Synaptic distribution of the AMPA-GluR2 subunit and its colocalization with calcium-binding proteins in rat cerebral cortex: an immunohistochemical study using a GluR2-specific monoclonal antibody.
Due to its role as the dominant AMPA receptor subunit in respect to regulation of calcium permeability, information on the neuronal localization of GluR2 is of particular importance, yet has been hampered by the lack of a GluR2-specific antibody. Monoclonal antibodies were raised against the putative N-terminal portion (amino acids 175--430) of GluR2, using the fusion protein linked to trpE as an antigen. Western blot analysis and immunocytochemistry of transiently transfected human embryonic kidney 293 cells unambiguously confirmed the specificity of monoclonal antibody 6C4 for GluR2, which did not recognize or cross-react with any other AMPA/Kainate GluR subunits expressed. 6C4 was used in immunohistochemical studies to characterize the regional, cellular, and subcellular distribution of the GluR2 subunit at the light and electron microscopic levels in rat hippocampus and somatosensory cortex and in colocalization studies with the three calcium-binding proteins: parvalbumin, calbindin, and calretinin. GluR2 was widely distributed in both pyramidal cells and interneurons. Asymmetric synapses were labeled on both spines and small dendritic shafts. In contrast to previous reports, our double labeling studies using monoclonal antibody 6C4 with polyclonal antisera against calcium-binding proteins demonstrated that 84--97% of parvalbumin and calbindin-immunoreactive and 45--66% of the calretinin-immunoreactive interneurons in CA1 and somatosensory cortex also contain GluR2. These data have important implications regarding heterogeneity in calcium permeability of AMPA receptors across cell types in neocortex and hippocampus, as well as for differential vulnerability to excitotoxic injury.
Vissavajjhala
P
P
Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Janssen
W G
WG
Hu
Y
Y
Gazzaley
A H
AH
Moran
T
T
Hof
P R
PR
Morrison
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000276:immunology; D002460:Cell Line / Q000737:chemistry / Q000502:physiology; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D007395:Interneurons / Q000737:chemistry / Q000648:ultrastructure; D007668:Kidney / Q000166:cytology; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D011153:Population; D051381:Rats; D018091:Receptors, AMPA / Q000032:analysis* / Q000276:immunology; D013003:Somatosensory Cortex / Q000737:chemistry* / Q000166:cytology; D013569:Synapses / Q000737:chemistry; D005680:gamma-Aminobutyric Acid / Q000502:physiology
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D018091:Receptors, AMPA; D005680:gamma-Aminobutyric Acid; D002118:Calcium