Male Colony-Bred C57BL/6J Mice Were Tested in Our 6-Day FPS Paradigm. One Group Was Presented

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Smith et al.

Male colony-bred C57BL/6J mice were tested in our 6-day FPS paradigm. One group was presented with a 30-s, 12-kHz, 85-db tone that had no initial rise time (No Rise Time, n=8) and another group was presented with the same tone that had an initial 3-s rise time (3-s Rise Time, n=8); the 3-s rise time is standard for all tone presentations in our modified FPS paradigm. Startle amplitude for each subject was measured during two 500 ms time bins: at the onset of the tone and again after 3 seconds of tone exposure. In the 3-s Rise Time group, this second measurement corresponded to when the tone reached its full intensity of 70 dB. Startle amplitude was measured with the MedAssociates Startle Viewer software program. Percent FPS values were calculated for PreTest and Test for each group of mice. Data were analyzed with a one-way ANOVA with a repeated measure of time and are presented as mean ± standard error of the mean; the significance level was set at p<0.05.

During PreTest, subjects presented with a tone that had no initial rise time exhibited a startle response at tone onset that was significantly higher than subjects presented with a tone that had a 3-s rise time (significant main effect of group, F(1,14)=7.16, p<0.05, Suppl. Fig. S1A). Subject reactivity in the No Rise Time group decreased significantly after 3 s of tone presentation when compared to responsivity at tone onset (significant time x group interaction, F(1,14)=10.51, p<0.01, Suppl. Fig. S1A) and was subsequently also not different from subjects in the 3-s Rise Time group. In contrast, subjects that were presented with a tone that had a 3-s rise time exhibited little reactivity both at tone onset and when the tone reached its full intensity. While there were no statistical differences in percent FPS values between the two groups either during PreTest or Test (Suppl. Fig. S1B), percent FPS during PreTest was 3 times greater for the No Rise Time group than the 3-s Rise Time group. Expression of fear potentiated startle during Test was robust and not different between groups (Suppl. Fig. S1B).

While the difference between groups at tone onset could be due to the fact that the tone intensity was initially less in the 3-s Rise Time Group, subject reactivity in this same group of mice subsequently remained the same when the tone reached 70 dB. Although there were no statistical differences when percent FPS values were examined, the pattern of responses suggests that an abrupt tone onset can generate unconditioned effects to the tone during PreTest that are not manifested until startle reactivity is assessed in response to a white noise startle burst following tone exposure. Use of a tone with or without an initial rise time does not affect the expression of fear-potentiated startle during Test.

Experiment 1A Colony-Bred Mice: Results and Discussion

Colony-bred C57BL/6J (n=15) patterns of responses in the 6-day FPS paradigm (Suppl. Fig. S2) were comparable to those exhibited by vendor-obtained mice (Fig. 2) although their overall startle amplitudes tended to be lower. Subjects showed an intensity-dependent increase in startle responding during each Acclimation day that did not habituate (Suppl. Fig. S2A) across experimental days. Results of a 2 x 3 factor ANOVA (test session x trial type) showed that PreTest startle amplitudes did not differ across the Leader, Startle Only, and Tone + Startle trials (F(2,47)=2.28, p=0.113; Fig. S2B). While the mean startle amplitude did not differ between Leader and Startle Only trials during Test, startle responding on the Tone + Startle trials was significantly potentiated compared to the other two trial types (F(2,47)=54.15, p<0.0001; Fig. S2B). Startle amplitudes for all three trial types during Test were also significantly elevated compared to the respective trial conditions during PreTest (significant test session x trial type interaction, F(5,47)=29.26, p<0.0001; Suppl. Fig. S2B).

As with vendor-obtained mice, startle amplitude across individual trials was similar for both trial types during the PreTest, indicative of an overall lack of an unconditioned effect of the tone on startle (Suppl. Fig. S2C). In contrast, there was a significant potentiation of startle on Tone + Startle trials compared to Startle Alone trials across the entire Test session even though startle amplitude tended to habituate across trials for both trial types (Suppl. Fig. S2C). Supplemental Figure S2D illustrates the percent change in mean startle amplitude on Tone + Startle trials compared with Startle Alone trials. There was a 16% change in startle amplitude in the PreTest reflecting a small overall unconditioned effect of the tone on startle, a value similar to that of vendor-obtained mice (Fig. 2). In contrast, after fear conditioning, animals showed significant FPS (paired t-test, t(15)=3.58, p=0.003; Suppl. Fig. S2D) compared to PreTest; this FPS value was also similar to that exhibited by vendor-obtained mice (Fig. 2D). While the scatterplot of individual responses before and after conditioning reveals the dynamic range and variability of responses between subjects (Suppl. Fig. S2E), this graph shows that the majority of subjects had a potentiation of startle on Test compared to PreTest.

Together, these data demonstrate that although C57BL/6J mice bred in our colony at McLean Hospital have different rearing and housing experiences than vendor-obtained mice, the overall patterns of responding and FPS values are comparable to vendor-obtained mice of the same strain.