THE SEGREGATION AND EXPRESSION OF GLUTAMATE-RECEPTOR SUBUNITS IN CULTURED HIPPOCAMPAL-NEURONS

The distribution and expression of 3-hydroxy-5-methyl-4-isoxazolepropi onate-selective glutamate receptor subunits (GluRl-4) were studied in cultured hippocampal neurons using antibodies generated against peptid es corresponding to the C-termini of GluR1-4, GluR2/3 and GluR4, and w ith a set of oligonucleotide probes designed complementary to specific pan, flip and flop GluR1-4 messenger RNA sequences. GluR1-4 subunit p roteins were localized in fixed hippocampal neurons (2 h to three week s after plating) by immunocytochemistry with light and electron micros copy. 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 Glu R1 and GluR2/3 and light with anti-GluR4 antibody in cell bodies and d endrites. 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 culture s matured over two and three weeks, staining was limited to the somato dendritic compartment. The intensity of glutamate receptor subunit sta ining increased and the extent of staining proceeded to the distal ext reme 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 ei ther not expressed on the surface of the neurons, or is present, but i naccessible to the antibody. Election microscopy demonstrated receptor localization similar to that found in brain, with staining of postsyn aptic membrane and density, dendritic cytoplasm and cell body, but not within the synaptic cleft. We examined the possible role of ''cellula r compartmentation'' in the pattern of glutamate receptor expression i n hippocampal neurons. Compartmentalization studies of the subcellular distribution of messenger RNAs encoding GluR1-4 subunits was determin ed in mature cultures by in situ hybridization. Significant silver gra in appearance was restricted to the cell body, indicating that the syn thesis of glutamate receptor subunits is limited largely to the neuron al cell body. The expression of microtubule-associated protein 2 was s tudied in parallel. Microtubule-associated protein 2 expression appear ed 6 h after plating, while glutamate receptor subunit expression was present at 2 h. This indicates that microtubule-associated protein 2 d oes not regulate the initial distribution of glutamate receptor subuni ts into neurites. Restriction of expression to the cell body raises im portant questions concerning the mechanisms governing the transport of GluR proteins to their appropriate compartments within neurons of the developing and mature nervous system.