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. 2007 Oct 9;104(41):16335-40.
doi: 10.1073/pnas.0701471104. Epub 2007 Oct 3.

Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens

Alexandra Jean  1 , Grégory Conductier, Christine Manrique, Constantin Bouras, Philippe Berta, René Hen, Yves Charnay, Joël Bockaert, Valérie Compan
Affiliations

Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens

Alexandra Jean et al. Proc Natl Acad Sci U S A. .

Abstract

Anorexia nervosa is a growing concern in mental health, often inducing death. The potential neuronal deficits that may underlie abnormal inhibitions of food intake, however, remain largely unexplored. We hypothesized that anorexia may involve altered signaling events within the nucleus accumbens (NAc), a brain structure involved in reward. We show here that direct stimulation of serotonin (5-hydroxytryptamine, 5-HT) 4 receptors (5-HT(4)R) in the NAc reduces the physiological drive to eat and increases CART (cocaine- and amphetamine-regulated transcript) mRNA levels in fed and food-deprived mice. It further shows that injecting 5-HT(4)R antagonist or siRNA-mediated 5-HT(4)R knockdown into the NAc induced hyperphagia only in fed mice. This hyperphagia was not associated with changes in CART mRNA expression in the NAc in fed and food-deprived mice. Results include that 5-HT(4)R control CART mRNA expression into the NAc via a cAMP/PKA signaling pathway. Considering that CART may interfere with food- and drug-related rewards, we tested whether the appetite suppressant properties of 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) involve the 5-HT(4)R. Using 5-HT(4)R knockout mice, we demonstrate that 5-HT(4)R are required for the anorectic effect of MDMA as well as for the MDMA-induced enhancement of CART mRNA expression in the NAc. Directly injecting CART peptide or CART siRNA into the NAc reduces or increases food consumption, respectively. Finally, stimulating 5-HT(4)R- and MDMA-induced anorexia were both reduced by injecting CART siRNA into the NAc. Collectively, these results demonstrate that 5-HT(4)R-mediated up-regulation of CART in the NAc triggers the appetite-suppressant effects of ecstasy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Influence of the 5-HT4R on food intake and CART mRNA expression into the NAc. (A and C) The data are means ± SEM of total food intake in fed and food-deprived WT mice treated with an intraaccumbal infusion of 1 μl/min NaCl (n = 11–12), BIMU8 (n = 6–11 per each dose), RS39604 (n = 5–11 per each dose), si5-HT4R control (siCt, 0.05 μg/μl, n = 5–6), or si5-HT4R (0.05 μg/μl, n = 5–7). Food intake was measured between 1 and 3 h after treatments. (B–D) Number of CART mRNA copies in the NAc of identical mice over 3 h. (E) si5-HT4R-mediated decreases in the levels of 5-HT4R mRNA over 3 and 6 h, which is restored for 24 h. (F) Autoradiographs of 5-HT4R-binding sites labeled with [125I]SB207710 in transverse midbrain sections (a) at the level of the NAc (b) from mice treated with siCt (c) and si5-HT4R (d) over 6 h, into the NAc. (e) Nonspecific binding (see Experimental Procedures). Arrows point to decreases in 5-HT4R binding sites (d) and the injection site in a midbrain section labeled with hematoxylin (f), which labeled the nuclei and illustrates that siRNA did not induce any tissue damage. A significant treatment effect is noted (§, P < 0.05; §§, P < 0.01 compared with NaCl. *, P < 0.05; **, P < 0.01 compared with siCt). (Scale bar: 1 mm.)
Fig. 2.
Fig. 2.
In the NAc, the 5-HT4R/cAMP/PKA signaling pathway positively controls CART mRNA levels. (A) The data are means ± SEM of tissue cAMP levels in the NAc of WT mice 30 min after intraaccumbal injection of BIMU8 compared with NaCl-treated mice (n = 4 each). (B) The data are means ± SEM of CART mRNA copies in the NAc of WT mice 3 h after intraaccumbal injection of NaCl (n = 8), BIMU8 (n = 8), H89 (n = 6), or BIMU8/H89 (n = 5). H89 inhibited BIMU8-induced increases in the levels of CART mRNA. Treatments that differ significantly from saline are marked (§, P < 0.05; §§, P < 0.01).
Fig. 3.
Fig. 3.
5-HT4R KO mice are less sensitive to MDMA-induced anorexia-like behavior. Data are means ± SEM of total food intake in starved WT and KO mice. Animals received an i.p. injection of NaCl (WT, n = 20; KO, n = 10) or MDMA (WT, n = 10; KO, n = 10). MDMA-treated WT mice displayed hypophagia over 1 h (A) and 3 h (B), which was less marked in 5-HT4R KO mice. Significant differences between saline and MDMA-treated animals were detected (§§§, P < 0.0001; §§, P < 0.01). The significance between genotypes and genotype × treatment interaction is noted (***, P < 0.0001; **, P < 0.01; ##, P < 0.01; #, P < 0.05, respectively).
Fig. 4.
Fig. 4.
MDMA lost its ability to increase CART mRNA expression in the NAc of 5-HT4R KO mice. Using QR-PCR (A and B), in eight MDMA-treated WT mice, the levels of CART mRNA were higher than in seven saline-treated WT animals (F1,13 = 4.7, P < 0.05). This effect was not apparent in 5-HT4R KO mice (F1,10 = 0.8, n = 5–7). A significant difference between NaCl and MDMA is noted (*, P < 0.05), and a significant genotype × treatment interaction is marked (# P < 0.05). Using in situ hybridization (C–F), MDMA-induced increases in CART mRNA levels in the NAc were also observed in coronal brain sections in WT (C and D), but not in KO mice (E and F). (Scale bar: 700 μm.)
Fig. 5.
Fig. 5.
Injection of CART 55–102 into the NAc diminished deprivation-induced eating. Data are means ± SEM of total food intake in starved WT mice treated with 1 μl of NaCl (n = 7) or CART 55–102 at a dose of 1 μg/μl (n = 9) or 5 μg/μl (n = 7) or untreated (controls, n = 7). Surgical manipulations did not modify feeding responses compared with mice not submitted to any surgical procedure (controls) (P = 0.19). Treatments that differ significantly from saline are noted (§§, P < 0.01; §§§, P < 0.001).
Fig. 6.
Fig. 6.
siCART effect on mice. (A) Overeating after intraaccumbal injection of siCART in fed WT mice. Reduced labeling of both CART mRNA and peptide on the third day plus 3 h after the final intracerebral injection of siCART into the NAc of WT compared with the siCART control-treated mice. Data are means ± SEM of total food intake in fed WT mice treated with NaCl (n = 6), siCART control (0.05 μg/μl, n = 5; 0.1 μg/μl, n = 7), or siCART (0.05 μg/μl, n = 6; 0.1 μg/μl, n = 7) measured 3 h after injection. Treatments that differ significantly from control are noted (§, P < 0.05; §§, P < 0.01; §§§, P < 0.001). (B) Darkfield photomicrographs show CART mRNA labeling on coronal sections taken at the level of NAc in control and siCART-treated mice. (Scale bar: 850 μm.) (a and b) Observed labeling after intraaccumbal injection of a fluorescent siRNA (siGlo) to visualize the diffusion of 1 μl. (c–f) CART peptide-Ir fibers on coronal sections at the levels of the NAc (c and e) and lateral hypothalamus (d and f). Arrows point to decreases in CART-Ir (e and f). Substance P-Ir fibers (g and h) were visualized at similar levels of the NAc from identical animals (arrow marks the injection site). aca, anterior commissure, anterior. (Scale bar: 300 μm.)
Fig. 7.
Fig. 7.
MDMA-induced anorexia-like behavior in starved mice was reduced when CART was knocked down in the NAc by using siCART. Data are means ± SEM of total food intake for 1 h. (A) WT mice treated with saline or BIMU8 injected alone (n = 14–14) or combined with the siCART control (n = 15) or siCART (n = 10) into the NAc. (B) WT mice treated with an i.p. injection of saline or MDMA plus an intraaccumbal infusion of siCART control (n = 8 and 14, respectively) or siCART (n = 14 and 12, respectively). Treatments that differ significantly from NaCl are noted (§, P < 0.05; §§§, P < 0.0001). Significant differences between siCART and siCART control are marked (*, P < 0.05; **, P < 0.01). There was a significant interaction between both modes of injection (#, P < 0.05).
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