Fig. 4.

Accumulation of D-aspartate immunoreactivity in terminals depends on the EAAT2-gene. Hippocampal slices were incubated with D-aspartate (50 μM, 20 min) and were protected with glutamate receptor blockers both during preincubation and incubation. (A) TEM of nerve terminals from juvenile wild type (+/+) mouse hippocampal slice preparations double labeled for D-aspartate (large particles) and EAAT1/EAAT2 transporters (small particles). As with the adult rat slices, terminals (t) containing vesicles can be seen to be heavily labeled for D-aspartate, but with low labeling for transporters. Glial profiles (g) are heavily labeled for the transporters and contain some labeling for D-Asp. (B) Hippocampal slice from a juvenile EAAT2-knockout (−/−) mouse. Note that vesicle containing terminals (t) are largely devoid of D-aspartate labeling, while glial elements express EAAT1 (small particles) and continue to show evidence of D-aspartate uptake by the presence of large particles over them. Antibodies: 482 D-Asp, anti-73 kDa (AB#171) and anti-A522 (AB#141). (C) Histogram showing relative distribution of D-aspartate labeling (gold particles) between nerve terminals (t) and astrocytes (g). Ten random TEMs were taken of the stratum radiatum in one slice from each of three +/+ and three −/− animals. Note that almost half of all the gold particles seen on the TEMs from the +/+ were located over nerve terminals. There was no significant difference between the proportions of labeling in terminals vs. glia (two-way ANOVA, P=0.94). It should be noted that no morphologically unambiguous terminals were labeled in the −/−, but all structures remotely resembling terminals were counted as such, implying that the number of particles in terminals in the −/− is likely to be overestimated. The difference in proportion of labeling between terminals and glia in the −/− was highly significant (P<0.001). NB: It was not possible to compare the uptake into glia directly between the +/+ and the −/− because of possible variations in labeling density between animals. Thus, the data show that glia account for most of the uptake still present after deletion of the EAAT2 gene, but not whether there is more or less uptake in +/+ or −/−. (D) TEM of L-glutamate labeling in the −/− mouse hippocampus. The picture shows heavy labeling for glutamate in terminals (t) and little labeling of dendrites (d). Thus, there are high concentrations of glutamate both in nerve terminals from −/− mice and in terminals from +/+ mice. Antibody: anti-L-Glu03. Scale bars=200 nm (A); 400 nm (B, D).