Topiramate protects against glutamate- and kainate-induced neurotoxicity in primary neuronal–astroglial cultures

Epilepsy Research 54 (2003) 63–71

Topiramate protects against glutamate- and kainate-induced neurotoxicity in primary neuronal–astroglial cultures Mikael Ängehagen∗ , Elinor Ben-Menachem, Lars Rönnbäck, Elisabeth Hansson Institute of Clinical Neuroscience, Göteborg University, P.O. Box 420, SE-405 30 Göteborg, Sweden Received 24 January 2003; received in revised form 28 February 2003; accepted 2 March 2003

Abstract Potential neuroprotective effects of the antiepileptic drug (AED) topiramate (TPM) were evaluated using primary neuronal– astroglial cultures or astroglial-enriched cultures from newborn rats exposed to excitotoxic concentrations of glutamate (Glu) or kainate. Neurons expressed functional Glu receptors of the NMDA and AMPA/kainate types as evaluated by immunocytochemistry and Ca2+ imaging. When Glu (10 mM) was added to 9–10-day cultures incubated with the fluorescent dye calcein/AM for 5 h, there was a marked cell loss in both culture types, but was more pronounced in the neuronal–astroglial cultures. When TPM (5–10 ␮M) was included in the medium together with Glu, the amount of surviving cells was significantly higher in the neuronal–astroglial cultures, but not in the astroglial-enriched cultures. Immuno-labeling of the cultures revealed an enhanced survival of MAP positive neuronal cells when TPM was included in the Glu containing medium. As TPM has a proven negative modulatory effect on kainate activated receptors, neuronal–astroglial cultures were further exposed to excitotoxic concentrations of kainate (100 ␮M) and analyzed immunohistochemically. Significantly more MAP positive neurons survived in the TPM containing medium and showed a morphology similar to untreated cells. Valproate and phenytoin were used as reference AEDs. In conclusion, our results demonstrate a protective effect of TPM upon neuronal cells in primary culture, exposed to excitotoxic levels of Glu or kainate. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Topiramate; Neuron; Glutamate; Kainate; Viability; Primary cultures

1. Introduction Topiramate [2,3:4,5-bis-O-(1-methylethylidene)␤d-fructo-pyranose sulfamate] (TPM) is an antiepileptic drug (AED) which has been available since 1994 and is now used worldwide. It is an interesting compound because of its multiple mechanisms of ac∗ Corresponding author. Tel.: +46-317733375; fax: +46-317733330. E-mail address: [email protected] (M. Ängehagen).

tion and its established broad-spectrum antiepileptic properties (Shank et al., 1994; Wauquier and Zhou, 1996; White et al., 1997). Among the pharmacological properties of TPM that probably contribute to its anticonvulsant activity are: suppression (negative modulation) of voltage-gated Na+ channels (DeLorenzo et al., 2000; McLean et al., 2000; Zona et al., 1997) and L-type high voltage-activated Ca2+ channels (Zhang et al., 2000), suppression of the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate subtypes of glutamate (Glu) receptors (Gibbs et al., 2000; Skradski and White,

0920-1211/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-1211(03)00039-1

64

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

2000), and enhancement of some types of GABAA receptor currents (White et al., 1997, 2000) and K+ channel currents (Herrero et al., 2002). TPM also inhibits some isozymes of carbonic anhydrase, particularly CA-II and CA-IV (Dodgson et al., 2000). These actions are all elicited by TPM in doses considered to be within the therapeutic range for the treatment of epilepsy (Shank et al., 2000). Interestingly TPM seems to have neuroprotective capabilities at least in animal models of status epilepticus and stroke-induced neurodegeneration (Edmonds et al., 2001; Niebauer and Gruenthal, 1999; Yang et al., 2000). A hypothesis is that this effect could be due in part to the suppressive activity of TPM on AMPA and kainate subtypes of Glu receptors. In the present study, the neuroprotective effects of TPM were evaluated using neuronal–astroglial, or astroglial-enriched primary cultures derived from the cerebral cortex of newborn rats. Cellular degeneration was induced by Glu or kainate to simulate excitotoxicity in vivo. Valproate and phenytoin were used as reference AEDs.

with freshly prepared poly-l-lysine (Sigma) 1.0 mg in 100 ml, 0.1 M boric acid–NaOH buffer at pH 8.4 (Pettmann et al., 1979). The medium was changed after 24 h of cultivation and thereafter three times a week. On day 6, the cells were treated with 5 ␮M cytosine-1-␤-d-arabinofuranoside (C-Ara; Sigma) to suppress the growth of dividing glial cells and to promote neuronal survival. The cultures were used on days 9–10 of cultivation (Blomstrand et al., 1999). 2.2. Astroglial cell cultures To obtain primary astroglial cell cultures, the cells were treated similar to the neuronal–astroglial cultures except that the concentration of glucose was changed to 7.5 mM, no insulin and cytosine-1-␤-darabinofuranoside were used and the glass cover-slips were not pre-coated. The medium was changed after 3 days of cultivation and thereafter three times a week. Cultivation was continued for 15–17 days in a humidified atmosphere of 5% CO2 in air at 37 ◦ C and pH 7.3 (Hansson et al., 1984). 2.3. Cell viability estimation

2. Materials and methods 2.1. Neuronal–astroglial cell cultures Primary neuronal–astroglial cortical cell cultures were obtained from newborn Sprague–Dawley rats (Charles River, Uppsala, Sweden) as previously described (Nilsson et al., 1991). Briefly, the animals were decapitated, the skulls opened and the brain removed. The cortex was dissected and mechanically passed through an 80 ␮m mesh nylon net into Eagle’s minimum essential medium (MEM; Life Technologies, Paisly, UK). This MEM was then supplemented up to the following final composition: double concentrations of amino acids (Life Technologies), quadruple concentrations of vitamins (Life Technologies), 2 mM l-glutamine (Biological Industries, Israel), 25 mM glucose (Sigma, St. Louis, MO), double concentrations of NaHCO3 (Merck, Darmstadt, Germany), 1% penicillin–streptomycin (PEST, Biological Industries), 20% (v/v) fetal calf serum (Harlan Sera-Lab, Sussex, UK), 5 ␮g/ml insulin (Sigma); pH 7.3. The cells were grown on pre-coated glass cover-slips, placed in Petri dishes (NUNC A/S, Roskilde, Denmark)

Neuronal–astroglial and astroglial-enriched cell cultures grown in non-fluorescent 96-well plates were used for viability measurements (Bozyczko-Coyne et al., 1993). For experiments on Glu-induced toxicity, the cultures were rinsed three times in a Hepes-buffered Hank’s balanced salt solution (HHBSS), pH 7.4, which contained 137 mM NaCl, 5.4 mM KCl, 0.41 mM MgSO4 , 0.49 mM MgCl2 , 1.26 mM CaCl2 , 0.64 mM KH2 PO4 , 3 mM NaCO3 , 5.5 mM glucose and 20 mM Hepes (Glaum et al., 1990). The cultures were treated with TPM (5 or 10 ␮M) or vehicle alone or together with 10 mM Glu (Okamoto et al., 1994; Planells-Cases et al., 2002) (n = 6–10) for 5 h. TPM was added 10 min before Glu. In some experiments phenytoin (50 or 100 ␮M) or valproate (0.1, 0.55 or 1.0 mM) were substituted for TPM as reference AEDs. Cultures were loaded with 1 ␮M calcein/AM (Molecular Probes, Leiden, The Netherlands) for 45 min at 37 ◦ C. Calcein/AM enters the cells through the membrane as a non-fluorescent ester and is cleaved by cellular esterases to a fluorescent product. Therefore, fluorescence occurs only in cells with functional cytoplasmic esterase activity.

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

After 45 min, the cells were rinsed three times with HHBSS. Fluorescence was measured with a Fluoroscan ascent (Labsystems Oy, Helsinki, Finland). The excitation light was set at 485 nm and the emission light at 530 nm. Surviving cells were defined as the fluorescence from drug-treated cells divided by the fluorescence from untreated cells (corrected for background fluorescence) × 100. Cultures were then treated with TPM (5 or 10 ␮M), vehicle alone or together with 100 ␮M kainate for 5 h. To estimate the number of surviving neurons the cultures were stained with antibodies and analyzed by immunocytochemistry. Eight fields on every cover-slip were chosen randomly, and the number of neurons were counted (n = 3 cover-slips). 2.4. Protein concentration estimation Protein concentrations were determined using a Pierce BCA Protein Assay (Pierce, Rockford, USA), which relies on the color change of a dye, bicinchoninic acid (Smith et al., 1985). Standards were prepared over the range 0–10 mg/ml bovine serum albumin. Samples were mixed with the BCA working reagent, incubated at 37 ◦ C for 30 min, read at 562 nm in a spectrophotometer (Molecular Devices, Sunnyvale, USA) and analyzed using a Macintosh LC 475.

65

GmbH) and MAP-2 (rabbit, 1:500) (Penisula Laboratories, Merseyside, UK) in order to immunostain astrocytes and neurons, respectively. The secondary antibodies used in the next step were Texas red (anti-mouse, 1:50) (Sigma), fluorescein isothiocyanate (anti-rabbit, 1:50) (Sigma) and Hoechst 33258 (Sigma). 3.2. Immunolabeling of membrane receptors Double immunolabeling with two primary antibodies (mouse), one IgM and one IgG was performed according to Berntson et al. (1998). After the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% saponin in PBS–BSA, the cells were incubated with primary IgG GluR5/6/7 antibody (5 ␮M/ml; BD Pharmingen, San Diego, USA) and then with the secondary Texas red-conjugated anti-mouse IgG antibody (diluted 1:100). Thorough rinsing was done before incubation with the primary IgM MAP-2 antibody and then with the secondary fluorescein isothiocyanate-conjugated anti-mouse IgM antibody (1:100). After the staining procedure, the cover-slips were mounted on microscope slides using fluorescent mounting medium (DAKO CA, USA) and viewed in a Nikon optiphot-2. The immuno-pictures were captured using a Hamamatsu C5810 color intensified 3CCD camera.

3. Immunocytochemistry

3.3. Ca2+ imaging

3.1. Identification of neuronal and astroglial cells

Intracellular Ca2+ transients (referred to as [Ca2+ ]i transients) in neuronal cells after stimulation with Glu, NMDA, or kainate were monitored using the Ca2+ -sensitive fluorophore probe fura-2 (8 ␮M for 30–45 min at 37 ◦ C) (Molecular Probes, Leiden, The Netherlands) according to a previously described procedure (Muyderman et al., 1998). The experiments were performed in room temperature (20–22 ◦ C) using calcium imaging system from Photon Technology System (PTI) with a 40× (0.85 N.A.) fluorescence dry lens. Calcium fluxes from neurons were video sampled. All images were sampled into a 96 MB random access memory on a 100 MHz Pentium computer and later stored on CD-ROM for analyses. Data are presented as the 340/380 nm ratio of the fluorescence intensities. Images were collected in time intervals of 240–420 s.

Immunostaining of the primary cultures was performed according to a previously described procedure (Nilsson et al., 1993). Briefly, immunostainings for glial fibrillary acidic protein (GFAP), microtubuleassociated protein 2 (MAP-2) and Hoechst 33258 to visualize cell nuclei were performed by incubating the cultures with antibodies diluted in Eagle’s MEM containing 20 mM HHBSS and 1% bovine serum albumin (EMEM-H-BSA). Rinsing was performed between every incubation step. The cells were first fixed with 4% paraformaldehyde at 4 ◦ C for 10 min and then permeabilized with 0.05% saponin in EMEM-H-BSA. The cultures were simultaneously incubated with the primary antibodies against GFAP (mouse, 1:20) (Boehringer Mannheim

66

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

3.4. Statistics All data in this study are expressed as mean±S.E.M. The level of significance between groups was calculated using ANOVA. The differences were considered significant at a P value of <0.05. 3.5. Chemicals Kainate was obtained from TOCRIS (Ballwin, MO, USA), topiramate was provided by Johnson & Johnson Pharmaceutical Research and Development, LLC (Raritan, NJ, USA), valproate, phenytoin, Glu, and NMDA and all other chemicals of analytic grade were purchased from Sigma.

4. Results 4.1. Cell viability The amount of surviving cells in neuronal–astroglial cultures and astroglial-enriched cell cultures exposed to 10 mM Glu for 5 h was 46 and 58%, respectively (Fig. 1). When TPM was included in the medium at 5 or 10 ␮M, the amount of surviving cells in the neuronal–astroglial cultures was 66 and 80%, respectively, which was statistically significant compared to the 46% found for incubation in Glu alone (P < 0.05) (Fig. 1). By comparison, the amount of surviving cells in astroglial cell cultures was not significantly improved by TPM at either concentration (68 and 67%, respectively) (Fig. 1). TPM at 5 or 10 ␮M did not affect the amount of surviving cells under control conditions (Fig. 1). Phenytoin (50 or 100 ␮M) or valproate (0.1, 0.55, or 1.0 mM) did not significantly affect cell survival for neuronal–astroglial or astroglial-enriched cell cultures exposed to 10 mM Glu for 5 h. There were no significant differences in protein content among the different wells that could effect the results.

Fig. 1. Cell viability in neuronal–astroglial (black) and astroglialenriched (grey) primary cultures after the cultures had been incubated for 5 h in 10 mM glutamate, 10 mM glutamate + 5 or 10 ␮M TPM, or 5 or 10 ␮M TPM only. In cell cultures exposed to glutamate, the amount of surviving cells was lower for both types of cultures, but more prominently for the neuronal–astroglial mixed cultures than in the astroglial-enriched cultures. After incubation with both glutamate and TPM present, the amount of surviving cells was significantly higher in the mixed neuronal–astroglial, but not in the astroglial-enriched cultures compared to the amount of surviving cells after incubation in glutamate alone. TPM alone did not affect cell survival of either type of cell culture. Values are means ± S.E.M. from 6 to 10 experiments ( P < 0.05). Each condition was tested on a minimum of three different cultures and each condition contained its own control group.

large array of dividing processes. These different cell types were MAP positive with immunohistochemical staining and were considered neurons. 4.2.2. Morphology of cells classified as astroglial cells Polygonal or spindle-shaped cells with a cell soma of approximately 16 ␮m, containing a central nucleus (approximately 6 ␮m in diameter) with two or more nucleoli, and with processes, were GFAP positive and were considered astroglial cells. 4.3. Immunocytochemistry

4.2. Morphology of the respective cultures 4.2.1. Morphology of cells classified as neurons The cells consisted of a cell soma with a diameter mostly of 6–10 ␮m (small neurons) to occasionally occurring cells with a soma of up to 20 ␮m. The cells extended processes, from two (bipolar cells) up to a

MAP positive cells in untreated 9–10-day-old neuronal–astroglial cultures had small somata with slender processes and were morphologically easy to distinguish from astroglial cells which had star-like shapes. Neither MAP positive cells nor GFAP positive cells, incubated for up to 5 h in 5 or 10 ␮M TPM

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

showed any altered morphology or cell death. The neuronal–astroglial cultures incubated with 10 mM Glu showed a MAP positive cell loss of 65%, and the remaining cells showed pyknotic nuclei and a loss of dentrites. The GFAP positive cells network were partly broken and the cell somata were swollen. In addition, the GFAP positive cell population was approximately 25% less than in control cultures, indicating a significant Glu-induced cell death and disintegration in these cultures. Immunostaining showed that for neuronal–astroglial cultures exposed to 10 mM Glu for 5 h, there were more surviving MAP positive cells when 5 or 10 ␮M TPM was also present in the medium, 69 or 75% (Figs. 2 and 3). MAP positive cells incubated in the Glu + TPM containing media had more dendrites than those incubated in only Glu. Immunostaining revealed that a smaller percentage of GFAP positive cells (Fig. 2) were killed by 10 mM Glu than MAP-2 positive cells (Figs. 2 and 3). The

67

immunostaining results indicated that neither valproate nor phenytoin, either alone or when combined with Glu, had a significant effect on the morphology of MAP positive cells or GFAP positive cells, or the number of dead cells. When neuronal–astroglial cultures were treated with 100 ␮M kainate for 5 h, only 24% of the MAP positive cells survived (Figs. 3 and 4). The surviving cells showed a loss of dendrites. When 5 ␮M TPM was present in the medium, 61% of the MAP positive cells survived, and when 10 ␮M TPM was present, 68% of the neurons survived (Figs. 3 and 4). MAP positive cells incubated in the TPM containing medium showed an identical morphology as that of untreated cells (Fig. 4). 4.4. Kainate receptor expression Immunofluorecent staining of the neuronal–astroglial cultures showed that most cells exhibited

Fig. 2. Neuronal–astroglial primary cultures immunocytochemically stained for the neuron-specific marker microtubule-associated protein (MAP-2; green), and the astrocyte-specific marker glial fibrillary acidic protein (GFAP; red). Scale bar = 50 ␮M. (A) In control cultures, neurons had small somata, approximately 15 ␮m in diameter, with slender processes and were morphologically easy to distinguish from astroglial cells which appeared more star-like. (B) After cultures had been incubated for 5 h in 10 mM glutamate, at least 60% of the neurons and 25% of the astroglial cells had died and the remaining neurons had fewer processes. The astroglial networks were morphologically partly broken and the somata of remaining cells were swollen. (C) When incubated in 10 mM glutamate and 5 ␮M TPM or (D) in 10 mM glutamate and 10 ␮M TPM, up to 75% of the neurons survived and had more processes compared to cultures incubated in 10 mM glutamate with no TPM.

68

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

Fig. 3. Cell viability measured from cultures immunocytochemically stained for the neuron-specific marker microtubule-associated protein (MAP-2) and the astrocyte-specific marker glial fibrillary acidic protein (GFAP). Significantly more neurons survived when TPM was added to the medium in combination with Glu (black) or kainate (grey). Neurons from eighth fields from three cover-slips were chosen randomly, and the number of neurons were counted. Values are presented as means ± S.E.M. with n = 2113 neurons ( P < 0.05; P < 0.01). Each condition was tested on three different cultures and each condition contained its own control group.

neuronal morphology and MAP-2 positive, expressed immunoreactivity for the GluR5/6/7 marker (Fig. 5A and B). 4.5. [Ca2+ ]i transients When neurons were exposed to Glu (1 mM), NMDA (100 ␮M) or kainate (100 ␮M), increases in [Ca2+ ]i were observed demonstrating that the cells express functional Glu receptors (Fig. 5C and D).

5. Discussion The results of this study provide the first reported evidence that TPM possesses neuroprotective properties in an in vitro system designed to reflect in vivo pathological conditions with Glu excitotoxicity. The conclusion that TPM protected against Glu-induced neuronal death was substantiated by the immunostaining experiments that revealed more MAP-2 positive cells in neuronal–astroglial cultures incubated with both Glu and TPM compared to those incubated

with Glu alone. It has earlier been reported that TPM has neuroprotective effects in animal models of status epilepticus and stroke-induced neurodegeneration (Edmonds et al., 2001; Niebauer and Gruenthal, 1999; Yang et al., 2000) although Cha et al. (2002) has recently presented results of a lack of increased neuronal survival by TPM in an in vivo epilepsy model. Working with in vitro model systems demand higher Glu concentrations, and therefore we used 10 mM Glu to induce excitotoxicity in our model system. A concentration this high is rarely found in pathological conditions in the brain. A hypothetical explanation for this could be that the serum used for culturing cells supply Glu at concentrations sufficient to kill subpopulations of primary cultured neurons (Ye and Sontheimer, 1998). Under these conditions, a natural selection occurs where only those neurons that are most capable to withstand high Glu concentration may ultimately survive. The neuroprotective effect of TPM observed in this study could be related to its inhibitory effect on kainate activated receptors and probably reflects an ability of TPM to reduce intracellular Ca2+ accumulation caused by Glu (Choi, 1990; Choi and Rothman, 1990; Greenamyre and Porter, 1994; Ängehagen et al., 2003). Glu-induced [Ca2+ ]i transients can be mediated via several pathways. These include an influx through Glu-gated ion channels of the NMDA receptor subtype and AMPA/kainate receptor subtypes that do not possess the GluR2 subunit. Also, secondarily, voltage-gated Ca2+ channels can be activated as a result of membrane depolarization caused by Na+ influx. Similarly, Ca2+ may be released from intracellular stores. Although astroglial cells express functional Glu receptors, they are generally more resistant to excitotoxic insults than neuronal cells as reported by Prieto and Alonso (1999), and confirmed in our results. The mechanism of action of TPM includes several pharmacological properties that can contribute to its neuroprotective effects (Brorson et al., 1994; Kim et al., 2000; Leski et al., 1999; Obrenovitch and Urenjak, 1998; Olney, 1971; Pizzi et al., 2000; Pollard et al., 1994; Rothman and Olney, 1995; Zorumski and Olney, 1993). These include a modulatory effect on GABAA receptors (White et al., 1997, 2000), a negative modulatory effect on L-type high voltage-activated Ca2+ channels (Zhang et al., 2000), voltage-activated Na+ channels (DeLorenzo et al., 2000; McLean et al., 2000; Zona et al., 1997),

Fig. 4. Neuronal–astroglial primary cultures immunocytochemically stained for the neuron-specific marker microtubule-associated protein (MAP-2; green) and the astrocyte-specific marker glial fibrillary acidic protein (GFAP; red). Scale bar = 50 ␮M. (A) In control cultures, neurons had small somata, approximately 15 ␮m in diameter, with slender processes and were morphologically easy to distinguish from astroglial cells, which appeared more star-like. (B) After cultures had been incubated for 5 h in 100 ␮M kainate, at least 70% of the neurons had died and the remaining neurons had fewer processes. The astroglial somata were swollen. (C) When incubated in 100 ␮M kainate and 5 ␮M TPM or (D) in 100 ␮M kainate and 10 ␮M TPM, up to 70% of the neurons survived and had more processes compared to cultures incubated in 100 ␮M kainate with no TPM.

Fig. 5. (A–E) Immunocytochemical double labeling of neurons with antibodies against the kainate receptor GluR5/6/7 (red) and the neuron-specific marker microtubule-associated protein (MAP-2; green). Approximately 90% of the total neuronal cell number stained both with antibodies against GluR5/6/7 (A) and against MAP-2 (B). Scale bar = 50 ␮M. Intracellular calcium transients in neurons after exposure to Glu (1 mM) (C), NMDA (100 ␮M) (D), and kainate (100 ␮M) (E). Drug application is indicated by upward arrows.

70

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71

K+ channel currents (Herrero et al., 2002) and the AMPA/kainate subtypes of Glu receptors (Gibbs et al., 2000; Skradski and White, 2000). It has not been established at the molecular level how TPM modulates the activity of AMPA/kainate receptors or any of the other receptor/channel systems noted above. Observations by Gibbs et al. (2000) that okadaic acid inhibits the effect of TPM on AMPA receptor currents and that dibutyryl-cAMP promotes the restoration of AMPA receptor currents after wash-out of TPM, provided the basis for a working hypothesis in which TPM is postulated to bind to phosphorylation sites on the proteins it modulates (Shank et al., 2000). The ability of TPM to suppress voltage-activated Na+ channel currents and high voltage-activated L-type Ca2+ channel currents, and enhance some GABAA receptor-mediated Cl− currents, and K+ currents may also contribute to the neuroprotective effect of TPM found in our study. From the present study it seemed that the TPM protection of the Glu-induced excitotoxity was mainly due to the interference with kainate receptors. In summary, the present study demonstrates that TPM protects neuronal cells against Glu- and kainate-induced excitotoxicity in mixed cortical cultures and supports the potential of TPM as a neuroprotective agent.

Acknowledgements We are grateful to Richard Shank at RW Johnson Pharmaceutical Research Institute for his help and support, and the skillful technical assistance of Ulrika Björklund and Barbro Eriksson is greatly appreciated. The work was supported in part by the Swedish Research Council (Grant No. 21X-13015 and 21BI-14586), by Edith Jacobsson’s Foundation, John and Brit Wennerström’s Foundation for Neurological Research, Göteborg Foundation for Neurological Research and the RW Johnson Pharmaceutical Research Institute, Spring House, PA, USA. References Ängehagen, M., Ben-Menachem, E., Shank, R., Rönnbäck, L., Hansson, E., 2003. Topiramate modulation of kainate-induced calcium currents is inversely related to channel phosphorylation level, in preparation.

Berntson, Z., Hansson, E., Rönnbäck, L., Fredman, P., 1998. Intracellular sulfatide expression in a subpopulation of astrocytes in primary cultures. J. Neurosci. Res. 52, 559–568. Blomstrand, F., Khatibi, S., Muyderman, H., Hansson, E., Olsson, T., Rönnbäck, L., 1999. 5-Hydroxytryptamine and glutamate modulate velocity and extent of intercellular calcium signalling in hippocampal astroglial cells in primary cultures. Neuroscience 88, 1241–1253. Bozyczko-Coyne, D., McKenna, B.W., Connors, T.J., Neff, N.T., 1993. A rapid fluorometric assay to measure neuronal survival in vitro. J. Neurosci. Methods 50, 205–216. Brorson, J.R., Manzolillo, P.A., Miller, R.J., 1994. Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells. J. Neurosci. 14, 187–197. Cha, B.H., Silveira, D.C., Liu, X., Hu, Y., Holmes, G.L., 2002. Effect of topiramate following recurrent and prolonged seizures during early development. Epilepsy Res. 51, 217–232. Choi, D.W., 1990. Methods for antagonizing glutamate neurotoxicity. Cerebrovasc. Brain Metab. Rev. 2, 105–147. Choi, D.W., Rothman, S.M., 1990. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13, 171–182. DeLorenzo, R.J., Sombati, S., Coulter, D.A., 2000. Effects of topiramate on sustained repetitive firing and spontaneous recurrent seizure discharges in cultured hippocampal neurons. Epilepsia 41 (Suppl. 1), S40–S44. Dodgson, S.J., Shank, R.P., Maryanoff, B.E., 2000. Topiramate as an inhibitor of carbonic anhydrase isoenzymes. Epilepsia 41 (Suppl. 1), S35–S39. Edmonds Jr., H.L., Jiang, Y.D., Zhang, P.Y., Shank, R., 2001. Topiramate as a neuroprotectant in a rat model of global ischemia-induced neurodegeneration. Life Sci. 69, 2265– 2277. Gibbs III, J.W., Sombati, S., DeLorenzo, R.J., Coulter, D.A., 2000. Cellular actions of topiramate: blockade of kainate-evoked inward currents in cultured hippocampal neurons. Epilepsia 41 (Suppl. 1), S10–S16. Glaum, S.R., Holzwarth, J.A., Miller, R.J., 1990. Glutamate receptors activate Ca2+ mobilization and Ca2+ influx into astrocytes. Proc. Natl. Acad. Sci. U.S.A. 87, 3454–3458. Greenamyre, J.T., Porter, R.H., 1994. Anatomy and physiology of glutamate in the CNS. Neurology 44, S7–S13. Hansson, E., Rönnbäck, L., Persson, L.I., Lowenthal, A., Noppe, M., Alling, C., Karlsson, B., 1984. Cellular composition of primary cultures from cerebral cortex, striatum, hippocampus, brainstem and cerebellum. Brain Res. 300, 9–18. Herrero, A.I., Del Olmo, N., Gonzalez-Escalada, J.R., Jose, M.S., 2002. Two new actions of topiramate: inhibition of depolarizing GABA(A)-mediated responses and activation of a potassium conductance. Neuropharmacology 42, 210–220. Kim, A.H., Sheline, C.T., Tian, M., Higashi, T., McMahon, R.J., Cousins, R.J., Choi, D.W., 2000. L-type Ca2+ channel-mediated Zn2+ toxicity and modulation by ZnT-1 in PC12 cells. Brain Res. 886, 99–107. Leski, M.L., Valentine, S.L., Coyle, J.T., 1999. L-type voltagegated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells. Brain Res. 828, 27–40.

M. Ängehagen et al. / Epilepsy Research 54 (2003) 63–71 McLean, M.J., Bukhari, A.A., Wamil, A.W., 2000. Effects of topiramate on sodium-dependent action-potential firing by mouse spinal cord neurons in cell culture. Epilepsia 41 (Suppl. 1), S21–S24. Muyderman, H., Nilsson, M., Blomstrand, F., Khatibi, S., Olsson, T., Hansson, E., Rönnbäck, L., 1998. Modulation of mechanically-induced calcium waves in hippocampal astroglial cells. Inhibitory effects of alpha 1-adrenergic stimulation. Brain Res. 793, 127–135. Niebauer, M., Gruenthal, M., 1999. Topiramate reduces neuronal injury after experimental status epilepticus. Brain Res. 837, 263–269. Nilsson, M., Eriksson, P.S., Rönnbäck, L., Hansson, E., 1993. GABA induces Ca2+ transients in astrocytes. Neuroscience 54, 605–614. Nilsson, M., Hansson, E., Rönnbäck, L., 1991. Adrenergic and 5-HT2 receptors on the same astroglial cell. A microspectrofluorimetric study on cytosolic Ca2+ responses in single cells in primary culture. Brain Res. Dev. Brain Res. 63, 33–41. Obrenovitch, T.P., Urenjak, J., 1998. Glutamate release inhibitors: a critical assessment of their action mechanism. Amino Acids 14, 143–150. Okamoto, M., Mori, S., Endo, H., 1994. A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate. Brain Res. 637, 57–67. Olney, J.W., 1971. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus. An electron microscopic study. J. Neuropathol. Exp. Neurol. 30, 75–90. Pettmann, B., Louis, J.C., Sensenbrenner, M., 1979. Morphological and biochemical maturation of neurons cultured in the absence of glial cells. Nature 281, 378–380. Pizzi, M., Benarese, M., Boroni, F., Goffi, F., Valerio, A., Spano, P.F., 2000. Neuroprotection by metabotropic glutamate receptor agonists on kainate-induced degeneration of motor neurons in spinal cord slices from adult rat. Neuropharmacology 39, 903– 910. Planells-Cases, R., Montoliu, C., Humet, M., Fernandez, A.M., Garcia-Martinez, C., Valera, E., Merino, J.M., Perez-Paya, E., Messeguer, A., Felipo, V., Ferrer-Montiel, A., 2002. A novel N-methyl-d-aspartate receptor open channel blocker with in vivo neuroprotectant activity. J. Pharmacol. Exp. Ther. 302, 163–173. Pollard, H., Charriaut-Marlangue, C., Cantagrel, S., Represa, A., Robain, O., Moreau, J., Ben Ari, Y., 1994. Kainate-induced apoptotic cell death in hippocampal neurons. Neuroscience 63, 7–18.

71

Prieto, M., Alonso, G., 1999. Differential sensitivity of cultured tanycytes and astrocytes to hydrogen peroxide toxicity. Exp. Neurol. 155, 118–127. Rothman, S.M., Olney, J.W., 1995. Excitotoxicity and the NMDA receptor: still lethal after eight years. Trends Neurosci. 18, 57–58. Shank, R.P., Gardocki, J.F., Streeter, A.J., Maryanoff, B.E., 2000. An overview of the preclinical aspects of topiramate: pharmacology, pharmacokinetics, and mechanism of action. Epilepsia 41 (Suppl. 1), S3–S9. Shank, R.P., Gardocki, J.F., Vaught, J.L., Davis, C.B., Schupsky, J.J., Raffa, R.B., Dodgson, S.J., Nortey, S.O., Maryanoff, B.E., 1994. Topiramate: preclinical evaluation of structurally novel anticonvulsant. Epilepsia 35, 450–460. Skradski, S., White, H.S., 2000. Topiramate blocks kainate-evoked cobalt influx into cultured neurons. Epilepsia 41 (Suppl. 1), S45–S47. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. Wauquier, A., Zhou, S., 1996. Topiramate: a potent anticonvulsant in the amygdala-kindled rat. Epilepsy Res. 24, 73–77. White, H.S., Brown, S.D., Woodhead, J.H., Skeen, G.A., Wolf, H.H., 1997. Topiramate enhances GABA-mediated chloride flux and GABA-evoked chloride currents in murine brain neurons and increases seizure threshold. Epilepsy Res. 28, 167–179. White, H.S., Brown, S.D., Woodhead, J.H., Skeen, G.A., Wolf, H.H., 2000. Topiramate modulates GABA-evoked currents in murine cortical neurons by a nonbenzodiazepine mechanism. Epilepsia 41 (Suppl. 1), S17–S20. Yang, Y., Li, Q., Shuaib, A., 2000. Enhanced neuroprotection and reduced hemorrhagic incidence in focal cerebral ischemia of rat by low dose combination therapy of urokinase and topiramate. Neuropharmacology 39, 881–888. Ye, Z.C., Sontheimer, H., 1998. Astrocytes protect neurons from neurotoxic injury by serum glutamate. Glia 22, 237–248. Zhang, X., Velumian, A.A., Jones, O.T., Carlen, P.L., 2000. Modulation of high voltage-activated calcium channels in dentate granule cells by topiramate. Epilepsia 41 (Suppl. 1), S52–S60. Zona, C., Ciotti, M.T., Avoli, M., 1997. Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells. Neurosci. Lett. 231, 123–126. Zorumski, C.F., Olney, J.W., 1993. Excitotoxic neuronal damage and neuropsychiatric disorders. Pharmacol. Ther. 59, 145–162.