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. 1998 Nov 1;18(21):8751-7.
doi: 10.1523/JNEUROSCI.18-21-08751.1998.

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

K P Lehre  1 , N C Danbolt
Affiliations

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

K P Lehre et al. J Neurosci. .

Abstract

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.

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Figures

Fig. 1.
Fig. 1.
Demonstration of the purity of the isolated glutamate transporter proteins and the lack of contamination with IgG.A, Purified GLT and GLAST proteins were separated by SDS-PAGE (10% acrylamide) and visualized by silver staining as described in Materials and Methods. Lanes 1–6 contain 30, 60, 100, 300, 600, and 1000 ng of GLT, respectively, whereaslanes 7–12 contain 1000, 600, 300, 100, 60, and 30 ng of GLAST, respectively. The positions of monomer (a, b) and dimer (arrowheads) bands of GLAST (a) and GLT (b) are marked.B, Purified GLAST protein and preimmune IgG (rabbit 68488) were run on 10% SDS-PAGE and stained with silver. The IgG has been treated with the low pH elution buffer and dithiothreitol, like the GLAST protein during isolation (see Materials and Methods).Lane 1, IgG alone (500 ng); lane 2, IgG and GLAST (500 and 300 ng, respectively); lane 3, GLAST alone (300 ng).
Fig. 2.
Fig. 2.
Electron micrographs of neighboring synapses in stratum radiatum of the hippocampus CA1 (A) and of the cerebellar molecular layer (B). The synapses shown have the typical morphology of glutamatergic synapses in the two regions: synapses with asymmetric postsynaptic specializations between boutons (b) and spines (s). Note that the (GLAST- and GLT-expressing) glial processes (asterisks) in hippocampus (A) surround the group of synapses and axons (a), and that one of the boutons shown is in close contact with the neighboring synaptic cleft (arrow). In contrast, the three cerebellar synapses (B) between parallel fibers and Purkinje cell spines are almost completely ensheathed and thereby separated from each other by (GLAST- and GLT-expressing) glial processes (asterisks). Scale bar, 400 nm.
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