Elsevier

Brain Research

Volume 831, Issues 1–2, 12 June 1999, Pages 273-282
Brain Research

Research report
Transient in vivo membrane depolarization and glutamate release before anoxic depolarization in rat striatum

https://doi.org/10.1016/S0006-8993(99)01481-XGet rights and content

Abstract

Increased extracellular glutamate ([GLU]e), under the condition of cerebral ischemia, anoxia or hypoxia, has been recognized as being associated with neuronal cell damage and death. We performed real-time monitoring of [GLU]e dynamics in vivo in the rat striatum during systemic acute anoxia or hypoxia, as well as monitoring the direct current potential (DC) and cerebral blood flow (CBF). Adult Wistar rats were orotracheally intubated and artificially ventilated with room air. A microdialysis electrode, temperature sensor probe, DC microelectrode and laser Doppler probe were then implanted. The inspired gas was changed to 100% N2 (anoxia), or to 3, 5 or 8% O2 (remainder N2) (hypoxia). With 100% N2, distinct biphasic [GLU]e elevations were observed. With 3% O2, a transient [GLU]e increase was seen before anoxic depolarization (AD). With 5% O2, however, the start of the transient [GLU]e increase was significantly delayed. Anoxia-induced depolarization started at about 100 s. The 3% O2-induced transient depolarization and AD began at nearly the same time as the transient and AD-induced increase in [GLU]e. Similarly, the responses to 5% O2 showed significant delays in the transient depolarization and AD-induced increase in [GLU]e. CBF during 3 or 5% O2 hypoxic insult was consistently maintained above the control level, i.e., prior to cardiac arrest. Our new dialysis electrode method employing both GOX and ferrocene-conjugated bovine serum albumin allowed evaluation of transient [GLU]e dynamics in the early phase of severe hypoxia in vivo.

Introduction

More than 80% of excitatory neurotransmission in the brain is mediated by l-glutamic acid (glutamate) 6, 7, 8. In recent years, increased extracellular space glutamate ([GLU]e), under conditions of global cerebral ischemia, anoxia and hypoxia, has been recognized as being intimately involved in the development of neuronal damage and cell death 6, 7, 26, 27. It is widely accepted that a slight increase in [GLU]e occurs in the early phase of oxygen deprivation, e.g., hypoxia [22]. To our knowledge, however, there are no reports demonstrating this slight increase in [GLU]e before anoxic depolarization during severe systemic hypoxia in vivo. We previously described a microdialysis electrode method 2, 24, 34, utilizing glutamate oxidase (GOX) and ferrocene-conjugated bovine serum albumin (FBSA), which allows excellent time resolution with real-time monitoring of [GLU]e during ischemia 3, 4, 30, 35, 36.

In this study, we subjected the rat striatum during severe or moderate systemic hypoxia to in vivo real-time monitoring of [GLU]e, direct current potential (DC), and cerebral hemodynamics and metabolism, assessed by measuring mean arterial blood pressure (MABP) and cerebral blood flow (CBF). We found that even in the absence of a decrease in CBF below the basal level, the neuronal membrane potential underwent transient depolarization, associated with a concurrent, transient increase in [GLU]e during severe systemic hypoxia. These transient phenomena were significantly prolonged in parallel with decreasing inspired oxygen concentration. Given our experimental concept and conditions, we consider the results to be entirely incompatible with spreading depression (SD) 10, 12, 21, 28 and hypoxic spreading depression (HSD) 5, 28, 29. We describe our interesting results herein.

Section snippets

Reagents

Glutamate oxidase was purchased from Yamasa, Chiba, Japan. Ferrocene carboxylic acid was obtained from Tokyo Kasei Kogyo, Tokyo, Japan; BSA (bovine serum albumin) and O-phenylenediamine from Sigma, St. Louis, MO; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) from Pierce Chemical, Rockford, IL; 2-(N-morpholino)-ethane sulfonic acid (MES) from Dojindo Laboratories, Kumamoto, Japan; and phosphate buffered saline (PBS) from Gibco/BRL, Grand Island, NY. Omega-conotoxin GVIA was

Results

The results of real-time monitoring of [GLU]e during anoxia induced by 100% N2 (Fig. 1A) and hypoxia induced by 3% O2/97% N2 (Fig. 1B) or 5% O2/95% N2 (Fig. 1C) are shown in Fig. 1. When the inspired gas was switched from room air to 100% N2 to induce anoxia, a sharp [GLU]e elevation (first phase) took place about 100 s later. This elevation shifted, continuing to rise throughout the anoxic period, and a slow rise persisted thereafter (second phase). On the other hand, when 3% O2 hypoxia was

Discussion

As shown in Fig. 1A, [GLU]e increased rapidly, peaking approximately 100 s after the induction of anoxia (first phase), then decreased, but showed a subsequent gradual increase (second phase) without recovering to the basal level. These findings indicate that the [GLU]e increase due to anoxia has a biphasic pattern similar to that induced by ischemia 3, 4, 35, 36, 37. It is well known that [GLU]e increases by two distinct mechanisms during cerebral ischemia [32]. One is rapid presynaptic

Conclusion

Under conditions of severe systemic hypoxia, MABP was maintained above 60 mmHg, and CBF was consistently maintained above the pre-cardiac arrest control value. Then, a transient DC depolarization and [GLU]e increase were observed before hypoxia-induced AD.

The transient increase in [GLU]e before hypoxia-induced AD was inhibited by local microinjection of a Ca2+-channel antagonist, omega-conotoxin GVIA, before the induction of 3% O2 hypoxia. With 8% O2 hypoxia, membrane depolarization was delayed

Acknowledgements

e thank Mr. Yuichi Matsumoto for his excellent technical advice on the dialysis electrode method. This work was supported by The Science Research Promotion Fund from the Japan Private School Promotion Foundation and a Special Research in Health Science Research Grant from The Ministry of Health and Welfare.

References (37)

  • S. Asai et al.

    Real time monitoring of biphasic glutamate release using dialysis electrode in rat acute brain ischemia

    NeuroReport

    (1996)
  • S. Asai et al.

    Minimal effect of brain temperature changes on glutamate release in rat following severe global brain ischemia: a dialysis electrode study

    NeuroReport

    (1998)
  • D.W. Choi et al.

    Glutamate neurotoxicity in cortical cell culture

    J. Neurosci.

    (1987)
  • G.L. Collingridge et al.

    Excitatory amino acid receptors in the vertebrate central nervous system

    Pharmacol. Rev.

    (1989)
  • A.J. Hansen

    The extracellular potassium concentration in brain cortex following ischemia

    Acta Physiol. Scand.

    (1978)
  • A.J. Hansen et al.

    Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex

    Acta Physiol. Scand.

    (1981)
  • A.J. Hansen et al.

    Anoxia increases potassium conductance in hippocampal nerve cells

    Acta Physiol. Scand.

    (1982)
  • A.J. Hansen et al.

    The role of spreading depression in acute brain disorders

    An Acad. Brasil Cliénc.

    (1984)
  • Cited by (21)

    • The earliest neuronal responses to hypoxia in the neocortical circuit are glutamate-dependent

      2016, Neurobiology of Disease
      Citation Excerpt :

      In our study, we measure the Ca2 + directly under the membrane, near the carboxy terminal region of the BK channel (Hou et al., 2016), and the high values suggest that the source is a Ca2 +-permeable channel nearby in the membrane. Since the neuron was not depolarized, and the rise is recorded in the presence of Cd2 +, which blocks voltage-dependent Ca2 + channels, and since one of the earliest consequences of hypoxia is an increase extracellular glutamate (Hagberg et al., 1985; Kunimatsu et al., 1999; Rossi et al., 2000), the simplest explanation is that the source is Ca2 +-permeable glutamate receptors. It is noteworthy that somatic excitatory synapses are very rare in cortical pyramidal cells (Gray, 1959), and AMPA receptors, most of which are impermeable to Ca2 +, have a significantly lower affinity to glutamate (Patneau and Mayer, 1990).

    • Spreading depolarizations mediate excitotoxicity in the development of acute cortical lesions

      2015, Experimental Neurology
      Citation Excerpt :

      The loss of electrochemical membrane gradients during SD (Hablitz and Heinemann, 1989; Hansen and Lauritzen, 1984; Kraig and Nicholson, 1978) may also imply that SD is a primary contributor to excitotoxic processes. Microdialysis studies have shown increases in extracellular glutamate in association with both SD and the related phenomenon of anoxic terminal spreading depolarization (ATSD) (Fabricius et al., 1993; Iijima et al., 1998; Kunimatsu et al., 1999; Satoh et al., 1999; Ueda et al., 1992). During sustained depolarizations, neurons may be particularly vulnerable to glutamate since the Mg2 + block of NMDARs is removed and the sodium- and voltage-dependence of excitatory amino acid transporters (EAATs) would limit glutamate clearance (Danbolt, 2001; Sarantis and Attwell, 1990; Szatkowski et al., 1990).

    View all citing articles on Scopus
    View full text