Computational protocol: Altered phosphorylation, electrophysiology, and behavior on attenuation of PDE4B action in hippocampus

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Protocol publication

[…] The specificity of the pCREB and pERK1/2 antibodies employed here has been reported by a number of other groups, working on mouse brain tissues [–]. Quantitation of the signal produced with the pCREB antibody in mouse brain has been validated by several other groups [, , , ]. Stimulus-dependent increases in pCREB and/or pERK1/2, as detected by these antibodies, has also been reported by these groups [–]. Appropriate regions of hippocampus were selected (see Figs. , for the specific areas) and grain intensity in each region was quantitated by ImageJ software (NIH, Bethesda, USA). Means ± standard errors were reported and comparisons made with a 2-way t-test (Excel, Microsoft, USA). [...] Mice were tested at 8–12 weeks of age. The experimenter was blind to genotype in all behavioral experiments. Mice were housed in cages containing from 2 to 5 animals (except for 1 male mouse in cohort 2, below, which was housed singly), on a 12-h light, 12-h dark cycle. All behavioral experiments were performed in the light phase, except as specified below. All mice were tested first in a basic behavioral battery (i.e., SHIRPA testing, which included Rotarod), followed by at least a 5–7-day rest period. Open field testing, followed about 7 days later by fear conditioning and then acoustic startle, were performed on a single cohort of mice (Cohort 1; 2 generations backcrossed into C57BL/6 J; several mice were added to this cohort after the SHIRPA data were collected); many of these mice were then sacrificed for electrophysiological studies. For the elevated plus maze, tail suspension and forced swim tests (each performed about 1 week apart, in that order; one mouse was lost during a “rest” interval), a different cohort of mice was used (Cohort 2; 3 generations back-crossed into C57BL/6 J). Males and females were tested throughout and results were analyzed separately for each sex; if the results (WT vs. TG) were not statistically significant when sexes were separated in this fashion, the results for both sexes were pooled and statistical tests performed on this pooled sample set. Graphs were generated with SigmaPlot (Systat, Inc., USA). […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Chemicals Nucleotides