NMDA-induced neuronal injury in cortical cell cultures from WT and HINT1-/-mice . (A) Cultures were exposed to increasing concentrations of NMDA for 24 h. Cell death was measured by LDH efflux into the medium. The data shown represent the mean ± S.E.M. from 20 wells per group. * Significant difference between WT and HINT1-/- cultured neurons, p < 0.05. (B) The cultures were exposed to a fixed concentration of 30 μM NMDA for 24 h in the absence (--) or presence of increasing concentrations of the cannabinoid agonist WIN55,212-2. To eliminate the possible participation of cannabinoid receptor 2 (CNR2), the assay was conducted in the presence of 3 μM of the CNR2 antagonist JTE907. Φ Significant difference compared to NMDA alone, p < 0.05. (C-H) Fluorescence photomicrograph of cortical cell cultures immunolabelled with an anti-MAP2 antibody (Ab) (green) following treatment with vehicle (C and D), 30 μM NMDA (E and F) or 30 μM NMDA plus 100 nM WIN55,212-2 (G and H); the nuclei were counterstained with 4,6-diamidino-2-phenylindole. Inset: Immunodetection study of CNR1 (antibody C terminal sequence 461–472; Cayman Chemical, Mi, USA, 10006590) and HINT1 (antibody from Abnova H00003094-A01. Abyntek, Spain) in HINT1-/- cultured neurons (40 μg/lane).