Contextual fear conditioning

In order to understand the ability of mice of the three lines to discriminate between a shock context where the animals experienced an electric footshock and a grid context where they received no shock, we assessed fear conditioning to a context and to a tone in a background contextual fear conditioning task. Mice were exposed to the grid context and to the shock context on days 1, 2 and 7 after conditioning. On day 8, mice were exposed to another neutral context where the tone of the conditioning procedure was presented to the mice. Furthermore, mice were conditioned either with 0.7 mA or with 1.5 mA footshock intensity.

On day 1 after conditioning with 0.7 mA, GABA-CB1-ko mice showed increased levels of freezing in the grid context (genotype: p < 0.05) whereas no significant differences could be found for total-CB1-ko and Glu-CB1-ko mice, compared to the respective wildtype controls (Figure 17A, C, E, Table 8). Glu-CB1-ko mice revealed higher levels of freezing in the shock context (genotype: p < 0.05) but total-CB1-ko and GABA-CB1-ko mice showed no significant differences here, compared with the wildtype controls, respectively (Figure 17A, C, E, Table 8). On day 2, total-CB1-ko and GABA-CB1-ko mice had increased levels of freezing in the grid context (genotype: p < 0.05 and p < 0.01, respectively) whereas Glu-CB1-ko mice showed no significant difference, compared to the respective wildtype littermates (Figure 17A, C, E, Table 8). However, Glu-CB1-ko mice showed augmented freezing-levels in the shock context (genotype: p < 0.05) and total-CB1-ko mice had a strong tendency to freeze more in the shock context (genotype: p < 0.07), but GABA-CB1-ko mice did not show any significant differences, compared to the wildtype littermates (Figure 17A, C, E, Table 8). On day 7, freezing of GABA-CB1-ko mice was elevated in the grid context (genotype: p < 0.01) but no significant elevation could be seen in total-CB1-ko and Glu-total-CB1-ko mice, compared to the wildtype controls, respectively (Figure 17A, C, E, Table 8). Total-CB1-ko mice had increased freezing levels in the shock context (interval x genotype: p < 0.01) and Glu-CB1-ko mice had a strong

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GABA-CB1-ko mice revealed no significant differences, always compared with the respective wildtype control animals (Figure 17A, C, E, Table 8).

On day 1 after conditioning with 1.5 mA, total-CB1-ko mice, GABA-CB1-ko mice and Glu-CB1-ko mice displayed increased levels of freezing in the grid context (genotype:

p < 0.001, p < 0.005 and p < 0.05, respectively), always compared with the respective wildtype animals (Figure 17B, D, F, Table 9). Total-CB1-ko mice revealed higher levels of freezing in the shock context (genotype x interval: p < 0.05), whereas no significant differences were found for GABA-CB1-ko mice and Glu-CB1-ko mice, compared with the wildtype littermates (Figure 17B, D, F, Table 9). On day 2, total-CB1-ko mice had increased levels of freezing in the grid context (genotype: p <

0.001), whereas GABA-CB1-ko and Glu-CB1-ko mice showed no significant differences, compared with the respective wildtype controls (Figure 17B, D, F, Table 9). GABA-CB1-ko mice had a strong tendency to freeze more in the shock context (genotype: p < 0.08), but total-CB1-ko mice and Glu-CB1-ko mice did not display any significant differences, compared to the wildtype control mice (Figure 17B, D, F, Table 9). On day 7, freezing of total-CB1-ko mice was elevated in the grid context (genotype: p < 0.001), but no significant difference was found for GABA-CB1-ko and Glu-CB1-ko mice, compared with the wildtype controls, respectively (Figure 17B, D, F, Table 9). Glu-CB1-ko mice had increased freezing in the shock context (genotype:

p < 0.01), but no significant differences were seen in total-CB1-ko and GABA-CB1-ko mice, compared to the wildtype controls (Figure 17B, D, F, Table 9).

We next tested whether mice of the three lines were able to discriminate between the two contexts. When being conditioned with 0.7 mA, on day 1, total-CB1-ko mice (genotype x context: p < 0.01), total-CB1-wt mice (genotype x context: p < 0.05), GABA-CB1-ko mice (genotype x context: p < 0.005) and GABA-CB1-wt mice (genotype x context: p < 0.001) had higher freezing levels in the shock context compared to the grid context, but Glu-CB1-ko and Glu-CB1-wt mice failed to do so (Table 12). On day 2, GABA-CB1-wt mice (genotype x context: p < 0.005) and Glu-CB1-ko mice (genotype x context: p < 0.05) displayed higher freezing levels in the shock context, but total-CB1-ko, total-CB1-wt, GABA-CB1-ko and Glu-CB1-wt mice failed to do so (Table 12). On day 7, GABA-CB1-wt mice were close to significance to reveal higher levels of freezing (genotype x context: p < 0.059) and Glu-CB1-ko mice (genotype x context: p < 0.01) indeed revealed higher levels of freezing in the shock context compared to the grid context, whereas total-CB1-ko, total-CB1-wt, GABA-CB1-ko and Glu-CB1-wt mice showed similar levels of freezing in both contexts (Table 12).

On day 1 after conditioning with 1.5 mA, GABA-CB1-wt mice showed more freezing in the shock context (context: p < 0.01), whereas total-CB1-ko, total-CB1-wt, GABA-CB1-ko, Glu-CB1-ko and Glu-CB1-wt mice failed to do so (Table 13). On day 2, total-CB1-wt mice (context: p < 0.05), CB1-ko mice (context: p < 0.01) and GABA-CB1-wt mice (context: p < 0.05) displayed more freezing in the shock context compared to the grid context, but there was no significant difference for total-CB1-ko, Glu-CB1-ko and Glu-CB1-wt mice (Table 13). On day 7, total-CB1-ko mice (context x interval: p < 0.05), Glu-CB1-ko mice (context: p < 0.01), Glu-CB1-wt mice (context: p

< 0.05), GABA-CB1-ko mice (context: p < 0.05) and GABA-CB1-wt mice (context: p <

0.05) had higher levels of freezing in the shock context compared to the grid context, but total-CB1-wt mice showed no significant differences (Table 13) .

Results

Figure 17: Background contextual fear conditioning. Freezing levels of total-CB1-ko mice (A-B), GABA-CB1-ko mice (C-D) and Glu-CB1-ko mice (E-F) and the respective wildtype littermates in the contextual fear conditioning paradigm on day 1, 2 and 7 after conditioning. Mice were conditioned with 0.7 mA (A, C, E) or with 1.5 mA (B, D, F). Grey insets and square symbols highlight the freezing values in the shock context compared to the grid context with diamond symbols. Data are shown in 1-min-intervals. Values are means +/- S.E.M. Arrows indicate significant main effects of genotype.

Asterisks indicate post-hoc comparisons following significant genotype x interval interaction. ↑ p <

0.05, ↑↑ p < 0.01 and ** p < 0.01. Effects close to significance are indicated by the given p value.

Sample sizes are indicated for genotypes. Please see Table 8+9 for statistical details.

If the development of the freezing response on the repeated test days was analysed separately for context, at 0.7 mA, total-CB1-ko mice and GABA-CB1-ko mice showed generally more freezing in the grid context than the wildtype control mice (genotype:

p < 0.05 and p < 0.01, respectively) independently of the testing days (genotype x day: p = 0.3 and p = 0.7, respectively) but there was no significant difference in

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Figure 18: Development of the fear response in the two contexts. Freezing levels of total-CB1-ko mice, GABA-CB1-ko mice and Glu-CB1-ko mice and the respective wildtype littermates in the shock context (A, B) or the grid context (C, D) following background contextual fear conditioning with 0.7 mA (A, C) or 1.5 mA (B, D), respectively over the three testing days. Values are means +/- S.E.M. Sample sizes are the same as in Fig. 17. Arrows indicate significant main effects of genotype. Asterisks indicate post-hoc comparisons following significant genotype x interval interaction. ↑ p < 0.05, ↑↑ p <

0.01, ↑↑↑ p < 0.001 and ** p < 0.01. Please see Table 14+15 for statistical details.

Glu-CB1-ko mice, compared with the wildtype littermates (Figure 18C, Table 14). On the other hand, Glu-CB1-ko mice revealed generally more freezing in the shock context (genotype: p < 0.01) which is not the case in total-CB1-ko and GABA-CB1-ko mice, compared to the wildtype mice (Figure 18A, Table 14).

At 1.5 mA, total-CB1-ko mice, GABA-CB1-ko mice and Glu-CB1-ko mice showed generally more freezing in the grid context than the wildtype control mice (genotype:

p < 0.001, p < 0.05 and p < 0.05, respectively) (Figure 18D, Table 15). In the shock context, no differences in the levels of freezing could be found in total-CB1-ko and GABA-CB1-ko mice, compared to their wildtype control mice, respectively, whereas there was augmented freezing in Glu-CB1-ko mice compared to the wildtype littermates (day x genotype: p < 0.01) (Figure 18B, Table 15).

Results

Figure 19: Conditioning response to the auditory cue. Freezing levels of total-CB1-ko mice, GABA-CB1-ko mice and Glu-CB1-ko mice and the respective wildtype littermates for the auditory cue in the background contextual fear conditioning paradigm on day 8. The same mice as shown in Fig. 15 were conditioned with 0.7 mA (A) or with 1.5 mA (B). Data are shown in 1-min-intervals. Values are means +/- S.E.M. Sample sizes are the same as in Fig. 17. Arrows indicate significant main effects of genotype. Asterisks indicate post-hoc comparisons following significant genotype x interval interaction.

↑ p < 0.05, ↑↑ p < 0.01 and ** p < 0.01, *** p < 0.001. Effects close to significance are indicated by the given p value. Please see Table 8+9 for statistical details.

Next, we tested whether mice of the three lines displayed different freezing behaviour to the tone used for fear conditioning. The animals were exposed to a neutral context and the conditioning tone was presented for 5 min.

Being conditioned with 0.7 mA, total-CB1-ko (interval x genotype and genotype: p <

0.001 and p < 0.05, respectively) showed higher freezing levels and Glu-CB1-ko (genotype: p < 0.092) mice showed a tendency to display more freezing to the tone, whereas there was no significant difference for GABA-CB1-ko mice, always compared to the wildtype control mice (Figure 19A, Table 8).

Being conditioned with 1.5 mA, total-CB1-ko (Interval x genotype and genotype: p <

0.05 each) showed higher freezing levels whereas there was no significant difference for GABA-CB1-ko mice and Glu-CB1-ko mice, always compared to the wildtype control mice (Figure 19B, Table 9). Additionally, we conditioned Glu-CB1 with 0.5 mA (these results are summarised in the Appendix section). Here, Glu-CB1-ko mice revealed higher freezing levels than their wildtype littermates when the tone was presented (Interval x genotype: p < 0.01) (Figure 35B, Table 17).

Eventually, we checked whether the mice of the three lines revealed altered freezing behaviour in a new environment, which was the neutral context on day 8 before tone presentation. For 0.7 mA, there was no difference in freezing for total-CB1-ko, GABA-CB1-ko and Glu-GABA-CB1-ko mice compared to the wildtype littermates (Figure 20A, Table 22). At 1.5 mA, no difference could be detected for total-CB1-ko and Glu-CB1-ko mice, but GABA-CB1-Glu-CB1-ko mice showed augmented levels of freezing (genotype: p

< 0.01), always compared to the wildtype animals (Figure 20B, Table 22).

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Figure 20: Response of fear in a neutral context. Freezing levels of total-CB1-ko mice, GABA-CB1-ko mice and Glu-CB1-GABA-CB1-ko mice and the respective wildtype littermates before the auditory cue in the contextual fear conditioning paradigm on day 8 after conditioning. Mice were conditioned with 0.7 mA (A) or with 1.5 mA (B). Data are shown in 1-min-intervals. Values are means +/- S.E.M. Sample sizes are the same as in Fig. 17. Arrows indicate significant main effects of genotype. ↑↑ p < 0.01. Please see Table 22 for statistical details.

3.3 Emotionality and Anxiety