We finally investigated whether the changes in neuronal structure induced by RNAi-mediated knockdown of VEGFD causes cognitive EPZ-6438 manufacturer deficits. Mice stereotaxically injected with rAAV-shVEGFD or rAAV-shSCR into the hippocampus were tested in two well-characterized hippocampus-dependent memory tests: Morris

water maze and contextual fear conditioning. In the hidden-platform version of the Morris water maze, mice learn the location of the platform by using distal visual cues ( Morris et al., 1982). We found that mice stereotaxically injected with rAAV-shSCR or rAAV-shVEGFD needed significantly less time to find the hidden platform across training trials (main effect of training session: F[7,91] = 11.30, p < 0.0001); however, no effect of treatment was observed (main effect of treatment: F[1,13] = 1.34, p = NS) ( Figure 8D). This suggests that both groups have developed a learning strategy to find the hidden platform. Multiple behavioral strategies may be employed by the mice to obtain the reward (escape from water) and some of these strategies may be comparably efficient but distinct in the requirement

for hippocampal function ( Lipp and Wolfer, 1998 and Gerlai, 2001). To assess spatial memory, we performed a probe trial during which the platform was removed from the water maze and the mice were given 60 s to search for it. The search pattern during the probe trial can reveal a spatial preference that is believed to represent spatial memory. We observed that rAAV-shSCR-injected mice show a spatial preference for OSI-906 the target quadrant, whereas rAAV-shVEGFD-injected mice did not ( Figure 8E). This was expressed as a significantly longer time spent in the target quadrant by the rAAV-shSCR-injected mice in comparison to the other quadrants (one-way analysis of variance [ANOVA]: F[3,28] = 9.84, p < 0.001; multiple post hoc comparisons: time in target quadrant versus time in adjacent or opposite quadrants; p < 0.05). In contrast, rAAV-shVEGFD-injected mice spent similar amounts of time in the different quadrants (F[3,24] = 1.2, p = NS). This suggests that the rAAV-shVEGFD-injected mice did not develop

a spatial searching strategy, pointing to spatial memory impairment in these mice. Swimming speed was not different between Terminal deoxynucleotidyl transferase the two groups ( Figure 8F, main effect of treatment: F[1,13] = 3.94, p = NS). To determine whether abnormalities in motivation, motor coordination, or vision could account for the deficit in spatial memory, we also trained mice on a visible-platform version of the water maze, a hippocampus-independent task. In this task, the mice use a proximal, visual cue to locate the platform (Figure 8G). rAAV-shSCR- and rAAV-shVEGFD-injected mice showed similar escape latencies to find the visible platform (main effect of training session: F[2,26] = 7.12, p < 0.01; main effect of treatment: F[1,13] = 0.36, p = NS), demonstrating that both groups acquired the task ( Figure 8G).