Axonal projection patterns visualized with horseradish peroxidase in organized cultures of cerebellum

Neurons of the cerebellum and brain stem region of organotypic cultures were injected intracellularly with horseradish peroxidase. The axonal pattern was analyzed for the three cell types studied—Purkinje neurons, deep cerebellar neurons, and brain stem neurons. There was a consistent pattern for each cell type. The axon of the Purkinje neuron was uniform in diameter throughout, sometimes with one recurrent collateral, and within the deep nuclear region branched to produce a terminal field with many axon bulbs measuring 2–4 μm in diameter. The deep cerebellar neuron gave rise to a single axon which branched into a number of major axons, most of which coursed for long distances along the margin of the cortical region. Where the cortical region was separated off from the deep nuclear area, the axon of the deep cerebellar neuron branched and many axons coursed through the intervening zone to the cortex. The terminals in the cortical region were seen either as expansions along the fibre or as grape-like clusters on small side branches of the axons. The brain stem neuron had multiple axons which branched profusely and produced a system of fine-calibre varicose fibres, many of which coursed far into the outgrowth region.This study characterized the distribution of the axons of each neuron type. The results corroborate the fibre patterns seen in the living, stained and fluorescence studies of these cultures. The axon pattern for each neuron type resembles that of the corresponding neuron in the animal. These results will be correlated with the electrophysiological studies of the connections formed by the same neurons in sister cultures.