A novel morphological technique to investigate a single climbing fibre synaptogenesis with a Purkinje cell in the developing mouse cerebellum: DiI injection into the inferior cerebellar peduncle.

To study morphologically the relationship between climbing fibre and Purkinje cell in the developing mouse cerebellum, we established a novel tract tracing using injection of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the inferior cerebellar peduncle, the half point of olivocerebellar projection. In this tracing method, only a certain number of climbing fibres were labelled with DiI and they revealed the single-fibre resolution, individually. These technical advantages enabled us to follow the projection of a climbing fibre to a Purkinje cell at the light microscopic level. To further investigate how a single labelled olivocerebellar axon interacts with a Purkinje cell, we introduced a photoconversion method into this tracing method and successfully observed the photo-oxidized climbing fibre terminals at the electron microscopic level. At postnatal days 7 and 9, a single DiI-labelled climbing fibre arborized around some adjacent Purkinje cell bodies in a distinguishable nest. At this pericellular nest stage, we first demonstrated that the terminal arborization stemmed from a single climbing fibre formed synapses simultaneously on both a soma and dendrites of a Purkinje cell. This finding suggests that the pericellular nest may be such an efficient form that a single climbing fibre innervates a Purkinje cell at both perisomatic and peridendritic sites. Thus, we succeeded in establishing an effective tracing method to investigate a single climbing fibre synaptogenesis with a Purkinje cell both at the light and electron microscopic levels.