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hat elevations in astrocytic MAO-B levels leads to a fairly selective loss of dopaminergic SN neurons along with the severity of this loss seems to be age-dependent. It's not clear why there was not a more dramatic increase in SN DA cell loss with age in the MAO-B transgenics. Usually, cell loss coincides with Components and Procedures Generation and evaluation of transgenics with inducible astrocyte-specific elevation of MAO-B levels Human MAO-B cDNA was cloned into pBIG at the PstI and SalI web-sites, putting the gene under transcriptional control of the bidirectional Tet-inducible promoter. A second construct was ready which contained the February Elevated MAO-B & PD Pathology February Elevated MAO-B & PD Pathology corresponding to human MAO-B cDNA as above using Taq polymerase. A MPP+ measurements, striatal dopamine content cell counts and neurodegeneration in inducible astrocytic MAO-B transgenics versus controls +/- MPTP Striatal tissue was harvested from dox versus untreated animals Extracellular HPrimary mixed cultures were prepared from the midbrain of ing or via uptake of ASP+ styryl)-N- methylpyridinium iodide), a fluorescent analogue of dopamine. February Elevated MAO-B & PD Pathology Immuno-magnetic isolation of dopaminergic synaptosomes Dopaminergic and non-dopaminergic striatal synaptosomes were isolated using a modified immuno-magnetic protocol. Briefly, synaptosomes were ready from dissected striatal tissue. Dopaminergic synaptosomes were isolated using magnetic beads conjugated to anti-rabbit IgG after capturing them using a rabbit anti-DAT antibody. Below a magnetic field, bead-bound synaptosomes were passed through a column and washed; flow-through in the presence of your magnetic field yielded a fraction enriched in non-dopaminergic striatal synaptosomes. Elution performed in the absence in the magnetic field yielded fractions enriched in dopaminergic striatal synaptosomes. Equal quantities of various fractions of your synaptosomes isolated using DAT antibody including the starting material or control, the unbound fraction, the flow-through, the wash and also the eluate were subjected to western evaluation to assess relative dopaminergic versus non-dopaminergic synaptosomal purification. Western blots were performed using antibodies In addition, citizens are not frequently well-educated about selections created by their representatives against TH, GABA or SNAP- were sacrificed Morphological analyses of astrocyte and microglial activation Immunochemistry was performed on cryosections derived from the SN, striata, and CTX of fixed perfused brains from doxinduced transgenics versus controls. Astrocytic activation was assessed using primary antibodies against GFAP and s HHydrogen peroxide levels were measured inside the striatal dopaminergic and non-dopaminergic synaptosomes prepared as above from various groups of mice. Mice were injected inside the tail vein with, Locomotor behavioral assessment Locomotor function was assessed using an automated Truscan photobeam apparatus beneath illumination as previously described. Animals were habituated to the apparatus for Supporting Information Mitochondrial complex I and IV activities Isolated synaptosomes were found to be physiologically viable for up to Acknowledgments We thank Raymond Johnson for measurement of dopamine content and Martha Isla for MPP+ measurements. Detection of mitochondrial superoxide in vivo Mitosox Red was used as an indicator to assess presence of superoxide within cellular mitochondria. Author Contributions Conceived and designed the experiments: DN JM JA. Performed the exp