Sm1 neuronal supplement

The cochleae of P3 wild-type mice were dissected from the inner ear and immersed in bioclean HBSS (Multicell, 311512011) using a stereo microscope. The spiral ganglion, spiral ligament, and stria vascularis of cochleae were removed, and the cochlear basilar membranes were put in dishes that were smeared with Corning^® Cell-Tak™ and then cultured with DMEM-F12 medium added with ampicillin (Beyotime, ST008), N2 Supplement (Stemcell, 07152), and SM1 neuronal supplement (Stemcell, 05711) for 12 h in an incubator at 37°C, 5% CO₂. About 0.01 μm PTV was given to the samples for 12 h, and then, 0.5 mm neomycin with 0.01 μm PTV was given together to the cochleae for another 12 h.

Human iPSC-derived DA neurons [iCell DopaNeurons, DNC-301-030-001, FUJIFILM Cellular Dynamics, Inc (FCDI)] were cultured at 8.0 × 10⁵ cells/cm² on 16-channels per well across 4-well MEA plates (MED-P5NF30, Alpha Med Scientific) and 24-well MEA plate (MED-Q2430M, Alpha Med Scientific Inc.) coated with Polyethyleneimine (Sigma) and Laminin-511 (Nippi). Two vials of DA neurons were cultured on different days, respectively. Human iPSC-derived astrocytes (iCell Astrocyte, ASC-100-020-001-PT, FCDI) were seeded at 5.4 × 10⁴ cells per well. After 1 day, the medium was replaced with BrainPhys Neuronal Medium (STEMCELL technologies) with iCell DopaNeurons Medium Supplement (FCDI), Laminin (Sigma), N2 Supplement (STEMCELL technologies), and 100 U/mL penicillin/streptomycin (168-23191, Wako). Human iPSC-derived Glutamatergic neuron (iCell GlutaNeurons, R1061, FCDI) and astrocytes (FCDI) at a ratio of 6:1 were seeded at 8.49 × 10⁴ cells per well on 4-well and 24-well MEA plate (M384-tMEA-24W, Axion BioSystems). After 8 days of culture, the medium was replaced with BrainPhys Neuronal Medium with SM 1 neuronal supplement (STEMCELL technologies, United States). Half the media was exchanged every 4 days. Cell seeding was performed twice using different vials.

α-Hemolysin (αHL) and tetrodotoxin (TTX) were purchased from Sigma-Aldrich. Both reagents were reconstituted in sterile water, and diluted to the specified concentration in BrainPhys culture media containing SM-1 neuronal supplement (StemCell Technologies); 30-min baseline recordings of mature [more than day in vitro (DIV)21] rat primary cortical cultures were performed, and arrays were assigned to cohorts with comparable activity (six to eight replicates per group) using the technique described above. Cultures were treated with a titration of TTX (0.001–1 μM), and recorded again for 30 min both immediately following treatment and 24 h following treatment. For array-level spike frequency analysis the magnitude of effect of each condition was determined from the coefficients of a linear mix-effect model, while for cluster-level spike frequency analysis, the magnitude of effect of each condition was determined from the coefficients of Γ-distributed generalized linear model (GLM).

All experiments were performed on E18 rat dissociated hippocampal culture except electrophysiology experiments that have been performed in mice P1 hippocampal culture. Banker culture from rat hipppocampal E18 culture neurons were prepared using a previously described protocol (Penn et al., 2017 (link)) with the following modifications: neuron cultures were maintained in Neurobasal medium (Cat# 12348017, Thermo Fisher Scientific) supplemented with 2 mM l-glutamine (Thermo Fisher Scientific, Cat# 25030-024) and SM1 Neuronal Supplement (Cat# 05711, STEMCELL Technologies).
The interactomics and knock-down-shRNA interference experiments were performed on rat E18 dissociated culture maintained in Neurobasal Plus medium supplemented with 0.5 mM GlutaMAX and 1× B-27 Plus supplement (Thermo Fisher Scientific).

Primary cranial or trunk NC cells were seeded at 10,000 cells/cm² onto tissue culture-treated glass slides coated with 50 µg/ml poly-D-lysine (Sigma-Aldrich) and 10 µg/ml laminin (Corning). Cells were grown in neuronal differentiation medium: Neurobasal-A Medium (Gibco), 2 mM GlutaMAX (Gibco), SM1 neuronal supplement (STEMCELL Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Corning). The medium was sterile filtered, then supplemented with 100 ng/ml mouse nerve growth factor 2.5S (NGF; MilliporeSigma) and 50 ng/ml recombinant human neurotrophin-3 (NT3; MilliporeSigma). Half the medium was exchanged every other day.