IMMUNOFLUORESCENCE LOCALIZATION OF THYROID-HORMONE RECEPTOR PROTEIN-BETA-1 AND VARIANT ALPHA-2 IN SELECTED TISSUES - CEREBELLAR PURKINJE-CELLS AS A MODEL FOR ALPHA-1 RECEPTOR-MEDIATED DEVELOPMENTAL EFFECTS OF THYROID-HORMONE IN BRAIN

Rat c-erbA-beta-1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T3) levels and T3-dependent brain development. These data suggest that the beta-1 receptor may mediate the T3 effect. However, in cerebellum c-erbA-beta-1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T3-sensitive process, we assessed the levels of the beta-1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta-1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T3 binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-beta-1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta-1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of beta-1 receptor protein despite the low beta-1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta-1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha-2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The alpha-2 fluorescence in testis was high, consistent with the high levels of alpha-2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta-1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.