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Research report

NMDA-sensitive neurons profoundly influence delayed

staurosporine-induced apoptosis in rat mixed cortical neuronal cultures

*

Craig E. Thomas , Douglas A. Mayle

Investigative Toxicology, Lilly Research Laboratories, A Division of Eli Lilly and Company, 2001 W. Main St., Greenfield, IN 46140, USA Accepted 8 August 2000

Abstract

We have investigated cell killing in cultured rat embryonic cortical neurons exposed to the protein kinase inhibitor staurosporine, the excitatory amino acid N-methyl-D-aspartate (NMDA), or a combination thereof. Our data indicate that there are several populations of

neurons that differ in their response to these agents. Cultures exposed to NMDA undergo cell death typified by lactate dehydrogenase (LDH) leakage which is likely primarily necrotic in that little caspase-3 activation or oligonucleosome formation is observed even when followed for 48 h. Cells exposed to staurosporine (STS) exhibit rapid, extensive activation of caspase-3 with coincident LDH leakage, oligonucleosome formation and TUNEL staining. Both LDH leakage and oligonucleosome content were significantly more elevated at 48 h than at 20 h with STS treatment while caspase-3 activity peaked early (8–20 h) and declined markedly by 48 h. Deletion of NMDA-responsive neurons by pre-treatment of the cultures with NMDA for 4 days prevented the late phase (20–48 h) increases in LDH leakage and oligonucleosomes in the remaining neuronal population. Caspase-3 activity was also completely abolished by NMDA pre-treatment. These results indicate that cells susceptible to acute NMDA-induced toxicity can be killed by non-apoptotic means when exposed to NMDA; however, they undergo a delayed, apoptotic death when exposed to STS. Interestingly, removal of NMDA-responsive cells prevents the processing of procaspase-3; thus, STS-induced apoptosis in cells resistant to NMDA-mediated killing proceeds independent of caspase-3 activation. The data indicate that nearly all neurons in these mixed cultures can undergo apoptosis in response to appropriate stimuli such as STS but that the temporal nature, and the pathways activated in response to STS, vary amongst the subpopulations of neurons. These findings may help to explain the simultaneous appearance of features of both apoptosis and necrosis observed in vivo following cerebral ischemia.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system Topic: Ischemia

Keywords: Neuron; Apoptosis; Necrosis; Staurosporine; N-Methyl-D-aspartate; Caspase

1. Introduction exhibiting features of apoptosis. Interestingly, caspase-8

activation occurred beginning at 6 h and was primarily Neuronal cell death following insult such as ischemia or localized to the large pyramidal neurons while caspase-3 trauma is a complex series of integrated events which can activation was not detected until 24 h and was localized to lead to neuronal loss via apoptosis and / or necrosis. Velier lamina II / III. In a model of controlled cortical impact et al. [28] have recently examined cell death and caspase injury in the rat, DNA damage indicative of both apoptotic activation in cortical regions following focal cerebral and necrotic cell death were observed at the site of impact ischemia. It was observed that there appeared to be an [20]. These findings indicate that delayed cell death early non-apoptotic, likely necrotic, loss of cortical neu- following ischemia is highly dependent upon the cell type rons followed by a progressive elimination of neurons and that conclusions concerning cell death can be strongly influenced by the time and region in which cell death is examined.

*Corresponding author. Lilly Development Centre, Parc Scientifique de

Ample in vitro evidence also suggests a multiplicity of Louvain-la-Neuve, Rue Granbonpre, 11, 1348 Mont-Saint-Guibert,

Bel-pathways leading to cell death in cultured neurons. Treat-gium. Tel.:132-10-47-6496; fax:132-10-47-6925.

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glutamate [2,24] to serum withdrawal [29] has provided apoptosis, such as STS, and necrosis, such as NMDA, may evidence for aspects of both apoptosis and necrosis. The affect cells differently depending upon cell type and protein kinase inhibitor staurosporine (STS) has been concentration of the activating agent. In an effort to more utilized by a number of investigators as an inducer of fully characterize the response of a mixed neuronal popula-neuronal apoptosis [13,15,23]. In cortical neurons, STS has tion to presumably apoptotic and necrotic insults we have been shown to activate apoptosis in both neurons [19,21] carefully studied STS and / or NMDA-induced cell death in and, at high concentration, in astrocytes [21]. DNA primary rat cortical neurons by monitoring LDH leakage, alterations and morphologic appearance were consistent caspase-3 activation and oligonucleosome formation. A with apoptosis. In PC12 cells, STS induced caspase-3 time-course experiment indicated that there appeared to be activity and, accordingly, the peptide inhibitor z-VAD.fmk, two phases to the STS-induced apoptotic process; the first a pan-selective inhibitor of caspases, prevented cell death occurred during the initial 24 h period of STS treatment [17]. In contrast, cell death elicited by the lipid peroxida- and was followed by increasing levels of oligonucleosome tion product 4-hydroxynonenal did not activate caspase-3 formation over an additional 24 h of treatment. NMDA nor did the caspase inhibitor prevent cell death. McManus treatment elicited cell death which showed little evidence et al. [19] compared STS-induced cell death to glutamate of apoptosis. Pre-treatment of cultures with NMDA prior toxicity in cortical cells and concluded that glutamate to STS demonstrated that the latter phase of cell death caused cell killing by non-apoptotic means as no evidence following STS was most likely due to the death of NMDA-for DNA laddering was observed and the appearance of the sensitive neurons which were exhibiting a delayed, apop-nuclei was distinct from that following STS treatment. totic death in response to STS. Furthermore, NMDA-While STS treated neurons are typified by features responsive neurons were obligatory for activation of associated with apoptosis, the exact mechanism of cell caspase-3 by STS. These findings indicate that: (1) most death remains unknown. In rat hippocampal neurons, the neurons undergo apoptosis in response to STS, (2) neurons caspase inhibitor DEVD-CHO prevented the STS-induced which die in a non-apoptotic fashion in response to NMDA increase in caspase-3 activity but did not ameliorate cell have the capacity to undergo delayed (20–48 h) apoptosis, death [16]. Choi et al. [10] have reported that z-VAD.fmk, (3) caspase-3 is activated specifically in NMDA-sensitive but not z-DEVD.fmk, a caspase-3-like protease inhibitor, neurons and (4) non-NMDA-sensitive neurons which can prevent STS-induced cell death in mouse neocortical undergo apoptosis with STS do so in a caspase-3 in-cultures. In SH-SY5Y cells exposed to STS, the protein dependent manner. Thus, it is clear that neuronal apoptosis synthesis inhibitor cycloheximide did not prevent cell in the in vivo setting is likely to involve both apoptosis and death [26] whereas it effectively does so in other cell types necrosis, and that the pattern observed is a reflection of the [10]. These disparate findings further suggest that induc- cell phenotypes and the nature of the activators of cell tion of apoptosis can be inducer and cell-type specific; death which are particular to the system under

inves-even in neurons. tigation.

An alternative mode of cell death occurring in neurons is necrosis which is generally considered to occur in

neurons following exposure to excitotoxins such as gluta- 2. Materials and methods

mate, NMDA and kainate [6,7,12,19,25]. As high

con-centrations of glutamate are achieved during ischemia, it 2.1. Cell culture was originally believed that cell death following ischemia

was primarily necrotic. In agreement, z-VAD.fmk was Rat cortical mixed neuronal and glial cultures were unable to block excitotoxicity induced with NMDA or prepared from gestational day 17 fetal rats. The cortex kainate [10]. Interestingly, they also showed that if the from each brain hemisphere was dissected under a micro-excitotoxicity was prevented by the NMDA channel scope and maintained in Hank’s balanced salt solution blocker MK-801, the surviving neurons gradually under- (HBSS) (Life Technologies, Gaithersburg, MD), on ice went death which was preventable by the caspase inhibitor. until the dissection was completed. Cortical tissue was In cultured cortical neurons, glutamate causes cell death then transferred to a tube containing room temperature which appears to be necrotic and distinct from that induced 0.025% (w / v) trypsin (Sigma, St. Louis, MO) in HBSS by STS [19]. However, in cerebellar granule cells, gluta- and mixed by gentle rocking for 10 min. Tissue was mate at low concentrations has been reported to induce allowed to settle, the trypsin-containing solution was caspase-3, with resultant apoptosis [8]. Neither NMDA nor aspirated, and the tissue was washed three times with MK-801 were shown to affect STS-induced cell death in minimum essential media (MEM) (Life Technologies, hippocampal neurons [23] suggesting that neurons suscep- Gaithersburg, MD). Cells were dissociated by trituration in tible to apoptosis are of a different phenotype than those MEM containing 25 mM glucose (Mallinckrodt Chemical, which undergo excitotoxicity. Paris, KY) and 2 mML-glutamine (Sigma, St. Louis, MO)

The conflicting reports which have emerged concerning through the mouth of a 10 ml glass pipette. Cells were cell death in in vitro cultures indicate that inducers of counted and plated in 24-well cell-culture plates (Costar,

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Corning, NY) or Lab Tek II 4-well chamber slides (Nalge nucleosome ELISA kit (Oncogene Research Products, Nunc International, Naperville, IL), which were previously Cambridge, MA) essentially as described by the manufac-coated with polyethylenimine (Sigma, St. Louis, MO) and turer. Briefly, the assay measured mono- and oligonucleo-conditioned with serum-containing media prior to cell somes in cell lysates by capture of free nucleosomes on the

culture initiation. Cells were plated at a density of 2.5310 surface of a DNA-binding protein coated microtiter plate cells / well in media composed of MEM, 25 mM glucose, 2 well. Anti-histone 3 biotin-labeled antibody was added, mM L-glutamine, 10% heat-inactivated horse serum and detection was accomplished with streptavidin-linked

(Sigma, St. Louis, MO), and 10% heat-inactivated fetal horseradish peroxidase conjugate which catalyzes the bovine serum (Life Technologies, Gaithersburg, MD) and conversion of a colorless substrate to yield a product maintained in a 5% CO , humidified, 372 8C incubator. Four which, following the addition of an acidified stop solution, days after plating, half of the culture media was removed absorbs at a wavelength of 450 nm.

and replaced with fresh culture media composed of MEM,

25 mM glucose, 2 mML-glutamine, and 10% heat-inacti- 2.4.2. Caspase-3 activity

vated horse serum. Cells were also treated at this time with Caspase-3 activity was measured in cell lysates using 15mg / ml 5-fluoro-29-deoxyuridine and 35mg / ml uridine the fluorogenic substrate N acetyl Asp Glu Val Asp -(both obtained from Sigma, St. Louis, MO) to minimize 7 - amino - 4 - trifluoromethylcoumarin (Ac-DEVD-AFC) non-neuronal cell growth. Additional media changes were (PharMingen International, San Diego, CA). Equal vol-conducted, as described above, at seven and eleven days in umes of cell lysate and reaction buffer containing 40 mM culture, and cells were treated for experimentation at HEPES, 200 mM NaCl, 2 mM EDTA, 0.2% (w / v)

twelve days in culture. CHAPS, 20% sucrose, 20 mM DTT, and 100 mM

Ac-DEVD-AFC were combined in a microtiter plate and 2.2. Treatment of cells with pharmacologic agents incubated at 378C for 1 h. Fluorescence was measured at an excitation wavelength of 380 nM and an emission 2.2.1. NMDA pre-treatment wavelength of 508 nm. Background fluorescence values, In order to effect deletion of NMDA-sensitive neurons, determined by measuring samples of cell lysis buffer cell cultures were exposed to 500 mM NMDA in cell without cell lysate and reaction buffer, were subtracted culture media for 4 days prior to STS treatment. When the from fluorescence units obtained from each sample. media change was conducted in pre-treated cultures on

culture day 11, NMDA was added to maintain a 500mM 2.4.3. Western blot analysis of caspase-3

concentration. Cells were lysed in 0.3 ml of insect cell lysis buffer

(PharMingen International, San Diego, CA) and samples

2.2.2. Treatment were stored at 2208C until analyzed. Samples were

Cells were exposed to treatment solutions by complete analyzed for protein content and 2.5mg total protein were aspiration of cell culture media and replacement with a mixed with 23Tris–glycine sample buffer (NOVEX USA, serum-free balanced salt solution (BSS) (116 mM NaCl, San Diego, CA) containing 3% b-mercaptoethanol and 5.4 mM KCl, 0.8 mM MgSO , 1.0 mM NaH PO , 0.94 2 4 heated to 958C for 5 min. Samples were loaded into a CaCl , 26 mM NaHCO , 25 mM glucose, pH 7.4).2 3 NOVEXEpre-cast 4–20% Tris–glycine gel and run on an Treatment agents (Sigma, St. Louis, MO) were dissolved electrophoresis apparatus at 125 constant volts for approxi-in BSS, except for STS which was dissolved approxi-in DMSO mately 2 h in NOVEXETris–glycine STS running buffer. (final treatment concentrations of DMSO did not exceed The gel was then removed from its casting and placed in

0.5%). BupHE (Pierce, Rockford, IL) Tris–glycine transfer

buf-fer. Proteins were transferred to a Hybond-P membrane at

2.3. Cytotoxicity 100 mA for 18 h at 88C. The membrane was blocked with

5% powdered milk, 0.1% Tween 20 in tris buffered saline. Cellular leakage of LDH was determined by collection Primary antibody for caspase-3, rabbit polyclonal IgG of an aliquot of the BSS treatment solution at the appro- (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted priate timepoint for analysis using the Modified Gay to a concentration of 0.2 mg / ml and incubated with the procedure [3,9] on a Hitachi 911 or Hitachi 914 analyzer membrane for 1 h. After washing, the membrane was (Roche Boehringer Mannhein, Indianapolis, IN). Total incubated with the secondary antibody, donkey anti-rabbit cellular LDH content was determined by lysis of control IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) at a cells with Triton X-100. concentration of 0.2 mg / ml for 45 min. Detection was accomplished with the use of ECL Plus Western blotting 2.4. Apoptosis related assays detection reagents (Amersham, Buckinghamshire, UK). 2.4.1. Oligonucleosome quantitation 2.4.4. TUNEL and DAPI staining

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from cells which had been cultured in 4-well chamber of oligonucleosomes was determined as an indicator of slides and cells washed once in phosphate buffered saline apoptosis. It can be seen that STS induced a 831% increase (PBS). Cells were then fixed for 30–45 min at room in cleavage of the substrate while NMDA produced a temperature in 4% paraformaldehyde followed by a single modest increase of 225%. The difference in the formation wash with PBS and storage at 48C until staining procedure of nucleosomes when comparing the two agents was much was performed. An in situ cell death detection kit from less, with STS treatment increasing nucleosome formation Boehringer Mannheim (Indianapolis, IN) was used to to 308% of control as compared to 225% for NMDA. conduct TUNEL staining. Briefly, terminal deoxynu- The finding that LDH leakage occurred with a pro-cleotidyl transferase is used to catalyze the polymerization apoptotic agent such as STS led us to examine the of nucleotides to free 39-OH DNA ends. Fluorescein labels timecourse of LDH leakage and caspase activation, and to incorporated into the nucleotide polymers are detectable by compare it to other inducers of cell death. In Fig. 1A it is fluorescence microscopy. Cells were permeabilized with shown that over a 20 h time period there is essentially no 0.1% Triton-X 100 for 2 min, washed twice with PBS, and temporal separation of LDH leakage and caspase-3 activa-incubated with the TUNEL reaction mixture at 378C in the tion in response to STS. Treatment of the cells with 100 dark for 1 h and rinsed with PBS. The chamber walls were mM hydrogen peroxide resulted in LDH leakage of ap-removed from each slide and one drop of Vectashield proximately 27% of total cellular LDH (Fig. 1B). Caspase-mounting media with DAPI (Vector Laboratories, Inc., 3 activity was also elevated but occurred more slowly than Burlingame, CA) was added to each well. A coverslip was LDH leakage. Exposure of the cultures to the nitric oxide placed on each slide and cells were examined using an oil donor, spermine NONOate, also lead to a similar degree of immersion lens on a fluorescent microscope at the follow- cell injury as assessed by LDH leakage (Fig. 1C). In ing wavelengths: TUNEL staining, excitation 450 nm, contrast to STS and peroxide treatment, no caspase-3 emission 565 nm; DAPI staining, excitation 360 nm, activity was detected. We have also determined that no

emission 460 nm. oligonucleosomes are generated via NONOate exposure

which also implies a non-apoptotic death with this agent (data not shown). These data suggest that caspase-3

3. Results activity is not simply activated non-specifically in response

to injury and that LDH leakage cannot be used to 3.1. Acute cytotoxicity distinguish necrosis from apoptosis. This finding is con-sistent with Grotton et al. [10] who utilized LDH leakage Treatment of rat cortical neurons with STS or NMDA in primary mouse cortical neurons as a marker of apop-for 20 h resulted in a similar 22–24% leakage of total tosis.

cellular LDH (Table 1). Immunostaining of similar

cul-tures and quantitation of fluorescence suggested that 40– 3.2. Cytotoxicity studied over 48 h 45% of the cells stained positive for neuron specific

enolase (results not shown); thus, roughly 50% of the total Considering the indications in the literature that STS neurons demonstrated LDH leakage during this time induced cell death via apoptotic mechanisms and NMDA frame. Caspase-3 activity was measured using cleavage of via necrotic mechanisms, it was surprising that the differ-Ac-DEVD-AFC while endonuclease-dependent formation ence in the magnitude of oligonucleosome formation Table 1

a Effect of 20 h treatment with staurosporine (STS) or NMDA on rat cortical cell cultures on LDH leakage, nucleosome formation, and caspase-3 activity

Treatment 500 nM 500mM 500 nM 500mM 500 nM 500mM

STS NMDA STS NMDA STS NMDA

Experiment % Total % Total % Control nucleosome % Control caspase-3

LDH LDH formation activity

1 24 20 293 208 697 172

2 19 13 258 209 977 353

3 24 24 373 243 864 221

4 25 28 325 216 980 224

5 28 24 289 249 635 137

Mean 24 22 308 225 831 221

S.E.M. 61 63 620 69 671 637

Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine, 500mM NMDA, or vehicle (0.5% DMSO) which remained for 20 h. LDH concentration in the media was monitored as a measure of cell membrane integrity, nucleosome formation was measured in cell lysates by an ELISA as a measure of apoptosis, and caspase-3 activation was measured in cell lysates by the use of the fluorogenic substrate Ac-DEVD-AFC.

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Fig. 2A, it can be seen that with NMDA, LDH leakage of 48 units that occurs between 20 and 48 h is comparable to the 57 unit increase in control cells. This contrasts with STS treatment where LDH leakage increases by 135 units during the 20–48 time period. In a similar manner, oligonucleosomes are not increased at 48 h relative to 20 h with NMDA treatment; whereas with STS, oligonucleo-some formation was 370% and 880% of control at 20 h

Fig. 1. The effect of staurosporine, hydrogen peroxide or spermine NONOate on LDH leakage and caspase-3 activation in cortical cultures. Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine (STS), 100mM hydrogen peroxide (H O ), 1 mM spermine NONOate,2 2 or vehicle (0.5% DMSO) which remained for up to 20 h. LDH concentration in the media was monitored as a measure of cell membrane integrity and caspase-3 activation was measured in cell lysates by the use of the fluorogenic substrate Ac-DEVD-AFC. (A) effect of STS treatment

over time; (B) effect of H O treatment over time; (C) effect of spermine2 2 _{Fig. 2. Comparison of cell damage elicited by NMDA or staurosporine at} NONOate treatment over time. Data points represent an average of

20 and 48 h of treatment. Cells were treated by removing the cell culture samples taken from 6 cell culture wells.

media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine, 500mM NMDA or vehicle (0.5% DMSO) which remained for 20 or 48 h. (A) LDH concentration in the media was between STS and NMDA-treated neuronal cultures was not

monitored as a measure of cell membrane integrity; (B) nucleosome greater. Also surprising was that the LDH leakage was

formation was measured in cell lysates by an ELISA as a measure of comparable, especially considering the magnitude of the _{apoptosis; (C) caspase-3 activation was measured in cell lysates by the} difference in caspase activity. Therefore, the effect of 48 h use of the fluorogenic substrate Ac-DEVD-AFC. Bars represent exposure to STS or NMDA on the cells was studied. In mean6standard deviation of three samples.

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control at 48 h. for 20 h as compared to 48 h NMDA treatment (Fig. 2A).

Morphological changes observed following STS treat- Furthermore, if cells were pretreated with the NMDA for 4 ment support the progression of apoptosis between 20 and days, washed and exposed again to NMDA, the cells which 48 h. Fig. 3 shows a dramatic increase in the relative survived the initial NMDA pre-treatment were refractory number of apoptotic bodies which demonstrated positive as indicated by similar levels of LDH leakage as control TUNEL staining between 20 and 48 h of STS treatment. cells at both 20 and 48 h, suggesting the complete Co-staining with DAPI showed that control cells retain a depletion of NMDA-sensitive neurons in those cultures normal nuclear morphology with no evidence of TUNEL (results not shown).

staining (data not shown). A detailed time course study was performed in cells which received NMDA pre-treatment and were sub-3.3. Effect of NMDA pre-treatment on cell death sequently exposed to STS for 48 h. Staurosporine treat-ment alone yielded the biphasic response with a 163 unit In an effort to ascertain the nature of the subpopulation increase in LDH leakage, while oligonucleosomes in-of neurons which undergoes an apparent delayed apoptotic creased from 558% to 1794% of control between 20 and

Fig. 3. TUNEL staining of neurons exposed to staurosporine for 20 or 48 h. Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine or vehicle (0.5% DMSO) which remained for 20 or 48 h. Cells were then fixed, permeabilized,and incubated with TUNEL reaction mixture as indicated in the Materials and methods section. Cells were visualized on a fluorescent microscope using a 603oil-immersion objective. (A) vehicle-treated control cells after 20 h of treatment; (B) staurosporine-treated cells after 20 h of treatment; (C) vehicle-treated control cells after 48 h of treatment; (D) staurosporine-treated cells after 48 h of treatment.

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Table 2

a Effect of 4-day pre-treatment with 500mM NMDA on rat cortical cell cultures

Pre-treatment condition LDH leakage (U / L) between day24 and day 0

Mean6S.D.

None 16.764.7

500mM NMDA 80.068.9

15mM MK8011500mM NMDA 10.060.0

15mM MK801 11.061.4

Cells were pretreated with 500mM NMDA, 15mM MK-801 and 500mM NMDA, 15mM MK-801 or vehicle which were added in a small volume directly to the culture media 4 days prior to the initiation of treatment. Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution which remained for 20 h. LDH concentration in the media was monitored as a measure of cell membrane integrity.

48 h (Fig. 4). NMDA pre-treatment had little effect on the STS-induced LDH leakage or oligonucleosome formation up to 20 h. Conversely, prior removal of NMDA-sensitive neurons totally abolished the delayed (20–48 h) apoptotic changes as evidenced by oliognucleosome formation (Fig. 4B). Caspase-3 activity was elevated 2300% in STS treated cells as early as 8 h, was maintained for at least 32 h, and declined by 48 h. Depletion of NMDA-sensitive neurons totally prevented the rise in caspase-3 like activity. Cells treated only with NMDA for 48 h showed little caspase-3 activity over the 48 h exposure. Analysis of similar treatment conditions for active caspase-3 by West-ern blot yielded similar results (Fig. 5). Staurosporine alone caused notable formation of active caspase-3 (17 kDa) and decreased levels of pro-caspase-3, but was unable to generate active caspase-3 in cultures which had been pretreated to remove NMDA-sensitive neurons. These data provide new evidence that NMDA-responsive neurons are required for activation of pro-caspase-3 by STS.

To further investigate the selective effects of NMDA pre-treatment on STS-induced apoptosis at 20 and 48 h, the effect of the NMDA antagonist MK-801 was tested in the experiment depicted in Fig. 6. Again, the delayed cell injury, as determined by LDH leakage and oligonucleo-some formation, was prevented by NMDA pre-treatment. The effects of NMDA pre-treatment on caspase-3 activa-tion and delayed apoptosis were totally prevented by the inclusion of 801 during the pre-treatment. Thus, MK-801 protected cells show a pattern comparable to control cells exposed to STS wherein there is a late phase release of LDH which is accompanied by oligonucleosome forma-tion. MK-801 treatment by itself had no effect on STS-induced cell killing.

Fig. 4. Timecourse effect of NMDA pre-treatment on

staurosporine-induced neuronal cell injury. Cells were pretreated with 500mM NMDA _{4. Discussion} or vehicle which were added in a small volume directly to the culture

media 4 days prior to the initiation of treatment. Cells were treated by

4.1. NMDA vs. staurosporine-induced neuronal cell removing the cell culture media and replacing with a serum-free balanced

death salt solution which contained 500 nM staurosporine or vehicle control

(0.5% DMSO) which remained for the indicated amount of time. (A)

LDH concentration in the media was monitored as a measure of cell _{Historically, it has been suggested that neurons can die} membrane integrity; (B) nucleosome formation was measured in cell _{by either apoptosis or necrosis; we have provided evidence} lysates by an ELISA as a measure of apoptosis; (C) caspase-3 activation

that specific populations of neurons, in this case, NMDA-was measured in cell lysates by the use of the fluorogenic substrate

sensitive neurons, can be killed by either mechanism. The Ac-DEVD-AFC. Bars represent mean6standard deviation of three

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Fig. 5. Effect of NMDA pre-treatment on staurosporine induced activation of caspase-3 as analyzed with Western blotting. Cells were pretreated with 500

mM NMDA or vehicle which were added in a small volume directly to the culture media 4 days prior to the initiation of treatment. Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution which contained 500 nM staurosporine or vehicle control (0.5% DMSO) which remained for 20 h. Cells were then lysed and processed for Western blot analysis of pro-caspase-3 and active caspase-3 protein levelsas described in the Methods. (A) control; (B) STS; (C) NMDA; (D) NMDA pre-treatment1STS; (E) blank; (F) Jurkat cell lysate.

(i.e. NMDA vs. STS) and there exists a distinct difference geneity in the response of the cultures to STS that might in the timeframe over which cell death ensues. When reflect killing of different subpopulations of neurons. As primary mixed cortical cultures are challenged with the our data, and that of others [6,7,11,19], has shown that excitotoxin NMDA, a sequence of events is rapidly excitotoxins can kill neurons containing the appropriate initiated such that the majority of cell death occurs over receptors via necrosis, we proceeded to eliminate the the next 24 h, with brief NMDA exposure eliciting cell NMDA-sensitive neurons and test for any effect on death equivalent to that observed with continuous applica- subsequent cell killing by STS. Depletion of NMDA-tion. This cell death is likely to be evoked as a result of sensitive neurons dramatically affected the response of the massive calcium influx and a loss of cell ion homeostasis remaining neurons to STS. First, the marked rise in

[7,12,25]. cleavage of Ac-DEVD-AFC in response to STS was

In our model, LDH leakage occurs with all injurious entirely absent (Fig. 4). This strongly implied that the agents tested and cannot be used to distinguish apoptosis enzyme that is responsible for this activity, presumably from necrosis. Similar observations have been made by caspase-3, resides within cells that are killed by NMDA. others in primary neocortical cultures [10]. Treatment with However, while Western blot analysis supported the ab-NMDA induced LDH leakage by 20 h which was not sence of STS stimulated formation of activated caspase-3 substantially increased between 20 and 48 h of treatment. in cultures pretreated with NMDA, it documented the At no timepoint did NMDA treated cells show evidence of continued presence of pro-caspase-3. Therefore, it is caspase-3 activation and there was no significant increase apparent that the presence of neurons (those that are in DNA fragmentation as judged by oligonucleosome susceptible to killing by 500 mM NMDA) is required for formation (Fig. 2). These data indicate, not surprisingly, activation of caspase-3 by STS. One view of these results that NMDA selectively kills a subpopulation of neurons could be that the non-activation of caspase-3 represents a via a non-apoptotic process. In contrast to NMDA, STS- basic phenotype of non-NMDA receptor-containing vs. induced LDH leakage was continuous over the 20–48 h receptor-containing cells. However, it should be considered treatment period. Cell death with STS was also typified by that we have not conducted immunohistochemical de-an early, robust increase in caspase-3 activity that declined tection of NMDA receptor-containing cells in these cul-after 32 h. Oligonucleosome formation was increased tures. Therefore, we cannot exclude the possibility that greater than 3-fold at 20 h and rose to 9-fold at 48 h. This some cells surviving the NMDA treatment do, in fact, marker of apoptosis was confirmed with a morphological contain some combination of NMDA receptor subunits; assessment which evaluated the formation of apoptotic thus, such a global statement cannot be made at this time. bodies with TUNEL staining. The relative number of cells The concordance between cell death induced by NMDA which had condensed nuclear material and intense TUNEL pre-treatment and that occurring from 20 to 48 h with STS staining increased dramatically between the 20 h and 48 h (as assessed by LDH leakage) can be interpreted, as have STS treatment timepoints which agrees with a delayed or we, that it is the NMDA-sensitive cells which account for late stage apoptosis. this delayed apoptosis with STS. However, one alternative explanation that cannot be discounted is that the NMDA-4.2. Effect of NMDA pre-treatment on staurosporine- sensitive cells represent only a minor percentage of the induced cell death cells which die in the late phase, and that the near total absence of apoptosis with STS following NMDA pre-The kinetics of LDH release, caspase-3 activation and treatment represents cells surviving the NMDA treatment oligonucleosome formation implied that there was hetero- experiencing a pre-conditioning or adaptive response

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injury. If the lack of caspase-3 activation following NMDA pre-treatment is an adaptive response, then one might expect that apoptosis would also be prevented. It was observed that the degree of LDH leakage and oligonucleo-some formation in response to STS treatment measured at 20 h was similar in the presence and absence of NMDA pre-treatment. This data indicated, first, that the population of neurons which undergoes early apoptosis with STS is not influenced by the absence of NMDA-sensitive cells. Second, it indicates that, during this early time period, those neurons sensitive to NMDA-induced toxicity are not undergoing apoptosis. Conversely, deletion of NMDA-sensitive neurons nearly totally abolished the late apoptotic changes that occurred over the 20–48 time frame. Clearly, additional work will be required to determine whether the lack of late phase apoptosis is attributable solely to abolition of these cells by NMDA, or to induction of an adaptive response in a subset of cells which are not sensitive to NMDA, but normally undergo only delayed apoptosis with STS. The ability of MK-801 to restore caspase-3 activation and the induction of delayed apoptosis with STS emphasizes the critical role that NMDA receptor-containing cells play, but does not definitively distinguish between these two possibilities.

If our data is interpreted that NMDA-sensitive neurons are susceptible to delayed injury with STS, the conclusion drawn is that NMDA-sensitive neurons can undergo either necrosis or apoptosis. Cell death elicited by NMDA occurs primarily over the first 20 h and is prevented by the channel blocker MK-801. This calcium-dependent killing is likely necrotic; nonetheless, our data suggests that these same cells are capable of participating in apoptosis when exposed to STS. McManus et al. [19] have compared cell killing by STS and glutamate in cultured rat cortical neurons and demonstrated that apoptotic cell death in response to STS was not mirrored by glutamate, which induced necrosis. Our results with NMDA and STS are in agreement with that work which was conducted only in the 24 h and less time period. The present work suggests that had these investigators monitored cell death for an addi-Fig. 6. Effect of MK-801 on NMDA or staurosporine induced cell injury. _{tional 24 h there would have been noted additional} Cells were pretreated with 500mM NMDA, 15mM MK801 and 500mM

occurrence of apoptosis upon treatment with STS. NMDA, 15mM MK801 or vehicle which were added in a small volume

The ability of NMDA-sensitive neurons to undergo directly to the culture media 4 days prior to the initiation of treatment.

apoptosis has been described previously [4]. When rat Cells were treated by removing the cell culture media and replacing with

a serum-free balanced salt solution which contained 500 nM stauro- _{neocortical cultures were exposed to 10 min of NMDA,} sporine or vehicle control (0.5% DMSO) which remained for 20 or 48 h. ₃₀₀_m_{M NMDA elicited a slowly evolving apoptosis while} (A) LDH concentration in the media was monitored as a measure of cell

2 mM NMDA generated a more rapid, necrotic cell death. membrane integrity; (B) nucleosome formation was measured in cell

Co-treatment with MK-801 prevented necrosis and apop-lysates by an ELISA as a measure of apoptosis; (C) caspase-3 activation

tosis; thus, both mechanisms were activated via the NMDA was measured in cell lysates by the use of the fluorogenic substrate

Ac-DEVD-AFC. Bars represent mean6standard deviation of three sam- _{receptor gated calcium channel. Our study of STS over a}

ples. _{48 h time period has demonstrated that NMDA-sensitive}

neurons likely undergo delayed apoptosis, which is in-which alters their response(s) to subsequent apoptotic dependent of receptor activation by an excitotoxin, and is stimuli. Induction of tolerance is well described for therefore insensitive to MK-801. Support for the ability of ischemia [5] and, very recently 3-nitropropionic acid [18], neurons to selectively undergo apoptosis or necrosis has both of which protect against subsequent ischemia-induced also been gained using cerebellar granule cells [1].

(10)

Gluta-mate exposure for 30 min induced a rapid loss of mito- suggests that caspase-3 is not involved in STS-induced cell chondrial membrane potential and death by necrosis within injury. However, the latest timepoint studied was 24 h and 3 h. In neurons which were able to restore a mitochondrial our findings implicate caspase-3 only in the more delayed membrane potential there was observed a delayed apop- apoptotic death of NMDA-sensitive neurons. In their tosis as judged by oligonucleosome formation and DNA studies the protein synthesis inhibitor cycloheximide pre-laddering. This substantiates the ability of excitotoxins to vented STS-induced cell death. We have seen no protection elicit either necrosis or apoptosis depending upon the with this agent at either 20 or 48 h which agrees with intensity of the insult and the time over which cell death is others whom have suggested that transcriptional activation monitored. Similar results using the calcium ionophore and new protein synthesis does not occur with STS-A23187 in striatal neurons have been recently reported induced apoptosis (for review see [27]). Interestingly, [22]. Our data extends this work by demonstrating that these authors determined that prevention of excitotoxic cell NMDA receptor containing neurons can undergo apoptosis death with MK-801 in cultures subjected to oxygen / glu-without receptor activation. cose deprivation led to an apoptotic injury. These results are analogous to our data which describes the ability of 4.3. Caspase involvement in cell killing by NMDA and MK-801 to prevent NMDA pre-treatment from ameliorat-staurosporine ing the late phase apoptosis. This suggests a commonality between STS and oxygen / glucose deprivation in the Our results suggest that the pathways leading to late activation of apoptosis in these cultures.

phase oligonucleosome formation in NMDA-sensitive neu- It is intriguing that Feuerstein et al. [28] have recently rons may be somewhat divergent from that in the neurons described that caspase-8 activity is observed within 6 h that undergo STS-induced apoptosis during the first 20 h. following focal ischemia in the rat and that caspase-3 is With STS treatment, caspase-3 activation was a relatively not activated until 24 h. Furthermore, the demonstration early event, reaching 2500% of control by 8 h. The loss of that caspase-8 protein was detectable predominately in caspase-3 activation with NMDA pre-treatment localized lamina V of the cortex while caspase-3 was in lamina II / III the activity to these neurons, yet they apparently did not provides evidence of both a temporal and regional (cell-undergo apoptosis until the latter phase of the experiment specific) pattern to neuronal apoptosis which is consistent (20–48 h). This implies that, in these neurons, caspase-3 with our in vitro findings. Together, these data highlight activation significantly precedes cell death. The STS-in- the need to fully characterize neuronal apoptosis when duced death of neurons which do not succumb to NMDA, multiple cell types are present. Further work is required to and are eliminated in the first 20 h, does not appear to determine if NMDA-sensitive cells account solely for the involve caspase-3 as NMDA pre-treatment prevented delayed apoptosis with STS, or if they in fact induce an caspase-3 activation, but had no impact on their apoptosis. adaptive response (vis a vis prevention of caspase activa-The alternative explanation for our data, as discussed tion) in other neurons. Such studies may provide new earlier, would suggest that elimination of NMDA-sensitive insight regarding intercellular control of apoptosis. neurons induced an adaptive response in other neurons.

The lack of pro-caspase-3 activation could be one

mecha-nism involved in this adaptation. _{Acknowledgements}

In hippocampal neurons, it has been demonstrated that

irradiation induces apoptosis and cleavage of caspase-3 _{We would like to thank Thomas K. Baker for} perform-substrates, yet cell killing was insensitive to z-DEVD.fmk, _{ing the Western blot for determination of caspase-3} activa-a peptide inhibitor of cactiva-aspactiva-ase-3 like proteactiva-ases [14]. Cleactiva-ar- _{tion and Dr. Phil Skolnick for helpful discussions and} ly, lack of absolute substrate specificity amongst the _{reading of the manuscript prior to its submission.}

caspases and difficulties with cell permeability complicates interpretations of activity-based measurements and further experimentation will be required to understand the role of

References

caspases, other than caspase-3, in the STS-induced apop-tosis in NMDA-insensitive neurons. Our preliminary data

[1] M. Ankarcrona, J.M. Dypbuki, E. Bonfoco, B. Zhivotovsky, S. has shown that caspase-8 is elevated 7.5-fold at 20 h with

Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal STS, and that NMDA pre-treatment prevents its activation. _{death: a succession of necrosis or apoptosis depending on}

mito-Choi’s laboratory has compared the effectiveness of chondrial function, Neuron 15 (1995) 961–973.

inhibitors of apoptosis on prevention of cell death follow- [2] M. Ankarcrona, Glutamate induced cell death: apoptosis or necro-sis?, Prog. Brain Res. 116 (1998) 265–272.

ing STS, excitotoxins, serum deprivation or oxygen /

glu-[3] H.U. Bergmeyer, Methods of Enzymatic Analysis, III, 3rd Edition, cose deprivation [10]. Z-VAD.fmk was capable of

inhib-Verlag Chemie, Weinheim, 1983.

iting cell death induced by many agents while z- _{[4] E. Bonfoco, D. Krainic, M. Ankarcrona, P. Nicotera, S.A. Lipton,} DEVD.fmk was without effect. Although cleavage of Apoptosis and necrosis: two distinct events induced, respectively, by fluorogenic substrates was not monitored, this data also mild and intense insults with N-methyl-D-asparate or nitric oxide /

(11)

superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. 92 (1995) neural cell apoptosis and necrosis, J. Neurochem. 72 (1999) 529–

7162–7166. 539.

[5] J. Chen, R. Simon, Ischemic tolerance in the brain, Neurology 48 [18] T. Kuroiwa, I. Yamada, S. Endo, Y. Hakamata, U. Ito, 3-Nitro-(1997) 306–311. propionic acid preconditioning ameliorates delayed neurological [6] D.W. Choi, M. Maulucci-Gedde, A.R. Kriegstein, Glutamate neuro- deterioration and infarction after transient focal cerebral ischemia in

toxicity in cortical cell culture, J. Neurosci. 7 (1987) 357–368. gerbils, Neurosci. Lett. 283 (2000) 145–148.

[7] F. Dessi, C. Charriaut-Marlangue, M. Khrestchatisky, Y. Ben-Ari, [19] J.P. MacManus, I. Rasquinha, M.A. Black, N.B. Laferriere, R. Glutamate-induced neuronal death is not programmed cell death in Monette, T. Walker, P. Morley, Glutamate-treated rat cortical cerebellar culture, J. Neurochem. 60 (1993) 1953–1955. neuronal cultures die in a way different from the classical apoptosis [8] Y. Du, K.R. Bales, R.C. Dodel, E. Hamilton-Byrd, J.W. Horn, D.L. induced by staurosporine, Exper. Cell Res. 233 (1997) 310–320.

Czilli, L.K. Simmons, B. Ni, S.M. Paul, Activation of a caspase [20] J.K. Newcomb, X. Zhao, B.R. Pike, R.L. Hayes, Temporal profile of 3-related cysteine protease is required for glutamate-mediated apoptotic-like changes in neurons and astrocytes following con-apoptosis of cultured cerebellar granule neurons, Proc. Natl. Acad. trolled cortical impact injury in the rat, Exp. Neurol. 158 (1999)

Sci. 94 (1997) 11657–11662. 76–88.

[9] R.J. Gay, R.B. McComb, G.N. Bowers Jr., Optimum reaction [21] M. Patel, Inhibition of neuronal apoptosis by a metalloporphyrin conditions for human lactate dehydrogenase isoenzymes as they superoxide dismutase mimic, J. Neurochem. 71 (1998) 1068–1074. affect total lactate dehydrogenase activity, Clin. Chem. 14 (1968) [22] A. Petersen, R.F. Castilho, O. Hansson, T. Wieloch, P. Brundin, 740–753. Oxidative stress, mitochondrial permeability transition and activa-[10] F.J. Gottron, H.S. Ying, D.W. Choi, Caspase inhibition selectively tion of caspases in calcium ionophore A23187-induced death of

reduces the apoptotic component of oxygen-glucose deprivation- cultured striatal neurons, Brain Res. 857 (2000) 20–29.

induced cortical neuronal cell death, Mol. Cell. Neurosci. 9 (1997) [23] J.H. Prehn, J. Jordan, G.D. Ghadge, E. Preis, M.F. Galindo, R.P. 21

159–169. Roos, J. Krieglstein, R.J. Miller, Ca and reactive oxygen species [11] B.J. Gwag, D. Lobner, J.Y. Koh, M.B. Wie, D.W. Choi, Slowly- in staurosporine-induced neuronal apoptosis, J. Neurochem. 68

triggered excitotoxicity occurs by necrosis in cortical cultures, (1997) 1679–1685.

Neuroscience 77 (1997) 393–401. [24] C. Portera-Cailliau, D.L. Price, L.J. Martin, Non-NMDA and [12] N. Hori, J.M.H. Ffrench-Mullen, D.O. Carpenter, Kainic acid NMDA receptor-mediated excitotoxic neuronal deaths in adult brain

responses and toxicity show pronounced Ca dependence, Brain are morphologically distinct: further evidence for an apoptosis-Res. 358 (1985) 380–384. necrosis continuum, J. Comp. Neurol. 378 (1997) 88–104. [13] M. Jacobson, M.C. Raff, Programmed cell death in Bcl-2 protection [25] M.T. Price, J.W. Olney, L. Sampson, J. Labruyere, Calcium influx

in very low oxygen, Nature 374 (1995) 814–816. accompanies but does not cause excitotoxin-induced neuronal [14] M.D. Johnson, H. Xiang, S. London, Y. Kinoshita, M. Knudson, M. necrosis, Brain Res. Bull. 14 (1985) 369–376.

Mayberg, S.J. Korsmeyer, R.S. Morrison, Evidence for involvement [26] J.A. Prince, L. Oreland, Staurosporine differentiated human SH-of Bax and p53, but not caspases, in radiation-induced cell death SH-of SY5Y neuroblastoma cultures exhibit transient apoptosis and trophic cultured postnatal hippocampal neurons, J. Neurosci. Res. 54 (1998) factor independence, Brain Res. Bull. 43 (1997) 515–523. 721–733. [27] L.L. Rubin, Neuronal cell death: when, why and how, Br. Med. [15] J.Y. Koh, B.J. Gwag, S.L. Sensi, L.M.T. Canzoniero, J. Demaro, C. Bull. 53 (1997) 617–631.

Czernansky, D.W. Choi, Staurosporine-induced neuronal apoptosis, [28] J.J. Velier, J.A. Ellison, K.K. Kikly, P.A. Spera, F.C. Barone, G.Z. Exp. Neurol. 135 (1995) 153–159. Feurestein, Caspase-8 and caspase-3 are expressed by different [16] A.J. Krohn, E. Preis, J.H. Prehn, Staurosporine-induced apoptosis of populations of cortical neurons undergoing delayed cell death after

cultured rat hippocampal neurons involves caspase-1-like proteases focal stroke in the rat, J. Neurosci. 19 (1999) 5932–5941. as upstream initiators and increased production of superoxide as a [29] M. Villalba, J. Bockaert, L. Journot, Concomitant induction of main downstream effector, J. Neurosci. 18 (1998) 8186–8197. apoptosis and necrosis in cerebellar granule cells following serum [17] I.I. Kruman, M.P. Mattson, Pivotal role of mitochondrial calcium in and potassium withdrawal, Neuroreport 8 (1997) 981–985.

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and 48 h, respectively (Fig. 2B). Caspase-3-like activity death in response to STS, the cells which were sensitive to was elevated 560% at 20 h in response to STS, but killing by NMDA were eliminated by treatment for 4 days declined to control level by 48 h (Fig. 2C). The decline at with 500 mM NMDA. Table 2 shows the effect of pre-the later timepoint could be due to loss of measurable treatment of the neurons with NMDA. LDH leakage was activated protease as a result of cell lysis and release of the completely prevented by inclusion of the NMDA channel protease into the media. A comparatively modest 82% blocker MK-801 which demonstrates elimination of only increase in caspase-3 activity was observed upon NMDA NMDA-responsive neurons. As described earlier, there is treatment after 20 h, this activity was decreased to 41% of little additional LDH leakage in cells exposed to NMDA control at 48 h. for 20 h as compared to 48 h NMDA treatment (Fig. 2A). Morphological changes observed following STS treat- Furthermore, if cells were pretreated with the NMDA for 4 ment support the progression of apoptosis between 20 and days, washed and exposed again to NMDA, the cells which 48 h. Fig. 3 shows a dramatic increase in the relative survived the initial NMDA pre-treatment were refractory number of apoptotic bodies which demonstrated positive as indicated by similar levels of LDH leakage as control TUNEL staining between 20 and 48 h of STS treatment. cells at both 20 and 48 h, suggesting the complete Co-staining with DAPI showed that control cells retain a depletion of NMDA-sensitive neurons in those cultures normal nuclear morphology with no evidence of TUNEL (results not shown).

(2)

Table 2

Effect of 4-day pre-treatment with 500mM NMDA on rat cortical cell cultures

Pre-treatment condition LDH leakage (U / L) between day24 and day 0

Mean6S.D.

None 16.764.7

500mM NMDA 80.068.9

15mM MK8011500mM NMDA 10.060.0

15mM MK801 11.061.4

Fig. 4. Timecourse effect of NMDA pre-treatment on

staurosporine-induced neuronal cell injury. Cells were pretreated with 500mM NMDA _{4. Discussion} or vehicle which were added in a small volume directly to the culture

media 4 days prior to the initiation of treatment. Cells were treated by

4.1. NMDA vs. staurosporine-induced neuronal cell removing the cell culture media and replacing with a serum-free balanced

death

salt solution which contained 500 nM staurosporine or vehicle control (0.5% DMSO) which remained for the indicated amount of time. (A)

that specific populations of neurons, in this case, NMDA-was measured in cell lysates by the use of the fluorogenic substrate

sensitive neurons, can be killed by either mechanism. The Ac-DEVD-AFC. Bars represent mean6standard deviation of three

(3)

Fig. 5. Effect of NMDA pre-treatment on staurosporine induced activation of caspase-3 as analyzed with Western blotting. Cells were pretreated with 500

[7,12,25]. cleavage of Ac-DEVD-AFC in response to STS was

induced cell death cells which die in the late phase, and that the near total absence of apoptosis with STS following NMDA pre-The kinetics of LDH release, caspase-3 activation and treatment represents cells surviving the NMDA treatment oligonucleosome formation implied that there was hetero- experiencing a pre-conditioning or adaptive response

(4)

occurrence of apoptosis upon treatment with STS. NMDA, 15mM MK801 or vehicle which were added in a small volume

The ability of NMDA-sensitive neurons to undergo directly to the culture media 4 days prior to the initiation of treatment.

apoptosis has been described previously [4]. When rat Cells were treated by removing the cell culture media and replacing with

a serum-free balanced salt solution which contained 500 nM stauro- _{neocortical cultures were exposed to 10 min of NMDA,} sporine or vehicle control (0.5% DMSO) which remained for 20 or 48 h. ₃₀₀_{mM NMDA elicited a slowly evolving apoptosis while} (A) LDH concentration in the media was monitored as a measure of cell

2 mM NMDA generated a more rapid, necrotic cell death. membrane integrity; (B) nucleosome formation was measured in cell

Co-treatment with MK-801 prevented necrosis and apop-lysates by an ELISA as a measure of apoptosis; (C) caspase-3 activation

tosis; thus, both mechanisms were activated via the NMDA was measured in cell lysates by the use of the fluorogenic substrate

Ac-DEVD-AFC. Bars represent mean6standard deviation of three sam- _{receptor gated calcium channel. Our study of STS over a}

ples. _{48 h time period has demonstrated that NMDA-sensitive}

(5)

ameliorat-staurosporine ing the late phase apoptosis. This suggests a commonality between STS and oxygen / glucose deprivation in the Our results suggest that the pathways leading to late activation of apoptosis in these cultures.

The lack of pro-caspase-3 activation could be one

mecha-nism involved in this adaptation. _{Acknowledgements} In hippocampal neurons, it has been demonstrated that

caspases and difficulties with cell permeability complicates interpretations of activity-based measurements and further experimentation will be required to understand the role of

References caspases, other than caspase-3, in the STS-induced

apop-tosis in NMDA-insensitive neurons. Our preliminary data

[1] M. Ankarcrona, J.M. Dypbuki, E. Bonfoco, B. Zhivotovsky, S. has shown that caspase-8 is elevated 7.5-fold at 20 h with

Orrenius, S.A. Lipton, P. Nicotera, Glutamate-induced neuronal STS, and that NMDA pre-treatment prevents its activation. _{death: a succession of necrosis or apoptosis depending on}

mito-Choi’s laboratory has compared the effectiveness of chondrial function, Neuron 15 (1995) 961–973.

inhibitors of apoptosis on prevention of cell death follow- [2] M. Ankarcrona, Glutamate induced cell death: apoptosis or necro-sis?, Prog. Brain Res. 116 (1998) 265–272.

ing STS, excitotoxins, serum deprivation or oxygen /

glu-[3] H.U. Bergmeyer, Methods of Enzymatic Analysis, III, 3rd Edition, cose deprivation [10]. Z-VAD.fmk was capable of

inhib-Verlag Chemie, Weinheim, 1983.

(6)

superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. 92 (1995) neural cell apoptosis and necrosis, J. Neurochem. 72 (1999) 529–

7162–7166. 539.

toxicity in cortical cell culture, J. Neurosci. 7 (1987) 357–368. gerbils, Neurosci. Lett. 283 (2000) 145–148.

Sci. 94 (1997) 11657–11662. 76–88.

reduces the apoptotic component of oxygen-glucose deprivation- cultured striatal neurons, Brain Res. 857 (2000) 20–29.

induced cortical neuronal cell death, Mol. Cell. Neurosci. 9 (1997) [23] J.H. Prehn, J. Jordan, G.D. Ghadge, E. Preis, M.F. Galindo, R.P.

triggered excitotoxicity occurs by necrosis in cortical cultures, (1997) 1679–1685.

Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol884.Issue1-2.2000:

Research report

NMDA-sensitive neurons profoundly influence delayed

staurosporine-induced apoptosis in rat mixed cortical neuronal cultures

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Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol884.Issue1-2.2000:

Research report

NMDA-sensitive neurons profoundly influence delayed

staurosporine-induced apoptosis in rat mixed cortical neuronal cultures

*

Craig E. Thomas , Douglas A. Mayle

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