FIGURE 9.

d-Serine induces ultrastructurally organized and functional synapses. Cerebral cortex neurons were cultured in the presence of Neurobasal medium alone, with 0.4 mm d-serine alone, and simultaneously with d-serine and NMDA receptor inhibitors MK-801 and DCK (D-ser + MK and D-ser + DCK). After 12 days, the number of synaptophysin/PSD-95 puncta was quantified (A–C), and levels of synaptic proteins were evaluated by Western blotting assays (D). Synapse formation was also evaluated by quantitative electron microscopy (E–J) and electrophysiological assays (K–M). Number of synapses (I) and length of PSD density (J) were evaluated by electron microscopy. The arrowheads (E and G) and brackets (F and H) indicate postsynaptic density. In both cases, structurally normal synapses are observed. d-serine increased the number of synapses, although their ultrastructure seemed to be indistinguishable from control synapses. Scale bars, 20 μm (A and B), 0.5 μm (E and G), and 0.2 μm (F and H); *, p < 0.001; **, p < 0.0001. K–M, electrophysiological effects of d-serine treatment. Representative raw current traces illustrate differences in spontaneous activity recorded in the control (Ctrl) and d-serine-treated group (K). The distributions of the amplitude and interval between events of the two groups were significantly different (p < 0.001; Kolmogorov-Smirnov test; n = 5,644 and 6,127 events, from 25 and 21 neurons, respectively) (L). Averaged voltage-activated Na⁺ current traces obtained from five randomly chosen neurons in each condition illustrate the effect of TGF-β1 (p < 0.001 versus control) (M). Error bars, S.E.