神经酰胺诱导小鼠皮层神经元凋亡

本实验以原代培养的小鼠大脑皮层神经元为模型,观察了天然神经酰胺在神经元凋亡中的作用。从测定ＬＤＨ漏出率、ＭＴＴ代谢率、ＤＮＡ凝胶电泳,Ｇｉｅｍｓａ染色以及电镜观察等各个方面,探讨了神经酰胺对神经元的作用模式。研究发现,神经酰胺在５００～１０００ｎＭ浓度范围内,作用１２ｈ以上,即可诱导原代培养的皮层神经元凋亡,而且此作用具有时间依赖性和剂量依赖性。表明神经酰胺不仅对ＨＬ－６０细胞有促凋亡的作用,对大脑皮层神经元同样具有促凋亡的作用。     

Rapid Communication: Oxidative Stress Induces Apoptosis in Embryonic Cortical Neurons

Abstract: Glutamate-induced glutathione depletion in immature embryonic cortical neurons has been shown to lead to oxidative stress and cell death. We have used this in vitro model to investigate the mechanism(s) by which free radicals induce neuronal degeneration. We find that glutathione depletion leads to hypercondensation and fragmentation of chromatin into spherical or irregular shapes, a morphologic signature of apoptosis. These morphologic changes are accompanied by laddering of DNA into multiple oligonucleosomal fragments and can be prevented by the antioxidants idebenone and butylated hydroxyanisole. Cell death induced by glutathione depletion can also be prevented by inhibitors of macromolecular synthesis. Taken together, these observations suggest that oxidative stress can induce apoptosis in neurons.     

Respiration in Primary Cultured Cerebellar Granule Neurons and Cerebral Cortical Neurons

Respiration was measured polarographically in primary cultures enriched with cerebellar granule neurons or cerebral cortical neurons. The basal respiratory rate, measured on the sixth day after culturing, was 12.00 natom equiv. O/mg protein/min for the cortical neurons and 12.70 natom equiv. O/mg protein/min for the granule neurons. Maximal stimulation by 2,4-dinitrophenol produced a 20-40% increase over the basal rate for both neuronal types. Oligomycin inhibited neuronal basal respiration by 45%. These respiratory rates in neurons from primary culture are markedly lower than those measured in astrocytes grown under similar conditions.     

Guattegaumerine Protects Primary Cultured Cortical Neurons Against Oxidative Stress Injury Induced by Hydrogen Peroxide Concomitant with Serum Deprivation

Guattegaumerine is a natural product with characteristics of being lipophilic and reaching high concentration in the brain, but its function in the central nervous system has not yet been observed. This study was designed to evaluate the neuroprotective effects of guattegaumerine on rat primary cultured cortical neurons. Following a 24-h exposure of the cells to combined serum-starvation and hydrogen peroxide, a significant augment in neuron damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release were observed. Preincubation of guattegaumerine dramatically improved the cell viability and inhibited LDH release. Preincubation of guattegaumerine also dramatically inhibited malondialhehyde (MDA) production and elevated the decreased total antioxidative capacity in cells caused by the combined injury. Results of flow cytometry and immunohistochemistry showed that pre-addition of guattegaumerine interrupted the apoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and the down regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerine suppressed the increase of intracellular calcium ([Ca²⁺]_i) stimulated by either H₂O₂ or KCl in Ca²⁺-containing extracellular solutions, and high concentration of 2.5 μM guattegaumerine also suppressed the increase of [Ca²⁺]_i induced by H₂O₂ in Ca²⁺-free solution. These observations suggested that guattegaumerine may possess potential protection against oxidative stress injury, which might be beneficial for neurons.     

Caspase-dependent Apoptosis Induced by Thapsigargin was Prevented by Glycogen Synthase Kinase-3 Inhibitors in Cultured Rat Cortical Neurons

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca²⁺-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9–10). Blockade of N-methyl-d-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3β activation in rat cortical neurons.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

Neurotrophic Effects of GnRH on Neurite Outgrowth and Neurofilament Protein Expression in Cultured Cerebral Cortical Neurons of Rat Embryos

The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.     

小脑颗粒神经元谷氨酸兴奋性毒性模型的优化

在急性、慢性神经退行性疾病和炎症引发的神经系统疾病的发病机制中,兴奋性毒性可能是造成后期神经元死亡的共同途径．小脑颗粒神经元谷氨酸兴奋性毒性模型是研究上述过程的重要实验手段,该模型的稳定性和可重复性是开展相关研究的重要基础．然而,文献报道的建模方法条件各异,说法不一,很难适从．本工作针对小脑颗粒神经元谷氨酸兴奋性毒性模型建立的关键环节,包括小脑颗粒神经元的培养、兴奋性毒性刺激条件的确定,毒性标志性指标的表征,分别进行了比较和优化, 从培养皿的包被、神经元消化、兴奋性刺激的溶液介质选择、神经元刺激的最佳时间及谷氨酸的最佳刺激浓度等方面分别给出了优化条件．通过特征性钙离子曲线、NMDA受体特异性抑制剂MK-801的干预作用以及c-fos基因转录水平的动力学变化等指标,确认了毒性模型的成功建立．本工作不仅对建立小脑颗粒神经元谷氨酸兴奋性毒性模型的实验室具有重要参考意义,而且,其针对不同条件分析比较的结果及优化原则,对其他神经毒性模型的建立也具有普遍参考意义．     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K⁺ (25 m M ) and then switched to physiological concentrations of K⁺ (5 m M ) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of G_s with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K⁺, whereas stimulation of G_o/G_i with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K⁺. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell-permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.     

Cocaine Induces Apoptosis in Cortical Neurons of Fetal Mice

Abstract: Exposure of fetal mouse brain cocultures to cocaine results selectively in the loss of neurites followed by neuronal death. By using enriched neuronal cultures, we here demonstrate that disappearance of neurons, when cultured with cocaine, is caused by apoptosis, based on (1) characteristic morphology of apoptotic nuclei at the level of neurons but not of glial cells by optic microscopy, and on total cell pellets by electron microscopy; (2) fragmentation of total DNA with a typical "ladder" pattern on agarose gels; (3) extensive in situ DNA fragmentation labeling (TUNEL method); and (4) prevention of cell loss by cycloheximide. The major metabolites of cocaine have no detectable effects on neurons, indicating that apoptosis is due to cocaine itself. Inappropriate neuronal apoptosis in cocaine-exposed fetal brain could perturb the neurodevelopmental program and contribute to the quantitative neuronal defects that are too frequently reported in the offspring of cocaine-abusing pregnant women.     

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Potentiate c-fos Expression Induced by Glutamate in Cultured Cortical Neurons

Abstract: Previous reports have demonstrated that glutamate stimulates c- fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c- fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK-801, therefore indicating that VIP stimulates c- fos expression in a glutamate-dependent manner. A similar effect was observed with pituitary adenylate cyclase-activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate-dependent stimulation of c- fos expression evoked by VIP, because either H-89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a "biochemical AND gate" mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c- fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c- fos expression, suggesting that both peptides can increase the "throughput" or "strength" of glutamate-containing circuits in the cerebral cortex.     

Activation of Metabotropic Glutamate Receptors Prevents Neuronal Apoptosis in Culture

Abstract: Cultured granule cells grown in serum-containing medium with a "low K⁺" concentration (10 m M ) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) prevented the development of low K⁺-induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1 S ,3 R -ACPD was prevented by the mGluR antagonist ( RS )-α-methyl-4-carboxyphenylglycine (MCPG) and was mimicked by N -methyl- d -aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li⁺—which reduced polyphosphoinositide response to concentrations of glutamate (5 µ M ) that approximate those physiologically present in the incubation medium—or MCPG, the development of low K⁺-induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against apoptosis during early stages of maturation.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Genetic Program of Neuronal Differentiation and Growth Induced by Specific Activation of NMDA Receptors

Glutamate and its receptors are expressed very early during development and may play important roles in neurogenesis, synapse formation and brain wiring. The levels of glutamate and activity of its receptors can be influenced by exogenous factors, leading to neurodevelopmental disorders. To investigate the role of NMDA receptors on gene regulation in a neuronal model, we used primary neuronal cultures developed from embryonic rat cerebri in serum-free medium. Using Affymetrix Gene Arrays, we found that genes known to be involved in neuronal plasticity were differentially expressed 24 h after a brief activation of NMDA receptors. The upregulation of these genes was accompanied by a sustained induction of CREB phosphorylation, and an increase in synaptophysin immunoreactivity. We conclude that NMDA receptor activation elicits expression of genes whose downstream products are involved in the regulation of early phases of the process leading to synaptogenesis and its consolidation, at least in part through sustained CREB phosphorylation. Special issue dedicated to Dr. Anthony Campagnoni.     

Quinolinic Acid Stimulates Somatostatin Gene Expression in Cultured Rat Cortical Neurons

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphonovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p less than 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

The Desglycinyl Metabolite of Remacemide Hydrochloride Is Neuroprotective in Cultured Rat Cortical Neurons

Abstract: The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR), a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 µ M ) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5–20 µ M ), but not its remacemide precursor (10–100 µ M ), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5–20 µ M ) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 µ M NMDA and reduced the NMDA-triggered increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by up to 70%. By contrast, remacemide (50 and 100 µ M ) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca²⁺]_i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca²⁺]_i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca²⁺]_i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.     

Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K⁺ concentration ([K⁺]_o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K⁺]_o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning ～4 h after the removal of high [K⁺]_o and developed to nuclear condensation 4 h later. Six hours after high [K⁺]_o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K⁺, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K⁺]_o. Similar to high [K⁺]_o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K⁺]_o removal. An ～40% decrease in RNA and protein synthesis was detected by 6 h of [K⁺]_o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.