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. 2020 May 5;9:e54781. doi: 10.7554/eLife.54781

Figure 4. Hippocampal neuronal knockdown of CIM6P/IGF2R in mice selectively impairs hippocampus-dependent memories.

Figure 4.

(a) Experimental timeline for b–e and g–i. Data are expressed as mean ± SEM, with blue datapoints representing male mice, and pink datapoints representing female mice. Igf2r-floxed mice received bilateral hippocampal injections of AAV-hSyn-Cre-eGFP (Cre) or control AAV-hSyn-eGFP (GFP), 2 weeks prior to behavioral experiments. (b) Open field of GFP- and Cre-injected mice measured total distance travelled, average travel velocity, and cumulative time spent in the center of the area (n = 8, four independent experiments). (c) Novel objection location memory and total exploration time, tested 4 hr after training, (n = 8, four independent experiments). (d) Percent (%) of time spent freezing in mice tested in the training context 1 day (1 d) and 1 week after Pavlovian conditioning (n = 8, four independent experiments). (e) Percent (%) of time spent freezing to a new context prior to onset of tone, and during the tone (n = 8, four independent experiments). (f) Contextual fear conditioning expressed as percent (%) of time spent freezing. Left: % of time spent freezing in mice tested 1 min, 1 day, and 1 week after training (n = 8, four independent experiments). Right: % of time spent freezing in mice tested 5 min after training. (g) Representative western blot and quantification obtained from dorsal hippocampi homogenates from Cre- or GFP-injected mice stained for CIM6P/IGF2R, Cre recombinase (Cre), and actin. Actin-normalized values were expressed as mean percentage ± SEM (n = 7–8, four independent experiments). (h) Upper panels: representative dorsal hippocampus composite tile scans in GFP-injected or Cre-injected mice immunostained for CIM6P/IGF2R (scale bar, 500 μm). Middle and lower panels: CA1 and DG (scale bar, 10 μm) are shown. Bar graphs on the right report immunostaining intensity quantifications for each sub-region (n = 8, four independent experiments). (i) Representative images of GFP- or Cre-injected mice, composite tile scans of dorsal hippocampus double staining with anti-GFP and anti-CIM6P/IGF2R antibodies (scale bar 500 μm). Two-tailed Student’s t-test or two-way repeated measures ANOVA followed by Sidak’s post-hoc tests. *p<0.05, ****p<0.0001; see Source data one for detailed statistical information.