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. 2014 Oct;124(10):4351-62.
doi: 10.1172/JCI74726. Epub 2014 Aug 26.

Estrogens stimulate serotonin neurons to inhibit binge-like eating in mice

Xuehong Cao, Pingwen Xu, Mario G Oyola, Yan Xia, Xiaofeng Yan, Kenji Saito, Fang Zou, Chunmei Wang, Yongjie Yang, Antentor Hinton Jr, Chunling Yan, Hongfang Ding, Liangru Zhu, Likai Yu, Bin Yang, Yuxin Feng, Deborah J Clegg, Sohaib Khan, Richard DiMarchi, Shaila K Mani, Qingchun Tong, Yong Xu

Estrogens stimulate serotonin neurons to inhibit binge-like eating in mice

Xuehong Cao et al. J Clin Invest. 2014 Oct.

Abstract

Binge eating afflicts approximately 5% of US adults, though effective treatments are limited. Here, we showed that estrogen replacement substantially suppresses binge-like eating behavior in ovariectomized female mice. Estrogen-dependent inhibition of binge-like eating was blocked in female mice specifically lacking estrogen receptor-α (ERα) in serotonin (5-HT) neurons in the dorsal raphe nuclei (DRN). Administration of a recently developed glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate designed to deliver estrogen to GLP1 receptor-enhanced regions effectively targeted bioactive estrogens to the DRN and substantially suppressed binge-like eating in ovariectomized female mice. Administration of GLP-1 alone reduced binge-like eating, but not to the same extent as the GLP-1-estrogen conjugate. Administration of ERα-selective agonist propylpyrazole triol (PPT) to murine DRN 5-HT neurons activated these neurons in an ERα-dependent manner. PPT also inhibited a small conductance Ca2+-activated K+ (SK) current; blockade of the SK current prevented PPT-induced activation of DRN 5-HT neurons. Furthermore, local inhibition of the SK current in the DRN markedly suppressed binge-like eating in female mice. Together, our data indicate that estrogens act upon ERα to inhibit the SK current in DRN 5-HT neurons, thereby activating these neurons to suppress binge-like eating behavior and suggest ERα and/or SK current in DRN 5-HT neurons as potential targets for anti-binge therapies.

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Figures

Figure 6
Figure 6. Estrogens inhibit SK-like currents in DRN 5-HT neurons.
(A) A voltage clamp protocol to induce SK-like currents. (B) Representative traces for SK-like currents recorded from a 5-HT neuron before (black) and after (red) apamin perfusion. SK currents were shown as an outward tail current following step depolarization of Vh. (C–E) Representative traces before (black) and after (red) PPT treatment from responsive 5-HT neurons in WT brain slice (C), from 5-HT neurons in KO brain slice (D), from 5-HT neurons in WT slices preincubated with apamin (100 nM, 2 hours) (E). (F) PPT-induced changes in SK-like current in WT responsive neurons, in WT irresponsive neurons, in KO 5-HT neurons, or in WT 5-HT neurons preincubated with apamin (100 nM for 2 hours). n = 7–13/group. Results are shown as mean ± SEM. ***P < 0.001. (G) Effects of intra-DRN preinjections of saline or apamin (50 nM, 0.5 μl) on binge-like eating (2.5-hour HFD intake) in OVXV or OVXE WT and KO female mice. n = 5/group. Results are shown as mean ± SEM. **P < 0.01. (H) Intake of 0.1 M NaCl solution in OVX WT female mice receiving intra-DRN injections of saline or apamin (50 nM, 0.5 μl) in the CTA tests. n = 4/group. Results are shown as mean ± SEM.
Figure 5
Figure 5. An ERα agonist activates DRN 5-HT neurons.
(A–C) Brightfield (A), fluorescent with FRITC filter (B), and with FITC filter (C) illuminations of a targeted DRN 5-HT neuron. Scale bars: 10 μm. (D and E) Post hoc identification of the recorded neuron within the DRN in the fixed brain slice. Scale bars: 100 μm (D); 20 μm (E). (F–H) Representative traces before and after PPT treatment in 5-HT neurons from WT mice (F), in 5-HT neurons from KO mice (G), and in WT 5-HT neurons preincubated with 100 nM apamin for 2 hours (H). Upper panels: repetitive firing traces; values at the start of each trace are RMs. Lower panels: 3 continuous APs zoomed in from the upper-panel repetitive firing traces; values at the bottom of each trace are AHPA. (I) Cell numbers of 5-HT neurons with (responsive) or without (irresponsive) RM depolarization (> 1 mV) by PPT in various groups. ***P < 0.001. (J) Magnitude of depolarization induced by PPT in various groups. n = 9–18/group. Results are shown as mean ± SEM. **P < 0.01; ***P < 0.001. (K) Changes in firing rate induced by PPT in various groups. n = 7–18/group. Results are shown as mean ± SEM. ***P < 0.001. (L) Changes in AHPA induced by PPT in various groups. n = 7–18/group. Results are shown as mean ± SEM. ***P < 0.001.
Figure 4
Figure 4. GLP-1–estrogen inhibits binge-like eating in female mice partly through estrogenic actions on ERα in 5-HT neurons.
(A) Real-time RT-PCR analyses for Trim25 mRNAs in the DRN from female OVX mice receiving s.c. implantation of placebo pellets (OVXV), 17β-estradiol pellets (0.5 μg/d for 2 weeks; OVXE), single injection of GLP-1 (4 μg/kg, s.c., 2 hours; OVX + GLP-1), or GLP-1–estrogen (4 μg/kg, s.c., 2 hours; OVX + GLP-1–estrogen). n = 4–6/group. Results are shown as mean ± SEM. *P < 0.05 and **P < 0.01 vs. OVXV; #P < 0.05 vs. OVX+GLP-1 in 1-way ANOVA analyses followed by post hoc Bonferroni’s tests. (B) WT and KO mice (24 weeks) were ovariectomized. After a 7-day recovery, mice were subjected to intermittent HFD exposure for 1 week, as described in Methods. At the end of that week, mice received s.c. injections of saline, GLP-1 (4 μg/kg), or GLP-1–estrogen (4 μg/kg) at 10:30 am, and HFD and chow diet were provided to cages at 11:00 am; 2.5-hour HFD intake was measured. n = 8 or 9/group. Results are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 in 1-way ANOVA analyses followed by post hoc Bonferroni’s tests.
Figure 3
Figure 3. ERα in 5-HT neurons mediates estrogenic actions to inhibit binge-like eating in female mice.
(A–D) Representative immunohistochemistry for ERα from female WT (A and C) and KO (B and D) mice (after tamoxifen inductions). Scale bars: 100 μm. 3V, third ventricle; ME, median eminence. (E) WT and KO mice received tamoxifen inductions at 8 weeks of age (3 mg/injections, i.p., 24 hours apart). At 24 weeks of age, mice were ovariectomized and implanted with s.c. 17β-estradiol pellets (0.5 μg/d for 60 days; OVXE) or vehicle pellets (OVXV). After a 7-day recovery, mice were subjected to intermittent HFD exposure for 1 week, as described in Methods. At the end of that week, HFD and chow diet were provided to cages at 11:00 am, and 2.5-hour HFD intake was measured. n = 7–10/group. Results are shown as mean ± SEM. ***P < 0.001, between OVXV and OVXE mice in 2-way ANOVA analyses followed by post hoc Bonferroni’s tests. (F) Body weight, fat mass, and lean mass of WT and KO mice measured when binge-like behavior was assessed. n = 16 or 18/group. Results are shown as mean ± SEM. (G) Plasma orexin A measured in WT and KO mice after assessment of binge-like eating behavior. n = 6–7/group. Results are shown as mean ± SEM.
Figure 2
Figure 2. 5-HT neurons express ERα.
Representative immunofluorescent images for 5-HT (left, green) and ERα (middle, red) in coronal mouse brain sections containing the DRN (A), MRN (B), and CRN (C). Yellow neurons in the right panels indicate 5-HT neurons that coexpress ERα. Scale bars: 100 μm. Aq, aqueduct; PAG, periaqueductal gray; py, pyramidal tract.
Figure 1
Figure 1. Estrogen replacement inhibits binge-like eating in female mice.
(A and B) C57BL/6 female mice (12 weeks) were ovariectomized and implanted with s.c. 17β-estradiol pellets (0.5 μg/d for 60 days; OVXE) or vehicle pellets (OVXV). After a 7-day recovery, mice were subjected to intermittent HFD exposure or continuous HFD exposure for 1 week, as described in Methods. At the beginning of the next week, HFD and chow diet were provided to cages at 11:00 am, and 2.5-hour HFD intake (A) and chow intake (B) was measured. (C) Body weight was measured on the same day when binge-like behavior was assessed. n = 6–7/group. Results are shown as mean ± SEM. **P < 0.01; ***P < 0.001 between OVXV and OVXE mice in 2-way ANOVA analyses followed by post hoc Bonferroni’s test.
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