Activity-dependent developmental maturation of the neocortical network is thought to involve the stabilization and potentiation of immature synapses. In particular, N-methyl-d-aspartate (NMDA) receptor-dependent long-term plasticity that is expressed presynaptically appears to be crucial for the selection of functionally adequate synapses. However, presynaptic expression of long-term plasticity in neocortical neurons has mainly been studied indirectly by electrophysiological techniques. Here we analyzed presynaptic plasticity directly by repeated imaging of actively cycling presynaptic vesicles with the styryl dye FM4-64 in cultured neocortical neurons at 34°C. To monitor long-term changes, stimulation-induced saturating FM4-64 staining and subsequent destaining was performed twice with an interval of 1.5 h between stainings and with the first staining serving as a plasticity stimulus. In the vast majority of presynaptic release sites, we found an increase in the mean fluorescence intensity after the second staining indicating an enhanced number of cycling synaptic vesicles. Most intriguingly, we additionally observed the appearance of new active release sites. As demonstrated by the addition of the NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid (d-AP5), both plasticity phenomena were strictly dependent on NMDA receptor activation. This suggests that a subpopulation of release sites was functionally silent during the first round of staining. Moreover, we studied a potential role of brain-derived neurotrophic factor (BDNF) in this type of presynaptic plasticity by imaging BDNF-deficient neocortical neurons. The increase in fluorescence intensity was strongly inhibited in BDNF-knockout neurons and was absent in wild-type neurons in the presence of BDNF scavenging trkB receptor bodies. These results indicate that BDNF might play an important role as a plasticity-related messenger molecule in neocortical neurons.
NMDA receptor activation is required for the manifestation and maintenance of ventilatory long‐term facilitation in awake rats
