Extracellular levels of amino acid neurotransmitters during anoxia and forced energy deficiency in crucian carp brain

Abstract

The crucian carp is one of the few vertebrates that has the ability to survive long periods of anoxia. A devastating event in the anoxic mammalian brain is a massive release of excitatory neurotransmitters, particularly glutamate. Using microdialysis to measure extracellular levels of several amino acid neurotransmitters and related compounds in the telencephalon of crucian carp in vivo, we show here that this species avoids a release of glutamate during anoxia, which is probably related to its ability to maintain energy charge. Instead, 6 h of anoxia produced a doubling of the extracellular level of GABA, the major inhibitory neurotransmitter in brain. The release of GABA may be a mechanism for lowering neuronal activity and energy use, thereby facilitating the maintenance of energy charge. Perfusing the microdialysis probe with a high-K⁺ Ringer showed that the telencephalon had the ability to release both glutamate and GABA. Moreover, if energy deficiency was produced during anoxia, by inhibiting glycolysis with iodoacetate (IAA), the resulting release of GABA was more rapid and profound than that of glutamate, possibly reflecting a second line of anoxia defence aimed at minimising the effect of a temporary energy failure.

Introduction

In the mammalian brain, anoxia rapidly leads to a series of catastrophic events, initially triggered by an inability to compensate the loss of aerobic ATP production by anaerobic ATP production alone. The falling ATP levels will cause Na⁺/K⁺-pumping to slow down, resulting in a net outward leakage of K⁺ from the neurons [9]. The rise in extracellular K⁺ soon makes the neurons progressively more depolarized, and within a few minutes there is a sudden and rapid increase in membrane conductance to inorganic ions, leading to a complete depolarization. Around the same time, the extracellular compartment is flooded with neurotransmitters, notably glutamate—the major excitatory neurotransmitter in brain. This leads to the opening of glutamate activated cation channels, an excitatory event that is thought to be a key mechanism behind anoxic/ischemic brain damage, primarily by causing a massive inflow of Ca²⁺ which activates numerous degenerating processes 18, 44, 46. Loss of ion-homeostasis, reflected by a steep increase in extracellular K⁺[36], as well as a massive release of amino acids [12], are also seen in anoxia-intolerant rainbow trout brain during anoxia. Thus, these events probably occur in most oxygen deficient vertebrate brains.

However, there are a few vertebrate species that show an extraordinary ability to survive prolonged anoxia. The crucian carp (Carassius carassius) readily survives anoxia for weeks at 10°C and even for months at temperatures close to 0°C [41]. Also the closely related goldfish (C. auratus), as well as freshwater turtles of the genera Chrysemys and Trachemys, show a similar degree of anoxia tolerance [52].

Mechanisms of brain survival in these anoxia-tolerant vertebrates has attracted a considerable interest [22]. In sharp contrast to the majority of vertebrates, Carassius and turtles have been found to maintain their brain ATP levels and avoid neural depolarization when exposed to anoxia 15, 21, 36, 53. Depressing the rate of ATP use (metabolic depression) is a principal strategy for anoxic survival in anoxia-tolerant vertebrates, allowing them to maintain ATP levels by matching ATP use to a reduced rate of ATP production 10, 22, 25. Brain slices from freshwater turtles and crucian carp display metabolic depression during anoxia, their heat production falling by approximately 40% 5, 16, 39. In situ, brain metabolic depression is probably even more substantial, at least in turtles: brain lactate measurements in freshwater turtles indicate an 80–90% reduction in metabolic rate during anoxia [21].

Still, much remains to be learnt about the mechanisms underlying the metabolic depression. In turtles, a down-regulation of ion permeability of the neuronal membrane is probably involved 1, 4, 40, but there is no evidence for this in crucian carp brain [16].

There are indications that neurotransmitters play a role in metabolic depression of anoxia-tolerant brains. In contrast to excitatory neurotransmitters, with their devastating actions in the anoxic mammalian brain, inhibitory neurotransmitters may promote anoxic survival by depressing neuronal activity and energy consumption 29, 30, 34. GABA is the dominating inhibitory neurotransmitter in the vertebrate brain, and one of the most striking neurochemical changes taking place during anoxia in the brain of crucian carp and freshwater turtles is a substantial increase in the brain tissue levels of GABA 29, 31.

However, if inhibitory neurotransmitters such as GABA shall be able to depress neuronal activity, their levels have to increase extracellularly, where they can interact with their receptors. Indeed, using microdialysis in vivo, Nilsson and Lutz [33]found that in the freshwater turtle brain, there is an 80-fold increase in the extracellular level of GABA during anoxia, whereas no substantial release of glutamate to the extracellular compartment occurs. Increases in the extracellular levels of glycine and taurine were also seen in the anoxic turtle brain. Glycine is an inhibitory neurotransmitter in the vertebrate CNS, while taurine has been suggested to act as an inhibitory neuromodulator that counteracts anoxic brain damage [45].

There has hitherto been no studies focusing on anoxia-induced changes in extracellular neurotransmitter levels in Carassius brain. However, by measuring the production of ethanol, which is the main end product of anaerobic metabolism in Carassius, it was found that an inhibition of GABA synthesis results in an increased systemic metabolic rate during anoxia (but not during normoxia) [30]. This indicates a role of GABA in metabolic depression of the brain as well as of the whole fish. It is important to point out that there are striking differences in the anoxic survival strategies utilized by Carassius and freshwater turtles [23]. While anoxic freshwater turtles become virtually comatic and depress their metabolic rate to 1/10 [14], anoxic Carassius are still responsive to mechanical stimuli and show spontaneous swimming activity, although at a lower level [37]. Moreover, their metabolic rate is only reduced to 1/3 [54]. These species differences may partly rely on differences in the release of neurotransmitters in brain during anoxia. The different degree of metabolic depression is also reflected in cerebral blood flow (CBF). In turtles, CBF is only elevated during the initial stage (ca. 1 h) of anoxia [13], while Carassius maintains a doubled CBF for many hours of anoxia [32].

Consequently, the aim of this present study was to use intracerebral microdialysis to examine the effect of anoxia on the extracellular levels of amino acids, particularly those with neurotransmitter function, in the crucian carp brain (telencephalon). Moreover, since the anoxic crucian carp brain normally maintains ATP levels and avoids depolarization [15], we also studied the effects of high extracellular K⁺ and iodoacetate (IAA), in order to establish which amino acids are released during depolarization and forced energy failure, respectively. IAA blocks glycolysis by inhibiting glyceraldehyde-3-phosphate dehydrogenase [27]. One rationale for blocking glycolysis was to examine if crucian carp possesses a second line of anoxia defence, aimed at counteracting the consequences of temporary energy failure by, for example, suppressing a release of glutamate.