Elsevier

Brain Research

Volume 851, Issues 1–2, 18 December 1999, Pages 20-31
Brain Research

Research report
In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons

https://doi.org/10.1016/S0006-8993(99)02035-1Get rights and content

Abstract

Calcium ions and calcium-dependent systems have been implicated in the pathophysiology of status epilepticus (SE). However, the dynamics of intracellular calcium ([Ca2+]i) levels during SE has not yet been studied. We have employed the hippocampal neuronal culture (HNC) model of in vitro SE that produces continuous epileptiform discharges to study spatial and dynamic changes in [Ca2+]i levels utilizing confocal laser scanning microscopy and the calcium binding dye, indo-1. During SE, the average [Ca2+]i levels increased from control levels of 150–200 nM to levels of 450–600 nM. This increased [Ca2+]i was maintained for the duration of SE. Following SE, [Ca2+]i levels gradually returned to basal values. The duration of SE was shown to affect the ability of the neuron to restore resting [Ca2+]i levels. Both N-methyl-d-aspartate (NMDA) receptor-gated and voltage-gated Ca2+ channels (VGCCs) contributed to the increased calcium entry during SE. Moreover, this elevation in [Ca2+]i occurred in both the nucleus and cytosol. These results provide the first dynamic measurement of [Ca2+]i during prolonged electrographic seizure discharges in an in vitro SE model and suggest that prolonged epileptiform discharges give rise to abnormal sustained increases in [Ca2+]i levels that may play a role in the neuronal cell damage and long-term plasticity changes associated with SE.

Introduction

Status epilepticus (SE) is a major medical and neurological emergency that affects approximately a quarter of a million Americans per year and is responsible for over 30,000 deaths per year in the United States alone 10, 13. Understanding the pathophysiology of this important neurological condition is a major objective of numerous research institutions and professional organizations [14]. Despite the importance of studying this condition, SE has been especially difficult to investigate since it has a complex clinical presentation and there is a lack of good experimental models of SE. Recent advances have led to the development of whole animal 6, 31 and in vitro models of SE 40, 49. Earlier studies have also indicated that elevation in [Ca2+]i may play an important role in the pathophysiology of SE 19, 20, 26. Despite the potential importance of [Ca2+]i dynamics during SE, it has not been possible to study the dynamics of [Ca2+]i during SE due to technical difficulties.

The hippocampal neuronal culture (HNC) model of SE [49] provides an ideal model system to study [Ca2+]i in isolated neurons during continuous electrographic epileptiform discharges. This model of electrographic SE characterized by continuous 3.0–20 Hz epileptiform discharges can be sustained for prolonged periods of time in the HNC model. Using confocal microscopy and simultaneous intracellular recording from multiple neurons, it has also been shown that the continuous epileptiform discharge activity during SE in the HNC model is a population phenomena [49]. Although continuous electrographic epileptiform discharges in interconnected neurons in culture are not a complete representation of SE in the intact animal, this cell culture model of SE provides a powerful tool to investigate the molecular mechanisms underlying the induction, maintenance, and termination of continuous epileptiform discharges in hippocampal neurons in vitro and demonstrates that neuronal networks in culture can be transformed to manifest continuous seizure-like activity.

Despite the importance of Ca2+ ions in the pathophysiology of SE, it has not been possible to directly quantitate [Ca2+]i during SE. It is important to determine the effect of SE on [Ca2+]i, since understanding the relationship of [Ca2+]i to SE duration and termination is essential in understanding the pathophysiology of SE. [Ca2+]i can be spatially and dynamically evaluated in single neurons or in neuronal networks using Ca2+-sensitive dyes like indo-1, fura-2, fura-red and calcium-green 21, 53 in conjunction with confocal laser scanning microscopy 2, 5, 23, 30, 44. The present research effort was initiated to utilize the HNC model of SE to evaluate the effect of prolonged seizures on the spatial and dynamic changes in [Ca2+]i. The results demonstrate that SE causes significant changes in the dynamics of [Ca2+]i which may contribute to long-term plasticity changes associated with SE.

Section snippets

Materials

Indo-1 AM was purchased from Molecular Probes (Eugene, OR). Sodium pyruvate, minimum essential media (MEM) containing Earle's salts, and 25 mM N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) were obtained from Gibco BRL (Gaithersburg, MD). 5-Methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801) and nifedipine were purchased from Sigma (St. Louis, MO). dl-2-Amino-5-phosphonovaleric acid (dl-APV) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) were

In vitro SE electrophysiology in HNC

Continuous epileptiform discharges were produced in the HNC preparation by exposure of the culture to media low in Mg [49]. Before low Mg treatment, whole-cell current clamp recordings showed spontaneous excitatory and inhibitory post-synaptic potentials (EPSPs, IPSPs) and occasional action potentials. Low Mg exposure caused the neurons to develop larger, longer duration synaptic potentials and multiple action potentials, evolving into continuous tonic high-frequency burst discharges (Fig. 1).

Discussion

This paper provides direct evidence that in vitro SE caused significant changes in [Ca2+]i levels in the HNC model of SE. Using this model, the resting average [Ca2+]i in hippocampal pyramidal neurons was 150–200 nM and rose up to 450–600 nM in neurons undergoing SE. During SE, [Ca2+]i spiking occurred with peak [Ca2+]i reaching 1 μM for brief durations. The morphology of the cells during SE was not significantly altered. This indicates that although [Ca2+]i levels during SE were elevated, they

Conclusion

We have provided evidence, for the first time, that there is a sustained increase in [Ca2+]i in the hippocampal neuronal cells during SE in vitro and this elevation is primarily due to influx from extracellular Ca2+. Both glutamate receptors as well as VGCCs play a role in this Ca2+ influx during SE. Although this rise in [Ca2+]i during SE durations of 1–2 h in culture was not high enough to cause cell death, the levels may be high enough to cause both short- and long-term plasticity changes in

Acknowledgements

We thank Drs. D. Coulter, T. Allen Morris and S.B. Churn for their suggestions during this research effort. This research was supported by NINDS grants RO1 NS23350 and PO1 NS25630 to R.J.D., the Nathan and Sophie Gumenick Neuroscience Research Fund and the Milton L. Markel Alzheimer's Disease Research Fund.

References (54)

  • E.W. Lothman et al.

    Functional anatomy of hippocampal seizures

    Prog. Neurobiol.

    (1991)
  • H. Miyakawa et al.

    Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels

    Neuron

    (1992)
  • D.A. Przywara et al.

    Sites of transmitter release and relation to intracellular Ca2+ in cultured sympathetic neurons

    Neuroscience

    (1993)
  • H.E. Scharfman et al.

    Consequences of prolonged afferent stimulation of the rat fascia dentata: epileptiform activity in area CA3 of hippocampus

    Neuroscience

    (1990)
  • S. Sombati et al.

    Neurotoxic activation of glutamate receptors induces an extended neuronal depolarization in cultured hippocampal neurons

    Brain Res.

    (1991)
  • S. Tanaka et al.

    Regional calcium accumulation and kainic acid (KA)-induced limbic seizure status in rats

    Brain Res.

    (1989)
  • R.S. Vick et al.

    GABAA alpha 2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal–entorhinal cortical slices

    Brain Res.

    (1996)
  • M.A. Abdul-Ghani et al.

    Metabotropic glutamate receptors coupled to IP3 production mediate inhibition of IAHP in rat dentate granule neurons

    J. Neurophysiol.

    (1996)
  • H. Bading et al.

    Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways

    Science

    (1993)
  • T.H. Brown et al.

    Calcium imaging in hippocampal neurons using confocal microscopy

    Ann. N.Y. Acad. Sci.

    (1994)
  • E.A. Cavalheiro

    The pilocarpine model of epilepsy

    Ital. J. Neurol. Sci.

    (1995)
  • D.A. Coulter et al.

    Electrophysiology of glutamate neurotoxicity in vitro: induction of a calcium-dependent extended neuronal depolarization

    J. Neurophysiol.

    (1992)
  • R.J. DeLorenzo et al.

    A prospective, population-based epidemiologic study of status epilepticus in Richmond, Virginia

    Neurology

    (1996)
  • R.J. DeLorenzo et al.

    Long-term modulation of gene expression in epilepsy

    The Neuroscientist

    (1999)
  • R.J. DeLorenzo et al.

    Prolonged activation of the N-methyl-d-aspartate receptor–Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • R.J. DeLorenzo et al.

    Epidemiology of status epilepticus

    J. Clin. Neurophysiol.

    (1995)
  • W.E. Dodson et al.

    The treatment of convulsive status epilepticus: recommendation of the Epilepsy Foundation of America's working group of status epilepticus

    JAMA

    (1993)
  • Cited by (77)

    • Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis

      2020, Experimental Neurology
      Citation Excerpt :

      Following this reasoning, most of the events that encompass the epileptogenesis, such as neurodegeneration and pathological plasticity, activate downstream pathways that are regulated by intracellular Ca+2 dynamics (isnt). In addition, intracellular Ca+2 concentrations are strongly modulated during epileptogenesis, remaining elevated during the progression of the disease (DeLorenzo et al., 1998; Delorenzo et al., 2005; Nagarkatti et al., 2010; Pal et al., 1999; Raza et al., 2004; Raza et al., 2001; Steinlein, 2014). It was determined that long-lasting intracellular Ca2+ elevation during epileptogenesis could be reverted by using the ryanodine receptor (RyR) blocker dantrolene in an in vitro model of status epilepticus-induced epilepsy (Nagarkatti et al., 2010).

    • Endocannabinoids and epilepsy

      2015, Cannabinoids in Neurologic and Mental Disease
    View all citing articles on Scopus
    View full text