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

Neurochemistry International 38 (2001) 293 – 301 www.elsevier.com/locate/neuint

Transient increase in the high affinity [3H]-L-glutamate uptake activity during in vitro development of hippocampal neurons in culture S. Gaillet *, C. Plachez, F. Malaval, M.-F. Be´zine, M. Re´casens CNRS UMR 5102, Laboratoire de Plasticite´ Ce´re´brale, Uni6ersite´ Montpellier II, CC90, Place E. Bataillon, 34095 Montpellier Cedex 5, France Received 2 March 2000; received in revised form 2 August 2000; accepted 3 August 2000

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, [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 mM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 mM [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-2,4PDC), dihydrokainate (DHK), and DL-threo-b-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. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: [3H]-glutamate uptake; Hippocampal neurons; In vitro development; Uptake inhibitors

1. Introduction Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It is involved in brain plasticity and many higher brain functions such as learning and memory (Fonnum, 1984; Bliss and Collingridge, 1993). However, its excessive accumulation could result in neuronal death (Choi et al., 1987). Consequently, both the physiology of the glutamatergic transmission and the non-occurrence of excitotoxicity depend on the maintenance of low concentrations of * Corresponding author. Tel.: + 33-4-67-14-36-23; fax: +33-4-6714-42-51. E-mail address: [email protected] (S. Gaillet).

this transmitter within the synaptic cleft, and more generally, within the extracellular space. The ending of glutamatergic transmission results from two main processes: diffusion from the synaptic cleft to the extracellular space and uptake into surrounding glial and neurons. Transporters provide the major pathway for regulating extracellular glutamate concentration. To date, five transporters have been cloned and named GLAST (EAAT1 in human; Storck et al., 1992), GLT1 (EAAT2; Pines et al., 1992), EAAC-1 (EAAT3; Kanai and Hediger, 1992), EAAT4 (Fairman et al., 1995) and EAAT5 (Arriza et al., 1997; for review see: Danbolt et al., 1998a). Glial cells have the highest glutamate uptake activity in the adult brain (Schousboe, 1981). Glial cells express two of the most abundant

0197-0186/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0197-0186(00)00098-X

294

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

and active transporters GLAST and GLT-1 (Haugeto et al., 1996; Rothstein et al., 1996). Knockout of selective glutamate transporters (GLT-1 in particular) reveals the major role played by glial transporters in the clearance of glutamate (Rothstein et al., 1996) and induces the appearance of glutamate transmission disturbance (Tanaka et al., 1997). On the contrary, the three other cloned transporters EAAC-1, EAAT4 and EAAT5 are primarily located on neurons. EAAC-1 is present in most brain regions (Rothstein et al., 1994), while EAAT4 and EAAT5 are predominantly expressed in cerebellum and retina, respectively. Thus, only the neuronal EAAC-1 transporter could also play an important general role in the clearance of glutamate in the whole brain. However, its somato-dendritic and nonsynaptic location rules out this possibility at the synapse level (Rothstein et al., 1994; Coco et al., 1997). Consequently, glial transporters are indeed essential to remove extracellular glutamate. In adult animals, the majority of excitatory synapses are surrounded by glial cell processes possessing GLAST and GLT-1 (Spacek, 1985). However, when synapses form during development, few glial cells are present around neurons. Moreover, it was suggested that glutamate has a morphogenetic action during development as particularly shown in Purkinje cells (Cohen-Cory et al., 1991; Mount et al., 1993; Meaney et al., 1998). Thus, extracellular glutamate concentration could have important impacts on the development of neurons. This raises the question of the mechanism(s) by which glutamate is removed from the extracellular space during developmental period. Interestingly, it was recently reported that the glial-localized glutamate transporter GLT-1 is also expressed in growing axons of spinal cord (Yamada et al., 1998) or along axonal pathways (Furuta et al., 1997). This indicates that during development GLT-1 could be expressed by neurons, which are known to possess GLT-1 mRNA (Schmitt et al., 1996; Torp et al., 1997). GLT-1 could also be expressed in vitro in hippocampal neuronal culture (Mennerick et al., 1998; Brooks-Kayal et al., 1998). Moreover, in specific brain areas, such as the retina, GLT-1 was also found in neurons (Rauen et al., 1996; Harada et al., 1998). This suggests that external conditions could determine or at least influence cell expression of GLT-1. Recently, we found that GLAST and GLT-1 are also transiently expressed in hippocampal neuronal culture as revealed by immunocytochemistry (Plachez et al., 2000). In all, the expression of glutamate transporters during development appears to be largely different from that found in adult animals, with the expression of glutamate transporters, usually located in glial cells, in neurons. This also appears to hold true for in vitro development. All these facts prompted us to study the developmental profile of glutamate uptake activity in

embryonic hippocampal neurons in vitro. Neuronal culture in a defined medium (serum free) is a privileged experimental model to observe development and explore the influence of external factors. In these studies, we found a global increase of glutamate uptake with the maturation of the culture. Importantly, we also demonstrated a transient increased dihydrokainate-insensitive uptake activity, at 2 mM [3H]-glutamate potentially related to the transient GLAST expression in neurons during in vitro development of hippocampal neurons.

2. Materials and methods

2.1. Materials Primary neuronal cultures were established from 18days old embryonic rat hippocampi, essentially as described elsewhere (Blanc et al., 1995, 1999). After 12 min preincubation with EDTA, hippocampal cells were mechanically dissociated and plated at a density of 0.75 million cells per well (Nunc, surface of one well 2 cm2), coated with poly-L-lysine (5 mg/ml, Sigma) in a medium containing basal DMEM/HAM F12, supplemented with: glucose (33 mM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), HEPES buffer (5 mM), sodium bicarbonate (13 mM), transferrine (50 mg/ml), insuline (87 mM), b-estradiol (1 pM), triiodothyronine (3 nM), progesterone (20 nM), sodium seleniate (46 nM), and putrescine (100 mM). L(− )-Threo-3-hydroxyaspartic acid (THA) and Ltrans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4PDC), from Tocris (Bristol, UK), dihydrokainate (DHK), from Sigma (St Quentin Fallavier, France), and DL-threo-b-benzyloxyaspartate (DL-TBOA), gently given by Dr Shimamoto K. (Japan), were used to perform the uptake inhibition. Glutamic acid, L-[2,33 H] (15–25 Ci/mmol) was obtained from New England Nuclear, Boston, MA, USA.

2.2. Imunocytochemistry and cell counting Immunochemistry experiments were performed directly on plastic wells of 2.2-mm-diameter plated at a density of 1.5× 106 per well. Cells were fixed in 4% paraformaldehyde for 20 min. After 30 min of preincubation at 37°C in phosphate buffer saline (PBS) containing 0.2% bovine serum albumin (BSA), 10% of either goat or horse serum (depending on the specie in which the secondary antibody was raised) and 0.1% Triton X-100, cells were incubated overnight with primary antibodies (mouse IgG to glial fibrillary acidic protein, G-9269, 1/500, Sigma, St Louis, MO, USA; anti-rabbit Rab 3A, K-15, 1/50, Santa Cruz, Tebu, Le Perray-en-Yvelines, France) diluted in PBS, containing

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

0.2% BSA and 1% goat or horse serum. After two washes in PBS–BSA 0.2%, cells were incubated in biotinylated secondary antibody from Vecstatin Elite ABC kit (Vector, Byosis, France) and samples were then processed as indicated by the manufacturer. Horse radish peroxidase activity was revealed using the ‘VIP’ substrate from Vector. Cell counting were performed from at least three independent experiments. For each experiment, five to seven randomly chosen fields were counted. Each field represents about 800 – 2000 neurons. Results were expressed as % 9S.E.M. Two types of ratio were calculated: (1) the ratio of the number of glial cells or neurons to the total number of plated cells (shown in Fig. 1A). (2) The ratio of the number of glial cells to the number of neurons at each age of development (see values indicated in the abstract and in the text).

2.3. Determination of [ 3H] -L -glutamate uptake Measurements of glutamate uptake velocity as a function of days in vitro (1, 2, 3, 6, 8, 10, 14, 18 and 23 DIV) were performed on primary cultures of hippocampal neurons.

295

Several [3H]-glutamate concentrations were used in this study 2, 20 and 200 mM, in order to modulate the contribution of each transporter to the total glutamate uptake. We have previously checked in several experiments that [3H]-glutamate uptake linearly increases (0– 2 min) whatever the [3H]-glutamate concentration used and reaches a plateau between 5 and 10 min (data not shown). Thus the subsequent glutamate uptake kinetics studies have been performed in the linear range activity, i.e. between 0 and 2 min. Briefly and in routine experiments, the 24-well plates were placed in a gently shaking circular 37°C water bath and the culture medium was replaced for 10 min by 500 ml of oxygenated Krebs–Ringer buffer containing (in mM): NaCl 125; KCl 3.5; Mg SO4 –7H2O 1.25; KH2PO4 1.25; NaHCO3 25; glucose 10; HEPES 10; CaCl2 1.5 and adjusted to pH 7.4. After washing, the cells were preincubated for 15 min in 400 ml Krebs–Ringer and the uptake assay started immediately following the addition of 100 ml per well of Krebs–Ringer buffer containing 0.2 mCi of glutamic acid, L-[2,3– 3H] (15–25 Ci/mmol, isotopic dilution: 1:178 000), with or without an antagonist treatment (Bridges et al., 1999), adjusted to the appropriate concentration with unlabeled L-glutamate. Glutamate uptake was stopped by placing the plates on ice and rapidly washing cell layers twice with 1 ml per well of ice-cold Krebs–Ringer buffer to remove the excess of radioactivity outside the cells. Cells were then immediately solubilized in 200 ml of 0.1 N NaOH and the radioactivity was determined on a Beckman liquid scintillation counter. Protein contents of samples were determined on 25 ml aliquots of solubilized cells by using the Bradford assay (1976).

2.4. Method of calculation

Fig. 1. Neuronal and glial cells during in vitro development. (A) The percentage of neurons (number of neurons/number of plated cells; solid circle) and glial cells (number of glial cells/number of plated cells; solid square) were counted after immunolabeling with Rab3A and GFAP antibodies, respectively. Each value represents the % 9 S.E.M. of at least three independent experiments, the number of plated cells being taken as 100%. One value resulted from the counting of five – seven fields, each containing from 800 to 2000 cells depending on culture stage. Insert shows a zoom of the percentage of glial cells during development: the concentration of total proteins (mg/well) was determined by using the Bradford assays. Each value represents the means 9S.E.M. from at least three cultures (*: PB 0.05; ***: P B 0.001 vs the protein concentrations at 1 DIV).

The initial velocity of glutamate uptake was determined from the mean of the slopes, obtained after linear regression analysis of uptake data performed at 0, 30, 60 and 120 s in triplicates. Data of glutamate uptake velocity were expressed as pmol/min/well. All the results are presented as mean9 S.E.M. Experiments were performed from at least three independent cultures (n]3). These data were then used to analyze [3H]-glutamate concentration dependence and inhibition displacement curves using adapted EBDA-ligand program derived from Munson and Rodbach (1980). Concentration dependence data were systematically fitted either with a single saturable uptake component, or two independent saturable uptake components, or finally with a combination of one saturable and one non-saturable uptake component. The significance of the various fits was

296

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

obtained by the F-test (DeLean et al., 1978; Munson and Rodbach, 1980). The concentration dependence for inhibition was only examined with compounds that inhibited more than 70% at 500 mM, assuming a single homogeneous population sites. For this curve fitting, the maximal inhibition was not constrained. Variations in glutamate uptake at different days in 6itro were analyzed by the one-way analysis of variance (ANOVA) followed by F Scheffe´ test; Statistical significance of the results are indicated as followed on graphs (ns: P\0.05; *: P B 0.05; **: P B0.01; ***: P B 0.001).

2.5. Western blots Hippocampal cultures were rinsed with PBS, homogenized in 50 mM NaPi containing 1 mM phenylmethyl-sulfonyl-fluoride and 1% sodium dodecyl-sulfate and centrifuged (4°C; 15 000 × g for 5 min). Supernatants were stored at − 80°C until used and then mixed with buffer (1% (w/v) sodium dodecyl-sulfate, 40 mM Tris– HCl (pH 6.8), 5% (w/v) glycerol, 280 mM mercaptoethanol and bromophenol blue) and heated at 100°C for 5 min. Samples were subjected to 10% (w/v) SDS– PAGE (Laemmli and Quittner, 1974) and blotted onto nitrocellulose (Towbin et al., 1979). Protein (20 mg) of protein was loaded in each lane. Nitrocellulose membranes were incubated with a primary antibodies (Lehre et al., 1995; Danbolt et al., 1998b) directed against EAAC-1 (1 mg/ml, anti-C491, rabbit 7D0993, C491523: CLDNEDSDTKKSYVNGGFSVDKS DTISFTQTSQF), GLT-1 (0.2 mg/ml, anti-B12, rabbit 26970, B12-26: KQVEVRMHDSHLSSE) and GLAST (0.2 mg/ml, anti-A522, rabbit 68488, A522-541: PYQLIAQDNEPEKPVADSET). Bound antibodies were detected with the ECL-system (Amersham, les Ulis, France). Densitometric analyses of western blot were performed by using Sigma Gel software.

3. Results

3.1. Neuronal 6ersus glial cell percentage during in 6itro de6elopment During the first 48 hour of culture about 45% of cells died, due likely in part to the mechanical dissociation. During the later in vitro development, between 2 and 10 DIV, the percentage of hippocampal neurons decreased by a further 32% (Fig. 1A) and then remained roughly constant up to 23 DIV (not shown). The number of glial cells increased by around 166% between 1 DIV and 7 DIV (Fig. 1A and insert). Consequently, the ratio of glial cells to neurons increased during the first week of culture (from 1.7% at 1 DIV to 8.1% at 7 DIV).

Fig. 2. Changes in the initial glutamate uptake velocity during development of hippocampal neurons in culture. Glutamate uptake initial velocity (pmol/min/well) was determined at 1, 2, 3, 6, 10, 14, 18 and 23 DIV with three different glutamate concentrations: 2 (A), 20 (B) and 200 mM (C). Each point represent the mean9S.E.M. of initial velocity determined from at least three independent cultures (ns: not significant; *: P B0.05; **: PB 0.01 vs the uptake velocity at 1 DIV). In each experiment, initial velocity was determined from the linear slope obtained by linear regression analysis of [3H]-glutamate uptake performed in triplicate at four times 0, 30, 60 and 120 s.

In parallel, we have also determined the protein concentration during the development of the culture (Fig. 1B) and we observed a regular increase between 1 and 7 DIV (41.8 9 1 vs 56.8 9 0.8) which then remained constant up to 14 DIV (599 1.1).

3.2. De6elopment of the glutamate transport acti6ities with the age of the culture The evolution of the glutamate uptake initial velocity was measured as a function of the age of the culture (1, 2, 3, 6, 10, 14, 18 and 23 DIV; Fig. 2). Three different glutamate concentrations were chosen 2, 20 and 200 mM for this study. This choice was made for gradually favoring the activity of distinct glutamate transporter subtypes. In fact, it was reported in Xenopus oocyte expressing system that the Km for murine GLT-1 ranges from 2 to 20 mM (Pines et al., 1992; Wang et al., 1998) and that for GLAST between 11 and 70 mM (Klo¨ckner

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

et al., 1994; Tanaka, 1993). Rabbit EAAC-1 expressed in Xenopus oocytes possesses a Km in the range of 12 – 15 mM (Kanai and Hediger, 1992; Dowd et al., 1996). However, the Km values reported are greatly variable according to the expression systems or the experimental model used (Bridges et al., 1999). In our hippocampal neuronal culture, we have used different glutamate concentrations (200, 20 and 2 mM), in order to identify the relative contributions of progressively higher affinity glutamate transport systems. The initial glutamate uptake velocity regularly increased with the development of the culture for uptake activities measured with 20 and 200 mM [3H]-glutamate (Fig. 2B and C, respectively). The initial velocity augmented 5.7 (49.596.2 vs 283.789 25.31) and 5.1 times (103.259 6.27 vs 526.33 949.7) between 1 and 10 DIV for uptake activity determined at glutamate concentrations of 20 and 200 mM, respectively. Interestingly, at a glutamate concentration of 2 mM, the glutamate uptake initial velocity showed a biphasic pattern (Fig. 2A). A rapid increase occurred between 1 and 2 DIV (12.99 0.77 vs 28.1991.35 pmol/min/well), followed by a regular decrease between 2 and 8 DIV (28.199 1.35 vs 11.0 91.0 pmol/min/well). At 8 DIV, the initial velocity was not significantly different from that found at 1 DIV (11.091.0 vs 12.990.77 pmol/ min/well). After 8 DIV, a second increase in uptake velocity took place. The maximum of uptake velocity is reached at 14 DIV, and then remained about constant up to 23 DIV (33.9390.33 vs 32.919 1.08 pmol/min/well).

297

was chosen. Under these conditions, the apparent Km values for saturable high affinity glutamate transport were 3.99 2.3 mM and 18.29 10.9 mM, at 2 and 10 DIV, respectively. The Vmax values were 68.3925.7 pmol/min/well at 2 and 3389 164 pmol/min/well at 10 DIV. Km and Vmax values are significantly different according to the stage of the culture (2 DIV vs 10 DIV). The apparent Km values for saturable low affinity glutamate transport were 1.792.4 and 4.1910.6 mM, at 2 and 10 DIV, respectively. The Vmax values were 16649 2474 pmol/min/well at 2 DIV and 20079 4566 pmol/ min/well at 10 DIV.

3.4. Potency of inhibitors of transport In order to further characterize the transient increased glutamate uptake activity measured with 2 mM glutamate, three uptake inhibitors were tested: DHK, THA, L-trans-2,4-PDC, both at 2 DIV (Fig. 4A) and 10 DIV (Fig. 4B). DHK is a selective, non-transported

3.3. Kinetics of glutamate transport in neuronal hippocampal cultures Functional activity of glutamate transport into neuronal culture was measured by using increasing concentrations of [3H]-glutamate at two developmental stages, 2 and 10 DIV (Fig. 3A and B, respectively). The amount of [3H]-glutamate radioactivity was maintained at a constant level, and glutamate concentrations were adjusted with unlabeled glutamate. F-test analysis showed that (i) two independent saturable uptake components give a significantly better fitting than one single saturable uptake component, F =6.18; P =0.045 for 2 DIV and F= 5.03; P = 0.039 for 10 DIV; (ii) a combination of one saturable and one non-saturable uptake also give a significantly better fitting than one single saturable uptake component, F =10.92; P =0.016 for 2 DIV and F=11.17; P =0.009 for 10 DIV; (iii) no significant improvement was observed when testing two independent saturable uptake components versus a combination of one saturable and one non-saturable uptake. Since it is unlikely that glutamate being passively transported against its concentration gradient, the assumption of the presence of two saturable sites

Fig. 3. Kinetic properties of [3H]-glutamate uptake into cultured hippocampal neurons at 2 (A) and 10 DIV (B). Uptake velocity (pmol/min/well) was measured with 0.2 mCi of glutamic acid, L-[2,3– 3 H] (15 – 25 Ci/mmol) and glutamate concentrations were adjusted with unlabeled glutamate. The data presented are the mean of at least three independent experiments, each value being determined in triplicate. The low concentrations of glutamate are shown in larger scale (top inserts). The best curve fittings using the EBDA ligand program are shown in the bottom inserts. F-test analysis showed that the best fitting for glutamate uptake was obtained when assuming the presence of two saturable uptake components, both at 2 and 10 DIV. Only the high affinity saturable site was represented in the bottom inserts.

298

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

We have also used, at 2 DIV, DL-TBOA a non-specific potent inhibitor of glutamate transporters. DLTBOA almost completely inhibited [3H]-glutamate uptake, in a dose-dependent manner (data not shown), like THA and L-trans-2,4-PDC.

3.5. Immunoreacti6ity of EAAC-1, GLT-1 and GLAST in culture

Fig. 4. The potencies of glutamate uptake inhibitors were assessed in hippocampal neurons in culture at 2 and 10 DIV. Uptake of 2 mM glutamate was measured in the presence of the indicated amount of inhibitors. Inhibitors were THA (solid squares; 5, 10, 25, 50 and 100 mM), L-trans-2,4-PDC (solid triangles; 10, 25, 50 and 100 mM) and DHK (solid circles; 50 and 100 mM) at 2 DIV (A) and 10 DIV (B). Each data set shown represents the mean 9S.E.M of at least three distinct experiments with triplicate determinations (ns: not significant; *: PB 0.05). Ki values were determined using EBDA program by assuming a single inhibitory displacement site. Details are indicated in the text.

inhibitor for GLT-1 (Ki =23 mM as compared to Ki for GLAST and EAAC-1\3 mM; for a review, see Robinson and Dowd, 1997). THA and L-trans-2,4PDC are non-specific competitive inhibitors of glutamate uptake (Balcar and Johnston, 1972) and of the main glutamate transporter subtypes. They are both slightly more effective for inhibiting GLT-1 than GLAST or EAAC-1 in some systems (Robinson and Dowd, 1997; Arriza et al., 1994). DHK did not inhibit glutamate uptake determined with 2 mM [3H]-glutamate either at 2 or 10 DIV, except at 50 mM at 2 DIV. At this concentration, a slight but significant inhibition (17.08%) was observed (*: PB 0.05; Fig. 4A and B). THA almost completely inhibited [3H]-glutamate uptake in a dose-dependent manner both at 2 and 10 DIV (Fig. 4A and B, respectively). At 2 DIV, THA inhibition, fitted with a one site inhibition, possessed a Ki of 3.19 1.0 mM, while at 10 DIV, THA showed a Ki of 8.39 2.5 mM. L-trans-2,4-PDC was also found to potently inhibit [3H]-glutamate uptake both at 2 and 10 DIV with Ki of 9.4 91.9 and 25.4 96.6 mM (data not shown).

Immunoblots, labeled with either anti-C491, antibodies to EAAC-1 (Fig. 5A), or anti-B12, antibodies to GLT-1 (Fig. 5B), or anti-A522, antibodies to GLAST (Fig. 5C); showed that the EAAC-1, GLT-1 and GLAST proteins are present in hippocampal culture both at 2 and 10 DIV. The three antibodies labeled polypeptides with electrophoretic mobilities similar to the corresponding proteins in adult rat hippocampus (not shown). No apparent EAAC-1 immunoreactivity difference was observed between 2 and 10 DIV (Fig. 5A). On the contrary, GLT-1 and GLAST immunoreactivities increased 4.579 0.58 and 2.649 0.54 times, respectively, from 2 to 10 DIV. In addition, GLAST proteins seem to be more expressed than GLT-1 proteins at any stage. However, the increase of GLT-1 proteins is stronger between 2 and 10 DIV than that observed with GLAST proteins.

4. Discussion Two main facts merge from our data. Firstly, glutamate transport into cells regularly increased with the age of the culture, when using high glutamate concentrations (20 or 200 mM). Secondly, superimposed to the regular increased uptake activity with development, a transient peak (1–6 DIV) of glutamate transport activity is observed during in vitro development of hippocampal neurons when initial uptake velocity is measured at lower glutamate concentration (2 mM).

Fig. 5. Immunoblots showing EAAC-1, GLT-1 and GLAST immunoreactivities. Each lane was loaded with 20 mg of proteins, extracted from neuronal hippocampal cultured during 2 DIV and 10 DIV. GLAST and GLT-1 SDS – PAGE were provided from the same gel whereas EAAC-1 SDS – PAGE was run separately. Antibodies: 1 mg/ml anti-C491 to EAAC-1; 0.2 mg/ml anti-B12 to GLT-1; 0.2 mg/ml anti-A522 to GLAST.

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

The main glutamate uptake activity, which regularly increases during development, may essentially be due to uptake into glial cells. Indeed, this increase occurs despite the death of numerous neurons (Fig. 1) during in vitro development of hippocampal neurons. About 77% of the neurons died between 1 and 10 DIV. On the contrary, the number of glial cells increases from 11 300 at 1 DIV to 19 800 at 10 DIV. Moreover, the development of glial extensions is considerable from 5 DIV and the membrane cell surface dramatically augments (Plachez et al., 2000). Glial cells express the two major glutamate uptake transporters, GLT-1 and GLAST, which are known to assume the major part of glutamate uptake (Rothstein et al., 1996). Some additional evidence are that (i) the ‘neuronal’ GLT-1 and GLAST expressions, present at the early developmental culture stages, disappear from 7 DIV (Plachez et al., 2000) and (ii) EAAC-1 which is located on neurons is usually considered as a minor component in total glutamate uptake in vivo (Rothstein et al., 1996), although the situation is less clear in in vitro experimental models. Nevertheless, Swanson et al., 1997, reported that coculturing astrocyte with neurons leads to a slight decrease in glutamate uptake as compared to glutamate uptake in pure astrocyte culture. In all, these data indicated that the major part of global glutamate uptake capacity, measured with high glutamate concentrations, likely results from transporters present in glial cells and not in neurons. Kinetic experiments revealed the presence of a single ‘high affinity’ uptake component both at 2 and 10 DIV, suggesting that either one glutamate carrier subtype is predominant in this culture or that all carrier subtypes possess similar affinity for glutamate. The first proposal appears as the most likely if we consider that GLT-1 and GLAST are the two transporters responsible for the majority of glutamate uptake. Indeed, we found that the uptake activity is blocked by L-trans-2,4-PDC, THA and DL-TBOA, but not by DHK, a rather GLT-1 specific inhibitor. This assertion is in agreement with two recent reports indicating that [3H] – glutamate uptake correlated with GLAST expression in astrocyte cultures at all developmental stages (Swanson et al., 1997; Stanimirovic et al., 1999). It is also interesting to note that the glutamate uptake high affinity values significantly changes between 2 and 10 DIV (Fig. 3). This could be attributed to a change either in the relative contribution of the glutamate carriers subtypes involved in the total uptake activity, or in the structure of the carrier with the age of the culture (for instance appearance of a regulatory protein associated with the carrier), or finally in the nature of the glutamate carrier implicated in [3H]–glutamate uptake activity. Our data also demonstrate the presence of a transient increased uptake activity during a narrow period of

299

development (1–6 DIV). This uptake activity is solely revealed when using low (2 mM) and not high glutamate concentrations for uptake measurements. This finding suggests that a concentration of 2 mM favors the contribution to the total uptake activity of a transporter which has a relatively high affinity for glutamate. Recently, we have reported that neurons can express the glial transporters GLT-1 and GLAST during a narrow period of in vitro development of hippocampal neurons in culture (Plachez et al., 2000). Consequently, from this coincidence, it is tempting to speculate that the transient high affinity glutamate uptake activity is associated with the transient presence of GLT-1 and GLAST in neurons. This supposition is substantiated by the fact that glial cells represent a minor part during the first days of culture. The ratio glial cells on neurons varies from 1.7% at 1 DIV to 8.1% at 7 DIV. In addition, during this period, glial cells possess few extensions. The major glial cell extension development occurs after 5 DIV, at a period when both the transient uptake activity and the neuronal GLAST and GLT-1 expressions disappear. Though the pharmacological tools, presently at our disposal, did not allow to specifically block each glutamate transporter subtype, four main uptake inhibitors, DHK, THA, L-trans-2,4-PDC and DL-TBOA were used. DHK is relatively specific for GLT-1 uptake, while THA, L-trans-2,4-PDC and DL-TBOA inhibit the three main transporters GLT-1, GLAST and EAAC-1 with about similar potency (data not shown for DLTBOA). The transient uptake activity present in the cultured hippocampal neurons is insensitive to DHK, while THA, L-trans-2,4-PDC and DL-TBOA are effective inhibitors. This suggests that the transient peak of activity is not associated with an activity due to the GLT-1 transporter. This transient activity could be associated either with GLAST or EAAC-1. Among these two last glutamate transporters, only GLAST was transiently expressed on neurons (Plachez et al., 2000). Moreover, immunostaining reveal that EAAC-1 remains localized in neurons between 3 and 10 DIV (Plachez et al., 2000). Immunoblots show that there is indeed no apparent difference in EAAC-1 expression between 2 and 10 DIV. In addition, at 2 DIV, GLAST appears much more expressed than EAAC-1. Thus, the most evident explanation to propose from these data is that the transient glutamate uptake could essentially be related to the transient GLAST expression in hippocampal neurons. As already indicated, the glutamate affinity found at 2 DIV is different from that at 10 DIV. If we consider that GLAST is the predominant carrier in this culture both at 2 and 10 DIV, then this would imply that the GLAST carrier, expressed in neurons at 2 DIV is distinct from the GLAST carrier expressed in glial cells at 10 DIV, or at least differently regulated in the two cell types. This hypothesis is also

300

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301

substantiated by the fact that the two main inhibitors THA and L-trans-2,4-PDC have distinct inhibitory constant (Ki ) at 2 and 10 DIV. The clearance of extracellular glutamate appeared to be primarily mediated by glial glutamate transporters, GLAST and GLT-1 in the adult hippocampus (Rothstein et al., 1996). The role of the transient presence of the glial glutamate transporters GLAST and GLT-1 in neurons is not yet known. However, their presence is associated with a period when astrocytes did not yet surround neuron terminals in culture. They might help neurons either to control the extracellular glutamate concentration or to sniff extracellular glutamate. In fact, in this neuronal culture containing a low density of glial cells, neurons might transiently express high affinity and high capacity glutamate transporter such as GLAST and GLT-1 either to protect themselves from glutamate toxicity until the arrival of appropriate glial extensions, equipped with a high density of transporters. These transporters may also serve to provide glutamate into neurons for the energy metabolism and the protein synthesis, which are essential at this period of growth and differentiation. These roles may also hold true during early in vivo development (Yamada et al., 1998). Glial cells may, in turn, repress the neuronal GLAST and GLT-1 expression, when they begin to surround neurons either by diffusible factor or cell–cell contacts and are able to assume the neuronal protection (Amin and Pearce, 1997) and to constantly provide neurons with energy suppliers (Hertz et al., 1999). Another possibility, not necessarily excluding the preceding ones, is that GLT-1 and/or GLAST may also transiently serve as a glutamate detector in growing axons. Indeed, it was shown that a glutamate gradient delivered by microiontophoresis in cell culture produced a chemotropic turning of nerve growth cone (Zheng et al., 1996) suggesting the presence of glutamate recognition sites (NMDA receptors in this case) in the filopodia of nerve growth cones. In summary, these data indicated the transient presence of a high affinity glutamate transport activity on hippocampal neuronal culture. This activity could be mainly due to GLAST expression in neurons at a period when glial processes are not yet fully developed suggesting an important role of this transporter during the neuronal network formation. They also brought an additional evidence on the high degree of plasticity of hippocampal neurons.

Acknowledgements The authors wish to thank Dr K. Shimamoto (Japan) for the gift of DL-threo-b-benzyloxyaspartate. We also thank Dr N.C. Danbolt for the gift of glutamate transporter antibodies, and for his fruitful comments. We

would like also to thank Dr M. Jallageas for determining by immunocytochemistry the number of neuronal and glial cells in the culture. References Amin, N., Pearce, B., 1997. Glutamate toxicity in neuron-enriched and neuron-astrocyte co-cultures: effect of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate. Neurochem. Int. 30, 271 – 276. Arriza, J.L., Fairman, W.A., Wadiche, J.I., Murdoch, G.H., Kavanaugh, M.P., Amara, S.G., 1994. Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J. Neurosci. 14, 5559 – 5569. Arriza, J.L., Eliasof, S., Kavanaugh, M.P., Amara, S.G., 1997. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. Acad. Sci. USA 94, 4155 – 4160. Balcar, V., Johnston, G.A.R., 1972. The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J. Neurochem. 19, 2657 – 2666. Blanc, E., Vignes, M.H., Re´casens, M., 1995. Excitatory amino acido, except 1S,3R-ACPD induced transient high stimulation of phosphoinositide metabolism during hippocampal neuron development. Int. J. Dev. Neurosci. 13, 723 – 737. Blanc, E., Jallageas, M., Re´casens, M., Guiramand, J., 1999. Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation. Eur. J. Neurosci. 11, 3377 – 3386. Bliss, T.V.P., Collingridge, G.L., 1993. A synaptic model of memory: long term potentiation in the hippocampus. Nature 361, 31–39. Bridges, R.J., Kavanaugh, M.P., Chamberlin, A.R., 1999. A pharmacological review of competitive inhibitors and substrates of highaffinity, sodium-dependent glutamate transport in the central nervous system. Curr. Pharmac. Design 5, 363 – 379. Brooks-Kayal, A.R., Munir, M., Jin, H., Robinson, M.B., 1998. The glutamate transporter, GLT-1, is expressed in cultured hippocampal neurons. Neurochem. Int. 33, 95 – 100. Choi, D.W., Maulucci-Gedde, M., Kriegstein, A.R., 1987. Glutamate neurotoxicity in cortical cell culture. J. Neurosci. 7, 357–368. Coco, S., Verderio, C., Trotti, D., Rothstein, J.D., Volterra, A., Matteoli, M., 1997. Non-synaptic localization of the glutamate transporter EAAC-1 in cultured hippocampal neurons. Eur. J. Neurosci. 9, 1902 – 1910. Cohen-Cory, S., Dreyfus, C.F., Black, I.B., 1991. NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured Purkinje cells. J. Neurosci. 11, 462 – 471. Danbolt, N.C., Chaudhry, F.A., Dehnes, Y., Lehre, K.P., Levy, L.M., Ullensvang, K., et al., 1998a. Properties and localization of glutamate transporters. Prog. Brain Res. 116, 23 – 43. Danbolt, N.C., Lehre, K.P., Dehnes, Y., Chaudhry, F.A., Levy, L.M., 1998b. Localization of transporters using transporter-specific antibodies. Meth. Enzymol. 296, 388 – 407. DeLean, A., Munson, P.J., Rodbach, D., 1978. Simultaneous analysis of families of sigmoidal curves. Am. J. Physiol. 235, E97–E102. Dowd, L.A., Coyle, A.J., Rothstein, J.D., Pritchett, D.B., Robinson, M.B., 1996. Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC-1). Mol. Pharmacol. 49, 465 – 473. Fairman, W.A., Vandenberg, R.J., Arriza, J.L., Kavanaugh, M.P., Amara, S.G., 1995. An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375, 599– 603.

S. Gaillet et al. / Neurochemistry International 38 (2001) 293–301 Fonnum, F., 1984. Glutamate: a neurotransmitter in mammalian brain. J. Neurochem. 42, 1–11. Furuta, A., Rothstein, J.D., Martin, L.J., 1997. Glutamate transporter protein subtypes are expressed differentially during rat CNS development. J. Neurosci. 17, 8363–8375. Harada, T., Harada, C., Watanabe, M., Inoue, Y., Sakagawa, T., Nakayama, N., et al., 1998. Functions of the two glutamate transporters GLAST and GLT-1 in the retina. Proc. Natl. Acad. Sci. USA 95, 4663 – 4666. Haugeto, Ø., Ullensvang, K., Levy, L.M., Chaudhry, F.A., Honore´, T., Nielsen, M., Lehre, K.P., Danbolt, N.C., 1996. Brain glutamate transporter proteins from homomultimers. J. Biol. Chem. 271, 27715 – 27722. Hertz, L., Dringen, R., Schousboe, A., Robinson, S.R., 1999. Astrocytes: glutamate producers for neurons. J. Neurosci. Res. 57, 417 – 428. Kanai, Y., Hediger, M.A., 1992. Primary structure and functional characterization of a high affinity glutamate transporter. Nature 360, 467 – 471. Klo¨ckner, U., Storck, T., Conradt, M., Stoffel, W., 1994. Functional properties and substrate specificity of the cloned L-glutamate/L-aspartate transporter GLAST-1 from rat brain expressed in Xenopus oocytes. J. Neurosci. 14, 5759–5765. Laemmli, U.K., Quittner, S.F., 1974. Maturation of the head of bacteriophage T4. IV. The proteins of the core of the tubular polyheads and in vitro cleavage of the head proteins. Virology 62, 483 – 499. Lehre, K.P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J., Danbolt, N.C., 1995. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J. Neurosci. 15, 1835–1853. Meaney, J.A., Balcar, V.J., Rosthstein, J.D., Jeffrey, P.L., 1998. Glutamate transport in cultures from developing avian cerebellum: presence of GLT-1 immunoreactivity in Purkinje neurons. J. Neurosci. Res. 54, 595–603. Mennerick, S., Dhond, R.P., Benz, A., Xu, W., Rothstein, J.D., Danbolt, N.C., et al., 1998. Neuronal expression of the glutamate transporter GLT-1 in hippocampal microcultures. J. Neurosci. 18, 4490 – 4499. Mount, H.T.J., Dreyfus, C.F., Black, I.B., 1993. Purkinje cell survival is differentially regulated by metabotropic and ionotropic excitatory amino acid receptors. J. Neurosci. 133, 3173–3179. Munson, P.J., Rodbach, D., 1980. Ligand: a versatile computerized approach for characterization of ligand binding system. Anal. Biochem. 107, 220 – 239. Pines, G., Danbolt, N.C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., et al., 1992. Cloning and expression of a rat brain L-glutamate transporter. Nature 360, 464–467. Plachez, C., Danbolt, N.C., Re´casens, M., 238. Transient expression of the glial glutamate transporters GLAST and GLT on hippocampal neurons in primary culture. J. Neurosci. Res. 59, 587 – 593. Rauen, T., Rothstein, J.D., Wassle, H., 1996. Differential expression of three glutamate transporter subtypes in rat retina. Cell Tissue Res. 286, 325 – 336.

.

301

Robinson, M.B., Dowd, L.A., 1997. Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv. Pharmacol. 37, 69 – 115. Rothstein, J.D., Martin, L., Levey, A.I., Dykes-Hoberg, M., Jin, L., Wu, D., et al., 1994. Localization of neuronal and glial glutamate transporters. Neuron 13, 713 – 725. Rothstein, J.D., Dykes-Hoberg, M., Pardo, C.A., Bristol, L.A., Jin, L., Kuncl, R.W., et al., 1996. Knockout of glutamate transporter reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675 – 686. Schmitt, A., Asan, E., Puschel, B., Jons, T., Kugler, P., 1996. Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry. Neuroscience 71, 989 – 1004. Schousboe, A., 1981. Transport and metabolism of glutamate and GABA in neurons and glial cells. Int. Rev. Neurobiol. 22, 1–45. Spacek, J., 1985. Three dimensional analysis of dendritic spines. III. Glial sheath. Anat. Embryol. 171, 245 – 252. Stanimirovic, D.B., Ball, R., Small, D.L., Muruganandam, A., 1999. Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocytes cultures differentiated in vitro. Int. J. Dev. Neurosci. 17, 173 – 184. Storck, T., Schulte, S., Hofmann, K., Stoffel, W., 1992. Structure, expression, and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain. Proc. Natl. Acad. Sci. USA 89, 10955 – 10959. Swanson, R.A., Liu, J., Miller, J.W., Rothstein, J.D., Farell, K., Stein, B.A., Longuemare, M.C., 1997. Neuronal regulation of glutamate transport subtype expression in astrocytes. J. Neurosci. 17, 932 – 940. Tanaka, K., 1993. Cloning and expression of a glutamate transporter from mouse brain. Neurosci. Lett. 159, 183 – 186. Tanaka, K., Watase, K., Manabe, T., Yamada, K., Watanabe, M., Takahashi, K., et al., 1997. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699 – 1702. Torp, R., Hoover, F., Danbolt, N.C., Storm-Mathisen, J., Ottersen, O.P., 1997. Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis. Anat. Embryol. (Berlin) 195, 317–326. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Wang, G.J., Chung, H.J., Schnuer, J., Pratt, K., Zable, A.C., Kavanaugh, M.P., Rosenberg, P.A., 1998. High affinity glutamate transport in rat cortical neurons in culture. Mol. Pharmacol. 53, 88 – 96. Yamada, K., Watanabe, M., Shibata, T., Nagashima, M., Tanaka, K., Inoue, Y., 1998. Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J. Neurosci. 18, 5706 –5713. Zheng, J.Q., Wan, J.J., Poo, M.M., 1996. Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient. J. Neurosci. 16, 1140 – 1149.