All the expression constructs were made by PCR. Internal deletions and point mutations were made either using QuikChange Site-Directed Mutagenesis Kit (Stratagene) or by megaprimer method. The sequence for each mutant will be supplied upon request. PCR products were cloned into expression vectors pEGFPC1 (Clontech), pGEX 4T2 (Pharmacia), and pRK5 (Genentech), with Myc or HA tags. All constructs were verified by sequencing.
Preso1 antibodies were generated in rabbits using the rat Preso1 C terminal 20 aa or 330 aa as antigens. The antibody from 20-aa antigen was used for immunoelectron microscopy (1:100), and the antibody from 330-aa antigen was used for western blot (1:2,000) and immunostaining (1:100). Group I mGluR phospho-Ser antibody was generated in two female rabbits using rat mGluR5 peptide (ELVALTPPpSPFRD) as antigen (1:3,000 for western blot). These rabbit antibodies were generated by Covance, except the Preso1 antibody against the 330-aa antigen, which was generated at Sichuan University under a rabbit protocol approved by Animal Care and Use Committee of Sichuan University. All other antibodies were previously described or were acquired commercially, and their dilutions are as follows: mGluR5 (Upstate, 06-451) at 1:10,000, mGluR1 (BD, 610965) at 1:10,000, IP3R (gift from Alan Sharp (Johns Hopkins University School of Medicine)) at 1:1,000, Shank (Neuromab, clone N23B/49) at 1:2, PSD95 (Neuromab, clone K28/43) at 1:5,000, GluA1 (JH1710) at 1:1000, GluA2 (JH1707) at 1:500, NR1 (Millipore, 05-432) at 1:1,000, pan-Homer (Santa Cruz, sc-17842) for immunoprecipitation, Homer1 (JH2621) at 1:5,000, Homer2 (JH2623) at 1:5,000, Homer3 (JH2793) at 1:5,000, ERK (Cell Signaling, 9102) at 1:1,000, CDK5 (Santa Cruz, sc-173) at 1:1,000, actin (Sigma-Aldrich, clone AC-74) at 1:10,000, c-Fos (Calbiochem, PC38) at 1:4,000, HA-HRP (Roche, clone 3F10) at 1:4,000, Myc-HRP (Santa Cruz, clone 9E10) at 1:3,000.
mice were generated as follows. A 13-kb NheI BAC fragment containing exon 3 of Preso1
was subcloned into the pBS vector. Exon 3 was released by Bsu36I and BstBI and subcloned into a loxP/PGK-neo vector. The Preso1
targeting construct was generated by inserting the loxP/PGK-neo/Exon 3 back into Bsu36I and BstBI sites of the 13-kb BAC fragment of the mouse Preso1
gene (Supplementary Fig. 7
). The resulting targeting construct was linearized and electroporated into ES cells. ES cells were selected with G-418, and clones were picked, screened by PCR, and confirmed by Southern blotting for homologous recombination. Correctly targeted ES cell clones were injected into blastocysts, and chimeras were mated to C57BL/6 mice to produce Preso1
floxed heterozygotes. These Preso1
floxed heterozygotes were mated to CMV-cre
mice to produce Preso1+/−
mice, which were crossed to generate WT and Preso1−/−
) mice were obtained from John Roder50
(mGluR5FR) mice replace the mGluR5 gene with a mutant that does not bind Homer, and were described previously33
mice were generated as previously reported22
, and are healthy, fertile and not different from WT mice in size or behavior by casual observation.
Mice were group housed in plastic mouse cages with free access to standard rodent chow and water. The colony room was maintained at 22 ± 2 °C with a 12 h:12 h light:dark cycle. Preso1−/− mice were backcrossed at least five generations onto C57/Bl6J mice, and all the experiments were performed on littermate controls and blind to mouse genotype. Grm5−/− mice, Grm5R/R mice or Homer2−/−Homer3−/− mice and their age-matched, same-background WT mice were used in all the experiments. Only adult male mice were used for behavioral experiments, and total 250 mice were used for all the experiments. All experiments were performed under protocols approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine for mice and rats.
Cell culture and transfection
HEK293T cells were cultured in DMEM medium with 10% FBS. Transfections were performed with Fugene 6 to manufacture’s specifications. Cells were harvested 2 d after transfection.
Neuronal culture and electroporation
Neuronal cortical cultures from embryonic day 18 pups were prepared as reported previously30
. Dorsal spinal cord neurons were prepared from embryonic day 15 pups. 5 × 105
neurons were added to each well of a 12-well plate (Corning) with cover slips coated with poly-L-lysine. Growth medium consisted of MEM (Invitrogen) supplemented with 5% horse serum (Hyclone), 2% B27, 1% glutamine (Invitrogen), 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen). Neurons were fed twice per week with glia-conditioned growth medium. DIV 14 neurons were used for calcium imaging. For the rescue experiment, electroporation was performed using a Nucleofector kit (Amaxa) after neuron dissociation. Calcium imaging was performed after 2 d.
Mouse brain tissues or HEK293T cells were used for the coimmunoprecipitation assay as previous reported30
. Briefly, 500 microliters of immunoprecipitation buffer (PBS, pH 7.4, with 5 mM EDTA, 5 mM EGTA, 1 mM Na3
, 10 mM sodium pyrophosphate, 50 mM NaF, and 1% Triton X-100) containing Complete EDTA-Free protease inhibitors (Roche) was added and samples were sonicated. After centrifugation, the supernatant (300 μl) was then mixed with 0.5–2 μg of the appropriate antibody for 3 h at 4 °C. Then 50 μl of 1:1 protein A– or protein G–Sepharose slurry (Amersham-Pharmacia Biotech) was added for an additional 1 h. The protein beads were washed three times with immunoprecipitation buffer containing 1% Triton X-100. The protein samples were eluted with SDS loading buffer and analyzed by gel electrophoresis and western blotting.
Surface biotinylation assay
DIV 14 cortical neurons were used for surface biotinylation assay as previously reported30
. Briefly, cortical neurons were cooled on ice, washed twice with ice-cold PBS++ (PBS, 1 mM CaCl2
, 0.5 mM MgCl2
) and then incubated with PBS++ containing 1 mg/ml sulfo-NHS-SS-biotin (Pierce) for 30 min at 4 °C. Unreacted biotin was quenched by washing cells three times with PBS++ containing 100 mM glycine (pH 7.4) (briefly once and for 5 min twice). Cultures were harvested in RIPA buffer and sonicated. Homogenates were centrifuged at 13,200 r.p.m. (16,100g
) for 20 min at 4 °C. Fifteen percent of the supernatant was saved as the total protein. The remaining 85% of the homogenate was rotated with streptavidin beads (Pierce) for 2 h. Precipitates were washed with RIPA buffer three times (5 min each time). All procedures were done at 4 °C.
Immunoelectron microscopy was performed by postfixation immunogold labeling as described51
, using rabbit Preso1 antibody against the Preso1 C-terminal 20 aa.
Mouse spinal cord L4–5 sections were washed in PBS and incubated with 0.3% H2O2 in PBS for 30 min. After washing with PBS, sections were then blocked for 2 h at room temperature in blocking solution. c-Fos antibody was diluted in blocking solution (1:4,000) and incubated with sections overnight at 4 °C. After washing with PBS six times, sections were processed using an ABC kit (Vector) according to the manufacturer’s instructions. Slices were mounted and photographed with a Nikon microscope.
HEK293T cells were loaded at 37 °C in aCSF buffer with 1 μM Fura-2/AM for 45 min. Dorsal spinal cord neurons were loaded at room temperature in aCSF buffer with 1 μM Fura-2/AM for 30 min. Ratiometric Ca2+ imaging was performed at 340 and 380 nm in 2 mM Ca2+ solution with a Nikon Eclipse 2000-U inverted microscope equipped with a fluorescence arc lamp, excitation filter wheel, and a Hamamatsu Orca CCD camera. Images were collected with Openlab (Improvision) and analyzed with Igor Pro.
Group I mGluR–coupled calcium current in the SCG neurons was measured as previously reported7
. Briefly, both ganglia were removed from adult male Wistar rats (175–225 g, total 14 rats) following CO2
euthanasia and decapitation, enzymatically and mechanically dissociated, and cultured overnight at 37 °C. Patch clamp experiments were performed the following day. All mGluR1 and mGluR5 constructs were injected at 100–130 ng μl−1
(pCDNA3.1+; Invitrogen); Preso1 was injected at 100 ng/μl where indicated. All neurons were coinjected with enhanced green fluorescent protein cDNA (0.02 μg / μl; pEGFPN1; BD Biosciences-Clontech, Palo Alto, CA) for identification of expressing cells.
Whole-cell current-clamp recordings were made using cultured DRG neurons. DRG neurons were cultured on cover slips for 1 d, and were transferred into a chamber with medium (the extracellular solution: ECS) of the following composition (in mM): NaCl 140, KCl 4, CaCl2 2, MgCl2 2, HEPES 10, glucose 5, with pH adjusted to 7.39 using NaOH and osmolarity adjusted to 310 mOsm with sucrose. The intracellular pipette solution (ICS) contained (in mM): KCl 135, Mg-ATP 3, Na2-ATP 0.5, CaCl2 1.1, EGTA 2, HEPES 10, glucose 5, with pH adjusted to 7.36 using KOH and osmolarity adjusted to 300 mOsm with sucrose. In current clamp recordings, action potential measurements were performed with an Axon 700B amplifier and the pCLAMP 9.2 software package (Axon Instruments). All experiments were performed at room temperature (~25 °C).
Inflammatory pain responsiveness was assessed using formalin, as described52
. Mice were injected subcutaneously with formalin (10 μl, 5% in saline) into the dorsal side of one hind paw. The total time spent licking or biting the injected hind paw was recorded during each 5-min interval for 60 min. For pharmacologic studies, MPEP (30 mg/kg in 10% Tween-80; Tocris) was administered subcutaneously 20 min before formalin injection.
CFA-induced pain response was performed as described53
. Briefly, the intra-plantar region of one hind paw of each mouse was injected with 6 μl 50% CFA solution in saline. Thermal-pain sensitivity was assessed by recording paw withdrawal latency on exposure to a defined radiant-heat stimulus (Hargreaves test) before CFA injection and 1–5 d after injection.
All data were analyzed by two-tailed Student’s t-test except the behavior data, which were analyzed by one-way ANOVA. Values are presented as means ± 95% confidence interval.