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Results: 7

1.
Fig. 7

Fig. 7. A-443654 selectively enhances LTM in WT mice but not in mTORC2-deficient mice. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a) Diagram of the experimental protocol. b) A single A-443654-injection (2.5 mg/kg i.p.) immediately after a weak training (a single pairing of a tone with a 1s, 0.7 mA foot-shock) enhanced contextual fear LTM in WT mice (n=18 per group; F(1, 34)=6.007, *p<0.05) but not in rictor fb-KO mice (c, n=10 per group; F(1, 18)=1.648, p=0.22). d) Similar freezing at 2 hr reflects normal contextual fear STM in vehicle-injected and A-443654-injected WT mice (n=9 for vehicle and n=10 for A-443654; F(1, 17)=0.6, p=0.449). Freezing was assessed 2hr (d) or 24 hr (a-c) after training, as described in Fig. 2.

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
2.
Fig. 6

Fig. 6. A-443654 promotes mTORC2 activity, actin polymerization and facilitates L-LTP in WT mice but not in mTORC2-deficient mice. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a-b) Treating WT hippocampal slices for 30 min with A-443654 (0.5 μM) increased the activity of both mTORC2 and PAK (a; n=5, U=0.00, **p<0.01; n=4, U=0.00, **p<0.01), as well as the F-actin/G-actin ratio (b; n=5, t=3.995, **p<0.01). c) A-443654 (0.5 μM) converted E-LTP elicited by single tetanic train into a sustained L-LTP in WT slices (n=6 for vehicle, n=8 for A-443654; LTP at 180 min: vehicle 17 ± 10.6%, A-443654 69 ± 7.1%, F(1, 12)=17.870, p<0.001). d-e) A-443654 (0.5 μM) had no effect on mTORC2 and PAK activities (d; n=4, t=0830, p=0.438; n=4; t=0.290, p=0.777) or on actin polymerization in rictor fb-KO slices (e; n=3, t=0.680, p=0.534). f) A-443654 failed to induce L-LTP in rictor fb-KO slices (n=6 for vehicle, n=7 for A-443654; LTP at 180 min: vehicle 24 ± 8.2%, A-443654 28 ± 6.2%, F(1, 11)=2.25, p=0.167). Insets in c and f are superimposed traces recorded before and 180 min after tetani. Calibration: 5 ms, 2 mV.

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
3.
Fig. 3

Fig. 3. In TOR2-deficient Drosophila, long-term spaced memory (but not massed) is impaired. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a) Western blotting for p-dAkt (Ser505), total dAkt and β-actin in the brain of control Canton-S and rictorΔ1 mutant flies. b) In olfactory conditioning, a single training trial consists of 12 electric shocks delivered during the presentation of an odor, while a second odor is explicitly unpaired with the shocks. LTM of the conditioned odor is generated when flies are given five such training trials at 15 min intervals (“spaced training”). However, if the five training trials are given with at much shorter (30 sec) intervals (“massed training”) only a shorter-lasting Anesthesia Resistant Memory (ARM) is formed, but not LTM. c-d) WT Canton-S and rictorΔ1 flies did not significantly differ in the avoidance of 0.12% methylcyclohexanol (MCH; c; t=0.47, p=0.647) and 0.2% octanol (OCT; d; t=1.86. p=0.99). e) Both Canton-S and rictorΔ1 flies similarly avoided the T-maze arm with 90V electric shocks (t=1.86, p=0.17). For each sensory control experiment, an “n” was at least 9 for either genotype. Performance index was calculated as described61. f) In Drosophila olfactory memory tests, spaced training-induced LTM was selectively impaired in rictorΔ1 flies (t=4.37, **p<0.01). In contrast, massed training elicited a similar performance in of both Canton-S (controls) and rictorΔ1 flies (t=1.10, p=0.3). Spaced training did not significantly improve the performance of rictorΔ1 mutants over that achieved through massed training protocols (t=019, p=0.85). n=6 for each group.

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
4.
Fig. 4

Fig. 4. Actin dynamics, Rac1-GTPase activity and signaling are impaired in CA1 of rictor fb-KO mice. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a-f) Western blotting shows that the ratio of F-actin/G-actin (a), Rac1-GTPase activities (c), p-PAK and p-Cofilin (e) are much reduced in CA1 of rictor fb-KO mice. Normalized data (b; n=4 per group, t=4.042, **p<0.01; d left; n=4 per group, t=2.762, *p<0.05; d right; n=4 per group, t=0.519, p=0.623; f; p-PAK n=4 per group, t=9.054, ***p<0.001; p-Cofilin n=4 per group, t=4.486, **p<0.01). g) We hypothesized that mTORC2 regulates Rac1-GTPase activity (and signaling) through the recruitment of a specific Rac1-GTPase GEF. To test this hypothesis, we co-transfected HEK293T cells with myc-tagged Rictor and flag-tagged GEFs or GAPs, and then performed co-immunoprecipitation (IP) experiments. myc-Rictor selectively pulled-down flag-Tiam1 (T-cell-lymphoma invasion and metastatis-1), a specific Rac1-GEF that is highly enriched in neurons62. Flag-tagged -Tiam1, -ephexin, -Abr and –Bcr were coexpressed in HEK293T cells with myc-Rictor and anti-flag immunoprecipitates were analyzed by anti-flag (top) and anti-myc (middle) immunoblotting. h) Diagram of the Tiam1 constructs (FL=full length, DH=point mutations in the DH domain; PDZ=deletion mutant encoding only the PDZ domain). Flag-tagged FL, DH or PDZ were co-expressed with myc-tagged Rictor in HEK293T cells. i) Anti-flag immunoprecipitates were analyzed by anti-myc (top) and anti-flag (middle) immunoblots whereas anti-myc immunoprecipitates (j) were analyzed by anti-flag (top) and anti-myc immunoblots (middle). k) Endogenous Tiam1 interacts with endogenous Rictor. Immunoprecipitates of anti-Tiam1 or anti-IgG (control) were prepared from adult hippocampal extracts and analyzed by Rictor (top) and Tiam1 (bottom) immunoblots. Arrows point to the interaction between Tiam1 and Rictor. l) Golgi-impregnation shows that spine density of apical CA1 pyramidal neurons is reduced in rictor fb-KO mice [scale bar 5 μm; n=70 (20-25 neurons/mouse; 3 mice per group), t=2.791, **p<0.01].

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
5.
Fig. 1

Fig. 1. L-LTP, but not E-LTP, is impaired in mTORC2-deficient slices. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a-c) Western blots show selective decrease in Rictor and mTORC2 activity (p-Akt Ser473) in CA1 (a) and amygdala (b) but not in midbrain (c) of rictor fb-KO mice. Below: normalized data (a; n=4 per group, t=9.794, **p<0.01; b; n=5 per group, t=2.976, *p < 0.05, c; n=4 per group, t=0.470, p=0.663). d-e) In CA1 extracts from control mice 30 min post-stimulation mTORC2 activity was consistently increased with four tetanic trains, but not a single train. Hippocampal slices were stimulated at 0.033 Hz (control), tetanized by one train (100 Hz for 1 s; d), or four such trains at 5 min intervals (e). f) Normalized mTORC2 activity (n=5 per group, 1 X 100 Hz: t=0.31, p=0.23; 4 X 100 Hz: t=6.01, **p<0.01). g) In CA1 from rictor fb-KO mice repeated trains failed to increase mTORC2 activity 30 min after stimulation. h) Normalized data (n=5 per group, U=5.00, p=0.151). i) Similar E-LTP was elicited in control (n=9) and rictor fb-KO slices (n=8) (LTP at 30 min: 41 ± 5.6% for controls and 44 ± 5.7% for rictor fb-KO, F(1, 14)=0.130, p=0.724; LTP at 180 min: 23.7 ± 5.3% for controls and 24.7 ± 8.5% for rictor fb-KO, F(1, 15)=0.011, p=0.917). j) L-LTP elicited by four trains in rictor fb-KO slices (n=11) was impaired vs. control slices (n=14; LTP was similar at 30 min, control 72 ± 11.3% and rictor fb-KO 67 ± 13.2%, F(1, 23)=0.811, p=0.368; but at 220 min L-LTP was only 21 ± 10.8% for rictor fb-KO slices vs. 70 ± 14.8% for controls; F(1, 23)=23.4, p<0.01). Superimposed single traces were recorded before and 180 min after tetani (i) or before and 220 min after tetani (j). Calibration: 5 ms, 2 mV.

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
6.
Fig. 2

Fig. 2. Long-term, but not short-term, fear memory is impaired in mTORC2-deficient mice. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a) In Western blots of control dorsal hippocampus, phosphorylation of both Akt at Ser473 and PAK is transiently enhanced 15 min after fear conditioning. b) Normalized data (top, n=6 per condition, t=2.599, *p<0.05; bottom, n=5 per condition, t=2.930, *p<0.05). c) Compared to home-cage mice, either context-alone (CS) or shock-alone (US) failed to increase mTORC2 activity (n=4 per group, F(2,9)=0.127, p=0.882). In the context-alone group (CS) mice were treated identically but were not given foot shocks whereas in the shock alone group (US) mice were given two foot-shocks and were immediately removed from the chamber. d) For contextual fear conditioning, freezing was assessed in control (n=22) and rictor fb-KO mice (n=14) during a 2 min period before conditioning (naïve) and then during a 5 min period at 2hr (STM) and 24 hr (LTM) after a strong training protocol (two pairings of a tone with a 0.7 mA foot-shock, 2s). e) For auditory fear conditioning, freezing was assessed 2 hr and 24 hr after training, for 2 min before the tone presentation (pre-CS) and then during a 3 min period while the tone sounded (CS). Decreased freezing at 24 hr after training indicates deficient fear LTM in rictor fb-KO mice (d, F(1, 34) =20.253, ***p<0.001; e, F(1,34) =4.704, *p<0.05). f-g) Spatial LTM is impaired in rictor fb-KO mice. f) In the hidden-platform version of the Morris water maze, on days 4, 5 and 6 escape latencies were significantly longer for rictor fb-KO mice (F(1,37)=8.585; **p<0.01; F(1,37)=14.651; ***p<0.001, F(1,37)=18.101, ***p<0.001). g) In the probe test on day 7, only control mice showed preference for the target quadrant (control vs. rictor fb-KO mice; F(1,37)=15.554, ***p<0.001; within control group F(3, 96)=28.840, ***p<0.001).

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.
7.
Fig. 5

Fig. 5. Restoring actin polymerization rescues the impaired L-LTP and contextual LTM caused by mTORC2 deficiency. From: mTORC2 controls actin polymerization required for consolidation of long-term memory.

a) Western blots show that jasplakinolide (JPK; 50 nM) increased the low F-actin/G-actin ratio in CA1 slices from rictor fb-KO mice (n=4 per group, t=3.821, *p<0.05) but not in control slices (n=4 per group, t=0.253, p=0.157). b) The same concentration of JPK restored L-LTP in rictor fb-KO slices (n=7 per group, LTP at 30 min, vehicle 67 ± 7.7%, JPK 73 ± 11.3%, H=0.0667, ANOVA on Ranks, p=0.852; LTP at 220 min, vehicle 23 ± 4.9%, JPK 73 ± 12.5%, F(1, 12)=9.81, p<0.01) but had no effect on L-LTP in WT slices (c; n=7 per group, LTP at 30 min: vehicle 74 ± 9.4%, JPK 72 ± 11.4%, F(1, 12)=0.989, p=0.784; LTP at 220 min: vehicle 64 ± 8.7%, JPK 68 ± 14.2%; F(1, 12)=0.010, p=0.921). The actin polymerization inhibitor Cytochalasin-D (Cyt-D; 100 nM) blocked L-LTP in WT slices (c; at 220 min 26 ± 7.8%, F(1,12)=9.215, p<0.01) but had no effect in rictor fb-KO slices (b, at 220 min 21 ± 7.9%, F(1, 13)=0.163, p=0.694). Insets in b and c are superimposed traces recorded before and 220 min after tetani. Calibration: 5 ms, 2 mV. d-e) Bilateral infusion of JPK (50 ng) into the dorsal hippocampus of rictor fb-KO mice (n=8 per group), immediately after a strong training protocol (two pairings of a tone with a 0.7 mA foot-shock, 2s), boosted contextual LTM (d; F(1, 14)=4.827, *p<0.05) but not auditory LTM (e; F(1, 14)=0.0407, p=0.843). Freezing was assessed 24 hr after training, as described in Fig. 2. f) In WT mice (n=8 per group) JPK bilateral infusion (50 ng) had no effect on contextual LTM (F(1, 14)=0.129, p=0.726). g) A single tetanic train elicits only E-LTP in vehicle-treated slices but a sustained L-LTP in JPK-treated slices (n=7 for vehicle, n=8 for JPK; LTP at 180 min: vehicle 19 ± 5.2%, JPK 81 ± 14.5%, ANOVA on Ranks H=10.59; p<0.001). Inset in g are superimposed single traces recorded before and 180 min after tetani. Calibration: 5 ms, 2 mV. h) Intra-hippocampal infusion of JPK immediately after a weak training (a single pairing of a tone with a 1s, 0.7 mA foot-shock) enhanced contextual fear LTM (n=15 for vehicle and n=16 for JPK; F(1, 29)=4.320, *p<0.05) but not contextual fear STM (i, n=9 for vehicle and n=10 for JPK, H=0.00167, ANOVA on Ranks, p=0.967).

Wei Huang, et al. Nat Neurosci. 2013 April;16(4):441-448.

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