Fig. 3.

Accumulation of D-aspartate immunoreactivity in rat slices. (A) Relative distribution of D-aspartate labeling in nerve terminals (solid bars) and astrocytes (open bars) in CA1 adult rat hippocampal slices from one representative experiment. The bars indicate mean±S.E.M. of number of gold particles per picture, and each bar represents eight pictures. Slices were incubated in Krebs’ solution with 50 μM D-aspartate or no D-aspartate (negative control) for 3 or 20 min as indicated. Excess L-glutamate (1 mM) or dihydrokainate (DHK; 0.5 mM) was added to the incubation medium as indicated. Note that the anti-D-aspartate antibodies do not significantly label tissue which has been incubated in the absence of D-aspartate (negative control), and that the accumulation of D-aspartate immunoreactivity in nerve terminals can be substantially reduced by dihydrokainate or L-glutamate. This attests to the fact that the antibodies are highly specific. A predominance of nerve terminal labeling is already observed at 3 min arguing against a saturation phenomenon. Two-way ANOVA showed that the difference between nerve terminal labeling at 20 min and 3 min was not significant. However, the presence of L-Glu and DHK significantly reduced terminal labeling (P<0.001). No differences between glial labeling in any condition were significant, although the trend was for reduction by L-Glu and DHK. (B, C) TEM of nerve terminals in adult rat hippocampal slices double labeled for D-aspartate (large particles) and EAAT1/EAAT2 transporters (small particles) after incubation in 50 μM D-aspartate. (B) Heavy labeling for D-aspartate occurs over terminals (t). The labeling for glutamate transporters is very low in the terminals. Some D-aspartate labeling is also detected over glial elements (g; the narrow process extending vertically between two other processes) which are heavily labeled for the transporters. (C) Micrograph taken from a slice treated as that in B except that 500 μM dihydrokainate was present during the incubation. There is little labeling for D-aspartate over the terminals (t). D-aspartate labeling is, however, still present over the glial elements (g). Antibodies: 482 D-Asp, anti-73 kDa (AB#171) and anti-A522 (AB#141). Scale bars=250 nm.