Persistent hippocampal CA1 LTP in mice lacking the C-terminal PDZ ligand of GluR1 Chong-Hyun Kim 1 , Kogo Takamiya 1 , Ronald S Petralia 2 , Rita Sattler 1 , Sandy Yu 1 , Weiguo Zhou 3 , Robert Kalb 3 , Robert Wenthold 2 & Richard Huganir 1 The C-terminal PDZ ligand of the AMPA receptor GluR1 subunit may be important for expression of CA1 hippocampal long-term potentiation. To test this directly in vivo, we generated a knock-in mouse lacking the last seven residues of GluR1, comprising the PDZ ligand. This deletion did not affect basal GluR1 synaptic localization, basal synaptic transmission, long-term potentiation or long-term depression, indicating that the ligand is not required for CA1 hippocampal synaptic plasticity. Recent studies have suggested that the regulated exocytosis and endocytosis of vesicles containing AMPA receptors or the lateral movement of AMPA receptors along the plasma membrane could be an important mechanism regulating the level of synaptic AMPA receptors after the induction of long-term potentiation (LTP) and long-term depression (LTD) 1 . A variety of data have indicated that AMPA receptor interactions with PDZ domain–containing proteins may be an important regulatory step during these cellular processes 1–7 . A recent study has reported that a single amino acid mutation (T887A) within the PDZ ligand of GluR1 blocks the synaptic delivery of virally overexpressed GluR1 during LTP induction in CA1 region of organo- typic hippocampal slices 8 . These results indicate a critical role of the GluR1 PDZ ligand in the membrane insertion of AMPA receptors during activity-dependent processes. To test the role of the GluR1 PDZ ligand in the synaptic targeting of GluR1 and synaptic plasticity in vivo directly, we generated a knock-in mouse (GluR1D7) that lacks the last seven amino acids of GluR1, which constitute the PDZ ligand (Fig. 1a and Supplementary Fig. 1 online). The GluR1D7 mutant mice are viable, breed normally and have no obvious behavioral or develop- mental phenotypes. We first examined whether the GluR1 PDZ ligand deletion mutation affected the expression of GluR1, by western blotting using an N-terminal GluR1 antibody. The expression of the mutated GluR1 was normal compared to that in wild-type mice (Fig. 1b), as was the expression of GluR2 and GluR3. Previous studies have shown that GluR1 interacts with the PDZ domain–containing synapse associated protein 97 (SAP97) through its C-terminal PDZ ligand 9–11 . We there- fore measured the expression of SAP97 in the GluR1D7 mice and found GluR1 Nissl stain α GluR1-N Wild-type GluR1– WT α GluR1N α GluR1-C80 α SAP97N (+/+) (–/–) (+/+) (–/–) SAP97N GluR1 GluR1 GluR2/3 KO Δ7 WT KO Δ7 Input IP:α SAP97 Stop HSSGM HSSGM P L G A T G L Stop (+/+) (–/–) (+/–) α SAP97N (+/+) (–/–) (+/–) α GluR1-C20 (+/+) (–/–) (+/–) α GluR2/3 (+/+) (–/–) (+/–) GluR1 Δ7– a c d e b Figure 1 Generation of GluR1 mutant mice lacking the last seven amino acids comprising the PDZ ligand. (a) Schematic representation of GluR1 protein and C-terminal amino acid sequence after modification of stop codon. (b) Immunoblot analysis of wild-type (+/+), heterozygous mutant (+/) and homozygous mutant (/) mice. GluR1 with the seven-amino-acid deletion can be detected by an antibody raised against the last 80 amino acids of the GluR1 C terminus (a GluR1-C80) but not by an antibody against the last 20 amino acids of the GluR1 C terminus (a GluR1-C20). aSAP97N, antibody against the N terminus of SAP97; aGluR2/3, antibody against GluR2/3. (c) Co-immunoprecipitation of GluR1 and SAP97 in vivo. Membrane fractions of brain homogenate of wild-type (WT), homozygous GluR1D7 mutant (D7) and GluR1 knockout mice (KO; Supplementary Fig. 4) were solubilized and immunoprecipitated with antibody to SAP97 (a SAP97) and detected with an antibody to the GluR1 N terminus (a GluR1N). Lower panel shows immunoprecipitated SAP97. (d) Gross hippocampal anatomy and localization of GluR1 immunoreactivity in wild-type and GluR1D7 mutant mice. Left, Nissl staining; right, GluR1 N-terminal immunocytochemistry. (e) Immunoelectron microscopic labeling of GluR1 receptor localization in wild-type and GluR1D7 mutant mice. ‘Pre’ indicates presynaptic terminal. Scale bar, 200 nm. All experiments were done in accordance with the policies of the Animal Care and Use Committee at the Johns Hopkins School of Medicine. Published online 10 July 2005; doi:10.1038/nn1432 1 Department of Neuroscience and Howard Hughes Medical Institute, The Johns Hopkins University, Baltimore, Maryland 21205, USA. 2 Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institute of Health, Bethesda, Maryland 20892, USA. 3 Children’s Hospital of Philadelphia, Joseph Strokes, Jr. Research Institute, ARC 814, 3615 Civic Center Blvd., Philadelphia, Pennsylvania, 19104, USA. Correspondence should be addressed to R.H. (rhuganir@jhmi.edu). NATURE NEUROSCIENCE VOLUME 8 [ NUMBER 8 [ AUGUST 2005 985 BRIEF COMMUNICATIONS © 2005 Nature Publishing Group http://www.nature.com/natureneuroscience