Endogenous perioxidatic activity in brain stem neurons as demonstrated by their staining with diaminobenzidine in...

In order to test for the presence or absence of endogenous peroxidatic activities within the brains of normal squirrel monkeys, sections from the brain stems were incubated in buffered media of diaminobenzidine (DAB) and hydrogen peroxide, according to the methods of Graham and Karnovsky20 or Novikoff and Goldfischer39. It was found that neurons within certain regions were consistently stained by both mediamthese reactive neurons belong to the extrapyramidal motor system (globus pallidus, substantia nigra (pars diffusa), red nucleus, pontine and mesencephalic reticular formation), to certain cranial nerve nuclei (oculomotor, trochlear, abducens, mesencephalic trigeminal, vestibular) and to relay nuclei of the auditory pathway (superior olivary nucleus and nucleus of the trapezoid body). Neurons within other regions of the brain stem, such as thalamus, hypothalamus and corpus striatum were not stained. On the other hand, the neuropil, excluding the neurons, in other motor areas (such as the subthalamic nucleus, inferior olivary nucleus and corpus striatum) and other auditory relay nuclei (inferior colliculus, nucleus of lateral lemniscus) were also significantly stained. Processes around the Purkinje cells of the cerebellum were particularly reactive. Under the electron microscope, reaction products were localized prominently along the cristae and limiting membranes of the mitochondria and within peroxisome-like bodies of the stained neurons. Non-reactive neurons have little or no reaction products in their organelles. The staining was absent when DAB was withheld, and it was weak or absent if H2O2 was omitted from the media. The addition of inhibitors of catalase, 3-amino-1,2,4-triazole and 2,6-dichlorophenolindophenol did not affect the staining intensity, whereas the addition of potassium cyanide, a strong inhibitor of metalloenzymes, especially heme-enzymes, almost completely abolished the reactions. In addition, a 3-day 'bleaching treatment' with 30% H2O2 did not affect the reactions. These results suggest that the reactivity in the above regions was due not to neuromelanin or other pigments, but rather to heme-enzymes and/or other hemoproteins. It was postulated that a higher concentration of the reactive agents was characteristic of certain neuronal types in the brain stem of the squirrel monkeys, and could be related to the cells' functional needs, energy requirements and perhaps to the metabolism of their specific neurotransmitters.