Poly l lysine (pll)

Primary hippocampal and cortical neurons were cultured from E18 Sprague-Dawley rat pups as described previously (Perez-Otano et al., 2006 (link); Chowdhury et al., 2018 (link)). For immunofluorescence, hippocampal neurons were plated at a density of 10,000–15,000 onto 12-mm coverslips coated with a mixture of poly-DL-ornithine (Sigma) and laminin (Corning), and maintained in Neurobasal medium (Life Technologies) supplemented with 2% (v/v) B27 (Life Technologies), 2 mM GlutaMAX-I (Life Technologies), 1 μg/ml Gentamicin (Life Technologies), and 5% fetal bovine serum (FBS) (Atlanta Biologicals). The anti-mitotic agent 5-fluoro-2’-deoxyuridine was added to the cultures to prevent glial overgrowth. For biochemistry, cortical neurons were plated onto 60 mm dishes coated with poly-L-lysine (Peptide Institute Inc.) at a density of 1 × 10⁶ cells per dish. Cultures were maintained for 17–19 days in vitro (DIV) before use. cLTD was induced by bath application of 50–75 μM NMDA in conditioned media for 5 min followed by washout for 15 min in conditioned media. For cLTP, cultures were treated for 5 min with 200 μM glycine in Mg²⁺ -free media in presence of 0.5 μM tetrodotoxin, 20 μM bicuculline, and 1 μM strychnine and then returned to Mg²⁺ -containing media without glycine for 15 min before harvesting.

U2OS, SH-SY5Y, and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% fetal calf serum. Primary rat hippocampal cells were obtained and cultured as described [31 (link)]. Animal use for this purpose was approved by the Dalhousie University Committee on Laboratory Animals (approval number 19–108). Rats were sacrificed by anesthesia with isofluorane (5% v/v) followed by CO2 asphyxiation. Hippocampi were dissected from E18 Sprague-Dawley rat embryos, incubated with 0.03% trypsin for 15 minutes, and dissociated using a fire-polished Pasteur pipette. Cells were then plated on coverslips coated with 0.1% (w/v) poly-L-lysine (Peptides International) at a density of 3-6x10³ cm^-1 in Neurobasal Medium (Invitrogen) supplemented with 2% B-27 (Invitrogen), 50 μM glutamine, 25 μM glutamate, 5% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 4 hours of plating, the medium was replaced with serum-free Neurobasal Medium supplemented with B-27 and 0.5 mM glutamine. One-third of the medium was replaced on a weekly basis until transfection.

Antibodies against P-Akt (#4060), P-mTORC1 (#5536), P-S6K (# 9234), P-PLCγ2 (#3871), and β-actin (#4967) were from Cell Signaling (Beverly, MA, USA); a pan histone antibody was from Sigma (#MABE71). Ficoll-Paque Plus was from GE Biosciences (Baie d’Urfé, Qc, Canada); Dextran T500 from Pharmacosmos (Holbæk, Denmark); endotoxin-free (< 2 pg/ml) RPMI 1640 from Wisent (St-Bruno, Qc, Canada); poly-L-lysine from Peptides International (Louisville, KY, USA). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA. Dimethyl sulfoxide (DMSO), N-formyl-methionyl-phenylalanine (fMLF), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma-Aldrich (St. Louis, MO, USA). All inhibitors were purchased through Cedarlane Labs (Missisauga, Canada). ProLong Gold Antifade, Hoechst 33342, propidium iodide, and 16% paraformaldehyde (PFA) were purchased from Thermo Fisher (Missisauga, Canada). PlaNET reagents (fluorescent chromatin-binding polymers) are no longer available from Immune Biosolutions or other suppliers; we therefore employed a close equivalent – fluorescent, 50-nm carboxylate microspheres (# 16661-10) from Polysciences Inc. (Warrington, PA, USA). These microspheres are referred to as PlaNET reagents throughout this study. All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.

A full-length human 5-HT_2AR was subcloned into pcDNA3.1, and a FLAG sequence (DYKDDDDK) was introduced at the N-terminus using the inverse PCR system (Toyobo biotech, Osaka, Japan). Mutant constructs were made by site-directed mutagenesis using the QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The sequences of all constructs were verified by DNA sequencing. The FreeStyle HEK 293-F cells (Thermo Fisher, Waltham, MA, USA) were grown under suspension culture in FreeStyle 293 Expression Medium (Thermo Fisher) at 37 °C under 5% CO₂. Cells were transfected using a mixture at a ratio of one μg plasmid DNA: polyethyleneimine MAX (Polysciences, Warrington, PA, USA) = 1:3 (weight) into 1 mL of cells at a density of 2 × 10⁶ cells/mL. The next day after transfection, the cells were inoculated on the surface of the 12.5 μm thick polyimide films (Kapton, Du Pont-Toray) that were treated with 0.001% poly L-lysine (Peptide institute, Osaka, Japan) at a density of 1 × 10⁶ cells/cm² and further cultured for 12 h. The viability of cells was examined by trypan blue dye exclusion.