The segregation and expression of glutamate receptor subunits in cultured hippocampal neurons

Abstract

The distribution and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-selective glutamate receptor subunits (GluR1-4) were studied in cultured hippocampal neurons using antibodies generated against peptides corresponding to the C-termini of GluR1, GluR2/3 and GluR4, and with a set of oligonucleotide probes designed complementary to specific pan, flip and flop GluR1-4 messenger RNA sequences. GluR1-4 subunit proteins were localized in fixed hippocampal neurons (2 h to three weeks after plating) by immunocytochemistry with light and electron microscopy. At early stages in culture, moderate staining with antibodies to GluR1 and GluR2/3 and very light staining with antibody to GluR4 was observed in cell bodies and proximal portions of all neurites of some neurons. Upon establishment of identified axons and dendrites by seven days in culture, staining was intense with specific antibodies to GluR1 and GluR2/3 and light with anti-GluR4 antibody in cell bodies and dendrites. Little or no staining was observed in axons. Cells at seven days in culture exhibited a variety of morphologies. However, we could not assign a pattern of staining to a particular type. As the cultures matured over two and three weeks, staining was limited to the somatodendritic compartment. The intensity of glutamate receptor subunit staining increased and the extent of staining proceeded to the distal extreme of many dendrites. Moreover, antibodies to GluR1-4 subunits were co-localized in neurons. Immunocytochemistry on living neurons did not result in any significant labeling, suggesting that the epitope is either not expressed on the surface of the neurons, or is present, but inaccessible to the antibody. Electron microscopy demonstrated receptor localization similar to that found in brain, with staining of postsynaptic membrane and density, dendritic cytoplasm and cell body, but not within the synaptic cleft. We examined the possible role of “cellular compartmentation” in the pattern of glutamate receptor expression in hippocampal neurons. Compartmentalization studies of the subcellular distribution of messenger RNAs encoding GluR1-4 subunits was determined in mature cultures by in situ hybridization. Significant silver grain appearance was restricted to the cell body, indicating that the synthesis of glutamate receptor subunits is limited largely to the neuronal cell body. The expression of microtubule-associated protein 2 was studied in parallel. Microtubule-associated protein 2 expression appeared 6 h after plating, while glutamate receptor subunit expression was present at 2 h. This indicates that microtubule-associated protein 2 does not regulate the initial distribution of glutamate receptor subunits into neurites. Restriction of expression to the cell body raises important questions concerning the mechanisms governing the transport of GluR proteins to their appropriate compartments within neurons of the developing and mature nervous system.