Horseradish peroxidase uptake in vivo by neuronal and glial lysosomes

Horseradish peroxidase (HRP) was injected intracerebrally to immature rats, once a day for 3 days. The animals were killed 24 hr after the last injection and the cellular and intracellular localization of the enzymatic tracer in the cerebral cortex was investigated by ultrastructural cytochemistry at the light and electron microscopic level as well as biochemically. By light microscopy it could be shown that the neuronal cell bodies and their dendritic processes took up the HRP tracer selectively and that its intracellular disposition remained confined to the cytoplasm. The HRP reaction product also appeared in neuroglial cells. Microperoxidase, a small molecular weight tracer, whose presence in cells can also be demonstrated by the cytochemical technique used for the demonstration of HRP activity, also entered neuronal cell bodies readily but, unlike HRP, it invaded their space rather indiscriminately including the nucleus. The segregation of HRP in the lysosomes of both neuronal cell bodies and neuroglial cells was demonstrated by electron microscopy. No evidence could be secured for the presence of HRP-positive reaction product in any other intraperikaryal organelle or inside nerve terminals. Oligodendrocytes appeared to concentrate HRP-reaction product in their lysosomes somewhat more avidly than the neurons. Additional evidence for the segregation of HRP in the neuronal lysosome was obtained by demonstrating its presence in neuronal cell bodies isolated in bulk by a recently developed technique. This was accomplished both at the level of the electron microscope and, concurrently, by measurements of HRP activity in homogenates and subcellular fractions prepared from the bulk-isolated neuronal cell bodies. Finally, the association of the injected HRP with a lysosome-rich subfraction obtained by differential and density gradient centrifugation of neuronal homogenates was also established. An incidental observation was the marked reduction, as a result of the repeated administration of HRP to rats 4 or 5 days old, of the specific activity of the neuronal lysosomal enzyme, N-acetyl-β- d-glucosaminidase. This reduction was found to be accompanied by a severe retardation of the animals' growth.