The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1

Abstract

Neuronal plasticity associated with learning, memory and development is controlled, in part, by NMDA receptors, which are complexes consisting of the subunit NMDAR1 (NR1) and one or more NMDAR2 subunits (NR2A- NR2D). We made a polyclonal antibody to a C-terminus peptide of NR2A. In analysis of transfected cell membranes, this antibody recognizes NR2A and NR2B, and to a slight extent, NR2C and NR2D. In Western blots of rat brain, the antibody labeled a single band that comigrated with NR2A and NR2B. This antibody (NR2A/B) did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained sections of rat brain showed significant staining throughout the nervous system, including olfactory bulb, cerebral cortex, hippocampus, caudate- putamen, and many brainstem nuclei, as well as in neurons of spinal cord and sensory ganglia. This widespread distribution of staining was similar to that found with an antibody to NR1, supporting the presence of functional NR1/NR2 complexes throughout the nervous system. In the cerebellum, in contrast to staining with NR1 antibody, Purkinje cell staining with NR2A/B antibody was low, indicating that these neurons may lack functional NMDA receptors. EM examination revealed dense staining in dendrites and postsynaptic densities in cerebral cortex and hippocampus, similar to those seen with antibody to NR1. Since functional NMDA receptor complexes at synapses appear to require both NR1 and NR2 subunit proteins for full function, this study provides structural evidence for functional NR1/NR2 receptors in vivo in the nervous system.

d-Serine as a Neuromodulator: Regional and Developmental Localizations in Rat Brain Glia Resemble NMDA Receptors

“…H. Snyder, unpublished observations). The antibody to NR2A / B used in this study was developed and used extensively in a previous study (Petralia et al, 1994a). The labeling patterns we obtain are in generally good agreement with this work, with the modest differences explained by our use of younger animals and a high-glutaraldehyde fixative.…”

“…Labeling for NR2A / B is strong in many neurons, especially in dendritic spines (Fig. 5, right), as reported previously (Petralia et al, 1994a). …”

“…The labeling patterns we obtain are in generally good agreement with this work, with the modest differences explained by our use of younger animals and a high-glutaraldehyde fixative. When we stain P50 animals using the fixative of Petralia et al, (1994a), we faithfully reproduce their results.…”

“…In dot blots and in tissue sections, immunoreactivity is abolished by preincubation with 200 M of the D-serine conjugate. The antibody recognizing NMDA receptor 2A and 2B subunits (Chemicon, Temechula, CA) was generated against a C-terminal peptide of NR2A and has been characterized thoroughly (Petralia et al, 1994a). The polyclonal antibody to glycine was raised against a reduced glutaraldehyde conjugate of glycine and BSA and has at least a 100-fold selectivity over ␤-alanine and a 1000-fold selectivity over other amino acids tested.…”

“…Both are most concentrated in the external plexiform layer. D-Serine concentrates in protoplasmic astrocytes near mitral and tufted cell dendrites, which are intensely stained for NR2A / B (Petralia et al, 1994a). Lesser amounts of both D-serine and NR2A / B occur in the periglomerular region, olfactory nerve layer, and inner plexiform layer.…”