Requirement for HippocampalCA3 NMDA Receptors inAssociative Memory RecallKazu Nakazawa,1,2,3Michael C. Quirk,1,3*Raymond A. Chitwood,4Masahiko Watanabe,5Mark F. Yeckel,4† Linus D. Sun,1,3Akira Kato,1,2,3‡ Candice A. Carr,1,2,3Daniel Johnston,4Matthew A. Wilson,1,3Susumu Tonegawa1,2,3§Pattern completion, the ability to retrieve complete memories on the basis ofincomplete sets of cues, is a crucial function of biological memory systems. Theextensive recurrent connectivity of the CA3 area of hippocampus has led tosuggestions that it might provide this function. We have tested this hypothesisby generating and analyzing a genetically engineered mouse strain in which theN-methyl-D-asparate (NMDA) receptor gene is ablated specifically in the CA3pyramidal cells of adult mice. The mutant mice normally acquired and retrievedspatial reference memory in the Morris water maze, but they were impaired inretrieving this memory when presented with a fraction of the original cues.Similarly, hippocampal CA1 pyramidal cells in mutant mice displayed normalplace-related activity in a full-cue environment but showed a reduction inactivity upon partial cue removal. These results provide direct evidence for CA3NMDA receptor involvement in associative memory recall.In both humans and animals, the hippocam-pus is crucial for certain forms of learningand memory (1, 2). Anatomically, the hip-pocampus can be divided into several majorareas: the dentate gyrus, CA3, and CA1 (3).In area CA3, the pyramidal cells, whichproject to CA1 pyramidal cells via Scha¨ffercollaterals, receive excitatory inputs fromthree sources: the mossy fibers of the dentategyrus granule cells, the perforant path axonsof the stellate cells in the superficial layers ofthe entorhinal cortex, and the recurrent col-laterals of the CA3 pyramidal cells them-selves, which are the most numerous type ofinput to the CA3 pyramidal cells (4 ). Theprominence of these recurrent collaterals hasled to suggestions that CA3 might serve as anassociative memory network. Associativenetworks, in which memories are storedthrough modification of synaptic strengthwithin the network, are capable of retrievingentire memory patterns from partial or de-graded inputs, a property known as patterncompletion (5–10). In CA3, the strength ofthe recurrent collateral synapses along withperforant path synapses can be modified in anNMDA receptor (NR)– dependent manner(11–14). In this study, we have examined therole of these synapses in memory storage,retrieval, and pattern completion by generat-ing and analyzing a mouse strain in which theNMDAR subunit 1 (NR1) is specifically andexclusively deleted in the CA3 pyramidalcells of adult mice.CA3 NMDA receptor knockout mice.To generate CA3 pyramidal cell–specificNR1 knockout mice (CA3-NR1 KO mice),we used the bacteriophage P1– derived Cre/loxP recombination system (15). Because theCA3 pyramidal cell layer is a robust site ofexpression of KA-1, one of the kainate recep-tor subunits (16 ), we created transgenic micein which the transcriptional regulatory regionof the KA-1 gene drives the expression of theCre transgene (17). In one transgenic line(G32-4), the level of Cre immunoreactivity(IR) was robust in the CA3 pyramidal celllayer in mice older than 4 weeks of age (17)(Fig. 1A). The spatial and temporal pattern ofCre/loxP recombination in the G32-4 Cretransgenic mouse line was examined bycrossing it with a lacZ reporter mouse (Ro-sa26) and staining brain sections derivedfrom the progeny with X-gal (17 ) (Fig. 1, Bto D). Cre/loxP recombination was first de-tectable at postnatal day 14 in area CA3 ofthe hippocampus. At 8 weeks of age, recom-bination had occurred in nearly 100% of py-ramidal cells in area CA3 (Fig. 1C). Recom-bination also occurred in a few other brainareas, but at distinctly lower frequencies: inabout 10% of dentate gyrus (Fig. 1C) andcerebellar granule cells (Fig. 1D) and in about1Picower Center for Learning and Memory, RIKEN-MITNeuroscience Research Center,2Howard HughesMedical Institute, and3Department of Biology andDepartment of Brain and Cognitive Sciences, Massa-chusetts Institute of Technology, Cambridge, MA02139, USA.4Division of Neuroscience, Baylor Col-lege of Medicine, Houston, TX 77030, USA.5Depart-ment of Anatomy, Hokkaido University School ofMedicine, Sapporo 060-8638, Japan.*Present address: Cold Spring Harbor Laboratories,Cold Spring Harbor, NY 11724, USA.†Present address: Department of Neurobiology, YaleUniversity School of Medicine, New Haven, CT06520–8001, USA.‡Present address: Shionogi Research Laboratories,Shionogi & Co., Ltd., Koka-gun, Shiga 520-3423, Japan.§To whom correspondence should be addressed. E-mail: [email protected]. 1. Distribution of Cre im-munoreactivity and Cre/loxPrecombination in G32-4mice. (A) A parasagittal Vi-bratome section from thebrain of a 4-week-old maleG32-4 mouse was stainedwith a rabbit antibodyagainst Cre, and Cre IR wasvisualized with fluoresceinisothiocyanate. Arrowheads,CA3 pyramidal cell layer.Scale bar, 50 ␮m. (B to D)Coronal sections from thebrain of an 8-week-old maleG32-4/Rosa26 double-trans-genic mouse stained with X-gal and Nuclear Fast Red. Inforebrain (B and C), arrow,CA3 cell layer; arrowhead,dentate granule cell layer. Inhindbrain (D), arrowheads,facial nerve nuclei. Scale bar,100 ␮m. (E and F) Parasagit-tal hippocampal sectionsfrom the brain of an 8-week-old G32-4/Rosa26 double-transgenic mouse subjected to double immunofluorescence stainingwith (E) antibodies against ␤-galactosidase (visualized by Alexa488) and GAD67 (visualized byCy3), or (F) with antibodies against ␤-galactosidase and calretinin (visualized by Cy3). Green,␤-galactosidase IR; red in (E) GAD67-IR; red in (F), calretinin-IR. DG, dentate gyrus; Th, thalamus;white arrows, somata of mossy cells; white arrowheads, axon terminals of mossy cells. Scale bar,10 ␮m.R ESEARCH A RTICLESwww.sciencemag.org SCIENCE VOL 297 12 JULY 2002 21150% of cells in the facial nerve nuclei of thebrain stem (Fig. 1D). Recombination fre-quency did not change in older mice. Norecombination was detected in the cerebralcortex or in the hippocampal CA1 and sub-icular regions (Fig. 1, B to D). We deter-mined the type of the recombination-positivecells in the hippocampus with double immu-nofluorescence staining using a set of anti-bodies specific for ␤-galactosidase (a markerfor the Cre/loxP recombination), glutamicacid decarboxylase (GAD)-67 (a marker forinterneurons), and