Transgenic Sca1(154Q-20) mice were used for behavioral and neurophysiological studies.
Whole brain or specific subregions were homogenized in Tris buffer containing Complete (Roche) proteinase inhibitors and the resulting supernate used for Western blot. For detection of Ataxin-1, samples were lysed and run on SDS-PAGE gel. Western blots were probed with polyclonal anti-ataxin-1 and monoclonal polyglutamine antibodies.
Coordination and motor skills acquisition was tested on the Rotorod type 7650 treadmill(Ugo Basile). Statistical analysis used ANOVA. Mice were trained on the visible platform test and after training given a 60 sec probe trial test. They were also tested with auditory stimulus and foot shock to study fear paradigm. Morris water tank was used to train mice to find the hidden escape platform. For testing the platform was removed and time and distance to search the pool recorded.
Hippocampal brain slices were obtained and recordings of Schaffer collateral synapse responses were taken. Sagittal cerebellar slices were prepared and whole cell recording was made from Purkinje cells. Stimulation and data acquisition were performed using the PULSA software(HEKA).
Immunohistochemistry and immunofluoresence staining was performed on brain tissue using rabbit polyclonal anti-atixin-1 (11NQ), monoclonal anti-HDJ2/HSDJ (Neomarkers), monoclonal anti-ubiquitin (Novacastra) and monoclonal anti-calbindin (Sigma). For quantitation of dendritic branching, frozen sections of cerebelli were stained with anti-calbindin and viewed with a confocal microscope(Biorad 1024). Using the NIH image J projection to plot profiles of fluorescent intensity the data was used to compare fluorescence and average change over the course of the dendrite branch.
Midsagittal brain sections were stained and the Purkinje and hippocampal pyramidal neurons were counted under light microscopy. Statistical analysis by ANOVA
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A New CAG Repeat Model Warns: Beware The Freely Floating Abnormal Protein!
