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astrocyte co-cultures
Neurochemistry International 62 (2013) 1020–1027
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Spatio-temporal spread of neuronal death after focal photolysis of caged glutamate in neuron/astrocyte co-cultures Sadahiro Iwabuchi, Tomoharu Watanabe, Koichi Kawahara ⇑ Laboratory of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan
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Article history: Received 29 August 2012 Received in revised form 13 March 2013 Accepted 15 March 2013 Available online 26 March 2013 Keywords: Caged glutamate NMDA receptor Acute neuronal death Delayed neuronal death Glutamate transporters
a b s t r a c t Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. In the infarct core region, massive neuronal death is observed, but neurons in the surroundings of the core (ischemic penumbra) seem viable at the time of stroke. Several hours or days after a stroke, however, many neurons in the penumbra will undergo delayed neuronal death (DND). The mechanisms responsible for such DND are not fully understood. In this study, we investigated whether and how glutamatemediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. Uncaging of the glutamate resulted in acute neuronal death in the flashed area. After that, DND was observed in the surroundings of the flashed area late after the uncaging. In contrast, DND was not observed in neuron-enriched cultures, suggesting that functional changes in astrocytes, not neurons, after focal acute neuronal death were involved in the induction of DND. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the nitric oxide (NO)-induced reduction in the expression of astrocytic GLT-1 was responsible for the occurrence of the DND. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. A reduction in cerebral blood flow (CBF) below certain thresholds is critical to a series of functional, biochemical and structural changes culminating in irreversible neuronal death, and can be used to distinguish between an irreversible infarct core, penumbra, and benign oligemia (penumbra that recovers spontaneously) (Bandera et al., 2006). In the core of brain tissue exposed to a dramatic reduction in CBF, the concentration of extracellular glutamate is markedly increased, resulting in massive acute neuronal death (Broughton et al., 2009). This infarct core is surrounded by a zone of less severely affected tissue which is rendered functionally silent by reduced CBF but remains viable (Astrup et al., 1981). This zone is known as the ‘‘ischemic penumbra’’. Previous studies have revealed that several hours or days after a stroke, many neurons in the ischemic penumbra will undergo delayed neuronal death (DND) (Broughton et al., 2009; Mattson, 2000). In general, the ischemic penumbra has been de-
scribed on the basis of CBF, suggesting a possible causal link between a decrease in CBF, the extent of which is not severe to induce acute neuronal death, and DND in the penumbra. However, the mechanisms responsible for the DND observed in the penumbra after a stroke are not fully understood. Elucidation of these mechanisms seems important, since the damages to the brain will become irreversible after hours or days unless treated effectively (Broughton et al., 2009). In this study, we investigated whether and if so how glutamatemediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the reduced expression of astrocytic GLT-1 was responsible for the induction of the DND.
2. Materials and methods ⇑ Corresponding author. Address: Laboratory of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo 0600814, Japan. Tel./fax: +81 11 706 7591. E-mail address: [email protected] (K. Kawahara). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.010
The animal experiments were carried out in accordance with The Guide for the care and use of laboratory animals, Hokkaido University School of Medicine.
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2.1. Cell culture Primary neuronal/astrocytic co-cultures were prepared exactly as described previously (Kawahara et al., 2002, 2005). The cortical hemispheres of 17- to 19-day-old embryonic rats were obtained and dissociated using a papain-cysteine solution. Cells were placed on poly-L-lysine-coated IWAKI glass-based dishes (AGC TECHNO GLASS, Chiba, Japan) at 30,000 cells/cm2, and maintained with a culture medium (CM) (80% Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA), 10% Ham’s F-12 nutrient mixture and 10% fetal bovine serum) supplemented with about 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator for 4 days. Then, neuronal/astrocytic co-cultures were fed a filtered (0.22 lm; Millipore, Bedford, USA) conditioned-CM twice a week. The conditioned CM was obtained from astrocytic cultures in which a cooled CM was incubated for 1 day. Astrocytic cultures were obtained from the cortical hemispheres of postnatal 2–3-day-old rats. The experiments were performed with cocultures maintained for 7–10 days.
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The methods used to obtain primary astrocyte-enriched cultures were described previously in detail (Iwabuchi and Kawahara, 2011; Kawahara et al., 2002). In brief, the cortical hemispheres of postnatal 2–3-day-old rats, were isolated and digested at 37 °C for 30 min with a 0.01% papain-cysteine solution. Primary astrocytes were plated onto polyethyleneimine-coated glass coverslips/plastic dishes at 20,000 cells/cm2. Cells were maintained with a culture medium (CM) (80% Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with 10% Ham’s F-12 nutrient mixture and 10% fetal bovine serum) with 1% penicillin/streptmycin at 37 °C in a 5% CO2 incubator. Cultured astrocytes were fed a cooled CM twice a week. The experiments were performed with cultures maintained for 14–21 days. The methods used to obtain primary neuron-enriched cultures were described previously in detail (Kosugi et al., 2008). Cortical neurons and glias were cultured for 3 days, then fed CM containing 10 lM cytosine arabinofuranoside (Ara-C). After 3 day’s incubation, the medium was replaced with fresh warmed CM. Mature cultures (9–12 days in vitro) were used for the experiments.
Fig. 1. Focal photolysis of caged glutamate induces acute neuronal death in the UV flashed area (FA) in neuron/astrocyte co-cultures. An immunofluorescence analysis revealed that there were anti-bIII tubulin-positive neurons (red) and GFAP-positive astrocytes (green), indicating a neuron/astrocyte co-culture (A). Figs. B1and B2 indicate the area in which the survival rate of neurons was measured and the experimental protocol, respectively. B3 shows the distribution of the survival rate of neurons depending on the distance from the center of the FA at 1 h after the washout of caged and uncaged glutamate. For example, the survival rate at r = 1.5 mm shows the mean survival rate in the concentric area 1.0–1.5 mm from the center as indicated by B1. Data are expressed as the mean + SEM (n = 9, different cultures). ⁄p < 0.05 vs sham-treatment. Figs. B4 and B5 show typical phase-contrast images of co-cultures in the FA before and 1 h after the flash, respectively. The scale bar indicates 50 lm. Figs. B6 and B7 show typical fluorescent images of co-cultures in the FA and outside of the FA, respectively. Blue: DNA-binding dye Hoechst 33342 (HE), red: propidium iodide (PI). The scale bar indicates 100 lm. Exposure of co-cultures to UV flash only or administration of caged glutamate without UV did not induce significant neuronal death (C). Data are expressed as the mean + SEM (n = 9, different cultures). ⁄p < 0.05 vs sham-treatment. In astrocyte-enriched cultures (D1), however, focal photolysis of caged glutamate did not induce any astrocytic death (D2). Abbreviations: FA, flashed area; PI, propidium iodide; ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.2. Identifying neurons from co-cultures
2.3. Ca2+ imaging and photolysis of the caged compound
The immunofluorescent analysis was performed with antibodies against anti-bIII tubulin (Promega, Madison, USA; 100 ng/ml). Briefly, cultures were fixed with 4% paraformaldehyde for 30 min at room temperature (RT) and washed more than three times with phosphate-buffered saline (PBS), and then a detergent Triton-X was added for 10 min. After fixed-cells were washed with PBS, a blocking solution (1% normal goat serum with PBS) was added to the cultures for 30 min at RT. Immunofluorescent labeling was done with anti-bIII tubulin for 1 day at 4 °C. The primary antibody was visualized with Alexa Fluor 532-conjugated anti-mouse antibodies (Invitrogen, Karlsruhe, Germany) for 60 min at RT. The fluorescent DNA-binding dye Hoechst 33342 (HE) was used to detect nuclei. Immunoreactivity was observed with a confocal laser scanning microscope at a magnification of 150 (IX70; Olympus, Tokyo, Japan), and images were taken from random fields from 6 different cultures (n = 6). The size of neurons in the processed immunofluorescent images was determined using Scion Image for Windows (Scion Corporation, Maryland, USA). The same analysis was performed with phase-contrast images of co-cultures taken with a fluorescence microscope at a magnification of 150 (IX70; Olympus). In a series of analyses, skilled experimenters could distinguish neurons from astrocytes in phase-contrast images.
The cultures were incubated for 50 min with 10 lM fluo-4acetoxy-methylester (Fluo-4 AM; Invitrogen) in CM, and washed with essential balanced salt solution (EBSS) containing 1.5 mM Ca2+ and 1.5 mM Mg2+, supplemented with 25 mM HEPES and 5.5 mM D-glucose. Then, 4-methoxy-7-nitroindolinyl-caged-Lglutamate (caged glutamate, Tocris Bioscience, Bristol, UK) was added to the cultures with EBSS. To uncage the caged compound, cultures were placed under an IX70 microscope warmed at 37 °C, and exposed to ultraviolet light (UV) filtered by a fluorescent cube (peak 360 nm) (U-MWU; Olympus) (Iwabuchi et al., 2002). The duration of the photolytic flash was set at 6 s. Intercellular Ca2+ changes were detected by a cooled CCD camera, and analyzed with an image processing system including the multifunction image analysis software Aquacosmos (Hamamatsu Photonics, Hamamatsu, Japan). First of all, skilled experimenters detected neurons in phase-contrast images. The change in Fluo-4 AM emission at 505 nm in response to excitation at 488 nm was measured. The ratio intensity of the control image (F0) was set as the target neurons 4 s before the focal photolytic flash, and changes in the fluorescent ratio intensity (F) of each cell were monitored. The intercellular Ca2+ changes were analyzed using a series of images taken at 4-s intervals. F/F0 was used for evaluating the response of the intracellular Ca2+ concentration ([Ca2+]i) of neurons.
Fig. 2. Activation of NMDA receptors crucial to an increase in intracellular Ca2+ ([Ca2+]i) and neuronal death in the flashed area (FA) by focal photolysis of caged glutamate. A and B: Representative images of changes in [Ca2+]i at 3 different time points: 10 s before the exposure to UV (A1 and B1), and 10 s (A2 and B2) and 30 s (A3 and B3) after the flash. Figs. B1–B3 show changes in [Ca2+]i when the co-cultures were treated with 100 lM AP5, a specific inhibitor of NMDA receptors. [Ca2+]i increases from dark blue to red through yellow. The color bar on the right indicates the scale of ratio intensity. The scale bar indicates 100 lm. Treatment of co-cultures with AP5 resulted in the significant suppression of both the [Ca2+]i increase (C) and neuronal death (D and E). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄p < 0.05. Focal photolysis of caged glutamate induced an increase in [Ca2+]i within the FA but not outside it (F). The scale bar indicates 100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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The maximum increase of [Ca2+]i was defined as the maximum value of F/F0 during the observation period. After UV exposure, EBSS was replaced with CM and cultures were incubated at 37 °C in 5% CO2 for 1 h–1 day.
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ysis of caged glutamate. The intensity of immunofluorescence was measured for at least 20 cells in each of the areas (FA, SFA, and outside) in 4 different cultures. The definition of the areas was stated in the text. The average fluorescent intensity of cells in the shamtreated cultures was set at 100%.
2.4. Immunocytochemistry 2.6. Survival rate of neurons Cells were fixed with 4% paraformaldehyde and washed with PBS, followed by 0.01% Triton-X. They were then treated with 1% normal goat serum. Immunofluorescent labeling was done with antibodies directed against glial fibrillary acidic protein (GFAP) (Sigma, Saint Louis, USA; 1 lg/ml), GLAST (EAAT1) or GLT-1 (EAAT2) (Cell Signaling Technology, Denver, USA; 20 lg/ml or 20 lg/ml) for 1 day at 4 °C. Negative controls without each primary antibody were performed. Primary antibodies were visualized with Alexa Fluor 488-conjugated anti-rabbit or Alexa Fluor 633-conjugated anti-mouse antibodies (Invitrogen). Hoechst 33342 was used to detect nuclei. The immunoreactivity was observed with a confocal laser scanning microscope (FV300; Olympus Corporation). The image analysis was identical for each of the cultures.
The analysis of neuronal cellular death was performed following observation of the nuclear morphology using the fluorescent DNA-binding dyes HE and propidium iodide (PI). Cells were incubated with these dyes for 15 min at 37 °C, and individual nuclei were observed under the IX70 fluorescence microscope. The region inside a radius of 2.5 mm from the center was defined as the flashed area (FA). To take images correctly, experimenters drew the line with a square on the back of the dish before it was coated with poly-L-lysine. More than 4 individual images were randomly taken in each area. HE or PI-positive cells were counted automatically using Scion Image for Windows. The survival rate of neurons was calculated as the percentage of (HE–PI)/HE.
2.5. Semi-quantitative analyses of GLT-1 and GLAST expression
2.7. Monitoring of cytosolic nitric oxide production
Changes in the expression of the astrocytic glutamate transporters GLT-1 and GLAST were analyzed at 24 h after the focal photol-
Changes in the cytosolic NO concentration were monitored using a fluorescent NO probe, DAF-FM (Kojima et al., 1999). Cells
Fig. 3. Delayed neuronal death in the surroundings of the flashed area. Figs. A1 and A2 show the spatial distribution of PI-positive neurons at 1 h and 24 h after the termination of UV exposure for the photolysis of caged glutamate, respectively. The scale bar indicates 300 lm. Delayed neuronal death was observed at a radius of 3.0, 3.5, and 4.0 mm from the center of the flashed area (FA) (B1–B6), but not at less than 2.5 mm or more than 4.5 mm. Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄p < 0.05. Fig. C provides a schematic illustration of the FA, SFA, and outside. Abbreviations are the same as those in Fig. 1.
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were loaded with a NO indicator by incubation with 10 lM of DAF-FM DA for 4 h (0.5% DMSO). We have previously demonstrated that high concentrations of DAF-FM, more than 20 lM, seemed toxic to cells, since loading of cultures with a high concentration of DAF-FM sometimes resulted in morphological changes (Kawahara et al., 2006). Fluorescent images were acquired at 2-s intervals with a cooled CCD camera (C4880-80; Hamamatsu Photonics, Hamamatsu, Japan). An analysis of the acquired images was made with an image processing and measuring system (AQUACOSMOS, Hamamatsu Photonics). Free cytoslic NO was monitored by comparing the changes in fluorescence intensity at an excitation wavelength of 490 nm with the initial fluorescence intensity (F/F0), using an emission wavelength of 515 nm. Because DAF-FM is not a quantitative probe like DAF-2 (Kojima et al., 1999), no attempt was made to calibrate DAF-FM fluorescence.
2.8. Chemicals D(-)-2-Amino-5-phosphonopentanoic/phosphonovaleric acid (AP5) and NG-monomethyl-L-arginine (L-NMMA) were obtained from Sigma–Aldrich. Other chemicals were obtained from Wako Chemicals (Tokyo, Japan).
2.9. Statistic analysis The data are expressed as the mean ± standard error of the mean (SEM). Group comparisons were made using an analysis of variance (ANOVA) with Fisher’s test. A P-value of less than 0.01 or 0.05 was considered significant.
3. Results We first tried to induce glutamate-mediated acute excitotoxic neuronal death in a spatially-restricted region by focal photolysis of caged glutamate in mixed cultures of neurons and astrocytes (Fig. 1A). To uncage the compound, cultures were exposed to ultraviolet light (UV) within a radius of 2.5 mm from the center of the dish, an area defined here as the flashed area (FA) (Fig. 1B1). The duration of the UV flash and the concentration of caged glutamate were set at 6 s and 100 lM, respectively (Suppl. Fig. 1). Focal photolysis of caged glutamate resulted in cell death in the FA (Fig. 1B3 and B4), but not outside it (Fig. 1B3, B5, and B6). Since the morphology of astrocytes remained the same and few dead cells were observed in the FA, dead cells were considered neurons (Fig. 1D1 and D2). In addition, neither exposure to UV without the
Fig. 4. NMDA receptor-mediated neurotoxicity involved in the genesis of delayed neuronal death. Treatment with AP5 during the exposure to UV for uncaging glutamate resulted in the significant suppression of both acute neuronal death in the flashed area (FA) and delayed death in the surroundings of the FA (SFA) (A1, A2, and B). Note that treatment with AP5 only after the washout of caged and uncaged glutamate also suppressed the induction of delayed neuronal death (B). In contrast, in neuron-enriched cultures, delayed neuronal death was not observed in the SFA (D). Figs. C1 and C2 show anti-bIII tubulin-positive neurons (red) and GFAP-positive astrocytes (green), respectively, indicating a neuron-enriched culture. The scale bars indicate 50 lm (C1) and 100 lm (C2). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄ p < 0.01; ⁄p < 0.05. Abbreviations are the same as those in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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administration of caged glutamate nor the administration of caged glutamate without the UV flash induced neuronal death (Fig. 1C), suggesting that neuronal death in the FA was caused by uncaged glutamate, not by the UV itself or by caged glutamate. We next investigated the mechanisms responsible for the glutamate-mediated neuron-specific death in the FA. Photolysis of caged glutamate by UV induced an increase in the concentration of intracellular Ca2+ ([Ca2+]i) (Fig. 2A) and the increase was mostly restricted to cells in the FA (Fig. 2F). UV exposure itself did not increase [Ca2+]i in the FA (data not shown), suggesting that glutamate uncaged by photolysis induced the increase of [Ca2+]i in neurons. We then examined whether the activation of Ca2+-permeable glutamate receptors, NMDA receptors, was involved in the uncaged glutamate-mediated increase in [Ca2+]i and neuronal death. Treatment with AP5, a specific antagonist of NMDA receptors, reversed both the Ca2+ increase (Fig. 2B and C) and the death of neurons (Fig. 2D and E) in the FA caused by the photolysis of caged glutamate, suggesting that the activation of NMDA receptors was
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involved in the flash-induced neuronal death. Taken together, these results demonstrated that focal photolysis resulted in glutamate-mediated excitotoxic neuronal death in a spatially restricted area 1 h after the uncaging of glutamate. We named this type of neuronal death, acute neuronal death. We next investigated whether and how the localized neuronal death induced by the focal photolysis of caged glutamate affected surrounding neurons and astrocytes. Although both uncaged and caged glutamate in neuron/astrocyte co-cultures were washed out about 5 min after the exposure to UV, PI-positive dead neurons were observed outside of the FA 24 h after the washout (Fig. 3A2). We named this type of neuronal death, delayed neuronal death (DND). DND was observed within a radius of 2.5–4.0 mm from the center (Fig. 3B1–B6), area referred to as the surroundings of the FA (SFA) or ‘‘in vitro penumbra’’ (Fig. 3C). Treatment with AP5 during the exposure to UV resulted in a significant increase in the survival rate of neurons in the SFA (Fig. 4A1, A2, and B), suggesting that the DND was caused by the activation of NMDA
Fig. 5. Photolytic flash-induced decrease in the expression of the astrocytic glutamate transporter GLT-1 crucial to the induction of delayed neuronal death. The expression of astrocytic GLT-1 was significantly reduced in the flashed area (FA) and the surroundings of the FA (SFA), but not outside these areas (A1–A5), 24 h after the washout. The scale bar indicates 100 lm. Fig. C indicates that GLT-1 (green) was not expressed in anti-bIII tubulin-positive neurons. The scale bar indicates 100 lm. Treatment of co-cultures with 1.0 mM L-NMMA, an inhibitor of nitric oxide synthase (NOS), reversed the photolytic flash-induced decrease in the expression of astrocytic GLT-1 (D and E). Figs. D1–D3 indicate the expression of GLT-1 in the FA, SFA, and region outside these areas in L-NMMA-treated co-cultures, respectively. The scale bar indicates 100 lm. The expression of astrocytic GLAST was significantly increased in the flashed area (FA) and the surroundings of the FA (SFA), but not outside these areas (F1–F5), 24 h after the washout. The scale bar indicates 100 lm. In the L-NMMA-treated co-cultures, the survival rate of neurons in the SFA was significantly increased, but the effect was antagonized by treatment with 100 lM DHK, a specific blocker of astrocytic GLT-1, during the period after the washout of caged and uncaged glutamate (H). Delayed neuronal death was not observed in the co-cultures treated with L-NMMA but was observed on treatment with DHK (I). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄ p < 0.05. Abbreviations are the same as those in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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receptors during the UV flash. Unexpectedly, treatment with AP5 only after the washout of uncaged glutamate also resulted in a significant reduction in DND (Fig. 4B). The question then arises as the origin of glutamate involved in the NMDA-mediated DND in the SFA. Although uncaged glutamate was washed out about 5 min after the UV exposure, the possibility that uncaged glutamate which rapidly diffused to the SFA in this 5 min contributed to the induction of DND cannot be completely excluded. If this is the case, DND might be also observed in neuron-enriched cultures. We next examined this possibility (Fig. 4C1 and C2). Unexpectedly, DND in the SFA was not observed in neuron-enriched cultures (Fig. 4D), suggesting that uncaged glutamate was not primarily responsible for DND, and that functional changes in astrocytes, not in neurons, after acute neuronal death in the FA contributed to the induction of DND in the SFA observed in neuron/astrocyte co-cultures. A question then arises as to what functional changes in astrocytes were responsible for the induction of DND. We next addressed this issue mainly focusing on changes in the expression of GLT-1 (EAAT2), one of the glutamate transporters expressed in astrocytes, since astrocytic GLT-1 is primarily responsible for the uptake of extracellular glutamate in the brain (Robinson, 1999), and glutamate-mediated toxicity was possibly involved in DND in neuron/asctrocyte co-cultures as indicated above. The expression of GLT-1 was significantly decreased in the FA and SFA 24 h after the UV flash (Fig. 5A1–A5), suggesting that the decrease was responsible for the DND in the SFA. Unexpectedly, the expression of astrocytic GLAST was significantly increased in the FA and in the SFA, but not outside these areas (Fig. 5F1–F5), 24 h after the washout, suggesting that the decrease in the expression of astrocytic GLT-1 in the SFA was not caused by the loss of astrocytes. We have previously demonstrated that the enhanced production of nitric oxide (NO) derived from the NMDA R-mediated activation of Ca2+-dependent neuronal nitric oxide synthase (nNOS) expressed in neurons is involved in a decrease in the expression of GLT-1 in astrocytes in neuron/astrocyte co-cultures (Yamada et al., 2006). The intensity of DAF fluorescence significantly increased with the photolysis of caged glutamate in the FA, and this increase was significantly suppressed by treatment with L-NMMA, an inhibitor of NOS, suggesting that the production of NO was actually enhanced by the focal photolysis of caged glutamate (Suppl. Fig. 2). Treatment of co-cultures with L-NMMA reversed the focal photolysis-induced decrease in the expression of GLT-1 in the SFA (Fig. 5D and E). In addition, the treatment of co-cultures with L-NMMA increased the survival rate of neurons in the SFA, but the increase was antagonized by treatment with DHK, a specific blocker of GLT-1, during the period after the washout of uncaged glutamate (Fig. 5H). Moreover, DND in the SFA was not observed on L-NMMA treatment, but was observed on treatment with DHK (Fig. 5I). These results suggested that the L-NMMA-induced increase in the survival rate of neurons was caused by suppression of a decrease in GLT-1 expression in astrocytes. 4. Discussion The present study demonstrated that delayed neuronal death (DND) was actually induced in the surroundings of the flashed area (SFA) late after acute neuronal death in the flashed area (FA). In general, the ischemic penumbra has been described on the basis of cerebral blood flow (CBF), suggesting a possible causal link between a decrease in CBF, the extent of which is not severe to induce acute neuronal death, and DND in the penumbra. However, the present in vitro study indicating that functional changes in astrocytes caused by the spatially-restricted excitotoxic neuronal death were primarily responsible for the occurrence of DND in the SFA raises a possibility that most of the DND observed in the ischemic
penumbra in vivo has little or nothing to do with the prior mild reduction of CBF in the penumbral region during ischemia. This study demonstrated the possibility that the focal photolysis of caged glutamate resulted in a significant decrease in the expression of GLT-1 in the SFA, leading to the decreased uptake of extracellular glutamate and the NMDA receptor-mediated DND (Figs. 4 and 5). Although functional changes in astrocytes, not neurons, are involved in the induction of DND in the SFA (Fig. 5), astrocytes also express the glutamate transporter GLAST (EAAT1) in addition to GLT-1 (EAAT2). Therefore, we examined whether the expression of GLAST changed after the focal photolysis of caged glutamate. Unexpectedly, GLAST expression was significantly increased both in the FA and in the SFA (Fig. 5F1–F5), raising the question of whether this enhanced expression contributes to the clearance of extracellular glutamate. Previous studies have revealed that following transient focal cerebral ischemia in rats, impaired functioning of GLT-1, not GLAST, contributed to the initiation of DND in the hippocampus (Rao et al., 2000, 2001). The expression GLAST did not change, or actually increased after transient ischemia (Rao et al., 2001). In addition, Duan et al. (1999) reported that the application of glutamate induced a rapid increase in the expression of GLAST in cultured astrocytes. However, it seems generally accepted that astrocytic GLT-1, not GLAST, is primarily responsible for the uptake of extracellular glutamate in the brain (Torp et al., 1995; Tanaka et al., 1997; Robinson, 1999). Therefore, further studies are needed to clarify the functional significance of the enhanced expression of GLAST after focal photolysis of caged glutamate observed in this study. Previous studies have demonstrated that the enhanced production of nitric oxide (NO) is involved in glutamate-induced, NMDA receptor-mediated excitotoxic neuronal death (Dawson et al., 1991; Garthwaite, 1991; Kawahara et al., 2004; Strijbos et al., 1996). Also in the present study, the focal photolysis of caged glutamate-induced enhanced production of NO was involved in the acute death of neurons and in the decreased expression of GLT-1 in the FA (Suppl. Fig. 3). At present, however, it remains unclear whether there exists a direct causal link between the decreased expression of astrocytic GLT-1 and the glutamate-mediated acute neuronal death in the FA. We previously demonstrated in neuron/astrocyte co-cultures that Ca2+-dependent nNOS-derived NO was critically involved in a decrease in the expression of GLT-1 in astrocytes (Yamada et al., 2006). Although neuron-derived NO seemed crucial to the reduction in astrocytic GLT-1 expression, these results raise a question as to what mechanisms are responsible for the NO-induced down-regulation of GLT-1 expression in astrocytes. The present study also revealed that the glutamate-mediated enhanced production of NO was responsible for the decreased expression of astrocytic GLT-1 in the SFA, and suggested that the decreased GLT-1 expression contributed to the induction of DND in the SFA (Fig. 5). However, there are other possible explanations linking the enhanced production of NO with the induction of DND in the SFA. A previous study has demonstrated that NO causes release of glutamate and ATP from astrocytes, and the NO-induced release of astrocytic glutamate and ATP may be important in physiological or pathological communication between astrocytes and neurons (Bal-Price et al., 2002). ATP released from astrocytes may activate ATP-sensitive purinoceptors, such as P2X7 receptors. P2X7 receptors are permeable to larger molecules up to 800– 900 Da (Surprenant et al., 1996), and several studies have been shown that prolonged activation of such receptors leads to cellular death (Atkinson et al., 2004; Schrier et al. 2002). Recent studies have shown that pannexin hemichannels are the P2X7 receptorsassociated protein (Huang et al., 2007; Pelegrin and Surprenant, 2009), and the opening of the channels may be regulated by P2X7 receptors (Pelegrin and Surprenant, 2009). In addition,
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P2X7 receptors mediate the release of glutamate and GABA from rat hippocampal slices (Sperlágh et al., 2002). However, we have recently demonstrated that ATP, released through pannexin-1 (Px1) hemichannels, activates P2X7 receptors, resulting in the closure of Px1 hemichannels in cultured astrocytes, and such a negative-feedback mechanism might contribute to the survival of astrocytes under ischemic stress (Iwabuchi and Kawahara, 2011). Further studies will be needed to clarify the possible involvement of the NO-induced release of astrocytic glutamate and ATP in the induction of DND in the SFA. Previous studies have demonstrated that the interleukin-1b-NO pathway is responsible for the reduction in the expression of the inwardly-rectifying potassium channel Kir4.1 in astrocytes (Casamenti et al., 1999; Olsen et al., 2010; Zurolo et al., 2012). Kir4.1 potassium channels are involved in the regulation of ionic homeostasis in astrocytes, in particular the extracellular concentration of potassium, which influences neuronal excitability. The activity of sodium and potassium coupled astrocytic glutamate transporter GLT-1and GLAST depends on the negative membrane potential provided by Kir4.1 establishing a sufficient electrochemical gradient for glutamate import. These findings raise a possibility that the NO-induced reduction in the expression of Kir4.1 is crucially involved in the induction of DND in the SFA. In the present study, the focal photolysis of caged glutamate resulted in a significant decrease in the expression of GLT-1, but increased the expression of GLAST in the SFA (Fig. 5). The NO-induced reduction in the expression of Kir4.1 might be responsible for a decrease in the uptake of glutamate leading to the induction of DND in the SFA, although the expression of astrocytic GLAST was increased. Acknowledgements The analysis of immunoreactivity was carried out with a confocal laser scanning microscope; FV300 at the OPEN FACILITY, Hokkaido University Sousei Hall. This study was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (22300148) to KK. There is no conflict-of-interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuint.2013. 03.010. References Astrup, J., Siesjo, B.K., Symon, L., 1981. Thresholds in cerebral ischemia-the ischemic penumbra. Stroke 12, 723–725. Atkinson, L., Batten, T.F.C., Moores, T.S., Varoqui, H., Erickson, J.D., Deuchars, J., 2004. Differential co-localization of the P2X7 receptor subunit with vesicular glutamate transporters VGLUT1 and VGLUT2 in rat CNS. Neuroscience 123, 761–768. Bal-Price, A., Moneer, Z., Brown, G.C., 2002. Nitric oxide induces rapid, calciumdependent release of vesicular glutamate and ATP from cultured rat astrocytes. Glia 40, 312–323. Bandera, E., Botteri, M., Minelli, C., Sutton, A., Abrams, K.R., Latronico, N., 2006. Cerebral blood flow threshold of ischemic penumbra and infarct core in acute ischemic stroke. Stroke 37, 1334–1339. Broughton, B.R.S., Reutens, D.C., Sobey, C.G., 2009. Apoptotic mechanisms after cerebral ischemia. Stroke 40, e331–e339. Casamenti, F., Prosperi, C., Scali, C., Giovannelli, L., Colivicchi, M.A., FaussonePellegrini, M.S., Pepeu, G., 1999. Interleukin-1b activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer’s disease. Neuroscience 91, 831–842.
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Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88, 6368–6371. Duan, S., Anderson, C.M., Stein, B.A., Swanson, R.A., 1999. Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST. J. Neurosci. 19, 10193–10200. Garthwaite, J., 1991. Glutamate, nitric oxide and cell-cell signaling in the nervous system. Trends Neurosci. 14, 60–67. Huang, Y.J., Maruyama, Y., Dvoryanchikov, G., Pereira, E., Chaudhari, N., Roper, S.D., 2007. The role of pannexin 1 hemichannels in ATP release and cell–cell communication in mouse taste buds. Proc. Natl. Acad. Sci. USA 104, 6436–6441. Iwabuchi, S., Kawahara, K., 2011. Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes. Neurochem. Intern. 58, 376–384. Iwabuchi, S., Kawahara, K., Makisaka, K., Sato, H., 2002. Photolytic flash-induced intercellular calcium waves using caged calcium ionophore in cultured astrocytes from newborn rats. Exp. Brain Res. 146, 103–116. Kawahara, K., Hachiro, T., Yokokawa, T., Nakajima, T., Yamauchi, Y., Nakayama, Y., 2006. Ischemia/reperfusion-induced death of cardiac myocytes: possible involvement of nitric oxide in the coordination of ATP supply and demand during ischemia. J. Mol. Cell. Cardiol. 40, 35–46. Kawahara, K., Hosoya, R., Sato, H., Tanaka, M., Nakajima, T., Iwabuchi, S., 2002. Selective blockade of astrocytic glutamate transporter GLT-1 with dihydrokainate prevents neuronal death during ouabain treatment of astrocyte/neuron co-cultures. Glia 40, 49–56. Kawahara, K., Kosugi, T., Tanaka, M., Nakajima, T., Yamada, T., 2005. Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning-induced ischemic tolerance of neurons in neuron/astrocyte cocultures. Glia 49, 349–359. Kawahara, K., Yanoma, J., Tanaka, M., Nakajima, T., Kosugi, T., 2004. Nitric oxide produced during ischemia is toxic but crucial to preconditioning-induced ischemic tolerance of neurons in culture. Neurochem. Res. 29, 797–804. Kojima, H., Urano, Y., Kikuchi, K., Higuchi, T., Hirata, Y., Nagano, T., 1999. Fluorescent indicators for imaging nitric oxide production. Angew. Chem. Int. Ed. 38, 3209– 3212. Kosugi, T., Kawahara, K., Tanaka, M., Watanabe, Y., Inanami, O., 2008. Neuron is the primary target of Ca2+ paradox-type insult-induced cell injury in neuron/ astrocyte co-cultures. Neurochem. Intern. 52, 887–896. Mattson, M.P., 2000. Apoptosis in neurodegenerative disorders. Nat. Rev. Mol. Cell Biol. 1, 120–129. Olsen, M.L., Campbell, S.C., McFerrin, M.B., Floyd, C.L., Sontheimer, H., 2010. Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17b-oestradiol treatment. Brain 133, 1013–1025. Pelegrin, P., Surprenant, A., 2009. The P2X(7) receptor-pannexin connection to dye uptake and IL-1 beta release. Purinergic Signal. 5, 129–137. Rao, V.L.R., Bowen, K.K., Dempsey, R.J., 2001. Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain. Neurochem. Res. 26, 497–502. Rao, V.L.R., Rao, A.M., Dogan, A., Bowen, K.K., Hatcher, J., Rothstein, J.D., Dempsey, R.J., 2000. Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global ischemia. Neurochem. Intern. 36, 531–537. Robinson, M.B., 1999. The family of sodium-dependent glutamate transporters: a focus on the GLT-1/EAAT2 subtype. Neurochem. Intern. 33, 479–491. Schrier, S.M., Florea, B.I., Mulder, G.J., Nagelkerke, J.F., IJzerman, A.P., 2002. Apoptosis induced by extracellular ATP in the mouse neuroblastoma cell line N1E-115: studies on involvement of P2 receptors and adenosine. Biochem. Pharmacol. 63, 1119–1126. Sperlágh, B., Köfalvi, A., Deuchars, J., Atkinson, L., Milligan, C.J., Buckley, N.J., Sylvester Vizi, E., 2002. Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus. J. Neurochem. 81, 1196–1211. Strijbos, P.J.L., Leach, M.J., Garthwaite, J., 1996. Vicious cycle involving Na+ channels, glutamate release, and NMDA receptors mediates delayed neurogeneration through nitric oxide formation. J. Neurosci. 16, 5004–5013. Surprenant, A., Rassendren, F., Kawashima, E., North, R.A., Buell, G., 1996. The cytosolic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272, 735–738. Tanaka, K., Watase, K., Manabe, T., Yamada, K., Watanabe, M., Takahashi, K., Iwama, H., Nishikawa, T., Ichihara, N., Kikuchi, T., Okuyama, S., Kawashima, N., Hori, S., Takimoto, M., Wada, K., 1997. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702. Torp, R., Lekieffre, D., Levy, L.M., Haug, F.M., Danbolt, N.C., Meldrum, B.S., Ottersen, O.P., 1995. Reduced postishemic expression of a glial glutamate transporter, GLT-1, in the rat hippocampus. Exp. Brain Res. 103, 51–58. Yamada, T., Kawahara, K., Kosugi, T., Tanaka, M., 2006. Nitric oxide produced during sublethal ischemia is crucial for the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures. Neurochem. Res. 31, 49–56. Zurolo, E., de Groot, M., Lyer, A., Anink, J., van Vliet, E.A., Heimans, J.J., Reijneveld, J.C., Gorter, J.A., Aronica, E., 2012. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 b. J. Neuroinflamm. 9, 280.
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Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Spatio-temporal spread of neuronal death after focal photolysis of caged glutamate in neuron/astrocyte co-cultures Sadahiro Iwabuchi, Tomoharu Watanabe, Koichi Kawahara ⇑ Laboratory of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan
a r t i c l e
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Article history: Received 29 August 2012 Received in revised form 13 March 2013 Accepted 15 March 2013 Available online 26 March 2013 Keywords: Caged glutamate NMDA receptor Acute neuronal death Delayed neuronal death Glutamate transporters
a b s t r a c t Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. In the infarct core region, massive neuronal death is observed, but neurons in the surroundings of the core (ischemic penumbra) seem viable at the time of stroke. Several hours or days after a stroke, however, many neurons in the penumbra will undergo delayed neuronal death (DND). The mechanisms responsible for such DND are not fully understood. In this study, we investigated whether and how glutamatemediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. Uncaging of the glutamate resulted in acute neuronal death in the flashed area. After that, DND was observed in the surroundings of the flashed area late after the uncaging. In contrast, DND was not observed in neuron-enriched cultures, suggesting that functional changes in astrocytes, not neurons, after focal acute neuronal death were involved in the induction of DND. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the nitric oxide (NO)-induced reduction in the expression of astrocytic GLT-1 was responsible for the occurrence of the DND. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Glutamate-mediated excitotoxicity is now accepted as a major mechanism of ischemic neuronal damage. A reduction in cerebral blood flow (CBF) below certain thresholds is critical to a series of functional, biochemical and structural changes culminating in irreversible neuronal death, and can be used to distinguish between an irreversible infarct core, penumbra, and benign oligemia (penumbra that recovers spontaneously) (Bandera et al., 2006). In the core of brain tissue exposed to a dramatic reduction in CBF, the concentration of extracellular glutamate is markedly increased, resulting in massive acute neuronal death (Broughton et al., 2009). This infarct core is surrounded by a zone of less severely affected tissue which is rendered functionally silent by reduced CBF but remains viable (Astrup et al., 1981). This zone is known as the ‘‘ischemic penumbra’’. Previous studies have revealed that several hours or days after a stroke, many neurons in the ischemic penumbra will undergo delayed neuronal death (DND) (Broughton et al., 2009; Mattson, 2000). In general, the ischemic penumbra has been de-
scribed on the basis of CBF, suggesting a possible causal link between a decrease in CBF, the extent of which is not severe to induce acute neuronal death, and DND in the penumbra. However, the mechanisms responsible for the DND observed in the penumbra after a stroke are not fully understood. Elucidation of these mechanisms seems important, since the damages to the brain will become irreversible after hours or days unless treated effectively (Broughton et al., 2009). In this study, we investigated whether and if so how glutamatemediated localized excitotoxic neuronal death affects surrounding neurons and astrocytes. To induce spatially-restricted excitotoxic neuronal death, a caged glutamate was focally photolyzed by a UV flash in neuron/astrocyte co-cultures. The present in vitro study showed that the spatially-restricted excitotoxic neuronal death resulted in DND in the surroundings of the flashed area, and suggested that the reduced expression of astrocytic GLT-1 was responsible for the induction of the DND.
2. Materials and methods ⇑ Corresponding author. Address: Laboratory of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo 0600814, Japan. Tel./fax: +81 11 706 7591. E-mail address: [email protected] (K. Kawahara). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.03.010
The animal experiments were carried out in accordance with The Guide for the care and use of laboratory animals, Hokkaido University School of Medicine.
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2.1. Cell culture Primary neuronal/astrocytic co-cultures were prepared exactly as described previously (Kawahara et al., 2002, 2005). The cortical hemispheres of 17- to 19-day-old embryonic rats were obtained and dissociated using a papain-cysteine solution. Cells were placed on poly-L-lysine-coated IWAKI glass-based dishes (AGC TECHNO GLASS, Chiba, Japan) at 30,000 cells/cm2, and maintained with a culture medium (CM) (80% Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA), 10% Ham’s F-12 nutrient mixture and 10% fetal bovine serum) supplemented with about 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator for 4 days. Then, neuronal/astrocytic co-cultures were fed a filtered (0.22 lm; Millipore, Bedford, USA) conditioned-CM twice a week. The conditioned CM was obtained from astrocytic cultures in which a cooled CM was incubated for 1 day. Astrocytic cultures were obtained from the cortical hemispheres of postnatal 2–3-day-old rats. The experiments were performed with cocultures maintained for 7–10 days.
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The methods used to obtain primary astrocyte-enriched cultures were described previously in detail (Iwabuchi and Kawahara, 2011; Kawahara et al., 2002). In brief, the cortical hemispheres of postnatal 2–3-day-old rats, were isolated and digested at 37 °C for 30 min with a 0.01% papain-cysteine solution. Primary astrocytes were plated onto polyethyleneimine-coated glass coverslips/plastic dishes at 20,000 cells/cm2. Cells were maintained with a culture medium (CM) (80% Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with 10% Ham’s F-12 nutrient mixture and 10% fetal bovine serum) with 1% penicillin/streptmycin at 37 °C in a 5% CO2 incubator. Cultured astrocytes were fed a cooled CM twice a week. The experiments were performed with cultures maintained for 14–21 days. The methods used to obtain primary neuron-enriched cultures were described previously in detail (Kosugi et al., 2008). Cortical neurons and glias were cultured for 3 days, then fed CM containing 10 lM cytosine arabinofuranoside (Ara-C). After 3 day’s incubation, the medium was replaced with fresh warmed CM. Mature cultures (9–12 days in vitro) were used for the experiments.
Fig. 1. Focal photolysis of caged glutamate induces acute neuronal death in the UV flashed area (FA) in neuron/astrocyte co-cultures. An immunofluorescence analysis revealed that there were anti-bIII tubulin-positive neurons (red) and GFAP-positive astrocytes (green), indicating a neuron/astrocyte co-culture (A). Figs. B1and B2 indicate the area in which the survival rate of neurons was measured and the experimental protocol, respectively. B3 shows the distribution of the survival rate of neurons depending on the distance from the center of the FA at 1 h after the washout of caged and uncaged glutamate. For example, the survival rate at r = 1.5 mm shows the mean survival rate in the concentric area 1.0–1.5 mm from the center as indicated by B1. Data are expressed as the mean + SEM (n = 9, different cultures). ⁄p < 0.05 vs sham-treatment. Figs. B4 and B5 show typical phase-contrast images of co-cultures in the FA before and 1 h after the flash, respectively. The scale bar indicates 50 lm. Figs. B6 and B7 show typical fluorescent images of co-cultures in the FA and outside of the FA, respectively. Blue: DNA-binding dye Hoechst 33342 (HE), red: propidium iodide (PI). The scale bar indicates 100 lm. Exposure of co-cultures to UV flash only or administration of caged glutamate without UV did not induce significant neuronal death (C). Data are expressed as the mean + SEM (n = 9, different cultures). ⁄p < 0.05 vs sham-treatment. In astrocyte-enriched cultures (D1), however, focal photolysis of caged glutamate did not induce any astrocytic death (D2). Abbreviations: FA, flashed area; PI, propidium iodide; ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.2. Identifying neurons from co-cultures
2.3. Ca2+ imaging and photolysis of the caged compound
The immunofluorescent analysis was performed with antibodies against anti-bIII tubulin (Promega, Madison, USA; 100 ng/ml). Briefly, cultures were fixed with 4% paraformaldehyde for 30 min at room temperature (RT) and washed more than three times with phosphate-buffered saline (PBS), and then a detergent Triton-X was added for 10 min. After fixed-cells were washed with PBS, a blocking solution (1% normal goat serum with PBS) was added to the cultures for 30 min at RT. Immunofluorescent labeling was done with anti-bIII tubulin for 1 day at 4 °C. The primary antibody was visualized with Alexa Fluor 532-conjugated anti-mouse antibodies (Invitrogen, Karlsruhe, Germany) for 60 min at RT. The fluorescent DNA-binding dye Hoechst 33342 (HE) was used to detect nuclei. Immunoreactivity was observed with a confocal laser scanning microscope at a magnification of 150 (IX70; Olympus, Tokyo, Japan), and images were taken from random fields from 6 different cultures (n = 6). The size of neurons in the processed immunofluorescent images was determined using Scion Image for Windows (Scion Corporation, Maryland, USA). The same analysis was performed with phase-contrast images of co-cultures taken with a fluorescence microscope at a magnification of 150 (IX70; Olympus). In a series of analyses, skilled experimenters could distinguish neurons from astrocytes in phase-contrast images.
The cultures were incubated for 50 min with 10 lM fluo-4acetoxy-methylester (Fluo-4 AM; Invitrogen) in CM, and washed with essential balanced salt solution (EBSS) containing 1.5 mM Ca2+ and 1.5 mM Mg2+, supplemented with 25 mM HEPES and 5.5 mM D-glucose. Then, 4-methoxy-7-nitroindolinyl-caged-Lglutamate (caged glutamate, Tocris Bioscience, Bristol, UK) was added to the cultures with EBSS. To uncage the caged compound, cultures were placed under an IX70 microscope warmed at 37 °C, and exposed to ultraviolet light (UV) filtered by a fluorescent cube (peak 360 nm) (U-MWU; Olympus) (Iwabuchi et al., 2002). The duration of the photolytic flash was set at 6 s. Intercellular Ca2+ changes were detected by a cooled CCD camera, and analyzed with an image processing system including the multifunction image analysis software Aquacosmos (Hamamatsu Photonics, Hamamatsu, Japan). First of all, skilled experimenters detected neurons in phase-contrast images. The change in Fluo-4 AM emission at 505 nm in response to excitation at 488 nm was measured. The ratio intensity of the control image (F0) was set as the target neurons 4 s before the focal photolytic flash, and changes in the fluorescent ratio intensity (F) of each cell were monitored. The intercellular Ca2+ changes were analyzed using a series of images taken at 4-s intervals. F/F0 was used for evaluating the response of the intracellular Ca2+ concentration ([Ca2+]i) of neurons.
Fig. 2. Activation of NMDA receptors crucial to an increase in intracellular Ca2+ ([Ca2+]i) and neuronal death in the flashed area (FA) by focal photolysis of caged glutamate. A and B: Representative images of changes in [Ca2+]i at 3 different time points: 10 s before the exposure to UV (A1 and B1), and 10 s (A2 and B2) and 30 s (A3 and B3) after the flash. Figs. B1–B3 show changes in [Ca2+]i when the co-cultures were treated with 100 lM AP5, a specific inhibitor of NMDA receptors. [Ca2+]i increases from dark blue to red through yellow. The color bar on the right indicates the scale of ratio intensity. The scale bar indicates 100 lm. Treatment of co-cultures with AP5 resulted in the significant suppression of both the [Ca2+]i increase (C) and neuronal death (D and E). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄p < 0.05. Focal photolysis of caged glutamate induced an increase in [Ca2+]i within the FA but not outside it (F). The scale bar indicates 100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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The maximum increase of [Ca2+]i was defined as the maximum value of F/F0 during the observation period. After UV exposure, EBSS was replaced with CM and cultures were incubated at 37 °C in 5% CO2 for 1 h–1 day.
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ysis of caged glutamate. The intensity of immunofluorescence was measured for at least 20 cells in each of the areas (FA, SFA, and outside) in 4 different cultures. The definition of the areas was stated in the text. The average fluorescent intensity of cells in the shamtreated cultures was set at 100%.
2.4. Immunocytochemistry 2.6. Survival rate of neurons Cells were fixed with 4% paraformaldehyde and washed with PBS, followed by 0.01% Triton-X. They were then treated with 1% normal goat serum. Immunofluorescent labeling was done with antibodies directed against glial fibrillary acidic protein (GFAP) (Sigma, Saint Louis, USA; 1 lg/ml), GLAST (EAAT1) or GLT-1 (EAAT2) (Cell Signaling Technology, Denver, USA; 20 lg/ml or 20 lg/ml) for 1 day at 4 °C. Negative controls without each primary antibody were performed. Primary antibodies were visualized with Alexa Fluor 488-conjugated anti-rabbit or Alexa Fluor 633-conjugated anti-mouse antibodies (Invitrogen). Hoechst 33342 was used to detect nuclei. The immunoreactivity was observed with a confocal laser scanning microscope (FV300; Olympus Corporation). The image analysis was identical for each of the cultures.
The analysis of neuronal cellular death was performed following observation of the nuclear morphology using the fluorescent DNA-binding dyes HE and propidium iodide (PI). Cells were incubated with these dyes for 15 min at 37 °C, and individual nuclei were observed under the IX70 fluorescence microscope. The region inside a radius of 2.5 mm from the center was defined as the flashed area (FA). To take images correctly, experimenters drew the line with a square on the back of the dish before it was coated with poly-L-lysine. More than 4 individual images were randomly taken in each area. HE or PI-positive cells were counted automatically using Scion Image for Windows. The survival rate of neurons was calculated as the percentage of (HE–PI)/HE.
2.5. Semi-quantitative analyses of GLT-1 and GLAST expression
2.7. Monitoring of cytosolic nitric oxide production
Changes in the expression of the astrocytic glutamate transporters GLT-1 and GLAST were analyzed at 24 h after the focal photol-
Changes in the cytosolic NO concentration were monitored using a fluorescent NO probe, DAF-FM (Kojima et al., 1999). Cells
Fig. 3. Delayed neuronal death in the surroundings of the flashed area. Figs. A1 and A2 show the spatial distribution of PI-positive neurons at 1 h and 24 h after the termination of UV exposure for the photolysis of caged glutamate, respectively. The scale bar indicates 300 lm. Delayed neuronal death was observed at a radius of 3.0, 3.5, and 4.0 mm from the center of the flashed area (FA) (B1–B6), but not at less than 2.5 mm or more than 4.5 mm. Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄p < 0.05. Fig. C provides a schematic illustration of the FA, SFA, and outside. Abbreviations are the same as those in Fig. 1.
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were loaded with a NO indicator by incubation with 10 lM of DAF-FM DA for 4 h (0.5% DMSO). We have previously demonstrated that high concentrations of DAF-FM, more than 20 lM, seemed toxic to cells, since loading of cultures with a high concentration of DAF-FM sometimes resulted in morphological changes (Kawahara et al., 2006). Fluorescent images were acquired at 2-s intervals with a cooled CCD camera (C4880-80; Hamamatsu Photonics, Hamamatsu, Japan). An analysis of the acquired images was made with an image processing and measuring system (AQUACOSMOS, Hamamatsu Photonics). Free cytoslic NO was monitored by comparing the changes in fluorescence intensity at an excitation wavelength of 490 nm with the initial fluorescence intensity (F/F0), using an emission wavelength of 515 nm. Because DAF-FM is not a quantitative probe like DAF-2 (Kojima et al., 1999), no attempt was made to calibrate DAF-FM fluorescence.
2.8. Chemicals D(-)-2-Amino-5-phosphonopentanoic/phosphonovaleric acid (AP5) and NG-monomethyl-L-arginine (L-NMMA) were obtained from Sigma–Aldrich. Other chemicals were obtained from Wako Chemicals (Tokyo, Japan).
2.9. Statistic analysis The data are expressed as the mean ± standard error of the mean (SEM). Group comparisons were made using an analysis of variance (ANOVA) with Fisher’s test. A P-value of less than 0.01 or 0.05 was considered significant.
3. Results We first tried to induce glutamate-mediated acute excitotoxic neuronal death in a spatially-restricted region by focal photolysis of caged glutamate in mixed cultures of neurons and astrocytes (Fig. 1A). To uncage the compound, cultures were exposed to ultraviolet light (UV) within a radius of 2.5 mm from the center of the dish, an area defined here as the flashed area (FA) (Fig. 1B1). The duration of the UV flash and the concentration of caged glutamate were set at 6 s and 100 lM, respectively (Suppl. Fig. 1). Focal photolysis of caged glutamate resulted in cell death in the FA (Fig. 1B3 and B4), but not outside it (Fig. 1B3, B5, and B6). Since the morphology of astrocytes remained the same and few dead cells were observed in the FA, dead cells were considered neurons (Fig. 1D1 and D2). In addition, neither exposure to UV without the
Fig. 4. NMDA receptor-mediated neurotoxicity involved in the genesis of delayed neuronal death. Treatment with AP5 during the exposure to UV for uncaging glutamate resulted in the significant suppression of both acute neuronal death in the flashed area (FA) and delayed death in the surroundings of the FA (SFA) (A1, A2, and B). Note that treatment with AP5 only after the washout of caged and uncaged glutamate also suppressed the induction of delayed neuronal death (B). In contrast, in neuron-enriched cultures, delayed neuronal death was not observed in the SFA (D). Figs. C1 and C2 show anti-bIII tubulin-positive neurons (red) and GFAP-positive astrocytes (green), respectively, indicating a neuron-enriched culture. The scale bars indicate 50 lm (C1) and 100 lm (C2). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄ p < 0.01; ⁄p < 0.05. Abbreviations are the same as those in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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administration of caged glutamate nor the administration of caged glutamate without the UV flash induced neuronal death (Fig. 1C), suggesting that neuronal death in the FA was caused by uncaged glutamate, not by the UV itself or by caged glutamate. We next investigated the mechanisms responsible for the glutamate-mediated neuron-specific death in the FA. Photolysis of caged glutamate by UV induced an increase in the concentration of intracellular Ca2+ ([Ca2+]i) (Fig. 2A) and the increase was mostly restricted to cells in the FA (Fig. 2F). UV exposure itself did not increase [Ca2+]i in the FA (data not shown), suggesting that glutamate uncaged by photolysis induced the increase of [Ca2+]i in neurons. We then examined whether the activation of Ca2+-permeable glutamate receptors, NMDA receptors, was involved in the uncaged glutamate-mediated increase in [Ca2+]i and neuronal death. Treatment with AP5, a specific antagonist of NMDA receptors, reversed both the Ca2+ increase (Fig. 2B and C) and the death of neurons (Fig. 2D and E) in the FA caused by the photolysis of caged glutamate, suggesting that the activation of NMDA receptors was
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involved in the flash-induced neuronal death. Taken together, these results demonstrated that focal photolysis resulted in glutamate-mediated excitotoxic neuronal death in a spatially restricted area 1 h after the uncaging of glutamate. We named this type of neuronal death, acute neuronal death. We next investigated whether and how the localized neuronal death induced by the focal photolysis of caged glutamate affected surrounding neurons and astrocytes. Although both uncaged and caged glutamate in neuron/astrocyte co-cultures were washed out about 5 min after the exposure to UV, PI-positive dead neurons were observed outside of the FA 24 h after the washout (Fig. 3A2). We named this type of neuronal death, delayed neuronal death (DND). DND was observed within a radius of 2.5–4.0 mm from the center (Fig. 3B1–B6), area referred to as the surroundings of the FA (SFA) or ‘‘in vitro penumbra’’ (Fig. 3C). Treatment with AP5 during the exposure to UV resulted in a significant increase in the survival rate of neurons in the SFA (Fig. 4A1, A2, and B), suggesting that the DND was caused by the activation of NMDA
Fig. 5. Photolytic flash-induced decrease in the expression of the astrocytic glutamate transporter GLT-1 crucial to the induction of delayed neuronal death. The expression of astrocytic GLT-1 was significantly reduced in the flashed area (FA) and the surroundings of the FA (SFA), but not outside these areas (A1–A5), 24 h after the washout. The scale bar indicates 100 lm. Fig. C indicates that GLT-1 (green) was not expressed in anti-bIII tubulin-positive neurons. The scale bar indicates 100 lm. Treatment of co-cultures with 1.0 mM L-NMMA, an inhibitor of nitric oxide synthase (NOS), reversed the photolytic flash-induced decrease in the expression of astrocytic GLT-1 (D and E). Figs. D1–D3 indicate the expression of GLT-1 in the FA, SFA, and region outside these areas in L-NMMA-treated co-cultures, respectively. The scale bar indicates 100 lm. The expression of astrocytic GLAST was significantly increased in the flashed area (FA) and the surroundings of the FA (SFA), but not outside these areas (F1–F5), 24 h after the washout. The scale bar indicates 100 lm. In the L-NMMA-treated co-cultures, the survival rate of neurons in the SFA was significantly increased, but the effect was antagonized by treatment with 100 lM DHK, a specific blocker of astrocytic GLT-1, during the period after the washout of caged and uncaged glutamate (H). Delayed neuronal death was not observed in the co-cultures treated with L-NMMA but was observed on treatment with DHK (I). Data are expressed as the mean ± SEM (n = 12, different cultures). ⁄⁄p < 0.01; ⁄ p < 0.05. Abbreviations are the same as those in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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receptors during the UV flash. Unexpectedly, treatment with AP5 only after the washout of uncaged glutamate also resulted in a significant reduction in DND (Fig. 4B). The question then arises as the origin of glutamate involved in the NMDA-mediated DND in the SFA. Although uncaged glutamate was washed out about 5 min after the UV exposure, the possibility that uncaged glutamate which rapidly diffused to the SFA in this 5 min contributed to the induction of DND cannot be completely excluded. If this is the case, DND might be also observed in neuron-enriched cultures. We next examined this possibility (Fig. 4C1 and C2). Unexpectedly, DND in the SFA was not observed in neuron-enriched cultures (Fig. 4D), suggesting that uncaged glutamate was not primarily responsible for DND, and that functional changes in astrocytes, not in neurons, after acute neuronal death in the FA contributed to the induction of DND in the SFA observed in neuron/astrocyte co-cultures. A question then arises as to what functional changes in astrocytes were responsible for the induction of DND. We next addressed this issue mainly focusing on changes in the expression of GLT-1 (EAAT2), one of the glutamate transporters expressed in astrocytes, since astrocytic GLT-1 is primarily responsible for the uptake of extracellular glutamate in the brain (Robinson, 1999), and glutamate-mediated toxicity was possibly involved in DND in neuron/asctrocyte co-cultures as indicated above. The expression of GLT-1 was significantly decreased in the FA and SFA 24 h after the UV flash (Fig. 5A1–A5), suggesting that the decrease was responsible for the DND in the SFA. Unexpectedly, the expression of astrocytic GLAST was significantly increased in the FA and in the SFA, but not outside these areas (Fig. 5F1–F5), 24 h after the washout, suggesting that the decrease in the expression of astrocytic GLT-1 in the SFA was not caused by the loss of astrocytes. We have previously demonstrated that the enhanced production of nitric oxide (NO) derived from the NMDA R-mediated activation of Ca2+-dependent neuronal nitric oxide synthase (nNOS) expressed in neurons is involved in a decrease in the expression of GLT-1 in astrocytes in neuron/astrocyte co-cultures (Yamada et al., 2006). The intensity of DAF fluorescence significantly increased with the photolysis of caged glutamate in the FA, and this increase was significantly suppressed by treatment with L-NMMA, an inhibitor of NOS, suggesting that the production of NO was actually enhanced by the focal photolysis of caged glutamate (Suppl. Fig. 2). Treatment of co-cultures with L-NMMA reversed the focal photolysis-induced decrease in the expression of GLT-1 in the SFA (Fig. 5D and E). In addition, the treatment of co-cultures with L-NMMA increased the survival rate of neurons in the SFA, but the increase was antagonized by treatment with DHK, a specific blocker of GLT-1, during the period after the washout of uncaged glutamate (Fig. 5H). Moreover, DND in the SFA was not observed on L-NMMA treatment, but was observed on treatment with DHK (Fig. 5I). These results suggested that the L-NMMA-induced increase in the survival rate of neurons was caused by suppression of a decrease in GLT-1 expression in astrocytes. 4. Discussion The present study demonstrated that delayed neuronal death (DND) was actually induced in the surroundings of the flashed area (SFA) late after acute neuronal death in the flashed area (FA). In general, the ischemic penumbra has been described on the basis of cerebral blood flow (CBF), suggesting a possible causal link between a decrease in CBF, the extent of which is not severe to induce acute neuronal death, and DND in the penumbra. However, the present in vitro study indicating that functional changes in astrocytes caused by the spatially-restricted excitotoxic neuronal death were primarily responsible for the occurrence of DND in the SFA raises a possibility that most of the DND observed in the ischemic
penumbra in vivo has little or nothing to do with the prior mild reduction of CBF in the penumbral region during ischemia. This study demonstrated the possibility that the focal photolysis of caged glutamate resulted in a significant decrease in the expression of GLT-1 in the SFA, leading to the decreased uptake of extracellular glutamate and the NMDA receptor-mediated DND (Figs. 4 and 5). Although functional changes in astrocytes, not neurons, are involved in the induction of DND in the SFA (Fig. 5), astrocytes also express the glutamate transporter GLAST (EAAT1) in addition to GLT-1 (EAAT2). Therefore, we examined whether the expression of GLAST changed after the focal photolysis of caged glutamate. Unexpectedly, GLAST expression was significantly increased both in the FA and in the SFA (Fig. 5F1–F5), raising the question of whether this enhanced expression contributes to the clearance of extracellular glutamate. Previous studies have revealed that following transient focal cerebral ischemia in rats, impaired functioning of GLT-1, not GLAST, contributed to the initiation of DND in the hippocampus (Rao et al., 2000, 2001). The expression GLAST did not change, or actually increased after transient ischemia (Rao et al., 2001). In addition, Duan et al. (1999) reported that the application of glutamate induced a rapid increase in the expression of GLAST in cultured astrocytes. However, it seems generally accepted that astrocytic GLT-1, not GLAST, is primarily responsible for the uptake of extracellular glutamate in the brain (Torp et al., 1995; Tanaka et al., 1997; Robinson, 1999). Therefore, further studies are needed to clarify the functional significance of the enhanced expression of GLAST after focal photolysis of caged glutamate observed in this study. Previous studies have demonstrated that the enhanced production of nitric oxide (NO) is involved in glutamate-induced, NMDA receptor-mediated excitotoxic neuronal death (Dawson et al., 1991; Garthwaite, 1991; Kawahara et al., 2004; Strijbos et al., 1996). Also in the present study, the focal photolysis of caged glutamate-induced enhanced production of NO was involved in the acute death of neurons and in the decreased expression of GLT-1 in the FA (Suppl. Fig. 3). At present, however, it remains unclear whether there exists a direct causal link between the decreased expression of astrocytic GLT-1 and the glutamate-mediated acute neuronal death in the FA. We previously demonstrated in neuron/astrocyte co-cultures that Ca2+-dependent nNOS-derived NO was critically involved in a decrease in the expression of GLT-1 in astrocytes (Yamada et al., 2006). Although neuron-derived NO seemed crucial to the reduction in astrocytic GLT-1 expression, these results raise a question as to what mechanisms are responsible for the NO-induced down-regulation of GLT-1 expression in astrocytes. The present study also revealed that the glutamate-mediated enhanced production of NO was responsible for the decreased expression of astrocytic GLT-1 in the SFA, and suggested that the decreased GLT-1 expression contributed to the induction of DND in the SFA (Fig. 5). However, there are other possible explanations linking the enhanced production of NO with the induction of DND in the SFA. A previous study has demonstrated that NO causes release of glutamate and ATP from astrocytes, and the NO-induced release of astrocytic glutamate and ATP may be important in physiological or pathological communication between astrocytes and neurons (Bal-Price et al., 2002). ATP released from astrocytes may activate ATP-sensitive purinoceptors, such as P2X7 receptors. P2X7 receptors are permeable to larger molecules up to 800– 900 Da (Surprenant et al., 1996), and several studies have been shown that prolonged activation of such receptors leads to cellular death (Atkinson et al., 2004; Schrier et al. 2002). Recent studies have shown that pannexin hemichannels are the P2X7 receptorsassociated protein (Huang et al., 2007; Pelegrin and Surprenant, 2009), and the opening of the channels may be regulated by P2X7 receptors (Pelegrin and Surprenant, 2009). In addition,
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P2X7 receptors mediate the release of glutamate and GABA from rat hippocampal slices (Sperlágh et al., 2002). However, we have recently demonstrated that ATP, released through pannexin-1 (Px1) hemichannels, activates P2X7 receptors, resulting in the closure of Px1 hemichannels in cultured astrocytes, and such a negative-feedback mechanism might contribute to the survival of astrocytes under ischemic stress (Iwabuchi and Kawahara, 2011). Further studies will be needed to clarify the possible involvement of the NO-induced release of astrocytic glutamate and ATP in the induction of DND in the SFA. Previous studies have demonstrated that the interleukin-1b-NO pathway is responsible for the reduction in the expression of the inwardly-rectifying potassium channel Kir4.1 in astrocytes (Casamenti et al., 1999; Olsen et al., 2010; Zurolo et al., 2012). Kir4.1 potassium channels are involved in the regulation of ionic homeostasis in astrocytes, in particular the extracellular concentration of potassium, which influences neuronal excitability. The activity of sodium and potassium coupled astrocytic glutamate transporter GLT-1and GLAST depends on the negative membrane potential provided by Kir4.1 establishing a sufficient electrochemical gradient for glutamate import. These findings raise a possibility that the NO-induced reduction in the expression of Kir4.1 is crucially involved in the induction of DND in the SFA. In the present study, the focal photolysis of caged glutamate resulted in a significant decrease in the expression of GLT-1, but increased the expression of GLAST in the SFA (Fig. 5). The NO-induced reduction in the expression of Kir4.1 might be responsible for a decrease in the uptake of glutamate leading to the induction of DND in the SFA, although the expression of astrocytic GLAST was increased. Acknowledgements The analysis of immunoreactivity was carried out with a confocal laser scanning microscope; FV300 at the OPEN FACILITY, Hokkaido University Sousei Hall. This study was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (22300148) to KK. There is no conflict-of-interest.
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