1. RS decreased autophagy in hippocampus
As shown in Fig. 1. B2-B3, 15 minutes and 60 minutes after RS, the p-ULK1-s757 increased significantly compared to control group (F(2,15) = 12.91, p = 0.001; Ctrl vs. RS-15: p = 0.001; Ctrl vs. RS-60: p = 0.01), p62 level was also increased significantly 15 minutes and 60 minutes compared to control (F(2,15) = 11.96, p = 0.001; Ctrl vs. RS-15: p = 0.009; Ctrl vs. RS-60: p = 0.001, Fig. 1. B2). 15 minutes after RS, the LC3-I level decreased significantly (t(10) = 2.22, p = 0.05, Fig. 1. B2). However, p-ULK1-s757 decreased significantly 30 minutes after the RS (t(10) = 4.28, p = 0.002) compared to control (Fig. 1. A2), and the LC3-II/LC3-I ratio showed an significant increase at 30 minutes after the RS (t(10) = 2.47, p = 0.033, Fig. 1. A4). These results indicate that autophagy in HPC was decreased at 15 and 60 min after the RS, but was transiently increased 30 min after the RS.
2. FS decreased autophagy in hippocampus.
After FS, there were no significant difference of p-ULK1-s757, p62 and LC3- I level between FS group and control group. However, the LC3-II/LC3-I ratio was decrease 15 minutes (t(10) = 2.775, p = 0.02) after stress (Fig. 2. A4). The LC3-II level decreased significantly 15 min (t(10) = 2.27, p = 0.047), 30 min (t(10) = 2.928, p = 0.015) and 60 (t(10) = 2.264, p = 0.047) minutes after FS (Fig. 2. A6, B6, C6). These data suggested that autophagy was inhibited in 1 hour after FS.
3. Acute corticosterone decreased autophagy in hippocampus.
After single CORT treatment, the p-ULK1-s757 increased significantly 15 (t(10) = 2.254, p = 0.048) and 60 minutes (t(10) = 3.055, p = 0.012) after injection (Fig. 3. A2, C2). The p62 level increased significantly 15 min (t(10) = 4.102, p = 0.021) and 60 min (t(10) = 12.19, p = 0 < 0.001) after CORT treatment (Fig. 3. A3, C4). There were no significant differences of LC3-II/LC3-I ratio, LC3-II/level and LC3-I level between CORT group and control group. These data suggested that autophagy was inhibited in 1 hour after single CORT injection.
Taken together, autophagy in hippocampus was inhibited by acute stress within 1 hour, except that RS transiently increased autophagy 30 minutes after the RS treatment. The inhibition showed different autophagy flow across stress pattern and time.
4. RS increased autophagy in mPFC.
mPFC plays crucial role in planning and decision making, which were impaired in stress related psychological disease. We then examined autophagy in the mPFC after restrain stress, foot shock and CORT treatment.
15 minutes after the RS, LC3-II (F(2,20) = 8.087, p = 0.004; Ctrl vs. RS-15: p = 0.032, Fig. 4. B5) and LC3-I (F(2,20) = 5.826, p = 0.004; Ctrl vs. RS-15: p = 0.014, Fig. 4. B6) level increased significantly in mPFC, while the p62 level decreased significantly compared to control (t(10) = 2.242, p = 0.049, Fig. 4. B3). No changes of autophagy flow was found 30 and 60 min after the RS in mPFC. However, the p-ULK1-s757 decreased significantly 60 minutes after the RS compared to 15 minutes after the RS (F(2,20) = 6.287, p = 0.01; RS-15 vs. RS-60: p = 0.008, Fig. 4. B2). These data suggested that autophagy was increased 15 min after RS.
5. FS decreased autophagy in the mPFC.
15 min after the foot shock, autophagy was significantly inhibited in mPFC. After FS, the p-ULK1-s757 decreased significantly 15 minutes (t(10) = 3.857, p = 0.018) compared to control group (Fig. 5. A2). The LC3-II/LC3-I ratio showed an significant decrease 15 (t(10) = 4.397, p = 0.012) minutesafter FS (Fig. 5. A4). The LC3-II level decreased significantly 15 minutes (t(10) = 4.708, p = 0.01, Fig. 5. A6) and increased significantly 60 minutes (t(10) = 3.391, p = 0.007, Fig. 5. C6) after FS. The p62 level decreased significantly 15 minutes (t(10) = 8.537, p = 0.001, Fig. 5. A3) and increased significantly 60 minutes (t(10) = 2.966, p = 0.014, Fig. 5. C6) after FS. These data suggested that autophagy was inhibited in 1 hour after FS.
6. CORT increased autophagy in the mPFC.
15 min after the single CORT treatment, LC3-II/LC3-I ratio were significantly increased (t(10) = 2.365, p = 0.04, Fig. 6. A4). However, The LC3-I level and LC3-II level were decreased significantly 30 minutes after the CORT injection (LC3-I : t(10) = 3.043, p = 0.016, Fig. 6. B5; LC3-II: t(8) = 2.63, p = 0.03, Fig. 6. B6), and the p62 level were decreased 30 min after the CORT administration (t(8) = 2.416, p = 0.042, Fig. 6. B3). 60 minutes after CORT treatment, LC3-II/LC3-I ratio were increased (t(10) = 4.654, p = 0.001, Fig. 6. B3), LC3-II level was also increased (t(10) = 2.805, p = 0.019, Fig. 6. C6). These data suggested that autophagy was slightly increased 15 min after the CORT, but decreased 30 min after the CORT, and then increased significantly 1 hour after the CORT.
In summary, autophagy in mPFC was increased within 1 hour after RS and single CORT injection, but autophagy in mPFC was inhibited by FS.
7. RS decreased autophagy in the BLA
After RS, the p-ULK1-s757 increased significantly 15 minutes and 60 minutes compared to control group (F(2,15) = 1.521, p = 0.021; Ctrl vs. RS-15: p = 0.03; Ctrl vs. RS-60: p = 0.01, Fig. 7. B2). Though the LC3-II/LC3-I ratio showed no significant change after stress, the LC3-I (F(2,15) = 18.01, p = 0.001; Ctrl vs. RS-15: p = 0.001; Ctrl vs. RS-60: p < 0.001, Fig. 7. B5) and LC3-II (F(2,15) = 6.478, p = 0.01; Ctrl vs. RS-15: p = 0.026; Ctrl vs. RS-60: p = 0.014, Fig. 7. B6) level decreased significantly 15 minutes and 60 minutes after stress. The p62 level increased significantly 15 minutes and 60 minutes compred to control (F(2,15) = 6.429, p = 0.01; Ctrl vs. RS-15: p = 0.004; Ctrl vs. RS-60: p = 0.049, Fig. 7. B3). These data suggested that RS decreased autophagy in BLA within 1 hour after the stress.
8. FS increased autophagy in BLA 15 min after the stress.
After FS, p62 level decreased significantly 15 minutes (t(10) = 4.159, p = 0.002) after the FS in BLA (Fig. 8. C), but no changes were found in the p-ULK1-s757 level, the LC3-II/LC3-I ratio, LC3-I level as well as LC3-II level. These data suggested that autophagy was slightly increased within 1 hour after FS.
9. CORT increased autophagy in BLA.
15 min after CORT treatment, LC3-II/LC3-I ratio and LC3-II was increased (LC3-II/LC3-I ratio: t(10) = 3.543, p = 0.005, Fig. 9. A4; LC3-II: t(10) = 3.489, p = 0.006, Fig. 9. A6). No changes of p-ULK1-s757, p62, LC3-II or LC3-I were found 30 min after the CORT administration. However, LC3-II/LC3-I ratio, as well as LC3-II level and LC3-I level were increased 60 min after the CORT treatment (LC3-II/LC3-I ratio: t(10) = 3.489, p = 0.006, Fig. 9. C4; LC3-I: t(10) = 3.58, p = 0.005, Fig. 9. C5; LC3-II: t(10) = 9.282, p < 0.001, Fig. 9. C6). These data suggested that autophagy was increased in 1 hour after CORT.
In summary, autophagy in BLA was increased within 1 hour after FS and single CORT injection, but was inhibited by RS in 1 hour after stress.
10. Chronic corticosterone increased autophagy in mPFC and BLA.
After chronic CORT treatment, there was no change of p-ULK1-s757, LC3-II/LC3-I ratio, LC3-II, LC3-I and p62 level between groups in HPC (Fig. 10A1, A2-A6). However, the p62 level was significantly lower in CORT group compared to control (t(8) = 2.901, p = 0.02, Fig. 10. B3), suggesting that chronic CORT increased autophagy in mPFC. In BLA, chonic CORT did not change p-ULK1-s757, p62, LC3-II and LC3-I levels, but the LC3-II/LC3-I ratio was significantly higher in CORT group compared to control (t(8) = 2.296, p = 0.05, Fig. 10. C4), indicating that autophagy was increased in BLA. Taken together, chronic CORT treatment increased autophagy in mPFC and BLA, but had no effect on autophagy in HPC.