Interleukin-1 beta enhances endocytosis of glial glutamate transporters in the spinal dorsal horn through activating protein kinase C.

Animals Young adult male

Sprague-Dawley rats or GFAP-GFP transgenic mice (6–8 weeks old) were used. GFAP-GFP transgenic mice were obtained from the Jackson Laboratory. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Georgia and were fully compliant with the National Institutes of Health Guidelines for the Use and Care of Laboratory Animals.

Ligation of the L5 spinal nerve and behavioral tests

Animals were randomly divided into partial sciatic nerve ligation (pSNL) or sham-operated groups. Briefly, under isoflurane-induced (2–3%) anesthesia, the left sciatic nerve at the upper thigh was exposed and ligated approximately two-thirds the thickness of the sciatic nerve with a 5-0 silk suture as previously described ( Seltzer et al., 1990 , Nie and Weng, 2010 ). In sham-operated animals, the left sciatic nerve was exposed but not ligated. Following surgery, the wound was closed with skin stables. Behavioral tests were performed to determine the hind paw mechanical sensitivity 1 day before operation, and on day 10 post-surgery, prior to electrophysiological and molecular experiments. Briefly, the animals were placed on wire mesh, loosely restrained under a Plexiglas cage, and allowed to accommodate for at least 30 min. Von Frey monofilaments with bending forces ranging from 0.1 to 12.4 g were applied from below through the mesh onto the mid-plantar side of each hind paw to evoke paw withdrawal responses. Each hind paw was stimulated 10 times with each Von Frey monofilament, and the frequency (percentage) of paw withdrawal responses to 10 stimulations was recorded. The least bending force that evoked withdrawal responses in more than half the trials was assigned as the 50% withdrawal threshold ( Weng et al., 2006 , Yan et al., 2013 ). Spinal slice preparations, recording and analysis of glial glutamate transporter currents (GTCs) from astrocytes in the spinal dorsal horn Adult male GFAP-GFP transgenic mice were used. These transgenic mice overexpressed Green Fluorescent Protein (GFP) under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Transverse mouse spinal cord slices of the L4–5 segment were prepared as described previously ( Weng et al., 2007 ). Transverse spinal cord slices (400 µm thick) were cut in the ice-cold sucrose aCSF and then pre-incubated in Krebs solution oxygenated with 95% O2 and 5% CO2 at 35°C. The Krebs solution contained (mM): 117.0 NaCl, 3.6 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , 1.2 NaH 2 PO4, 11.0 glucose, and 25.0 NaHCO 3 at 35°C. To record GTCs, the spinal slice was placed in a recording chamber perfused with Krebs solution. Visualized whole-Cell patch clamp recordings were obtained from the spinal dorsal horn laminae I and II astrocytes identified by GFP under the fluorescent microscope. Borosilicate glass recording electrodes (resistance, 4–6 ML) were filled with (mM)145 Kgluconate, 5 NaCl, 1 MgCl 2 , 0.2 EGTA, 10 HEPES, 2 Mg-ATP and 0.1 Na-GTP (pH 7.3, 290–300 mOsm) ( Zhang et al., 2009 ). GTCs were recorded at a holding potential of −80 mV in voltage clamp mode in the presence of blockers of GABA A receptor (10 µM bicuculline), glycine receptor (5 µM strychnine), AMPA/kainate receptors (10 µM DNQX), NMDA receptor (25 µM D-AP5), and tetrodotoxin (1 µM) at 35°C ( Bergles and Jahr, 1997 , Zhang et al., 2009 ). GTCs were evoked by puffing 50 µM L-glutamate onto the recorded astrocyte through a glass pipette connected to a Picospritzer controlled by a computer. Access resistance within the range of 10–20 MΩ was monitored continuously throughout the experiments. Recordings were abandoned when the access resistance changed more than 20%. In order to make comparison of GTCs between the controls and neuropathic mice, care was taken to ensure that the distance between the recorded astrocyte and the puffing pipette tip (about 15 µm), the pipette opening tip (3–4 µm), the puffing pressure (3 p.s.i), the puffing duration (25 ms, repeated every 30 s) ( Nie and Weng, 2009 ), and the depth of the cell in the slice (about 50 µm below the surface) were kept constant across all experiments. In addition, the experimenters who collected the data were blinded to treatments given to the mice. All the drugs were applied through bath-perfusion unless otherwise indicated. Data were recorded using Axopatch 700B amplifiers, digitized at 10 kHz, and analyzed off-line. Four sweeps of GTCs were averaged and the mean amplitude and charge transfer of GTCs ( Devaraju et al., 2013 ) were measured. To measure time constants for the rise phase and the decay phase of GTCs, the GTC rise or decay phase was fitted with monoexponential function, and time constants were measured ( Nie and Weng, 2009 ). The commercial computer software Clampfit (Molecular Device, CA) was used for data analysis.

Calcium imaging

Calcium imaging procedures established by other labs ( Ge et al., 2006 , Gordon et al., 2009 , Barabas et al., 2012 ) were followed. Spinal slices from GFAP-GFP mice were prepared in the same manner as those used for electrophysiological experiments stated above. A spinal slice was placed in the recording chamber perfused with Krebs solution bubbled with 95% O2 and 5% CO2 at 35°C. In order to selectively study calcium activities in astrocytes, an astrocyte identified by the green fluorescent protein in the superficial dorsal horn was patched under the microscope with a pipette filled with the intracellular solution containing the red-fluorescent calcium dye Rhod-2 (the cell impermeant tripotassium salt, Invitrogen) (200 µM) ( Ge et al., 2006 , Gordon et al., 2009 ). Twenty minutes were allowed to let Rhod-2 microdialyze into the recorded astrocyte and diffuse into the adjacent astrocytes through gap-junctions ( Andersson and Hanse, 2010 ). The fluorescence of Rhod-2 was excited at 514 nm using a helium-neon laser line ( Ge et al., 2006 , Sztretye et al., 2011 ). Calcium imaging was performed on an Olympus fluorescent microscope under a 60X objective (numerical aperture: 1.2). Images of fluorescence signals of 548–665 nm were acquired with a Hamamatsu CDD camera ( Ge et al., 2006 , Sztretye et al., 2011 ). Metafluor imaging software was utilized in order to detect and analyze intracellular calcium changes throughout the experiment (Molecular Devices, Sunnyvale, CA) ( Barabas et al., 2012 ).

Western blot experiments

For measuring IL-1β and phosphorylated PKC levels in neuropathic and sham-operated rats, animals were deeply anesthetized with urethane (1.3–1.5g/kg, i.p.) 10 days after the surgery. The L4 and L5 spinal segments were exposed by surgery and removed from the rats. The dorsal quadrant of the spinal cord of each segment ipsilateral to the operated side was isolated. The dorsal quadrants of the spinal cord were quickly frozen in liquid nitrogen and stored at −80 °C for later use. The frozen tissues were homogenized with a hand-held pellet pestle in lysis buffer for 0.5 hr at 37°C ( Weng et al., 2014 ). For measuring GLT-1 and GLAST expressions in the plasma membrane and cytosol in spinal slices after IL-1β treatments, rat spinal slices of the spinal L4–5 segment were obtained in the same way as those for mouse spinal slices described above. Spinal slices were incubated with the plain aCSF or aCSF plus IL-1β (10 ng/ml) bubbled with 95% O2 and 5% CO2 at 35°C for 15 min. The dorsal halves of the spinal cord were then isolated and quickly frozen in liquid nitrogen and stored at −80 °C. The tissue was fractionated into cytosolic and membrane fractions with the cytoplasmic, nuclear, and membrane compartmental protein extraction kit (Biochain Institute, Inc.). Homogenates were then centrifuged at 14000 × g for 20 min at 4 °C and the supernatant were collected. Protein concentrations were determined using bicinchoninic acid (kit from Pierce). Protein samples (40 µg) were electrophoresed in 10 % SDS polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against IL-1β (1:500, Millipore, Bedford, MA), phospho PKC (1:1000, Sigma-Aldrich, St. Louis, USA), GLT-1 (1:1000, Millipore, Bedford, MA), GLAST (1: 2000, Millipore, Bedford, MA ) or a monoclonal mouse anti-β-actin (1:2000, Sigma-Aldrich, St. Louis, USA) primary antibody as a loading control. The cytosolic and membrane fractions were checked for specificity by Western blotting with anti-tubulin (1:200), anti-EGFR (1:200; Santa Cruz Biotechnology). Then the blots were incubated for 1 hr at room temperature with a corresponding HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology, CA, USA), visualized in ECL solution (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL, USA).) for 1 min, and exposed onto FluorChem HD2 System. The intensity of immunoreactive bands was quantified using ImageJ 1.46 software (NIH). Results were expressed as the ratio to control protein ( Weng et al., 2014 ). In vitro measurement of glutamate uptake activity To investigate effects of IL-1β and PKC on glutamate transporter activities, the L4–L5 spinal cord was exposed by laminectomy and the spinal dura was excised in rats anesthetized with urethane (1.3–1.5 g/kg, i.p). The rate of heart beat and breathing, and the core temperature of the animals were constantly monitored and maintained in normal limits ( Weng et al., 2003 ). IL-1β or the PKC activator (phorbol 12-myristate 13-acetate, PMA) was applied onto the L4-L5 spinal segment through a piece of cotton soaked with IL-1β (concentration: 20 ng/ml) or PMA (4 µM) in artificial CSF (aCSF) at 35°C for 30 min. Rats in the control group receiving aCSF in the same fashion. Immediately after the treatment, the dorsal half of the L4-L5 spinal segment was isolated. To determine glutamate uptake activities in the spinal dorsal horn in neuropathic rats and sham-operated rats, the dorsal quadrant of the L4-L5 spinal segment ipsilateral to the surgery side was isolated 10 days after the surgery as described above for the Western blot experiments. Synaptosome preparations were prepared immediately after the spinal tissue was isolated according to the protocol published ( Rothstein et al., 1992 ). The spinal tissue was homogenized in an ice-cold buffer solution containing: 0.5 mM EDTA, 0.5 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.32 M sucrose, 5 µg/ml pepstatin, 5 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, 4 µg/ml leupeptin, and 0.01 M phosphate-buffered saline. The homogenates were centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant collected. The remaining pellets were resuspended in the same buffer solution and re-centrifuged at 15,000 rpm for 10 min at 4°C. The two supernatants were combined and centrifuged again at 13,000 rpm for 10 min at 4°C to obtain the synaptosomal pellets, which contained both neuronal and glial glutamate transporters ( Azbill et al., 2000 ). The synaptosomal pellets were suspended in Locke’s buffer. The glutamate uptake activity was determined by incubating the synaptosome preparation with a solution containing [ 3 H] L-glutamic acid (0.4 µCi/mmol) at 37°C for 5 min. The reaction was terminated by filtering the synaptosomes through a Whatman GF/B filter pre-soaked with the same buffer solution. The filter was then transferred to a vial containing scintillation cocktail and the radioactivity, which reflects glutamate uptake activities carried by both glial and neuronal glutamate transporters ( Azbill et al., 2000 ), in the final sample was measured by a liquid scintillation counter (Beckman, LS6500). Materials Bicuculline, strychnine, phorbol 12-myristate 13-acetate (PMA), tetrodotoxin (TTX), L-glutamic acid, cytochalasin D, and 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were obtained from Sigma (St. Louis, MO, USA). D-aminophosphonovaleric acid (D-AP5), DNQX, GF 109203X, phalloidin and dynamin inhibitory peptide were obtained from Tocris Bioscience (Minneapolis, MN, USA). Recombinant human IL-1β and IL-1ra proteins were purchased from R&D Systems (Minneapolis, MN). Rhod-2 was purchased from Invivogen (San Diego, CA). [ 3 H] L-glutamic acid was obtained from Perkin Elmer.

Data Analysis

All data are presented as the mean ± SE. Student’s t-test was used to determine the statistical difference between data obtained from the same group (paired t-test) or between groups (non-paired t-tests). A p value less than 0.05 was considered statistically significant.

Materials Bicuculline, strychnine, phorbol 12-myristate 13-acetate (PMA), tetrodotoxin (TTX), L-glutamic acid, cytochalasin D, and 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were obtained from Sigma (St. Louis, MO, USA). D-aminophosphonovaleric acid (D-AP5), DNQX, GF 109203X, phalloidin and dynamin inhibitory peptide were obtained from Tocris Bioscience (Minneapolis, MN, USA). Recombinant human IL-1β and IL-1ra proteins were purchased from R&D Systems (Minneapolis, MN). Rhod-2 was purchased from Invivogen (San Diego, CA). [ 3 H] L-glutamic acid was obtained from Perkin Elmer.