Overexpression of 5-HT2C receptors in forebrain leads to elevated anxiety and hypoactivity

Abstract

The 5-HT(2C) receptor has been implicated in mood and eating disorders. In general, it is accepted that 5-HT(2C) receptor agonists increase anxiety behaviours and induce hypophagia. However, pharmacological analysis of the roles of these receptors is hampered by the lack of selective ligands and the complex regulation of receptor isoforms and expression levels. Therefore, the exact role of 5-HT(2C) receptors in mood disorders remain controversial, some suggesting agonists and others suggesting antagonists may be efficacious antidepressants, while there is general agreement that antagonists are beneficial anxiolytics. In order to test the hypothesis that increased 5-HT(2C) receptor expression, and thus increased 5-HT(2C) receptor signalling, is causative in mood disorders, we have undertaken a transgenic approach, directly altering the 5-HT(2C) receptor number in the forebrain and evaluating the consequences on behaviour. Transgenic mice overexpressing 5-HT(2C) receptors under the control of the CaMKIIalpha promoter (C2CR mice) have elevated 5-HT(2C) receptor mRNA levels in cerebral cortex and limbic areas (including the hippocampus and amygdala), but normal levels in the hypothalamus, resulting in > 100% increase in the number of 5-HT(2C) ligand binding sites in the forebrain. The C2CR mice show increased anxiety-like behaviour in the elevated plus-maze, decreased wheel-running behaviour and reduced activity in a novel environment. These behaviours were observed in the C2CR mice without stimulation by exogenous ligands. Our findings support a role for 5-HT(2C) receptor signalling in anxiety disorders. The C2CR mouse model offers a novel and effective approach for studying disorders associated with 5-HT(2C) receptors.

Fig. 1

Overexpression of 5-HT_2C receptors in forebrain of CaMKIIα–2CR transgenic (C2CR) mice. (A) A schematic illustration of the transgene construct used for overexpressing 5-HT_2C receptors in forebrain; HA, haemagglutinin epitope tag. (B) Representative autoradiograph of mouse brains (control, C2CR.10 and C2CR.33) showing 5-HT_2C receptor mRNA levels and distribution detected by in situ mRNA hybridisation histochemistry. Extensive enhancement of 5-HT_2C receptor mRNA density was observed in the C2CR.33 mice and a more restricted increase (mainly in the dorsal hippocampus) of 5-HT_2C receptor mRNA level was observed in the C2CR.10 mice. (C) Elevated 5-HT_2C receptor binding sites in the membrane fraction isolated from C2CR.33 mouse forebrain compared with the littermate controls (n = 5–6; *P < 0.01). There was no difference between genotypes in the hindbrain membrane fraction. Data are mean ± SEM.

Fig. 2

C2CR mice showed decreased activity. (A) Voluntary wheel-running activity: mice were individually placed in a cage with free access to a running wheel connected to a system for recording the number of wheel revolutions (a measurement of activity levels). The average number of wheel revolutions (over 7 days) generated by the C2CR mice was significantly lower than for the controls. (B) Open-field (novel environment) test: mice were individually placed in an open-field arena and their movement monitored for 5 min. Total distance travelled within the open-field arena in the C2CR mice was significantly less than for the controls. White bar, control mice; black bar, C2CR mice. Data are mean ± SEM; n = 6–8, *P < 0.05 genotypic comparison.

Fig. 3

C2CR mice showed increased anxiety-like behaviour. (A) EPM test: mice were placed individually in the centre of the EPM and their movement monitored for 5 min. The % distance travelled in the more anxiogenic open arms in the C2CR mice (black bar) was significantly lower than the controls (white bar). (B) Open-field test: mice were placed individually in one corner of the open-field arena and their movement monitored for 5 min. The ratio of central to total crossings in the open-field arena was used for quantifying the anxiety component in this test. The percentage crossing into the more anxiogenic central zone in the C2CR mice (black bar) was lower than for the controls (white bar). Data are mean ± SEM; n = 8–11, *P < 0.05 genotypic comparison.

Fig. 4

Effect of 5-HT_2C receptor agonists on locomotor activity in 2CR mice in a novel environment. (A) Distance travelled in the open-field arena was determined in control and C2CR mice following saline or mCPP (0.3 mg/kg) injection (i.p.). The distance travelled by control mice was significantly decreased following the mCPP treatment. However, mCPP had no effect on the distance travelled in C2CR mice compared with saline treatment. (B) The effect of RO 60-0175 on the inhibition of activity in the open-field test was compared with the effect of saline alone in the control or C2CR mice. Mice were administered RO 60-0175 (3 mg/kg, black bar; 5 mg/kg, striped bar) or vehicle (open bar) i.p., 30 min prior to testing activity in the open-field arena for 5 min. The higher dose of RO 60-0175 (5 mg/kg) reduced the activity of both control and C2CR mice, whereas the lower dose (3 mg/kg) was only efficacious in the C2CR mice. Data are mean ± SEM; n = 9–11; *P < 0.05 compared with saline in respective genotypes; **P < 0.05 compared to saline injection in control mice.