Animals
Our study was carried out in accordance with French standard ethical guidelines for laboratory animals (Agreement 02376.01) and with ARRIVE guidelines (https://arriveguidelines.org). All experiments involving mice were approved by the ethics committee for animal experimentation of Pays de la Loire (Apafis 6751) as Nantes University CHU, of which our INSERM unit is part, is geographically located in the Pays de la Loire. Adult male C57BL/6 mice (6-7 weeks; Janvier Labs, Le Genest-Saint-Isle, France) weighing 25-30g were used. Mice were housed in cages with free access to food and tap water. Animals were quarantined under controlled conditions illumination (12h light/dark cycle), temperature, and humidity.
Water avoidance stress (WAS) protocol
For stress exposure, a single 1h WAS in between 8 AM to 9 AM in the morning, was performed daily at PD35 for 4 consecutive days, corresponding to the repeated acute stress protocol. Mice were placed on an elevated circular platform (10 cm height ; 4 cm diameter), positioned at the center of a plastic tank (52 x 37 x 27 cm3) filled with water at room temperature up to 1cm of the top of the platform adapted from previous study (3, 25).
Pharmacological treatment protocol
The specific GR antagonist, CORT108297 (kindly provided by Dr Azel Hunt, Corcept, Drive Menlo Park, CA, USA) or the vehicle (sesame oil, Millipore Sigma, Burlington, MA, USA) were injected intraperitoneally at the dose of 85 mg/kg (26) 1 hour before the WAS procedure.
In vivo measurement of colonic motility
As previously described (27, 28), to assess the fecal pellet output (FPO), control mice were placed individually in clean cages without bedding, food, or water for fecal pellet collection during 1 h. For mice subjected to WAS, the fecal pellet collection was performed for 1 h during the WAS time period. Fecal pellets were collected and counted immediately after expulsion.
Ex vivo measurement of colonic motility
As previously described (27, 29), at the end of the fecal pellet collection for the control mice and after the WAS treatment the fourth day, animals were killed by cervical dislocation. Tissues were immediately transferred in cold HBSS (Eurobio, Courtaboeuf, France) after surgical resection and brought to the laboratory. Strips of longitudinal muscle were dissected and placed in organ chamber (Radnoti, California, USA) with 15 mL of Krebs solution at 37°C, continuously bubbled with 95% O2 and 5% CO2. The contractile response of muscle strip was continuously recorded using isometric force transducers (No. TRI202PAD, Panlab, Cornellã, Spain) coupled to a computer equipped with the PowerLab 8/30 System and the Labchart data analysis software (AD Instruments, Spechbach, Germany). Strips were stretched with a preload of 1 g, which was maintained during an equilibration period of 60 minutes. Then, strips were subjected to electrical field stimulation (EFS) using a STG 4008 MCS electrical stimulator (Multi Channel Systems, Reutlingen, Germany). EFS parameters were as follows: train duration, 10 seconds; pulse frequency, 20Hz; pulse duration, 400µs; pulse amplitude 11V. This procedure was repeated 3 times spaced 4 minutes. Then the following drugs were added in the baths and after a 10 minutes incubation period the same EFS stimulation protocol was repeated. The following drugs were added sequentially, nitro-L-arginine methyl ester (L-Name, 50 µM; Sigma-Aldrich, Merck, Darmstadt, Germany), an inhibitor of the nitric oxide synthase (NOS); and then atropine (1 µM; Sigma-Aldrich, Merck, Darmstadt, Germany), a muscarinic receptor antagonist. Next, tissues were washed 5 times over a 10 min period and was then allowed to recover for an additional 20 min. At the end of each experiment, using bethanechol (10-9-10-2 M), a selective muscarinic receptor agonist (Millipore Sigma, Burlington, MA, USA), a dose-response curve was created by measuring the area under the curve of the bethanechol-induced contraction for 2 min after bethanechol was added. All values were normalized to the tissue weight.
Blood sampling and corticosterone assessment
Mice were bled from their tail, at J0 and at J4, at the end of the 1h WAS test (as described in figure 1). Samples were obtained by quickly clipping the distal tip of the tail with a razor blade and collecting ~ 50µL of blood into EDTA containing tubes.
In order to minimize hormonal variability due to circadian fluctuations, all procedures were performed during circadian cycle of the diurnal corticosterone rhythm, before 11:00 AM. After collection, blood samples were centrifuged at 3200 tr/min, for 15 min at 4°C and then plasma samples were kept at -20°C. Corticosterone plasma levels were measured by enzymatic immunoassay according to the manufacturer’s instructions (Laboratoire IDS, Pouilly en Auxois, France).
Acetylcholine assay
After mice killing, portions of the distal colon were removed and lysed in RIPA buffer (Merck Millipore, Fontenay-sous-Bois, France) containing phosphatase inhibitor cocktail III (Sigma-Aldrich, Merck, Darmstadt, Germany) and protease inhibitors cocktail (Roche, Neuilly-sur-Seine, France). Briefly, tissues were crushed in the RIPA buffer using a "Precellys 24" tissue homogenizer (Bertin technologies, St Quentin-en-Yvelines, France), followed by sonication with "vibracell 75 186" device (Sonics, Newton CT, USA). The amount of proteins was assessed with Bradford reagent using a BioPhotometer D30 spectrophotometer (Eppendorf, Montesson, France). Acetylcholine (ACh) concentration was determined in tissue homogenates using Amplex Red, acetylcholine/ acetylcholinesterase assay kit (Invitrogen Thermofisher Scientific, Waltham, MA USA) and normalized with protein amount.
Western Blot analysis
As previously described (30), proteic extraction from colonic tissues was performed with NucleoSpin RNA/Protein Kit (Macherey-Nagel, Hoerdt, France, Cat# 740966) according to the manufacturer instructions. Samples were further prepared for electrophoresis by diluting with NuPAGE sample buffer (Life Technologies, Saint-Aubin, France, Cat# NP0008) then heated at 98°C for 5 min. Lysates were separated using the NuPAGE 4–12% Bis-Tris gels (Life Technologies, Cat# NP0336BOX) together with the 2-(N-morpholino)ethanesulfonic acid/sodium dodecyl sulfate running buffer (Life Technologies, Cat# IB23002) before electrophoretic transfer to nitrocellulose membranes (Life Technologies, Cat# NP0002) with the iBlot2 Dry Blotting System (Life Technologies, Cat# IB21001). Membranes were then blocked for 1 h at 21°C in Tris-buffered saline (Sigma, Cat# T5912) with 0.1% (v/v) Tween-20 (Sigma, Cat# P1379) and 5% (w/v) non-fat dry milk and incubated overnight at 4°C with the following primary antibodies: rabbit anti-GR (D8H2, 3660S 1:500, Cell Signaling, Danvers, Massachusetts, USA), mouse monoclonal anti-β-actin (1 : 10000; Sigma, Cat#A5441, RRID:AB_476744). Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (Life Technologies Cat# 31460, diluted 1 : 5000) or anti-mouse antibodies (Sigma, Cat# A9044, diluted 1 : 5000) and visualized by enhanced chemiluminescent detection (Biorad, Clarity ECL, Marnes-la-Coquette, France, Cat# 170-5061).
Quantitative PCR analysis
As previously described (31), one µg purified mRNA by Nucleospin RNA/Protein kit was denatured and was converted to cDNA using the SuperScript III Reverse Transcriptase (Life Technologies). qPCR was performed using a StepOnePlus RealTime PCR Instrument (Life Technologies) with a FastSYBR Green Master Mix kit (Applied Biosystems, Foster City, CA, USA). Ribosomal protein S6 (RPS6) transcript was used as a reference. The relative expression of the gene of interest was measured by the 2−∆∆Ct method. The following primers were used:
• NR3C1 # NM_008173, forward: 5’-GCAGTGGAAGGACAGCACAAT-3’ ; reverse: 5’- CGTTTTTCGAGCTTCCAGGTTC-3’
• RPS6 forward: 5’-GAAGCGCAAGTCTGTTCGTG-3’ ; Reverse : 5’- GTCCTGGGCTTCTTACCTTCT-3’
Immunofluorescence staining
Distal colon segments were fixed in 0.1M PBS containing 4% paraformaldehyde at room temperature for 3h. Whole mounts of longitudinal muscle and myenteric plexus were obtained by microdissection and were permeabilized with PBS containing 10% horse serum (HS) and 4% Triton X-100 for 2h at room temperature. Tissues were then incubated with the following primary antibodies: rabbit anti-ChAT (1:1000, a gift from Professor M. Schemann, Hannover, Germany (32), mouse anti-neuronal NOS (nNOS; 610308, 1:500; BD Biosciences), rabbit anti-GR (D8H2, 3660S 1:500, Cell Signaling), human anti-Hu (gift from the CHU of Nantes; 1:5000) diluted in PBS containing 10% horse serum and 0,5% Triton X-100 for 24 or 48h at room temperature. After washing, tissues were incubated for 2h at room temperature with the appropriate secondary antibodies, respectively, anti-rabbit CY5 (1:500), anti-rabbit CY3 (1:500) and anti-human FP488 (1:200), and mounted Glycerol 60% (vol/vol) (Thermo Fisher Scientific). Nuclei were stained with 4′ 6-Diamidino‐2‐phenylindole dihydrochloride (DAPI D9542; 1:10000; Sigma Aldrich, Paris, France).
Image analysis and quantification
Images from immunostained tissues were acquired with a digital camera (Axiozoom, Carl Zeiss, United States). The number of Hu-, ChAT-, nNOS- and GR-neurons was counted in 20 ganglia per animal. The data were expressed as the percentage of ChAT-, nNOS- or GR-neurons normalized to the total number of Hu-neurons.
For GR immunostaining, confocal microscopy was performed using Nikon A1R confocal inverted microscope (Nikon France SAS, Champigny sur Marne, France) with a Nikon X60 Plan-Apo numerical aperture (NA) 1.4 oil-immersion objective (MicroPICell core facility).
Among these 20 ganglia, 10 were used to measure the intensity of cytoplasmic and nuclear expression of GR receptor. The DAPI-GR co-labelling was used to determine the intensity of nuclear expression of the GR receptor within each neuron of a ganglion, as the Hu-GR co-labelling was used to determine the total intensity of the GR receptor expression within each neuron of a ganglia. The intensity of GR cytoplasmic expression could then be deduced. The measurement of the nucleo-cytoplasmic ratio of GR expression was determined.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Data are represented as means +/- SD. Group comparisons were made using the Mann–Whitney U test or by 2-way ANOVA and Dunnett’s multiple comparisons test as indicated. Values of p < 0.05 were considered statistically significant.