G2C::Gene Targetting

Target Selection

Proteomic study of the composition of mammalian neuronal synapses has identified more than 1100 separate proteins localised at the synapse (Fig. 1) (16635246). Neurotransmitter receptor complexes, such as the N-methyl-D-aspartate receptor complex (NRC/MASC), are major components of the synaptic proteome (10862698). One major goal of our research programme is to understand the functional organisation of the signalling complexes at the synapse and their role in behaviour and disease.

We are using gene targeting technology to analyse the function of proteins expressed at the synapse and which form part of the MASC complex. Using this technology we can produce mutant mice lacking a gene of interest. These mice can be used in behavioural test to assess the contribution of a specific gene to learning and memory in an intact animal.

The initial targets selected were proteins which form part of the MASC complex, interacting synaptically localised proteins and potential disease gene candidates.

General Gene Targeting Methods

Gene targeting and generation of knockout mouse lines is performed largely according to standard protocols . Targeting vectors are constructed of a selectable marker and 2-5kb of homology on both sides (Fig. 2). Targeting vectors are designed to significantly impair the gene of interest by selecting important exons of the gene to be modified. These could be exons coding for the active site of an enzyme, or alternatively exons are deleted causing a frameshift in the transcript after removal. Targeting vectors are generated in the lab by recombination in E. coli using a modification of standard protocols (17426124; 12618378).

In brief, small homology arms are generated by PCR from a BAC containing the gene of interest and are cloned into pTargeter (Fig. 10). The modified pTargeter is linearised by restriction digest and electroporated into EL350 E.coli containing the BAC of interest. Recombination results in 8-15kb of the genomic sequences of the gene of interest cloned into pTargeter. Simultaneously small homology arms corresponding to the DNA sequences to be modified are cloned into a selectable marker plasmid such as pIRESlacZneoflox (Fig. 13). The modified pIRESlacZneoflox is introduced into EL350 cells containing the modified pTargeter. Recombination results in the selectable marker being inserted into the gene of interest in pTargeter and the targeting construct is now complete.

Constructs are subsequently electroporated into E14Tg2a embryonic stem cells, originally derived from 129P2 mice (Fig. 3). After electroporation cells are selected and individual colonies are isolated (Fig. 4). Genomic DNA from individual clones is screened by Southern blot or PCR for appropriate gene targeting. Importantly these probes or PCR primers are located outside the homology arms of the targeting vector. Example data for the generation and analysis of the Dlg3 mutant mouse is shown in Figure 5. After checking that recombination is appropriate, embryonic stem cells carrying the desired genetic modification are injected into blastocysts (Fig. 6). The resultant chimeras are then bred to generate a transgenic line (Fig. 7). Intercrossing of transgenic offspring can generate knockout mice deficient in the targeted gene (Fig. 8). The absence of the targeted gene protein product can be demonstrated by western blotting (Fig. 5).

Generation of specialised alleles

As well as generating our own mouse lines we also make use of mouse lines generated by other researchers as well as high throughput gene targeting and gene trapping resources which produce both targeted and conditional mutations.

In addition specialised types of alleles not generated by high throughput projects listed above. These include point mutations to make single amino acid changes in the targeted protein (Fig. 9). Other types of vectors allow modification of by insertion of protein tags, such as TAP tags, which allow the protein product of the gene of interest to be purified (Fig. 10).

G2C Programme targeting vectors

  • pTARGETER (Fig. 11)
  • pNeoflox (Fig. 12)
  • ploxPneoflrt (Fig. 13)
  • pIRESlacZneoflox (Fig. 14)

Selected G2C mouse lines

Gene targeting resources

References

  • Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies.

    Cuthbert PC, Stanford LE, Coba MP, Ainge JA, Fink AE, Opazo P, Delgado JY, Komiyama NH, O'Dell TJ and Grant SG

    Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.

    Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

    Funded by: NIMH NIH HHS: R01 MH060919, R01 MH060919-10; Wellcome Trust: 077155

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;10;2673-82

  • FAK is required for axonal sorting by Schwann cells.

    Grove M, Komiyama NH, Nave KA, Grant SG, Sherman DL and Brophy PJ

    Centre for Neuroscience Research, University of Edinburgh, Edinburgh EH9 1QH, Scotland, UK.

    Signaling by laminins and axonal neuregulin has been implicated in regulating axon sorting by myelin-forming Schwann cells. However, the signal transduction mechanisms are unknown. Focal adhesion kinase (FAK) has been linked to alpha6beta1 integrin and ErbB receptor signaling, and we show that myelination by Schwann cells lacking FAK is severely impaired. Mutant Schwann cells could interdigitate between axon bundles, indicating that FAK signaling was not required for process extension. However, Schwann cell FAK was required to stimulate cell proliferation, suggesting that amyelination was caused by insufficient Schwann cells. ErbB2 receptor and AKT were robustly phosphorylated in mutant Schwann cells, indicating that neuregulin signaling from axons was unimpaired. These findings demonstrate the vital relationship between axon defasciculation and Schwann cell number and show the importance of FAK in regulating cell proliferation in the developing nervous system.

    Funded by: Wellcome Trust

    The Journal of cell biology 2007;176;3;277-82

  • A recombineering based approach for high-throughput conditional knockout targeting vector construction.

    Chan W, Costantino N, Li R, Lee SC, Su Q, Melvin D, Court DL and Liu P

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK.

    Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies.

    Funded by: Intramural NIH HHS; Wellcome Trust

    Nucleic acids research 2007;35;8;e64

  • Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins.

    Elias GM, Funke L, Stein V, Grant SG, Bredt DS and Nicoll RA

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.

    Trafficking of AMPA receptors (AMPA-Rs) to and from synapses controls the strength of excitatory synaptic transmission. However, proteins that cluster AMPA-Rs at synapses remain poorly understood. Here we show that PSD-95-like membrane-associated guanylate kinases (PSD-MAGUKs) mediate this synaptic targeting, and we uncover a remarkable functional redundancy within this protein family. By manipulating endogenous neuronal PSD-MAGUK levels, we find that both PSD-95 and PSD-93 independently mediate AMPA-R targeting at mature synapses. We also reveal unanticipated synapse heterogeneity as loss of either PSD-95 or PSD-93 silences largely nonoverlapping populations of excitatory synapses. In adult PSD-95 and PSD-93 double knockout animals, SAP-102 is upregulated and compensates for the loss of synaptic AMPA-Rs. At immature synapses, PSD-95 and PSD-93 play little role in synaptic AMPA-R clustering; instead, SAP-102 dominates. These studies establish a PSD-MAGUK-specific regulation of AMPA-R synaptic expression that establishes and maintains glutamatergic synaptic transmission in the mammalian central nervous system.

    Neuron 2006;52;2;307-20

  • Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro.

    Charlesworth P, Komiyama NH and Grant SG

    Centre for Neuroscience Research, University of Edinburgh, Edinburgh, UK. pc3@sanger.ac.uk

    Background: Genetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice.

    Results: Mouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases.

    Conclusion: FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

    Funded by: Wellcome Trust

    BMC neuroscience 2006;7;47

  • Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.

    Collins MO, Husi H, Yu L, Brandon JM, Anderson CN, Blackstock WP, Choudhary JS and Grant SG

    Genes to Cognition, The Wellcome Trust Sanger Institute, Hinxton, UK.

    Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

    Funded by: Wellcome Trust

    Journal of neurochemistry 2006;97 Suppl 1;16-23

  • Synaptic Ras GTPase activating protein regulates pattern formation in the trigeminal system of mice.

    Barnett MW, Watson RF, Vitalis T, Porter K, Komiyama NH, Stoney PN, Gillingwater TH, Grant SG and Kind PC

    Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.

    The development of ordered connections or "maps" within the nervous system is a common feature of sensory systems and is crucial for their normal function. NMDA receptors are known to play a key role in the formation of these maps; however, the intracellular signaling pathways that mediate the effects of glutamate are poorly understood. Here, we demonstrate that SynGAP, a synaptic Ras GTPase activating protein, is essential for the anatomical development of whisker-related patterns in the developing somatosensory pathways in rodent forebrain. Mice lacking SynGAP show only partial segregation of barreloids in the thalamus, and thalamocortical axons segregate into rows but do not form whisker-related patches. In cortex, layer 4 cells do not aggregate to form barrels. In Syngap(+/-) animals, barreloids develop normally, and thalamocortical afferents segregate in layer 4, but cell segregation is retarded. SynGAP is not necessary for the development of whisker-related patterns in the brainstem. Immunoelectron microscopy for SynGAP from layer 4 revealed a postsynaptic localization with labeling in developing postsynaptic densities (PSDs). Biochemically, SynGAP associates with the PSD in a PSD-95-independent manner, and Psd-95(-/-) animals develop normal barrels. These data demonstrate an essential role for SynGAP signaling in the activity-dependent development of whisker-related maps selectively in forebrain structures indicating that the intracellular pathways by which NMDA receptor activation mediates map formation differ between brain regions and developmental stage.

    Funded by: Medical Research Council: G0300466; Wellcome Trust

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;5;1355-65

  • Differential expression of two NMDA receptor interacting proteins, PSD-95 and SynGAP during mouse development.

    Porter K, Komiyama NH, Vitalis T, Kind PC and Grant SG

    Centre for Neuroscience Research, University of Edinburgh, Edinburgh UK.

    Patterns of neural activity mediated by N-methyl-D-aspartate (NMDA) receptors are known to play important roles in development of the central nervous system. However, the signalling pathways downstream from NMDA receptors that are critical for normal neuronal development are not yet clearly understood. NMDA receptors interact with various signalling proteins via scaffolding proteins, which are important in adult neuronal and behavioural plasticity. For example, the NR2B subunits of the NMDA receptor interact with postsynaptic density 95 (PSD-95), which in turn binds to synaptic ras GTPase-activating protein (SynGAP). Interestingly, the developmental phenotype of mice carrying null mutations in these genes differ. NR2B and SynGAP homozygote mice die within the first week of birth whereas PSD-95 homozygote mice survive to adulthood. We therefore examined the expression patterns of PSD-95 and SynGAP genes from embryonic stages to adult using lacZ (beta-galactosidase) marker gene knock-in mice. Dramatic changes of expression were observed throughout development in brain and other tissues. Although SynGAP binds PSD-95, both genes had distinct, as well as overlapping expression. SynGAP expression peaked at times of synaptogenesis and developmental plasticity in contrast to PSD-95, which was expressed throughout the brain from early embryonic stages. Furthermore, SynGAP showed a more spatially restricted pattern as illustrated by its restriction to forebrain in contrast to PSD-95, which was also found in mid- and hindbrain. These data support the model that synaptic signalling complexes are heterogeneous and individual components show temporal and spatial specificity during development.

    The European journal of neuroscience 2005;21;2;351-62

  • Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression.

    McLean GW, Komiyama NH, Serrels B, Asano H, Reynolds L, Conti F, Hodivala-Dilke K, Metzger D, Chambon P, Grant SG and Frame MC

    The Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, G61 1BD, United Kingdom. g.mclean@beatson.gla.ac.uk

    We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

    Genes & development 2004;18;24;2998-3003

  • Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity.

    Yao WD, Gainetdinov RR, Arbuckle MI, Sotnikova TD, Cyr M, Beaulieu JM, Torres GE, Grant SG and Caron MG

    Howard Hughes Medical Institute Laboratories, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

    To identify the molecular mechanisms underlying psychostimulant-elicited plasticity in the brain reward system, we undertook a phenotype-driven approach using genome-wide microarray profiling of striatal transcripts from three genetic and one pharmacological mouse models of psychostimulant or dopamine supersensitivity. A small set of co-affected genes was identified. One of these genes encoding the synaptic scaffolding protein PSD-95 is downregulated in the striatum of all three mutants and in chronically, but not acutely, cocaine-treated mice. At the synaptic level, enhanced long-term potentiation (LTP) of the frontocortico-accumbal glutamatergic synapses correlates with PSD-95 reduction in every case. Finally, targeted deletion of PSD-95 in an independent line of mice enhances LTP, augments the acute locomotor-stimulating effects of cocaine, but leads to no further behavioral plasticity in response to chronic cocaine. Our findings uncover a previously unappreciated role of PSD-95 in psychostimulant action and identify a molecular and cellular mechanism shared between drug-related plasticity and learning.

    Funded by: NIDA NIH HHS: DA13511

    Neuron 2004;41;4;625-38

  • A highly efficient recombineering-based method for generating conditional knockout mutations.

    Liu P, Jenkins NA and Copeland NG

    Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.

    Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.

    Genome research 2003;13;3;476-84

  • Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice.

    Garry EM, Moss A, Delaney A, O'Neill F, Blakemore J, Bowen J, Husi H, Mitchell R, Grant SG and Fleetwood-Walker SM

    Division of Preclinical Veterinary Sciences, R(D)SVS, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom.

    Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

    Current biology : CB 2003;13;4;321-8

  • SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor.

    Komiyama NH, Watabe AM, Carlisle HJ, Porter K, Charlesworth P, Monti J, Strathdee DJ, O'Carroll CM, Martin SJ, Morris RG, O'Dell TJ and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8-9JZ, United Kingdom.

    At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;22;9721-32

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.

    Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ and Grant SG

    Centre for Genome Research, University of Edinburgh, UK.

    Specific patterns of neuronal firing induce changes in synaptic strength that may contribute to learning and memory. If the postsynaptic NMDA (N-methyl-D-aspartate) receptors are blocked, long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and the learning of spatial information are prevented. The NMDA receptor can bind a protein known as postsynaptic density-95 (PSD-95), which may regulate the localization of and/or signalling by the receptor. In mutant mice lacking PSD-95, the frequency function of NMDA-dependent LTP and LTD is shifted to produce strikingly enhanced LTP at different frequencies of synaptic stimulation. In keeping with neural-network models that incorporate bidirectional learning rules, this frequency shift is accompanied by severely impaired spatial learning. Synaptic NMDA-receptor currents, subunit expression, localization and synaptic morphology are all unaffected in the mutant mice. PSD-95 thus appears to be important in coupling the NMDA receptor to pathways that control bidirectional synaptic plasticity and learning.

    Funded by: Wellcome Trust

    Nature 1998;396;6710;433-9

  • nagy -year 2003 -isbn 978-0879695910 -author Nagy A -title Manipulating the mouse embryo : a laboratory manual, 3rd Ed -journal Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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