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A flow cytometric study ofN-methyl-d-aspartate effects on dissociated cerebellar cells

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A flow cytometric study ofN-methyl-d-aspartate effects on dissociated cerebellar cells
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  BR IN RESE RCH ELSEVIER Brain Research 723 (1996) 110-114 Research report A flow cytometric study of N-methyl-D-aspartate effects on dissociated cerebellar cells Francesc X. Sureda a, *, Antoni Camins a, Ramon Trullas b, Jordi Camarasa a, Elena Escubedo a a Laborato~ of Pharmacology and Pharmacognosy, Facul O of Pharmacy, Uni~,ersity of Barcelona, 08028 Barcelona, Spain b Neurobiology Unit, CID/CSIC, 08034 Barcelona, Spain Accepted 7 February 1996 Abstract The effects of N-methyl-D-aspartate (NMDA) on the generation of intracellular reactive oxygen species (ROS) and intracellular calcium in rat dissociated cerebellar cells were examined by flow cytometry. Flow cytometry allows the selection of a specific viable neuronal population with high sensitivity. We used 2',7'-dichlorofluorescin diacetate (DCFH-DA) as a marker of intracellular oxidative stress, and intracellular calcium was measured using Indo-1 as a calcium-sensitive indicator. The cerebellar cell population was isolated by size, granularity and NMDA-sensitivity by cell-sorting. In this cerebellar cell preparation, in which no glial cells were found, NMDA induced a concentration-dependent increase in ROS and intracellular calcium levels. These effects were inhibited by the non-competitive NMDA antagonist (+)MK-801. These results indicate that flow cytometry could be a useful tool to study the effect of neuroprotective drugs on NMDA receptor in isolated cerebellar neurons. Moreover, due to its high speed of analysis and the possibility to detect simultaneously a variety of fluorescent markers, we stated the utility of this technique in the pharmacology and physiology of the central nervous system. Keywords: NMDA; Flow cytometry; MK-801; Cerebellum; Granule cell; Oxidative stress; Calcium; Indo-I 1. Introduction Substantial evidence suggests that alterations in calcium homeostasis play a key role in the mechanism of neurotox- icity produced by excitatory amino acids [17]. Different mechanisms have been proposed to be involved in the cytotoxicity induced by intracellular calcium overload. This calcium overload can activate several key enzymes (e.g. proteases) such as calpain-1 and phospholipases and trig- ger some toxic processes responsible for neuronal degener- ation [5]. It is well known that N-methyl-D-aspartate (NMDA) produces neurotoxic effects by an excessive Ca 2+ influx through CaZ+-permeable NMDA receptor-gated channels [4]. In cerebellar granule cells, high levels of intracellular calcium induce oxidative stress, possibly through an acti- vation of phospholipase A 2 [14]. Furthermore, NMDA also induces free radical production in vivo [10]. * Corresponding author. Fax: (34) (3) 4021886. 0006-8993/96/ 15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0006- 8993(96)00230-2 The relationship between the neurotoxicity induced by excitatory amino acids and oxidative stress has been pro- posed by different authors [5,6,18,19]. On the other hand, it has been demonstrated that 21-aminosteroids, which are potent inhibitors of lipid peroxidation, protect neurons from NMDA-induced neurotoxicity [15] and hypoxia [8], The effects of excitatory amino acids on free radical production have been studied in different mice and rat brain preparations, including cell cultures of cerebellar granule cells [13] and synaptoneurosomes [2]. The aim of the present study was to investigate whether NMDA recep- tor activation results in production of ROS and whether this effect can be blocked by an NMDA receptor antago- nist. At the same time we intended to set up a suitable preparation of dissociated viable cerebellar neurons that would allow investigating NMDA receptor function 'in vitro' using two flow cytometry methods. Cell sorting was performed to assess the neuronal nature of the cell suspen- sion by electron microscopy. Cytosolic calcium increase was measured by using Indo-1, and the formation of reactive oxygen species (ROS) was detected by measuring the increase in fluorescence of 2',7'-dichlorofluorescein.  F.X. Sureda et al. / Brain Research 723 1996) 110-114 111 We now report that NMDA increases ROS production in dissociated cerebellar neurons, and that this effect can be blocked by (+)MK-801. In this work we propose that dissociated cerebellar neurons in combination with flow cytometry can be a useful tool to study the mechanisms of neuronal injury induced by excitatory amino acids. 2. Materials and methods 2.1. Materials Indo-1 acetoxymethyl ester, propidium iodide and NMDA were obtained from Sigma Chemical (St. Louis, MO, USA). 2',7'-Dichlorofluorescin diacetate (DCFH DA) was purchased from Serva (Heidelberg, Germany). Colla- genase A was from Boehringer Mannheim (Mannheim, Germany). (+)MK-801 was obtained from Research Bio- chemicals (Natick, MA, USA). All other chemicals were of analytical grade. 2.4. Measurement of cytoplasmatic Ca e + increase The cell suspension (1 × 10 6 cells/ml) was loaded with a final concentration of 1 /xM Indo-1 acetoxymethyl ester for 30 min at 37°C in a shaking water bath, under an atmosphere of 95% 02/5% CO 2. After loading, cells were kept at room temperature until calcium measurements were performed. Fluorescence measurement of live cell popula- tion in the cell suspension was performed on a Coulter Epics Flow Cytometer, equipped with an argon laser. The excitation wavelength was 364 nm and the emission was detected at both 525 and 395 nm using a ratiometric method. Fluorescence data were analyzed using the Elite software (Coulter, USA). Glycine (15 /xM) was added to the cell suspension 2 min before the cytometric analysis. Measurements of fluorescence were made before (basal values) and 5 min after (stimulated values) NMDA addi- tion, when the ratio reached a plateau. When used, differ- ent concentrations of (+)MK-801 were added simultane- ously with glycine. 2.2. Isolation of dissociated brain cells 2.5. Measurement of free radical production Experiments were performed with cerebellar cells disso- ciated from 9- to 13-day-old Sprague-Dawley rats (C.E.R.J., Le Grnest, France). Briefly, following animal decapitation, cerebella were rapidly dissected and cut lon- gitudinally (thickness: 400 /xm) in a McIlwain tissue chopper (The Mickle Laboratory Engineering, Gomshall, Surrey, UK). Slices from three cerebella (wet weight: = 0.3 g) were incubated in 30 volumes of previously gassed (95% 02/5% CO 2) Tyrode-Hepes MgZ+-free buffer (pH = 7.4) with collagenase A (0.33 mg/ml) at 37°C for 30 min, under vigorous shaking. Thereafter, the slices were gently aspirated by fire-polished Pasteur pipettes with tip diameter of 0.5 mm and 0.3 mm, 10 and 5 fold respec- tively. Afterwards, the cell suspension was filtered by gravity through a nylon mesh screen of 63 /zm (Maissa, Barcelona, Spain). After centrifugation (3 min, 300 × g), the pellet was resuspended in fresh buffer. 2.3. Cell sorting Propidium iodide was added to the cell suspension (final concentration: 10/xg/ml) in order to discard non-vi- able cells. This fluorescent dye is taken up only by dam- aged cells. Thus, all the propidium iodide-positive cells were rejected from all of our assays. Dissociated cerebellar neurons were isolated by size (forward-scatter) and granu- larity (side-scatter) parameters, as well as NMDA-sensitiv- ity. The sorted cell suspension was morphologically char- acterized by transmission electron microscopy (Philips EM-301). The formation of intracellular ROS was measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA) [1]. This method measures the formation of H202 generated by an increase in neuronal oxidative metabolism [16]. Viable cells can deacetylate DCFH-DA to 2',7'-dichlorofluo- rescin, which is not fluorescent. This compound reacts quantitatively with oxygen species to produce the fluores- cent dye 2',7'-dichlorofluorescein, which remains trapped | g g j rn 0 10 20 8@ 40 60 80 F C Fig. 1. Bivariate histogram of side light scatter SSC) versus forward light scatter FSC) of dissociated rat cerebellar cells. Arrow indicates the area corresponding to the population selected for analysis and cell sorting. Total number of cells analyzed in the sorting area was 5000.  112 F.X. Sureda et al. / Brain Research 723 1996) 110-114 within the cell and can be measured to provide an index of intracellular oxidation. One ml of the cell suspension (1 × 10 6 cells/ml) was loaded for 1 h with DCFH-DA (final concentration: 100 /xM) with horizontal agitation in a water bath at 37°C in an atmosphere of 95% 02/5% CO 2. After loading, different concentrations of NMDA were added to cell preparations for 90 min. Finally, the increase in 2',7'-dichlorofluo- rescein fluorescence was measured by flow cytometry. All data are expressed as mean _+ S.E.M. (standard error of the mean) from n different experiments. 3. Results Cell sorting was used to separate the viable neuronal population. Fig. 1 shows the sorting area selected to characterize the cerebellar cells morphologically. Fig. 2 illustrates the morphological characteristics of the cerebel- lar cells after sorting. As can be observed, no glial cells were present in the population selected. Different concentrations of NMDA (ranging from 10-7 M to 5 × 10 4 M) were tested for their ability to increase [Ca2+] i in the presence of 15 /xM of glycine. Maximal response was 21.4 _+ 2.8% over basal (n = 3 in duplicate, _+ S.E.M.). Fig. 3 (upper panel) shows the concentration- dependent [Ca 2+ ]i increase elicited by NMDA in dissoci- ated cerebellar cells. Data analysis yielded an ECs0 esti- mate of 12.9 +_ 1.8 /xM (n = 3). The effect of (+)MK-801 (ranging from 10 -9 M to 10 -4 M), a non-competitive NMDA antagonist, was evaluated on intracellular calcium increase evoked by NMDA (320 /xM) in the presence of 15 ~M of glycine (Fig. 3, lower panel). (+)MK-801 induced a concentration-dependent inhibition of the maxi- mal response. Non-linear regression analysis of the con- centration-response curves yielded an ICs0 of 36 + 15 nM (n = 3). NMDA was able to increase ROS generation within dissociated cerebellar cells in a concentration-dependent manner (Fig. 4, upper panel). After 90 min of incubation with different concentrations of NMDA (ranging from 10 6 Mto 10 2 M) in the presence of15 /xMofglycine, Fig. 2. Photomicrograph of rat cerebellar granule cells. This figure illustrates the transmission electron microscopic characteristics of the neurons of the sorting area indicated in Fig. 1. Calibration bar equals 1 /xm.  F.X. Sureda et al. / Brain Research 723 1996) 110-114 113 100 80 60 40 I -8 100 I I I -7 -6 -5 -4 -3 log [NMDA] M) 80 -20 60 20 O I I -4 -3 I -10 -9 -8 -7 -6 -5 log I +)MK-S011 0VI) Fig. 3. Upper panel: concentration-response curve of NMDA-induced [ Ca2+ ]i increase in cerebellar granule cells in the presence of 15 ,u,M of glycine. Data are the mean+S.E.M, from three separate experiments. Lower panel: inhibition by different concentrations of (+)MK-801 of NMDA (320 /zM)-induced [Ca 2+ ]i increase in the same cells in the presence of 15 /xM of glycine. Data are the mean S.E.M. from three separate experiments. a maximal increase of 15.5 ___ 2.4% in the generation of ROS, with respect to basal values was found. (+)MK-801 was tested for its ability to prevent and reverse the effects of NMDA (Fig. 4, lower panel). (+)MK-801 was added 1 h before NMDA addition and was able to inhibit the NMDA-induced ROS generation in a concentration-depen- dent manner, with an IC50 of 671 + 201 nM (n = 3). 4. Discussion The results obtained in the present studies show that NMDA increases [Ca 2+ ]i and ROS in dissociated cerebel- lar neuronal cells, and that these effects can be blocked by (+)MK-801. High levels of cytoplasmatic calcium and free radicals are involved in brain damage and could be a key step in the development of some neurodegenerative disorders such as Parkinson's and Alzheimer's diseases [12]. Although the effects of NMDA on [Ca 2+ ]i have been well established in cell cultures [3,17] and in dissociated brain cells [7], in this study we have determined the effect of NMDA on intracellular calcium increase and free radi- cal production in rat pup neurons using a flow cytometry method. Flow cytometry allows to select different cell populations by their cell size (forward light-scatter) and internal and external cell structure (side light-scatter). The ability to discard neurons which are damaged during the dissociation procedure (propidium iodide positive cells) is also relevant. The effects of NMDA on intracellular cal- cium increases (ECs0) and the IC50 found for (+)MK-801 are in agreement with those obtained in cell cultures [17] 120 ~ ioo 8 O 6O 4~ O 2(3 O .e 213 i -6 -5 -4 -3 -2 log [NMDAI M) I00 80 m ~ 4o 20 0 I -10 -9 -8 -7 -6 -5 -4 -3 log [ +)MK-8011 lVl) Fig. 4. Upper panel: effects of NMDA on DCF oxidation on dissociated cerebellar cells. Experiments were performed in the presence of 15 /xM of glycine. Data are the mean + S.E.M. from three separate experiments. Lower panel: concentration-response curve for (+)MK-801 in decreas- ing DCF fluorescence evoked by NMDA (1 mM) in the same cells in the presence of 15 /xM of glycine. Data are the mean+S.E.M, from three separate experiments.  114 F.X. Sureda et al. / Brain Research 723 (1996) 110- 114 and in dissociated brain cells [7] using fluorometric meth- ods. Thus, the present method provides simplicity, high sensitivity and reliability, and is suitable to study the NMDA receptor functionalism. Moreover, due to the high variety of fluorescent markers, there is a wide range of potential uses concerning neurochemical analysis, such as intracellular sodium measurement, intracellular pH or membrane potential detection. We have also evaluated the intracellular free radical production in rat neurons mediated by NMDA. The effects of excitatory amino acids on free radical production in cerebellar granule cells [14] has been previously described. While these authors measured free radicals released into the medium, we evaluated the intracellular ROS produc- tion by measuring DCF fluorescence. Previous studies have investigated the increase in DCF fluorescence medi- ated by NMDA in synaptoneurosomes. In these studies, kainate enhanced ROS generation. However, this effect could not be blocked by CNQX, a kainate receptor antago- nist [2]. The detection of intracellular ROS production in brain neurons using DCFH-DA and H20 2 has been well estab- lished [16]. In the present work we have shown that NMDA is able to induce an intracellular oxidative stress in rat cerebellar dissociated neurons and that this effect can be inhibited by (+)MK-801 in a concentration-dependent manner. Although there is some controversy about the link between cytosolic calcium increase and oxidative stress [11], recent studies have shown that in cerebellar granular cells there is a relationship between cytoplasmatic calcium and phospholipase activation linked to arachidonic acid release, finally leading to ROS generation, which is re- sponsible of neurotoxicity [9,14]. Considering this, we propose that this method can be a sensitive and reliable way to evaluate the effects of poten- tially neuroprotective drugs on NMDA-induced ROS pro- duction in rat neurons. In summary, we propose the utility of flow cytometry on the isolation of viable cerebellar neurons and in the evaluation of the effects of NMDA on neuronal cells. Acknowledgements We thank to Scientific-Technical Services of the Uni- versity of Barcelona. In particular we are grateful to Mr. Jaume Comes and to Mrs. Nuria Cortadellas for their helpful technical assistance in flow cytometry and electron microscopy, respectively. To Dra. M. Durfort for cytologi- cal characterization and to Mr. R. Rycroft for revising the language of the manuscript. References [l] Bas, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C. and Thomas, M., Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimula- tion, J. Immunol., 130 (1983) 1910-1917. [2] Bondy, S.C. and Lee, D.K., Oxidative stress induced by glutamate receptor agonists, Brain Res., 610 (1993) 229-233. [3] Bouchelouche, P., Belhage, B., Frandsen, A., Drejer, J. and Schous- boe, A., Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca 2+ by mobilization of cellular Ca 2+ and activation of Ca 2+ influx, Exp. Brain. Res., 76 (1989) 281-291. [4] Burgoyne, R.D., Pearce, I.A. and Cambray-Deakin, M., N-Methyl- D-aspartate raises cytosolic calcium concentration in rat cerebellar granule cells in culture, Neurosci. Lett., 91 (1988) 47-52. [5] Choi, D.W., Excitotoxic cell death, J. Neurobiol., 23 (1992) 1261- 1276. [6] Coyle, J.T. and Punfarcken, P., Oxidative stress, glutamate, and neurodegenerative disorders, Science, 262 (1993) 689-695. [7] Dildy, J. and Leslie, S.W., Ethanol inhibits NMDA-induced in- creases in free intracellular Ca 2+ in dissociated brain cells, Brain Res., 499 (1989) 383-387. [8] Domenici, M.R., Longo, R., Scotti de Carolis, A., Frank, C. and Sagratella, S., Protective actions of 21-aminosteroids and MK-801 on hypoxia-induced electrophysiological changes in rat hippocampal slices, Eur. J. Pharmacol., 233 (1993) 291-293. [9] Fagni, L., Lafon-Cazal, M., Lerner-Natoli, M., Rondouin, G. and Bockaen, J., Role of nitric oxide and free-radicals in the glutamate mediated neurotoxicity, J. Pharm. Belg., 50 (1995) 204-212. [10] Hammer, B., Parker, W.D. and Bennet, J.P., NMDA receptors increase OH radicals in vivo by using nitric oxide synthase and protein kinase C, NeuroReport, 5 (1993) 72-74. [11] Harman, A.W. and Maxwell, M.J., An evaluation of the role of calcium in cell injury, Annu. Ret,. Pharmacol. Toxicol., 35 (1995) 129-144. [12] Jesberger, J.A. and Richardson, J.S., Oxygen free radicals and brain dysfunction, Int. J. Neuroscience, 57 (1991) 1-17. [13] Lafon-Cazal, M., Culcasi, M., Gaven, F., Pietri, S. and Bockaert, J, Nitric oxide, superoxide and peroxynitrite: putative mediators of NMDA-induced cell death in cerebellar granule cells, Neurophar- macology, 32 (1993) 1259-1266. [14] Lafon-Cazal, M., Pietri, S., Culcasi, M. and Bockaert, J., NMDA- dependent superoxide production and neurotoxicity, Nature, 364 (1993) 535 537. [15] Monyer, H., Hartley, D.M. and Choi, D.W., 21-Aminosteroids atten- uate excitotoxic neuronal injury in cortical cell cultures, Neuron, 5 (1990) 121-126. [16] Oyama, Y., Hayashi, A., Ueha, T. and Maekawa, K., Characteriza- tion of 2',7'-dichlorofluorescin fluorescence in dissociated mam- malian brain neurons: estimation on intracellular content of hydro- gen peroxide, Brain Res., 635 (1994) 113-117. 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