Mitochondrial responses to intracellular Ca2+ release following infrared stimulation.

Many studies of Ca2+ effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca2+], and have reported increases in [NADH] and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1,863 nm, 8-10°C for ∼5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular [Ca2+] revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) [Ca2+]. IR stimulation increased O2 consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O2 consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes [tetramethyl rhodamine methyl ester (TMRM) and R123] indicated a depolarizing shift in ΔΨm that persisted in high [K+] medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with the opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca2+-dependent increase in superoxide production (MitoSox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus, the intracellular Ca2+ release that follows pulses of infrared light offers new insights into Ca2+-dependent processes controlling respiration and reactive oxygen species in intact cells.NEW & NOTEWORTHY Pulses of infrared light (IR) provide a novel method for rapidly transferring Ca2+ from the endoplasmic reticulum to mitochondria in intact cells. In PC12 cells the resulting ETC activation was not driven by increased ATP synthase activity or NADH. IR stimulation produced a Ca2+-dependent, reversible depolarization of ΔΨm that was partially blocked by cyclosporin A, and a Ca2+-dependent increase in superoxide that did not require transition pore opening.

Pulsed infrared releases Ca2+ from the endoplasmic reticulum of cultured spiral ganglion neurons.

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200-250 Hz for 2-5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6-12°C (above a baseline of 24-30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca2+ concentration ([Ca2+]) of 0.20-1.4 µM, with a simultaneous reduction of [Ca2+] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca2+] continued in medium containing no added Ca2+ (±Ca2+ buffers) and low [Na+], indicating that the [Ca2+] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca2+] response was prolonged and eventually blocked by inhibition of ER Ca2+-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP3)-mediated Ca2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca2+] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca2+] elevations is conferred by TRP channels on ER membranes, which facilitate Ca2+ efflux into the cytosol and thereby contribute to Ca2+-induced Ca2+-release via IP3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca2+-induced Ca2+-release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca2+, studying Ca2+ regulatory mechanisms, and influencing neuronal excitability.

Mitochondrial dysfunction induced by heat stress in cultured rat CNS neurons.

Previous work demonstrated that hyperthermia (43°C for 2 h) results in delayed, apoptotic-like death in striatal neuronal cultures. We investigated early changes in mitochondrial function induced by this heat stress. Partial depolarization of the mitochondrial membrane potential (ΔΨ(m)) began about 1 h after the onset of hyperthermia and increased as the stress continued. When the heat stress ended, there was a partial recovery of ΔΨ(m), followed hours later by a progressive, irreversible depolarization of ΔΨ(m). During the heat stress, O(2) consumption initially increased but after 20-30 min began a progressive, irreversible decline to about one-half the initial rate by the end of the stress. The percentage of oligomycin-insensitive respiration increased during the heat stress, suggesting an increased mitochondrial leak conductance. Analysis using inhibitors and substrates for specific respiratory chain complexes indicated hyperthermia-induced dysfunction at or upstream of complex I. ATP levels remained near normal for ∼4 h after the heat stress. Mitochondrial movement along neurites was markedly slowed during and just after the heat stress. The early, persisting mitochondrial dysfunction described here likely contributes to the later (>10 h) caspase activation and neuronal death produced by this heat stress. Consistent with this idea, proton carrier-induced ΔΨ(m) depolarizations comparable in duration to those produced by the heat stress also reduced neuronal viability. Post-stress ΔΨ(m) depolarization and/or delayed neuronal death were modestly reduced/postponed by nicotinamide adenine dinucleotide, a calpain inhibitor, and increased expression of Bcl-xL.

The Psi(m) depolarization that accompanies mitochondrial Ca2+ uptake is greater in mutant SOD1 than in wild-type mouse motor terminals.

The electrical gradient across the mitochondrial inner membrane (Psi(m)) is established by electron transport chain (ETC) activity and permits mitochondrial Ca(2+) sequestration. Using rhodamine-123, we determined how repetitive nerve stimulation (100 Hz) affects Psi(m) in motor terminals innervating mouse levator auris muscles. Stimulation-induced Psi(m) depolarizations in wild-type (WT) terminals were small (<5 mV at 30 degrees C) and reversible. These depolarizations depended on Ca(2+) influx into motor terminals, as they were inhibited when P/Q-type Ca(2+) channels were blocked with omega-agatoxin. Stimulation-induced Psi(m) depolarization and elevation of cytosolic [Ca(2+)] both increased when complex I of the ETC was partially inhibited by low concentrations of rotenone (25-50 nmol/l). This finding is consistent with the hypothesis that acceleration of ETC proton extrusion normally limits the magnitude of Psi(m) depolarization during mitochondrial Ca(2+) uptake, thereby permitting continued Ca(2+) uptake. Compared with WT, stimulation-induced increases in rhodamine-123 fluorescence were approximately 5 times larger in motor terminals from presymptomatic mice expressing mutations of human superoxide dismutase I (SOD1) that cause familial amyotrophic lateral sclerosis (SOD1-G85R, which lacks dismutase activity; SOD1-G93A, which retains dismutase activity). Psi(m) depolarizations were not significantly altered by expression of WT human SOD1 or knockout of SOD1 or by inhibiting opening of the mitochondrial permeability transition pore with cyclosporin A. We suggest that an early functional consequence of the association of SOD1-G85R or SOD1-G93A with motoneuronal mitochondria is reduced capacity of the ETC to limit Ca(2+)-induced Psi(m) depolarization, and that this impairment contributes to disease progression in mutant SOD1 motor terminals.

Repetitive nerve stimulation transiently opens the mitochondrial permeability transition pore in motor nerve terminals of symptomatic mutant SOD1 mice.

Mitochondria in motor nerve terminals temporarily sequester large Ca(2+) loads during repetitive stimulation. In wild-type mice this Ca(2+) uptake produces a small (<5 mV), transient depolarization of the mitochondrial membrane potential (Ψ(m), motor nerve stimulated at 100 Hz for 5s). We demonstrate that this stimulation-induced Ψ(m) depolarization attains much higher amplitudes in motor terminals of symptomatic mice expressing the G93A or G85R mutation of human superoxide dismutase 1 (SOD1), models of familial amyotrophic lateral sclerosis (fALS). These large Ψ(m) depolarizations decayed slowly and incremented with successive stimulus trains. Additional Ψ(m) depolarizations occurred that were not synchronized with stimulation. These large Ψ(m) depolarizations were reduced (a) by cyclosporin A (CsA, 1-2 µM), which inhibits opening of the mitochondrial permeability transition pore (mPTP), or (b) by replacing bath Ca(2+) with Sr(2+), which enters motor terminals and mitochondria but does not support mPTP opening. These results are consistent with the hypothesis that the large Ψ(m) depolarizations evoked by repetitive stimulation in motor terminals of symptomatic fALS mice result from mitochondrial dysfunction that increases the likelihood of transient mPTP opening during Ca(2+) influx. Such mPTP openings, a sign of mitochondrial stress, would disrupt motor terminal handling of Ca(2+) loads and might thereby contribute to motor terminal degeneration in fALS mice. Ψ(m) depolarizations resembling those in symptomatic fALS mice could be elicited in wild-type mice following a 0.5-1h exposure to diamide (200 µM), which produces an oxidative stress, but these depolarizations were not reduced by CsA.

Mitochondria in motor nerve terminals: function in health and in mutant superoxide dismutase 1 mouse models of familial ALS.

Mitochondria contribute to neuronal function not only via their ability to generate ATP, but also via their ability to buffer large Ca(2+) loads. This review summarizes evidence that mitochondrial Ca(2+) sequestration is especially important for sustaining the function of vertebrate motor nerve terminals during repetitive stimulation. Motor terminal mitochondria can sequester large amounts of Ca(2+) because they have mechanisms for limiting both the mitochondrial depolarization and the increase in matrix free [Ca(2+)] associated with Ca(2+) influx. In mice expressing mutations of human superoxide dismutase -1 (SOD1) that cause some cases of familial amyotrophic lateral sclerosis (fALS), motor terminals degenerate well before the death of motor neuron cell bodies. This review presents evidence for early and progressive mitochondrial dysfunction in motor terminals of mutant SOD1 mice (G93A, G85R). This dysfunction would impair mitochondrial ability to sequester stimulation-associated Ca(2+) loads, and thus likely contributes to the early degeneration of motor terminals.

Rapid, stimulation-induced reduction of C12-resorufin in motor nerve terminals: linkage to mitochondrial metabolism.

The Alamar blue (resazurin) assay of cell viability monitors the irreversible reduction of non-fluorescent resazurin to fluorescent resorufin. This study focused on the reversible reduction of C12-resorufin to non-fluorescent C12-dihydroresorufin in motor nerve terminals innervating lizard intercostal muscles. Resting C12-resorufin fluorescence decreased when the activity of the mitochondrial electron transport chain (ETC) was accelerated with carbonyl cyanide m-chloro phenyl hydrazone, and increased when ETC activity was inhibited with cyanide. Trains of action potentials (50 Hz for 20-50 s), which reversibly decreased NADH fluorescence and partially depolarized the mitochondrial membrane potential, produced a reversible decrease in C12-resorufin fluorescence which had a similar time course. The stimulation-induced decrease in C12-resorufin fluorescence was blocked by inhibitors of ETC complexes I, III, and IV and by carbonyl cyanide m-chloro phenyl hydrazone, but not by inhibiting mitochondrial ATP synthesis with oligomycin. Mitochondrial depolarization and the decreases in C12-resorufin and NADH fluorescence depended on Ca2+ influx into the terminal, but not on vesicular transmitter release. These results suggest that the reversible reduction of C12-resorufin in stimulated motor nerve terminals is linked, directly or indirectly, to the reversible oxidation of NADH and to Ca(2+) influx into mitochondria, and provides an assay for rapid changes in motor terminal metabolism.

Overexpression of Cdk5 or non-phosphorylatable retinoblastoma protein protects septal neurons from oxygen-glucose deprivation.

Activation of cyclin dependent kinases (Cdks) contributes to neuronal death following ischemia. We used oxygen-glucose deprivation (OGD) in septal neuronal cultures to test for possible roles of cell cycle proteins in neuronal survival. Increased cdc2-immunoreactive neurons were observed at 24 h after the end of 5 h OGD. Green fluorescent protein (GFP) or GFP along with a wild type or dominant negative form of the retinoblastoma protein (Rb), or cyclin-dependent kinase5 (Cdk5), were overexpressed using plasmid constructs. Following OGD, when compared to controls, neurons expressing both GFP and dominant negative Rb, RbDeltaK11, showed significantly less damage using microscopy imaging. Overexpression of Rb-wt did not affect survival. Surprisingly, overexpression of Cdk5-wild type significantly protected neurons from process disintegration but Cdk5T33, a dominant negative Cdk5, gave little or no protection. Thus phosphorylation of the cell cycle regulator, Rb, contributes to death in OGD in septal neurons but Cdk5 can have a protective role.

Cellular mechanisms of neuronal damage from hyperthermia.

Hyperthermia can cause brain damage and also exacerbate the brain damage produced by stroke and amphetamines. The developing brain is especially sensitive to hyperthermia. The severity of, and mechanisms underlying, hyperthermia-induced neuronal death depend on both temperature and duration of exposure. Severe hyperthermia can produce necrotic neuronal death. For a window of less severe heat stresses, cultured neurons exhibit a delayed death with apoptotic characteristics including cytochrome c release and caspase activation. Little is known about mechanisms of hyperthermia-induced damage upstream of these late apoptotic effects. This chapter considers several possible upstream mechanisms, drawing on both in vivo and in vitro studies of the nervous system and other tissues. Hyperthermia-induced damage in some non-neuronal cells includes endoplasmic reticular stress due to denaturing of nascent polypeptide chains, as well as nuclear and cytoskeletal damage. Evidence is presented that hyperthermia produces mitochondrial damage, including depolarization, in cultured mammalian neurons.

Preservation of neuromuscular function in symptomatic SOD1-G93A mice by peripheral infusion of methylene blue.

In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400µM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1µM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease.

Dysfunctional mitochondrial Ca(2+) handling in mutant SOD1 mouse models of fALS: integration of findings from motor neuron somata and motor terminals.

Abundant evidence indicates that mitochondrial dysfunction and Ca(2+) dysregulation contribute to the muscle denervation and motor neuron death that occur in mouse models of familial amyotrophic lateral sclerosis (fALS). This perspective considers measurements of mitochondrial function and Ca(2+) handling made in both motor neuron somata and motor nerve terminals of SOD1-G93A mice at different disease stages. These complementary studies are integrated into a model of how mitochondrial dysfunction disrupts handling of stimulation-induced Ca(2+) loads in presymptomatic and end-stages of this disease. Also considered are possible mechanisms underlying the findings that some treatments that preserve motor neuron somata fail to postpone degeneration of motor axons and terminals.

Calcium dependence of damage to mouse motor nerve terminals following oxygen/glucose deprivation.

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca(2+) contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca(2+)] indicator demonstrated an increase in intra-terminal [Ca(2+)] following OGD onset. When OGD was terminated within 20-30min of the increase in resting [Ca(2+)], these changes were sometimes reversible; in other cases [Ca(2+)] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca(2+)-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca(2+), but increased to 81% when Ca(2+) was absent. Removal of Ca(2+) only during OGD was more protective than removal of Ca(2+) only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca(2+) entry into motor terminals, including voltage-dependent Ca(2+) channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na(+)/Ca(2+) exchanger (KB-R7943). Inhibition of a Ca(2+)-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca(2+)-dependent, and that inhibition of Ca(2+) entry or Ca(2+)-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.

Stimulation-induced changes in NADH fluorescence and mitochondrial membrane potential in lizard motor nerve terminals.

To investigate mitochondrial responses to repetitive stimulation, we measured changes in NADH fluorescence and mitochondrial membrane potential (Psi(m)) produced by trains of action potentials (50 Hz for 10-50 s) delivered to motor nerve terminals innervating external intercostal muscles. Stimulation produced a rapid decrease in NADH fluorescence and partial depolarization of Psi(m). These changes were blocked when Ca2+ was removed from the bath or when N-type Ca2+ channels were inhibited with omega-conotoxin GVIA, but were not blocked when bath Ca2+ was replaced by Sr2+, or when vesicular release was inhibited with botulinum toxin A. When stimulation stopped, NADH fluorescence and Psi(m) returned to baseline values much faster than mitochondrial [Ca2+]. In contrast to findings in other tissues, there was usually little or no poststimulation overshoot of NADH fluorescence. These findings suggest that the major change in motor terminal mitochondrial function brought about by repetitive stimulation is a rapid acceleration of electron transport chain (ETC) activity due to the Psi(m) depolarization produced by mitochondrial Ca2+ (or Sr2+) influx. After partial inhibition of complex I of the ETC with amytal, stimulation produced greater Psi(m) depolarization and a greater elevation of cytosolic [Ca2+]. These results suggest that the ability to accelerate ETC activity is important for normal mitochondrial sequestration of stimulation-induced Ca2+ loads.

PGC-1alpha/beta upregulation is associated with improved oxidative phosphorylation in cells harboring nonsense mtDNA mutations.

We have studied the functional effects of nonsense mitochondrial DNA (mtDNA) mutations in the COXI and ND5 genes in a colorectal tumor cell line. Surprisingly, these cells had an efficient oxidative phosphorylation (OXPHOS); however, when mitochondria from these cells were transferred to an osteosarcoma nuclear background (osteosarcoma cybrids), the rate of respiration markedly declined suggesting that the phenotypic expression of the mtDNA mutations was prevented by the colorectal tumor nuclear background. We found that there was a significant increase in the steady-state levels of PGC-1alpha and PGC-1beta transcriptional coactivators in these cells and a parallel increase in the steady-state levels of several mitochondrial proteins. Accordingly, adenoviral-mediated overexpression of PGC-1alpha and PGC-1beta in the osteosarcoma cybrids stimulated mitochondrial respiration suggesting that an upregulation of PGC-1alpha/beta coactivators can partially rescue an OXPHOS defect. In conclusion, upregulation of PGC-1alpha and PGC-1beta in the colorectal tumor cells can be part of an adaptation mechanism to help overcome the severe consequences of mtDNA mutations on OXPHOS.

Stimulation-induced mitochondrial [Ca2+] elevations in mouse motor terminals: comparison of wild-type with SOD1-G93A.

Changes in mitochondrial matrix [Ca2+] evoked by trains of action potentials were studied in levator auris longus motor terminals using Ca2+-sensitive fluorescent indicator dyes (rhod-2, rhod-5F). During a 2500 impulse 50 Hz train, mitochondrial [Ca2+] in most wild-type terminals increased within 5-10 s to a plateau level that was sustained until stimulation ended. This plateau was not due to dye saturation, but rather reflects a powerful buffering system within the mitochondrial matrix. The amplitude of this plateau was similar for stimulation frequencies in the range 15-100 Hz. Plateau amplitude was sensitive to temperature, with no detectable stimulation-induced increase in fluorescence at temperatures below 17 degrees C, and increasing magnitudes as temperature was increased to near-physiological levels (38 degrees C). When stimulation ended, mitochondrial [Ca2+] decayed slowly back to prestimulation levels over a time course of hundreds of seconds. Similar measurements were also made in motor terminals of mice expressing the G93A mutation of human superoxide dismutase 1 (SOD1-G93A). In mice > 100 days old, all of whom exhibited hindlimb paralysis, some terminals continued to show wild-type mitochondrial [Ca2+] responses, but in other terminals mitochondrial [Ca2+] did not plateau, but rather continued to increase throughout most of the stimulus train. Thus mechanism(s) that limit stimulation-induced increases in mitochondrial [Ca2+] may be compromised in some SOD1-G93A terminals.

Bone morphogenetic proteins and neurotrophins provide complementary protection of septal cholinergic function during phosphatase inhibitor-induced stress.

Cultures of embryonic rat septum were exposed for 24-48 h to 2-5 nm okadaic acid (OA), an inhibitor of pp1A and pp2A phosphatases. This stress killed approximately 75% of neurons. A neurotrophin (NT) combination (nerve growth factor and brain-derived neurotrophic factor, each 100 ng/mL) plus a bone morphogenetic protein (BMP6 or BMP7, 5 nm) reduced the death of both cholinergic and non-cholinergic neurons, and preserved choline acetyltransferase (ChAT) activity assayed 2-6 days post-stress. This NT + BMP combination preserved ChAT activity better than either NTs or BMPs alone, and was effective even if trophic factor addition was delayed until 12 h after stress onset. A general caspase inhibitor (qVD-OPH, 10 micro g/mL) also increased survival of stressed cholinergic neurons, but its protection of ChAT activity was shorter lived than that produced by the NT + BMP combination. Neither the NT + BMP combination nor the caspase inhibitor reduced the OA-induced increase in tau phosphorylation. These findings indicate that NTs and BMPs have synergistic protective effects against an OA stress, and suggest that at least some of these protective effects occur upstream of caspase activation.

Caspase activation contributes to delayed death of heat-stressed striatal neurons.

Hyperthermia can contribute to brain damage both during development and post-natally. We used rat embryonic striatal neurons in culture to study mechanisms underlying hyperthermia-induced neuronal death. Heat stress at 43 degrees C for 2 h produced no obvious signs of damage during the first 12 h after the stress, but more than 50% of the neurons died during the next 3 days. More than 40% of the neurons had activated caspases 24 h following the heat stress. Caspase-3 activity increased with a delay of more than 10 h following cessation of the heat stress, reaching a peak at approximately 18 h. Neuronal death measured 1-3 days after the stress was reduced by the general caspase inhibitors qVD-OPH (10-20 microm) and zVAD-fmk (50-100 microm). These inhibitors were protective even when added 9 h after cessation of the heat stress, consistent with the delayed activation of caspases. In contrast, blockers of Na+ channels and ionotropic glutamate receptors did not reduce the heat-induced death, indicating that glutamate excitotoxicity was not required for this neuronal death. These results show that the neuronal death produced by heat stress has characteristics of apoptosis, and that caspase inhibitors can delay this death.