Cannula implantation was performed as previously described (Carvalho-Netto et al, 2007). Briefly, mice were implanted with stainless steel guide cannula (32-gauge) under anesthesia. Stereotaxic coordinates (Paxinos and Franklin, 2001) for the PAG were, respectively, 4.2 mm posterior to bregma, 1.3 mm lateral to the midline, and 2.2 mm ventral to the skull surface, with the guide cannula angled 26° to the vertical. A dummy cannula was inserted into each guide-cannula immediately after each surgery to reduce the incidence of occlusion. Five to seven days after surgical recovery, solutions were injected into the PAG by microinjection unit which extended 1.0 mm beyond the tips of the guide cannula. Each microinjection unit was attached to a 5-µl Hamilton microsyringe via polyethylene tubing and administration was controlled by an infusion pump programmed to deliver a volume at rate of 0.1 µl over a period of 30 s. At the conclusion of the experiments 1% Evans blue dye was administered to mice according to the microinjection procedure described above for intra-PAG administration. A post-mortem histological control of the injection site was performed on cryostat sections of unfixed brains previously frozen. The data of any mice were excluded from statistical analysis if the cannula tip was outside the PAG or if the region had sustained extensive damage. The brains from mice which were further submitted to Western blot were extracted leaving the cannula implanted in the brain. The location of cannula inside PAG was observed under stereomicroscope (Leica MZ12.5, Leica, Solms, Germany). Only specimens from mice with cannula path inside the PAG were used for Western blot experiments.

Mice were placed inside a stainless steel container, which was set thermostatically at 52.5±0.1 °C in a precision water-bath. Here we have used lower temperature in hot plate test (52°C instead of 54°C) to reveal possible subtle alterations that may occur in basal nociception. The licking latency was measured immediately prior i.p. morphine injection. Hot plate test started 15 min after morphine administration and licking latencies were measured at 15 min intervals for 60min after starting time. A 30s cut-off to prevent tissue damage was used. The endpoint for the licking response was the first paw lick of the front paw. Increased nociception was seen as shorter latencies to the responses evaluated. The test was performed in a blind fashion. Mice pretreated with drugs administered as previously described, were evaluated for licking latency basal value.

For both tests, animals were pretreated with saline (i.p.), intra-PAG vehicle or M119(40ng/mouse), 1µg/Kg morphine (i.p.) in presence or absence of M119 and submitted to rota-rod and hole-board (Ghelardini et al, 2008). Twelve mice per group were tested. The rota-rod test apparatus consists of a base platform and a rotating rod (3×30cm) placed at a height of 15 cm from the base and divided into five equal sections by six disks. Up to five mice were tested simultaneously on the apparatus, with a rod-rotation speed of 16 r.p.m. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 30 s, according to Vaught et al. (1985). Performance time was measured before and 15, 30 and 45 min after the administration of the investigated compounds. The hole board test consisted of a 40 cm square plane with 16 flush-mounted cylindrical holes (3 cm diameter) distributed four by four in an equidistant, grid-like manner. Mice were placed on the center of the board one by one and allowed to move about freely for a period of 10 min each. Two electric eyes, crossing the plane from midpoint to midpoint of opposite sides, thus dividing the plane into four equal quadrants, automatically signaled the movement of the animal (counts in 5 min) on the surface of the plane (spontaneous motility). Miniature photoelectric cells, in each of the 16 holes, recorded (counts in 5 min) the exploration of the holes (inspection activity) by the mice. Mice pretreated with 1µg/Kg morphine (i.p.) in presence or absence of intra-PAG CTOP (80ng/mouse) were previously submitted to both tests (Ghelardini et al, 2008).

Mice used for these experiments were sacrified 15 min after 1µg/Kg or 7mg/Kg morphine administration at which time maximum thermal hyperalgesic/analgesic effect is obtained (Galeotti et al, 2006) or seven days after repeated morphine administration at the doses previously described. μOR-G protein coupling assay data after acute and chronic high-dose morphine administration are largely known (Sánchez-Blázquez et al, 2001; Askari et al, 2008; Wang et al, 2005) but were used to support the method utilized in this study. The animals were anesthetized with CO₂, cervically dislocated, decapitated and the brain dissected, put immediately in liquid nitrogen and then stored at −80°C. PAG brain area from control and treated mice was dissected on a cold plate. Enriched synaptic membranes were prepared from PAG of mice from each treatment group as described by Gray and Whittaker (Gray and Whittaker, 1962). Protein concentration was determined according to Lowry (Lowry et al, 1951). The association of G protein coupled receptors with G proteins was investigated using coimmunoprecipitation procedure as previously described (Wang et al, 2005). Briefly, PAG membranes were incubated with Krebs–Ringer and then solubilized in immunoprecipitation buffer (25mM HEPES, pH 7.5, 200mM NaCl, 2mM MgCl₂, 1mM EDTA, 0.2% 2-mercaptoethanol, 50µg/ml leupeptin, 25µg/ml pepstatin A, 0.01 U/ml soybean trypsin inhibitor, 0.04mM phenylmethylsulfonyl fluoride) containing 0.5% digitonin, 0.2% sodium cholate and 0.5% NP-40 emulsifying agent with end-over-end rotation for 60 min at 4°C and further centrifugated at 50,000xg. The supernatant was divided for separate passage through immunoaffinity columns containing immobilized antibodies to Gαs, Gαi, Gαo, Gα11, Gαq or Gβ proteins. Anti-G protein antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were covalently cross-linked to protein-G-conjugated resin in Seize-X protein G immunoprecipitation kit (Pierce-ENDOGEN, Rockford, IL, USA) according to the manufacturer instructions. G protein complexes in solubilized brain lysates were isolated by immunoprecipitation in which 200µg solubilized brain membrane extracts were incubated with immobilized anti-G-protein G-resin at 4°C overnight. After centrifugation and three washes with phosphate-buffered saline (pH 7.2) at 4°C, the G protein complexes were eluted with 190 µl of antigen elution buffer (pH 2.8) and immediately neutralized by adding 20µl of 1.5M Tris buffer (pH 8.8). The neutralized G complexes were combined with 180µl of 2× PAGE sample preparation buffer (62.5mM Tris–HCl, pH 6.8; 20% glycerol, 4% SDS; 10% 2-mercaptoethanol, 0.1% Bromophenol Blue), boiled for 5 min and then submitted to Western blotting using a specific antibody directed against the amino-terminal region of the μOR for Gα and PLCβ_1–4 for Gβ (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 18% and 4–15% or 4–20%Tris-HCl gels were used respectively for G-protein-μOR complex and specificity assay of antibodies or Gβ-PLCβ_1–4 coimmunoprecipitation experiments. The specificity of the anti-Gα, anti-Gβ and PLCβ_1–4 antibodies was determined by Western blotting using 100µl of mouse whole brain homogenate with or without antigen peptide (25µg) pre-absorption for 30 min. The specificity of anti-μOR antibody was assayed on brain tissue from knockout brain mouse (GR21−/−; a generous gift of Dr Brigitte Kieffer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département Neurobiologie et génétique, Illkirch, France). A highly acidic (pH 2.8) or neutral elution buffer was used to elute antigens from the Gα immuno-complexes in order to establish if the experimental procedure yielded μOR with an approximate molecular weight of respectively 53 and 67 kD (Chen et al, 1995; Wang et al, 2005).

Western blot was performed as previously described in detail (Pan et al, 1995). In summary, immunoprecipitates (from 1µg/µl protein lysate) of Gαi, Gαo, Gαq, Gα11, Gαs and Gβ protein from PAG of morphine, CTOP, M119, saline and vehicle pretreated mice were solubilized in SDS buffer and separated on polyacrylamide gels (1.5mm). Proteins were transferred to nitrocellulose (1.5 h at 190 mA) and the membranes were blocked in PBS containing 3% BSA for 1h before addition of anti-μOR or anti-Gβ antibody at 1:500 dilution. The blots were stripped and reprobed with antibodies against various G proteins using the antisera against Gαi, Gαo, Gαq, Gα11, Gαs and Gβ protein as probes at 1:1,000 dilution. The blotting was visualized using a chemiluminescence detection system (Super Signal West Fento, Pierce Biotechnology Inc., Rockford, IL, USA) and quantified with the Versa Doc 1000 Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Three independent experiments were done at the same protein concentration for each experimental condition. Specific bands were quantitated by densitometric scanning.

PLC enzyme activity in anti-Gβ immunoprecipitates was measured as described in Allan (Allan et al, 1997). Immune complexes for Gβ (30µL) were assayed for 15 min at 37°C in the presence of 35 mM sodium phosphate, pH 6.8, 70 mM KCl, 0.8 mM EGTA, 0.8 mM CaCl₂, 0.20mM [³H]phosphatidylinositol 4,5-bisphosphate (8C_i/nmol, Perkin-Helmer Massachusetts, USA), and 2.86 mM (0.18%, v/v) Triton X-100 in a final volume of 50 µL. Enzyme activity was quantitated as the release of [³H] Ins(1,4,5)P₃ measured by liquid scintillation spectroscopy. Isozyme-specific activity was calculated by subtracting background [³H] Ins(1,4,5)P₃ (release present in no antibody control samples) from the activity measured in immune complexes. Data were calculated as nanomoles Ins(1,4,5)P₃ product formed per minute per milligram protein present in the fraction from which the enzyme was immunoprecipitated. All conditions were run in triplicate.

Morphine hyperalgesia induced by i.p. 1 µg/Kg dose in the mouse hot plate test was completely prevented by intra-PAG pre-treatment with M119 at 5–40 ng/ mouse (fig.2a). This effect appeared to be dose dependent. The M119 compound, when administered alone at the same doses, did not induce any significant change in licking latency (fig.2b).

The spontaneous motility as well as inspection activity of mice were unmodified by pre-treatment with drugs in comparison with control group (fig. 3a). The number of falls from the rotating rod evaluated before and 15, 30 and 45 min after the beginning of the rota-rod test showed the lack of any impairment in motor coordination of mice submitted to the above treatments in comparison with control group (fig. 3b).

In order to test the hypothesis that Gβγ associates with PLCβ in mouse PAG, anti-Gβ immune complexes were isolated from PAG of mice 15 min following 1 µg/Kg morphine administration and probed for PLCβ_1–4 immunoreactivity. We found that Gβ immunoprecipitates with PLCβ₃ whereas immunoreactivity associated with anti-PLCβ₁, β₂ and β₄ was not different from the saline background (fig.4a). Pre-absorption with 25µg of their respective antigen peptides drastically abolished the detection of PLCβ_1–4 and Gβ by Western blotting in mouse brain homogenate (fig.4b). Anti-Gβ immune complexes were isolated from PAG of mice 15 min following low dose morphine administration and assayed for associated PLC activity. These results demonstrate that Gβ associated with PLCβ₃ in low dose morphine administration and this was catalytically active (fig.5). PLC activity could not be detected in anti-Gβ immune complexes isolated from PAG of mice previously administered with low morphine dose in presence of M119 or CTOP at the higher effective doses (fig.5).