The experimental protocols were approved by the Animal Care and Use Committee of the University of Tokyo and that of the National Institute for Environmental Studies. Pregnant female and adult male C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan). Ahr−/− mice with a B6.129S-Ahr < tm1Yfk > mouse strain (BRC01710) (Mimura et al. 1997) were provided by RIKEN BioResource Research Center (Tsukuba, Japan). Ahr+/− male and female B6.129S-Ahr < tm1Yfk > mice were bred to obtain Ahr−/− progeny. These mice were housed singly and in groups (three per cage), respectively, in an animal facility at a temperature of 22–24 °C and humidity of 40–60% on a 12:12-h light/dark cycle (lights on from 08:00 to 20:00). Laboratory rodent chow (Lab MR Stock; Nosan, Yokohama, Japan) and distilled water were provided ad libitum. Offspring were selected for transcript and protein expression analyses as described in sections of RT-PCR, western blotting, quantitative RT-PCR, and immunohistochemistry below. The number of animals used for these analyses is described in the legends to figures.
To produce and maintain the Ahr−/− mouse strain, genotyping of the Ahr gene was performed as follows: genomic DNA was extracted from tail tips by lysis in 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulphate, and proteinase K (Wako Pure Chemicals, Osaka, Japan) at 55 °C for 4 h. The lysate was centrifuged at 17,400×g at 4 °C for 3 min. The genomic DNA in the supernatant was purified using phenol and chloroform, followed by washing with 70% ethanol. The genomic DNA (dissolved in Tris–EDTA buffer) was used as the template for PCR using the Takara LA Taq PCR kit (Takara Bio, Kusatsu, Japan) on a Veriti thermal cycler (Applied Biosystems, Foster City, CA, USA). The amplification conditions were as follows: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 35 s. The PCR primers to amplify the genomic Ahr locus were 5′-GCCCGAGTCTCCTCTGTCG-3′/5′-CTCACGGCAGCGGAGATCT-3′ for the wild-type Ahr allele and 5′-GCCCGAGTCTCCTCTGTCG-3′/5′-CGCCGAGTTAACGCCATCAA-3′ for the Ahr-null allele. The 25-μl reaction contained 400 nM of each primer, 1× GC buffer II, 320 μM deoxynucleoside triphosphate (dNTP) mixture, and 0.5 U of LA Taq DNA polymerase. PCR products were separated by electrophoresis on agarose gels, which were stained with Midori Green Advance (Nippon Gene, Tokyo, Japan). The PCR products of the wild-type Ahr allele and Ahr-null allele were expected to be 439 and 671 bp in size, respectively.
Developing mice at P3 and P5 were decapitated, and several organs, including the brain, liver, lung, kidney, thymus, and spleen, were quickly removed and stored at −80 °C until RT-PCR analysis. The total RNA was isolated from each organ using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). The cDNA for a given mRNA was synthesized using oligo-dT and random hexamers with a Primescript RT reagent kit (Takara Bio). Expression levels of Ahr and Gapdh transcripts were determined using a Veriti thermal cycler (Applied Biosystems) with a KOD Plus kit (Toyobo, Osaka, Japan). The amplification conditions were as follows: 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 30 s. The PCR primers for amplifying the murine Ahr and Gapdh transcripts were 5′-AGGATTTGCAAGAAGGAGAG-3′/5´-TTGGTTCGAATTTCCAGGAT-3´ and 5′-ACCCAGAAGACTGTGGATGG-3′/5′-CACATTGGGGGTAGGAACAC-3′, respectively. The 20-μl reaction solution contained 400 nM of each primer, 1× KOD Plus buffer, 200 μM dNTP mixture, 1 mM MgSO4, and 0.5 U of KOD Plus DNA polymerase. PCR products were separated by electrophoresis on agarose gels, which were stained with Midori Green Advance (Nippon Gene). The PCR products of the Ahr and Gapdh transcripts were expected to be 508 and 171 bp in size, respectively.
Developing mice at P3, P5, and P14 were decapitated, and several organs (brain, liver, lung, kidney, thymus, and spleen) were quickly collected and stored at −80 °C until western blotting analysis. Protein was extracted at 4 °C in an ice bath unless stated otherwise. Each type of organ was homogenized with 4 mM HEPES–NaOH buffer, pH 7.3, containing 0.32 M sucrose and 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), using a Potter-type homogenizer. The homogenates were centrifuged at 1000×g at 4 °C for 10 min, and the supernatants were used for western blotting. Protein concentration in the supernatants was measured with the Quick Start Bradford Protein Assay (BioRad, Hercules, CA, USA). Proteins in the supernatants were separated on a 7.5% polyacrylamide gel and blotted onto immobilon-P transfer membranes (Millipore, Bedford, MA, USA). The proteins adsorbed to membranes were allowed to react with mouse monoclonal anti-AhR antibody (1:1000; sc-398877, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Tris-buffered saline, pH 7.4, containing 0.1% Tween-20 (TBST), overnight at 4 °C, followed by incubation in TBST containing anti-mouse IgG-horseradish peroxidase (HRP)-conjugated antibody (1:5000; 7076S, Cell Signaling Technology, Beverly, MA, USA), for 1 h at room temperature. Chemi-Lumi One (Nacalai Tesque, Kyoto, Japan) was used to visualize the protein bands, which were detected on Hyperfilm ECL (GE Healthcare Ltd., Tokyo, Japan) and developed and fixed with GBX developer and GBX fixer (Kodak, Rochester, NY, USA), respectively. Following deactivation of endogenous HRP by incubation in TBST containing 15% hydrogen peroxide for 30 min at room temperature, the membranes were immersed in TBST containing rabbit polyclonal anti-GAPDH antibody (1:5000; ab9485, Abcam, Cambridge, UK), overnight at 4 °C, followed by incubation in TBST containing anti-rabbit IgG-HRP-conjugated antibody (1:5000; 7074S, Cell Signaling Technology). Then, targeted protein bands were visualized in the same manner as described for AhR detection. The intensity of AhR and GAPDH bands was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; purity > 99.5%) was purchased from Cambridge Isotope Laboratory (Andover, MA, USA). Corn oil and n-nonane were purchased from Wako Pure Chemicals and Nacalai Tesque, respectively. Twelve-week-old male C57BL/6J mice were divided to control and TCDD groups, and they were orally administered with vehicle (corn oil containing 0.6% n-nonane) or TCDD dissolved in vehicle (20 μg/kg body weight).
Brain and liver tissues of 12-week-old male mice treated with vehicle or TCDD were collected quickly and stored at −80 °C until analysis. Total RNA was isolated from the brain and liver using an RNeasy Mini Kit (Qiagen). The cDNA for a given mRNA was synthesized using oligo-dT and random hexamer primers with a PrimeScript RT reagent kit (Takara). Gene expression levels were determined quantitatively using a LightCycler System (Roche Molecular Biochemicals, Indianapolis, IN, USA) with Thunderbird SYBR qPCR Mix (Toyobo). The genes and primers are summarized in Supplementary Table 1. No-template reactions were analyzed in every PCR to monitor for cross-contamination. To verify the specificity of amplification, melting curve analyses of the products were performed at the end of every PCR. The Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr) mRNA expression levels were calculated using the ΔΔCt method and normalized to the 18S rRNA expression.
Developing and adult mice were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) under anesthesia with sodium pentobarbital (conducted at the University of Tokyo) or three types of mixed anesthetic agents containing medetomidine hydrochloride, midazolam, and butorphanol (at the National Institute for Environmental Studies). Brains were collected, fixed in 4% paraformaldehyde overnight, immersed in a series of 0.1 M PBS containing 5%, 15%, and 30% sucrose, frozen in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan), and stored at −80 °C until histological sectioning. Frozen brains were sliced in the sagittal plane using a cryostat (Model 3050S; Leica Microsystems, Tokyo, Japan). Brain sections were cut at 50 μm thickness for immunofluorescence analysis.
Brain tissue sections were immunohistochemically stained for AhR, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), or NeuN. In brief, the tissue sections were washed in PBS containing 0.1% Triton X-100 (PBST), soaked in 0.01 M citrate buffer (pH 6.0) (Muto Pure Chemicals, Tokyo, Japan), and incubated at 90 °C (developing mouse brains) or 65 °C (adult mouse brains) in a water bath for 10 min. The sections were blocked with PBST containing 5% bovine serum albumin (A3059; Sigma-Aldrich) (blocking solution) and allowed to react with mouse monoclonal anti-AhR antibody (1:500; sc-398877, Santa Cruz Biotechnology) and rabbit polyclonal anti-TH antibody (1:1000; ab112, Abcam), anti-DBH antibody (1:1000; 22,806, Immunostar, Hudson, WI, USA), or anti-NeuN (1:1000; ab177487, Abcam) in blocking solution overnight at 4 °C. Then, the signals of AhR and TH, DBH, or NeuN were visualized with the respective secondary antibodies anti-mouse IgG AlexaFluor 488 (Life Technologies, Gaithersburg, MD, USA) and anti-rabbit IgG AlexaFluor 568 (Life Technologies) in PBST (1:1000). Furthermore, the nucleus was stained with PBST containing Hoechst 33342 (1:1000; Dojin Laboratories, Kumamoto, Japan), followed by mounting with VECTASHIELD (H-1400; Vector Laboratories, Burlingame, CA, USA) for confocal microscopy. Immunostained images were captured using an inverted Leica DMi8 microscope, equipped with the Leica TCS SP8 confocal module (Leica Microsystems). Specific objective lens (HC PL APO CS 10×/NA = 0.40 and HC PL APO CS2 20×/NA = 0.75; Leica Microsystems) and LAS X 3.1.5 software (Leica Microsystems) were used to capture images (x = 2048 pixels and y = 2048 pixels, bit depth = 8 in each RGB color).
Intracellular localization of AhR
Cellular morphology and immunostaining intensity of AhR- and TH-double-positive cells in the locus coeruleus (LC) and AhR- and NeuN-double-positive cells in the island of Calleja major (ICjM) were analyzed by applying the ImageJ software to the confocal microscopy images. In analyses of AhR- and TH-double-positive cells in the LC, we outlined the nucleus and soma of TH-positive cells [i.e., noradrenergic (NA) neurons] and measured their nuclear and soma sectional areas, and then calculated the nuclear area percentage by dividing the nuclear area by the soma area in each cell. In addition, we determined AhR immunostaining intensity per nucleus and soma (AhRNuc intensity and AhRSoma intensity, respectively) of TH-positive cells. After the background subtraction of AhR-stained images, the AhRNuc intensity percentage was calculated by dividing the AhRNuc intensity by the AhRSoma intensity. In order to analyze the intracellular localization of AhR, AhRNuc intensity percentage data were normalized depending on the soma size of TH-positive cells. We divided the AhRNuc intensity percentage by the nuclear area percentage to calculate the ratio in locus coeruleus-noradrenergic (LC-NA) neurons (ratioLC−NA, thereafter). In order to compare ratioLC−NA of individual mice between the control and TCDD groups, we used the distribution of ratioLC−NA values in each mouse as a surrogate parameter and analyzed the percentage of ratioLC−NA that was divided by the arbitrarily chosen value of 0.2. For each mouse, 57 to 103 cells in developing mice and 51 to 96 cells in adult mice were subjected to analyses in intracellular localization of AhR in TH-positive cells in the LC. In analyses of AhR- and NeuN- double-positive cells in the ICjM, we outlined the nucleus of NeuN-positive cells and measured nuclear sectional areas and AhRNuc intensity in each cell. To adjust the variability of luminance among images, AhRNuc intensity was normalized to the mean value of immunostained AhR intensity in the whole ICjM area. The parameter ratio in ICjM neurons (ratioICjM, thereafter) was calculated by dividing AhRNuc intensity by the nuclear area in each cell. Furthermore, we analyzed the percentage of the ratioICjM that was divided by the arbitrarily chosen value of 0.005. The numbers of cells that were used to analyze the intracellular localization of AhR in NeuN-positive cells in the ICjM ranged from 65 to 263 cells in developing mice and from 133 to 291 cells in adult mice.
Statistical analysis was performed using BellCurve for Excel software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Protein expression, cellular morphology, immunostaining intensity, and ratio values were analyzed using Student’s t-test or one-way analysis of variance (ANOVA), followed by the Tukey–Kramer post hoc test, and p-values < 0.05 were considered statistically significant. Because the mention of F- and p-values for each statistical analysis in the main text is very complicated, statistically significant differences are shown by asterisks in each figure.