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Physiol. Genomics 35: 5-17, 2008. First published July 1, 2008; doi:10.1152/physiolgenomics.90221.2008
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Received 14 April 2008; accepted in final form 26 June 2008.
Physiological Genomics 35:5-17 (2008)
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Call For Papers: Comparative Genomics

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

Stian Ellefsen 1,2, Guro K. Sandvik 1, Helene K. Larsen 1, Kåre-Olav Stensløkken 3, Dag Are S. Hov 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 months without oxygen. During anoxia it needs to keep energy expenditure low, particularly in the brain, with its high rate of ATP use related to neuronal activity. This could be accomplished by reducing neuronal excitability through altered expression of genes involved in excitatory neurotransmission. Through cloning and the use of a recently developed real-time RT-PCR approach, with an external RNA control for normalization, we investigated the effect of 1 and 7 days of anoxia (12°C) on the expression of 29 genes, including 8 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, 6 N-methyl-D-aspartate (NMDA) receptor subunits, 7 voltage-gated sodium and calcium channels, 4 glutamate transporters, and 4 genes involved in NMDA receptor-mediated neuroplasticity. The subunits of the majority of the gene families had expression profiles similar to those observed in the mammalian brain and showed remarkably stable expression during anoxia. This suggests that the genes may have similar functions in crucian carp and mammals, and that the excitatory abilities of the crucian carp brain are retained during anoxia. Although the data generally argue against profound neural depression ("channel arrest"), NMDA receptor subunit (NR) expression showed features that could mediate reduced neural excitability. Primarily, the NR2 subunit expression, which was dominated by NR2B and NR2D, resembled that seen in hypoxia-tolerant neonatal rats, and decreased anoxic expression of NR1, NR2C, and NR3A indicated reduced numbers of functional NMDA receptors. We also report the full-length sequence of crucian carp NR1 mRNA and a novel NR1 splice cassette introducing an N-glycosylation site into the extracellular S1S2 domain.

N-methyl-D-aspartate/3-hydroxy-5-methyl-4-isoxazole propionate receptors; voltage-gated ion channels; glutamate transporter; anoxia; external RNA control


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
VERTEBRATE BRAINS ARE particularly sensitive to oxygen depletion, and even minutes of anoxia causes a fall in ATP levels, membrane depolarization, accumulation of cytosolic Ca2+, and cell death (2, 37, 41). The proximal cause of the anoxia intolerance is the high intrinsic rate of ATP consumption of brain tissue, which is largely devoted to maintaining ion gradients constantly challenged by ion fluxes through ion channels such as voltage-gated Na+ and Ca2+ channels (NaVs and CaVs) (23). The anoxic death process is accelerated by a coinciding release of glutamate, leading to the opening of ionotropic glutamate receptors such as 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors (AMPARs) and N-methyl-D-aspartate (NMDA) receptors (NMDARs), spreading the depolarization from neuron to neuron while enhancing it in single neurons (67). Indeed, the extent of neuronal cell death in ischemic and excitotoxic neural systems can be substantially decreased by blocking NMDARs and AMPARs (13, 51, 64). In particular, the NMDARs seem to play a detrimental role, being responsible for a large Ca2+ influx that eventually kills the cells, and blocking them in cultivated neurons can in some cases reduce the occurrence of induced excitotoxic cell death by 83–93% (65).

Ion channels are made up of one or more protein subunits, depending on the ion channel family. Whereas ion channels such as NMDARs and AMPARs require four subunits (36, 63), the ion pores of NaVs and CaVs require only one protein (9, 10). Also, each of the different ion channel families contains a multitude of subunits or variants, providing diversity in ion channel properties (8, 14, 19, 36, 55, 63). For example, whereas AMPARs containing the subunit GluR2 are impermeable to Ca2+, those lacking the GluR2 subunit are Ca2+ permeable. Accordingly, changes in ion channel composition have been observed during periods of physiological changes, such as during mammalian development. Whereas NMDARs are dominated by the NR2D subunit in neonatal mammalian brains, the same subunit is only slightly expressed in adult brains (44). Altered subunit composition can also be seen in response to physiological insults. For example, in mammalian neurons ischemia/hypoxia results in decreased expression of the AMPAR subunit GluR2 (hippocampal neurons) and the NaV protein NaV1.1 (neurons of the ischemic area) (28, 53, 74) and increased expression of the NMDAR subunit NR2C (hippocampal and cortical neurons) (56, 60). These changes indicate a role for ion channel plasticity in the response to oxygen deprivation. Still, they do not necessarily reflect adaptive responses aimed at increasing neuronal survival, but may reflect pathophysiological processes. This represents an ever-present problem in the interpretation of studies of anoxia-intolerant systems. With regard to excitatory neurotransmission, ion channels are not the only proteins that are affected by anoxia. Ischemia in mammalian brains has been shown to lead to reduced expression of the glutamate transporter EAAT2 (15, 20), an event that may play an important role in hypoxic preconditioning (20).

Some vertebrates, particularly fish of the genus Carassius and freshwater turtles of the genera Chrysemys and Trachemys, have evolved the ability to survive prolonged periods of anoxia (days to months, depending on temperature) (4, 46). In contrast to mammals, neurons from these organisms avoid anoxic ATP depletion and exhibit a stable rather than increased glutamate release (31, 47). An important part of their survival strategy has been suggested to involve reductions in the permeability of excitatory ion channels ("channel arrest") (30). Such channels would include Na+ and Ca2+ channels like AMPARs, NMDARs, NaVs, and CaVs, and could potentially involve changes in subunit composition. Studies on freshwater turtles have indicated a role for anoxia-inducible changes in ion channel properties. For example, AMPARs in turtle cortical neurons show a 60% reduction in evoked peak currents during anoxia (52), while NMDARs show a 50–65% reduction in opening probability (5, 7) and a 67% reduction in Ca2+ current amplitude (5). Also, the anoxic turtle cortex shows a 60% reduction in the abundance of the obligatory NMDAR subunit NR1 (5), while the anoxic turtle cerebellum shows a 42% reduction in the abundance of NaVs (54). These results suggest that a lowering of the ionic permeability of neuronal membranes plays a role in the anoxic survival of turtles. Still, this strategy may not be used by other anoxia-tolerant vertebrates, since the crucian carp (Carassius carassius) handles anoxia without displaying any reductions in the K+ and Ca2+ permeability of neurons, as quantified by monitoring changes during induced energy deficiency or ion pump inhibition (35, 45). Furthermore, in contrast to turtles, which are more or less comatose during anoxia, the crucian carp survives anoxia in an active state (45).

We have examined here how anoxia affects the expression of genes involved in excitatory neurotransmission in the crucian carp brain, with the aim of disclosing anoxia-induced changes that would affect neuronal excitability. This was done by partially cloning 29 genes involved in excitatory neurotransmission and by quantifying their expression with real-time RT-PCR. Since the expression of internal RNA control genes (reference genes) changes in crucian carp during anoxia (22), normalization of the real-time RT-PCR data was performed with a novel approach, achieving high-resolution normalization by adding an external RNA control gene (mw2060, an mRNA fragment from a blue-green alga) (22). The following genes were investigated: eight subunits of the AMPAR (GluR1a,b–4a,b), six subunits of the NMDAR (NR1, NR2A–D, and NR3A), three {alpha}-protein variants of NaVs (NaV1.1, NaV1.3, and NaV1.6), four {alpha}-protein variants of CaVs (CaV2.1, CaV2.3, CaV3.1, and CaV3.2), four glutamate transporters (EAAT2a, EAAT2b, EAAT3a, and EAAT3b), and four genes involved in NMDAR-mediated neuroplasticity (CREB-1, BDNF, and the BDNF receptors TrkB1 and TrkB2) (42). Also, we report the full-length sequence of the NMDAR subunit NR1 in crucian carp, including a novel splice cassette (aa sequence: NTSG), representing the first report of a splice cassette outside the COOH terminal of the NR1 subunit.


   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 community, Norway, in September. They were kept in 750-liter tanks continuously supplied with dechlorinated, aerated Oslo tap water for 3–4 mo at a temperature of 12 ± 1°C. The photoperiod was held at 12 h of darkness-12 h of daylight, and the fish were fed commercial carp food on a daily basis.

Before experiments crucian carp, weighing 35 ± 12 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 bubbled with either air (normoxia and reoxygenation) or nitrogen (anoxia). Oxygen concentrations and temperatures were continuously monitored with 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 12 ± 1°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 4 days of reoxygenation (R4). Fish were killed by stunning with a sharp blow to the head, followed by cutting of the spinal cord and dorsal aorta and removal of the brain. Within 30 s of the initiation of fish handling, brains were frozen in liquid nitrogen. The brains were stored at –80°C.

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

Obtaining sequences for genes involved in neurotransmission.
Gene-specific primers for cloning of crucian carp genes were designed from sequences of zebrafish (Danio rerio), which belongs to the same family as crucian carp (Cyprinidae), with Primer3 (59). Sequences were retrieved with the NCBI 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) (66). Gene-specific primers are listed in Table 1.


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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 with TRIzol reagent (Invitrogen, Carlsbad, CA). A 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) was used to ensure that the RNA was of sufficient quality, and a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Rockland, DE) was used to quantify the RNA.

For expressed sequence tag (EST) cloning, 1 µg of total RNA was DNase I treated (Sigma-Aldrich, St. Louis, MO) and reverse transcribed with oligo(dT)18 and Superscript III in reaction volumes of 20 µl (Invitrogen). PCR was performed on 1:25 dilutions of the resulting cDNA with 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 44 times, 6) 72°C for 10 min, 7) hold at 4°C. The resulting PCR products were cloned with 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 with PCR and were sequenced with T7 primers (ABI-lab, University of Oslo). All procedures were carried out according to manufacturers' protocols.

For full-length cloning of the NMDAR subunit NR1, which is a obligatory component of all functional NMDARs, 1 µg of total RNA was reverse transcribed with GeneRacer (Invitrogen). Rapid amplification of cDNA ends (RACE) PCR was performed on 1:25 dilutions of the resulting RACE cDNA with Advantage 2 Polymerase mix (Clontech, Mountain View, CA). The following PCR program was used (5'-RACE/3'-RACE): 1) 95°C for 1 min, 2) 95°C for 30 s, 3) 72°C for 3/8 min, 4) repeat steps 2 and 3 5 times, 5) 95°C for 30 s, 6) 70°C for 3/8 min, 7) repeat steps 5 and 6 5 times, 8) 95°C for 30 s, 9) 68°C for 3/8 min, 10) repeat steps 8 and 9 25 times, 11) 72°C for 10 min, 12) hold at 4°C. The resulting RACE products were cloned and sequenced as described above. All procedures were carried out according to manufacturers' protocols.

All primer sequences used for cloning are listed in Table 1. These include those used for cloning of subunits of AMPARs and NMDARs, {alpha}-protein variants of CaVs and NaVs, as well as glutamate transporters (EAATs) and genes involved in NMDAR-mediated neuroplasticity. The resulting crucian carp sequences were submitted to the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database (www.ebi.ac.uk/). Accession numbers are listed in Table 1.

RNA extraction and cDNA synthesis for real-time RT-PCR.
Total RNA for real-time RT-PCR experiments was extracted from whole brain homogenate corresponding to 42 mg of brain tissue. Fifteen microliters of TRIzol reagents was used per milligram. The extractions were performed in accordance with the protocol given by Ellefsen et al. (22), adding 20 pg of external RNA control gene (mw2060) per milligram of tissue. The quality and quantity of the total RNA were assessed with the 2100 Bioanalyzer and the NanoDrop ND-1000 UV-Vis Spectrophotometer.

One microgram of total RNA was DNase I treated and reverse transcribed with oligo(dT)18 and Superscript III as described above. 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 with autoclaved MilliQ water. All procedures were carried out according to manufacturers' protocols.

Real-time RT-PCR.
Real-time RT-PCR was performed on a Lightcycler 2.0 (Roche Diagnostics, Basel, Switzerland). Relative gene expression data were obtained from real-time RT-PCR raw data with Eq. 1:

Formula 1(1)
where Tar is target gene, Con is control gene (mw2060), E is priming efficiency, and Cp is crossing point. mw2060 was used as an RNA control gene in all real-time RT-PCR data sets. E values were calculated for each real-time RT-PCR reaction with LinRegPCR software (57), and an Emean was calculated for each real-time RT-PCR primer pair. These Emean values were used in calculations of relative gene expression. Cp values were obtained for each reaction with the Lightcycler 2.0 software and were calculated as the second derivative maximum. Sequences of real-time RT-PCR primers are presented in Table 1.

All real-time RT-PCR reactions were performed with a reaction volume of 10 µl, using Lightcycler Faststart DNA MasterPLUS SYBR Green I (Roche Diagnostics) and Lightcycler Capillaries (Roche Diagnostics). Five microliters of 1:25 diluted cDNA was used as template (prepared as described above), and the following real-time RT-PCR program was used: 1) 94°C for 10 min, 2) 94°C for 10 s, 3) 60°C for 12 s, 4) 72°C for 8 s, 5) repeat steps 2–4 39 times. 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. All real-time RT-PCR reactions were performed with 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 described above). Primer efficiencies and average Cp values for the different primer pairs are summarized in Table 1. It should be noted that three primer pairs were tested for each gene. Those displaying the most distinct melting curves and the highest Cp values under the described reaction conditions were preferred. All procedures were carried out according to manufacturers' protocols.

NR1 protein analysis.
A tentative three-dimensional (3D) structure of the S1S2 region of the crucian carp NR1 protein (aa: 412-564 and 682-820) was generated with Deepview (au.expasy.org/spdbv/), with the rat NR1 S1S2 crystal structure (Protein Data Bank ID 1PB7) acting as template. The NR1 S1S2 fragment includes the entire extracellular part of the protein, including the glycine-binding domain, except the NH2-terminal domain. In addition, the transmembrane topology of the crucian carp NR1 was verified with the TMHMM Server v2.0 (www.cbs.dtu.dk/services/TMHMM-2.0/), and the N-glycosylation and phosphorylation sites were predicted with the Prosite server (release 20.13, expasy.org/prosite/), the NetPhos 2.0 server (www.cbs.dtu.dk/services/NetPhos/), and the NetPhosK 1.0 Server (www.cbs.dtu.dk/services/NetPhosK/).

Statistical analyses.
To evaluate whether differential oxygen regimes (N7, A1, A7, and R4) had an effect on gene expression patterns, statistical analyses were performed with GraphPad InStat (3.06; Graphpad Software, San Diego, CA). All data sets were assessed for statistically significant variation by one-way ANOVA, followed by Tukey-Kramer posttest. The data sets that illustrate percentage distribution were arcsine-transformed before analyses. P ≤ 0.05 was considered significant. All data are presented as means ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gene expression analyses.
Functional AMPARs consist of four subunits (denoted GluR) and are either homo- or heterotetrameric (63). Eight AMPAR subunits have been identified in the zebrafish genome (GluR1a,b–4a,b), and mRNA for all eight GluRs were found in crucian carp brain. Only GluR2b displayed changes in response to treatment, increasing from 1.0 in N7 to 1.3 in A1 (P < 0.05) (Fig. 1A). However, comparing the expression of each of the different GluR subunits to the collective expression of GluRs revealed that the expression of GluR2a decreased from constituting 32.9 ± 2.5% in N7 to 26.2 ± 1.6% in A7 and 29.7 ± 0.8% in R4 (P < 0.001 and P < 0.05) and the expression of GluR3a decreased from constituting 2.4 ± 0.2% in N7 to 1.9 ± 0.4% in A7 and 2.0 ± 0.2% in R4 (P < 0.05) (Fig. 1B). Furthermore, the GluR2a and GluR2b subunits displayed the highest expression in the crucian carp brain, constituting a total of 64–70% of the collective GluR expression (Fig. 1B). All AMPAR subunits contain either a flip or a flop splice cassette in their 3' coding region (62). The crucian carp GluRs were found to contain these splice cassettes, and the expression of GluR2a-flip and GluR2a-flop did not change in response to anoxia or anoxia-reoxygenation (Fig. 1C). Moreover, the flop variant of GluR2a constituted 87.4 ± 1.0% of the overall GluR2a flip/flop expression in N7, a fraction that decreased slightly, but significantly, to 85.0 ± 0.9% in R4 (P < 0.001) (Fig. 1D). Interestingly, we found a variant of GluR2a that contained neither flip nor flop. To our knowledge this has not been previously described. The expression of this transcript did not change in response to anoxia or anoxia-reoxygenation and typically made up 0.5% of the total GluR2a expression (Fig. 1, C and D).


Figure 1
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  		Fig. 1. A: expression of 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor (AMPAR) subunits GluR1a,b–4a,b 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 GluR subunits in N7, A1, A7, and R4. C: expression of the flip/flop variants of the GluR2a subunit. i, Flip variant; o, flop variant; –io, variant lacking flip/flop. D: expression profile for the GluR2a splice variants. N7, normoxia 7 days (n = 5); A1, anoxia 1 day (n = 5); A7, anoxia 7 days (n = 5); R4, anoxia 7 days followed by reoxygenation 4 days (n = 6). *P < 0.05, ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 
Functional NMDARs consist of two NR1 subunits and two NR2 subunits (36). In mammals four different NR2 subunits have been identified (NR2A–D), and it has been established that the NR2 composition of NMDARs is an important determinant of their electrophysiological properties, providing developmental and regional diversity (18). In addition, two NR3 subunits have been identified (NR3A and -B), acting as modulators of NMDAR function (18). In this study all NMDAR subunits were cloned and investigated in the crucian carp brain (except NR3B, where no clone was found). Anoxia and/or anoxia-reoxygenation resulted in decreased expression of NR1, NR2B, NR2C, and NR3A (Fig. 2A). Compared with N7 data, being normalized to 1.0, the NR1 abundance decreased to 0.53 in A7 (P < 0.001) and to 0.60 in R4 (P < 0.01), the NR2B abundance decreased to 0.67 in R4 (P < 0.05), the NR2C abundance decreased to 0.76 in A1 (P < 0.05), to 0.66 in A7 (P < 0.001), and to 0.66 in R4 (P < 0.001), and the NR3A abundance decreased to 0.50 in A7 (P < 0.001) (Fig. 2A). However, when all NR2 subunits were added together the NR2 expression did not change significantly in anoxia and anoxia-reoxygenation (Fig. 2A). While the NR1 expression constituted 72% of the overall NMDAR subunit expression (NR1 + NR2) in N7, it constituted 62% in A7 (P < 0.001) (Fig. 2B). The NR2 subunit composition showed a minor but significant change in response to anoxia: whereas NR2C expression constituted 8.1 ± 1.3% of the overall NR2 expression in N7, it constituted 6.7 ± 0.8% in A7 (P < 0.05) (Fig. 2C).


Figure 2
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  		Fig. 2. A: expression of N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunits NR1, NR2A-D, and NR3A 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 NR1/NR2 subunits in crucian carp brain in N7, A1, A7, and R4. C: expression profile for the NR2 subunits. n = 11 (N7), 11 (A1), 11 (A7), and 12 (R4). *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA followed by Tukey-Kramer posttest).

 
In mammals, the ion pore of NaVs and CaVs consists of a single {alpha}-protein (9, 10). In each of the two channel families 10 {alpha}-protein variants have been identified, providing a large span in electrophysiological properties (9, 10). Of the seven {alpha}-proteins we were able to clone in this study (NaV1.1, NaV1.3, NaV1.6, CaV2.1, CaV2.3, CaV3.1, and CaV3.1), NaV1.1 and CaV3.1 displayed increased expression in response to anoxia, changing from 1.0 in N7 to 1.5 and 1.4 in A7, respectively (P < 0.05) (Fig. 3).


Figure 3
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  		Fig. 3. Expression of voltage-gated sodium- and calcium channels (NaV1.1, –1.3, and –1.6; CaV2.1, –2.3, –3.1, and –3.2) 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; n = 5 (N7), 5 (A1), 5 (A7), and 6 (R4). *P < 0.05 (1-way ANOVA followed by Tukey-Kramer posttest).

 
The expression of the four investigated glutamate transporters, EAAT2a, EAAT2b, EAAT3a and EAAT3b, did not change in response to anoxia, but the expression of EAAT2a decreased during anoxia-reoxygenation, going from 1.0 in N7 to 0.35 in R4 (Fig. 4A). No changes were seen in the relative composition of the four investigated EAATs (Fig. 4B). Furthermore, the expression of the genes involved in NMDAR-mediated neuroplasticity did not change in the crucian carp brain in response to anoxia (Fig. 4C), but changes were seen in anoxia-reoxygenation, with TrkB1 and TrkB2 decreasing from 1.0 in N7 to 0.48 and 0.47 in R4, respectively (P < 0.05) (Fig. 4C). In addition, the neuroplasticity genes showed differences in expression between anoxia and anoxia-reoxygenation: BDNF levels were suppressed from 1.2 in A7 to 0.45 in R4 (P < 0.05) (Fig. 4C; the statistic for the A7-R4 comparison is not presented), and expression of the BDNF receptors TrkB1 and TrkB2 was suppressed from 0.95 and 0.90 in A1 to 0.48 and 0.47 in R4 (P < 0.05) (Fig. 4C; the statistic for the A1-R4 comparison is not presented).


Figure 4
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  		Fig. 4. A: expression of glutamate transporters (EAAT2a, -2b, -3a, and -3b) in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and referenced to the control group N7. Values are means + SD. B: expression profile for the EAAT genes in N7, A1, A7, and R4. C: expression of neuroplasticity genes CREB-1, BDNF, TrkB1, and TrkB2. n = 6 for all groups. *P < 0.05 (1-way ANOVA followed by Tukey-Kramer posttest).

 
Full-length sequence of NR1.
The NR1 subunit is an obligatory component of vertebrate NMDARs (36). A comparison of the amino acid sequence of crucian carp NR1 to amino acid sequences from other vertebrates revealed no differences in the transmembrane topology or the number of predicted phosphorylation sites (data not shown). Moreover, the simulated 3D structure of the crucian carp NR1 S1S2 region (Fig. 5A) did not show any amino acid substitutions that would affect key properties such as glycine binding. However, a novel splice cassette, containing the amino acids N, T, S, and G, was discovered in the S1S2 region (Fig. 5A), in a part denoted loop 1 (residues 439-449 in human NR1) (33). This NTSG cassette introduces an N-glycosylation site into the protein (Fig. 5A) and represents the first splice cassette to be found outside the COOH- or NH2-terminal ends of NR1 (67). It is located in a part of the S1S2 region that seems to be exposed to the extracellular environment (Fig. 5A), making it a likely target for in vivo glycosylation. Interestingly, loop 1 of rat NR1 has a high degree of structural flexibility, as indicated by the difficulties in obtaining its crystal structure (Fig. 5A) (58, 26). Its crystal structure was only recently resolved, when the crystallization procedure was performed in the presence of the NMDAR agonist 1-aminocyclopropane-1-carboxylic acid (ACPC) (Fig. 5B) (33). Analogous sequences to the crucian carp NTSG cassette were found in the genomes of zebrafish, torafugu (Takifugu rubripes), and spotted green pufferfish (Tetraodon nigroviridis) (Fig. 5C). When these sequences were compared with the crucian carp sequence, zebrafish showed high degree of similarity, whereas the two pufferfish species showed moderate degrees of similarities to crucian carp (Fig. 5C). Interestingly, also when the two pufferfish species were compared to each other, only moderate degrees of similarity were found (Fig. 5C). The pufferfish aa sequences did not represent N-glycosylation sites. When the expression of NR1 transcripts containing and lacking the NTSG splice cassette (NR1 + NTSG and NR1 – NTSG) were measured separately, they did not change significantly in response to anoxia but decreased in response to anoxia-reoxygenation, going from 1.0 in N7 to 0.58 and 0.49 in R4, respectively (P < 0.05) (Fig. 5D). The NTSG cassette was found to be present in 17–20% of all NR1 transcripts, a pattern that did not change in response to different oxygen regimes (Fig. 5E).


Figure 5
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  		Fig. 5. A: tentative tertiary structure of the extracellular glycine-binding S1S2 segment of crucian carp NR1. Crystal structure for rat NR1 S1S2 was used as template (Protein Data Bank ID 1PB7) (26). Red sidechains/letters indicate differences in amino acid (aa) composition between crucian carp and rat. Orange regions indicate the parts of S1S2 that leave/enter the cell membrane. Blue fragment represents glycine. Green region (backbone + amino acids) indicates loop 1 of the S1S2 fragment, which had only been partially resolved in rat (1PB7). In the unresolved region of loop 1, a novel splice cassette (NTSG cassette, highlighted by red box) was found in some crucian carp NR1. B: tertiary structure of loop 1 obtained through crystallography in the presence of the NMDAR agonist 1-aminocyclopropane-1-carboxylic acid (ACPC) (Protein Data Bank ID 1y20) (33). C: comparisons of amino acid sequences from different fish species homologous to the part of loop 1 that had not been characterized in the rat crystal structure 1PB7 (see A). Sequences homologous to the NTSG sequence are framed by a red box and are also shown as nucleotide sequences. All sequences are compared with the crucian carp sequence, and differences are indicated by red letters. rn, Rattus norvegicus; cc, Carassius carassius; dr, Danio rerio; tr, Takifugu rubripes; tn, Tetraodon nigroviridis. D: expression of NR1 containing or lacking the NTSG cassette in brain tissue of crucian carp exposed to normoxia, anoxia, and reoxygenation. Data sets were normalized to the external RNA control mw2060 and referenced to the control group N7. Values are means + SD. E: expression profile for the NR1 + and –NTSG splice variants in crucian carp brain in N7, A1, A7, and R4. n = 6 for all groups. *P < 0.05 (1-way ANOVA followed by Tukey-Kramer posttest).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Of the 29 genes involved in excitatory neurotransmission that were quantified in brain tissue of normoxic and anoxic crucian carp, 6 genes displayed changes in expression in response to anoxia and 6 displayed changes in response to anoxia-reoxygenation. Although these changes may result in alterations in neuronal activity patterns, they were relatively small (ranging from a 70% fall to a 50% increase), indicating that profound anoxia-induced reduction in gene expression, compatible with "channel arrest," does not occur in crucian carp. Thus after 7 days of anoxia the expression of genes involved in excitatory neurotransmission was still strikingly well preserved. Still, NMDAR expression showed changes that could mediate a reduced neural excitability.

While previous large-scale studies of hypoxia-induced changes in gene expression in hypoxia-tolerant vertebrates have employed the microarray technique, using arrays optimized for hypoxia-induced changes or containing a relatively arbitrary selection of genes, this is the first large-scale study to map hypoxia-induced changes in a hypothesis-driven way. We have made a comprehensive attempt to include the most relevant gene families and genes that are involved in excitatory neurotransmission, the most energy-consuming process in the brain. In the results of microarray studies these genes are often strikingly absent, possibly because of low expression levels compared with other genes, making their appearance in arrays less probable. For example, of the 29 genes investigated in this study, only 6 were found in carpBASE 5.0 (http://legr.liv.ac.uk/searchDB/search_carpbase_5_0.php), a database containing over 22,500 annotated carp sequences originating from microarray-related clones produced by the Laboratory for Environmental Gene Regulation (LEGR), University of Liverpool. Also, intrinsic constraints of the microarray technique, such as nonspecific hybridization, may prohibit analyses of closely related genes and splice variants, features that are more easily resolved with real-time RT-PCR. Like all studies assessing mRNA levels, we need to keep in mind that these may not necessarily reflect the protein levels, which can also be controlled at the level of translation and degradation (29). Still, conventional antibody-based protein approaches were never a real alternative in such a broad investigation as the present one, because the necessary battery of antibodies are not available for fish in general, and crucian carp in particular. Future studies of the protein biology require development of such antibodies. Indeed, to avoid misinterpretations in studies of nonmammalian organisms, development of novel and more specific antibodies is a general and urgent requirement. However, it would still be difficult to separately quantify closely related paralogs such as EAAT2a and EAAT2b with antibody-based strategies, since most of the mutations that distinguish them do not alter the amino acid composition. Nevertheless, studies have indicated that proteins with modes of actions similar to those studied here, acting as components of multiprotein complexes (e.g., AMPARs and NMDARs), are likely to show a correlation between mRNA levels and protein levels (34). This is probably because the stoichiometric availability of such subunits determines the properties of the protein complexes (29, 34). Still, it should be noted that protein function will be affected by additional factors such as phosphorylation status and subcellular localization. Furthermore, the present study includes data from entire ion channel families, enabling analyses of subunit composition. We would also like to emphasize that the real-time RT-PCR data were normalized with a recently developed external RNA control approach (22), the first to add an external RNA to tissue on a per unit weight basis and subsequently use it for normalization. This enables high-resolution estimates of gene expression in experiments involving severe physiological insults (anoxia), where global changes in gene expression must be expected to occur, making internal RNA control genes highly unreliable (22).

Intrinsic patterns of gene expression.
The normoxic levels of gene expression observed in the crucian carp brain correlate with the gene expression patterns observed in the mammalian brain. For example, the flop variant of the GluR2a subunit dominated over the flip variant (63), and the NR1 expression exceeded the overall NR2 expression (27). Moreover, the NaV1.1 expression well exceeded the NaV1.3 expression (20-fold higher, data not shown) (24), and the EAAT2 glutamate transporter was more highly expressed than the EAAT3 glutamate transporter (16). This indicates vital and preserved roles for these proteins in vertebrate brain function.

Still, other expression patterns were less well conserved between crucian carp and mammals. For example, the AMPAR subunit expression of the crucian carp brain was dominated by GluR2-like subunits, and although this resembles the situation in mammals (63) the dominance was much more prominent in crucian carp, in which GluR2 constituted 67% of the overall subunit expression compared with 34% in rat (39). Since AMPARs that contain the GluR2 subunit are impermeable to Ca2+, this may represent a way to reduce Ca2+ influx during anoxia and thus to promote neuronal survival. Indeed, in mice neurons, knockout of the GluR2 gene has been shown to result in increased excitotoxic vulnerability (32). Furthermore, the NMDAR expression pattern displayed interesting differences. The NR2 composition was found to be rather different in crucian carp compared with adult rats. Primarily, the occurrences of NR2B and NR2D mRNA were considerably higher, constituting 45% and 34%, respectively, of the overall NR2 expression in crucian carp compared with 34% and 19% in rat (27). Consequently, expression of NR2A and NR2C was considerably lower, constituting 12% and 8%, respectively, in crucian carp compared with 26% and 21% in rat. Interestingly, neonatal rats show an NR2 composition that resembles that of crucian carp, being dominated by NR2B and NR2D (44). Neonatal rats also show a relatively high degree of hypoxia tolerance that well exceeds that of adult rats. Notably, the hypoxia tolerance of neonatal rats seems to decrease in parallel with the replacement of NR2B and NR2D with NR2A and NR2C (21, 71–73), making the NR2 composition a potential determinant of hypoxia tolerance (3, 6). Indeed, whereas NMDARs containing the NR2D subunit exhibit reduced ion currents during hypoxia, those containing the NR2C subunit exhibit increased currents (6). Therefore, it is tempting to suggest that the observed dominance of NR2B and NR2D expression in the crucian carp brain plays a role in its anoxia tolerance, representing some sort of "constitutive preconditioning" (46). Functional studies are needed to strengthen this hypothesis.

Anoxia-induced changes in gene expression.
The expression of the genes investigated in this study was either strikingly well preserved during anoxia/anoxia-reoxygenation or they showed only minor changes. Changes were generally <50% (up or down), which may be too small to be picked up by other procedures such as microarrays. Still, such changes may of course have functional consequences, particularly for genes involved in neurotransmission, which should be expected to be tightly controlled. However, the general picture is that the crucian carp brain seems to retain its excitatory capacity during anoxia, arguing against a major role for anoxia-induced "channel arrest." This is in line with other studies on crucian carp, demonstrating that cells of brain and heart tissues do not exhibit decreased ion permeability during anoxia (35, 45, 50, 68). Still, we observed interesting changes for NMDARs.

The subunit expression and composition of NMDARs changed in the crucian carp brain during anoxia, showing three traits, 1) decreased NR1 expression, 2) decreased NR2C expression, and 3) decreased NR3A expression. 1) The expression of NR1 decreased by 50% after 7 days of anoxia. This is consistent with a study on freshwater turtles, which found a 60% reduction in the expression of NR1 protein in the cortex during anoxia (5). The level of NR1 protein has been shown to control the release of NMDAR assemblies from endoplasmic reticulum (25), which likely means that the observed reduction in crucian carp NR1 mRNA during anoxia results in a reduction in the number of functional NMDARs. This may be of particular importance, since NMDARs are responsible for the Ca2+ influx that eventually kills mammalian cells. 2) The expression of NR2C was reduced from 1.0 in N7 to 0.8 in A1 and to 0.7 in A7, and it also constituted a reduced part of the overall NR2 expression during anoxia. This response contrasts with that of hypoxic rat neuron, which shows increased NR2C expression (56). Interestingly, NMDARs containing the NR2C subunit are the only NMDARs that respond to hypoxia by increasing their ion permeability (6). Indeed, when immature neonatal neurons start expressing NR2C they seem to become more vulnerable to hypoxic insults (6). Thus the observed decrease in NR2C expression in anoxic crucian carp brain could represent a strategy to reduce Ca2+ influx, thereby saving energy on ion pumping. 3) The expression of NR3A was reduced by 50% after 7 days of anoxia. In mammals, NR3A has been found to interact with NR2-containing NMDARs, providing channels with modified vestibules, smaller unitary conductances, shorter opening times, and lower Ca2+ permeabilities (17, 69). The reduced NR3A expression may thus act to increase the ion conductance through NMDARs. However, the physiological role of NR3A in the vertebrate central nervous system is largely unknown, and NR3A has also been indicated to coassemble with NR1 to form glycine-sensitive excitatory receptors (11). The observed reduction in NR3A expression may act to decrease the occurrence of these channels, but the functional significance of this, if any, for anoxia tolerance is difficult to deduce.

It is interesting to note how paralogous genes such as the AMPAR subunits GluR2a and GluR2b showed different patterns of expression in response to anoxia in the crucian carp brain. This suggests that these paralogs have evolved into proteins that to some extent differ in function. Still, the physiological significance of these changes is difficult to interpret since mammals have only one variant of these genes and current knowledge is based on this single variant.

The expression of voltage-gated ion channels appeared to remain unchanged in the crucian carp brain during anoxia. If anything, their abundance increased (NaV1.1 and CaV3.1 in A7, P < 0.05), which is in contrast to previous reports in turtles and mammals (54, 74) and may be linked to the fact that crucian carp endure anoxia in a physically active state (48). Indeed, the increased NaV1.1 expression observed during anoxia may result in a lowered threshold for action potential firing, thus ensuring that a given stimulus results in neuronal communication.

The expression of glutamate transporters was maintained in the anoxic crucian carp brain, which agrees with a previous study on freshwater turtles suggesting that glutamate reuptake mechanisms are maintained during anoxia (43). However, it contrasts with previous studies on mammals, in which a drastic decrease in EAAT2 expression has been demonstrated during hypoxia (15, 20). For anoxia-tolerant animals a fall in glutamate transport capacity during anoxia would probably be maladaptive as it could lead to a rise in extracellular glutamate levels. The maintained glutamate transporter expression may thus be prerequisite for the stable extracellular glutamate levels observed in crucian carp brain during anoxia (31).

Surprisingly, the expression of the neuroplasticity genes CREB-1 and BDNF remained unchanged in the anoxic crucian carp brain. Since CREB-1 and BDNF are known to be activated downstream of NMDAR activation (42), this suggests the maintenance of a relatively high level of neuronal activity. Indeed, this agrees well with the observed maintenance of the glutamatergic system. Furthermore, CREB-1 and BDNF have also been ascribed vital roles in neuronal protection during ischemia (38, 70). One may speculate that their sustained expression in the anoxic crucian carp brain reflects their neuroprotective functions.

Reoxygenation-induced changes in gene expression.
Some genes showed suppressed expression in the reoxygenation group compared with normoxia and/or anoxia. These changes could indicate that the anoxic episode functions as a cue for preparing the brain for further and longer anoxic exposures. In nature, the crucian carp can be exposed to anoxia for several months at close to 0°C during the long winter. This period is likely to be preceded by several shorter bouts of hypoxia or anoxia. Therefore, a reoxygenation group in crucian carp is probably not analogous to a recovery group in a species in which anoxia is a rare or pathological phenomenon. This may also be the major reason why the gene expression during reoxygenation in several cases (NR1, NR2B, NR2C, EAAT2-3, and TrkB2) looked like a continuation of the anoxia response.

NTSG—a novel NR1 splice cassette.
An NTS amino acid sequence is a typical N-glycosylation site (40), and the NTSG cassette found in a variant of the crucian carp NR1 protein thus represents an N-glycosylation site. It is openly positioned in an extracellular part of the protein and appears to be readily available for in vivo glycosylation. Consequently, in addition to introducing new amino acids into the NR1 protein, the NTSG cassette is likely to introduce a glycan residue, increasing the structural impact. In general, protein glycosylation is a potent way of modifying protein interactions with other molecules (49). Indeed, N-glycosylation of mammalian NR1 has been shown to be required to obtain functional assemblies of NMDAR subunits (12). The NTSG cassette is inserted into a loop that has been ascribed a role in subunit assembly and in the allosteric coupling of NR1 glycine binding and NR2 glutamate binding (1, 33, 58). Thus the crucian carp NTSG cassette potentially modifies the interaction between NR1 and NR2 subunits.

Gene sequences homologous to the crucian carp NTSG cassette were found in the genomes of zebrafish, torafugu, and spotted green pufferfish. Whereas the zebrafish nucleotide sequence was 100% conserved compared with crucian carp (i.e., it also contained the NTSG cassette), the two pufferfish sequences were only moderately conserved and lack the NTSG amino acid sequence. That the NTSG cassette is conserved between the two cyprinid species implies that it plays an important physiological role. In crucian carp brain the NTSG cassette is expressed in 17–20% of all NR1 transcripts, but a similar degree of expression in normoxia and anoxia does not immediately indicate a particular role for it during anoxia. However, this needs further study.

Conclusions.
The remarkably stable expression of genes involved in excitatory neurotransmission suggests that anoxia tolerance in crucian carp is not related to extensive "channel arrest" of excitatory ion channels. This is in line with the fact that the crucian carp survives anoxia in an active state (45), e.g., still showing spontaneous movements although at a reduced rate compared with normoxia (48). The sustained levels of gene expression are also in agreement with a study showing maintained protein synthesis rates in the anoxic crucian carp brain (61). However, the data indicate that some degree of neural depression occurs. First, the NMDAR subunits NR2B and NR2D were relatively highly expressed in the crucian carp brain. This resembles the situation in neonatal rat brains and may promote anoxia tolerance (3). Second, the NR1 expression decreased in the crucian carp brain during anoxia. This resembles the situation in anoxic turtles and may result in a reduced number of functional NMDARs (5). Third, NR2C showed a relatively low expression in normoxic crucian carp, and it was further lowered during anoxia. This resembles the situation in neonatal rats and may cause reduced ion fluxes through NMDARs.

Several of the responses seen in the anoxic crucian carp brain, such as the decreased NR2C expression, the maintained NaV1.1 expression, and the maintained EAAT2 expression, are quite dissimilar to those seen in the anoxic/ischemic mammalian brain. A reason for this may be that the changes observed in the mammalian brain largely reflect pathophysiological events rather than adaptive mechanisms.


   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
 

  1. ArmstrongN, Sun Y, Chen GQ, Gouaux E. Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature 395: 913–917, 1998.[CrossRef][Web of Science][Medline]
  2. ArundineM, Tymianski M. Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium 34: 325–337, 2003.[CrossRef][Web of Science][Medline]
  3. BicklerPE. Clinical perspectives: neuroprotection lessons from hypoxia-tolerant organisms. J Exp Biol 207: 3243–3249, 2004.[Abstract/Free Full Text]
  4. BicklerPE, Buck LT. Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu Rev Physiol 69: 145–170, 2007.[CrossRef][Web of Science][Medline]
  5. BicklerPE, Donohoe PH, Buck LT. Hypoxia-induced silencing of NMDA receptors in turtle neurons. J Neurosci 20: 3522–3528, 2000.[Abstract/Free Full Text]
  6. BicklerPE, Fahlman CS, Taylor DM. Oxygen sensitivity of NMDA receptors: relationship to NR2 subunit composition and hypoxia tolerance of neonatal neurons. Neuroscience 118: 25–35, 2003.[CrossRef][Medline]
  7. BuckLT, Bickler PE. Adenosine and anoxia reduce N-methyl-D-aspartate receptor open probability in turtle cerebrocortex. J Exp Biol 201: 289–297, 1998.[Abstract]
  8. CatterallWA. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16: 521–555, 2000.[CrossRef][Web of Science][Medline]
  9. CatterallWA, Goldin AL, Waxman SG. International Union of Pharmacology. XXXIX. Compendium of voltage-gated ion channels: sodium channels. Pharmacol Rev 55: 575–578, 2003.[Abstract/Free Full Text]
  10. CatterallWA, Striessnig J, Snutch TP, Perez-Reyes E. International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels. Pharmacol Rev 55: 579–581, 2003.[Abstract/Free Full Text]
  11. ChattertonJE, Awobuluyi M, Premkumar LS, Takahashi H, Talantova M, Shin Y, Cui J, Tu S, Sevarino KA, Nakanishi N, Tong G, Lipton SA, Zhang D. Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 415: 793–798, 2002.[Web of Science][Medline]
  12. ChazotPL, Cik M, Stephenson FA. An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor. Mol Membr Biol 12: 331–337, 1995.[Web of Science][Medline]
  13. ChoiDW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci 13: 171–182, 1990.[CrossRef][Web of Science][Medline]
  14. Cull-CandyS, Kelly L, Farrant M. Regulation of Ca2+-permeable AMPA receptors: synaptic plasticity and beyond. Curr Opin Neurobiol 16: 288–297, 2006.[CrossRef][Web of Science][Medline]
  15. DallasM, Boycott HE, Atkinson L, Miller A, Boyle JP, Pearson HA, Peers C. Hypoxia suppresses glutamate transport in astrocytes. J Neurosci 27: 3946–3955, 2007.[Abstract/Free Full Text]
  16. DanboltNC. Glutamate uptake. Prog Neurobiol 65: 1–105, 2001.[CrossRef][Web of Science][Medline]
  17. DasS, Sasaki YF, Rothe T, Premkumar LS, Takasu M, Crandall JE, Dikkes P, Conner DA, Rayudu PV, Cheung W, Chen HS, Lipton SA, Nakanishi N. Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature 393: 377–381, 1998.[CrossRef][Web of Science][Medline]
  18. DingledineR, Borges K, Bowie D, Traynelis SF. The glutamate receptor ion channels. Pharmacol Rev 51: 7–61, 1999.[Abstract/Free Full Text]
  19. DissJKJ, Fraser SP, Djamgoz MBA. Voltage-gated Na+ channels: multiplicity of expression, plasticity, functional implications and pathophysiological aspects. Eur Biophys J 33: 180–193, 2004.[Web of Science][Medline]
  20. DouenAG, Akiyama K, Hogan MJ, Wang F, Dong L, Chow AK, Hakim A. Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebral cortex plasma membranes. J Neurochem 75: 812–818, 2000.[CrossRef][Web of Science][Medline]
  21. DunahAW, Yasuda RP, Wang Yh Luo J, Davila-Garcia MI, Gbadegesin M, Vicini S, Wolfe BB. Regional and ontogenic expression of the NMDA receptor subunit NR2D protein in rat brain using a subunit-specific antibody. J Neurochem 67: 2335–2345, 1996.[Web of Science][Medline]
  22. EllefsenS, Stenslokken KO, Sandvik GK, Kristensen TA, Nilsson GE. Improved normalization of real time RT PCR data using an external RNA control. Anal Biochem 376: 83–93, 2008.[Web of Science][Medline]
  23. ErecinskaM, Silver IA. Ions and energy in mammalian brain. Prog Neurobiol 43: 37–71, 1994.[CrossRef][Web of Science][Medline]
  24. FeltsPA, Yokoyama S, DibHajj S, Black JA, Waxman SG. Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. Mol Brain Res 45: 71–82, 1997.[Medline]
  25. FukayaM, Kato A, Lovett C, Tonegawa S, Watanabe M. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice. Proc Natl Acad Sci USA 100: 4855–4860, 2003.[Abstract/Free Full Text]
  26. FurukawaH, Gouaux E. Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. EMBO J 22: 2873–2885, 2003.[CrossRef][Web of Science][Medline]
  27. GoebelDJ, Poosch MS. NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Mol Brain Res 69: 164–170, 1999.[Medline]
  28. GorterJA, Petrozzino JJ, Aronica EM, Rosenbaum DM, Opitz T, Bennett MVL, Connor JA, Zukin RS. Global ischemia induces downregulation of Glur2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil. J Neurosci 17: 6179–6188, 1997.[Abstract/Free Full Text]
  29. GreenbaumD, Colangelo C, Williams K, Gerstein M. Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4: 117, 2003.[CrossRef][Medline]
  30. HochachkaPW. Defense strategies against hypoxia and hypothermia. Science 231: 234–241, 1986.[Abstract/Free Full Text]
  31. HyllandP, Nilsson GE. Extracellular levels of amino acid neurotransmitters during anoxia and forced energy deficiency in crucian carp brain. Brain Res 823: 49–58, 1999.[CrossRef][Web of Science][Medline]
  32. IiharaK, Joo DT, Henderson J, Sattler R, Taverna FA, Lourensen S, Orser BA, Roder JC, Tymianski M. The influence of glutamate receptor 2 expression on excitotoxicity in GluR2 null mutant mice. J Neurosci 21: 2224–2239, 2001.[Abstract/Free Full Text]
  33. InanobeA, Furukawa H, Gouaux E. Mechanism of partial agonist action at the NR1 subunit of NMDA receptors. Neuron 47: 71–84, 2005.[CrossRef][Web of Science][Medline]
  34. JansenR, Greenbaum D, Gerstein M. Relating whole-genome expression data with protein-protein interactions. Genome Res 12: 37–46, 2002.[Abstract/Free Full Text]
  35. JohanssonD, Nilsson G. Roles of energy status, KATP channels and channel arrest in fish brain K+ gradient dissipation during anoxia. J Exp Biol 198: 2575–2580, 1995.[Web of Science][Medline]
  36. KohrG. NMDA receptor function: subunit composition versus spatial distribution. Cell Tissue Res 326: 439–446, 2006.[CrossRef][Web of Science][Medline]
  37. KristianT, Siesjo BK. Calcium-related damage in ischemia. Life Sci 59: 357–367, 1996.[CrossRef][Web of Science][Medline]
  38. LarssonE, Nanobashvili A, Kokaia Z, Lindvall O. Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rats. J Cereb Blood Flow Metab 19: 1220–1228, 1999.[CrossRef][Web of Science][Medline]
  39. LeeJC, Greig A, Ravindranathan A, Parks TN, Rao MS. Molecular analysis of AMPA-specific receptors: subunit composition, editing, and calcium influx determination in small amounts of tissue. Brain Res Brain Res Protoc 3: 142–154, 1998.[CrossRef][Medline]
  40. LehleL, Strahl S, Tanner W. Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew Chem Int Ed Engl 45: 6802–6818, 2006.[CrossRef]
  41. LiptonP. Ischemic cell death in brain neurons. Physiol Rev 79: 1431–1568, 1999.[Abstract/Free Full Text]
  42. LynchMA. Long-term potentiation and memory. Physiol Rev 84: 87–136, 2004.[Abstract/Free Full Text]
  43. MiltonSL, Thompson JW, Lutz PL. Mechanisms for maintaining extracellular glutamate levels in the anoxic turtle striatum. Am J Physiol Regul Integr Comp Physiol 282: R1317–R1323, 2002.[Abstract/Free Full Text]
  44. MonyerH, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12: 529–540, 1994.[CrossRef][Web of Science][Medline]
  45. NilssonGE. Surviving anoxia with the brain turned on. News Physiol Sci 16: 217–221, 2001.[Abstract/Free Full Text]
  46. NilssonGE, Lutz PL. Anoxia tolerant brains. J Cereb Blood Flow Metab 24: 475–486, 2004.[CrossRef][Web of Science][Medline]
  47. NilssonGE, Lutz PL. Release of inhibitory neurotransmitters in response to anoxia in turtle brain. Am J Physiol Regul Integr Comp Physiol 261: R32–R37, 1991.[Abstract/Free Full Text]
  48. NilssonGE, Rosen PR, Johansson D. Anoxic depression of spontaneous locomotor activity in crucian carp quantified by a computerized imaging technique. J Exp Biol 180: 153–162, 1993.[Abstract]
  49. OhtsuboK, Marth JD. Glycosylation in cellular mechanisms of health and disease. Cell 126: 855–867, 2006.[CrossRef][Web of Science][Medline]
  50. PaajanenV, Vornanen M. Effects of chronic hypoxia on inward rectifier K+ current (IK1) in ventricular myocytes of crucian carp (Carassius carassius) heart. J Membr Biol 194: 119–127, 2003.[CrossRef][Web of Science][Medline]
  51. PalmerGC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies. Curr Drug Targets 2: 241–271, 2001.[CrossRef][Web of Science][Medline]
  52. PamenterME, Shin DSH, Buck LT. AMPA receptors undergo channel arrest in the anoxic turtle cortex. Am J Physiol Regul Integr Comp Physiol 294: R606–R613, 2008.[Abstract/Free Full Text]
  53. Pellegrini-GiampietroDE, Zukin RS, Bennett MV, Cho S, Pulsinelli WA. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. Proc Natl Acad Sci USA 89: 10499–10503, 1992.[Abstract/Free Full Text]
  54. Perez-PinzonMA, Rosenthal M, Sick TJ, Lutz PL, Pablo J, Mash D. Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain. Am J Physiol Regul Integr Comp Physiol 262: R712–R715, 1992.[Abstract/Free Full Text]
  55. Perez-ReyesE. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83: 117–161, 2003.[Abstract/Free Full Text]
  56. Perez-VelazquezJL, Zhang L. In vitro hypoxia induces expression of the NR2C subunit of the NMDA receptor in rat cortex and hippocampus. J Neurochem 63: 1171–1173, 1994.[Web of Science][Medline]
  57. RamakersC, Ruijter JM, Deprez RH, Moorman AF. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62–66, 2003.[CrossRef][Web of Science][Medline]
  58. RegaladoMP, Villarroel A, Lerma J. Intersubunit cooperativity in the NMDA receptor. Neuron 32: 1085–1096, 2001.[CrossRef][Web of Science][Medline]
  59. RozenS, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386, 2000.[Medline]
  60. SmallDL, Poulter MO, Buchan AM, Morley P. Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices. Neurosci Lett 232: 87–90, 1997.[CrossRef][Web of Science][Medline]
  61. SmithRW, Houlihan DF, Nilsson GE, Brechin JG. Tissue-specific changes in protein synthesis rates in vivo during anoxia in crucian carp. Am J Physiol Regul Integr Comp Physiol 271: R897–R904, 1996.[Abstract/Free Full Text]
  62. SommerB, Keinanen K, Verdoorn TA, Wisden W, Burnashev N, Herb A, Kohler M, Takagi T, Sakmann B, Seeburg PH. Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249: 1580–1585, 1990.[Abstract/Free Full Text]
  63. SprengelR. Role of AMPA receptors in synaptic plasticity. Cell Tissue Res 326: 447–455, 2006.[CrossRef][Web of Science][Medline]
  64. SzenasiG, Harsing LG. Pharmacology and prospective therapeutic usefulness of negative allosteric modulators of AMPA receptors. Drug Discov Today 1: 69–76, 2004.[CrossRef]
  65. TaiKK, Blondelle SE, Ostresh JM, Houghten RA, Montal M. An N-methyl-D-aspartate receptor channel blocker with neuroprotective activity. Proc Natl Acad Sci USA 98: 3519–3524, 2001.[Abstract/Free Full Text]
  66. ThompsonJD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882, 1997.[Abstract/Free Full Text]
  67. VillmannC, Becker CM. On the hypes and falls in neuroprotection: targeting the NMDA receptor. Neuroscientist 13: 594–615, 2007.[Abstract/Free Full Text]
  68. VornanenM, Paajanen V. Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.). Am J Physiol Regul Integr Comp Physiol 287: R1263–R1269, 2004.[Abstract/Free Full Text]
  69. WadaA, Takahashi H, Lipton SA, Chen HS. NR3A modulates the outer vestibule of the "NMDA" receptor channel. J Neurosci 26: 13156–13166, 2006.[Abstract/Free Full Text]
  70. WaltonM, Woodgate AM, Muravlev A, Xu R, During MJ, Dragunow M. CREB phosphorylation promotes nerve cell survival. J Neurochem 73: 1836–1842, 1999.[Web of Science][Medline]
  71. WangYH, Bosy TZ, Yasuda RP, Grayson DR, Vicini S, Pizzorusso T, Wolfe BB. Characterization of NMDA receptor subunit-specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum. J Neurochem 65: 176–183, 1995.[Web of Science][Medline]
  72. WenzelA, Fritschy JM, Mohler H, Benke D. NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. J Neurochem 68: 469–478, 1997.[Web of Science][Medline]
  73. WenzelA, Villa M, Mohler H, Benke D. Developmental and regional expression of NMDA receptor subtypes containing the NR2D subunit in rat brain. J Neurochem 66: 1240–1248, 1996.[Web of Science][Medline]
  74. YaoC, Williams AJ, Cui P, Berti R, Hunter JC, Tortella FC, Dave JR. Differential pattern of expression of voltage-gated sodium channel genes following ischemic brain injury in rats. Neurotox Res 4: 67–75, 2002.[CrossRef][Medline]



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