Behavioral characterization of Tent2 KO and WT mice. Anxiety, exploratory behavior, and locomotion were assessed using open field (A–E) and elevated plus maze (F–J) tasks. Learning and memory were assessed using contextual fear conditioning (CFC) (K). (A) Population-averaged heatmaps showing mice activity in the open arena. No significant differences in time spent in the center (B), time spent in borders (C), velocity (D), or distance moved (E) were observed (P-value >0.05, unpaired t-test, n = 8–9). (F) Population averaged heatmaps showing mice activity in the elevated plus maze. Tent2 KO mice spent significantly more time in the open arms and in the center as compared to WTs (G, [*] P-value = 0.0156, unpaired t-test, n = 8–9) and significantly less time in the closed arms (H, P-value = 0.017, unpaired t-test, n = 8–9). Similarly, as in the open field task, no significant differences in velocity (I) or distance traveled (J) were observed (P-value >0.05, unpaired t-test, n = 8–9). (K) Experimental timeline (upper panel) and summary of data showing freezing levels during CFC and test sessions. Mice were conditioned in a novel context (3US presentations, 2 sec, 0.7 mA). Next, the mice were re-exposed three times to the same context: 1 h after CFC to assess short-term contextual fear memory (STM, 5 min); 24 h after CFC to test long-term contextual fear memory and to extinguish contextual fear (EXT1, 15 min); and 48 h after CFC to test fear extinction memory (EXT2, 15 min). Tent2 KO mice showed increased locomotion before the training (pre-US) as compared to WT animals ([**] P-value = 0.01), but did not differ in freezing behavior post-US (P-value >0.05), 1 h after training (STM) nor 24 h after training (LTM). Interestingly, Tent2 KO mice showed decreased freezing levels during EXT2 ([**] P-value = 0.009, [*] P-value = 0.039, [*] P-value = 0.012). Data are presented as mean ± SEM; n = 18–22; two-way ANOVA, post hoc Sidak's multiple comparisons test. (L) Levels of amino acids in the hippocampi of fear-conditioned Tent2 KO and WT mice. Directly after the last test in the CFC experiment shown in panel K, mice were sacrificed and the levels of amino acids were measured using mass spectrometry. Significantly decreased levels of GABA neurotransmitter were detected in the hippocampi of Tent2 KO mice as compared to WT ([**] P-value = 0.0038). Additionally, increased levels of glutamine were observed in Tent2 KO hippocampi ([*] P-value = 0.0267). Data are presented as mean ± SEM; n = 8–9; two-way ANOVA, post hoc Sidak's multiple comparisons test.
