Ifenprodil

Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium (MEM), deoxyribonuclease (DNase), poly-L-lysine, poly-D-lysine hydrobromide, cytosine arabinoside, NMDA, protease inhibitor cocktail, MK-801, and ifenprodil were purchased from Sigma (St. Louis, MO, USA). Pluronic acid and Fluo-3 AM were purchased from Molecular Probes (Eugene, OR, USA). TCN-201, IPA-3, and NSC23766 were purchased from Tocris (Bristol, UK). Pierce ECL kits (Thermo Fisher Scientific, Rockford, IL, USA), Neurobasal, and B-27 supplements were purchased from Invitrogen Corporation (Carlsbad, CA, USA); the p-PAK1 antibody (Thr 212) from Santa Cruz Biotechnology (Dallas, TX, USA); and the PAK1 antibody anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA). Brevetoxin-2 (PbTx-2) was isolated and purified from Karinia breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC, USA). QNZ-46 was a gift from SF Traynelis, Department of Pharmacology, Emory University, Atlanta, GA. The GluN2D subtype of NMDA receptor knockout mice was obtained from Daniel T. Monaghan, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska.

The mitogen-activated protein kinase (MEK1) inhibitor PD98059 (Cell Signaling Technology, EMD Millipore, Billerica, MA, USA), the CCR2 antagonist, RS504393 (Tocris Bioscience, Bristol, UK), CCL2 (R & D Systems, Minneapolis, MN, USA), and the selective blocker of the NMDAR GluN2B subunit, Ifenprodil, (Sigma, New York, NY) were used in this study. For intrathecal injection, under brief anesthesia with isoflurane a lumbar puncture was made at L5–L6 with a 30-gauge needle as previously described [20 (link)].

Other pharmacological agents were bath applied at final concentrations of: DTT (0.5 mM, Sigma), DTNB (0.5 mM, Sigma), 2-amino-5-phosphonopentanoic acid (AP5, Sigma, 100 µM), NVP-AAM077 (NVP, 0.4 µM, Sigma), ifenprodil (5 µM, Sigma), Ro 25-6981 (5 µM, Tocris), ZnCl₂ (1 µM, Sigma), ZX1 (100 µM, Strem Chemicals), and 4-[(2S)-2-[(5-isoquinolinylsulfonyl)-methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester (KN-62, 10 µM, Tocris). DTT, Ro 25-6981, NVP, ZnCl₂, and ZX1 were directly dissolved in the recording medium. DNQX was initially dissolved in DMSO (Sigma) and diluted in recording medium to a final DMSO concentration of <0.01%. PTX, DTNB, and ifenprodil were dissolved in ethanol and diluted in recording medium to a final ethanol concentration of 0.0001%.

HEK293T cells were used for ICC. Cells were fixed 48 h post-transfection, washed with 1× PBS, and then blocked with 2% bovine serum albumin (BSA) (Sigma, A9647). Incubation of primary antibody (20 µg/mL), either G11 or control B1, diluted in 2% BSA was done overnight at 4 °C.
Primary hippocampal neurons were made^{70 (link)} with minor modifications (see Supplementary Methods). Pharmacological treatment occurred at DIV14. Final concentrations of antagonists were 3 µM: TCN-201 (AdooQ Bioscience, A11947), MPX-004 (Alomone Labs, M280), and Ifenprodil (Sigma, I2892). ICC was performed 24-h post treatment on DIV15. Coverslips were washed with 1× PBS, fixed, and washed again with 1× PBS and blocked/permeabilized with antibody blocking solution, consisting of 2% (w/v) BSA, 0.25% (v/v) Triton-X100 in 1xPBS, for 60 min. Coverslips were then incubated in primary antibodies, rabbit anti-activated caspase-3 (1:250, Cell Signaling, 9661), and mouse anti-β tubulin III (Tubb3) (1:400, Millipore-Sigma, MAB1637), both in antibody blocking solution overnight in 4 °C. Coverslips were washed with 1xPBS, and incubated in fluorescence-conjugated secondary antibodies, Goat anti-rabbit Alexa-647 (1:1000, ThermoFisher, A21245) and Goat anti-mouse Alexa-488 (1:1000, ThermoFisher, A21121) for 1 h at RT and mounted as described above. Post-processing and image analysis are detailed in Supplementary Methods.