There Was No Difference Between the Stay and Removal Groups in Contextual Fear

Supplementary Materials

There was no difference between the stay and removal groups in contextual fear

All animal groups were matched to the total exposure time (44 min), which was the time that the long-spaced group (600 s) spent in the conditioning box; 2 min before training, 40 min during training and 2 min after the last footshock. Thus, after training, the animals of the massed (12 s) and spaced (122 s) groups were required to stay in the conditioning box until they had spent a total of 44 min in the box. However, it is unclear whether contextual fear memory would be different between staying in and removal from the conditioning box after training. Thus, we compared the stay (12 s-s and 122 s-s) and removal (12 s-m and 122 s-m) of the groups in the memory tests 24 h and 7 d after training. After the animals were placed into the conditioning box for 2 min and the freezing levels were set to a baseline (BL), 5 footshocks at an intensity of 0.8 mA for 1 s were given to the animals, and the freezing levels within 2 min after each footshock were measured as the learning curve. For the removal groups, the animals were removed from the conditioning box 2 min after the last footshock; however, for the stay groups, they were kept in the conditioning box until they had spent a total of 44 min in the box and were then returned to their home cages, similarly to the removal groups. We found that at the same ITIs (massed or spaced), there was no difference between the stay and removal groups in contextual fear examined 24 h and re-examined 7 d after the training (Supplementary S1).

Injections of CN04-A after but nor before contextual fear conditioning had no effect on contextual fear

We found that intrahippocampal injection of the Rac1 activator CN04-A immediately after contextual fear conditioning had no effect on contextual fear 24 h and 7 d after training. According to the manufacturer’s specifications, the Rac1 activator CN04-A could enhance Rac1 activity 50% by 1 h and 100% by 2 h after administration. Thus, we examined whether intrahippocampal injection of CN04-A immediately after contextual fear conditioning could produce any differing effects on contextual fear. We found that injections of CN04-A after the training had no effect on contextual fear examined 24 h and 7 d after training (Supplementary S2). Because Rac1 activity sharply dereased at 1 h after spaced training, and the training time of contextual fear conditioning was 44 min, so we adjusted the timepoint of CN04-A administration, we injected CN04-A into bilateral CA1 area before spaced training, and found contextual fear was obviously decreased at 24 h and 7 d after training.

Figure Legends

S1. Contextual fear memory was not different between the stay and removal groups at the same inter-trial intervals (ITIs). (a) Diagram for the experimental procedures. The stay groups: 12 s-s and 122 s-s; the removal groups: 12 s-m and 122 s-m. (b) In the massed training groups, there was no difference between the stay (12 s-s) and removal (12 s-m) groups in contextual fear examined 24 h and re-examined 7 d after training (12 s-s, n = 9; 12 s-m, n = 10; P = 0.281 for 24 h; P = 0.320 for 7 d). Similarly, there was no difference between the stay (122 s-s) and removal (122 s-m) groups by spaced training in contextual fear examined 24 h and re-examined 7 d after training (122 s-s, n = 9; 122 s-m, n = 8; P = 0.410 for 24 h; P = 0.667 for 7 d).

S2. Immunofluorescence straining of Rac-GTP in the CA1 area in the hippocampus. The straining of Rac-GTP sharply weakened in the spaced training (122 s and 600 s) groups at 1 h after contextual fear conditioning.

S3. Injections of CN04-A after but nor before contextual fear conditioning had no effect on contextual fear memory. (a1 and a2) Diagram for experimental procedures. A1, intrahippocampal injection of CN04-A immediately after contextual fear conditioning; A2, intrahippocampal injection of CN04-A before contextual fear conditioning. (b) Injections of CN04-A after contextual fear conditioning had no effect on contextual fear examined 24 h and re-examined 7 d after training (n = 11, P = 0.750 for 24 h, P = 0.933 for 7 d). Injections of CN04-A before contextual fear conditioning obviously decreased contextual fear at 24 h and 7 d after training (c) (n = 8, P = 0.005 for 24 h, P = 0.002 for 7 d).