Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous D-aspartate.
Nerve terminals as well as glial cells are thought to possess high-affinity Na(+)-dependent transport sites for excitatory amino acids. However, recent immunocytochemical results with antibodies against such a transporter isolated from rat brain showed a selective labelling of glial cells [Danbolt et al. (1992) Neuroscience 51, 295-310]. Critical evaluation of the literature indicates that previous evidence for nerve terminal uptake of acidic amino acids might possibly be attributed to glia. To find out whether there is indeed a glutamate transporter in nerve endings, we incubated hippocampal slices with D-aspartate (10 and 50 microM), a metabolically inert substrate for the high-affinity glutamate transport system. After fixation by glutaraldehyde/formaldehyde the slices were processed immunocytochemically with specific polyclonal antibodies raised against D-aspartate coupled to albumin by glutaraldehyde/formaldehyde. The electron-microscopic postembedding immunogold technique demonstrated a large accumulation of gold particles in nerve terminals making asymmetrical synapses, compared to their postsynaptic dendritic spines, as well as in glial cell processes. The labelled terminals include those of the glutamatergic Schaffer collaterals. Axosomatic boutons appeared unlabelled. Comparison with a test conjugate with known concentration of fixed D-aspartate (94 mM) suggests that the concentration attained in the terminals after incubation with 50 microM D-aspartate was in the lower millimolar range. The uptake was totally dependent on Na+, blocked by L-threo-3-hydroxyaspartate, and had a high affinity for D-aspartate (apparent Km about 20 microM). There was no labelling in slices incubated without D-aspartate. Compared to glia, the nerve terminals had a higher D-aspartate density and accounted for a much higher proportion of the total tissue uptake, but this relationship may be different in vivo. At the light-microscopic level the D-aspartate-like immunoreactivity showed a distinct laminar distribution, identical to that shown autoradiographically for D-[3H]aspartate and L-[3H]glutamate uptake sites [Taxt and Storm-Mathisen (1984) Neuroscience 11, 79-100], and corresponding to the terminal fields of the major excitatory fibre systems in the hippocampal formation. The novel approach described here establishes that glutamatergic nerve terminals as well as glia do sustain sodium-dependent high-affinity transport of excitatory amino acids, implying that more than one glutamate transporter must be present in the brain. Immunogold detection of D-aspartate gives a much higher anatomical resolution than electron microscopic autoradiography of D-[3H]aspartate or L-[3H]glutamate uptake, the only method that has been available previously for ultrastructural demonstration of uptake activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Gundersen
V
V
Anatomical Institute, University of Oslo, Norway.
Danbolt
N C
NC
Ottersen
O P
OP
Storm-Mathisen
J
J
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001224:Aspartic Acid / Q000276:immunology / Q000378:metabolism*; D001345:Autoradiography; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006624:Hippocampus / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods; D007536:Isomerism; D008854:Microscopy, Electron; D009411:Nerve Endings / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014018:Tissue Distribution
D000906:Antibodies; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid