The Journal of Neuroscience, September 2, 2009, 29(35):10869-10882; doi:10.1523/JNEUROSCI.5531-08.2009
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Cellular/Molecular
NMDA Receptor GluN2B (GluR
2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
Kaori Akashi,1 *
Toshikazu Kakizaki,1,5 *
Haruyuki Kamiya,2
Masahiro Fukaya,3
Miwako Yamasaki,3
Manabu Abe,1
Rie Natsume,1,4
Masahiko Watanabe,3 and
Kenji Sakimura1,4
1Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan, Departments of 2Neurobiology and 3Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan, 4Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 331-0012, Japan, and 5Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan
Correspondence should be addressed to either Masahiko Watanabe or Kenji Sakimura at the above address. Email: watamasa{at}med.hokudai.ac.jp or Email: sakimura{at}bri.niigata-u.ac.jp
GluN2B (GluR
2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluR
1/NR1) was moderately reduced, whereas GluN2A (GluR1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.
Received Nov. 18, 2008;
revised July 19, 2009;
accepted July 22, 2009.
Correspondence should be addressed to either Masahiko Watanabe or Kenji Sakimura at the above address. Email: watamasa{at}med.hokudai.ac.jp or Email: sakimura{at}bri.niigata-u.ac.jp
