, 1993; Vickers et al., 2003). Accordingly, m-cpp is considered a useful probe of 5-HT 2C receptor function in both laboratory and clinic (Kennett and Curzon, 1988b; Sargent et al., 1997). In particular, studies using the mixed 5-HT 2C/1B receptor agonist, m-cpp, indicated that its hypophagic effects in rats are primarily mediated via activation of 5-HT 2C receptors and that stimulation of 5-HT 1B receptors plays only a minor role in m- CPP-induced hypophagia (De Vry and Schreiber, 2000; Hewitt et al., 2002; Kennett and Curzon, 1988a, 1991). Moreover, the use of 5-HT receptor antagonists such as mianserin, metergoline and mesulergine, which show some selectivity for the 5- HT 2C sites, strongly suggested that the 5-HT 2C receptors were largely responsible for mediating the anorectic effects of m- CPP (De Vry and Schreiber, 2000; Kennett and Curzon, 1988a,b, 1991; Prinsen et al., 1996, 2000). The use of more selective 5-HT 2C receptor antagonists in recent years, such as SB and SB , which also caused a remarkable blockade of m-cpp-induced hypophagia in rats, have strengthened this argument (Bickerdike et al., 1999; Kennett et al., 1994, 1997; Vickers et al., 2003). Assessment of the effects of serotonin (5-HT) receptor agonists and antagonists on feeding behaviour has been generally restricted to measurement of the consumption of solid standard food, i.e., pellets, whereas studies using macronutrient or diet selection have been scarce (De Vry and Schreiber, 2000). Unfortunately, diet selection paradigm studies are fraught with methodological problems that in turn result in a lack of definitive results. Contextual variables, such as quality of the test diets, form/type of macronutrient chosen, route of drug administration, acute or chronic treatment, respectively, nutritional state of the animals, deprivation or free feeding protocol, or even the isocaloric value of chosen diets among other things, could have a marked effect on any observed results (Halford et al., 2000; Leibowitz and Alexander, 1998). Although recent studies have shown that 5-HT drugs suppress carbohydrate and/or fat intake while sparing protein consumption, it is not yet fully clear that any specific 5-HT receptor subtype mediates the intake of a specific macronutrient (Halford et al., 2000; Meguid et al., 2000). The objectives of the present study were threefold. First, to investigate the effects of both the mixed 5-HT 2C/1B receptor agonist, m-cpp, and the non-selective 5-HT 2C antagonist, mesulergine, on macronutrient selection. m-cpp is mentioned to be a 5-HT 2C receptor ligand in vivo and its hypophagic effects are mainly, but not exclusively, mediated by agonistic activity at 5-HT 2C receptors (De Vry and Schreiber, 2000; Kaplan et al., 1998; Kennett and Curzon, 1988b; Schreiber and De Vry, 2002; Vickers et al., 2003). Specifically, the hypophagic effects of m-cpp are thought to reflect a druginduced acceleration of satiety processes at low doses (i.e., mg/kg) and may also involve an inhibition of appetitive processes at the same dose range (De Vry and Schreiber, 2000; De Vry et al., 1999, 2003; Kitchener and Dourish, 1994). On the other hand, mesulergine has been reported to stimulate food intake in rats (De Vry and Schreiber, 2000; Dourish et al., 1989; Kaplan et al., 1998; Kennett and Curzon, 1988b; Schreiber and De Vry, 2002). Second, to assess the precise role of 5-HT 2C receptors in macronutrient selection using combined treatment of m-cpp, at its highest effective dose, with mesulergine. A series of studies have revealed the marked, but not the complete, blockade of m-cpp-induced hypophagia by the non-selective 5-HT 2C antagonist mesulergine. (De Vry and Schreiber, 2000; Kaplan et al., 1998; Kennett and Curzon, 1988b). Third, to clarify the effects of mesulergine on dietary choices given that this compound produced an unexpected increase in protein intake in rats according to our first findings. Consequently, we should try apomorphine, a dopamine agonist at D 2 receptors, since mesulergine also possesses dopamine properties at D 2 receptors besides its antagonist activity at 5-HT 2C sites (Galanopoulou and Giannakopoulos, 1999; Giannakopoulos et al., 1998). It has been postulated that dopamine (DA) through D 2 receptors located in the lateral hypothalamus and adjacent areas inhibits markedly or even completely feeding behaviour (Inoue et al., 1995; Parada et al., 1988; Schwartz et al., 2000; Yang et al., 1997). Systemic administration of D 2 agonists (i.e., apomorphine, lisuride) that reduce dopaminergic neurotransmission led to a substantial hypophagic effect (Ferrari et al., 1992; Inui, 2000; Muscat et al., 1986; Treit and Berridge, 1990). Particularly, dopamine (DA) receptors have been related to the preference of protein and/or fat ingestion (Leibowitz, 1991, 1992). A two-isocaloric diet paradigm was used. These two isocaloric diets were prepared in our laboratory (Paleologos, 1967; Patston et al., 1984). Experimental animals were treated under a fooddeprivation schedule (16 h deprivation and consequently 32 h free access to food) in order to increase feeding motivation (Blundell and Latham, 1979; De Vry and Schreiber, 2000). All compounds and the combined treatment were given immediately prior to a 4-h refeeding period. It has been reported that the effects of acute administration of a compound on ingestive behaviour may be more pronounced during this short time interval (De Vry and Schreiber, 2000).

3 114 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Methods 2.4. Experimental procedure 2.1. Animals Male Wistar rats 2 months T10 days of age and mean body weight 210T5 g were used. Rats were randomly assigned to treatment groups (usually n = 6 8/group) and individually housed in a quiet environment under a normal 12-h light/dark period (light on at 8:00 a.m.). Room temperature was maintained at 21 T1- C and relative humidity at 50 T 15%. Animals were allowed free access to a pair of isocaloric diets enriched in protein or carbohydrate, respectively, and had ad libitum access to water. All animal experiments were reviewed and approved by the local committee and all studies have been carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No ) revised Effects were made in order to use the minimal amount of rats and reduce their suffering Drugs m-cpp (1-[3-chlorophenyl]piperazine) was obtained from Research Biochemicals International(Natick, MA). Mesulergine hydrochloride and apomorphine hydrochloride were purchased from Sandoz Pharma Company, Basle, Switzerland. All compounds were dissolved in 0.9% saline and were administered via the intraperitoneal route in a volume of 1 ml/kg body weight. The doses of all drugs were based on our previous tests (unpublished data) and published studies of the others (Clark et al., 1988; De Vry and Schreiber, 2000; De Vry et al., 2003; Dourish et al., 1989; Hewitt et al., 2002; Schreiber and De Vry, 2002; Treit and Berridge, 1990) Preparation of isocaloric diets Animals were singly housed in cages, supported by two feeders. They were supplied with the following pair of powdered isocaloric diets prepared in our laboratory (Paleologos, 1967). These two diets in their protein and carbohydrate content but fat content was held constant. Specifically, protein enriched diet (PED) in feeder A contained 80% casein, whereas carbohydrate enriched diet (CED) in feeder B contained 80% d-glucose with starch (Patston et al., 1984). The precise proportions of the components in both diets are shown in Table 1. Feeders were alternated daily to minimize place preference development. Table 1 Precise proportions of the components in Feeders A and B Feeder A (PED) Feeder B (CED) 80% casein 0% casein 4.65% d-glucose 44.65% d-glucose 4.65% starch 44.65% starch 5% minerals 5% minerals 5% corn oil 5% corn oil 0.5% vitamin mixture 0.5% vitamin mixture 0.25% cholinochloride 0.25% cholinochloride PED: protein enriched diet, CED: carbohydrate enriched diet. Experimental study was performed according to fooddeprivation schedule which consists of 16-h food deprivation and consequently free access to food for the next 32 h(blundell and Latham, 1979). A maximum of six groups (usually n = 6 8 per group) was tested in parallel, always including control group(s) treated with the corresponding vehicle of the particular drug(s) tested. Rats were once tested. Before testing animals were acclimatized to 3 periods of deprivation-refeeding and received a single injection of vehicle (0.9% saline) at times corresponding to subsequent drug administration. On the day of the experiment, rats received a single injection of a particular dose of a given drug or its vehicle 15 min before the refeeding period. When cotreatment was used, the antagonist was given 30 min before testing, i.e., 15 min before the second injection (agonist or saline). Then, animals were given a simultaneous free access to each pair of isocaloric diets. Feeders, which contained preweighed food, were removed 4 h after the last injection. Finally, protein (PED) and carbohydrate enriched diet (CED) selected by animals were measured and total food intake (TFI) was calculated too. All testing was conducted during the light phase of the cycle between 9:00 a.m. and 14:00 p.m. (Dourish et al., 1989; Kennett and Curzon, 1988b, 1991) Statistical analysis For data presentation, food intake was expressed in grams per 4 h of refeeding period and means and S.E.s were calculated for all animal groups. For agonist testing (Experiments 1 and 3), data were analysed by a one-way ANOVA with the factor dose and the dependent variable macronutrient intake. For antagonist testing (Experiment 2), data were analysed by a two-way ANOVA with the factors agonist and antagonist and the dependent variable macronutrient intake. The Tukey HSD test was done for post hoc multiple comparisons with P <.05 considered to indicate statistical significance. 3. Results 3.1. Effects of m-cpp on feeding and dietary choices in rats under food-deprivation schedule Administration of all three doses of m-cpp (Experiment 1) led to a dose-dependent hypophagia (Fig. 1). Specifically, protein consumption was spared while carbohydrate and total food intake were significantly suppressed in a dosedependent manner. All doses of m-cpp were effective in this test. Accordingly, 2.50 mg/kg of m-cpp (maximum effective dose) was selected for the subsequent antagonist study with mesulergine. ANOVA showed a significant effect for agonist on carbohydrate consumption, F(3,29)=2.237, P <.001; total food intake, F(3, 29) = , P <.001. The protein consumption was unaffected by m-cpp, F(3, 29) = 0.824, P < 1.0.

4 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) m-cpp 10 PED CED TFI Mean (S.E.) foodintake (g/4hr) ,62 1,25 2, ,25 2,50 0 0,62 1,25 2,50 Treatment (mg/kg, IP) Fig. 1. Effects of the 5-HT 2C/1B receptor agonist, m-cpp, on feeding behaviour and dietary choices at 4-h basis. Rats were deprived of food for 16 h. Data represent mean S.E.; n =8 per group. P <.05, P <.01 as compared with the corresponding vehicle-treated group for that particular time period Effects of serotonin antagonist mesulergine on m-cppinduced hypophagia and dietary choices in rats under fooddeprivation schedule The results for this experimental testing (Experiment 2) are presented in Table 2. The dose of 2.50 mg/kg of m-cpp induced a significant reduction in carbohydrate and total food intake, respectively whereas protein intake remained unchanged again. Pretreatment with the non-selective 5-HT 2C antagonist mesulergine, dose-dependently reversed m-cpp-induced hypophagia during the 4-h test period. In great detail, protein consumption was significantly increased while the hypophagic Table 2 Effects of the non-selective 5-HT 2C receptor antagonist mesulergine on m-cppinduced hypophagia and macronutrient intakes, respectively, at 4-h basis Treatment PED (g/4 h) CED (g/4 h) TFI (g/4 h) Vehicle+vehicle 1.50T T T0.50 Mesulergine 2.60T T T1.10 (1.0 mg/kg) +vehicle Mesulergine 3.0T T T1.20 (3.0 mg/kg) +vehicle Vehicle+m-CPP 1.70T T T0.80 (2.50 mg/kg) Mesulergine (1.0 mg/kg) + m-cpp (2.50 mg/kg) 2.50T0.50,. 7.20T0.40,. 9.70T0.70,. Mesulergine 2.95T0.70, T0.25, T0.15,.. (3.0 mg/kg) + m-cpp (2.50 mg/kg) Rats were deprived of food for 16 h. Data represent mean S.E.; n =8 per group. P <.05, P <.01 as compared with the corresponding vehicle vehicle-treated group for that particular time period.. P <.05,.. P <.01; comparison of mesulergine m-cpp-treated group with the corresponding vehicle m-cpptreated group. All compounds were administered i.p. PED: protein enriched diet, CED: carbohydrate enriched diet, TFI: total food intake. effects induced by m-cpp on carbohydrate and total food intake were blocked by pretreatment with mesulergine in a dose-dependent manner. Interestingly, mesulergine treatment on its own caused a significant dose-dependent hyperphagia, since a substantial increase in both protein and carbohydrate intakes were found with significant increase of total food intake as a consequence. ANOVA indicated a significant interaction effect between the factors agonist and antagonist on protein ingestion, F(5, 42) = , P <.001; carbohydrate ingestion, F(5,42)= , P <.001; and total food intake, F(5,42)=32.340, P < Effects of dopamine agonist apomorphine on feeding and dietary choices in rats under food-deprivation schedule The results for this experimental testing (Experiment 3) are presented in Fig. 2. Apomorphine, a selective D 2 agonist, caused a dose-dependent increase in protein intake whereas carbohydrate and total food intake remained nearly unchanged. Treatment with mesulergine produced the expected feeding behaviour. According to our findings, mesulergine led to a significant hyperphagia, due to an increased intake of both protein and carbohydrate enriched isocaloric diets. ANOVA gave a significant effect for Agonist on Protein intake, F(3,21)=11.410, P <.001. The carbohydrate and total food intake were unaffected by apomorphine. 4. Discussion A number of studies have demonstrated that the administration of the mixed 5-HT 2C/1B receptor agonist, m-cpp, causes a significant hypophagia through a clear serotonin-type action (Hewitt et al., 2002; Kennett and Curzon, 1988b). The results obtained in the present food-deprivation protocol replicated the hypophagic effects of m-cpp in a dose-dependent manner. Particularly, a substantial decrease in carbohydrate ingestion was noted without any important influence on protein

5 116 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Apomorphine 12,00 PED CED TFI 10,00 Mean (S.E.) foodintake (g/4hr) 8,00 6,00 4,00 2,00 0,00 0 0,5 1,0 Mesulergine 0 0,5 1,0 Mesulergine 0 0,5 1,0 Mesulergine Treatment (mg/kg, IP) Fig. 2. Effects of the D 2 receptor agonist, apomorphine, on feeding behaviour and dietary choices at 4-h basis. Rats were deprived of food for 16 h. Data represent mean S.E.; n =6 per group. P <.05, P <.01 as compared with the corresponding vehicle-treated group for that particular time period. 3 mg/kg of the non-selective 5-HT 2C receptor antagonist with dopaminergic properties, mesulergine, was used as positive control in the apomorphine experimental testing. consumption. However, it is well known that serotonin (5-HT) and compounds which enhance serotonergic neurotransmission, like fenfluramine and fluoxetine, induce a reduction in caloric intake attributed to a decrease in carbohydrate and/or fat consumption, while protein intake remains relatively unaffected (Halford et al., 2000; Meguid et al., 2000). The hypophagic effects of m-cpp are predominantly mediated by agonistic activity at 5-HT 2C receptors (De Vry and Schreiber, 2000; Kaplan et al., 1998; Kennett and Curzon, 1988b; Schreiber and De Vry, 2002; Vickers et al., 2003). A bulk of studies have recommended a role of 5-HT 2C and a lesser extend 5-HT 1B receptors in the control of ingestive behaviour (De Vry and Schreiber, 2000; De Vry et al., 2003; Hewitt et al., 2002). It has also been suggested that 5-HT 2C knockout mice (mutant mice lacking functional 5-HT 2C receptors) are less sensitive to the hypophagic effects of m-cpp, supporting the suggestion that 5- HT 2C receptors are involved in the hypophagic effect of m-cpp (Hewitt et al., 1998, 2002; Tecott et al., 1995). Particular attention has focused on the hypothalamus as the locus of serotonetgic effects on feeding. Systemic administration of indirect agonists such as d-fenfluramine and fluoxetine increases extracellular hypothalamus serotonin (5-HT) levels and suppresses food intake in animals (Giorgetti and Tecott, 2004; Vickers et al., 2001). Microinjections of serotonin into the paraventricular nucleus (PVN) of hypothalamus have been found to suppress feeding by reducing meal size and feeding rate (Hutson et al., 1988; Schwartz et al., 1989; Shor-Posner et al., 1986). Similar effects were also observed with microinjection of serotonin into the ventromedial (VMN) and dorsomedial (DMN) nuclei of the hypothalamus (Leibowitz et al., 1988). Local perfusion of m-cpp into ventromedial hypothalamic nucleus but not into lateral area of hypothalamus (LHA) or frontal cortex, inhibits intake in rats (Hikiji et al., 2004). 5- HT 2C receptors are expressed in these hypothalamic regions principally in ventromedial hypothalamic nucleus (VMN) implicated in the regulation of energy balance (Giorgetti and Tecott, 2004; Hoffman and Mezey, 1989; Kaplan et al., 1998; Wright et al., 1995). According to our findings in the present study, 5-HT 2C receptor activation seems to make the major contribution to the hypophagic actions of m-cpp. Regarding the pharmacological mechanisms underlying m-cpp-induced hypophagia in the rats, it has been reported that m-cpp has little affinity for dopamine (DA) receptors and does not produce significant alterations to dopaminergic system (Invernizzi et al., 1981; Kennett and Curzon, 1988b), which is also in agreement with our neurochemical findings (unpublished data). The above data and results indicate that hypophagic effects of m-cpp is mainly mediated by 5-HT 2C receptors, especially in carbohydrate suppressing and protein sparing effect of serotonin (5-HT). In addition, it is generally accepted that non-selective 5- HT 2C receptor antagonists, such as mianserin, metergoline and mesulergine appear to induce a dose-dependent hyperphagia (De Vry and Schreiber, 2000; Dourish et al., 1989; Kennett and

6 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Curzon, 1991; Prinsen et al., 1996, 2000). Consequently, only hyperphagic response on carbohydrate isocaloric diet could be explained as a single antagonist activity of mesulergine at hypothalamic 5-HT 2C receptors. On the contrary, the mesulergine-induced increase in protein consumption ought to be further clarified Agonism study with apomorphine Our previous suggestion is based on the fact that although apomorphine, produced a weak reduction in total food intake, it caused a dose-dependent significant enhancement of protein intake. In fact, a decrease in carbohydrate almost equal to the increase in protein intake was observed. This could imply a role of dopaminergic function in regulating the ratio of protein to carbohydrate intake. It has recently been postulated using microdialysis and pharmacological approaches, that the ventrolateral striatum is closely involved in the motor control of oral activity and feeding behaviour (Inoue et al., 1995; Salamone et al., 1993). Furthermore, dopaminergic system through D 2 receptors located in the lateral hypothalamus (LHA) and the adjacent area of ventrolateral striatum inhibits feeding behaviour at a wide range (Inoue et al., 1995; Parada et al., 1988; Schwartz et al., 2000; Yang et al., 1997), and the above mentioned receptors are implicated in the preference of protein and/or fat ingestion (Leibowitz, 1991, 1992). Mesulergine is considered a dopamine (DA) agonist at D 2 sites besides its affinity at 5-HT 2C receptors (Galanopoulou and Giannakopoulos, 1999) and this dual mode of its action is in line with our neurochemical results (unpublished data). So, a possible explanation is that activation of D 2 receptors underlies the potency of mesulergine in increasing protein intake. The results in this study are consistent with our previous findings in free feeding rats treated with mesulergine. Those animals were fed two diets, differing in protein and carbohydrate content, but not isocaloric, where, a dose dependent increase of PED and total food intake was observed 4 h after its administration (Giannakopoulos et al., 1998). Therefore, it is possible that both food intake and diet selection elicited by mesulergine in the present study can be also explained as an interaction between D 2 and 5-HT 2C receptors located, at least in part, in adjacent hypothalamic areas. Indeed, the use of apomorphine was determinant in explaining the effects of mesulergine on protein consumption as well Antagonism study with mesulergine potential role for 5- HT 2C receptors in diet selection Pretreatment with mesulergine abolished the hypophagia induced by m-cpp. Such data provide further pharmacological support that activation of 5-HT 2C receptors induces a suppression of total food intake due to a significant reduction in a carbohydrate ingestion, while protein intake is not altered. Interestingly, among the most remarkable findings from earlier antagonism studies are the marked blockade of m-cpp-induced hypophagia by the non-selective 5-HT 2C receptor antagonists mianserin, metergoline and mesulergine (Curzon et al., 1997; De Vry and Schreiber, 2000; Kaplan et al., 1998; Kennett and Curzon, 1988b; Prinsen et al., 1996, 2000). In corroboration of this finding, the hypophagic effect of m-cpp is completely blocked by low doses of the selective receptor antagonists SB and SB , respectively (Bickerdike et al., 1999; Hewitt et al., 2002; Kennett et al., 1994, 1997; Vickers et al., 2003). The relationship between 5-HT and nutrient intake has been questioned. Unfortunately, diet selection paradigm studies are fraught with methodological problems that in turn end in a lack of definitive results. Contextual variables, such as form/type of macronutrient chosen, caloric value of chosen diet, acute or chronic treatment regime, central or peripheral administration of serotonergic drugs could have a noticeable effect on any observed results in animal studies (Halford et al., 2000). The differing paradigm methodology produced strikingly different results. Nonetheless, many studies employing the three-choice diet selection paradigm have shown uniformly that stimulation of serotonin activity, through either microinjection of 5-HT and 5-HT agonists (i.e., fluoxetine, d-fenfluramine) into the paraventricular nucleus of the hypothalamus or peripheral administration of 5-HT agonists and 5-HT antagonists (i.e., metergoline), leads to the selective suppression of carbohydrate consumption with no change in the consumption of protein or fat (Halford et al., 2000; Leibowitz et al., 1993; Weiss et al., 1991). In most cases, the effects of acute administration of a serotonergic compound on cumulative food consumption over a short time interval (i.e., 30 min 4 h) was measured in food-deprivation schedules than in free feeding rats (De Vry and Schreiber, 2000). In particular, the design of the studies contained a period of food deprivation, typically varying between 12 and 23 h, in order to increase feeding motivation. In our testing session, animals were treated with a food-deprivation protocol and had a 4-h period free access to a pair of isocaloric diets, which differed in protein or carbohydrate proportions but fat levels were held constant. Our results replicated and extended earlier findings got under similar experimental conditions. It seems likely that hypothalamic 5-HT 2C receptors play a dominant, but not the exclusive, role in hypophagic effects of m- CPP. Specifically, the activation of 5-HT 2C receptors is associated with both protein sparing and carbohydrate suppressing effects of m-cpp. Although m-cpp is reported to be a potent 5-HT 2C receptor ligand in vivo, it is finally a mixed 5-HT 2C/1B receptor agonist. Therefore, it remains to be confirmed by studies using more selective 5-HT 2C receptor agonists. The extend to which 5-HT 2C receptors located in hypothalamic regions influence energy balance ought to be further investigated. Concerning hyperphagia induced by mesulergine, the blockade of 5-HT 2C receptors produces a significant increase in carbohydrate increase, mesulergine led to a substantial enhancement of protein intake, attributed to its dopaminergic properties. 5. Conclusions In the present study, results indicate that mesulergine, a serotonin (5-HT) antagonist at 5-HT 2C sites and dopamine D 2 agonist, leads to food intake and diet selection by a dual mode of action: due to the simultaneous antiserotonergic and

7 118 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) dopaminergic activity causes hyperphagia, which goes in parallel with an increase in CED and PED intake, respectively. It is also suggested that the activation of hypothalamic 5- HT 2C receptors may be involved in both protein sparing and carbohydrate suppressing effects of 5-HT (m-cpp-like effect). On the contrary, an important role in increase of protein consumption seems to possess the dopaminergic system probably through D 2 receptors (apomorphine-like and mesulergine-like effect, respectively). In conclusion, hypothalamic 5-HT 2C receptors play a dominant, not the exclusive, role in food intake and diet selection. These findings extend our understanding on neurobiological substrate of appetite and contribute to the studies related to new drugs against obesity, especially those referred to 5-HT 2C compounds with agonistic properties (Bickerdike, 2003; Clifton et al., 2000; Hewitt et al., 2002; Vickers et al., 2003). Acknowledgements This work was supported by a grant of the Athens University to Panagiota Galanopoulou. We thank Dr. Katherina Antoniou for her contribution and careful notices on the manuscript. The generous gifts of test compounds are gratefully acknowledged. References Bickerdike, M.J., HT 2C receptor agonists as potential drugs for the treatment of obesity. Curr. Top. Med. Chem. 3, Bickerdike, M.J., Vickers, S.P., Dourish, C.T., HT 2C receptor modulation and the treatment of obesity. Diabetes Obes. Metab. 1, Blundell, J.E., Serotonin and appetite. Neuropharmacology 23, Blundell, J.E., Pharmacological approaches to appetite suppression. Trends Pharmacol. Sci. 12, Blundell, J.E., Halford, J.C.G., Serotonin and appetite regulation: implications for the pharmacological treatment of obesity. CNS Drugs 9, Blundell, J.E., Latham, C.J., Serotonergic influences in food intake: effect of 5-hydroxytryptophan on parameters of eating behaviour in deprived and free feeding rats. Pharmacol. Biochem. Behav. 11, Brownell, K.D., Fairburn, C.G., Eating Disorders and Obesity: A Comprehensive Textbook, second edr Guilford Press, New York. Chapter 4. Clark, M.L., Dourish, C.T., Fletcher, A., Iversen, S.D., Blockade at postsynaptic 5-HT 1 receptors elicits feeding in satiated rats. Br. J. Pharmacol. 94, S316 (Suppl.). Clifton, P.G., Lee, M.D., Dourish, C.T., Similarities in the action of Ro , a 5-HT 2C receptor agonist and d-fenfluramine on feeding patterns in the rat. Psychopharmacology 152, Curzon, G., Serotonin and appetite. Ann. N. Y. Acad. Sci. 600, Curzon, G., Gibson, E.L., Oluyomi, A.O., Appetite suppression by commonly used drugs depends on 5-HT receptors but not on 5-HT availability. Trends Pharmacol. Sci. 28, De Vry, J., Schreiber, R., Effects of selected serotonin 5-HT 1 and 5-HT 2 receptor agonists on feeding behavior: possible mechanisms of action. Neurosci. Biobehav. Rev. 24, De Vry, J., Jentzsch, K.R., Schreiber, R., Effects of selective 5-HT 1/2 receptor agonists on operant food intake in rats. Behav. Pharmacol. 10 (1), S26. De Vry, J., Eckel, G., Kuhl, E., Schreiber, R., Effects of selective 5-HT 1/2 receptor agonists in conditioned taste aversion paradigm in rats. Pharmacol. Biochem. Behav. 66, De Vry, J., Schreiber, R., Daschke, A., Jentzsch, K.R., Effects of serotonin 5-HT 1/2 receptor agonists in a limited-access operant food intake paradigm in the rat. Eur. Neuropsychopharmacol. 13, Dourish, C.T., Clark, M.L., Fletcher, A., Iversen, S.D., Evidence that blockade of 5-HT 1 receptors elicits feeding in satiated rats. Psychopharmacology 97, Ferrari, F., Pelloni, F., Giuliani, D., Effects of the D 2 dopamine agonists lisuride and CQ, on rat feeding behaviour. Pharmacol. Biochem. Behav. 41, Fletcher, P., Increased food intake in satiated rats induced by the 5-HT antagonists methysergide, metergoline and ritanserin. Psychopharmacology 96, Galanopoulou, P., Giannakopoulos, G., Mesulergine: a review. CNS Drugs 5, Garattini, S., Bizzi, A., Caccia, S., Mennini, T., Progress report on the anorectic effects of dex-dexfenfluramine, fluoxetine and sertraline. Int. J. Obes. 16 (Suppl. 3), S43 S50. Giannakopoulos, G., Galanopoulou, P., Daifotis, Z., Couvaris, C., Effects of mesulergine treatment on diet selection, brain serotonin (5-HT) and dopamine (DA) turnover in free feeding rats. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 22, Giorgetti, M., Tecott, L.H., Contributions of receptors to multiple actions of 5-HT 2C central serotonin systems. Eur. J. Pharmacol. 488, 1 9. Halford, J.C.G., Blundell, J.E., Pharmacology of appetite suppression. Prog. Drug Res. 54, Halford, J.C.G., Smith, B.K., Blundell, J.E., Serotonin (5-HT) and serotonergic receptors in the regulation of macronutrient intake. In: Berthoud, H.R., Seeley, R.J. (Eds.), Neural and Metabolic Control of Macronutrient Intake. CRC Press, New York, pp Hewitt, K.N., Dourish, C.T., Clifton, P.G., HT 2C receptors and ingestive behaviour in mice. J. Psychopharmacol. 12, A38. Hewitt, K.N., Lee, M.D., Dourish, C.T., Clifton, P.G., Serotonin 2C receptor agonists and behavioural satiety sequence in mice. Pharmacol. Biochem. Behav. 71, Hikiji, K., Inoue, K., Iwasaki, S., Ichihara, K., Kiriike, N., Local perfusion of into ventrolateral hypothalamic nucleus but not into lateral hypothalamic area and frontal cortex, inhibits intake in rats. Psychopharmacology 174, Hoffman, B.J., Mezey, E., Distribution of serotonin 5-HT1C receptor mrna in adult rat brain. FEBS Lett. 247, Hoyer, D., Hannon, P.J., Martin, G.R., Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, Hutson, P.H., Donohoe, T.P., Curzon, G., Infusion of the 5-hydroxytryptamine agonists RU and TFMPP into the paraventricular nucleus of the hypothalamus causes hypophagia. Psychopharmacology 95, Inoue, K., Kiriike, N., Fujisaki, Y., Okuno, M., Ito, H., Yamagami, S., D 2 receptors in the ventrolateral striatum are involved in feeding behaviour in rats. Pharmacol. Biochem. Behav. 50, Inui, A., Trangenic approach to the study of body weight regulation. Pharmacol. Rev. 52, Invernizzi, R., Cotecchia, S., De Blasi, A., Mennini, T., Pataccini, R., Samanin, R., Effects of m-chlorophenylpiperazine on receptor binding and brain metabolism of monoamines in rats. Neurochem. Int. 3, Kaplan, J.E., Song, S., Grill, H.J., Serotonin receptors in the caudal brainstem are necessary and sufficient for the anorectic effect of peripherally administered m-cpp. Psychopharmacology 137, Kennett, G.A., HT 1C receptors and their therapeutic relevance. Curr. Opin. Investig. Drugs 2, Kennett, G.A., Curzon, G., 1988a. Evidence that m-cpp may have behavioural effects mediated by central 5-HT 1C receptors. Br. J. Pharmacol. 94, Kennett, G.A., Curzon, G., 1988b. Evidence that hypophagia induced by m- CPP and TFMPP requires 5-HT 1C and 5-HT 1B receptors, hypophagia induced by RU24969 only requires 5-HT1B receptors. Psychopharmacology 96, Kennett, G.A., Curzon, G., Potencies of antagonists indicate that 5-HT 1C receptors mediate 1-3 (chlorophenyl) piperazine-induced hypophagia. Br. J. Pharmacol. 103,

8 S. Antonatos, P. Galanopoulou / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) Kennett, G.A., Wood, M.D., Glen, A., Grewal, S., Forbes, I., Gadre, A., Blackburn, T.P., In vivo properties of SB A, a 5-HT 2C/2B receptor antagonist. Br. J. Pharmacol. 111, Kennett, G.A., Wood, M.D., Bright, F., Trail, B., Riley, G., Holland, K.Y., Avenell, K.Y., Stean, T., Upton, N., Bromidge, S., Forbes, I.T., Brown, A.M., Middlemiss, D.N., Blackburn, T.P., SB , a selective and brain penetrant 5-HT 2C receptor antagonist. Neuropharmacology 36, Kitchener, S.J., Dourish, C.T., An examination of the behavioural specificity of hypophagia induced by 5-HT 1B, 5-HT 1C and 5-HT 2 receptor agonists using the post-prandial satiety sequence in rats. Psychopharmacology 113, Leibowitz, S.F., The role of serotonin in eating disorders. Drugs 39 (Suppl. 3), S33 S48. Leibowitz, S.F., Brain neurochemical system controlling appetite and body weight gain. In: Rothwell, N.J., Stock, M.J. (Eds.), Obesity and Cachexia. John and Wiley and Sons, London, pp Leibowitz, S.F., Neurochemical neuroendocrine systems in the brain controlling macronutrient intake and metabolism. Trends Neurosci. 15, Leibowitz, S.F., Alexander, J.T., Hypothalamic serotonin in control of eating behavior, meal size, and body weight. Biol. Psychiatry 44, Leibowitz, S.F., Weiss, G.F., Shor-Posner, G., Hypothalamic serotonin: pharmacological, biochemical, and behavioural analyses of its feedingsuppressive action. Clin. Neuropharmacol. 11, S51 S71. Leibowitz, S.F., Alexander, J.T., Cheung, W.K., Weiss, G.F., Effects of serotonin and the serotonin blocker metergoline on meal patterns and macronutrient selection. Pharmacol. Biochem. Behav. 45, Meguid, M.M., Fetissov, S.O., Varma, M., Sato, T., Zhang, L., Laviano, A., Rossi-Fanelli, F., Hypothalamic dopamine and serotonin in the regulation of food intake. Nutrition 16, Muscat, R., Willner, P., Towell, A., Apomorphine anorexia: a further pharmacological characterization. Eur. J. Pharmacol. 123, Paleologos, G., Free amino acid content of tissues from rats with accelerated and depressed gluconeogenesis. In: Dissertation from PhD degree, New York, pp Parada, M., Hernandez, L., Hoebel, B.G., Sulpiride injections in the lateral hypothalamus induce feeding and drinking in rats. Pharmacol. Biochem. Behav. 30, Patston, P.A., Espinal, J., Randle, P.J., Effects of diet and of alloxandiabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues. Biochem. J. 222, Prinsen, E.P.M., Kleven, M.S., Koek, W., Further evidence for the involvement of 5-HT 2C receptors in m-cpp-induced hypophagia. Behav. Pharmacol. 7, S91. Prinsen, E.P.M., Koek, W., Kleven, M.S., The effects of antipsychotics with 5-HT 2C receptor affinity in behavioural essays selective for 5- HT 2C receptor antagonist properties of compounds. Eur. J. Pharmacol. 388, Salamone, J.D., Mahan, K., Rogers, S., Ventrolateral striatal dopamine depletions impair feeding and food handling in rats. Pharmacol. Biochem. Behav. 44 (3), Sargent, P., Sharpley, A.L., Williams, C., Goodall, E.M., Cowen, P.J., HT 2C receptor activation decreases appetite and body weight in obese subjects. Psychopharmacology 133, Schreiber, R., De Vry, J., Role of 5-HT 2C receptors in the hypophagic effect of m-cpp, ORG and CP-94,253 in the rat. Prog. Neuro- Psychopharmacol. Biol. Psychiatry 26, Schwartz, D., Hernandez, L., Hoebel, B.G., Fenfluramine administered systemically or locally increases extracellular serotonin in the lateral hypothalamus as measured by microdialysis. Brain Res. 482, Schwartz, M.W., Woods, S.C., Porte, D.J., Seeley, R.J., Baskin, D.G., Central nervous system control of food intake. Nature 404, Shor-Posner, G., Grinker, J.A., Marinesku, C., Brown, O., Leibowitz, S.F., Hypothalamic serotonin in the control of meal patterns and macronutrient selection. Brain Res. Bull. 17, Simansky, K.J., Serotonergic control of the organization of feeding and satiety. Behav. Brain Res. 73, Simansky, K.J., Serotonin and the structure of satiation. In: Smith, G.P. (Ed.), Satiation: Gut Brain. Oxford University Press, New York, pp Simansky, K.J., Vaidya, A.H., Behavioral mechanisms for the anorectic action of the serotonin (5-HT) uptake inhibitor sertraline in rats: comparison with directly acting 5-HT agonists. Brain Res. Bull. 25, Tecott, L.H., Sun, L.M., Akana, S.F., Strack, A.M., Lowenstein, D.H., Dallman, M.F., Julius, D., Eating disorder and epilepsy in mice lacking 5-HT2C serotonin receptors. Nature 374, Treit, D., Berridge, K.C., A comparison of benzodiazepine, serotonin and dopamine agents in the taste reactivity paradigms. Pharmacol. Biochem. Behav. 37, Vickers, S.P., Dourish, C.T., Kennett, G.A., Evidence that hypophagia by d-fenfluramine and d-norfenfluramine in the rat is mediated by 5-HT 2C receptors. Neuropharmacology 41, Vickers, S.P., Easton, N., Webster, L.J., Wyatt, A., Bickerdike, M.J., Dourish, C.T., Kennett, G.A., Oral administration of the 5-HT 2C receptor agonist, mcpp, reduces body weight gain in rats over 28 days as a result of maintained hypophagia. Psychopharmacology (Berl) 167 (3), Weiss, G.F., Rogacki, N., Fueg, A., Buchen, D., Suh, J.S., Wong, D.T., Leibowitz, S.F., Effect of hypothalamic and peripheral fluoxetine injection on natural patterns of macronutrient intake in the rat. Psychopharmacology 105, Wright, D.E., Seroogy, K.B., Lundgren, K.H., Davis, B.M., Jennes, L., Comparative localization of serotonin 1A, 1C, and 2 receptor subtype mrnas in rat brain. J. Comp. Neurol. 351, Yang, Z.J., Meguid, M.M., Chai, J.K., Chen, C., Oler, A., Bilateral hypothalamic dopamine infusion in male Zucker rat suppresses feeding due to reduced meal size. Pharmacol. Biochem. Behav. 58,