1. Volume 4, Issue 3, Pages 405-412 (August 2013)
Genetic Reduction of the α1 Subunit of Na/K-ATPase Corrects Multiple Hippocampal Phenotypes in Angelman Syndrome 
Hanoch Kaphzan, Shelly A. Buffington, Akila B. Ramaraj, Jerry B. Lingrel, Matthew N. Rasband, Emanuela Santini, Eric Klann 
Cell Reports 
Volume 4, Issue 3, Pages (August 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 405-412DOI: (10.1016/j.celrep.2013.07.005)
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3. Figure 1
Genetic Reduction of α1-NaKA Corrects Increased Expression of Ankyrin-G and NaV1.6 in AS Model Mice
Western blot analysis of hippocampal homogenates from wild-type (WT), Angelman syndrome (AS), α1-NaKA heterozygous knockout (α1-NaKA), and AS/α1-NaKA heterozygous knockout (dKO) mice.
(A) Representative blots of ankyrin-G (480 kDa), NaV1.6, α1-NaKA, and actin.
(B) Cumulative data for the blots probed for α1-NaKA and actin.
(C) Cumulative data for the blots probed with antibodies to ankyrin-G and actin.
(D) Cumulative data for the blots probed for NaV1.6 and actin.
For all panels, actin was used as a loading control and quantification was done as a ratio to wild-type on the same blot (WT: n = 6; AS: n = 7; α1-NaKA: n = 7; dKO: n = 7). Asterisks (∗p < 0.05 and ∗∗p < 0.01) denote statistical significance from wild-type (ANOVA followed by Tukey’s post-hoc test). Results are displayed as mean ± SEM.
Cell Reports 2013 4, DOI: ( /j.celrep )
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4. Figure 2
AIS Length in AS Model Mice Is Restored to Wild-Type Length in Hippocampal CA1 and CA3 Pyramidal Neurons by Genetically Reducing α1-NaKA Levels
(A–C) Immunostaining analysis of ankyrin-G expression and AIS length in hippocampal and cortical tissue from wild-type (WT), Angelman syndrome (AS), α1-NaKA heterozygous knockout (α1-NaKA), and AS/α1-NaKA heterozygous knockout (dKO) mice. Coronal brain slices containing the hippocampal formation and somatosensory cortex were immunostained with antibodies targeted against ankyrin-G. AIS length was quantified in CA1 (A), CA3 (B), and layer II/III somatosensory cortex (C) in tissue from each of the four genotypes. WT: n = 240 cells per region, 2 mice; AS: n = 239 cells per region, 2 mice; α1-NaKA: n = 240 cells per region, 2 mice; dKO: n = 239 cells per region, 2 mice. Average, region-specific AIS length data are provided in box and whisker plots. ∗∗∗p < (ANOVA). Fluorescence intensity (F.I.) plots provide a comparison of representative AIS ankyrin-G immunosignal strength and AIS length of cells contained in the expanded views, shown to the right of the contextual image.
Cell Reports 2013 4, DOI: ( /j.celrep )
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5. Figure 3
Genetic Reduction of α1-NaKA Corrected Alterations in the Intrinsic Membrane Properties of Hippocampal CA1 Pyramidal Neurons in AS Model Mice
(A–H) Intrinsic membrane properties of hippocampal CA1 pyramidal neurons in WT, AS, α1-NaKA heterozygous knockout (α1-NaKA), and dKO mice measured by current clamp without current injection (I = 0).
(A) Resting membrane potential.
(B) Membrane time constant.
(C) Membrane input resistance.
(D) Threshold potential.
(E) Action potential amplitude.
(F) Action potential full width at half-maximum.
(G) Maximal rate of rise (dV/dt) of the action potential.
(H) Rheobase (amount of current injection needed to induce an action potential at the 5 ms time point).
WT: n = 28 cells, 8 mice; AS: n = 28 cells, 8 mice; α1-NaKA: n = 24 cells, 7 mice; dKO: n = 27 cells, 8 mice. ∗∗∗p < (ANOVA). Results are displayed as mean ± SEM. See also Figure S1.
Cell Reports 2013 4, DOI: ( /j.celrep )
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6. Figure 4
Genetic Reduction of α1-NaKA Corrects Deficits in Hippocampal LTP, Contextual Fear Memory, and Spatial Memory Displayed by AS Mice
(A) Hippocampal slices from WT, AS, α1-NaKA heterozygous knockout mice (α1-NaKA) and dKO mice were stimulated with two trains of high-frequency stimulation (HFS, indicated by the arrows). WT: n = 10 slices, 5 mice; AS: n = 10 slices, 5 mice; α1-NaKA: n = 10 slices, 5 mice; dKO: n = 16 slices, 8 mice. p < 0.05 for group, p < for time, and p < for interaction (RM-ANOVA).
(B) Sample traces of typical field excitatory postsynaptic potentials (fEPSPs) recorded before (black) and 60 min after (red) HFS.
(C) Mice were trained using a standard contextual fear conditioning paradigm and tested for long-term memory measured as % time freezing seven days after training. WT: n = 12 mice; AS: n = 11 mice; α1-NaKA: n = 11 mice; dKO: n = 11 mice. Results are displayed as the average of percent freezing during the entire 5 min test. ∗∗∗p < (ANOVA followed by Tukey’s post-hoc test).
(D) Results are displayed as percent freezing along the entire test, minute by minute. ∗∗∗p < for interaction of group and treatment (RM-ANOVA).
(E) Mice were trained using a standard Morris water maze paradigm and tested for spatial memory of platform location in the probe test (platform removed). n = 8 for WT, AS, and dKO; n = 5 for α1-NaKA. Results are displayed as percentage of time spent in each quadrant along the entire probe test (quadrant time occupancy). p < 0.05 for interaction of genotype and quadrant (two-way ANOVA); ∗∗p < 0.01 for the trained quadrant (Bonferroni post-hoc test).
(F) Results are displayed as a number of platform location crosses during the probe test. ∗∗p < 0.01 (ANOVA followed by Tukey’s post-hoc test).
Results are displayed as mean ± SEM. See also Figures S2 and S3.
Cell Reports 2013 4, DOI: ( /j.celrep )
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7. Figure S1
Action Potentials from Hippocampal CA1 Pyramidal Neurons, Related to Figure 3
(A and B) Sample traces of action potentials and their first derivative from hippocampal CA1 pyramidal neurons in AS (A) and dKO (B) mice. The deflection point for threshold was set as the rate of rise at 30V/s. The action potential waveform is shown in blue for AS mice and in black for dKO mice, and the first derivative of the action potential is shown in green for AS mice and in red for dKO mice. The deflection point is marked by the yellow dot.
(C) The two traces (AS mice and dKO mice) and their first derivatives are superimposed for comparison.
(D) Sample traces of action potentials in response to injection of 10pA depolarizing current steps in current clamp. Active intrinsic properties were taken from the action potential with the closest peak to 5ms from start of injection.
Cell Reports 2013 4, DOI: ( /j.celrep )
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8. Figure S2
Paired-Pulse Facilitation, Acquisition of Contextual Fear Memory, and Spatial Learning in the Morris Water Maze Task, Related to Figure 4
(A) Paired-pulse facilitation PPF before and after LTP-inducing HFS in hippocampal slices from dKO mice is unchanged. n = 16 slices, 8 mice.
(B) Differences in contextual fear memory between the four genotypes are not due to alterations in memory acquisition. % freezing during acquisition was measured at the following time intervals: 1st = pre-US (2 min), 2nd = 1US (1 min), 3rd = 2US (1 min) and 4th = post-US (1 min). The learning acquisition curves were similar between all four genotypes.
(C) Impaired spatial learning of the Morris water maze task by the AS mice is rescued by decreasing the expression of α1-NaKA. Escape latencies to reach the platform in the Morris water maze during the training sessions show differences between the mice. Results are the escape latencies of the 4th trial in each training session of each day. p < 0.01 for genotype in 2-way-RM-ANOVA. ∗ denotes statistical significance p < 0.05 and ∗∗ denotes statistical significance p < 0.01 in 2-way-ANOVA for the specific time points.
Results are displayed as mean ± SEM.
Cell Reports 2013 4, DOI: ( /j.celrep )
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9. Figure S3
Visible Morris Water Maze, Swim Speed in the Water Maze, and Open Field Tests, Related to Figure 4
Hippocampus-dependent memory deficits in AS mice are not due to sensory, motor, or emotional (anxiety) impairments, and decreasing the expression of α1-NaKA does not induce such deficits.
(A) There are no differences in the visible Morris water maze task among the four genotypes. n = 8 for WT, AS, and dKO mice; n = 5 for α1-NaKA mice.
(B) There are no differences between the four genotypes in the swim speed in the hidden platform probe trials of the Morris water maze. n = 8 for WT, AS, and dKO mice; n = 5 for α1-NaKA mice.
(C–E) There are no differences between the four genotypes in the various parameters of the open field test. n = 8 for WT, AS, and dKO mice; n = 5 for α1-NaKA mice. These results suggest that there are no differences between the four genotypes that are related to either motor impairments (C and D) or anxiety (E).
(F) There are no differences in the context-independent novel object recognition test. n = 8 for WT, AS, and dKO mice; n = 5 for α1-NaKA mice.
Results are displayed as mean ± SEM.
Cell Reports 2013 4, DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions