Mice. We crossed Tph2-CreER (Jackson Laboratory, #016584) transgenic mice and Esr1fl/fl mice (30). This cross produced KO mice (those that are homozygous for Esr1fl/fl and also carry the Tph2-CreER transgene) and WT mice (those that are homozygous for Esr1fl/fl, but do not carry the Tph2-CreER transgene). At 8 weeks of age, female KO mice received i.p. injections of tamoxifen (3 mg/mouse, twice, 24 hours apart) to induce Cre activity and therefore delete ERα only in 5-HT neurons. Female WT also received the same tamoxifen injections to rule out any possible effects of tamoxifen itself. Weekly body weight of these mice was monitored from weaning till the end of the study.

In parallel, we also crossed the Rosa26-tdTOMATO allele onto Tph2-CreER mice or Esr1fl/fl Tph2-CreER mice. This cross produced Tph2-CreER Rosa26-tdTOMATO (WT) and Esr1fl/fl Tph2-CreER Rosa26-tdTOMATO (KO) mice. Upon tamoxifen inductions similar to those described above, both mice expressed TOMATO exclusively in 5-HT neurons, and KO mice had Esr1 (ERα) deleted specifically in 5-HT neurons. These mice were used for electrophysiology recordings.

All the breeders have been backcrossed to C57BL/6 background for more than 12 generations. In addition, some C57BL/6 mice were purchased from the mouse facility of Baylor College of Medicine. Mice were housed in a temperature-controlled environment in groups of 2 to 5 at 22ÚC to 24ÚC using a 12-hour light/12-hour dark cycle. The mice were fed standard chow (6.5% fat, #2920; Harlan-Teklad) until training and assessment of binge-like eating behavior. Water was provided ad libitum.

Histology. We used dual immunofluorescence to examine the colocalization of ERα and 5-HT in mouse brain. Briefly, C57BL/6 female mice were perfused with 10% formalin, and brain sections were cut at 25 μm. The sections were incubated at room temperature in primary goat anti–5-HT antibody (1:5,000, #20079; Immunostar) overnight, followed by the secondary donkey anti-goat Alexa Fluor 488 (1:500; #A-11055; Invitrogen) for 1.5 hours. Then, the sections were incubated in the primary rabbit anti-ERα antibody (1:10,000, #06-935; Millipore) overnight, followed by secondary donkey anti-rabbit Alexa Fluor 594 (1:500; #A-21207; Invitrogen) for 1.5 hours. Slides were coverslipped with DAPI-containing mounting media (H1500; Vector Laboratories). Fluorescence images were analyzed using a Leica DM5500 fluorescence microscope with OptiGrid structured illumination configuration.

Similarly, we performed immunofluorescence for 5-HT in Tph2-CreER Rosa26-tdTOMATO mice to confirm colocalization of TOMATO and 5-HT. Briefly, mice (after tamoxifen inductions) were perfused with 10% formalin, and brain sections were cut at 25 μm. The sections were incubated at room temperature in primary goat anti–5-HT antibody (1:5000, #20079; Immunostar) overnight, followed by the secondary donkey anti-goat Alexa Fluor 488 (1:500, #A-11055; Invitrogen) for 1.5 hours. Slides were coverslipped with DAPI-containing mounting media (H1500; Vector Laboratories) and analyzed using a Leica DM5500 fluorescence microscope with OptiGrid structured illumination configuration. TOMATO signals were observed directly with a red fluorescence channel. As a negative control, TOMATO signals were also examined in Tph2-CreER Rosa26-tdTOMATO mice without tamoxifen induction.

We also performed immunohistochemistry for ERα in WT and KO mice to validate selective deletion of ERα in 5-HT neurons. Briefly, mice (after tamoxifen inductions) were perfused with 10% formalin, and brain sections were cut at 25 μm. The sections were incubated at room temperature in the primary rabbit anti-ERα antibody (1:20,000; Upstate) overnight followed by biotinylated anti-rabbit secondary antibody (1:1,000; Vector) for 2 hours. Sections were then incubated in the avidin-biotin complex (1:500, ABC; Vector Elite Kit) and incubated in 0.04% 3, 3′-diaminobenzidine and 0.01% hydrogen peroxide. After dehydration through graded ethanol, the slides were then immersed in xylene and coverslipped. Images were analyzed using a brightfield Leica microscope.

Training and assessment of binge-like eating behavior. We used the published protocol (29) to train and assess binge-like eating behavior in mice. Briefly, mice were randomly assigned into “intermittent” or “continuous” group. “Intermittent” mice were exposed to both regular chow pellets (6.5% fat, #2920; Harlan) and HFD pellets (40% fat, TD.95217; Harlan) for 48 hours (from Monday 11:00 am to Wednesday 11:00 am) and then exposed to only chow for the rest of the week. On the binge assessment day (Monday of the 2nd week), HFD was given back to cages at 11:00 am, and HFD and chow intake were measured for 2.5 hours (from 11:00 am to 1:30 pm). The “continuous” group was exposed to chow and HFD for the entire study, and HFD and chow intake were measured for 2.5 hours at the same time as in the “intermittent” group. Mice were housed in their original home cages for the entire training and study period. These cages (width: 7.25 inch, length: 11.5 inch, height: 5 inch; # RC71U-UD; Alternative Design) were made of polysulfone, with a gridded metal top holding a water bottle and pellet diets. A metal board was vertically inserted into the food holder to separate chow and HFD pellets.

Binge-like eating behavior in OVXV and OVXE mice. Female littermates were anesthetized with isoflurane. As previously described (42, 43), bilateral OVX was performed, followed by s.c. implantations of a pellet containing 17β-estradiol (0.5 μg/d for 60 days, OVXE) or containing vehicle (OVXV). These pellets were purchased from Innovative Research of America. After a 7-day recovery, mice were subjected to the intermittent or continuous (as control) exposure to HFD for 1 week in order to induce binge-like eating behavior. Two weeks after the surgery, binge-like eating behavior was assessed as detailed above. Body weight and food intake were monitored every other day during the entire study period. Body composition was determined using quantitative magnetic resonance on the same day when binge-like behavior was assessed. After assessment of binge-like eating behavior, mice were deeply anesthetized with isoflurane, and blood was collected through cardiac aspiration. Plasma was obtained by centrifugation, and plasma orexin A was measured with a mouse orexin A ELISA kit (MBS815052; MyBioSource).

Effects of GLP-1–estrogen on binge-like eating behavior in OVX female mice. First, to determine whether stable GLP-1–estrogen delivers bioactive estrogens into mouse DRN, we examined effects of GLP-1–estrogen on expression of Trim25 (a known estrogen target) in the DRN. To this end, female C57BL/6 mice (12 weeks of age) received OVX surgery as described above. After a 7-day recovery, these mice received s.c. injections of GLP-1 (4 μg/kg) or GLP-1–estrogen (4 μg/kg). Two hours after injections, mice were sacrificed and the DRN was quickly microdissected and stored at –80ÚC. As control groups, another cohort of female C57BL/6 mice (12 weeks of age) received OVXV or OVXE surgery as described above. After a 7-day recovery, the DRN was quickly microdissected and stored at –80ÚC. As described previously (44), total mRNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol, and reverse transcription reactions were performed from 2 μg of total mRNA using a High-Capacity cDNA Reverse Transcription Kits (Invitrogen). Samples were amplified on a CFX384 Real-Time System (Bio-Rad) using SsoADV SYBR Green Supermix (Bio-Rad). Correct melting temperatures for all products were verified after amplification. Results were normalized against the expression of housekeeping gene cyclophilin. Primer sequences were as follows: cyclophilin, forward: TGGAGAGCACCAAGACAGACA; cyclophilin, reverse: TGCCGGAGTCGACAATGAT; Trim25, forward: TGATGTGGCTGTGCATGATA; Trim25, reverse: AAGACCTGCTCCCCTACGAC.

Further, we tested to determine whether GLP-1–estrogen can inhibit binge-like eating behavior via acting upon ERα in 5-HT neurons. To this end, WT and KO female mice received tamoxifen inductions as described above at 8 weeks of age. At 24 weeks of age, these WT and KO mice were anesthetized with isoflurane and received bilateral OVX surgery. After a 7-day recovery period, mice were subjected to the 1-week intermittent HFD exposure to induce binge-like eating behavior as described above. On the binge assessment day, vehicle (saline), GLP-1 (4 μg/kg), or GLP-1–estrogen (4 μg/kg) was s.c. injected at 10:30 am, followed by assessment of binge-like eating behavior (11:00 am to 1:30 pm). The doses of GLP-1 and GLP-1–estrogen were chosen based on the previous report (31) and our preliminary studies.

Effects of intra-DRN injections on binge-like eating behavior in OVX female mice. WT and KO female mice received tamoxifen induction as described above at 8 weeks of age. At 24 weeks of age, mice were anesthetized with i.p. injections of the ketamine/xylazine cocktail (100 mg/kg ketamine and 10 mg/kg xylazine) and received OVXV or OVXE treatment as described above. During the same period under anesthesia, an indwelling microinjection cannula was stereotaxically inserted to target the DRN (midline, 4.36 mm posterior and 3.1 ventral to the bregma). After a 7-day recovery, mice were subjected to the 1-week intermittent HFD exposure to induce binge-like eating behavior as described above. On the binge assessment day, vehicle (saline) or apamin (50 nM, 0.5 μl, # STA-200; Alomone Labs) was injected into the DRN at 10:30 am, followed by assessment of binge-like eating behavior (11:00 am to 1:30 pm). The dose of apamin was chosen based on preliminary studies.

Conditioned taste-aversion tests. To rule out the possibility that intra-DRN injections of apamin inhibit binge-like eating behavior through nonspecific adverse effects, we performed conditioned taste aversion (CTA) tests as described in the literature (45). Briefly, C57BL/6 female mice (12 weeks of age) were bilaterally ovariectomized, and during the same period under anesthesia, an indwelling cannula was inserted to target the DRN as described above. After a 7-day recovery, these mice were housed individually and acclimated to intra-DRN injections (saline solutions) and a 2-hour daily water supply (3:00 pm–5:00 pm) over 8 days. On a test day, mice were given access to 0.2 m NaCl solution for 2 hours (3:00 pm–5:00 pm) in the same type of bottle with which they were usually presented with water. Immediately after exposure to the NaCl solution, mice received intra-DRN injections of saline or apamin (50 nM, 0.5 μl). The amount of solution intake was measured. The same tests were repeated in another 5 trials every other day.

Anxiety tests (light-dark test and EPM). To determine whether intermittent HFD exposure causes anxiety in mice, an independent cohort of C57BL/6 female mice (12 weeks) were subjected to intermittent or continuous HFD exposure for 1 week as described above. Binge-like eating behavior was confirmed in the “intermittent” HDF group in the second week. At 11:00 am on Monday of the third week (the same time when mice would be tested for binge-like eating behavior), these mice were subjected to the light-dark tests using published protocol (46). Briefly, the test consisted of a polypropylene chamber (44 × 21 × 21 cm) unequally divided into a larger, brightly illuminated open compartment (clear polypropylene) and a smaller, dark compartment (in dark polypropylene), connected by a small opening. Mice were placed in the illuminated chamber and allowed to move freely between the 2 chambers for 10 minutes. The latency to enter the light and dark chambers, the time spent in the chambers, the total number of transitions, and distance traveled in each chamber was measured using the VersaMax Animal Activity Monitoring System (AccuScan Instruments Inc.) and analyzed. Transfer of all 4 paws of an experimental animal from one chamber to the other was considered as 1 transition event.

At 11:00 am on Tuesday of the third week, these mice were subjected to the EPM using published protocol (46). The EPM was constructed of Plexiglas, with 2 open arms (30 × 5 cm) and 2 enclosed black arms (30 × 5 × 15 cm) at an elevation of 50 cm above the floor. The arms of the maze form a cross, with the 2 open arms facing each other. The maze was cleaned with 70% ethanol solution after each session and allowed to dry between the sessions. Anxiety-like behavior was measured by placing the mice in the center of the junction of the arms of the maze facing an open arm, and the behavior was analyzed for 10 minutes. The number of entries into the open and closed arms, the time spent exploring the open and closed arms, and the distance traveled were recorded and analyzed using the ANY-maze software (Stoelting Co.). The changes in anxiety-like behavior were calculated by dividing the number of entries into the open arms by the total number of entries into all 4 arms (open to total ratio for entries) or by dividing the amount of time spent in the open arms by the amount of time spent in all 4 arms (OTR for time). The time spent in the center platform not exploring any of the arms was not included in these calculations.

Forced swim tests. To determine whether the intermittent HFD exposure causes depression in mice, an independent cohort of C57BL/6 mice was subjected to the intermittent or continuous HFD exposure for 1 week as described above. Binge-like eating behavior was confirmed in the intermittent HDF group in the second week. At 11:00 am on Monday of the third week (the same time when mice would be tested for binge-like eating behavior), these mice were subjected to the forced swim tests using published protocol (47–49). Briefly, the mice were individually placed into a glass cylinder (25 cm tall × 10 cm diameter) containing 8 cm of water, maintained at 23°C to 25°C for 6 minutes. The mouse movement was digitally recorded from the side using a camcorder. Water in the cylinder was replaced after each recording. The starting and total time of immobility for each mouse was recorded. Immobility is defined as the absence of active floating with only minimal movements needed to keep head above water. The immobility was scored every 30 seconds for the last 4 minutes. During each 30 seconds, 1 immobility score was recorded if the immobility time was more than 10 seconds. Data from mice that had difficulty keeping heads above water were excluded from analyses. All the videos were blindly rated by a different experimenter.

Electrophysiology. Mice were deeply anesthetized with isoflurane and transcardially perfused (50) with a modified ice-cold artificial cerebral spinal fluid (aCSF) in: 10 mM NaCl, 25 mM NaHCO 3 , 195 mM sucrose, 5 mM glucose, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM sodium pyruvate, 0.5 mM CaCl 2 , and 7 mM MgCl 2 ) (51). The mice were then decapitated, and the entire brain was removed. Brains was quickly sectioned in ice-cold aCSF solution (126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl 2 , 2.4 mM CaCl 2 , 1 mM NaH 2 PO 4 , 11.1 mM glucose, and mM 21.4 NaHCO 3 ) (52) saturated with 95% O 2 and 5% CO 2 . Coronal sections containing the DRN (270 μm) were cut with a Microm HM 650V vibratome (Thermo Scientific) and then preincubated in the aCSF (52) at 34°C for at least 1 hour before recording.

Whole-cell patch clamp recordings were performed in the target neurons in the DRN visually identified by an upright microscope (Eclipse FN-1; Nikon) equipped with IR-DIC optics (×40 NIR; Nikon). Signals were processed using Multiclamp 700B amplifier (Axon Instruments), sampled using Digidata 1440A, and analyzed offline on a PC with pCLAMP 10.3 (Axon Instruments). The slices were bathed in oxygenated aCSF (52) (32°C–34°C) at a flow rate of approximately 2 ml/min. Patch pipettes with resistances of 3 to 5 M⁄ were filled with solution containing 126 mM K gluconate, 10 mM NaCl, 10 mM EGTA, 1 mM MgCl 2 , 2 mM Na-ATP, and 0.1 mM Mg-GTP (adjusted to pH 7.3 with KOH) (34). Voltage clamp was used to record SK-like currents. Current clamp was engaged to test neural firing, RM, and input resistance. Based on previous reports and our preliminary data, PPT (100 nM, perfusion up to 6 minutes at 2 ml/min; Sigma-Aldrich, H6036) (53), acute apamin perfusion (100 nM, perfusion upon to 8 minutes at 2 ml/min), and chronic apamin incubation (100 nM, incubation about 2 hours) (34) were used. Alexa Fluor 488 (0.01 mM) was included in the pipette solution to trace the recorded neurons. After recordings, slices were fixed with 4% formalin overnight and mounted onto slides. Cells were then visualized with the a Leica DM5500 fluorescence microscope to identify post hoc the anatomical location of the recorded neurons in the DRN.

Statistics. The data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism. Most data were analyzed by 1- or 2-way ANOVA, followed by post hoc Bonferroni’s tests. Comparisons between 2 groups were analyzed by t tests. Numbers of responsive neurons in each group were analyzed by χ2 tests. P < 0.05 was considered statistically significant.

Study approval. Care of all animals and procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.