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7 protocols using rs 3 5 dihydroxyphenylglycine dhpg

1

Immunoblotting and Immunocytochemistry Protocols

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Alexa Fluor 488-coupled goat anti-rabbit, Alexa Fluor 568-coupled goat anti-mouse, and anti-rabbit phospho-eIF2αS52 antibodies were from Invitrogen. Rabbit anti-PKR-like endoplasmic reticulum kinase (PERK) antibody was from Rockland. Mouse anti-eIF2α (L57A5), and rabbit anti-PERK (C33E10) and rabbit anti-mGluR1 antibodies were from Cell Signaling Technology. Anti-puromycin (12D10) antibody was kindly provided by Dr. P. Pierre. Anti-mGluR5 antibody was from R&D Systems. 3,3′-Diaminobenzidine (DAB) substrate staining kit was from Vector Laboratories. (RS)-3,5-Dihydroxyphenylglycine (DHPG) and anisomycin were obtained from Tocris Bioscience. Puromycin (P8833) and mouse anti-actin and anti-MAP2 antibodies were from Sigma.
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2

Immunoblotting of Glutamate Receptors

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We used the following primary antibodies: a mouse antibody against mGlu1a (BD Biosciences, San Jose, CA) or transferrin receptors (TfR, ThermoFisher), or a rabbit antibody against mGlu5 (MilliporeSigma), pY416 (Cell Signaling, Danvers, MA), Src (Cell Signaling), or β-actin (MilliporeSigma). Pharmacological agents used in this study include D-amphetamine sulfate (MilliporeSigma) and (RS)-3,5-dihydroxyphenylglycine (DHPG) (Tocris, Minneapolis, MN). At the day of experiments, all drugs were freshly prepared.
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3

Inhibition of Neuronal Synaptic Signaling

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Infected cortical neurons cultured on 6-well plates were treated with an inhibitor cocktail (1 µM tetrodotoxin (TTX, Bio Trend), 40 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Sigma-Aldrich), 100 µM 2-amino-5-phosphonopentanoic acid (AP-5, Tocris), and 5 µM nimodipine (Sigma-Aldrich)) for 4 h to inhibit/reduce synaptic signaling. This was followed by treatment with 100 µM (RS)-3,5-dihydroxyphenylglycine (DHPG, Tocris) for 5 min under inhibitory conditions. Cells were lysed in RIPA buffer, and lysates were mixed with Laemmli buffer and analyzed by Western blotting.
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4

Selective mGlu5 Receptor PAM Protocol

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VU0360172 (N-clyclobutyl-6-[2-3(fluorophenyl) ethynyl] pyridine-3-carboxamine), a selective mGlu5 receptor positive allosteric modulator (PAM), was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). VU0360172 was dissolved in 10% Tween 80 and injected subcutaneously (s.c.). Fresh drug solution was prepared daily. VU0360172 was administered at the centrally active dose of 3 mg/kg (Rodriguez et al., 2010) ; control animals received equal volumes of 10% Tween 80. U73122 was purchased from Sigma (Milan, Italy). For the incubation of acute thalamic slices, VU0360172 and U73122 were dissolved in dimethyl sulfoxide (DMSO) at the initial concentration of 10 mM. Aminooxyacetic acid (AOAA) was from SIGMA Aldrich (Milan, Italy). (RS)-3,5-dihydroxyphenylglycine (DHPG) was purchased from Tocris Bioscience (Bristol, United Kingdom); DHPG was dissolved in Krebs buffer at the initial concentration of 10 mM.
GABAzine and kynurenic acid were obtained from Hello Bio (Bristol, UK).
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5

Pharmacological Modulation of Synaptic Plasticity

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The N-methyl-D-aspartate (NMDA), the NMDA receptor (NMDAR) specific competitive antagonist D(-)-2-amino5-phosphonopentanoic acid (D-APV), and the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) were obtained from Tocris Cookson (UK) or Ascent Scientific Ltd (UK). Cycloheximide (4-[(2R)-2-[(1S,3S,5S)-3,5-Dimethyl-2-oxocyclohexyl]-2-hydroxyethyl]piperidine-2,6-dione), anisomycin (2-[p-methoxybenzyl]-3,4,pyrrolidinediol-3-acetate), penicillin-streptomycin solution, and dimetylsulfoxide (DMSO) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). [3H]leucine was obtained from Amersham, Buckinghamshire, UK. Milli-Q deionized water (Millipore, Bedford, MA, USA) was used in all preparations of buffer solutions. Other chemicals used were all of highest grade commercially available.
The drugs were made up as stock solutions in either double-distilled water (D-APV, NMDA, anisomycin and DHPG) or 99% v/v. DMSO (Cycloheximide) at 10-1000-fold their final concentration and stored at -20°C. DMSO at final concentration of 0.01–0.1% was added to the solution of the control group as vehicle to balance any slight effect it might have on LTD. These stocks were diluted in aCSF to achieve their desired final concentrations and were applied by switching the perfusion from control aCSF to drug-containing aCSF.
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6

Neuromodulation drug administration protocols

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NMDAR antagonists D-(−)-2-amino-5-phosphonopentanoic acid (D-AP5; Cat# 0106) and 3,5-dimethyl-tricyclo-(3.3.1.13,7) decan-1-amine hydrochloride (memantine, Cat# 0773), group 1 mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; Cat# 0342), and kainate (KA; Cat# 0222) were purchased from Tocris Bioscience (Bristol, UK).
In vitro, drugs were applied to the slice perfusion bath, as previously described.4 (link) In vivo, drug or vehicle control was administered by intraperitoneal (IP) injection or, for experiments requiring cortical drug microinjection (1 hour prior to ctDCS) by a 30G injection tube coupled to a 10μl Hamilton syringe and a perfusion pump (NE-1000, New Era Pump Systems, Farmingdale, NY).
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7

Arachidonic Acid Metabolite Assay

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All chemicals were analytical grade and were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Arachidonic acid was obtained from BIOMOL (Plymouth, PA). 20-HETE (20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid), HET0016 N-hydroxy-N′-(4-butyl-2-methylphenyl)formamidine, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E), MSPPOH and indomethacin was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). RS-3,5-dihydroxyphenylglycine (DHPG) was obtained from Tocris Bioscience (Bristol, BS11 0QL United Kingdom).
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