The present proposal is designed to address questions related to the time of axon arrival, the specificity of target recognition and the expression of putative neurotransmitters (serotonin, 5-Hydroxytryptamine - 5HT, and enkephalin - ENK) in the developing cerebellum. The following hypotheses will be tested: 1) During early development, two systems of 5HT axons arise from different populations of neurons in anatomically distinct areas of the brainstem and the first system is transient. 2) There is an early expression of enkephalin in axonal projections to the cerebellum. However, there will be a delay before specific axon terminal phenotypes are developed and synaptic contacts are made with postsynaptic targets. 3) Populations of climbing fibers are heterologous in their putative transmitter content during the developmental stage when Purkinje cells receive multiple climbing fibers. The presence of ENK and non-ENK terminals will be determined. 4) Climbing fibers showing ENK-like immunoreactivity do not originate within the inferior olive. Light and electron microscopic immunohistochemistry and a double-labeling paradigm will be employed to test these hypotheses. The results of this study should document the existence of a transient 5HT pathway to the cerebellum present during early development, and further our general understanding of mechanisms used to establish neural circuits. Moreover, the data generated should provide additional evidence for a differential timetable of 5HT development in the cerebellum when compared to the forebrain. The proposed temporal analysis of axons and their terminals, labeled by enkephalinlike immunoreactivity should provide a timetable for transmitter expression, maturation of axon terminal phenotypes and target recognition. A study of these maturation phases may provide insights into the sequence of events growing axons go through in making the appropriate synaptic connections necessary for normal brain function.
