Gene expression in anoxic crucian carp brain

Abstract

Crucian carp, Carassius carassius, survives days to months without oxygen (anoxia). Still, during anoxia, it needs to keep the energy expenditure low, particularly in the brain, which has a high rate of ATP use mainly related to neuronal activity. We hypothesized that the anoxic crucian carp brain reduces its ATP use by suppressing neuronal excitability, and that this is reflected by the expression of genes involved in excitatory and inhibitory neurotransmission. Real-time RT PCR has become a dominating technique for analyses of gene expression. It enables large-scale, hypothesis-driven analyses of gene expression, and should be well-suited for studies in anoxic crucian carp. However, so far, the use of real-time RT PCR has been limited by the lack of a proper procedure for data normalization, with existing procedures depending on the assumption that internal control genes show constitutive expression and do not vary between experimental groups. This is a particular problem in experiments involving severe physiological stress, such as anoxia, where the expression of control genes must be expected to change. Paper 1 reports a novel procedure for normalization of real-time RT PCR data, using an external RNA control gene (mw2060). It is the first to report the addition of an external RNA to tissue on a per-unit-weight basis. The procedure was demonstrated to be suitable for normalization of real-time RT PCR data in crucian carp heart and brain, and provided more accurate normalization than internal RNA control genes. For example, in anoxic hearts, β-actin failed to detect a 2.5-fold increase in the expression of the stress-response gene HSC70. Papers 2 and 3 use the real-time RT PCR procedure to investigate the effects of 1 and 7 days of anoxia on the expression of 29 genes involved in excitatory glutamatergic neurotransmission and 22 genes involved in inhibitory GABAergic neurotransmission, respectively. In general, paper 2 talks against profound neural depression caused by reduced expression of excitatory ion channels in anoxic crucian carp brains. Still, the NMDA receptor-subunits (NR) showed expression patterns that could mediate reduced neuronal excitability. Primarily, the NR2 subunit expression was dominated by NR2B and NR2D, which resembles that seen in hypoxia-tolerant neonatal rats, but also, the expression of NR1, NR2C and NR3A decreased during anoxia, which suggests a reduced number of functional NMDA receptors. Paper 3 indicates that the GABAergic system in the crucian carp brain is dominated by extrasynaptic components. While the expression of GABAA-receptors subunit was dominated by α4, α6, and δ 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 and GAT3, which also show extrasynaptic location in mammals. The majority of the investigated genes were largely unaltered by anoxia, but the expression of GAT2 (a and b) and GAT3 was reduced by up to 80%. This suggests reduced GABA transport in the anoxic crucian carp brain, which may explain the previously reported elevation in extracellular GABA levels, and could underlie the previously observed GABAergic inhibition of anoxic metabolic rate.