In the present proposal the role of peptides in developing and mature cerebellar circuits will be analyzed. Experiments are designed to determine the effects of enkephalin (ENK) on Purkinje cell activity in the adult and developing cerebellum. In addition, analysis of the developmental history of corticotropin releasing factor (CRF) and cholecystokinin (CCK) in the cerebellum will be undertaken. Because of the unique distribution of CRF ad CCK in the adult animal we plan to use these peptides as markers to determine the origin, the time course of maturation, and cellular interaction(s) of cerebellar afferents at early stages of development, before individual fibers have the morphological features that permit their identification as mossy or climbing fibers. The following hypotheses will be tested: 1) ENK and CRF are co-localized in the same olivary neurons, but they have opposite effects on the firing rates of Purkinje cells. 2) A transient population of ENK climbing fibers evident during cerebellar maturation will be eliminated by collateral withdrawal. Further, the transient ENK fibers will have a different effect on the firing rate of Purkinje cells than those that are maintained in the adult animal. 3) CRF will be expressed very early in cerebellar maturation an in axons derived initially from the inferior olive and subsequently from precerebellar nuclei that give rise to mossy fibers. 4) Immature axons within the cerebellum with anatomical features of both mossy and climbing fibers that are present during development, do not represent """"""""developmental errors"""""""" in target recognition, but are part of the normal circuitry in the adult animal. CCK will be used as an immunohistochemical marker to test the fourth hypothesis, as it is localized almost exclusively in mossy fibers in the adult animal. Iontophoresis of peptides, immunohistochemistry, in situ hybirdization, and double labeling paradigms using retrograde transport and immunohistochemistry will be employed to analyze the expression of peptides in the developing and adult cerebellum. The proposed experiments that employ the iontophoresis of ENK will provide an initial assessment of the physiological effects of this peptide during cerebellar maturation. The anatomical experiments on transient connections during development are designed to provide information on possible developmental mechanisms involved in the loss of ENK in the cerebellar hemispheres. The use of specific peptide markers will provide insights into the possible temporal relationships between the expression of a putative neuromodulator, neuronal migration, axogenesis, and target recognition.
