Transient increase in the high affinity [3H]-l-glutamate uptake activity during in vitro development of hippocampal neurons in culture

Abstract The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587–593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [ 3 H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 μM [ 3 H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 μM [ 3 H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, l - threo -3-hydroxy-aspartic acid (THA), l - trans -pyrrolidine-2,4-dicarboxylic acid ( l - trans -2,4-PDC), dihydrokainate (DHK), and dl - threo -β-benzyloxyaspartate (TBOA) were tested. THA, l - trans -2,4-PDC and dl -TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.