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Comparative Study
. 2008 Mar 12;28(11):2933-40.
doi: 10.1523/JNEUROSCI.5723-07.2008.

Serotonin 5-HT2B receptors are required for 3,4-methylenedioxymethamphetamine-induced hyperlocomotion and 5-HT release in vivo and in vitro

Stéphane Doly  1 , Emmanuel Valjent, Vincent Setola, Jacques Callebert, Denis Hervé, Jean-Marie Launay, Luc Maroteaux
Affiliations
Comparative Study

Serotonin 5-HT2B receptors are required for 3,4-methylenedioxymethamphetamine-induced hyperlocomotion and 5-HT release in vivo and in vitro

Stéphane Doly et al. J Neurosci. .

Abstract

The "club drug" 3,4-methylenedioxymethamphetamine (MDMA; also known as ecstasy) binds preferentially to and reverses the activity of the serotonin transporter, causing release of serotonin [5-hydroxytryptamine (5-HT)] stores from nerve terminals. Subsequent activation of postsynaptic 5-HT receptors by released 5-HT has been shown to be critical for the unique psychostimulatory effects of MDMA. In contrast, the effects of direct activation of presynaptic and/or postsynaptic receptors by MDMA have received far less attention, despite the agonist actions of the drug itself at 5-HT(2) receptors, in particular the 5-HT(2B) receptor. Here we show that acute pharmacological inhibition or genetic ablation of the 5-HT(2B) receptor in mice completely abolishes MDMA-induced hyperlocomotion and 5-HT release in nucleus accumbens and ventral tegmental area. Furthermore, the 5-HT(2B) receptor dependence of MDMA-stimulated release of endogenous 5-HT from superfused midbrain synaptosomes suggests that 5-HT(2B) receptors act, unlike any other 5-HT receptor, presynaptically to affect MDMA-stimulated 5-HT release. Thus, our findings reveal a novel regulatory component in the actions of MDMA and represent the first demonstration that 5-HT(2B) receptors play an important role in the brain, i.e., modulation of 5-HT release. As such, 5-HT(2B) receptor antagonists may serve as promising therapeutic drugs for MDMA abuse.

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Figures

Figure 1.
Figure 1.
Effect of 5-HT2B receptor inhibition on MDMA-induced hyperlocomotion. a, MDMA-induced locomotion in WT mice. WT mice were injected (i.p.) with either MDMA (10 mg/kg; squares) or saline solution (triangles) (arrow) after 1 h of habituation. Data, between 0 and 120 min, were analyzed using two-way ANOVA (means ± SEM; n = 12 per group): effect of MDMA, F(1,480) = 22.25; p = 0.0002. b, RS127445 abolishes MDMA-induced locomotion in WT mice. WT mice were injected with RS127445 [0.5 mg/kg (dark squares), 0.1 mg/kg (light gray squares), or 0.05 mg/kg (white squares)] solution 1 h before MDMA (10 mg/kg) injection (arrow). Data, between 0 and 120 min, were analyzed using two-way ANOVA (means ± SEM; n = 12 per group): effect of RS127445 compared with saline injection, 0.5 mg/kg, F(1,480) = 10; p = 0.005; 0.1 mg/kg, F(1,480) = 6.7; p = 0.02; 0.005 mg/kg, F(1,480) = 12.5; p = 0.003. c, MDMA-induced locomotion failed in 5-HT2B−/− mice. MDMA (10 mg/kg) or saline solutions were injected after 1 h of habituation (arrow). Effect of MDMA on 5-HT2B−/− mice (n = 12), F(1,528) = 1.31; p = 0.265. A Bonferroni posttest was also applied on each graph. *p < 0.05; **p < 0.01; ***p < 0.001; ns, nonsignificant.
Figure 2.
Figure 2.
Effect of 5-HT2B receptor inhibition on MDMA-induced 5-HT release as measured by in vivo microdialysis. a, Effect of MDMA (10 mg/kg; triangles) or saline injection (squares) on 5-HT concentrations in dialysates from NAcc of WT mice. b, c, Effect of RS127445 (5-HT2B receptor antagonist; b) or 5-HT2B receptor genetic ablation (c) on MDMA-induced 5-HT release in NAcc. d, Effect of saline (squares) or MDMA (triangles) in WT mice on 5-HT level in the VTA. e, f, Effect of MDMA in RS127445-treated mice (diamonds; e) or in 5-HT2B−/− mice (open circles; f) on 5-HT level in the VTA. MDMA or saline solutions were injected 35 min after testing began (arrow). Data (means ± SEM; n = 5 per group) were analyzed using two-way ANOVA: effect of MDMA on 5-HT level in the NAcc WT mice, F(1,144) = 6822.46; p < 0.0001; effect of MDMA on 5-HT level in 5-HT2B−/− NAcc or VTA mice, ns; effect of MDMA on 5-HT level in RS127445-treated mice NAcc or VTA mice, ns; effect of MDMA on 5-HT level in the VTA, F(18,162) = 110.29; p < 0.0001. A Bonferroni posttest was also applied on each graph. ***p < 0.001; ns: nonsignificant.
Figure 3.
Figure 3.
Effect of 5-HT2B receptor inhibition on MDMA-induced DA release as measured by in vivo microdialysis in NAcc. a, Effect of MDMA (10 mg/kg; open squares) or saline injection (filled squares) on DA concentrations in dialysates from NAcc of WT mice. b, Effect of RS127445 (0.5 mg/kg; diamonds) on MDMA-induced DA release in NAcc. c, Effect of 5-HT2B receptor genetic ablation (open circles) on MDMA-induced DA release in NAcc. MDMA or saline solutions were injected 35 min after testing began (arrow). Data (means ± SEM; n = 5 per group) were analyzed using two-way ANOVA: effect of MDMA on DA level in the NAcc WT mice, F(1,144) = 9162.79; p < 0.0001; effect of MDMA on DA level in RS127445-treated mice, ns; effect of MDMA on DA level in NAcc 5-HT2B−/− mice, ns. A Bonferroni posttest was also applied on each graph. *p < 0.05; **p < 0.01; ***p < 0.001; ns, nonsignificant.
Figure 4.
Figure 4.
SERT and DAT binding site and uptake analysis. a, SERT expression in 5-HT2B−/− and WT mice using radioligand saturation binding assays with [3H]citalopram on synaptosome membranes prepared from whole brain. [3H]citalopram binding analysis did not reveal differences in the Bmax. b, [3H] 5-HT uptake in 5-HT2B−/− and WT mice synaptosomal preparation from whole brain. Saturation isotherms of [3H]5-HT uptake were similar for the WT and 5-HT2B−/− mice, and nonlinear regression analysis did not reveal differences in the Vmax. c, Radioligand binding assays with [3H]GBR12935 (selective ligand for DAT) on membranes prepared from whole brain. Neither Bmax nor KD was altered in 5-HT2B−/− mice compared with wild-type mice. d, DAT function was assessed in wild-type and 5-HT2B−/− mice by measuring [3H]DA uptake in synaptosomes prepared from whole brain. The resulting saturation isotherms revealed no differences in either Vmax or Km, demonstrating that DAT uptake function is not altered in 5-HT2B−/− mice compared with wild-type mice.
Figure 5.
Figure 5.
5-HT2B receptor mRNA and protein expression in raphe nucleus. a, Total RNA were isolated from 5-HT2B+/+ or 5-HT2B−/− mice raphe nucleus. Putative contaminating genomic DNA was removed by digestion with RNase-free DNase. RT was omitted in samples indicated RT(−) as a control. On these samples, we used β-actin mRNA amplification, as positive control. We found 5-HT2B receptor mRNA only in WT mice raphe nucleus (first panel). b, BW723C86 (preferential 5-HT2B receptor agonist) injection (arrow) through microdialysis probe (10 nmol) in the raphe nucleus produced an increase in 5-HT extracellular concentration (open squares). Pretreatment with RS127445 (100 nm; selective 5-HT2B receptor antagonist) completely blocked BW723C86-induced 5-HT increase (filled circles), whereas RS127445 alone (open circles) had no effect on basal 5-HT concentration (filled squares). Data (means ± SEM; n = 5 per group) were analyzed using two-way ANOVA (repeated measures); each drug's effects were compared with saline. Effect of BW723C86 on 5-HT level, F(3,70) = 52.31; p < 0.0001. A Bonferroni posttest was also applied. ***p < 0.001.
Figure 6.
Figure 6.
MDMA-induced 5-HT release in vitro is 5-HT2B receptor dependent. MDMA (10 μm) induced 5-HT release from a superfused midbrain synaptosome preparation of WT mice (75 ± 5.7 fmol/sample), whereas it had no effect in 5-HT2B−/− mice (15 ± 1.8 fmol/sample). Data (means ± SEM) were analyzed using unpaired t test (two tailed): t = 9.9; df, 6. ***p < 0.001; ns, nonsignificant.
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