Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system

Abstract

Immunohistochemical distributions of tyrosine hydroxylase and calmodulin in the rat forebrain were analyzed quantitatively to confirm our previous results that the activities of central catecholamine-synthesizing enzymes are regulated by a calcium-calmodulin-dependent system. The adjacent slices of adult rat brain were stained immunohistochemically for tyrosine hydroxylase and for calmodulin, and the distributions and amounts of these proteins were measured by a fluorescence microphotometry system that was developed in our laboratory. Immunohistochemical fluorescence intensity was measured stepwise at 40 μm intervals through a 6 μmφ (on the slice) pin hole. Each stained brain slice was divided into approximately 100,000 areas, and measured for fluorescence intensity and displayed two- and three-dimensionally. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all areas of the brain, but its intensity varied. The relatively high levels of calmodulin could be observed in brain regions with high levels of tyrosine hydroxylase distribution, though high levels of tyrosine hydroxylase could not always be observed in brain regions where high levels of calmodulin were distributed. In the present study, high levels of tyrosine hydroxylase and calmodulin were distributed in the nucleus accumbens septi and the lateral part of the neostriatum regions in which the amount of dopamine was increased by the intraventricular administration of calcium. These findings suggest that the synthesis of central catecholamines is regulated by a calcium-calmodulin-dependent system.