Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons

Abstract

Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate-induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.

In Vivo Protection against NMDA-induced Neurodegeneration by MK-801 and Nimodipine: Combined Therapy and Temporal Course of Protection

“…Early neurotoxicity during the first 2 h after NMDA-exposure, exerted by the initial massive Ca 2ϩ influx may explain this early irreversible cell damage. Studies in which single neurons in culture were lethally challenged with excitatory amino acids showed an initial Ca 2ϩ influx through NMDA-channels followed by a delayed secondary, sustained and irreversible Ca 2ϩ rise indicating imminent cell death (Randall & Thayer, 1992;Tymianski et al, 1993). Early blockade of Ca 2ϩ influx by NMDA-antagonists was able to prevent this early cell death (Hartley et al, 1993;Tymianski et al, 1993).…”

“…Based on the observations of delayed enhancement of voltage-dependent Ca 2ϩ currents after NMDAoverstimulation (Randall & Thayer, 1992;Tymianski et al, 1993), we would expect a longer lasting neuroprotective potential of nimodipine. Surprisingly, nimodipine alone applied 2 h after NMDA-exposure revealed no significant neuroprotection, compared to a still strongly protective effect established by MK-801.…”

“…During cerebral ischaemia excessive release of the excitatory neurotransmitter glutamate is observed (Benveniste et al, 1984), which leads to an uncontrolled rise of intracellular free Ca 2ϩ (Dubinsky, 1993) and ultimately to neuronal necrosis (Randall & Thayer, 1992). Massive entry through various channels of Ca 2ϩ into neurons is considered to be the final common pathway leading to irreversible neuronal damage, since persistent high levels of cytosolic free Ca 2ϩ may evoke dysregulations of Ca 2ϩ activated processes which are detrimental to neurons (Clapham, 1995).…”

Determination of the Time Course and Extent of Neurotoxicity at Defined Temperatures in Cultured Neurons Using a Modified Multiwell Plate Fluorescence Scanner

“…Electrophysiologic mea surements of cell membrane potential may indicate cell death in some preparations (Scharfman and Schwartzkroin, 1989). Also, measurements of the ability to maintain intracellular calcium homeostasis have been correlated with impending cell death (Tsien, 1981;Randall and Thayer, 1992;Tymianski et aI., 1993a). However, these approaches are labor intensive and difficult to apply on a large scale.…”

Ca2+-Dependent, Stimulus-Specific Modulation of the Plasma Membrane Ca2+Pump in Hippocampal Neurons

“…We were not able to detect PMCA internalization using the 10-min stimulus protocol employed in this study; whether this was due to insufficient resolution to detect small changes in PMCA distribution or whether pump inactivation precedes internalization is not clear. Both NMDA and low [Mg 2ϩ ] o stimuli are potentially toxic, so the loss of PMCA activity following these treatments may contribute to the [Ca 2ϩ ] i dysregulation that is a hallmark of excitotoxicity (Randall and Thayer 1992). The depolarization-induced slowing of PMCA-mediated Ca 2ϩ clearance follows more rapid acceleration and thus may be a homeostatic process.…”