Neurochemistry International 40 (2002) 661–671 Developmental expression and activity of high affinity glutamate transporters in rat cortical primary cultures B. Guillet a,b , S. Lortet a,b , F. Masmejean a , D. Samuel a , A. Nieoullon a , P. Pisano a,b,∗ a Laboratoire de Neurobiologie Cellulaire et Fonctionnelle, UPR CNRS 9013, 31 chemin Joseph Aiguier, 13009 Marseille, France b Laboratoire de Pharmacodynamie, UFR Pharmacie, 27 bd Jean Moulin, 13005 Marseille, France Received 19 July 2001; accepted 23 July 2001 Abstract The expression and activity of glutamate transporters (EAAC1, GLAST and GLT1) were examined during the development of cortical neuron-enriched cultures. Protein content and mitochondrial respiration both increased during the first 7 days, later stabilized and decreased from DIV14. Glutamate transport and extracellular concentration were relatively constant from DIV3 to 18. The kinetic parameters of glutamate transport were at DIV7: K m = 19 ± 3 M and V max = 1068 ± 83 pmol/mg protein/min and at DIV14: K m = 40.8 ± 9.3 M and V max = 1060 ± 235 pmol/mg protein/min. The shift in K m towards higher values suggest a more important participation of GLAST after DIV14. At DIV7 and 14, glutamate transport was poorly sensitive to dihydroka¨ ınate (DHK) suggesting a weak participation of GLT1 in glutamate transport. Western blot experiments and immunocytochemistry showed that EAAC1 was expressed by neurons whatever the stage of the culture. GLAST was found in astrocytes as soon as DIV3 and labeling increased during the development of the culture. There was little neuronal GLT1 immunoreactivity at DIV7, only detected by immunocytochemistry. From DIV10 to 18, an increasing astrocytic expression of GLT1 was observed, also detected by Western blotting. These results show that: (1) glutamate uptake remains stable all along the development of the cultures although the pattern of expression of the different transporters is changing, suggesting that glutamate transport is highly regulated; (2) neuronal EAAC1 may play a critical role during the early stages of the culture when it is expressed alone; and (3) the developmental expression pattern of glutamate transporters in cortical neuron-enriched cultures is quite similar to that observed in vivo during early postnatal development. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Development; Glutamate transport; Cultured cortical neurons 1. Introduction Glutamate is the main excitatory amino acid transmitter in the mammalian central nervous system but represents a potent neurotoxin at high concentrations. Such a con- centration is thought to be maintained at a low level by a specific high affinity glutamate uptake into nerve terminal and/or more likely by surrounding glial cells. Considerable attention have focussed recently on glutamate transport system because inhibition, impairment or reversal of this transport may contribute to increase the extracellular level of glutamate, thereby inducing acute or progressive neu- rodegenerative processes (Rothstein et al., 1992, 1993; Abbreviations: DHK, Dihydroka¨ ınate; NF, Neurofilament; GFAP, Gliofibrillar acidic protein ∗ Corresponding author. Tel.: +33-4-91-83-56-24; fax: +33-4-91-80-29-24. E-mail address: pisano@pharmacie.univ-mrs.fr (P. Pisano). Robinson et al., 1993; Hazell et al., 1997; Rossi et al., 2000; Trotti et al., 2000). Glutamate and possibly glutamate trans- porters are also considered to play important roles during the development of the mammalian central nervous system (Komuro and Rakic, 1993). Up to now, five members of sodium-dependent glutamate transporters have been cloned (Pines et al., 1992; Kanai and Hediger, 1992; Storck et al., 1992; Fairman et al., 1995; Arriza et al., 1997). The human subtypes are designated as excitatory amino acid transporters EAAT1 to 5. EAAT1, 2 and 3 are the human homologues of respectively GLAST, GLT1 and EAAC1, the three main glutamate transporters in the brain. In the adult, EAAC1 is considered to be ex- pressed by neurons selectively (Kanai and Hediger, 1992; Rothstein et al., 1994; Shashidharan et al., 1997; Furuta et al., 1997a) whereas GLAST and GLT1 are expressed by astrocytes (Danbolt et al., 1992; Torp et al., 1994; Rothstein et al., 1994; Chaudhry et al., 1995; Lehre et al., 1997; Furuta et al., 1997a). Nevertheless, this segrega- 0197-0186/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0197-0186(01)00110-3