Animal Care And Experimental Procedures

919 WordsJun 2, 20164 Pages
Animals: 8-12 week old male C57Bl/J6 mice (Jackson Laboratories) were housed in groups of four on a 12 hour light/dark cycle with ad libitum access to food and water. All experiments were performed during the light part of the diurnal cycle. Housing, animal care and experimental procedures were consistent with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute. Fear Conditioning: Contextual fear conditioning experiments were performed using a set of four modified Noldus Phenotyper (Model 3000) chambers (Leesburg, VA) with shock floors (Lattal et al., 2007). The Phenotyper Model 3000 chamber has a 30 × 30 cm floor and is 40 cm in height. The…show more content…
During this period of time, the mice were also acclimated to the injection procedure performing dummy cannula removal and infusers placement using infusion needles (Plastics One) shorter than the guide cannula, and to the sound of the infusion pump that would be used for intracranial injections. For those animals that received siRNA injection, after recovery from surgery and three days before the contextual fear conditioning, animals received 3 days of acclimation to transport from their home cages to the procedure rooms. Mice were injected during the first day (72 hours before training) and then handled during the following 2 days. Behavioral data were analyzed using ANOVA and Student’s T test (GraphPad Prism). Polyribosome isolation and RNAseq analysis: Polyribosomes were prepared as described in Stefani et al., 2004. 20-50% w/w linear sucrose gradients were prepared in 10 mm HEPES-KOH, pH 7.4, 150 mm KCl 5 mm MgCl2, 1 mM DTT and 0.1 mg/ml cycloheximide using a Hoeffer SG 50 Gradient Maker (Amersham Biosciences) and MINIPULS® 3 peristaltic pump (Gilson Inc.). 5.6 ml of a 20% sucrose solution was pipetted into the outlet side of the gradient maker, tiled to fill the connecting passage between the chambers and tapped to remove any bubbles. The valve between two chambers was closed with the 20% solution filling the passage. 5 ml of a 50%
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