Fig 1: Dorsal raphe (DR) neurons were resistant to chronic meth-induced degeneration. (A) Sample images (left) depicting tryptophan hydroxylase expressing (TPH2+) neurons in DR of saline (ctrl; 10 ml/kg)-and meth (+meth; 5 mg/kg)-treated mice; scale bars denote 200 μm; TPH stained in red with NeuN counterstained in green. Quantified data (right) indicate that chronic meth had no effect on number of TPH2+ cells in the DR; ctrl n = 5 and +meth n = 5 mice. Data analyzed using an unpaired t-test [t(8) = 0.6708, p = 0.5212, two-tailed]. (B) Sample images (left) depicting SERT+ axons in the primary motor cortex of ctrl and +meth mice; scale bars denote 20 μm. Quantified data (right) show no difference in axon length between ctrl and +meth mice; ctrl n = 5 and +meth n = 5 mice. Data analyzed using an unpaired t-test [t(8) = 0.07813, p = 0.9396, two-tailed].
Fig 2: Reciprocal aggravation of serotonin and neurotrophin pathways via 5-HT7 activation in neurons.Human SH-SY5Y cells were stimulated with NGF or BDNF (100 ng/ml) for 48 h and the neurite length and protein levels were measured. A Representative immunostaining images of PGP9.5, TPH2, and 5-HT7 in cells with or without neurotrophin stimulation. Bar: 50 μm. B Longer nerve fibre length was observed after neurotrophin stimulation. a Average nerve fibre length and b percentage of neurons with neurite longer than 50 μm. *P < 0.05 vs. w/o. C Increased immunofluorescent intensity of (a) TPH2 and (b) 5-HT7 in neurons after neurotrophin stimulation. *P < 0.05 vs. w/o. A total of 250–300 neurons from each group were used for quantification. The bar graph represented the mean ± SEM of each group. D Transcript levels of tryptophan hydroxylases (TPH1 and TPH2) and 5-HT receptor subtypes (HTR3, HTR4, and HTR7) in neurons after neurotrophin stimulation. The neuroblastoma cells which are untreated (UT) or those differentiated with retinoic acid (RA) served as controls. *P < 0.05 vs. UT; #P < 0.05 vs. RA. N = 6/group. E Knockdown of NTRK1 gene in SH-SY5Y cells by lentiviral shRNA targeting TrkA (shTrkA) suppressed the NGF-induced TPH2 and HTR7 overexpression. The mock plasmid control is labeled as pLKO. a Gel images of semi-quantitative PCR band products showing gene silencing of NTRK1. b, c Quantitative PCR results showing reduction of TPH2 and HTR7 mRNA levels in the NTRK1-knockdown cells upon NGF stimulation. F Knockdown of NTRK2 gene by lentiviral shRNA targeting TrkB (shTrkB) decreased the BDNF-induced TPH2 and HTR7 overexpression. a Gel images of semi-quantitative PCR band products showing gene silencing of NTRK2. b, c Quantitative PCR results showing reduction of TPH2 and HTR7 mRNA levels in the NTRK2-knockdown cells upon BDNF stimulation. *P < 0.05 vs. pLKO. N = 6/group. G Neurotrophin-mediated nerve fibre elongation was inhibited by pretreatment with 5-HT7 antagonists CYY and SB7. *P < 0.05 vs. UT; #P < 0.05 vs. vehicle. A total of 250–300 neurons from each group were used for quantification. The bar graph represented the mean ± SEM of each group. H Proposed schema of a positive-feedback loop between serotonin and neurotrophin pathways via 5-HT7 activation for induction of neurite outgrowth and visceral hypersensitivity.
Fig 3: Effect of melatonin on tryptophan hydroxylase 2 (TPH2) expression in dorsal raphe nucleus (DRN) neurons.(A) Western blots showing TPH2 expression in DRN neurons with melatonin treatment (0.1, 1, and 10 μM) for 4 h. (B) Averaged optical density of TPH2 expression, at 4 h melatonin incubation, expressed relative to control level of TPH2. Each column represents the mean and SEM of data from 15 experiments. *p < 0.05 compared with control.
Fig 4: Distribution of melatonin receptors (MT) and tryptophan hydroxylase 2 (TPH2)-containing serotonergic neurons in the dorsal raphe nucleus (DRN).Fluorescent images of melatonin receptors (including both MT1 and MT2, red) and TPH2-containing neurons (TPH, green). The bottom right images was taken from the rat brain atlas. The red square indicates the location of DRN. Scale bars: 200 μm.
Fig 5: SCFAs trigger PPARγ to mediate the antidepressant responses in CRS-exposure mice via TPH2. (A) The mRNA levels of PPARγ in SH-SY5Y cells with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 12 h. Data were normalized with GAPDH mRNA levels. Scale bars represent means values, and error bars represent SEM of triplicate samples. Data are shown as means ± SEM (n = 4 per group) and were analyzed using one-way ANOVA, F4,15 = 11.7, P = 0.0002, and Tukey’s multiple comparison test, *P < 0.05, **P < 0.01, and ****P < 0.0001. (B) The lysates of SH-SY5Y cells with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h were subjected to Western blot with indicated antibodies. The levels of TPH2, PPARα, PPARβ, and PPARγ were quantitatively analyzed (n = 3 biological replicates). Data are shown as means ± SEM (n = 3 per group) and were analyzed using one-way ANOVA, TPH2 (F4,10 = 8.281, P = 0.0032), PPARα (F4,10 = 0.1347, P = 0.9658), PPARβ (F4,10 = 0.1479, P = 0.9597), and PPARγ (F4,10 = 13.1, P = 0.0005), and Tukey’s multiple comparison test, **P < 0.01 and ***P < 0.001. (C) SH-SY5Y cells were incubated with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h. ChIP analyses using an anti-ACSS2, anti-CTD, and anti-Sin3A antibodies were performed. The histogram shows the amount of immunoprecipitated DNA expressed as a percentage of the total input DNA. The data are presented as the means ± SEM of quadruplicate samples. Data are shown as means ± SEM (n = 4 per group) and were analyzed using one-way ANOVA, PPARγ (F4,15 = 29.04, P < 0.0001), p-Ser2/5 pol II CTD (F4,15 = 33.26, P < 0.0001), and Sin3a (F4,15 = 23.71, P < 0.0001), and Tukey’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (D) Representative BiFC fluorescent images of SH-SY5Y cells transfected with 2 μg of plasmid encoding ACSS2 or PPARγ fused to the fluorescent protein fragments indicated in each panel in response to 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h. DAPI stain demonstrated nuclear locus. The intensity YFP signal indicates the amounts and localization of BiFC complex (ACSS2–PPARγ). (E) Two weeks after injecting the shPPARγ virus into the hippocampus of mice using stereotaxic injection techniques, the CRS modeling experiment was initiated for 4 weeks, during which mice had free access to drinking SCFAs or water. Behavioral tests were then conducted for 1 week (n = 10 per group). (F) Immobility time in the TST in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM (n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.946, df = 18, P = 0.0086. **P < 0.01. (G) Immobility time in the FST in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM (n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.522, df = 18, P = 0.0213. *P < 0.05. (H) SPTs in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM (n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.258, df = 18, P = 0.0366. *P < 0.05. (I) Raw traces of mice in the OFT were shown. Total distance traveled in the OFT and time spent exploring the center area in the OFT from mice in individual animals from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM (n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, time (t = 3.185, df = 18, P = 0.0051) and locomotion (t = 1.329, df = 18, P = 0.2004). **P < 0.01. (J) Raw traces of mice in the EPM were shown. Time spent in the open arms and probability of entering open arms in the EPM test from mice in individual animals from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM (n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, time (t = 2.923, df = 17, P = 0.0095) and entries (t = 4.064, df = 17, P = 0.0008). **P < 0.01 and ***P < 0.001. (K) Analysis of 5-HT content of hippocampus by ELISA in male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM (n = 6 per group) and were analyzed using unpaired 2-tailed Student’s t test, **P < 0.01. (L) RT-PCR analysis of PPARγ and TPH2 expression levels in hippocampus of male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups (n = 6 per group). Data were normalized with GAPDH mRNA levels and presented as fold changes compared with control group. Scale bars represent means values, and error bars represent SEM. Data are shown as means ± SEM (n = 6 per group) and were analyzed using unpaired 2-tailed Student’s t test, *P < 0.05 and ****P < 0.0001. (M) Representative immunoblots and quantification of PPARγ and TPH2 protein levels normalized to loading controls in hippocampal male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM (n = 3 per group) and were analyzed using unpaired 2-tailed Student’s t test, PPARγ (t = 6.951, df = 4, P = 0.0023) and TPH2 (t = 7.074, df = 4, P = 0.0021). **P < 0.01.
Supplier Page from Abcam for Anti-TPH2 antibody