Male C57BL/6 mice and FVB/N-Tg(GFAPGFP)14Mes/J (GFAP-GFP; #003257, RRID:IMSR_JAX:003257) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). As described previously (7), the mice were around 3 months old (~25 g) and were kept in standard housing conditions with food and water freely available. All operations were carried out in accordance with the USA National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023) and its 1978 revision, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University.

Most chemicals, including fluoxetine (fluoxetine hydrochloride; F132), SB204741 [N-(1-Methyl-1H-5-indolyl)-N′-(3-methyl-5-isothiazolyl)urea; S0693], U0126 [1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene monoethanolate; U120], LY294002 [2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; L9908], and a primary antibody raised against β-actin (A5441) were purchased from Sigma (MO, USA). Other primary antibodies were acquired from Millipore (MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). MCC950 (CP-456773; HY-12815) was purchased from MedChem Express (Shanghai, China). WP1066 [(E)-3(6-bromopyridin-2-yl)-2-cyano-N-(S0-1phenylethyl)acrylamide] was from EMD Chemicals (San Diego, CA, USA). Recombinant murine leptin (obesity protein; 450-31) was obtained from Preprotech Inc. (Jiangsu, China). BDNF enzyme-linked immunosorbent assay (ELISA) kit was from R&D Systems (MN, USA).

FVB/N-Tg(GFAPGFP)14Mes/J mice were used for SD models. SD was induced by “gentle handling” according to standard protocols (20), including the introduction of new objects into the cage or gentle touching with a brush to keep the mice awake. As described previously (7), SD was induced for 6 h, from 7 a.m. to 1 p.m. During the treatment of SD, the mice were offered food and water ad libitum. Animals in the sham group were kept undisturbed in a separate room with the same light/dark cycle. The mice were treated with sham or SD stimulation for 3 weeks. During the third week, MCC950 (50 mg/kg for intraperitoneal injection), SB204741 (0.5 μmM in 2 μL for intracerebroventricular (ICV) infusion), U0126 (10 μM in 2 μL for ICV infusion), LY294002 (10 μM in 2 μL for ICV infusion), or vehicle (artificial cerebrospinal fluid, ACSF) was injected each day, then fluoxetine (10 mg/kg/day), leptin (4 mg/kg/day) or PBS (phosphate buffered saline) used as control group was injected intraperitoneally.

FVB/N-Tg(GFAPGFP)14Mes/J mice were used for collecting astrocytes. A purified astrocyte suspension isolated from the cortex and hippocampus was prepared as described previously (7). Briefly, tissue from two transgenic mice were pooled for one sample. Wavelengths of GFP excitation and emission were 488 and 530/30 nm, respectively. The purity of the sorted astrocytes has been confirmed in our previous study (21) by checking mRNA expression of cell markers of astrocytes, neurons and oligodendrocytes.

Astrocytes were cultured according to our previous description (22, 23). In brief, primary cultures of mouse astrocytes were prepared from the cerebral hemispheres of newborn C57BL/6 mice. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 7.5 mM glucose. For the entire third week, 0.25 mM dibutyryl cyclic AMP (dBcAMP) was included in the medium. All dishes of primary cultured astrocytes were randomly separated into different experimental groups.

As described previously (24), the cultured astrocytes were incubated in DMEM without serum for half day before transfection. A transfection solution containing 2 μl oligofectamine (Promega, Madison, WI, USA), 40 μl Opti-MEMI, and 2.5 μl siRNA (666 ng) was added to the culture in every well for 8 h. In the siRNA-negative control cultures, transfection solution without siRNA was added. Thereafter, DMEM with three times serum was added to the cultures. The siRNA duplex chains of leptin receptor (LepR), 5-HT_2B receptor (5-HT_2BR) and c-fos were purchased from Santa Cruz Biotechnology (CA, USA).

Twenty-four mice were randomly separated into different groups, and every mouse was scheduled for tests, and the test sequences were randomly assigned to avoid interference from different tests.

Western blotting was performed as previously described (23, 25). The protein content was determined by using BSA as the standard. Samples containing 50 μg protein were added into 10% polyacrylamide slab gels. After protein transfer to nitrocellulose membranes, the membranes were blocked by 5% skimmed milk powder in TBS-T for 2 h and then incubated with the primary antibody for 2 h at room temperature. After washing of the membrane, the specific binding was detected by goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated secondary antibodies. Staining was visualized with ECL detection reagents, and images were acquired with an electrophoresis gel imaging analysis system. Band density was measured in Windows AlphaEase FC 32-bit software.

According to our previously described (25, 27), BDNF level in the sorted astrocytes from GFAP-GFP transgenic mice or in the cultured astrocytes was measured via a total BDNF immunoassay kit methods. In brief, the measurements were performed according to the manufacturer's protocol. Samples were collected in pyrogen/endotoxin-free tubes. The lower limit of detection was <10 pg/ml.

Total RNA was reverse transcribed, and PCR amplification was performed with a Robocycler thermocycler, as our previously described (28). In brief, the relative quantities of the transcripts were assessed using 5-fold serial dilutions of RT product (200 ng). The RNA quantities were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) before calculation of the relative expression of 5-HT_2BR, c-fos, and LepR. Values were first calculated as the ratio of the relative expression of 5-HT_2BR and GAPDH, then the values were normalized by control group.

Differences between multiple groups with one or two variables were evaluated by one-way or two-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) or a Tukey-Kramer post-hoc multiple comparison test for unequal replications using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA). Sample size was not predetermined by formal power calculation, and no samples or data were excluded from the analysis. All statistical data in the text are expressed as the mean ± SEM; the level of significance was set at p < 0.05.

Three weeks of SD triggered depressive-like behaviors, as shown in Figures 1A,B. Exposure to SD decreased the percentage of sucrose preference by 44 ± 3.5% (n = 12) of control group (the left panel of Figure 1A). Meanwhile, 3-weeks of SD significantly elevated the immobility time by 126 ± 7.3 and 53 ± 9.5% (n = 12) of control group, in the forced swimming and tail suspension tests, respectively (the middle and right panels of Figure 1A). The depressive-like behaviors induced by SD required the activation of NLRP3 inflammasomes. Treatment with MCC950 (an inhibitor of NLRP3 inflammasomes) alleviated the depressive-like behaviors induced by SD: the percentage of sucrose preference was significantly increased by about 40 ± 4.2% (n = 12) compared with SD group (the left panel of Figure 1A), and the immobility time was reduced to 64 ± 6.3 or 80 ± 10.7% (n = 12) of SD group in the forced swimming or tail suspension test, respectively (the middle and right panels of Figure 1A).

Antidepressant fluoxetine effectively remedied the depressive-like behaviors induced by SD, as shown in Figure 1B. This effect of fluoxetine on depressive-like behaviors required the involvement of 5-HT_2B receptor. Treatment with SB204741 (an antagonist of 5-HT_2B receptor) inhibited effects of fluoxetine in behavioral tests: the pre-treatment with SB204741 decreased sucrose preference to the 73 ± 3.3% (n = 12) of the fluoxetine group (the left panel of Figure 1B), significantly prolonged the immobility time by 75 ± 6.1% (n = 12) in forced swimming test (the middle panel of Figure 1B), and by 35 ± 8.3% (n = 12) in tail suspension test compared with fluoxetine treated-SD group (the right panel of Figure 1B). However, sole treatment with SB204741 did not affect results of behaviors tests compared with neither control nor SD groups.

As have been shown previously, fluoxetine inhibits the activation of NLRP3 inflammasomes in astrocytes, which was induced by 3 weeks of SD of 3 weeks (7). Here, we further corroborated that inhibition of fluoxetine effect on NLRP3 inflammasome was mediated by 5-HT_2B receptor. In astrocytes isolated from transgenic mice fluoxetine reduced the protein level of NLRP3 and caspase-1 to 35 ± 4.3 and 72 ± 2.9% (n = 6) of SD group, respectively (Figure 1D). However, the pretreatment with SB204741 eliminated effects of fluoxetine on the expression of NLRP3 and caspase-1 in SD models (Figure 1D). Meanwhile, SD with or without fluoxetine had no effects on the protein level of ASC or procaspase-1 (Figure 1D).

The level of BDNF was significantly decreased in the astrocytes isolated from SD-treated transgenic mice, as shown in Figures 2A,B. Exposure to SD suppressed the protein expression of BDNF to 43 ± 3.3% (n = 6) of control group measured by western blotting (the upper panel of Figure 2B), the level of BDNF measured by ELISA was decreased by 39 ± 4.3% (n = 6), as compared with control group (the lower panel of Figure 2B). Furthermore, MCC950 (the inhibitor of NLRP3 inflammasomes) partly reversed this suppressive effect of SD on the level of BDNF (Figures 2A,B). In the isolated astrocytes, the protein expression of BDNF was elevated by 90 ± 2.7% (n = 6; the upper panel of Figure 2B), whereas the release of BDNF was increased by 30 ± 3.9% (n = 6; the lower panel of Figure 2B), as compared with SD group.

As shown in Figure 2C, fluoxetine increased the expression of BDNF by 92 ± 4.2 or 36 ± 5.7% (n = 6), with or without SD treatment, respectively. Pretreatment with SB204741 (an antagonist of 5-HT_2B receptors), completely abolished the effect of fluoxetine on BDNF. Similarly, as compared with PBS group exposed to SD in animals, fluoxetine increased the level of BDNF by 50 ± 7.5% (n = 6), but after the pretreatment with SB204741, the elevated BDNF by fluoxetine was decreased by 10 ± 7.2% (n = 6; Figure 2D).

Administration of fluoxetine to the primary cultured astrocytes increased the expression of BDNF by 180 ± 5.1% (n = 6; Figure 3A). Similarly to in vivo experiments, SB204741 completely abolished the fluoxetine induced increase in BDNF expression (by 60 ± 4.2% (n = 6) of fluoxetine group). Pretreatment with U0126 (a selective inhibitor of MEK) completely abolished the fluoxetine-induced increase in expression of BDNF by 66 ± 4.7% of fluoxetine group (n = 6; Figure 3A). However, after the pretreatment with LY294002 [a selective inhibitor of phosphatidylinositol-3-kinase (PI3K)], fluoxetine still significantly increased the expression of BDNF by 176 ± 5.9% (n = 6) of control (PBS) group (Figure 3A). As shown in the lower panel of Figure 3C, the level of BDNF measured by ELISA had a similar tendency as the expression of BDNF measured by western blotting. SB204741 and U0126 dramatically abolished the fluoxetine induced increase in the level of BDNF by 65 ± 5.2 and 57 ± 4.7% (n = 6) of fluoxetine group, respectively. After pretreatment with LY294002, fluoxetine still increased the level of BDNF by 171 ± 6.2% (n = 6) of PBS group (the lower panel of Figure 3C).

Meanwhile, after treatment with interfering RNA of 5-HT_2B receptor, the protein level of BDNF increased by fluoxetine was suppressed to 111 ± 4.5 or 117 ± 6.9% (n = 6) of PBS group, as shown in Figures 3B,D. We also found that transcription factor c-fos was required for the fluoxetine induction of the expression of BDNF. Pretreatment with the siRNA duplex chains of c-fos eliminated differences between PBS group and fluoxetine group (Figures 3B,D). The relative expression of 5-HT_2BR and c-fos were shown in Supplementary Figures 1A,B.

The administration of leptin significantly induced the phosphorylation of STAT3 by about two times in the isolated astrocytes from GFAP-GFP mice (Figure 4A). However, in the SD-treated group, the ratio of p-STAT3/STAT3 was decreased to 46 ± 3.2% (n = 6) of control group (Figure 4A). Leptin re-elevated the phosphorylation of STAT3 decreased by SD to 177 ± 3.5% (n = 6) of SD-treated PBS group, but it was still only 80 ± 3.3% (n = 6) of PBS group without SD treatment (Figure 4A).

In the primary cultured astrocytes, treatment with leptin significantly increased the protein and mRNA expression of 5-HT_2B receptors by 100 ± 5.5 and 52 ± 4.7% (n = 6), respectively (Figure 4B). Treatment with RNA interfering with the expression of leptin receptors with siRNA duplex chains, completely the abolished effect of leptin on the expression of 5-HT_2B receptors. Pretreatment with WP1066 (an inhibitor of JAK2/STAT3) similarly decreases the protein and mRNA expression of 5-HT_2B receptors to the level of PBS group (Figure 4B). The relative expression of LepR was shown in Supplementary Figure 1C.

After pretreatment of the primary cultured astrocytes with leptin, fluoxetine significantly increased the phosphorylation of ERK_1/2 by 120 ± 7.2 and 106 ± 7.7% (n = 6), and enhanced the protein expression of c-fos or BDNF by 57 ± 6.5 or 57 ± 7.5% (n = 6), as compared with fluoxetine group (Figures 5A–C). The effect of leptin was mediated through leptin receptors, because after RNA interfering the expression of leptin receptors, leptin lost its ability to augment effects of fluoxetine on expression of p-ERK_1/2, c-fos and BDNF (Figures 5A–C). At the same time after suppressing the expression of 5-HT_2B receptors, the effects of fluoxetine and leptin on the level of p-ERK_1/2, c-fos, and BDNF were all abolished (Figures 5A–C).

In experiments in vivo, leptin augments effects of fluoxetine on the depressive-like behaviors. In sucrose preference test, SD decreased the sucrose uptake ratio by 49 ± 2.9% (n = 12), the administration of leptin alone had no effect. However, while fluoxetine increased this ratio by 59 ± 3.1% (n = 12) of SD group, treatment with leptin and fluoxetine combined increased the sucrose preference by 92 ± 5.7% (n = 12) of SD group, and by 21 ± 3.3% (n = 12) of SD plus fluoxetine group (Figure 6A). Similarly, in forced swimming test, leptin did not change the immobility time increased by SD, whereas treatment with leptin and fluoxetine decreased the immobility time to 54 ± 3.7% (n = 12) of SD group, and to 73 ± 3.1% (n = 12) of SD plus fluoxetine group (Figure 6B). Similar effect of leptin in tail suspension test is shown in Figure 6C, the immobility time of leptin plus fluoxetine in SD-treated group was decreased by 31 ± 2.9% (n = 12) in SD group, and by 12 ± 2.2% (n = 12) in SD plus fluoxetine group.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.