Confocal imaging of N-methyl- d-aspartate receptors in living cortical neurons

The fluorescence-conjugated N-methyl- d-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl- d-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17–20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10–15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl- d-aspartate receptor-associated polyamine site. The N-methyl- d-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl- d-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl- d-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of fluorescently labeled clusters compared with the control group, neurons in the high potassium group exhibited a higher number of fluorescently labeled receptor clusters. These results suggest that more active neurons may tend to form more N-methyl- d-aspartate synapses during early development.