1. Volume 150, Issue 5, Pages 1055-1067 (August 2012)
Role of Leaky Neuronal Ryanodine Receptors in Stress- Induced Cognitive Dysfunction 
Xiaoping Liu, Matthew J. Betzenhauser, Steve Reiken, Albano C. Meli, Wenjun Xie, Bi-Xing Chen, Ottavio Arancio, Andrew R. Marks 
Cell 
Volume 150, Issue 5, Pages (August 2012)
DOI: /j.cell
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Chronic Stress Response in WT and in the RyR2-S2808A+/+ Mice
(A) Mice were randomly divided into groups as indicated. Stressed mice were subjected to chronic restraint stress in Plexiglas restraint tubes overnight daily for 3 weeks. S107 (75 mg/kg/day) in drinking water was begun 3 days before stress and throughout the experiment.
(B) Plasma corticosterone levels were assayed in the morning. Data are mean ±SEM, n = 4.
(C) Representative immunoblots showing pCREB and pERK in control and stressed mice. Hippocampal samples were isolated immediately after 3 weeks of stress from WT and RyR2-S2808A+/+ mice.
(D and E) Summary data (mean ±SEM, n = 5) are shown for pCREB (C) and pERK (D). Total CREB, total ERK, and GAPDH are loading controls.
See also Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 2
Stress-Induced Remodeling of Neuronal RyR2 Macromolecular Complexes
(A) Hippocampal RyR2 was immunoprecipitated and immunoblotted to detect PKA hyperphosphorylation, oxidation, and Cys S-nitrosylation of RyR2 and calstabin2 as previously described (Marx et al., 2000; Reiken et al., 2003b; Shan et al., 2010a, 2010b; Ward et al., 2003).
(B–E) Summary data for RyR2-pS2808, Cys S-nitrosylation, DNP (oxidation), and calstabin2. Data are mean ±SEM, ∗p < 0.05, #p < 0.05, n = 5. The mean for the unstressed control was set = 1.0.
(F) ER microsomes were resuspended and treated with PKA (40 units), H2O2 (1 mM), and Noc-12 (100 μM) separately or in combination. Treated microsomes were immunoprecipitated and immunoblotted with the antibodies described in (A)–(E).
(G) Summary data for F; immunoblot data for calstabin2/RyR2.
(H) Kds for 35S-calstabin2 binding to RyR2.
Data are mean ± SEM, ∗p < 0.05, #p < 0.05, ##p < 0.01, n = 5. See also Figures S3, S4, and S5.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3
Single-Channel Recordings of Hippocampal RyR Channels from Stressed Mice
(A–E) Representative hippocampal RyR single-channel current traces from control (WT, n = 6) (A), stress (WTStr, n = 5) (B), S107-treated stress (WTStr+S107, n = 5) (C), RyR2-S2808A+/+ (S2808A, n = 4) (D), and stressed RyR2-S2808A+/+ mice (S2808AStr, n = 6) (E) examined with 150 nmol/l (nM) free cytosolic [Ca2+] in the cis chamber. Channel openings are upward deflections, and horizontal bars at the left of each trace indicate the closed (c-) state of the channels. For each group, channel activity is illustrated with four traces, each 5 s. RyR identity was confirmed by addition of 5 μmol/l (μM) ryanodine at the end of each experiment. The single-channel open probability (Po), mean open time (To), and mean closed time (Tc) at 150 nmol/l free cytosolic [Ca2+] are above the upper trace.
(F) Summary of RyR2 single-channel Po with 150 nmol/l free cytosolic [Ca2+] from RyR2-WT, RyR2-WTStr, WTStr+S107, S2808A, and S2808AStr. Data are mean ±SEM, ∗p < 0.05 versus WT.
(G) Representative traces of Ca2+ leak from brain microsomes induced by addition of thapsigargin (3 μM).
(H) Ca2+ leak was calculated as the percentage of uptake. Data (mean ±SEM) analysis was performed by one-way ANOVA, p = Bonferroni posttest revealed ∗p < 0.05 versus WTStr (WT, n = 9; WTStr, n = 7; WTStr+S107, n = 7; S2808A, n = 4; S2808A+ST, n = 4).
See also Figure S3H.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4
Effects of Stress on Cognitive Function and Hippocampal Synaptic Plasticity
(A) MWM escape latency for WT nonstressed control (WT, n = 21), S107-treated WT nonstressed (WT+S107, n = 14), WT stressed (WTStr, n = 21), and S107-treated stressed (WTStr+S107, n = 22).
(B and C) Probe trials measuring time spent in the target quadrant and number of target crossings. p < 0.05.
(D and E) EPM test on the same groups of mice. (D) Summary of ratios of time spent on the open arm versus closed arm is shown. (E) Summary data of ratios of number of entries to the open arm versus closed arm are shown.
(F) Open-field test from the same group of mice as shown in MWM and EPM tests. ∗p < 0.05 versus nonstressed controls within the first 3 min, #p < 0.05 versus nonstressed controls within the second 3 min.
(G) Potentiation following theta burst stimulation in the CA1 region of hippocampal slices from WT mice (WT, n = 7), S107-treated control (WT+S107, n = 7), stress (WTStr, n = 7), and S107-treated stress (WTStr+S107, n = 7).
(H) Summary of field input-output relationships in the same slices as in (G).
All data are mean ±SEM. See also Figure S3G.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5
Effects of Stress on Cognitive Function and Hippocampal Synaptic Plasticity in RyR2-S2808A+/+ Mice
(A) MWM escape latency for WT (WT, n = 15), RyR2-S2808A+/+ nonstressed (S2808A, n = 13), and RyR2-S2808A+/+ stressed (S2808AStr, n = 16).
(B and C) Probe trials after escape platform removed, ∗p < 0.05.
(D and E) EPM test in the same groups. (D) Summary data of ratios of time spent on the open arm versus closed arm. (E) Summary data of ratios of number of entries to the open arm versus closed arm.
(F) Open-field test from the same group of mice. Ratios of total time spent in the center area versus periphery area within first 3 min and second 3 min.
(G) Potentiation following theta burst stimulation in the CA1 region of hippocampal slices from WT mice (WT, n = 7), RyR2-S2808A+/+ (S2808A, n = 7), and stressed RyR2-S2808A+/+ (S2808AStr, n = 7).
(H) Field input-output relationships in the same slices as in (G).
See also Figures S3G, S4, S5, S6, and S7. All data are mean ±SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6
Stress-Induced Remodeling of RyR1 Complexes and Cognitive Function in RyR1-S2844A+/+ Mice
(A) RyR2-S2844A+/+ mice were subjected to the restraint stress protocol in Figure 1A. Morning plasma corticosterone levels collected (n = 4).
(B–D) (B) Representative immunoblots showing levels of hippocampal pCREB and pERK in control and stressed mice. Summary data for the levels of pCREB (C) and pERK (D). Total CREB, total ERK, and GAPDH were loading controls.
(E) Representative immunoblots of RyR1 immunoprecipitated from whole-brain samples from control (WT), WT stressed (WTStr), RyR1-S2844A+/+ (S2844A), and RyR1-S2844A+/+ stressed (S2844AStr) probed with antibodies showing PKA hyperphosphorylation, oxidation (DNP), Cys S-nitrosylation (CysNO) of RyR1, and coimmunoprecipitated calstabin1.
(F–I) Summary data for phosphorylated RyR1, oxidized and nitrosylated RyR1, and calstabin1. ∗p < 0.05, n = 4. The mean for the unstressed WT control was taken as 1.0.
All data are mean ±SEM. See also Figures S5E–S5G.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

9. Figure 7
Effects of Stress on Cognitive Function in RyR1-S2844A+/+ Mice
(A) Mice were divided into WT (WT, n = 30), stressed WT (WTStr, n = 15), RyR1-S2844A+/+ (S2844A, n = 30), and stressed RyR1-S2844A+/+ (S2844AStr, n = 12). Learning curves of MWM tasks are presented as the time to reach the platform (escape latency). Mice were trained for 5 days, three trials each day, 60 s per trial.
(B and C) On day 6 after 5 day training trials, the escape platform was removed, and each mouse was allowed to swim in the pool for 60 s. Total time spent in the target quadrant and the number of target crossings were recorded. The escape latency and the ratios of swimming time spent in the target quadrant were recorded and analyzed as mean ±SEM, ∗p < The results were analyzed using one-way ANOVA and comparison t test.
(D and E) Results of EPM test from the same groups of mice as shown in MWM test. (D) Summary data of ratios of time spent on the open arm versus closed arm are shown. (E) Summary data of ratios of number of entries to the open arm versus closed arm are shown. The results of EPM were analyzed using two-way ANOVA and comparison t test.
(F) Results of open-field test from the same group of mice as shown in MWM and EPM tests. Ratios of total time spent in the center area versus periphery area within first 3 min and second 3 min are shown. Data were analyzed by one-way ANOVA and comparison t test.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

10. Figure S1
Poststress Time Course of HPA Axis Activity and c-Fos Expression following Stress, Related to Figure 1
(A–C) WT and RyR2-S2808A+/+ mice were subjected to the same chronic restraint stress protocol as shown in Figure 1A. S107 (75 mg/Kg/day) was delivered through drinking water 3 days before stress. Mice were treated with drug throughout the entire experimental period. Post-stress plasma CRF (by ELISA) (A), ACTH (by ELISA) (B) and corticosterone (by EIA) (C) levels were measured at different time points as indicated in the figure. One group of animals was subjected to a single episode of 2 hr restraint indicated as acute stress. Another group of animals was subjected to one episode of 2 hr restraint 24 hr after the last episode of 3-week chronic restraint stress indicated as chronic plus acute stress. Data were analyzed by mean ± S.E.M., n = 4.
(D) Representative immunoblots of c-Fos in cortical samples from WT and RyR2-S2808A+/+ mice: non-stressed control (control), single episode of 2 hr restraint stress (Acute), 3-week restraint stress (Chronic), and single episode of 2 hr restraint 24 hr after the last episode of 3-week restraint stress (Chronic+Acute).
(E and F) Quantification. Mean ±SEM, n = 4.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

11. Figure S2
Levels of pCREB and pERK in Cortical and Whole Brain from Stressed WT and RyR2-S2808A+/+ Mice and Hippocampal PKA Activity, Related to Figure 1
(A–F) Representative immunoblots of pCREB and pERK in the cortex (A) and whole brain (B) from control and stressed mice. Mice were subjected to chronic restraint stress protocol as shown in Figure 1A for 3 weeks. S107 (75 mg/kg/day) was given in the drinking water 3 days before stress. Tissues were removed immediately after 3 weeks of stress from WT and RyR2-S2808A+/+ mice and lysed. Quantification (mean ±SEM, n = 3) of pCREB and pERK in cortex (C and D) and in whole brain (E and F). Loading controls were total CREB, total ERK, and GAPDH.
(G) Protein kinase A (PKA) activity in hippocampal lysates (100 μg). Quantification of 32P incorporation into the substrate quantified by filter binding (mean ±SEM, n = 4). ∗p < 0.05.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

12. Figure S3
Stress-Induced Remodeling of RyR2 from Whole Brain, SERCA Activity, Swimming Speed, and RyR Single-Channel Recordings from Whole-Brain Microsomes, Related to Figures 2, 3, 4, 5, and 7
(A) RyR2 was immunoprecipitated from whole brain and immunoblotted to detect PKA hyperphosphorylation, oxidation and Cys S-nitrosylation of RyR2 as previously described (Marx et al., 2000; Reiken et al., 2003; Shan et al., 2010a, 2010b; Ward et al., 2003).
(B–E) Quantification of RyR2-pS2808, Cys S-nitrosylation, DNP (oxidation), and calstabin2. Data are mean ± S.E.M., ∗, ∗∗, # p < 0.05, n = 3. The mean for the unstressed control was set = 1.0.
(F) Related to Figure 2: SERCA2a activity was unchanged by stress in WT mice.
(G) Related to Figures 4, 5, and 7: swimming speed. On day-6 probe trial in the Morris water maze (MWM) tests, swimming speeds were recorded and analyzed. Data are mean ± SEM for WT non-stressed (WT, n = 21), S107 treated WT non-stress (WT+S107, n = 14), WT stressed (WTStr, n = 21), and S107 treated stress (WTStr+S107, n = 22), RyR2-S2808A+/+ (S2808A, n = 13), stressed RyR2-S2808A+/+ (S2808AStr, n = 16), RyR2-S2844A+/+ (S2844A, n = 30), stressed RyR2-S2844A+/+ (S2844AStr, n = 12).
(H) Related to Figure 3: RyR single-channel recordings from whole brain microsomes. Representative RyR2 single-channel current traces are shown from RyR2-WT, RyR2-WTStr, and WTStr+S107 mice, examined with 150 nmol/l (nM) free cytosolic [Ca2+] in the cis chamber. Channel openings are upward deflections; the closed (c) state of the channel is indicated by horizontal bars at the beginning of each tracing. For each group, channel activity is illustrated by 4 different traces, each of 5 s in length as indicated by dimension bars. RyR identity was confirmed by addition of 5 μmol/l (μM) ryanodine at the end of each experiment. The single channel open probability (Po), To (mean open time) and Tc (mean closed time) of the 2 min recording with 150 nmol/l free cytosolic [Ca2+] are shown above the upper trace. Bar graph summarizes RyR2 single-channel Po with 150 nmol/l free cytosolic [Ca2+] from RyR2-WT (n = 5), RyR2-WTStr (n = 4) and WTStr+S107 treated (n = 3) samples.
Data are mean ±SEM, ∗p < 0.05 versus WT.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

13. Figure S4
Effects of S36 and of Administering S107 after Stress Protocol, Related to Figures 2, 4, and 5
Mice were subjected to the 3 week restraint stress protocol in Figure 1A. S36 (75 mg/kg/day) was delivered in the drinking water.
(A) RyR2 was immunoprecipitated from whole brain samples and immunoblotted to detect PKA hyperphosphorylation, oxidation and Cys S-nitrosylation of RyR2 as described above.
(B–E) Bar graphs show RyR2-pS2808, Cys S-nitrosylation, DNP (oxidation), and calstabin2. Data are mean ± S.E.M., ∗, ∗∗, # p < 0.05, n = 3. The mean for the unstressed control was set = 1.0.
(F) Learning curves of MWM tasks in WT mice for the time to reach the platform defined as the “escape latency.” Mice were trained for 5 days, 3 trials each day, 60 s per trial. Mice were divided into WT non-stressed control (WT, n = 10), WT stress (WTStr, n = 12), and S36 treated stress (WTStr+S36, n = 12).
(G and H) Probe trials of MWM tasks are presented as time spent in the target quadrant and number of target crossings. After 5-day training trials, on day 6, the escape platform was removed, each mouse was allowed to swim in the pool for 60 s. Total time spent in the target quadrant and the number of target crossings were recorded. The escape latency and the ratios of swimming time spent in the target quadrant and the number of target crossings were recorded and analyzed as mean ± S.E.M., ∗p < The results were analyzed using one-way ANOVA and comparison t test.
(I) Related to Figure 4: Administering S107 after stress protocol showed no improvement in learning and memory. Male, 3-month old WT mice were subjected to 3-week restraint stress protocol in Figure 1A. S107 (75 mg/kg/day) was administered through drinking water after 3-week restraint, from the same day of MWM test and throughout the 6-day testing period. Learning curves of MWM tasks in WT mice are presented as the time to reach the platform defined as “escape latency.” Mice were trained for 5 days, 3 trials each day, 60 s per trial. Mice were divided into WT non-stressed control (WT, n = 15), WT stress (WTStr, n = 15), and S107 treated stress [WTStr+S107 (late), n = 15].
(J and K) Probe trials of MWM tasks presented as time spent in the target quadrant and number of target crossings. After 5-day training trials, on day 6, the escape platform was removed, each mouse was allowed to swim in the pool for 60 s. Total time spent in the target quadrant and the numbers of target crossings were recorded. The escape latency and the ratios of swimming time spent in the target quadrant and the number of target crossings were recorded and analyzed as mean ± SEM. The results were analyzed using one-way ANOVA and comparison t test.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

14. Figure S5
Calstabin Binding Assay and Stress-Induced Remodeling of RyR1 and IP3R1 in Whole Brain, Related to Figures 2 and 6
(A and B) 35S-calstabin binding to RyR2 in samples treated with PKA, H2O2, Noc-12, or in combination. Radioactive counts were normalized to the untreated control samples.
(C) Co-immunoprecipitation with calstabin and IgG. IP with anti-calstabin antibody and IgG from brain microsomes of WT mice, stressed, with and without S107 treatment.
(D) Total RyR and calstabin. Whole brain lysates were prepared from WT, stressed WT, stressed WT+S107, RyR2-S2808A+/+ and stressed RyR2-S2808A+/+ mice. Lysates (50 mg) were separated by SDS-PAGE and immunoblotted using antibodies against RyR or calstabin.
(E) related to Figure 6: Representative immunoblots of immunoprecipitated RyR1 from whole brain samples showing PKA hyperphosphorylation of RyR1.
(F and G) Quantification of pRyR1, and calstabin1. Mean ± S.E.M., ∗, ∗∗, # p < 0.05, n = 4. The mean for the unstressed control was set as 1.0.
(H) Representative immunoblot of immunoprecipitated IP3R1 from whole brain from stressed (WTStr, n = 4) and non-stressed (WT, n = 4) mice showing S-nitrosylation of IP3R1.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

15. Figure S6 Novel Object Recognition, Related to Figures 4 and 5
(A) Exploration duration (T) of two-identical objects (T1 and T2) recorded during 5 min exposure period of phase-1.
(B) Exploration duration (T) of the constant object (T1) and the novel object (TN) recorded during 5 min exposure period of phase-2. Discrimination index [DI = TN/(T1+TN)] after 1 hr interval. Mean ± S.E.M., ∗, ∗∗p < Experimental groups include WT non-stressed (WT, n = 12), S107 treated WT non-stress (WT+S107, n = 12), WT stressed (WTStr, n = 11), S107 treated stress (WTStr+S107, n = 9), RyR2-S2808A+/+ (S2808A, n = 12), and stressed RyR2-S2808A+/+ (S2808AStr, n = 13).
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions

16. Figure S7 Elevated Plus Maze, Related to Figures 4 and 5
(A–H) Results of EPM test from the same groups of mice as shown in Figures 4 and 5. Summary data of total time-spent on the closed-arm (A) and open-arm (B) for WT (n=21), S107 treated WT (WT+S107, n=14), stressed WT (WTStr, n=21) and S107 treated stressed WT (WTStr+S107, n=22) are shown. Summary data of total number of closed-arm entries (C) and open-arm entries (D) for WT, S107 treated WT (WT+S107), stressed WT (WTStr) and S107 treated stressed WT (WTStr+S107) are shown. Summary data of total time-spent on the closed-arm (E) and open-arm (F) for WT (n=15), non-stressed RyR2-S2808A+/+ (S2808A, n=13), and stressed RyR2-S2808A+/+ (S2808AStr, n=16) are shown. Summary data of total number of closed-arm entries (G) and open-arm entries (H) for WT, non-stressed RyR2-S2808A+/+ (S2808A), and stressed RyR2-S2808A+/+ (S2808AStr) are shown. The results of EPM were analyzed using two-way ANOVA and comparison t-test, ∗p < All data are mean ± SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2012 Elsevier Inc. Terms and Conditions