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. 2020 Apr 18;23(5):101078. doi: 10.1016/j.isci.2020.101078

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© 2020 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Normal Context-Object Recognition but Affected Neutral Context Recognition in Trkb^Gad1-KO Mice

(A–C) Context-object recognition test. (A) Mean discrimination ratio was calculated as a ratio of the time spent exploring an object not previously paired with the context over the total time spent observing both paired and unpaired objects. No difference was found between genotypes (Trkb^Gad1-WT [n = 7] 0.61 ± 0.04; Trkb^Gad1-KO [n = 8] 0.59 ± 0.03, mean ± SEM, unpaired Student's t test, p = 0.7195). (B) Mean total object exploration time during the 4-day acquisition phase was similar between Trkb mutant and control mice and decreased significantly over time (two-way ANOVA repeated measures: F_(3,39) = 3.276, p = 0.031, main effect of time). (C) The mean total object exploration time analyzed during the test session was also not significantly different between genotypes (Trkb^Gad1-WT, 19.61 ± 4.9; Trkb^Gad1-KO, 26.78 ± 4.23; mean ± SEM; unpaired Student's t test: t₍₁₃₎ = 1.112, p = 0.2861).

(D–F) Fear conditioning test. (D) Acquisition phase. Two-way ANOVA repeated measures (Trkb^Gad1-WT, n = 10; Trkb^Gad1-KO, n = 13) revealed a main effect of genotype in freezing behavior (F_(1,23) = 10.56, p = 0.0035), a main effect of test stage (F_(4,92) = 22.1, p < 0.0001), and an interaction between genotype and test stage (F_(4,92) = 3.072, p = 0.0201). To explore the observed interaction, Sidak's multiple corrected comparisons showed a statistically significant difference between genotypes in the percentage time spent freezing during the CS-US1 (Trkb^Gad1-WT, 30.00% ± 6.03%; Trkb^Gad1-KO, 14.29% ± 3.43%; mean ± SEM, p = 0.0166), during the CS-US2 (Trkb^Gad1-WT, 41.82% ± 5.85%; Trkb^Gad1-KO, 25.71% ± 3.59; p = 0.013), and in the post-US2 (Trkb^Gad1-WT, 44.09% ± 2.68%; Trkb^Gad1-KO, 25.18% ± 3.39%; p = 0.002). (E) Twenty-four hours after conditioning, mice were first tested for contextual fear conditioning. A statistically significant difference in the time spent freezing was found between control (n = 10), 58.72% ± 5.22%, and Trkb^Gad1-KO (n = 13) mice, 30.04% ± 2.84%; mean ± SEM, unpaired Student's t test, p < 0.0001, two-tailed. (F) Twenty-four hours later mice were tested for cued fear conditioning. Two-way ANOVA repeated measures (Trkb^Gad1-WT, n = 8; Trkb^Gad1-KO, n = 12) revealed a main effect of test stage (F_(1,18) = 92.3, p < 0.0001) and a main effect of genotype (F_(1,18) = 5.446, p = 0.0314), but no interaction (F_(1,18) = 0.7558, p = 0.3961). Sidak's multiple comparisons test showed no significant difference between control and mutant mice in freezing responses (%) (Trkb^Gad1-WT, 81.67% ± 4.40%; Trkb^Gad1-KO, 67.22% ± 5.77%, p = 0.07).

(G–I) Context and cued fear conditioning are unaffected in BAC-Gad1-Cre mice. (G) Percentage of time spent freezing during conditioning. Two-way ANOVA repeated measures (BAC-Gad1-Cre mice, n = 13; WT control, n = 10) revealed a main effect of genotype in freezing behavior (F_(1,21) = 8.477, p = 0.0083) and a main effect of the stage of the test (F_(4,84) = 24.43, p < 0.0001), but no interaction between genotype and test stage (F_(4,84) = 1.865, p = 0.1241). Sidak's multiple corrected comparisons showed a statistically significant difference between genotypes in the percentage of time spent freezing during the second tone-foot shock presentations (CS-US2, WT: 28.00% ± 4.16%, BAC-Gad1-Cre: 45.38% ± 6.06%, p = 0.0305) compared with the other stages (CS-US1, WT 13.00% ± 5.17%, BAC-Gad1-Cre: 27.69% ± 5.68%, p = 0.09; post-US2, WT: 33.25% ± 1.24%, BAC-Gad1-Cre: 49.42% ± 5.15%, p = 0.052). (H) Twenty-four hours after conditioning, mice were first tested for contextual fear conditioning, and no difference was found in freezing responses (%) between WT (n = 10) and BAC-Gad1-Cre mice (n = 13) (% of time spent freezing, WT: 52.53% ± 4.17%, BAC-Gad1-Cre: 54.72% ± 3.92%; unpaired Student's t test, p = 0.697, two tailed). (I) Cued fear conditioning was tested 24 h later. Two-way ANOVA repeated measures revealed a main effect of test stage (F_(1,18) = 266.3, p < 0.0001), but no effect of genotype (F_(1,18) = 0.2818, p = 0.6020). Freezing responses (%) (WT, n = 7, BAC-Gad1-Cre, n = 13) (% of time spent freezing, CS, WT: 76.43% ± 6.48%, BAC-Gad1-Cre: 71.54% ± 5.25%, p = 0.76). Values are mean ± SEM. ITI, intertrial interval; Hb, habituation; CS, conditioned stimulus; US, unconditioned stimulus.

See also Figures S7.