Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius)

Abstract

Ellefsen S, Stensløkken K, Fagernes CE, Kristensen TA, Nilsson GE. Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius). Physiol Genomics 36: 61-68, 2009. First published October 28, 2008 doi:10.1152/physiolgenomics.90301.2008.-The crucian carp, Carassius carassius, survives days to months without oxygen, depending on temperature. In the anoxic crucian carp brain, increased GABAergic inhibition, mediated by increased extracellular levels of GABA, has been shown to suppress electric activity and ATP consumption. To investigate an involvement of gene expression in this response, we utilized real-time RT-PCR to test the effect of 1 and 7 days anoxia (8°C) on the expression of 22 genes, including nine GABAA receptor subunits (␣1-6, ␤2, ␦, and ␥2), three GABAB receptor subunits (GB1a-1b and GB2), three enzymes involved in GABA metabolism (GAD65 and GAD67, GABAT), four GABA transporters (GAT1, 2a-b and 3), two GABAA receptor-associated proteins (GABARAP 1 and 2), and the K ϩ /Cl Ϫ cotransporter KCC2. While the expression of GABAA receptor subunits was dominated by ␣4-, ␣6-, and ␦-subunits, all of which are located to extrasynaptic sites in mammalian brains and respond to elevations in extracellular levels of GABA by showing tonic activity patterns, the expression of GABA transporters was dominated by GAT2 (a and b) and GAT3, which also show extrasynaptic location in mammals. These expression patterns differ from those observed in mammals and may be a prerequisite for GABAergic inhibition of anoxic metabolic rate in crucian carp. Furthermore, while the expression of the majority of the genes was largely unaltered by anoxia, the expression of GAT2 and GAT3 decreased to 20%. This suggests impairment of GABA transport, which could be a mechanism behind the accumulation of extracellular GABA and the increased GABAergic inhibition.GABA; GABA receptor; GABA transporter; external RNA control SINCE GABA IS THE DOMINANT inhibitory neurotransmitter in vertebrate brains, GABAergic neurotransmission has been suggested to function as an endogenous defense against excitotoxic events, such as the excessive release of glutamate during periods of ischemia/anoxia (26). However, GABAergic activity seems to be lowered rather than enhanced in ischemic mammalian brains, resulting in neuronal hyperexcitability and cell death (41). Thus, efforts have been made to reverse this process, and several studies have found that artificially increased GABAergic activity results in increased neuroprotection (41).Interestingly, anoxia induces increased GABA levels and GABAergic activity in brains of vertebrate species that tolerate prolonged periods of oxygen depletion, such as fish of the genus Carassius and freshwater turtles of genera Chrysemys and Trachemys. For example, while crucian carp (Carassius carassius) brains show a doubling of extracellular GABA levels after 6 h of anoxia and a fivefold increase in tissue GABA levels after prolonged anoxia (32, 19), freshwater turtle brains show a ...

Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition

“…Compared with levels in turtle brain, the anoxia-mediated increase in extracellular [GABA] in crucian carp brain is quite modest and doubles after 5 h of anoxia (Hylland and Nilsson, 1999). Furthermore, with anoxia, GABA removal from the extracellular space is significantly reduced because the expression of GABA transporter proteins decreases by 80%, increasing the effective extracellular [GABA] further (Ellefsen et al, 2009). We observed a significant increase in neuronal V m with anoxia.…”

“…Cl − flow occurs through both synaptic ( phasic) and extrasynaptic (tonic) GABA receptors (Blaesse et al, 2009). Localization of GABA A receptors has not been investigated in goldfish neurons; however, crucian carp neurons express the gamma subunit, which is necessary for phasic currents, and delta subunits, which in mammalian brain are extrasynaptic (Mody, 2001;Ellefsen et al, 2009). By applying GZ, we likely inhibited the activity of phasic currents as GZ has a greater affinity for synaptic GABA A receptors (Mody, 2001).…”

First Aid Kit for Hypoxic Survival: Sensors and Strategies

“…In contrast, there is a slight fall in mRNA levels of GABA A -receptor subunits in crucian carp exposed to 1-7 d of anoxia at 8ЊC, suggesting a more modest and modulated GABAergic inhibition in this species (Ellefsen et al 2009). Nevertheless, pharmacological inhibition of GABA receptors or GABA synthesis causes a threefold increase in ethanol release to the water by anoxic crucian carp, suggesting a significant GABAergic component in anoxic whole-body metabolic depression (Nilsson 1992).…”

“…However, two contributing mechanisms have been suggested. A study has shown that the mRNA levels of transporter proteins of the GAT2 family, which is responsible for a major part of GABA reuptake, fall by ∼75% during anoxia (Ellefsen et al 2009). Thus, there may be a transcriptionally induced suppression of GABA reuptake from the extracellular space during anoxia.…”

Hypoxia Tolerance in Animals: Biology and Application

“…Oddly, turtles appear to maintain vision during anoxia (Stensløkken et al 2008b). A moderate increase in extracellular GABA levels (Hylland and Nilsson 1999), possibly mediated by a reduced expression of GABA reuptake proteins (Ellefsen et al 2009), is probably involved in depressing energy use in the crucian carp (Nilsson 1992), but only to a level that is compatible with some physical activity. Other mechanisms that may be involved in defending brain and heart energy charge in crucian carp include phosphorylation of AMPactivated kinase that in turn will initiate mechanisms that increase energy supply and reduce energy use (Stensløkken et al 2008a).…”