Animals and antibiotics treatment protocol

All procedures were conducted according to Model Animal Research Center and were approved by the Experimentation Ethics Review Committee of Nanjing University. Twenty male C57BL/6 mice (6–7 weeks old) were obtained from Model Animal Research Center at Nanjing University and adapted to environmental conditions for one week before experiments. Mice were housed in a temperature and humidity-controlled room with a 12 h light/dark cycle (19.0 ± 1 °C, 55% humidity, and fed on a maintenance diet). For the induction of gut dysbiosis, ten mice were treated with broad-spectrum antibiotics in drinking water for 4 weeks (ampicillin, 1 g/L, Sigma; neomycin sulfate, 1 g/L, Sigma; metronidazole, 1 g/L, Sigma; vancomycin, 500 mg/L, Sigma) [18].

Analysis of gut microbiota

After antibiotics, stool samples of mice were collected in a sterile micro-tube, which were frozen immediately in liquid nitrogen and stored in a −80 °C freezer until analysis. Total DNA was isolated using a Wizard Genomic DNA Purification Kit, which was following the manufacturer’s instructions (Promega, Madison, USA). The relative abundance of bacteria was measured using 16S rRNA analysis, which was performed at the laboratory of BGI (Huada Gene Institute). Primers were designed to amplify the V3–V4 hypervariable region according to the manufacturer’s recommendations [21]. Then bioinformatics analysis was performed as described [22]. The mother v.1.27.0 Standard Operation Procedure (SOP) was used to analyze high quality sequence reads [23].

Total gastrointestinal transit

After fasted overnight with free access to water, mice were administrated by gavage with a semiliquid solution (0.1 mL) containing Evans blue (5%) and methyl cellulose (1.5%) (M0262, Sigma) [13]. Then fecal pellets were monitored at 10 min intervals for the presence of the first blue pellet, and the time for expulsion of the first blue pellet was measured [24].

Measuring colonic transit

After fasted overnight with free access to water, the colonic transit of mice was measured with a bead expulsion test. A 3-mm glass bead was inserted into the colon (2 cm proximal to the anal) using a plastic Pasteur pipette lightly lubricated with lubricating jelly as described [25]. The time until bead expulsion was measured.

Fecal parameter measurements

Freely feeding mice were observed for 8 h, and the number of pellets was counted every 2 h. Fecal water content was measured by comparing the weight of the pellets at the end of the experiment and after drying (24 h at 60 °C). Finally, fecal dimensions of each mice were measured.

Macroscopic assessment

Animals were weighted every week from the beginning to the end of experiments. Mice were euthanized by cervical dislocation, and the whole intestine (from stomach to anus) were excised. The length of the whole intestine and colon was measured. The weight of cecum with their contents was also measured.

Analysis of smooth muscle contractility

Strips of longitudinal (6 mm in length) muscle from proximal colon were mounted in a 37 °C organ bath with a force transducer (MLT0202; AD Instruments, Spain) connected to a PowerLab (AD Instruments, Australia) recording device. Briefly, Ca2+-free HEPES-Tyrode (H-T) buffer (140.6 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl 2 , 10 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4) was used to wash out the lumen contents of proximal colon segments. Then, segments were transferred to the organ bath and equilibrated in H-T buffer (137.0 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl 2 , 1.8 mmol/L CaCl 2 , 10 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4) for 15 min. The resting tension was set to 0.5 g prior to force measurement. The isotonic contractions of longitudinal muscle was recorded for 1 h, with H-T buffer being changed every 15 min. The mean amplitude of the basal tension and the frequency of phasic contractions were measured for 30 min after experiments. The detailed force assays were performed according to our previously described methods [26, 27].

Serotonin measurements

ELISA was used to assess the serotonin in supernatant of tissue homogenates according to the manufacturer’s instructions (DEE5900, Germany). Readings from tissue samples were normalized to total protein content as detected by BCA assay as described [12].

RNA preparation and qRT-PCR

The entire length of the mouse colon were washed in H-T buffer, flushed with H-T buffer to remove luminal contents. Then the total RNA was extracted from 1 cm regions of proximal mouse colon using TRIzol reagent (Invitrogen). The qRT-PCR assay was performed by a ABI Prism 7100 Sequence Detection System and 96-well optical plates (Applied Biosystems). The PCR primer sequences were as follows, M-TPH1: forward, 5′TCA AGC CCT TTG ATC CCA AG-3′; reverse, 5′-GAC ATC AAG GTC ATA CCG CAA C-3′.

Immunofluorescence

Whole frozen sections were processed for indirect immunofluorescence and confocal microscopy as described [12], which were prepared from paraformaldehyde-fixed tissues. Briefly, 10-μm frozen sections of proximal colon samples were collected on coated slides. Then it was washed three times with PBS and blocked with 5% bovine serum albumin (sigma Aldrich) at room temperature for 30 min. After staining using the primary antibodies, rabbit anti-mouse CgA (1:500; Abcam), rat anti-mouse 5-HT (1:50; Abcam), and secondary antibodies conjugated to Alexa fluor 488 or 594 (Molecular Probes). Images were examined using a confocal microscope (Olympus, Germany).

Cecal bile acids determination

Cecal contents from mice were collected, freeze-dried, and pulverized. BAs were analyzed using the LC–MS–MS (Liquid Chromatography–Mass Spectrometry and Liquid Chromatography–Tandem Mass Spectrometry). Each bile acid was identified by its relative retention time compared with that of standard BAs.

Statistical analysis

Statistical analysis was performed using SPSS for windows version 19. Data were measured for normal distribution and plotted in the figures as mean ± SEM. The significance of the differences between groups was determined with Student t test (two comparisons) or one-way ANOVA (multiple comparisons). P < 0.05 was considered significant.