Autoradiographic quantitation of β-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells

Abstract

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether β-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). β-Adrenergic receptors were labeled with [ 125I]pindolol or [ 125I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of β-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of 125I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of β-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of β-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the β-adrenergic receptor is lost from this population of astroglia during development. This method provides a novel approach to the problem of measuring cell-type specific, membrane-associated receptors on individual, well-characterized cells in mixed primary neural cultures.