Selective distribution of kainate receptor subunit immunoreactivity in monkey neocortex revealed by a monoclonal antibody that recognizes glutamate receptor subunits GluR5/6/7

Abstract

A monoclonal antibody (4F5) was generated against a portion of the putative extracellular domain of glutamate receptor subunit GluR5. Western blot analyses and immunocytochemistry of transfected human embryonic kidney 293 cells confirmed that monoclonal antibody 4F5 was specific for GluR5, -6, and -7 (the three identified members of the kainate receptor subunit class), but did not recognize GluR1, -2, or -3 (the AMPA/kainate receptor subunit class). The antibody was subsequently used to examine immunocytochemically the regional, laminar, and cellular distribution of GluR5/6/7 receptor subunits at the light and electron microscopic levels in monkey neocortex. Receptor subunit immunoreactivity was present throughout all cortical areas examined, but exhibited marked cellular, laminar, and regional specificity. Typically, pyramidal cell somata and apical dendrites were well stained. Electron microscopy revealed an extensive cytoplasmic localization of GluR5/6/7 immunoprecipitate, with intense staining of many postsynaptic densities, all of which were associated with asymmetric synapses located on dendritic shafts or dendritic spines. There was no evidence of stained glial cells or presynaptic axon terminals. In most areas, labeled cells and dendrites were concentrated in layers II, III, and V while layers I, IV, and VI typically possessed the fewest and/or least intensely stained elements. A consistent feature in many areas was groups of clustered layer V pyramidal cells and bundles of ascending apical dendrites. Regionally, motor areas and higher-order association areas of the frontal, parietal, and occipital lobes were more densely stained than primary sensory areas (somatic sensory and visual cortex), which was confirmed quantitatively. These data indicate a high degree of selectivity in the distribution of kainate receptors composed of GluR5/6/7 subunits, and suggest that functional specificity and diversity in the ubiquitous excitatory amino acid-utilizing axonal systems in neocortex are achieved in part by the differential association of particular glutamate receptor subunits with specific cortical circuits. In addition, the regional, laminar, and morphological characteristics of GluR5/6/7-immunoreactive neurons bear a strong similarity to those of the neocortical neurons with heightened vulnerability in certain neurodegenerative disorders.

Excitotoxicity in the Enteric Nervous System

“…Some were very intensely stained; others were more lightly stained. The cytoplasmic localization of kainate receptors in enteric neurons agrees with previous studies that have visualized these receptors in fixed and permeabilized central neurons, using C-terminal antibodies (Huntley et al, 1993). At least part of the internal labeling may represent receptor subunits in synthesis or in transport to and from the cell membrane, similar to the proposals for AMPA-selective glutamate receptor subunits (Wenthold et al, 1990).…”

“…To locate proteins in the tissue by immunocytochemistry, we exposed the preparations to PBS containing 0.5% Triton X-100 and 4% horse serum for 30 min to permeabilize the tissue and reduce background staining. Immunoreactivity was then demonstrated by incubating the tissues with affinitypurified rabbit polyclonal antibodies (24 -48 hr; 4°C) to GluR1 and GluR2/3 (0.5 g /ml; Petralia and Wenthold, 1992), EAAC1 (0.06 g /ml; Rothstein et al, 1993), GluR5/6/7 (Huntley et al, 1993), or NSE (1:3000; Polysciences, Warrington, PA); mouse monoclonal antibodies to calbindin (CBP; 1:100; Sigma) or nonphosphorylated neurofilament H (SM I-32; 1:500; Sternberger Monoclonals); or goat antibodies to calretinin (1:6000; Chemicon, Temecula, CA) (see Table 1). In most experiments, bound antibody was visualized by incubating tissues for 3 hr with indocarbocyanine (C y3)-labeled or FI TC -labeled species-specific secondary antibodies to IgG (diluted 1:4000; Jackson ImmunoResearch, West Grove, PA); however, SM I-32 immunoreactivity was demonstrated with the Elite kit (Vector Laboratories, Burlingame, CA) and visualized with diaminobenzidine (DAB substrate kit; Vector Laboratories).…”

Kainate receptors in hippocampal CA3 subregion: evidence for a role in regulating neurotransmitter release

“…The use of antibodies recognizing GluR6/7 (Petralia et al, 1994), GluRs/6/7 (Huntley et al, 1993;Siegel et al, 1995), or KA2 subunits (Petralia et al, 1994;Roche and Huganir, 1995) revealed a similar distribution of glutamate receptor subunits and of [3H]kainate binding sites. In the rat and monkey hippocampus, the immunoreactivity with these antibodies occurs mainly in the hilus and in the CA3 subregion (Petralia et al, 1994;Siegel et al, 1995).…”

“…In the rat and monkey hippocampus, the immunoreactivity with these antibodies occurs mainly in the hilus and in the CA3 subregion (Petralia et al, 1994;Siegel et al, 1995). At the synaptic level, the localization of kainate receptor subunits appears to be principally at the postsynaptic membranes (Petralia et al, 1994;Huntley et al, 1993;Siegel et al, 1995). However, presynaptic KA2 subunits were also identified in nerve terminals of the rat cerebral and cerebellar cortex (Petralia et al, 1994).…”

Reciprocal homeostatic reactions to chronic excitatory synaptic receptor inactivation in developing cerebral cortex networks

“…Kainate receptors are densely distributed along cortical dendrites [7], namely, and might therefore require a critical amount of morphological substrate in order to display clearcut signs of plasticity. Furthermore, organotypic co-cultures -especially of the recently developed 'mega' variety -exhibit firing patterns which much more closely approximate the in vivo picture than do simpler preparations [9].…”