A combined immunohistochemical and autoradiographic method to detect midbrain dopaminergic neurons and determine their time of origin

The present paper describes an efficient tyrosine hydroxylase immunohistochemical method combined with [3H]thymidine autoradiography to identify, in the same tissue section, substantia nigra pars compacta dopaminergic neurons and quantitatively determine their neurogenetic timetables both in control animals and in homozygous weaver mice. The experimental animals were the offspring of pregnant dams injected with [3H]thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. Preservation of tyrosine hydroxylase immunoreactivity, as well as the establishment of the best conditions for the [3H]thymidine autoradiography, were attained after many trials in which fixatives, buffers, antibody dilution and conditions for photographic emulsion, developer and fixer were tested. The proposed combined technique reveals that the detection of tyrosine hydroxylase positive neurons is accurate, unambiguous and has a low interassay variability. Moreover, it did not influence posterior [3H]thymidine autoradiography, which was highly reproducible and presented very low background. The method described here allows us to demonstrate that the neurogenetic timetables of midbrain dopaminergic neurons were different between control and homozygous weaver mice in the mesencephalic area studied. © 2002 Elsevier Science B.V. All rights reserved.