We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.

For the last twenty years, the Bussey-Saksida touchscreen-based operant conditioning platform has evolved in close parallel alongside the Cambridge Neuropsychological Test Automated Battery (CANTAB) to produce batteries of tests for studying complex cognitive functions in rodents that are increasingly analogous to human diagnostic tests and greatly narrow the translational gap in cognition research. Naturally, with this increasing usefulness comes increasing use, and with it, a need for a greater understanding of and controlling for confounding factors that may limit the system's ability to optimally detect cognitive deficits. In the present study, we show a strong image preference bias in a standard pairwise discrimination task with a widely used spider-plane image pairing. This bias greatly influenced the performance of our experimental mice, significantly affecting the length of time it took mice to complete the task, their progress over time, and several accessory measures usefully recorded by the Bussey-Saksida touchscreen system. We further show that this bias can be corrected by using more highly similar image pairings without sacrificing the animal's ability to learn to distinguish the stimuli. This eliminated all significant stimuli specific differences seen with the spider-plane pairing. We then analyze the pixel composition of the various stimuli to suggest that the bias is due to a difference in image brightness. These findings highlight the importance of carefully modulating paired touchscreen stimuli to ensure equivalence prior to learning and the need for more studies of visual perception in mice, particularly as it relates to their performance in cognitive assays.