Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: CaMKIIβ activation in the vCA1 drives stress resilience and has antidepressant-like effect (A) AAV vectors engineered to overexpress a control construct (AAV-GFP), CaMKIIβ (AAV-CaMKIIβ), or a constitutively active form of CaMKIIβ (AAV-CaMKIIβ-CA). ITR, inverted terminal repeats; P Camk2a , Camk2a promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) Experimental timeline for chronic stress and behavioral testing. CUMS, chronic ultra-mild stress; SIT, social interaction test; SPT, sucrose preference test; FST, forced swim test. (C) Schematic representation showing the microinjection of AAV into the vCA1 region of mice. Region-specific expression of GFP in vCA1 but not dCA1 is shown. Scale bar, 500 μm. (D–F) BALB mice overexpressing CaMKIIβ or CaMKIIβ-CA show significantly increased social interaction time in the SIT (D), increased sucrose preference in the SPT (E), and decreased immobility time in the FST (F) under stress. n = 12–13 mice per group. (G) CaMKIIβ-CA overexpression prevents CUMS-induced reduction of Fos expression in the vCA1 of BALB mice. n = 6 mice per group. (H) AAV-mediated spatiotemporal gene expression strategy using a cocktail of AAV- Camk2a -tTA and AAV-TRE-CaMKIIβ-CA or AAV-TRE-GFP. (I) Experimental timeline for CaMKIIβ-CA overexpression after the termination of CUMS episodes. BALB mice were treated with doxycycline (dox) after AAV injection and exposed 21 days later to CUMS for 6 weeks; they were then taken off dox, and 4 days later behavioral tests were performed. (J) AAV microinjection into the vCA1 region. Region-specific and dox-regulated expression of GFP in the vCA1 is shown. Scale bar, 200 μm. (K–M) CaMKIIβ activation after CUMS leads to a significant increase in social interaction time in the SIT (K), a tendency for increased sucrose preference in the SPT (L), and reduced immobility time in the FST (M). n = 15–16 mice per group. One-way ANOVA followed by a Tukey's post hoc test (in D-G) and two-tailed Student's t-test (in K-M) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. See also Figure S2 . Complete statistical summaries are provided in .

Article Snippet: Free-floating sections were incubated with blocking solution (3% normal goat serum, 3% bovine serum albumin, and 0.3% TritonX-100 in PBS) for 1 h. Primary antibodies against Fos (1:500, Cell Signaling Technology, #2250), GFP (1:1000, Thermo Fisher Scientific, #A11122), and mCherry (1:1000, Takara Bio Clontech, #632543) were diluted in blocking solution and sections were incubated overnight at 4°C with gentle shaking.

Techniques: Activation Assay, Construct, Expressing, Over Expression, Injection, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: CaMKIIβ inhibition in the vCA1 increases stress susceptibility (A) AAV vectors used for the control construct (AAV-shControl) and CaMKIIβ knockdown (AAV-shCaMKIIβ). ITR, inverted terminal repeats; P CMV , cytomegalovirus promoter; P Camk2a , Camk2a promoter; U6, U6 promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) Experimental paradigm for behavioral testing. CUMS, chronic ultra-mild stress; SIT, social interaction test; SPT, sucrose preference test; FST, forced swim test. (C) AAV microinjection into the vCA1. Region-specific expression of GFP in vCA1 is shown. Scale bar, 100 μm. (D) Specific knockdown of CaMKIIβ but not CaMKIIα by the AAV-shCaMKIIβ. (E–G) CaMKIIβ knockdown in the vCA1 of B6 mice leads to significantly decreased social interaction time in the SIT (E), decreased sucrose preference in SPT (F), and increased immobility time in FST (G) following CUMS exposure. n = 12–15 mice per group. (H) AAV vectors engineered to overexpress a constitutively active form of CaMKIIβ (CaMKIIβ-CA) with resistance against shCaMKIIβ (AAV-CaMKIIβ-CA Res ) or a control construct (AAV-mCherry). (I) Experimental paradigm for behavioral testing. (J) AAV microinjection into the vCA1. Region-specific expression of GFP and mCherry in the vCA1 is shown. Scale bar, 100 μm. (K and L) Overexpression of CaMKIIβ-CA Res together with shCaMKIIβ increases time in the interaction zone in the SIT (K) and tends to increase sucrose preference in the SPT (L) in B6 mice, when compared to the mice overexpressing mCherry together with shCaMKIIβ. n = 14–15 mice per group. (M) Experimental paradigm for behavioral testing. smSDS, subchronic and mild social defeat stress. (N) Decreased time in the interaction zone in B6 mice given AAV-shCaMKIIβ following smSDS exposure is prevented by overexpression of CaMKIIβ-CA Res . n = 15–16 mice per group. (O) AAV vectors engineered to overexpress a dominant-negative form of CaMKIIβ (AAV-CaMKIIβ-DN) or a control construct (AAV-GFP). (P) Experimental paradigm for behavioral testing. (Q) AAV microinjection into the vCA1. Region-specific expression of GFP in vCA1 is shown. Scale bar, 100 μm. (R–S) Overexpression of CaMKIIβ-DN decreases time in the interaction zone in the SIT (R) and sucrose preference in the SPT (S) in B6 mice following smSDS. n = 13–15 mice per group. (T) Overexpression of CaMKIIβ-DN decreases Fos levels in the vCA1 of B6 mice following smSDS exposure. n = 6 for all groups. Two-way ANOVA followed by a Tukey's post hoc test (in E-G, N, R, and S), one-way ANOVA followed by a Tukey's post hoc test (in N), and two-tailed Student's t-test (in K, L, and T) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. See also Figure S3 . Complete statistical summaries are provided in .

Article Snippet: Free-floating sections were incubated with blocking solution (3% normal goat serum, 3% bovine serum albumin, and 0.3% TritonX-100 in PBS) for 1 h. Primary antibodies against Fos (1:500, Cell Signaling Technology, #2250), GFP (1:1000, Thermo Fisher Scientific, #A11122), and mCherry (1:1000, Takara Bio Clontech, #632543) were diluted in blocking solution and sections were incubated overnight at 4°C with gentle shaking.

Techniques: Inhibition, Construct, Expressing, Over Expression, Dominant Negative Mutation, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet:

Article Snippet: Free-floating sections were incubated with blocking solution (3% normal goat serum, 3% bovine serum albumin, and 0.3% TritonX-100 in PBS) for 1 h. Primary antibodies against Fos (1:500, Cell Signaling Technology, #2250), GFP (1:1000, Thermo Fisher Scientific, #A11122), and mCherry (1:1000, Takara Bio Clontech, #632543) were diluted in blocking solution and sections were incubated overnight at 4°C with gentle shaking.

Techniques: Produced, Plasmid Preparation, Recombinant, SYBR Green Assay, Multiplex Assay, Mutagenesis, Staining, Western Blot, Real-time Polymerase Chain Reaction, Software