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Physiol. Genomics 36: 61-68, 2009. First published October 28, 2008; doi:10.1152/physiolgenomics.90301.2008
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Received 4 August 2008; accepted in final form 28 October 2008.
Physiological Genomics 36:61-68 (2009)
1094-8341/09 $8.00 © 2009 American Physiological Society

Call For Papers: Comparative Genomics

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

Stian Ellefsen 1,2, Kåre-Olav Stensløkken 3, Cathrine E. Fagernes 1, Tom A. Kristensen 4 and Göran E. Nilsson 1

1 Physiology Programme, Department of Molecular Biosciences, University of Oslo, Oslo
2 Lillehammer University College, Lillehammer
3 Cancer and Surgical Division, Ullevål University Hospital, Oslo
4 Gene Programme, Department of Molecular Biosciences, University of Oslo, Oslo, Norway


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 ({alpha}1–6, β2, {delta}, and {gamma}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+/Cl– cotransporter KCC2. While the expression of GABAA receptor subunits was dominated by {alpha}4-, {alpha}6-, and {delta}-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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 80-fold increase in extracellular GABA levels after 6 h of anoxia (34). Also, pharmacological inhibition of GABA synthesis or GABA receptors has been found to increase the whole body metabolic rate of anoxic crucian carp by threefold (31), and similar treatment has been found to increase the extracellular levels of glutamate in brain tissue of anoxic freshwater turtles (43). Thus, these vertebrates seem to rely on increased GABAergic activity to depress neuronal activity, thereby reducing the ATP consumption, during periods of oxygen deprivation.

The action of extracellular GABA is primarily mediated by GABAA and GABAB receptors (GABAARs and GABABRs). While GABAARs are ionotropic and represents the principal sites for neural inhibition (12), GABABRs are metabotropic and also mediate neural inhibition (1). In mammals, 19 different GABAAR subunits have been described ({alpha}1–6, β1–3, {gamma}1–3, {delta}, {epsilon}, {theta}, {pi}, and {rho}1–3), and functional receptors consist of five subunits that make up an anion-permeable pore (12). The typical stoichiometry of synaptic GABAARs is 2{alpha}-2β-1{gamma}, with the most abundant combination in mammals being {alpha}1β2{gamma}2 (12). Still, within this stoichiometrical composition, GABAARs can display wide variations in subunit composition, bringing along variations in receptor functionality. For example, the {alpha}-subunit composition may vary and will largely determine the desensitization rate and the GABA affinity (defined as the GABA concentration needed to produce a half-maximal response: EC50) (12). While the human {alpha}1β3{delta}-receptor show an EC50 of 5.2 µM (13), the human {alpha}4β3{delta}- and {alpha}6β3{delta}-receptors show EC50s of 0.3–0.7 µM (4, 13). However, GABAARs also show stoichiometric compositions other than 2{alpha}-2β-1{gamma}. This further increases the flexibility of receptor function. For example, by replacing the {gamma}-subunit with a {delta}-subunit, the extent of receptor desensitization can be dramatically reduced (12). Whereas {alpha}1β3{gamma}2-receptors are desensitized by ~90% during sustained stimulation by high GABA levels, {alpha}1β3{delta}-receptors show only ~35% desensitization (2). Thus, given that the GABA levels are high enough to provide receptor opening, GABAARs containing the {delta}-subunit seem well suited for mediating tonic inhibition of neurons (12). In line with this, these GABAARs are primarily expressed at extrasynaptic sites in the mammalian brain and are controlled by the ambient, extracellular GABA levels (12). The {delta}-subunit is often coexpressed with {alpha}4- or {alpha}6-subunits (12).

With regard to the GABABR, two different mammalian subunits have been described (GB1 and GB2), and functional GABABRs contain both subunits, making up G protein-coupled receptors (36). GABABRs can be found at both presynaptic and postsynaptic locations.

In tissues, the GABA availability can be regulated by changing the activity of the GABA synthesizing glutamate decarboxylases (GAD65 and GAD67), the GABA degrading GABA aminotransferase (GABAT), and the GABA transport proteins (GAT1–3 and BGT-1) (6, 41). The efficiency of the GABAergic system is also affected by interactions/coexpression with various proteins. For example, the presence of the GABAA receptor-associated protein (GABARAP) gives GABAARs with increased single-channel conductance (11). Also, the presence of the neuron-specific K+/Cl– cotransporter, KCC2, is vital for restoring the Cl– homeostasis after GABAAR opening, making KCC2 activity essential for GABAergic activity (40).

The observed plasticity in the GABAergic system allows regional and developmental difference in GABA function but may also allow for defense against severe physiological insults such as excitotoxicity and ischemia. For example, GAD65 and GAD67 show increased expression during excitotoxic seizures (10), presumably increasing the GABA production in an activity-dependent manner, and the GABA transporters GAT1 and GAT3 show reduced expression during excitotoxic seizures and ischemic insults (27, 44), presumably leading to reduced GABA uptake and increased extracellular GABA levels. Also, substantial alterations in the subunit expression of GABAARs and GABABRs have been reported in response to excitotoxic and ischemic events (14, 38, 42), and decreased KCC2 expression has been found during kindling-induced seizures, where it may contribute to neuronal death (39). Still, in mammalian tissues, the physiological/pathophysiological responses to ischemia and excitotoxicity are not easy to interpret. For example, the GABA transporters GAT1 and GAT3 have also been indicated to show increased expression during excitotoxic seizures and ischemic insults (18, 27).

Little is known about the mechanisms that underlie the increased GABAergic inhibition in crucian carp and turtles during anoxia. However, anoxic turtle brains have been reported to show increased densities of GABAARs (25), arguably promoting GABAergic inhibition. Still, important traits such as {alpha}-subunit composition of GABAARs and the processes that underlie the increased extracellular GABA levels in anoxic crucian carp and turtle brains have not been investigated.

Here we have investigated the expression of genes involved in GABAergic neurotransmission in the crucian carp brain, aiming to disclose anoxia-induced changes in expression, as well as innate patterns of expression. This was done by quantifying the expression of 22 genes, 16 of which were partially cloned, by real-time RT-PCR. Since the expression of internal RNA control genes (reference genes) changes in crucian carp during anoxia (9), normalization of the real-time RT-PCR data were performed using our recently developed approach, achieving high-resolution normalization by including an external RNA control gene (mw2060) during the extraction procedure (9). The following genes were investigated: nine GABAAR subunits ({alpha}1–6, β2, {delta} and {gamma}2), three GABABR subunits (GB1a-1b and GB2), GAD65 and GAD67, GABAT, four GABA transporters (GAT1, 2a-b, and 3), two GABARAPs (1 and 2), and KCC2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal handling and experimental design.
Crucian carp were caught in Tjernsrud pond, Oslo municipality, Norway, in September and were kept in 750-liter tanks continuously supplied with dechlorinated, aerated Oslo tap water for 3–4 mo at water temperatures of 9 ± 2°C. The photoperiod was held at 12 h darkness/12 h daylight, and the fish were fed commercial carp food on a daily basis.

Prior to experiments, crucian carp, weighing 40 ± 16 g, were transferred to flow through (2–4 l/h) circular 25-liter tanks (16 fish in each) and left to acclimate for 18 h in the dark. Subsequently, the tanks were sealed with a tight lid, and the water was either bubbled with air (normoxia and reoxygenation) or nitrogen (anoxia). Oxygen concentrations and temperatures were continuously monitored using a galvanometric oxygen electrode, Oxi 340i (WTW, Weilheim, Germany), connected to a computer. Water with no detectable oxygen (<0.1 mg O2 l–) was considered anoxic.

Anoxia exposures were performed at 8°C and included four groups of fish: 7 days of normoxia (N7), 1 day of anoxia (A1), 7 days of anoxia (A7) and 7 days of anoxia followed by 7 days of recovery (R7). We killed fish by stunning them with a sharp blow to the head, before cutting the spinal cord and dorsal aorta, and removing the brain. Within 1 min of collecting the fish, brains were snap-frozen in liquid nitrogen. The brains were stored at –80°C until use.

The animal protocol was evaluated and approved by The National Animal Research Authority of Norway.

Obtaining sequences for genes involved in GABAergic neurotransmission.
For GABAAR{alpha}1, GAD65, GAD67, GABAT, GAT1, and GAT3 no cloning was necessary as their sequences were available for goldfish (Carassius auratus), a close relative to crucian carp (accession numbers are listed in Table 1). Gene-specific primers (GSPs) for cloning of the remaining genes are listed in Table 1, and were designed from zebrafish (Danio rerio) sequences, using Primer3 (http://frodo.wi.mit.edu/primer3/input.htm). Zebrafish belongs to the same family as crucian carp (Cyprinidae). Moreover, GSPs were located to gene regions that showed high degree of sequence conservation between zebrafish and other vertebrates. Sequences were retrieved from the National Center for Biotechnology Information homepage (www.ncbi.nlm.nih.gov/) or the Ensembl Genome Browser (www.ensembl.org/index.html). Sequence alignments were performed with GeneDoc (version 2.6.0.2, www.psc.edu/biomed/genedoc/) and ClustalX (version 1.81, ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/).


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  		Table 1. Primer sequences used for cloning and real-time RT-PCR

 
Total RNA was extracted from untreated crucian carp brain tissue using TRIzol reagent (Invitrogen, Carlsbad, CA), and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) was used to assure the quality of the RNA, whereas a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Rockland, DE) was used to quantify the RNA.

For expressed sequence tag cloning, 1 µg of total RNA was DNase I treated (Sigma-Aldrich, St. Louis, MO) and reverse transcribed using oligo(dT)18 and Superscript III in reaction volumes of 20 µl (Invitrogen). PCR was performed on 1/25 dilutions of the resulting cDNA using Platinum Taq (Invitrogen). The following PCR program was used: 1) 94°C for 10 min, 2) 94°C for 30 s, 3) 50°C for 1 min, 4) 72°C for 1 min, 5) repeat steps 2–4 44x, 6) 72°C for 10 min, 7) hold 4°C. The resulting PCR products were cloned using pGEM-T Easy Vector System I (Promega, Madison, WI) and CaCl2-competent cells (TOP10 F', Invitrogen). Positive clones were checked for inserts of correct size using PCR and were sequenced using T7 primers (ABI-lab, University of Oslo, Oslo, Norway). All procedures were carried out according to manufacturers’ protocols.

The resulting crucian carp sequences were submitted to The European Molecular Biology Laboratory Nucleotide Sequence Database (www.ebi.ac.uk/); the accession numbers are listed in Table 1. Duplicate paralogs were found for GABAA subunits {alpha}2, {alpha}4–6, β2, {delta}, and {gamma}2, the GABAB subunit GB1, and GAT2. The two paralogs for each of these genes were labeled a and b, with the exception of the {delta}-paralogs, which were labeled 1 and 2.

RNA extraction and cDNA synthesis for real-time RT-PCR.
Total RNA for real-time RT-PCR experiments was extracted from 42 mg brain tissue using 15 µl TRIzol/mg. The extractions were performed in accordance with the detailed protocol outlined by Ellefsen et al. (9), adding 20 pg external RNA control gene (mw2060) per mg tissue. The quality and quantity of the total RNA were assessed using 2100 Bioanalyzer and NanoDrop ND-1000 UV-Vis Spectrophotometer.

Total RNA (1 µg) was DNase I treated and reverse transcribed using oligo(dT)18 and Superscript III, as previously described. For each fish, duplicate cDNA syntheses were performed. Negative RT controls were performed as random checks on ~10% of the samples. cDNA reactions were diluted to 1:25 using autoclaved MilliQ water. All procedures were carried out according to manufacturer's protocol.

Real-time RT-PCR.
Real-time RT-PCR was performed on a Lightcycler 2.0 (Roche Diagnostics, Basel, Switzerland). Calculations of normalized levels of gene expression were performed with Eq. 1:

Formula 1(1)
Tar = target gene, Con = control gene (mw2060), E = priming efficiency, and Cp = crossing point. mw2060 was used as RNA control gene in all real-time RT-PCR data sets. E values were calculated for each real-time RT-PCR reaction using LinRegPCR software (37), but in the final calculations, average priming efficiencies (Emean) were used, calculated separately for each primer pair, and based on all real-time RT-PCR reactions. Cp values were obtained for each reaction using the Lightcycler 2.0 software and were defined as the second derivative maximum. Real-time RT-PCR primers were designed using Primer3 and are presented in Table 1.

All real-time RT-PCR reactions were performed with reaction volumes of 10 µl, using Lightcycler Faststart DNA MasterPLUS SYBR Green I (Roche Diagnostics) and Lightcycler Capillaries (Roche Diagnostics). We used 5 µl 1:25 diluted cDNA as template (prepared as previously described), and the following real-time RT-PCR program was used: 94°C for 10 min, 94°C for 10 s, 60°C for 12 s, 72°C for 8 s, repeat steps 2–4 39x. Two real-time RT-PCR reactions were performed for each gene for each cDNA synthesis. Since two cDNA syntheses were performed for each total RNA sample, a total of four real-time RT-PCR reactions were performed on each gene for each sample of total RNA. The mean value of all four reactions was used in the gene expression analyses. All real-time RT-PCR reactions were performed using primer concentrations of 100 nM, and all primer pairs were found to amplify the desired cDNA species. The latter was verified by melting curve analyses (Lightcycler 2.0 software), gel electrophoresis, and cloning/sequencing (performed as previously described). Primer efficiencies and average Cp values for the different primer pairs are summarized in Table 1. It should be noted that, to find primers that worked well with the outlined real-time RT-PCR protocol (primer concentration of 100 nM and annealing temperature of 60°C), three primer pairs were tested for each gene. The primer pairs that displayed distinct melting curves and the highest Cp values were chosen. This was done as an alternative to optimization of primer concentrations/annealing temperatures. All procedures were carried out according to manufacturer's protocol.

Statistical analyses.
To evaluate whether treatments (N7, A1, A7, and R7) had an effect on gene expression patterns, statistical analyses were performed using GraphPad InStat (3.06; GraphPad Software, San Diego, CA). All data sets were assessed for statistically significant variation using one-way ANOVA, followed by Tukey-Kramer posttest. The data sets in Fig. 1 that illustrate percentage distribution were arcsine-transformed prior to analyses. P ≤ 0.05 was considered significant. Possible differences, indicated by P values between 0.1 and 0.05, are commented upon as tendencies. All data are presented as means + SD.


Figure 1
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  		Fig. 1. A: expression of the GABAAR subunits {alpha}1–6, β2, {delta}, and {gamma}2 in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and were referenced to the control group N7. Values are means +SD. B: expression profile for the GABAAR {alpha} subunits in N7, A1, A7, and R7. C: expression profile for the GABAAR {delta}/{gamma}2 subunits in N7, A1, A7, and R7. N7, normoxia 7 days (n = 5); A1, anoxia 1 day (n = 5); A7, anoxia 7 days (n = 6); R7, anoxia 7 days followed by reoxygenation 7 days (n = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The cloning of the GABAergic genes from crucian carp brain revealed paralogous variants for the GABAAR subunits {alpha}2, {alpha}4–6, β2, {delta}, and {gamma}2, for the GABABR subunit GB1, and for GAT2. Although the majority of these paralogs showed a close relationship (94–95% homologies) and were treated as single genes during gene expression analyses, the GB1 and GAT2 paralogs showed a more distant relationship (84 and 78% homologies, respectively), presumably having evolved different functions, and were treated as separate genes. Thus, for the majority of the paralogs real-time RT-PCR primers were directed at conserved gene region, but for GABABR1 and GAT2 paralogs, real-time RT-PCR primers were directed at poorly conserved gene regions.

Exposure of crucian carp to anoxia and reoxygenation resulted in decreased expression of the GABAAR subunits {alpha}2, {alpha}3, and {alpha}6 (Fig. 1A; P = 0.04, P = 0.008, and P = 0.03, respectively). Compared with N7 levels, being set to 1.0, the {alpha}2-subunit decreased to 0.59 in R7 (P < 0.05), the {alpha}3-subunit decreased to 0.57 in A1 (P < 0.05), to 0.63 in A7 (P < 0.05), and to 0.49 in R7 (P < 0.05), and the {alpha}6-subunit decreased to 0.64 in A7 (P < 0.05). Other GABAAR subunits, such as {alpha}1, β2, and {delta}, also showed tendencies toward decreased expression, with ANOVA analyses resulting in P values <0.1.

However, although the expression of several GABAAR subunits decreased during anoxia (A1 or A7), the subunit composition remained largely unchanged. For example, the {alpha}-subunit composition changed only slightly, with {alpha}1 being the only subunit to show changed abundance, increasing from 5.6 ± 0.7% in N7 to 7.3 ± 1.2% in A1 (P < 0.05) (Fig. 1B). Reoxygenated fish showed somewhat larger changes in {alpha}-subunit composition, displaying decreased abundance of {alpha}1 (from 5.6 ± 0.7% to 4.0 ± 0.2%, P < 0.05), {alpha}2 (from 8.5 ± 0.5% to 5.9 ± 0.8%, P < 0.001), and {alpha}3 (from 2.8 ± 0.5% to 1.7 ± 0.1%, P < 0.01). Interestingly, the {alpha}4- and {alpha}6-subunit, which have previously been described to play vital roles in extrasynaptic GABA signaling (12), dominated the {alpha}-subunit expression, constituting between 76.0 ± 2.2% and 84.0 ± 1.0% of the collective expression (Fig. 1B). A corresponding dominance of an extrasynaptic subunit was found in the {delta}-/{gamma}2-subunit expression profile (Fig. 1C), where the extrasynaptic {delta}-subunit constituted 72.5 ± 1.9% of the collective expression in N7. Although the abundance of the {delta}-subunit decreased to 67.6 ± 1.0 in A7 (P = 0.01), it still remained the most abundant.

The GABABR subunits showed no significant changes in expression in the anoxic crucian carp brain (Fig. 2). However, in resemblance with several of the GABAAR subunits, the GABABR subunits GB1a and GB1b showed a tendency toward decreased expression, with ANOVA giving P values <0.1. The expression of KCC2, GABARAP1, and GABARAP2 did not change significantly (Fig. 3).


Figure 2
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  		Fig. 2. Expression of the GABABR subunits 1a-b and 2 in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and were referenced to the control group N7. Values are means + SD. N7 n = 5; A1 n = 5; A7 n = 6; R7 n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 

Figure 3
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  		Fig. 3. Expression of GABARAP1–2 and KCC2 in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and were referenced to the control group N7. Values are means + SD. N7 n = 5; A1 n = 5; A7 n = 6; R7 n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 
Of the enzymes involved in the metabolism of GABA, only GAD65 showed a significantly altered expression in response to anoxia (P = 0.0006), changing from 1.0 in N7 to 0.74 in A7, and to 0.70 in R7 (P < 0.05 and P < 0.01, respectively) (Fig. 4). This indicated that the rate of GABA production was decreased, rather than increased, in brains of anoxic crucian carp.


Figure 4
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  		Fig. 4. Expression of GAD65, GAD67, and GABAT in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and were referenced to the control group N7. Values are means + SD. N7 n = 5; A1 n = 5; A7 n = 6; R7 n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 
With regard to GABA transporters, the expression of GAT1 appeared unaffected by anoxia (Fig. 5A, P = 0.30), while the expression of GAT2a, GAT2b, and GAT3 was significantly reduced (compared with N7 levels, being set to 1.0) (P = 0.0005, P = 0.0113, and P = 0.0001, respectively). The GAT2a expression decreased to 0.44 in A1 (P < 0.01), to 0.23 in A7 (P < 0.001), and to 0.52 in R7 (P < 0.05), the GAT2b expression decreased to 0.49 in A7 (P < 0.01), and finally the GAT3 expression decreased to 0.39 in A1 (P < 0.01), to 0.20 in A7 (<0.001), and to 0.40 in R7 (P < 0.01). Moreover, this resulted in a shift in the GAT composition. Whereas GAT1 mRNA constituted 16.2 ± 3.8% of the collective expression of GAT mRNA in N7 and did not change significantly during anoxia, GAT2a, GAT2b, and GAT3 constituted 26.8 ± 1.8%, 6.6 ± 2.1%, and 50.4 ± 4.1% in N7 and decreased to 6.1 ± 1.7%, 3.3 ± 1.8%, and 10.1 ± 2.1% in A7 (Fig. 5B). Thus, while GAT2a+b and GAT3 dominated the expression in N7, GAT1 showed the highest expression in A7. Furthermore, compared with N7 (set to 100%), the overall GAT expression decreased to 47.3 ± 18.9% in A1, to 31.3 ± 7.3 in A7, and to 50.8 ± 22.4 in R7 (P < 0.001) (Fig. 5B).


Figure 5
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  		Fig. 5. A: expression of GAT1, GAT2a-b, and GAT3 in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and were referenced to the control group N7. Values are means + SD. B: expression profile for GATs in N7, A1, A7, and R7. Data were referenced to the control group N7, which were set to 100%. N7 n = 5; A1 n = 5; A7 n = 6; R7 = n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA followed by Tukey Kramer's posttest).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To our knowledge, this is the first report of a large-scale analysis of the expression of genes underlying GABAergic neurotransmission in a nonmammalian vertebrate. The technical procedures used were in accordance with our previous studies (8, 9), and the approach allowed us to evaluate several components of the GABAergic system, including receptors, synthesis, and transport. GABA receptor properties are largely dependent on subunit stoichiometry (12), and GABAAR subunits are likely to show close correlations between mRNA and protein levels, since they act as parts of multiple-protein complexes (21). Still, mRNA levels may not reflect protein levels, which can be controlled at the level of translation and degradation (16).

GABA receptors.
A general, but relatively small decrease (~30%) was seen in the expression of subunits for GABAARs and GABABRs in brains of anoxic crucian carp (Figs. 1A 2). This is in contrast to a previous report from turtles, which showed increased densities of GABAAR in brains during anoxia (25), and could be the result of a negative feedback response to the elevated GABA levels, serving to limit the inhibitory tonus during anoxia and allow an appropriate level of neural activity. However, the observed decrease in anoxic crucian carp did not result in substantial changes in the composition of the subunits. For example, the {alpha}-subunit composition of GABAAR remained largely unchanged during anoxia (Fig. 1B), and the {delta}-subunit remained much more abundantly expressed than the {gamma}2-subunit also in anoxia (Fig. 1C).

Sustained properties of GABA receptors during anoxia were also supported by the unchanged expression of KCC2 and GABARAP1–2 (Fig. 3). The KCC2 data were of particular interest, since the KCC2 expression has been shown to decrease in mammals in response to excitotoxic events (39), seemingly playing a role in subsequent cell death.

Compared to the GABAAR subunit composition of mammals, which is dominated by the synaptic {alpha}1- and {gamma}2-subunits (12), GABAARs in crucian carp were dominated by {delta}- and {alpha}4/{alpha}6-subunits (Fig. 1, B and C), which are primarily extrasynaptic in mammals (12). This suggests that extrasynaptic GABAARs play a more prominent role in crucian carp, which may have to do with its ability to withstand anoxia. Indeed, the EC50 values of {alpha}4/{alpha}6-{delta}-containing receptors (~0.3–0.7 µM) (4, 13) correlate well with the extracellular GABA levels measured in the crucian carp brain during anoxia (0.5 µM) (19), indicating that these receptors are likely to be in an open state during anoxia. Moreover, since {delta}-subunit-containing GABAARs do not desensitize during sustained binding to GABA (2), they appear well suited for providing continuous GABAergic depression of neurons. Overall, the observed subunit composition of GABAARs in the crucian carp brain supports the notion that the anoxic survival of crucian carp does not require anoxia-induced regulation of receptor function, a regulation that indeed seems to be rather limited (as previously discussed). The normoxic subunit composition can thus be argued to represent some sort of constitutive preconditioning, representing a selective advantage during anoxic insults. Interestingly, we recently found evidence for similar subunit-dependent preconditioning in glutamate receptors (8). Together, these findings indicate that constitutive preconditioning may be a general trait of anoxia-tolerant vertebrates, as was also suggested by Nilsson and Lutz (33). Such preconditioning would, in addition to involving genes controlling neuronal activity, likely involve genes responsible for cellular housekeeping and survival (e.g., heat shock proteins). Collectively, our data underline the need for studies that provide broad overviews of biological processes rather than solely focusing on particular genes.

GABAergic enzymes and transporters.
Only minor changes were seen in the expression of genes involved in GABA synthesis and GABA degradation in anoxic crucian carp brain (GAD65, GAD67, and GABAT) (Fig. 4). Although GAD65 showed decreased expression (Fig. 4), which might reflect a negative feedback response to elevated GABA levels, this indicates sustained GABA synthesis. Indeed, tissue GABA levels rise severalfold in the anoxic crucian carp brain (32).

The most prominent changes in gene expression in response to anoxia involved large decreases in the expression of GAT2a-b and GAT3 and also a decrease in the overall expression of GATs, indicating a reduced ability to remove GABA from the extracellular space (Fig. 5). This could be a mechanism behind the increased extracellular GABA levels observed in the crucian carp brain during anoxia (19), which is likely to increase the degree of tonic GABAergic activity (15). In this way, decreased GABA transport could cause the increased GABAergic inhibition observed in anoxic crucian carp (31), thereby representing a vital adaptation for anoxic survival. GAT-related control of GABA levels has been found to play roles in controlling GABA responses in mammals (6). Interestingly, a recent study on rats suggests that GAT3, and not GAT1, is important for regulating tonic GABAergic activity in hypothalamic neurons (35).

In the mammalian brain, the different GATs have been ascribed different roles (6). GAT1 has a main responsibility for the termination of the synaptic response, while GAT2 has been suggested to play roles in GABA transport across the blood-brain barrier, and GAT3 has been suggested to be involved in controlling the GABA levels in the extracellular space. These roles have primarily been inferred from ultrastructural studies, which have found GAT1 to be most highly expressed in the vicinity of synapses (29), whereas GAT2 and GAT3 are mainly found in cells lining the leptomeninges/ventricles and blood vessels, and in astrocytic processes (30, 22). However, different functional roles for the different GATs have also been indicated by differences in transport properties. For example, different GABA affinities have been described. In mouse and rat, GAT1 shows a Km of 6–7 µM (17, 23), GAT2 shows a Km of 10–18 µM (3, 24), and GAT3 shows a Km of 0.8–2.3 µM (5, 24). Thus, GAT3 has the highest affinity for GABA, which supports a role for it in controlling extrasynaptic levels of GABA, being able to bind GABA at lower concentrations than GAT1. Indeed, the extracellular levels of GABA observed in the mammalian brain, typically being between 0.2 and 2.5 µM (15), indicate that GAT3 is required to obtain sufficient removal of GABA.

It is suggestive that GAT2 and GAT3 dominate GAT expression in an anoxia-tolerant vertebrate like the crucian carp, while the collective GAT expression in mammalian brains is dominated by GAT1 (6). In the normoxic crucian carp brain, GAT2 (a and b) and GAT3 amounted to a total of 84% of the overall GAT expression (Fig. 5B), indicating that GABA transport is primarily allocated to extrasynaptic sites where GABA typically induce tonic activity (12). Interestingly, a similar GAT composition is seen in neonatal rats, with GAT3 dominating the expression and accounting for the largest fraction of GABA transport (28). Such a "neonatal expression pattern" potentially mediates hypoxia tolerance (6).

Although controlling GAT function has been suggested to be an efficient way of controlling GABAergic function in mammals (7), few studies have examined this mechanism in detail (6), and in existing reports GAT1 has been the chief target, mainly because it is the only GAT for which selective antagonists have been available (7). Taking into account that evolution has solved the problem of anoxic survival in crucian carp, it is tempting to suggest that GAT2a-b and GAT3 are better targets for stimulating anoxic/ischemic GABAergic activity. Indeed, studies to elucidate the physiological significance of GAT activity during excitotoxic and ischemic insults in mammals have provided contradictive results. Whereas some studies have reported decreased GAT1 and GAT3 expression (27, 44), others have reported increased expression (18, 27). These contradictions may reflect different GATs serving different roles in different biological systems or contexts. This observation gains support from studies on different anoxia-tolerant species. While anoxic crucian carp shows modest elevations of extracellular [GABA] (19), anoxic freshwater turtles show massive elevations (34), indicating that extracellular GABA levels are regulated by different mechanisms. Indeed, this difference in GABA regulation, which presumably results in different levels of GABA action, is likely to underlie the differences seen in survival strategies: the crucian carp remains in an active state during anoxia, while freshwater turtles become more or less comatose (33).

Reoxygenated crucian carp brain.
Genes that showed altered expression during anoxia largely failed to recover during reoxygenation (Figs. 1, 2, 4, 5). This resembles our previous observation (8), and a likely reason for this is that the anoxic episode functions as a cue for preparing the brain for further and longer anoxic exposures. In nature, crucian carp can be exposed to anoxia for several months at temperatures close to 0°C during the long winter (20). This period is likely to be preceded by several shorter bouts of hypoxia or anoxia. Therefore, reoxygenated crucian carp is probably not analogous to a recovery group of a species where anoxia is a rare or pathological phenomenon.

Conclusions
The current study suggests that the GABAergic system in crucian carp brain is dominated by an extrasynaptic component. For example, the expression of GABAAR subunits is dominated by {alpha}4, {alpha}6, and {delta}, and the expression of GATs is dominated by GAT2a-b and GAT3, all of which are primarily extrasynaptic in mammals. Moreover, these GABAAR subunits are those that are likely to be best suited for responding tonically to the elevated extracellular GABA levels previously observed in anoxic crucian carp. Furthermore, anoxia resulted in reduced expression of the GABA transporters GAT2a-b and GAT3, which could be a mechanism behind the increased extracellular GABA levels seen in anoxic crucian carp. The resultant elevation of GABAergic neural inhibition could underlie the previously observed GABAergic inhibition of anoxic metabolic rate (31).


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was financed by grants from The Research Council of Norway (to G. E. Nilsson).


   ACKNOWLEDGMENTS
 
We thank Tove K. Larsen for technical assistance.


   FOOTNOTES
 
Address for reprint requests and other correspondence: S. Ellefsen, Lillehammer Univ. College, PO Box 952, N-2604 Lillehammer, Norway (e-mail: stian.ellefsen{at}hil.no).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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The major contribution of brain GABAergic function to anoxic survival
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