Properties of Contextual Memory Formed in the Absence of αCaMKII Autophosphorylation
© Irvine et al; licensee BioMed Central Ltd. 2011
Received: 18 October 2010
Accepted: 28 January 2011
Published: 28 January 2011
The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM.
A major goal in neuroscience is to understand the molecular and cellular mechanisms underlying learning and memory. Synaptic plasticity, in particular NMDA receptor-dependent long-term potentiation (LTP), is thought to be an important cellular mechanism of memory formation that can be induced by behavioral training [1–4]. An essential signaling molecule downstream of NMDA receptor activation is the alpha-isoform of calcium/calmodulin-dependent protein kinase II (αCaMKII), the major post-synaptic density protein in the hippocampus . After activation αCaMKII can autophosphorylate at threonine-286 (T286), switching the kinase into a calcium/calmodulin-independent, autonomous activity state. This T286 autophosphorylation is essential for NMDA receptor-dependent LTP at excitatory synapses in hippocampal area CA1 and neocortex, as indicated by studies with αCaMKII autophosphorylation-deficient knockin (αCaMKIIT286A) mice [6–9].
Hippocampal area CA1 is essential for memory formation after contextual fear conditioning, a task in which a rodent learns to associate a neutral environment (context) with an aversive foot shock [10, 11]. The αCaMKIIT286A mutant mice have impaired contextual fear long-term memory (LTM) formation after a single training trial or a massed session of 3 trials . However, unexpectedly αCaMKIIT286A mutants can form contextual LTM after a massed training session of 5 trials . This finding posed several mechanistic questions, which we have addressed here. We studied whether a) short-term memory (STM) formation depends on αCaMKII autophosphorylation in the same way as LTM formation, b) spaced training also enables contextual LTM formation without αCaMKII autophosphorylation, c) contextual LTM formation in the absence of αCaMKII autophosphorylation requires the hippocampus, d) multiple tetani can induce L-LTP in hippocampal area CA1 in the absence of αCaMKII autophosphorylation, e) remote contextual LTM requires αCaMKII autophosphorylation, f) LTM formation in the absence of αCaMKII autophosphorylation is more sensitive to aging than LTM formation with intact αCaMKII autophosphorylation.
αCaMKII autophosphorylation is necessary for contextual STM formation after 3, but not 5, tone-shock pairings
There is evidence to suggest that STM may be an independent process to LTM . Our previous studies have shown that αCaMKII autophosphorlyation is essential for contextual LTM formation after training with either 1 or 3 tone-shock pairings but is not needed after a more intense training protocol of 5 tone-shock pairings . Therefore, we wanted to investigate whether the αCaMKIIT286A mice have the same contextual STM phenotype as that observed for contextual LTM.
Spaced training does not enable contextual LTM formation in the absence of αCaMKII autophosphorylation
Contextual LTM formation in the absence of αCaMKII autophosphorylation is hippocampus-dependent
Multi-tetanus-dependent late-LTP in hippocampal area CA1 cannot be induced in the absence of αCaMKII autophosphorylation
αCaMKII autophosphorylation is not necessary for remote contextual LTM
In normal rodents contextual LTM becomes hippocampus-independent over a period of approximately 30 days [19, 20]. Studies with heterozygous αCaMKII null mutants have suggested that neocortical NMDA receptor-dependent LTP is essential for the reorganization of contextual LTM . We were able to test this hypothesis with the αCaMKIIT286A mutants as these animals form contextual LTM 24 hours after 5 conditioning trials  and because they have fully blocked NMDA receptor-dependent LTP in neocortex .
Contextual LTM formation in the absence of αCaMKII autophosphorylation is impaired at 18 months of age
Here, we have used the αCaMKIIT286A mutant mice to investigate several mechanistic questions of contextual LTM formation. Firstly, we have shown that like contextual LTM formation, contextual STM formation is impaired in the absence of αCaMKII autophosphorylation after 3, but not 5, conditioning trials. In principle, this result is consistent with the idea that STM may be a prerequisite for LTM formation. However, in earlier studies we found that αCaMKII autophosphorylation is required for the induction of immediate-early gene (IEG) expression by contextual fear conditioning [; Radwanska et al., unpublished data]. IEG expression, which is essential for LTM formation [e.g., ], is induced 'immediately' after training. Therefore, it seems more likely that STM and LTM formation occur in parallel as independent processes as suggested by Izquierdo and colleagues . In this case αCaMKII autophosphorylation is needed for both types of memory formation by triggering distinct molecular processes. However, we did not observe a genetic dissociation between STM and LTM formation indicating that αCaMKII autophosphorylation is required for both.
We found that massed, but not spaced, training enables contextual LTM formation in αCaMKIIT286A mutants. This is the opposite to contextual LTM formation in CREBαΔ-/- mutants that is enabled by spaced, but not massed, training . It is not known why spaced training enables LTM formation in the CREB hypomorphic mutants. In this case, a process needs to be 'remembered' at the second training trial one hour after the first trial. Consequently, the activation of such a process seems to require the αCaMKII autophosphorylation. However, the nature of the process remains unclear. The comparison of the contextual LTM phenotypes in the CREB mutants and the αCaMKIIT286A mutants shows that spaced training is not always more beneficial than massed training for LTM formation. It suggests that massed training may be more beneficial for LTM formation when synaptic signaling is impaired, whereas spaced training may be more beneficial for LTM formation when transcription in the nucleus is affected.
We have shown that contextual fear conditioning in the αCaMKIIT286A mutants is hippocampus dependent and that multi-tetanus-dependent L-LTP cannot be induced in hippocampal area CA1 in these mice. Together, with previous hippocampal slice recordings [6, 8, 9] this strongly suggests that in the absence of αCaMKII autophosphorylation there is no hippocampal CA1 NMDA receptor-dependent LTP and therefore that contextual LTM does not necessarily require this type of plasticity. This is an unexpected proposal because an earlier study showed that CA1-restricted knockout of the essential NMDA receptor subunit NR1 impairs contextual LTM formation . However, Rampon et al. investigated contextual LTM formation after a single training trial . Consistently, the absence of αCaMKII autophosphorylation blocks contextual LTM formation after a single training trial . Thus, we suggest that CA1 NMDA receptor-dependent LTP has a specific role in contextual fear conditioning after one trial, but it is not required for LTM formation after 5 massed trials.
We also found that αCaMKII autophosphorylation is not required for remote contextual LTM after massed training. As NMDA receptor-dependent LTP in the neocortex is blocked in the αCaMKIIT286A mutants, the finding of normal remote contextual LTM in these animals suggests that NMDA receptor-dependent LTP in neocortex is not required for the reorganization of contextual LTM. This suggestion is in contrast with an earlier suggestion from a study with heterozygous αCaMKII null mutants that have impaired NMDA receptor-dependent LTP in neocortex . However, these heterozygous null mutants also have altered neurogenesis in the adult dentate gyrus , which has recently been implicated in regulating the reorganization of contextual LTM . Consistently, the αCaMKIIT286A mutants have normal neurogenesis in adult dentate gyrus  and normal remote contextual LTM. It should be noted that our findings do not claim that αCaMKII is dispensable for remote contextual LTM. This is because αCaMKII has various functions and only a subset of these functions is impaired in the αCaMKIIT286A mutants. For example, αCaMKII has a structural function that is independent from the T286 autophosphorylation . Thus, it is not surprising that heterozygous αCaMKII null mutants, but not αCaMKIIT286A mutants, have impaired remote contextual LTM.
Finally, we provide some evidence that 18 month-old αCaMKIIT286A mutants are impaired in contextual, but not cued, LTM formation. However, more systematic aging studies are required to further establish whether aged αCaMKIIT286A mutants have impaired contextual LTM formation. Nonetheless, our preliminary findings suggest that αCaMKII autophosphorylation-dependent plasticity in the hippocampus is important to delay age-dependent memory impairments. This idea may be of relevance for understanding Alzheimer's disease that has been suggested to be associated with impaired αCaMKII autophosphorylation [29–31].
One of the main conclusions from our studies is that contextual fear conditioning in the absence of αCaMKII autophosphorylation is hippocampus-dependent. Thus, hippocampal plasticities that do not require αCaMKII autophosphorylation appear to be sufficient for contextual LTM formation. Further, NMDA receptor-dependent LTP in hippocampal area CA1 that requires αCaMKII autophosphorylation seems to have a specific role in contextual LTM formation: it is needed for one-trial LTM formation but not for LTM formation after a session of massed training trials. Additionally, our findings suggest that NMDA receptor dependent LTP in neocortex is not required for reorganization of contextual LTM as previously proposed , as remote contextual LTM is intact in the absence of αCaMKII autophosphorylation and indeed autophosphorylation is essential for this type of LTP . Finally, we suggest that αCaMKII autophosphorylation may be important for delaying age-related hippocampus-dependent memory decline.
The subjects were housed in groups of two to five and maintained on a 12 h light/dark cycle with food and water ad libitum. Male and female homozygous αCaMKIIT286A mutants and control wild-type (WT) littermates were obtained in the 129B6F4-6 background by intercrossing heterozygous mutants. Genotyping was carried out by PCR analysis, as described previously  with DNA obtained from tail biopsies on postnatal day 21, the day of weaning. All the young, adult mice were 10-16 weeks of age, and all aged mice were 18 months old, at the time of training. All experiments used approximately equal numbers of male and female mice, and were undertaken in accordance with the UK Animals (Scientific Procedures) Act 1986.
For all of the experiments background contextual fear conditioning was used. Background contextual fear conditioning involves the hippocampus more strongly than conditioning without a tone presentation . Background conditioning took place in a conditioning chamber (Med Associates Inc, St Albans, USA) that was situated in a soundproof box. The conditioning chamber floor was made up of 36 stainless steel rods that were used for shock delivery. A speaker was mounted on one side of the chamber for delivery of the tone (80 dB, 3.0 kHz). In order to camouflage any noise in the behavioural room background noise was supplied to the chamber by a white noise generator positioned in the side of the soundproof box. Prior to training and contextual fear memory test the chamber was cleaned with 70% ethanol and a paper towel soaked in the ethanol was placed under the grid floor. The cued fear memory test was conducted in a novel chamber that was structurally different from the conditioning chamber. This chamber was semi-circular, had a plastic floor and prior to test, the chamber was cleaned with a lemon-scented solution.
On the conditioning day, the mice were brought from the housing room into a holding room where they were allowed to acclimatise for 30 min before training. During training, the mice were placed individually in the chamber and after a 120 s introductory period a tone (80 dB, 3.0 kHz) was presented for 30 s, the last 2 s of which coincided with a foot shock (0.7 mA). Depending on the intensity of the training protocol the mice received a further 2 or 4 tone-shock pairings at 60 s intervals, and for spaced training they received two training trials with a single tone-shock pairing and an inter-trial interval of 1 h. After training mice were returned to their home cage.
In order to test for short-term contextual memory mice were re-exposed to the conditioning chamber for a period of 5 min 2 h after training. Long-term contextual fear memory was assessed by re-exposing the mice to the conditioning chamber for 5 min 24 h after training. Long-term cued fear memory was assessed by placing each mouse in a novel chamber 48 h after training. Following 180 s without a tone (Pre-CS) the tone was presented for 180 s (CS) to assess cued fear memory. In order to test for the stability of the contextual and cued fear memories the mice were re-tested 30 and 31 days respectively after training.
The behaviour of the mice was recorded and freezing behaviour (defined as complete lack of movement, except for respiration) was scored for 2 s in every 5 s.
Stereotaxic coordinates for lesioning the dorsal hippocampus.
After completion of behavioural testing all lesioned and sham-lesioned mice were given a lethal injection of sodium pentobarbitone (Euthatal; Animal Care Ltd, York, UK) and were perfused with physiological saline and 4% paraformaldehyde in PBS. The brains were removed and stored in 4% paraformaldehyde until they were coronally sectioned at 40 μm and stained with cresyl violet.
Hippocampal slices were perfused with artificial cerebrospinal fluid (ACSF) with the following composition: 124 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.24 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4, 10 mM D-glucose, bubbled with a mixture of 95% O2 and 5% CO2. Mice were anesthetized and decapitated. The hippocampus was dissected and cut in 450 μm-thick slices with a tissue chopper. The slices were transferred into the recording chamber and kept in interface at 28°C for 1.5 h. The perfusion rate of ACSF was 1 ml/min. Bipolar twisted nickel-chrome electrodes (50 μm each) were used to stimulate Schaffer's collaterals.
Extracellular field excitatory postsynaptic potentials (fEPSP) were recorded in the stratum radiatum of the CA1 region with low resistance (2-5 megaOhm) glass microelectrodes filled with ACSF. Test stimuli were biphasic (0.08 ms for each pulse) constant-voltage pulses delivered every minute with an intensity adjusted to evoke an approximate 40% maximal response. The slope of the fEPSP was measured on the average of four consecutive responses. For each slice, an input-output curve was established from the responses obtained for various stimuli. LTP was induced by applying four trains (50 Hz, 1s, 5 min interval). In both protocols, the potentiated response was recorded for 4 h.
For each slice, the fEPSP slopes were normalized with respect to the mean slope of the fEPSPs recorded during the 30-minute period, preceding induction of LTP. To determine whether or not the normalized fEPSP of a group of slices submitted to the same experimental conditions was significantly potentiated (p < 0.05), the percentages of baseline measured after induction of LTP were compared by a two-way ANOVA and several student t tests at different times after LTP induction.
Normally distributed data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc analysis. Not normally distributed data were analyzed by ANOVA on ranks.
List of abbreviations
alpha-isoform of the calcium/calmodulin-dependent kinase II
We thank Dr Jeff Vernon for helpful advice during the course of these studies. This work was generously supported by the Alzheimer's Research Trust, UK, British Medical Research Council and the Belgian Queen Elisabeth Medical Fundation. L.R. is senior scientist of the Belgian Fund for Scientific Research (F.R.S.-FNRS).
- Bliss TV, Collingridge GL: A synaptic model or memory: long-term potentiation in the hippocampus. Nature. 1993, 361: 31-39. 10.1038/361031a0.View ArticlePubMedGoogle Scholar
- Martin SJ, Grimwood PD, Morris RG: Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci. 2000, 23: 649-711. 10.1146/annurev.neuro.23.1.649.View ArticlePubMedGoogle Scholar
- Gruart A, Muñoz MD, Delgado-García JM: Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci. 2006, 26: 1077-1087. 10.1523/JNEUROSCI.2834-05.2006.View ArticlePubMedGoogle Scholar
- Whitlock JR, Heynen AJ, Shuler MG, Bear MF: Learning induces long-term potentiation in the hippocampus. Science. 2006, 313: 1093-1097. 10.1126/science.1128134.View ArticlePubMedGoogle Scholar
- Lisman J, Schulman H, Cline H: The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev Neurosci. 2002, 3: 175-190. 10.1038/nrn753.View ArticlePubMedGoogle Scholar
- Giese KP, Fedorov NB, Filipkowski RK, Silva AJ: Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science. 1998, 279: 870-873. 10.1126/science.279.5352.870.View ArticlePubMedGoogle Scholar
- Hardingham N, Glazewski S, Pakhotin P, Mizuno K, Chapman PF, Giese KP, Fox K: Neocortical long-term potentiation and experience-dependent synaptic plasticity require alpha-calcium/calmodulin-dependent protein kinase II autophosphorylation. J Neurosci. 2003, 23: 4428-4436.PubMedGoogle Scholar
- Yasuda H, Barth AL, Stellwagen D, Malenka RC: A developmental switch in the signaling cascades for LTP induction. Nat Neurosci. 2003, 6: 15-16. 10.1038/nn985.View ArticlePubMedGoogle Scholar
- Cooke SF, Wu J, Plattner F, Errington M, Rowan M, Peters M, Hirano A, Bradshaw KD, Anwyl R, Bliss TV, Giese KP: Autophosphorylation of alphaCaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse. J Physiol. 2006, 574: 805-818. 10.1113/jphysiol.2006.111559.PubMed CentralView ArticlePubMedGoogle Scholar
- Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien JZ: Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nat Neurosci. 2000, 3: 238-244. 10.1038/72945.View ArticlePubMedGoogle Scholar
- Hunsaker MR, Kesner RP: Dissociations across the dorsal-ventral axis of CA3 and CA1 for encoding and retrieval of contextual and auditory-cued fear. Learn Mem. 2008, 89: 61-69. 10.1016/j.nlm.2007.08.016.View ArticleGoogle Scholar
- Irvine EE, Vernon J, Giese KP: AlphaCaMKII autophosphorylation contributes to rapid learning but is not necessary for memory. Nat Neurosci. 2005, 8: 411-412.PubMedGoogle Scholar
- Izquierdo I, Barros DM, Mello e Souza T, de Souza MM, Izquierdo LA, Medina JH: Mechanisms for memory types differ. Nature. 1998, 393: 635-636. 10.1038/31371.View ArticlePubMedGoogle Scholar
- Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ: Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. Cell. 1994, 79: 59-68. 10.1016/0092-8674(94)90400-6.View ArticlePubMedGoogle Scholar
- Kogan JH, Frankland PW, Blendy JA, Coblentz J, Marowitz Z, Schütz G, Silva AJ: Spaced training induces normal long-term memory in CREB mutant mice. Curr Biol. 1997, 7: 1-11. 10.1016/S0960-9822(06)00022-4.View ArticlePubMedGoogle Scholar
- Silva AJ, Kogan JH, Frankland PW, Kida S: CREB and memory. Annu Rev Neurosci. 1998, 21: 127-148. 10.1146/annurev.neuro.21.1.127.View ArticlePubMedGoogle Scholar
- Anagnostaras SG, Gale GD, Fanselow MS: Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus. 2001, 11: 8-17. 10.1002/1098-1063(2001)11:1<8::AID-HIPO1015>3.0.CO;2-7.View ArticlePubMedGoogle Scholar
- Maren S: Pavlovian fear conditioning as a behavioral assay for hippocampus and amygdala function: cautions and caveats. Eur J Neurosci. 2008, 28: 1661-1666. 10.1111/j.1460-9568.2008.06485.x.View ArticlePubMedGoogle Scholar
- Kim JJ, Fanselow MS: Modality-specific retrograde amnesia of fear. Science. 1992, 256: 675-677. 10.1126/science.1585183.View ArticlePubMedGoogle Scholar
- Frankland PW, Bontempi B: The organization of recent and remote memories. Nat Rev Neurosci. 2005, 6: 119-130. 10.1038/nrn1607.View ArticlePubMedGoogle Scholar
- Frankland PW, O'Brien C, Ohno M, Kirkwood A, Silva AJ: Alpha-CaMKII-dependent plasticity in the cortex is required for permanent memory. Nature. 2001, 411: 309-313. 10.1038/35077089.View ArticlePubMedGoogle Scholar
- Fukushima , et al: Upregulation of calcium/calmodulin-dependent protein kinase IV improves memory formation and rescues memory loss with aging. J Neurosci. 2008, 28: 9910-9919. 10.1523/JNEUROSCI.2625-08.2008. 2008View ArticlePubMedGoogle Scholar
- von Hertzen LS, Giese KP: Alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation is required for memory consolidation-specific transcription. Neuroreport. 2005, 16: 1411-1414. 10.1097/01.wnr.0000175244.51084.bb.View ArticlePubMedGoogle Scholar
- Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, Charnay P, Bozon B, Laroche S, Davis S: A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat Neurosci. 2001, 4: 289-296. 10.1038/85138.View ArticlePubMedGoogle Scholar
- Yamasaki N, Maekawa M, Kobayashi K, Kajii Y, Maeda J, Soma M, Takao K, Tanda K, Ohira K, Toyama K, Kanzaki K, Fukunaga K, Sudo Y, Ichinose H, Ikeda M, Iwata N, Ozaki N, Suzuki H, Higuchi M, Suhara T, Yuasa S, Miyakawa T: Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders. Mol Brain. 2008, 1: 6-10.1186/1756-6606-1-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Kitamura T, Saitoh Y, Takashima N, Murayama A, Niibori Y, Ageta H, Sekiguchi M, Sugiyama H, Inokuchi K: Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory. Cell. 2009, 139: 814-827. 10.1016/j.cell.2009.10.020.View ArticlePubMedGoogle Scholar
- Kee N, Teixeira CM, Wang AH, Frankland PW: Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci. 2007, 10: 355-362. 10.1038/nn1847.View ArticlePubMedGoogle Scholar
- Hoijati MR, van Woerden GM, Tyler WJ, Giese KP, Silva AJ, Pozzo-Miller L, Elgersma Y: Kinase activity is not required for alphaCaMKII-dependent presynaptic plasticity at CA3-CA1 synapses. Nat Neurosci. 2007, 10: 1125-1127. 10.1038/nn1946.View ArticleGoogle Scholar
- Amada N, Aihara K, Ravid R, Horie M: Reduction of NR1 and phosphorylated Ca2+/calmodulin-dependent protein kinase II levels in Alzheimer's disease. Neuroreport. 2005, 16: 1809-1813. 10.1097/01.wnr.0000185015.44563.5d.View ArticlePubMedGoogle Scholar
- Townsend M, Mehta T, Selkoe DJ: Soluble Abeta inhibits specific signal transduction cascades common to the insulin receptor pathway. J Biol Chem. 2007, 282: 33305-33312. 10.1074/jbc.M610390200.View ArticlePubMedGoogle Scholar
- Zeng Y, Zhao D, Xie CW: Neurotrophins enhance CaMKII activity and rescue amyloid-β-induced deficits in hippocampal synaptic plasticity. J Alzheimers Dis. 2010, 21: 823-831.PubMed CentralPubMedGoogle Scholar
- Phillips RG, LeDoux JE: Lesions of the dorsal hippocampal formation interfere with background but not foreground contextual fear conditioning. Learn Mem. 1994, 1: 34-44.PubMedGoogle Scholar
- Paxinos G, Franklin KJ: The Mouse Brain in Stereotaxic Coordinates. 2001, Academic PressGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.