D glucose

Neurons were isolated from 0 to 1 days old postnatal rats of either sex of which brains were placed in cold dissection medium consisting of HBSS (Invitrogen, Carlsbad, CA), 1 mM sodium pyruvate (Sigma‐Aldrich, USA), 0.1% w/v D‐glucose (Riedel‐de Haën, Germany) and 10 mM HEPES (Invitrogen, USA). Cortices were dissociated in DMEM (Invitrogen, USA), containing 2.5% trypsin (Difco Laboratories, USA) and 10 mg/ml DNAse 1 (Roche, Germany) at 37°C for 20 min. Dissociated tissue was passed through a 100 μm cell strainer and centrifuged at 500 g for 5 min. After which the pallet was resuspended in plating medium consisting of DMEM containing pyruvate, glucose and glutamine (Invitrogen, USA), penicillin (100 U/ml, Lonza, Switzerland), streptomycin (100 mg/ml, Lonza, Switzerland) and 10% FCS (Invitrogen, USA). Finally, primary neurons were plated on top of a confluent astrocyte feeder layer consisting of U373 astrocytes. Non‐adhered cells were removed 2 hr after plating. Culturing of primary neurons was performed in culture medium consisting of DMEM (Invitrogen, USA); penicillin (100 U/ml, Lonza, Switzerland), streptomycin (100 mg/ml, Lonza, Switzerland) glutamic acid (25uM),1 mM sodium pyruvate and 10% FCS (Invitrogen, USA) for 12–14 days in a humidified incubator at 37°C and 5% CO².

Mixed glia cells were isolated from 0‐ to 1‐day‐old postnatal rats of either sex of which brains were placed in cold dissection medium consisting of HBSS (Invitrogen, Carlsbad, CA, USA), 1 mM sodium pyruvate (Sigma‐Aldrich, USA), 0.1% w/v D‐glucose (Riedel‐de Haën, Germany) and 10 mM HEPES (Invitrogen, USA). Cortices were dissociated in DMEM (Invitrogen, USA), containing 2.5% trypsin (Difco Laboratories, USA) and 10 mg/ml DNAse 1 (Roche, Germany) at 37°C for 20 min. Dissociated tissue was passed through a 100 μm cell strainer and centrifuged at 500g for 5 min. After which the pallet was resuspended in plating medium consisting of DMEM containing pyruvate, glucose and glutamine (Invitrogen, USA), penicillin (100 U/ml), streptomycin (100 mg/ml) (Lonza, Switzerland) and 10% FCS (Invitrogen, USA). Cells were cultured for 7–10 days. Microglia was removed from the mixed glia cell culture using an orbital shaker at 230 rpm for 3 hr.