STOP null mice were generated on a 50:50 BALBc/129 SvPas background as previously described , with gene targeting being used to replace exon 1 of the STOP gene with a non-functional construct. Experiments were performed on 37 day-old STOP null male mice and littermates, weighing 30 ± 3 g. Animals were given free access to food and water, with a 12 h light-dark cycle at a temperature of 22 ± 2°C. All experiments were carried out in accordance with the European Community Council Directive (86/609/EEC) of November 24, 1986 and were approved by the regional ethics committee and French ministry of agriculture (license No. 67-116, to P.P.).
Drugs were purchased from the following sources: morphine hydrochloride (Sigma Aldrich, Saint Louis, USA), Naloxone hydrochloride (Ascent Scientific, Princeton, USA). Morphine hydrochloride and naloxone hydrochloride were dissolved in saline (NaCl 0.9%), for subcutaneous injections. The effects of morphine and naloxone on nociception were tested 20 min after the injections. [N-methyl-3H]-morphine was purchased from American Radiolabeled Chemicals (80 Ci/mmol St. Louis, USA).
All nociceptive tests were preceded by at least 5 days of habituation to handling and testing procedures, in order to obtain stable basal values.
Mechanical nociceptive threshold measurement
The mechanical threshold leading to nociceptive hindpaw withdrawal was determined using von Frey hairs . Briefly, mice were placed in clear Plexiglas® boxes (7 cm × 9 cm × 7 cm) on an elevated mesh screen for a habituation time of 15 min. Calibrated von Frey filaments (Bioseb, Chaville, France) were then applied to the plantar surface of each hindpaw in a series of ascending forces (ranging between 0.4 and 26 g). Each filament was tested five times per paw. The mechanical threshold corresponded to the force of the filament inducing three or more hindpaw nociceptive withdrawals out of five consecutive trials.
Thermal nociceptive tests were made using a computer-controlled dynamic hot and cold-plate (Bioseb, Vitrolles, France) following our recently-described procedure . The animals were placed in a Plexiglas cylinder (10 cm diameter, 15 cm height) with a drilled cover for a habituation time of 15 min. For dynamic hot plate test (DHP), animals were placed on the plate at 30 ± 0.1°C, and the plate temperature increased up to 44°C, with a 1°C.min-1 speed (r2 = 1). For dynamic cold plate test (DCP), animals were placed on the plate at 20 ± 0.1°C, and the plate temperature was decreased down to 1°C, with a 1°C.min-1 speed (r2 = 0.99). During each degree interval, for DHP or DCP, we counted the number of jumps displayed by the mouse. A cutoff value of 30 jumps was used to remove the mice from the test (always over 43°C or below 2°C). The thermal nociceptive threshold corresponded here to the temperature of first jump for each mouse. The total number of jumps is also given for hot (from 30°C to 43°C) and cold temperature ramps (20°C to 2°C). Note that only mice exhibiting jumps in DHP/DCP tests were kept for analysis. If all mice were producing jumps during the DHP test, only a small percentage of them were having this behavior in the DCP test.
The presence of endogenous morphine-like compounds in mouse brain of STOP and WT animals was first revealed via an immunohistochemistry approach on sagittal brain slices (global morphine presence).
Tissue preparation for immunohistochemistry studies
Mice were deeply anesthetized by intraperitoneal injection of 0.1 ml of a 5.6% (w/v) pentobarbital sodium solution (CEVA Santé Animale, Libourne, France) and perfused transcardially with 4% formaldehyde (EMS, Hatfield, USA) in NaCl/Pi buffer (0.9% NaCl and 25 mM sodium phosphate, pH 7.4) using a peristaltic pump. Fixative solutions were chilled, and then injected for 10 min with a peristaltic pump at a flow rate of 10 ml/min. The brain was quickly removed and incubated for 2 h at 4°C in the same fixative. Coronal and sagittal brain sections (70 μm thick) were cut with a vibratome (Leica VT 1000 S, Nanterre, France) and collected in Tris-buffered saline (TBS: 50 mM Tris-HCl, 0.9% NaCl, pH 7.4).
Immunostaining was performed on sections free-floating in TBS as previously described . Brain slices were washed in TBS and incubated for 1 h in bovine serum albumin (BSA; Sigma-Aldrich) diluted in TBS (3%, w/v) in order to saturate nonspecific immunoreactive sites. After six TBS washes of 5 min each, sections were incubated overnight with a mouse monoclonal antibodies (6D6, dilution 1:1000, Aviva System Biology, San Diego, USA) raised against morphine-like compounds (morphine, M3G, and M6G, based on supplier specifications and our own experiments) [21, 23].
After incubation with the primary antibody, brain slices were washed six times with TBS (5 min) and incubated with a horseradish-conjugated specific secondary antisera (HRP-conjugated donkey anti-mouse IgG, dilution 1:400; P.A.R.I.S., Compiegne, France) for 2 h at room temperature, followed by six TBS washes (5 min).
Several controls were carried out to assess antibody specificity and nonspecific immunoreactivity. The primary antibody was omitted, and the secondary antibody was tested individually or in a mixture in the presence of tissue sections or cells. Controls for morphine immunoreactivity were carried out by incubating the antibody with morphine (2 h, 25°C, 50:1, w/w)  prior to immunocytochemistry experiments. Anti-morphine antibody was tested by ELISA in order to determine cross reactivity with morphine, morphine-6-glucuronide (M6G), morphine-6-glucuronide (M6G), dopamine, adrenaline, noradrenaline and norlaudanosoline/tetrahydropapaveroline, showing a specificity for morphine, M6G and M3G. No cross reactivity was found for dopamine, adrenaline, noradrenaline and norlaudanosoline/tetrahydropapaveroline . In order to assess whether morphine binds to proteins nonspecifically, extracts mouse hippocampus were submitted to Western blot analysis (50 μg of RIPA-extract, SDS-PAGE 4-12% acrylamide Criterion XT precast gel 12%, sample buffer 60 mM Tris-HCl pH6.8, 2% SDS, 4 M Urea, 5% glycerol, 1% β-mercaptoethanol, 5 min at 100°C); the results show no anti-morphine antibody labeled proteins in these extracts (data not shown).
Light microscopy immunocytochemistry studies
Peroxidase activity was observed after 20 min of incubation in a freshly prepared solution of 4-chloro 1-naphtol (0.2 mg/ml) in TBS containing 0.006% (w/v) hydrogen peroxide. After washing with TBS, the sections were mounted in glycerol/TBS (1:1, v/v) before analysis with a Leica DMRB microscope equipped with a digital camera (Axiocam, Zeiss;objectives 10×, 20× and 40×). Brain pictures were reconstructed using the Photostich 3.1 software (Canon).
Homogenized brain areas (cerebellum, brainstem and brain) were sonicated at 4°C (3 × 10 sec) in water. Extracts were centrifuged (30 min, 10.000 g, 4°C), and the supernatant containing the intracellular material was extracted with methanol (1:3, v:v final ratio). After centrifugation (15 min, 10.000 g, 4°C), the supernatant was dried with a SpeedVac evaporator (Thermo Fisher Scientific, Brebières, France) and then dissolved in water prior to ELISA analysis. For mouse serum analysis, aliquots of 40 μl were tested in duplicates.
The morphine-specific enzyme-linked immunosorbent assay (ELISA) kit from Immunalysis Corporation (Pomona, USA provided by AgriYork 400 Limited, Pocklington, UK) was used for the quantification of morphine present in brain tissue extracts (n = 6) . The specificity of the test for morphine was confirmed by testing different amounts of dopamine, adrenaline, noradrenaline, norlaudanosoline/tetrahydropapaveroline, morphine, M6G, M3G, and codeine (0.01 to 25 ng/ml, data not shown). For all tests, morphine standards were diluted in the appropriate buffer.
Gel electrophoresis and Western blot analysis
Proteins were separated on SDS-PAGE gradient gels (4%-12% acrylamide; Criterion XT, BioRad) and electrotransferred onto polyvinyldifluorene membranes (GE Healthcare Bioscience, Sweden) . In order to immunodetect the mu receptors, 50 μg of brain RIPA-extracts from WT and STOP null mice (n = 3). The MOR receptor was detected using a goat anti-MOR1 antibody (N-20; Santa Cruz Biotechnology, USA; dilution 1:1,000) and revealed using HRP-conjugated anti-goat antisera (Jackson immunoresearch, England; dilution 1:50,000) and a Supersignal West Femto Kit (Pierce, Rockford, USA). Apparent molecular weights were evaluated by comparison with molecular weight standards (Bio-Rad). Control experiments omitting the primary antibody confirmed the specificity of the label. Quantifications of the intensity of immunoreactive bands were done using the ImageJ software Version 1.43 http://rsbweb.nih.gov
HPLC-MS/MS Instrumentation and Analytical Conditions
Few mass spectrometry (MS) methods have been reported for the qualification and the quantification of morphine and its glucuronide in biological sample [59–61]. HILIC chromatography coupled to a triple quadrupole mass spectrometry was used to develop a method to accurately detect the presence of morphine (MOR), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in the selected reaction monitoring (SRM). Identification of the compounds was based on precursor ion, one selective fragment ions and their relative retention times.
Prior to LC-MS-MS analysis, methanol extracted samples were dissolved in 0.1% trifluoroacetic acid in H2O (v:v) and purified using an Äkta purifier HPLC system (GE Healthcare Bioscience) as previously described . Fractions corresponding to morphine were dried with a SpeedVac evaporator prior to LC-MS-MS analysis.
LC separations were carried out with an Agilent LC 1100 binary pump, autosampler, vacuum degasser, and column oven coupled with an Agilent 6410 Triple Quad LC/MS (Agilent Technologies, Palo Alto, USA).
LC separations were carried out with an Agilent LC 1100 binary pump, autosampler, vacuum degasser, and column oven coupled with an Agilent 6410 Triple Quad LC/MS (Agilent Technologies, Palo Alto, USA). The dry sample were resolvated in 10 μl of acetonitrile 70%, vortex-mixed for 1 min and injected on an acrylamido-type column (TSKgel Amide-80, TOSOH, Tokyo) at 25°C. The solvent system consisted of 100% water, 0.15% formic acid and 5 mM ammonium acetate (NH OAc; solvent A) and 100% acetonitrile (solvent B). Elution was performed at a flow rate of 220 μl/min with a 70-40% linear gradient (solvent B) over the 8 first minutes, followed by a 80% stage (solvent B) over 2 min before the reconditioning of the column at 70% of solvent B. The system was fully controlled by MassHunter software (Agilent Technologies).
Electrospray ionization was achieved in the positive mode with the spray voltage set at 4000 V. Nitrogen was used as nebulizer gas and nebulizer pressure was set at 20 psi with a source temperature of 100°C. Desolvation gas (nitrogen) was heated to 350°C and delivered at a flow rate of 10 L/min. Qualification was performed in selected reaction monitoring (SRM) mode with the following transitions: m/z 286.2 → 165.1 for morphine. The details of the optimized SRM parameters for Morphine are shown in additional file 1.
Binding assay to brain membranes was conducted in a 96-well format. Animals were decapitated, brains were rapidly removed and homogenized with 10 volumes (w/v) of ice-cold 0.32 M sucrose using a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenate was sonicated and then centrifuged at 4°C (10 min at 1000 g). The pellet was discarded and the supernatant was centrifuged at 4°C for 20 min at 20,000 g. The supernatant was decanted and the pellet was suspended in ice-cold 50 mM Tris-HCl buffer (pH 7.4) and sonicated. After 30 min at 4°C, the suspension was resuspended and centrifuged at 4°C for 20 min at 20,000 g. The resulting pellet was further suspended in Tris-HCl buffer (pH 7.4) and gently sonicated. Protein concentrations were determined by the BCA method. Proteins were stored at -70°C until used.
The binding assays were performed on membrane extract (100 μg of protein in Tris-HCl buffer, pH 7.4) from a pool of STOP mice (5 brains in the pool) or WT mice (7 brains in the pool) in the presence of a concentration of 0.1 nM to 50 nM of [3H]-morphine ([N-methyl-3H]-morphine, 80 Ci/mmol, American Radiolabeled Chemicals). Nonspecific binding was determined in the presence of 10 μM morphine. Incubations were performed at 37°C during 30 min. The binding was terminated by rapid filtration of the mixture under vacuum through Whatman GF/B filters presoaked for 30 min in Tris-HCl buffer (pH 7.4). The filters were washed three times with 0.2 ml of ice-cold Tris-HCl buffer (pH 7.4) and transferred to 5 ml vials with 3 ml scintillation liquid. Radioactivity was counted 24 h later by using a Beckman scintillation counter. All experiments were conducted in triplicate. Specific binding was determined as the difference between radioligand bound in the absence (total binding) and presence (nonspecific binding) of 10 μM morphine. The GraphPad Prism program (GraphPad-Prism, San Diego, CA), was employed, with a nonlinear curve fitting analysis, to fit the nM/mg of proteins data to a saturation binding curve.
Morphine ELISA analysis
In order to assess if a difference of morphine amount exist in brain tissues, determined morphine concentrations were subjected to post hoc analysis using a Mann-Whitney test. (STOP, n = 10; WT, n = 13). Statistical data analysis was performed using MINITAB 13.20 (Minitab Inc.). For multiple comparisons, the significance level was adjusted using Bonferroni correction. The significant level was set at P < 0.001.
Morphine binding assay
These statistical processes were performed using the GraphPad statistical software. B
values used for statistics were calculated from the non-linear regression analysis which provides more reliable estimations (GraphPad Prism Program). However, linear Scatchard plot regression analyses were presented for rapid visual interpretation of the data. Deviation from the model non-linear regression analysis was checked with the runs test and was never significant for all experiments. The r² were always better than 0.96 in all cases. Statistical comparison between experimental conditions was assessed by analysis of Student's t- test after the confirmation that the variance aren't significantly different. A probability level of 0.05 or smaller was used to indicate statistical significance.
Data are expressed as mean ± SEM. Statistical analyses were performed with KyPlot (KyensLab, Tokyo, Japan). The Wilcoxon - Mann-Whitney U test for unpaired data was used to compare non-parametric data from the von Frey hair test or the hot/cold dynamic plate. For the thermal nociceptive score as well as for the global activity tests, Student's t-test was used. The significant level was set at p < 0.05.