Combination of immunocytochemistry and radioligand receptor assay to identify beta-adrenergic receptor subtypes on astroglia in vitro.

There is an increasing need to assess the distribution of receptors for neuroactive substances on specific neural cell types. This study describes the establishment of methodology that combines the quantification of beta-adrenergic receptor subtypes by radioligand binding assays with immunocytochemical analysis of the contribution of astroglia (identified by the presence of glial fibrillary acidic protein) and fibroblasts (identified by the presence of fibronectin) to cultures prepared from neonatal rat cerebral cortex. The effects of subtle changes in culture methodology on the cellular composition of cerebral cortical cultures and the distribution of beta-adrenergic receptor subtypes were examined. The data indicate that (1) a decrease in the density of the initial plating suspension, (2) an increase in the age of the animals, or (3) supplementation of the cortical cell suspension with meningeal fibroblasts all result in an increase in fibronectin staining and a decrease in glial fibrillary acidic protein antibody staining. This change in the cellular composition of the cortical cultures correlated with an increase in the number of beta 2-adrenergic receptors and a corresponding decrease in the number of beta 1-adrenergic receptors. These observations point out the care which must be exercised when preparing primary astroglial cultures of sufficient purity for large-scale biochemical and pharmacological studies.