DOR-eGFP knock-in mice expressing the delta opioid receptor fused to its C terminus to a green fluorescent protein were generated by homologous recombination. In these mice, the eGFP cDNA was introduced into exon 3 of the delta opioid receptor gene, in frame and 5′ from the stop codon (Scherrer et al. 2006). MOR-mcherry knock-in mice expressing the mu opioid receptor fused its C-terminus to the red protein mcherry were generated by homologous recombination following a procedure similar to the one used for DOR-eGFP knock-in mice. A targeting construct in which the Oprm1 stop codon has been replaced by a Gly-Ser-Ile-Ala-Thr-mcherry encoding cDNA followed by a neomycin resistance gene flanked by FRT sites was transfected into ES cells (Fig. 1). Two independent homologous recombinants were electroporated with a FLP recombinase expressing plasmid to excise the neomycin gene and microinjected into C57Bl6/J blastocysts. Chimeric mice were crossed with C57Bl6/J mice to obtain F1 heterozygous progenies. Heterozygous animals were intercrossed to generate mice homozygous for Oprm1-mcherry that are fertile and develop normally. DOR-eGFP mice were crossed with MOR-mcherry mice to obtain mice homozygous for both constructs. Wild-type mice were used as control in behavioral experiments. The genetic background of all mice was C57/Bl6/J: 129svPas (50:50 %). Mice genotyping was performed by standard PCR technique using a 5′ oligonucleotide located on the fourth exon of the oprm1 gene (BAZ 43 tgacgtgacatgcagttgagattt) and a 3′ oligonucleotide located in the 3′ UTR untranslated region (BAZ 44 tcccacaaaccctgacagcaac). Introduction of the coding sequence for mcherry increased the size of the amplified fragment by about 800 bp enabling identification of wild type oprm1
+/+, heterozygous oprm1
+/mch and homozygous oprm1
mch/mch animals by PCR.
Mice were housed in a temperature- and humidity-controlled animal facility (21 ± 2 °C, 45 ± 5 % humidity) on a 12-h dark–light cycle with food and water ad libitum. Male and female mice aged 8–14 weeks were used in all protocols. All the experiments were conducted during the light period. All experiments were performed in accordance with the European Communities Council Directive of 26 May 2010 and approved by the local ethical committee (Com’Eth 2012-006).
Morphine chlorhydrate (Francopia, Lyon, France) was administered s.c. or i.p. at doses of 5, 10, 25 or 30 mg/kg. Naloxone hydrochloride (Sigma) was used at 1 mg/kg (s.c.) for the pharmacological induction of morphine withdrawal. SNC 80 (Tocris) was used in vivo at 10 mg/kg (s.c.). All drugs were administered at 10 mL/kg and dissolved in 0.9 % NaCl (solution used for control animals).
The delta agonist AR-M100390 (N, N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide) is a SNC80 derivative synthesized at AstraZeneca R&D Montreal (Canada). The kappa agonist U50-488H (2-(3,4-dichlorophenyl)-N-methyl-N-[(2R)-2-pyrrolidin-1-ylcyclohexyl] acetamide) was purchased from Sigma. [3H] Diprenorphine (50 Ci/mmol), [3H] DAMGO (35 Ci/mmol) and [35S] GTPγS (1,250 Ci/mmol) were from Perkin Elmer Life and Analytical Sciences (Boston, MA, USA).
Rabbit polyclonal antibodies raised against eGFP (Cat. Nr A-6455, Molecular Probes, Paisley, UK, dilution 1:1,000), mcherry (Cat Nr 632496, Clontech, dilution 1:1,000) were used for fluorescent protein detection when indicated. MORs were detected using a rabbit polyclonal antibody raised against the C-terminus (1:100, generous gift from Dr C. Evans). Primary antibodies used for co-localization with neuronal markers are mouse monoclonal antibodies raised against calbindin D-28K (Cat. Nr 300, Swant, Bellinzona, Switzerland, dilution 1:1,000), or parvalbumin (Cat. Nr 235, Swant, Bellinzona, Switzerland, dilution 1:1,000), rat monoclonal antibodies raised against somatostatin (Cat. Nr MAB 354, Millipore, Billerica, MA, USA, dilution 1:1,000). The following AlexaFluor-conjugated secondary antibodies (Molecular Probes, Paisley, UK) were used: goat anti rabbit AlexaFluor 488 conjugated (Cat. Nr A-11034, dilution 1:2,000), goat anti rabbit IgG AlexaFluor 594 conjugated (Cat. Nr A-11012, dilution 1:2,000), goat anti mouse IgG AlexaFluor 594 conjugated (Cat. Nr A-11005, dilution 1:500), goat anti rat IgG AlexaFluor 594 conjugated (Cat. Nr 1-11007, dilution 1:500), goat anti mouse IgG AlexaFluor 350 conjugated (Cat. Nr 1-21049, dilution 1:500). Absence of cross-reactivity (rabbit/mouse, rabbit/rat, mouse/rat) was systematically checked in control experiments for each antibody. Immunohistochemistry was also performed without primary antibodies to verify absence of non-specific staining by the secondary antibody alone.
Real-time quantitative PCR analysis
RNA extraction, cDNA synthesis and qPCR assays were carried out as described previously (Befort et al. 2008). Total brain tissue was collected from three animals for each genotype to isolate RNA using Trizol reagent (Invitrogen, Cergy Pontoise, France) according to the manufacturer’s instructions. Briefly, 2.5 μg of total RNA was reverse transcribed using Superscript II (200 or 400 U, Invitrogen, Cergy Pontoise, France) with anchored-oligodT primer (8 mmol/L), random Hexamer (16 mmol/L), and deoxynucleotide triphosphates (500 μmol/L each). Real-time PCR was performed in triplicate on a MyIQ BioRad instrument using iQSYBRGreen supermix (Bio-Rad, Marnes-la-Coquette, France), cDNA (0.5 μL), and gene-specific primers (200 nmol/L) in a 25 μL reaction as recommended by the manufacturer. Gene-specific primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3/). Sequences of primers are provided below. Thermal cycling parameters were 2 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 15 s at 60 °C and 30 s at 72 °C. Relative expression ratios were normalized to the level of HPRT reference gene, and the 2-DDCt method was used to evaluate the differential expression level. Two reference genes (β-actin, Rplp0) were tested in each run as an internal control.
Primers for RT PCR
Ex vivo tissue analysis of MOR-mcherry mice
Membrane preparations were carried out as described previously (Befort et al. 2001). Briefly, whole brain were removed, immediately frozen on dry ice, and stored at −80 °C prior to use. Whole brain membranes were prepared by homogenizing the brain in ice-cold 0.25 M sucrose solution 10 vol (ml/g wet weight of tissue). Samples were then centrifuged at 1,100g for 10 min. Supernatants were collected and diluted five times in buffer containing 50 mM Tris–HCl (pH 7.4) and 1 mM EDTA, following which they were centrifuged at 35,000g for 30 min. The pellets were homogenized in 2 ml ice-cold sucrose solution (0.32 M) and aliquots kept at −80 °C until further use.
50 μg of membrane proteins was incubated in the presence of variable concentrations (3 10−9 to 2 10−10 M) of [3H] DAMGO for 1 h at 25 °C. Membranes were washed and filtered, and radioactivity was quantified using a liquid scintillation counter. Assays were performed in triplicates in eight experiments using six different membrane preparations.
[35S] GTPγS binding assay
5 μg of membrane proteins was used per well. Samples were incubated with the mu agonist DAMGO, the delta agonist AR-M1000390 or the kappa agonist U50-488H (10−4 to 10−11 M) for 1 h at 25 °C in assay buffer 50 mM Tris–HCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, 0.2 mM EGTA containing 30 μM GDP and 0.1 nM [35S] GTPγS. Incubation was terminated by rapid filtration and washing in ice-cold buffer (50 mM Tris–HCl, 5 mM MgCl2, 50 mM NaCl, pH 7.4). Bound radioactivity was quantified using a liquid scintillation counter. Non-specific binding was defined as binding in the presence of 10 μM GTPγS, and basal binding was assessed in the absence of agonist. Assays were performed in triplicates in nine experiments using six different membrane preparations.
Membrane preparations (500 μg) were solubilized in Tris–HCl 50 mM pH 7.4, 100 mM NaCl, 10 % CHAPS, complete protease inhibitor cocktail (Roche applied Bioscience, Mannheim, Germany) for 1 h at 4 °C, immunoprecipitated with either 1 μg anti-eGFP or 1 μg anti-mcherry antibodies for 1 h at 4 °C and isolated by incubation with G protein Sepharose for 1 h at 4 °C. Samples were washed three times with Tris–HCl 50 mM pH 7.4 and resuspended in SDS-PAGE sample buffer.
Western blot analysis
Total protein content of brain membranes was determined by Bradford assay. Samples were heated in loading buffer (62.5 mM Tris–HCl, pH 6.8, 5 % (wt/vol) ß-mercaptoethanol, 2 % (wt/vol) SDS, 10 % (vol/vol) glycerol, 0.1 % (wt/vol) Bromophenol blue) for 5 min at 95 °C. 50 µg proteins were loaded onto an 8 % SDS-PAGE gel. Proteins were transferred onto Immobilon P polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Following blocking in 5 % (wt/vol) non-fat dry milk in 50 mM Tris–HCl pH 8, 150 mM NaCl, 0.2 % (vol/vol) Tween 20 (TBST) for 1 h, PVDF membranes were incubated overnight at 4 °C with a 1:1,000 dilution of the anti mu opioid receptor or a 1:1,000 dilution of the anti mcherry antibody. PVDF membranes were washed three times for 10 min with 5 % (wt/vol) non-fat dry milk in TBST, incubated for 2 h with a 1: 10 000 dilution of HRP-conjugated anti-mouse (Fab′2) fragment antibody in 5 % (wt/vol) non-fat dry milk in TBST. PVDF membranes were washed three times for 10 min in TBST. Chemiluminescence was detected using ECL+ according to the manufacturer’s instructions.
Experiments were performed in stable conditions: 21 ± 2 °C, 45 ± 5 % humidity, 40 ± 2 lux. All experiments were preceded by 2 days of animal handling. Tail immersion and hot plate tests were used to evaluate antinociceptive responses.
Tail immersion test
The mouse was maintained in a cylinder and the tail immersed into a heated water bath set at 52 °C. Morphine (5 or 10 mg/kg) or a saline solution were injected i.p. Tail withdrawal latencies were measured 45 min later with a 10 s cutoff time. Baseline responses were measured 1 h prior drug injection.
Hot plate test
Morphine (5 or 10 mg/kg) or a saline solution was injected i.p. The mouse was placed on a 52 °C hot plate 45 min later and latencies to jump were recorded with a 300 s cutoff time.
Conditioned place preference test
Place conditioning experiments were performed in unbiased computerized boxes (Imetronic, Pessac, France) formed by two Plexiglas chambers (15.5 × 16.5 × 20 cm) separated by a central alley (6 × 16.5 × 20 cm). Two sliding doors (3 × 20 cm) connected the alley with the chambers. Two triangular prisms of transparent polycarbonate were arranged in one chamber, and one rectangular prism in the other to form different shape patterns (covering the same surface). Distinct-textured removable floors made of translucent polycarbonate provided additional contextual cues. The activity and location of mice were recorded using five photocells located throughout the apparatus. Behavioral data were collected by an interface connected to a PC. Light intensity in the chambers was set at 30 Lux.
Animals were naive when conditioning started. Morphine conditioning consisted of 3 phases. On day 1, naive mice were placed in the central alley and allowed to freely explore the apparatus for 20 min for a pretest session. Based on the individuals’ spontaneous preference during this pretest phase, the drug-paired chamber was assigned in such a way that saline and morphine groups were counterbalanced and unbiased toward contextual cues. Statistical analysis on pre-test data indicated no bias between the two chambers (p = 0.99). Conditioning phase lasted 3 days. Mice underwent two daily conditioning sessions, vehicle and drug paired, 7 h apart. Drug pairings were performed in the morning (10:00 AM). The animals were injected with either morphine (10 mg/kg, s.c.) or saline (controls) immediately before being confined in the “drug-paired” chamber. Vehicle pairings were performed in the afternoon (4:00 PM). All the animals received an injection of saline and were confined in the vehicle-paired compartment. Testing phase was conducted on day 5. The animals, in a drug-free state, were placed in the neutral central alley and allowed to explore the apparatus for 20 min with the two sliding doors opened. The time spent in each chamber was recorded.
Mice daily received escalating doses (20, 40, 60, 80, 100 mg/kg) morphine i.p. or a saline solution for 6 days. Physical dependence to morphine was verified by measuring withdrawal syndrome precipitated by a naloxone (1 mg/kg, s.c.) injection 2 h after the last morphine injection. A global withdrawal score was calculated as previously described (Berrendero et al. 2003).
Locomotor activity was assessed in clear Plexiglas boxes (21 × 11 × 17 cm) placed over a white Plexiglas infrared-lit platform. Light intensity of the room was set at 15 lux. The trajectories of the mice were analyzed and recorded via an automated tracking system equipped with an infrared-sensitive camera (Videotrack; View Point, Lyon, France). Behavioral testing started when the animals were placed in the activity boxes for a 60-min habituation period. They were then injected with saline and locomotor activity was measured for another 1 h. Animals were then injected with morphine (25 mg/kg) or saline and activity was measured for 2 h. Locomotor activity was assessed during five consecutive days (Contet et al. 2008).
Tissue preparation and immunohistochemistry
Mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i. p.) and perfused intracardiacally with 50 ml of 4 % paraformaldehyde (PFA) (at 2–4 °C) in PB 0.1 M or PBS 1X (Dulbecco’s Phosphate Buffer Saline, Sigma Aldrich), pH 7.4. Brains were post-fixed for 24 h at 4 °C in 4 % PFA solution, cryoprotected at 4 °C in a 30 % sucrose, PB 0.1 M pH 7.4 solution, embedded in OCT (Optimal Cutting Temperature medium, Thermo Scientific), frozen and kept at −80 °C. 30-μm thick brain sections were cut with a cryostat (CM3050, Leica) and kept floating in PB 0.1 M pH 7.4.
Immunohistochemistry was performed according to standard protocols (Erbs et al. 2012). Briefly, 30-μm thick sections were incubated in blocking solution (PB 0.1 M pH 7.4, 0.5 % Triton X100 (Sigma, St. Louis, MO, USA), 5 % normal goat or donkey serum (Invitrogen, Paisley, UK) depending on the secondary antibody) for 1 h at room temperature (RT). Sections were incubated overnight at 4 °C in the blocking solution with appropriate primary antibodies. Sections were washed three times with PB 0.1 M pH 7.4, 0.5 % Triton X100, incubated for 2 h at RT with appropriate AlexaFluor-conjugated secondary antibodies. Sections were washed three times and mounted on SuperfrostTM glass (Menzel-Glaser) with Mowiol (Calbiochem, Darmstadt, Germany) and 4′, 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostic, Mannheim, Germany) (0.5 μg/ml).
DOR-eGFP fluorescence was enhanced by detection with an anti-GFP antibody and a secondary antibody coupled to the AlexaFluor 488. MOR-cherry fluorescence was enhanced by detection with an anti-mcherry antibody and a secondary antibody coupled to AlexaFluor 594. Double labeling was performed to co-localize DOR-eGFP or MOR-mcherry with the chosen neuronal marker. Antibodies specific for the neuronal markers were detected with a secondary antibody coupled to the AlexaFluor 594 or 488 depending on amplification of the DOR-eGFP or MOR-mcherry signal, respectively, or with secondary antibody coupled to the AlexaFluor 350 for triple labeling.
Immunocytochemistry on MOR-mcherry primary neuronal cultures
Primary neuronal cultures were performed as previously described (Pradhan et al. 2009). Briefly, P0 mice pups were decapitated, and hippocampi were dissected and digested with papain (15 U/ml, Worthington). Cells were plated on glass coverslips coated with poly-l-lysine (PLL, Sigma) in B27/NeurobasalA medium (Invitrogen) completed with 0. 5 mM glutamine and antibiotics. Cells were plated at a density of 8 × 104 cells/cm2. Medium was replaced 60 min after plating, and half the medium changed every 5–7 days. Cultures were maintained for 15 days in vitro (DIV). Fully matured primary neurons (DIV 10–14) were used for DAMGO-induced receptor internalization studies. Cells were fixed with 4 % PFA in PBS before or at various time points after 1 μM DAMGO addition. Immunological detection with an anti-mcherry antibody was then performed as described previously (Massotte 2006). Briefly, cells were incubated in blocking solution (PB 0.1 M pH 7.4, 0.2 % Tween 20 (Sigma, St. Louis, MO, USA), 5 % normal goat serum (Invitrogen, Paisley, UK) for 1 h at room temperature (RT). Coverslips were incubated overnight at 4 °C in the blocking solution with anti-mcherry antibodies (1:1,000), washed three times with PB 0.1 M pH 7.4, 0.2 % Tween 20 and incubated for 2 h at RT with goat anti rabbit AlexaFluor 594-conjugated secondary antibodies. Coverslips were washed three times and mounted with Mowiol (Calbiochem, Darmstadt, Germany) and 4′, 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostic, Mannheim, Germany) (0.5 μg/ml).
Image acquisition was performed with the slide scanner NanoZoomer 2 HT and fluorescence module L11600-21 (Hamamatsu Photonics, Japan). The light source LX2000 (Hamamatsu Photonics, Japan) consisted in an ultra high-pressure mercury lamp coupled to an optical fiber. Single RGB acquisition was made in the epifluorescence mode with the 3-chip TDI camera equipped with a filter set optimized for DAPI, fluorescein and tetramethylrhodamine detection. The scanner was equipped with a time delay integration camera and performed line scanning that offered fast acquisition at high resolution of the fluorescent signal. The acquisition was performed using a dry 20× objective (NA 0.75). The 40× resolution was achieved with a lens converter. The latter mode used the full capacity of the camera (resolution 0.23 μm/pixel). Neurons expressing a given fluorescent marker are visualized using the NDP viewer system with an integrated high-resolution zoom and possibility to separate the different fluorescent components.
Observations with a confocal microscope (SP2RS, Leica) using 40× (NA 1.25) and 63× (NA 1.4) oil objectives were used to validate mu and delta opioid receptor co-localization. Images were acquired with the LCS (Leica) software. Confocal acquisitions were performed in the sequential mode (single excitation beams 405, 488 and 568 nm) to avoid potential cross talk between the different fluorescence emissions.
Brain regions were identified using the Mouse Brain Atlas (2nd edition) from G. Paxinos and K.B.J. Franklin.
Images corresponding to each brain section were individualized using the NDP toolkit program (Hamamatsu Photonics, Japan) and arranged according to the rostrocaudal axis for coronal sections and lateromedial axis for the sagittal sections.
Statistical analysis was performed with Graph-Pad Prism v4 (GraphPad, San Diego, CA) and Statistica v9 (StatSoft, Maisons-Alfort, France). In vitro pharmacology experiments were analyzed using a one-way ANOVA. Behavioral experiments were analyzed using a two-way ANOVA. Multiple comparisons were made using Newman-Keuls or Tukey tests for post hoc analysis. A paired t test was performed to verify that the apparatus used in the conditioned place preference test was unbiased. Place conditioning data were expressed as percentage of time spent in the drug-paired compartment. Four-way ANOVA was performed with gender, genotype and treatment as between-group factors and conditioning (pretest versus test session) as a within-group factor.