J Neurosci Res. and mGlu3 receptors in recombinant cells. To determine whether astrocytes played any role in the neuroprotection mediated by group-II mGlu receptors, we uncovered pure cultures of cortical astrocytes to DCG-IV, 4C3HPG, or L-CCG-I for 10 min. The astrocyte medium collected 2C20 hr after the exposure to any of these drugs was highly neuroprotective when transferred to mixed cultures treated with NMDA. This protective activity was reduced when CHX was applied to astrocyte cultures immediately after the transient exposure to group-II mGlu receptor (S)-3-Hydroxyisobutyric acid agonists. We conclude that neuroprotection mediated by group-II mGlu receptors in cultured cortical cells requires new protein synthesis and involves an conversation between neurons and astrocytes. Mixed cortical cell cultures made up of both neurons and astrocytes were prepared from fetal mice at 14C16 d of gestation, as described previously (Rose et al., 1992). Briefly, dissociated cortical cells were plated in 15 mm multiwell vessels (Falcon Primaria, Lincoln Park, NJ) on a layer of confluent astrocytes (7C14 d Glial cell cultures were prepared as described previously (Rose et al., 1992) from postnatal mice (1C3 d after birth). Dissociated cortical cells were produced in 15 mm multiwell vessels (Falcon Primaria) using a plating medium of MEM-Eagles salts supplemented with 10% heat-inactivated horse serum, 10% fetal bovine serum, glutamine (2 mm), and glucose (final concentration 21 mm). Cultures were kept at 37C in a humidified CO2 atmosphere until they reached confluence (7C14 dBrief exposure to NMDA (10 min), in the presence or absence of mGlu receptor agonists and/or antagonists or protein synthesis inhibitors, was carried out in mixed or pure cortical cultures at room temperature in a HEPES-buffered salt solution made up of (in mm): 120 NaCl, 5.4 Rabbit Polyclonal to APOL1 KCl, 0.8 MgCl2, 1.8 CaCl2, 20 HEPES, 15 glucose. After 10 min the drugs were washed out, and cultures were incubated at 37C for the following 24 hr in medium stock (MS) (MEM-Eagles supplemented with 15.8 mm NaHCO3 and glucose 25 mm). In some experiments, mixed cortical cultures were uncovered for 10 min to mGlu receptor agonists, 6 hr before the NMDA pulse. In another set of experiments, a glial-conditioned medium (GCM) was added to the cultures immediately after the NMDA pulse and was kept for the following 24 hr of incubation. GCM was prepared as follows: glial cortical cultures were uncovered for 10 min to mGlu receptor agonists, and then the drugs were washed out and cultures were kept in MS at 37C for the following 20 hr. After this incubation, the medium was collected and used immediately for the experiments. Neuronal injury was estimated in (S)-3-Hydroxyisobutyric acid all experiments by examination of cultures with phase-contrast microscopy at 100C400, 1 d after the insult, when the process of cell death was largely complete. Neuronal damage was quantitatively assessed in all experiments by estimation of dead neurons by trypan-blue staining. Stained neurons were counted from three random fields per well. Neuronal injury was also assessed by measuring the activity of lactate dehydrogenase (LDH) released from damaged or destroyed cells into the extracellular medium 1 d after the addition of the excitotoxins. Because results obtained with cell count or measurement of LDH activity were similar, only the former method was used in most of the experiments. Mixed cortical cultures at 13C14 dwere stained for mGlu2/3 receptors using immunopurified polyclonal antibodies raised against a synthetic peptide corresponding to the following C-terminal amino acid sequence of rat mGlu2 receptor (one-code letter): VPTVCNGREVVDSTTSSL (for a detailed description of the preparation of the antibody, see Koulen et al., 1996). The specificity of the antibody was assessed in HEK 293 cells transfected with mGlu2 or -3 subtype. In brief, (S)-3-Hydroxyisobutyric acid (S)-3-Hydroxyisobutyric acid HEK 293 cells were transfected with 4 g of plasmid DNA/dish by calcium phosphate precipitation (Chen and Okayama, 1987). Forty-eight hours later, cells were lysed, separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary and secondary antibodies. The primary antibody was diluted.