Intracellular injection of lucifer yellow into lightly fixed cerebellar neurons

Lucifer yellow was injected intracellularly into mouse cerebellar neurons in 200 micron thick slices of tissue that had been lightly fixed by cardiac perfusion with paraformaldehyde. Direct observation of fluorescence at the time of injection established that dye diffusion was very rapid and was confined to an intracellular distribution, so that the detailed shapes of neuron cell bodies and dendrites, and some features of axons, were visualized. Different types of cerebellar neurons were identified. With sequential penetration of different neuron types near one another within a given tissue slice, synaptic relationships could be visualized. The validity of the method was tested with cells whose form and relationships are well known. The presence of axonal baskets, for example, around the somata of a row of Purkinje cells after penetration of a single basket cell axon identify cells that are related by a common inhibitory input. Once the baskets were visualized, it was possible to penetrate and fill these related postsynaptic neurons. By filling several Purkinje cells spaced along a given folium, variations in cell form can be 'correlated with' position along the changing curvature of the folium. Immature forms of cerebellar neurons could be penetrated and filled with dye in neonatal animals so that the morphology of cerebellar neurons can be compared at different stages of development.