Transient increase in the high affinity [H-3]-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., 200 0. J. Neurosci. Res., 59, 587-593). In order to test if this transient expr ession is associated to a transient glutamate uptake activity. [H-3]-glutam ate uptake was studied during the in vitro development of embryonic hippoca mpal 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 f rom 1.7 to 11.3% during the first 10 days of culture, while 77% of the neur ons died. The number of neurons then remains stable up to 23 days of cultur e. The initial glutamate uptake velocity at 20 and 200 muM [H-3]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). In terestingly, at 2 muM [H-3]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum b eing 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 hi gh and low affinities, at both 2 and 10 DIV. To pharmacologically character ize the transient increased glutamate uptake activity, four uptake inhibito rs, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarbox ylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta -benzy loxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibite d glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake i nhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. Thes e data indicated that. besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glut amate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression o f the glial GLAST transporter in neurons. (C) 2001 Elsevier Science Ltd. Al l rights reserved.