Elsevier

Brain Research

Volume 1006, Issue 1, 23 April 2004, Pages 8-17
Brain Research

Research report
Spontaneous synchronized calcium oscillations in neocortical neurons in the presence of physiological [Mg2+]: involvement of AMPA/kainate and metabotropic glutamate receptors

https://doi.org/10.1016/j.brainres.2004.01.059Get rights and content

Abstract

Primary cultures of neocortical neurons exhibit spontaneous Ca2+ oscillations under zero or low extracellular [Mg2+] conditions. We find that mature murine neocortical neurons cultured for 9 days also produce spontaneous Ca2+ oscillations in the presence of physiological [Mg2+]. These Ca2+ oscillations were action potential mediated inasmuch as tetrodotoxin eliminated their occurrence. AMPA receptors were found to regulate the frequency of Ca2+ oscillations. In contrast, Ca2+ oscillations were independent of activation of L-type Ca2+ channels, and NMDA receptors provided only a minor contribution. Release of intracellular Ca2+ stores was involved in the oscillatory activity since thapsigargin reduced the amplitude and frequency of the oscillations. S-4-carboxyphenylglycine ((S)-4CPG), an antagonist of group I metabotropic glutamate receptor (mGluR), also reduced the amplitude of oscillations. In addition, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), a group I mGluR agonist, increased the oscillation frequency, suggesting a critical role for mGluR in the generation of Ca2+ oscillations. The mGluR-mediated release of intracellular Ca2+ stores appeared to be mediated by phospholipase C (PLC) since the PLC inhibitor U73122 eliminated the Ca2+ oscillations. These results indicate that Ca2+ oscillations in neocortical cultures in the presence of physiologic [Mg2+] are primarily initiated by excitatory input from AMPA receptors and involve mobilization of intracellular Ca2+ stores following activation of mGluR.

Introduction

Oscillations in cytoplasmic Ca2+ levels are a common mode of signaling both in excitable and nonexcitable cells and can increase the efficiency and specificity of gene expression [7]. Ca2+ oscillations act as a biological Morse code in that alterations of the temporal features of the oscillations produce differential activation of certain genes [8], [16]. Neurons in culture exhibit synchronous spontaneous Ca2+ oscillations that are represented by rhythmic activation of populations of neurons in characteristic temporal and spatial patterns. It has been shown that neurons in culture form functional synapses [27] and rhythmic neurotransmitter release in a neuronal network may drive the synchronous oscillatory activity.

Ca2+ oscillations play an important physiologic role in the nervous system. Spontaneous Ca2+ transients have been implicated in regulating neuronal plasticity in developing neurons [33]. Ca2+ spikes also promote neurotransmitter receptor expression and channel maturation in Xenopus embryonic neurons [13] and play a critical role in regulating the advancement of the leading process of migrating cerebellar granule neurons [21].

Synchronized Ca2+ oscillations in cultured neurons were first reported in hippocampal neurons [29], and similar oscillatory behavior has been reported in other neuronal cell types including neocortical neurons [30], [35] and cerebellar granule neurons [22], [28]; however, these oscillations were typically observed upon removal or lowering of extracellular Mg2+. Mg2+ is a critical ion in the regulation of NMDA receptors [30]. NMDA receptors are ligand-gated channels that are blocked by submillimolar concentrations of extracellular Mg2+ at resting membrane potential [24]. Removal of Mg2+ from the extracellular environment will consequently favor Ca2+ influx through NMDA receptors due to relief of Mg2+ block of these ion channels. Accordingly, it has been reported that NMDA receptors are indeed necessary for generation of Ca2+ oscillations in the absence of extracellular Mg2+[28], [36]. Although Ca2+ oscillations have also been reported in mature cultures of neocortical neurons in the presence of physiologic [Mg2+] [26], the molecular mechanisms responsible for the generation of these oscillations have not been thoroughly investigated. Specifically, the role of mGluR-mediated signaling in spontaneous Ca2+ oscillations occurring in a physiologic medium remains to be explored.

The mechanisms involved in generation of Ca2+ oscillations vary in different cell types. In general, Ca2+ oscillations may be initiated by influx through the plasma membrane or mobilization of intracellular Ca2+ stores. In nonexcitable cells, formation of inositol triphosphate (IP3) and cyclical release of Ca2+ from IP3-sensitive stores is believed to be responsible for the oscillatory behavior [2]. In sympathetic neurons, caffeine induces oscillations through a mechanism that involves Ca2+-induced Ca2+ release (CICR) [12]. In HEK cells transfected with different metabotropic glutamate receptor (mGluR) subtypes, it has been shown that cyclic phosphorylation and dephosphorylation of mGluR5 by protein kinase C (PKC) is responsible for generation of Ca2+ oscillations [20]. However, in coronal slices from neocortex endogenous glutamate acting on the group I mGluR is responsible for generation of Ca2+ oscillations [9]. Intracellular Ca2+ waves have been observed in cortical slices and in neocortical cultures that are plated at a high cell density. In cultured neocortical neurons, the Ca2+ waves propagate at a rate of 100–200 μm/s [4]. These Ca2+ waves involve communication through gap junctions rather than synaptic transmission [4], [19].

Approximately 13% of neurons in neocortical cultures possess Ca2+-permeable AMPA receptors [18], [35], and Ca2+ entry through these receptors may serve as the initial trigger for generation of calcium oscillations. Neocortical neurons also express mGluR receptors [10], and activation of these mGluR receptors by endogenous glutamate can induce the release of Ca2+ from the intracellular Ca2+ stores. Thus, both AMPA and mGluR-mediated signaling events may contribute to the generation of Ca2+ oscillations.

We find that neocortical neuronal culture (10–13 days in vitro) obtained from embryonic mouse brains produce spontaneous Ca2+ oscillations in the presence of physiological [Mg2+]. The role of glutamate receptors in the generation of these Ca2+ oscillations in neocortical neurons was therefore explored. AMPA receptors and mGluR were both found to play a critical role in the generation of oscillations, and a combination of Ca2+ influx from extracellular media and mobilization from intracellular stores served as a source of Ca2+ to oscillations. Furthermore, we found that Ca2+ oscillations are tonically regulated by adenosine receptor signaling.

Section snippets

Mice neocortical primary cell cultures

Primary cultures of neocortical neurons were obtained from embryonic day 16–17 Swiss-Webster mice. Briefly, pregnant mice were euthanized by CO2 asphyxiation, and embryos were removed under sterile conditions. Neocortices were collected, stripped of meninges, minced by trituration with a Pasteur pipette and treated with trypsin for 25 min at 37 °C. The cells were then dissociated by two successive trituration and sedimentation steps in soybean trypsin inhibitor and DNase containing isolation

Spontaneous synchronized Ca2+ oscillations at physiological Mg2+

Neocortical neurons in culture develop extensive processes and form a synaptically connected network [6], [27]. We found that these murine neocortical cultures (10–13 days in vitro) reliably produce spontaneous Ca2+ oscillations in the presence of physiological Mg2+ (1 mM). The mechanisms underlying the Ca2+ oscillations were explored inasmuch as they are likely to differ from those elicited by low or zero extracellular Mg2+ concentration. These Ca2+ oscillations were highly synchronized in

Discussion

In the present study, we have examined the sources of Ca2+ required to support the generation of Ca2+ oscillations in neocortical neurons under physiological Mg2+. We found that the mechanisms of Ca2+ oscillations in the presence of physiological Mg2+ differ from those described earlier in preparations with low or zero extracellular Mg2+. At low or zero extracellular Mg2+, concomitant activation of NMDA receptors and L-type Ca2+ channels have been reported to be necessary for the generation of

References (37)

  • I.J. Reynolds

    Modulation of NMDA receptor responsiveness by neurotransmitters, drugs and chemical modification

    Life Sci.

    (1990)
  • K. Rose et al.

    Magnesium removal induces paroxysmal neuronal firing and NMDA receptor-mediated neuronal degeneration in cortical cultures

    Neurosci. Lett.

    (1990)
  • D.M. Turetsky et al.

    Cortical neurones exhibiting kainate-activated Co2+ uptake are selectively vulnerable to AMPA/kainate receptor-mediated toxicity

    Neurobiol. Dis.

    (1994)
  • X. Wang et al.

    Mechanism of synchronized Ca2+ oscillations in cortical neurons

    Brain Res.

    (1997)
  • M.J. Berridge

    Inositol trisphosphate and calcium signalling

    Nature

    (1993)
  • K.K. Dev et al.

    Pharmacology and regional distribution of the binding of 6-[3H]nitro-7-sulphamoylbenzo[f]-quinoxaline-2,3-dione to rat brain

    J. Neurochem.

    (1996)
  • R.E. Dolmetsch et al.

    Calcium oscillations increase the efficiency and specificity of gene expression

    Nature

    (1998)
  • R.D. Fields et al.

    Action potential-dependent regulation of gene expression: temporal specificity in Ca2+, cAMP-responsive element binding proteins, and mitogen-activated protein kinase signaling

    J. Neurosci.

    (1997)
  • Cited by (63)

    • Schekwanglupaside C, a new lupane saponin from Schefflera kwangsiensis, is a potent activator of sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase

      2019, Fitoterapia
      Citation Excerpt :

      The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.7 μM [1.22–2.50 μM, 95% Confidence Interval (95% CI)] and 2.5 μM (1.22–5.17 μM, 95% CI), respectively (Fig. 3B&C). Many channels affecting membrane excitability and Ca2+ permeation can alter the spatiotemporal pattern of SCOs [21,26]. The spatiotemporal pattern of Sch C response on SCOs is distinct to that of tetrodotoxin, a voltage-gated sodium channel (VGSC) blocker, which, upon addition, immediately abolishes SCOs [26].

    View all citing articles on Scopus
    View full text