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

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. [3H]-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 [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 μM [3H]-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.