Use of quantitative ultrastructural immunoperoxidase labeling for analysis of catecholamine neurotoxicity and...

Levels of catecholamines and the synthesizing enzyme, tyrosine hydroxylase (TH) are markedly decreased in the dorsal striatum, caudate-putamen nuclei, following neurotoxic lesions with 6-hydroxy-dopamine (6-OHDA). We examined whether pre-embedding immunoperoxidase labeling of TH could be standardized for quantitatively examining the density and ultrastructure of spared dopaminergic terminals in the striatum of lesioned rats. The peroxidase-antiperoxidase (PAP) method was used to localize rabbit antiserum against TH in caudate-putamen nuclei of adult rats given unilateral nigral injections of either vehicle or 6-hydroxydopamine in the early postnatal period. Experimental differences in fixation and immunocytochemical labeling were minimized by limiting comparisons of immunoreactivity to co-processed sections from the same litters of animals. Imaging software and a Phillips CM-10 electron microscope were used to quantitatively examine immunoreactive profiles in a narrow zone of tissue in contact with the embedding resin. Under these conditions variables attributed to differences in penetration were minimized. There were no significant differences in numbers or mean-cross sectional diameter of immunoreactive terminals in striatum ipsilateral versus contralateral to vehicle injections. Ipsilateral to the 6-OHDA injections, the density (numbers/area) of striatal TH-immunoreactive terminals was reduced by 50-90% in the majority of animals. In the most extensively lesioned rats, the cross-sectional areas of the remaining immunoreactive axons were significantly larger than in the contralateral striatum of the same animal or either hemisphere of vehicle injected controls. These results confirm and extend earlier findings on the plasticity of residual dopaminergic terminals in adult animals after neurotoxic damage. They also establish a quantitative method for ultrastructural analysis of the density of immunoreactivity in thick sections of tissue labeled prior to plastic embedding. The method has broad applicability to quantitative studies of neurotoxicity and plasticity in brain.