Figure 6. The RAM system in rats and flies.
(a–d) RAM labels active neuronal ensembles in rats exposed to inescapable stress (IS). (a) Schematic timeline of the experimental procedure. Rats were injected with AAV-RAM-NLS-mKate2 and AAV-Ef1α-EGFP vectors in the medial prefrontal cortex (mPFC). After 10 days they were either subjected to IS or left undisturbed in their home cages (HC). (b) Schematic drawing of the rat brain with the red region indicating the target area (PL: prelimbic cortex of the mPFC) for virus infection and quantification of RAM+ cells. (c) Percentage of RAM+ cells among total EGFP+ cells in the prelimbic cortex in HC and IS animals. Data are mean ± SEM, n = 3–4 animals per group, Student’s t-test, **p<0.01. (d) Representative images of prefrontal cortex showing mKate2 (red) and EGFP (green) fluorescence in rats subjected to IS or HC conditions. Areas in purple squares are enlarged in the right image for each condition. CC: corpus callosum. Scale bars are 500 and 100 μm for the left and right images, respectively. (e–h) The Drosophila RAM reporter system. (e) Schematic diagram of the Drosophila RAM reporter system. The RAM-luc transgene can be turned on in specific cell types by the targeted expression of Flp recombinase using the GAL4-UAS system (left) or a cell-type specific driver (right). (f) Drosophila RAM reporter activity has low baseline levels and high fold induction. The specificity of the RAM-luc to Flp recombinase was tested using a UAS-Gal4 system, in which Flp recombinase expression is under the control of a heat-shock HS promoter. Flies in the no heat-shock (No HS) condition were maintained at 20°C throughout development and experimental conditions. For the heat-shock (HS) condition, flies were exposed to a 37°C heat shock for 30 min and allowed to recover for a full day at 20°C before measuring reporter expression. To ensure that results were not due to insertional effects, the UAS-flp transgene was combined with fly lines with the reporter transgene on either chromosome II (RAM-luc;UAS-flp) or chromosome III (UAS-flp;RAM-luc). n = 40–47 flies per group, Student’s t-test, ***p<0.001. (g) Pan-neuronal RAM-luc reporter expression displays circadian rhythm. The RAM-luc reporter transgene was combined with a transgene expressing FLP recombinase in all adult neurons and luciferase activity measured in live flies over time. Bars under plots indicate day (light) and night (dark). (h) Pan-neuronal RAM-luc reporter expression is sensitive to memory formation in Drosophila. Flies as described in g were trained in an olfactory memory task. 24 hr after training, flies exposed to Forward Spaced (FS) training showed significantly higher RAM-luc expression than control flies exposed to Backward Spaced (BS) training. Bars under plots indicate day (light) and night (dark). n = 23–24 flies per group, Student’s t-test, **p<0.01.