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. 2018 Jul 18;7:e37993. doi: 10.7554/eLife.37993

Figure 6. Effects of p18 KD in hippocampal CA1 region on LTP in WT and AS mice.

(A) Representative images of dendrites of CA1 pyramidal neurons stained with anti-p18 (red) and -PSD95 (green) antibodies. Arrowheads indicate co-localized puncta. Scale bar = 10 µm. (B) Representative images of CA1 pyramidal neurons stained with anti-p18 (red) and -GFP (green) antibodies. Scale bar = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity (MFI) of p18-immunoreactive puncta in GFP-positive CA1 pyramidal neurons. N = 6 mice, p<0.001, WT-siScrambled vs. WT-siP18; p<0.001, WT-siScrambled vs. AS-siScrambled; p<0.001, AS-siScrambled vs. AS-siP18; p=0.043, WT-siP18 vs. AS-siP18, two-way ANOVA with Tukey’s post-test. (D, E) Effects of AAV siRNA-mediated p18 KD on LTP in WT and AS mice. (D) Slopes of fEPSPs were normalized to the average values recorded during the 10 min baseline. (E) Means ± SEM of fEPSPs measured 40 min after TBS in different groups. N = 7–14 slices from four to eight mice, p=0.005, WT-siScrambled vs. WT-siP18, p<0.001, WT-siScrambled vs. AS-siScrambled, p=0.001, AS-siScrambled vs. AS-siP18, p=0.305, WT- siScrambled vs. AS-siP18, two-way ANOVA with Tukey’s post-test. Inset shows representative traces of evoked fEPSPs before and 40 min after TBS. Scale bar 0.5 mV/10 ms. See also Figure 6—figure supplements 1 and 2 and Figure 6—source data 1.

Figure 6—source data 1. Source data for Figure 6 and Figure 6—figure supplement 1.
elife-37993-fig6-data1.xlsx (29.7KB, xlsx)
DOI: 10.7554/eLife.37993.021

Figure 6.

Figure 6—figure supplement 1. Effects of p18 knockdown in hippocampal CA1 region on mTOR signaling in WT and AS mice.

Figure 6—figure supplement 1.

(A) Quantitative analysis of the number of p18- (left, p=0.001) and PSD95-immunoreactive puncta (right, p=0.929), as well as percentage of p18 and PSD95 dually stained puncta/synapses (middle, p=0.004) in hippocampal CA1 region. N = 6 mice, unpaired t-test. These data are paired with Figure 6A. (B) The coordinates of the injection sites were (mm): AP −1.94, ML ±1.4, DV −1.35 from Bregma; AP −2.2, ML ±1.8, DV −1.5 from Bregma, in the CA1 region of hippocampus and are indicated by red circles. (C) Representative tile scan confocal image of GFP expression in hippocampal CA1 region 4 weeks following injection of AAV with GFP reporter gene. Scale bar = 200 μm. (D) Representative images of Western blots labeled with Ube3a, p18, p-mTOR, mTOR, p-S6K1, p-S6, S6, and PKCα (GAPDH as a loading control). Protein lysates from hippocampal CA1 region infected with the indicated AAV were prepared for Western blot analysis. (E) Effects of p18 knockdown in hippocampal CA1 region on mTOR signaling in WT and AS mice. For p-mTOR, p=0.010, WT-siScrambled vs. WT-siP18, p=0.002, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; For p-S6, p<0.001, WT-siScrambled vs. WT-siP18, p=0.002, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; For PKC, p=0.012, WT-siScrambled vs. WT-siP18, p=0.001, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; n = 4 mice for WT-siScrambled, WT-siP18, and AS-siScrambled, n = 3 mice for AS-siP18, two-way ANOVA with Tukey’s post-test.
Figure 6—figure supplement 2. Effects of p18 knockdown in hippocampal CA1 region on input/output curves and paired-pulse facilitation in WT and AS mice.

Figure 6—figure supplement 2.

Related to Figure 6. (A–C) Input/output curves. Amplitudes of field EPSPs (A) and the slope of the field EPSP (B) were determined for various intensities of stimulation. (C) Relationship between the slope of the evoked fEPSPs and the corresponding fiber volley amplitude. The results are means ± SEM; n = 3 mice; there were no significant differences among the four groups of mice. (D) Paired-pulse facilitation. The amplitude of the second response of a paired-pulse was calculated as a percentage of the amplitude of the first response for various inter-pulse intervals. The results are means ± SEM; n = 5 mice; there were no significant differences among the four groups of mice.