The proposal is aimed at the study of the olivocerebellar circuit of the mouse using a unique transgenic mouse as a model system. The transgene carried by this line of animals consists of the toxic alpha subunit of the diphtheria toxin gene under the regulatory control of the Purkinje cell specific gene, L7. Because of the peculiarities of its temporal regulation, the L7 regulatory regions drive marker gene expression in an initially heterogeneous fashion a series of sagittal bands that are first apparent at the midline and subsequently spread laterally. By adulthood, all Purkinje cells are positive. When the L7 regulatory sequences are used to drive the a- diphtheria toxin gene (ADT), they produce a Purkinje cell autonomous, early onset cell death that begins at the midline and spreads to the hemispheres. As an apparent consequence of this cell death, the presynaptic inferior olive and granule cells suffer a retrograde degeneration. In the end, virtually no granule cells and very few inferior olive neurons remain. To pursue this model system, the applicant proposes five specific aims: To study the effects of the Purkinje cell loss on granule cell behavior, including the nature and timing of granule cell death and changes in proliferative behavior. The PI will attempt to correlate gaps in the external granule cell layer (EGL) with the growing gaps in the Purkinje cell layer. The state of maturation of the EGL cells will be assessed by in situ hybridization of two molecular markers, TAG-1 (maturing/migrating granule cells) and the GABA receptor. Proliferation will be measured by BrdU incorportation by the EGL cells with qualitative and, perhaps, quantitative analysis of the relationship between Purkinje cells and dividing granule cells. Granule cell deaths will be detected in part via the TUNEL technique. To study the potential role of the Bergmann glial cell as an intermediary between the Purkinje cell loss and the granule cell changes. Preliminary studies show that the Bergmann glia become thickened and distorted as early as P8 and by P24, there are no longer any GFAP positive Bergmann glia visible in the mutant. L7ADT mice will be stained with GFAP and a transient, Bergmann glial-specific cell marker, BLBP. Using these reagents, the state of the glia will be compared with the state of the EGL to look for correlations. Potential in vivo paradigms are listed, including disrtuption of the glial cells with araC. To examine the timing and spatial pattern of cell loss in the inferior olive in the mutants with particular regard to """"""""heterogeneous effects on olive structures that could be correlated with specific sets of Purkinje cells that have undergone degeneration"""""""". TUNEL labeling will be used in this system as well. To construct additional lines of L7:ADT mice with shorter 5' regions. Experience with such promoters in lacZ fusion genes suggests that the resulting Purkinje cell death should be at earlier times than in the current line of animals. To create and analyze an L7 gene knockout. Based on the localization of the L7 protein and mRNA, the applicant hypothesizes that these animals should have defects in Purkinje cell dendritic development. To facilitate the analysis of the null mice, the PI has assembled a list of several collaborators, including Drs. Richard Smeyne, of Hoffman- LaRoche (who will create the knockout mice), Karl Schilling (who will perform """"""""various tests"""""""" on cells from the L7 knockout brain), and Dr. David Linden (who will test the electrophysiological properites of the L7-/- cells in brain slices.
