Figure 5 Basal Freezing Long After TMT

Figure 5.1: Timeline of experimental proceedings: 10 days after the single 5-minute exposure to 10% TMT, mice were placed in each side of the conditioning apparatus for 5 minutes and basal levels of freezing were measured (A). Nor-BNI injected mice froze more than saline mice on the TMT-paired side, but not on the neutral side (B). Both drug conditions exhibited a robust increase in freezing relative to their baseline data from 10 days earlier (C). Saline mice decreased freezing between the two sessions whereas NorBNI-treated mice did not (C). The change in freezing behaviour between the two sessions was significantly reduced by NorBNI administration (D). The frequency of freezing was significantly higher for NorBNI injected mice on the TMT-paried side, but not the neutral side (E). Saline but not NorBNI injected mice reduced the frequency of freezing between the two sessions (F). The magnitude of the change between the two sessions was significantly reduced by NorBNI (G). Data presented as mean +/- SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

5.1 Statistical Analyses

5.1.1 Time Spent Freezing

We computed a 2x2 ANOVA with side (Neutral or TMT-paired) as the within subjects factor and Drug condition (Saline or NorBNI) as the between groups factor. This model indicated a significant side * Drug interaction (F(1,38) = 5.69, p = 0.02)

Effect	DFn	DFd	F	p	p<.05	ges
Drug	1	38	11.071	0.002	*	0.195
variable	1	38	2.542	0.119		0.011
Drug:variable	1	38	5.693	0.022	*	0.025

Follow-up comparisons of the significant interaction indicated that the difference in time spent freezing between Saline and Nor-BNI treated mice was not significant on the neutral size (p = 0.059), but Nor-BNI injected mice spent significantly more time freezing in the the TMT-paired compartment (p < 0.001).

variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
Neutral side	value	Saline	Nor-BNI	19	21	0.05780	ns	0.05780	ns
TMT side	value	Saline	Nor-BNI	19	21	0.00015	***	0.00015	***

Saline-treated mice exhibited a decrease in time spent freezing between the first and the second session of the long after test (p = 0.003) whereas TMT-treated mice did not decrease freezing between the two sessions (p =0.62).

Drug	.y.	group1	group2	n1	n2	statistic	df	p	p.adj	p.adj.signif
Saline	value	Neutral side	TMT side	19	19	3.4548388	18	0.003	0.003	**
Nor-BNI	value	Neutral side	TMT side	21	21	-0.4979122	20	0.624	0.624	ns

Additionally, the change in freezing behaviour between the two sessions was significantly greater for Saline-treated mice than for NorBNI (t(38) = 2.43, p = 0.01)

## 
##  Welch Two Sample t-test
## 
## data:  change by Drug
## t = -2.4301, df = 35.605, p-value = 0.02027
## alternative hypothesis: true difference in means between group Saline and group Nor-BNI is not equal to 0
## 95 percent confidence interval:
##  -27.018872  -2.430953
## sample estimates:
##  mean in group Saline mean in group Nor-BNI 
##            -12.281579              2.443333

5.1.2 Freezing Frequency

We also computed a 2x2 ANOVA on freezing frequency, which also indicated a Drug*Timepoint interaction (F(1,38) = 7.26, p = 0.01).

Effect	DFn	DFd	F	p	p<.05	ges
Drug	1	38	4.708	0.036000	*	0.093
variable	1	38	12.972	0.000902	*	0.055
Drug:variable	1	38	7.257	0.010000	*	0.032

Follow-up comparisons of the significant interaction indicated that there was no effect of Drug treatment on the frequency of freezing on the neutral size (p = 0.45), but Nor-BNI injected mice spent significantly more time freezing than saline-treated mice in the the TMT-paired compartment (p < 0.001).

variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
Neutral side	value	Saline	Nor-BNI	19	21	0.45600	ns	0.45600	ns
TMT side	value	Saline	Nor-BNI	19	21	0.00063	***	0.00063	***

Saline-treated mice exhibited a decrease in the frequency of freezing between the first and the second session of the long after test (p = <0.001) whereas TMT-treated mice did not decrease freezing between the two sessions (p = 0.53).

Drug	.y.	group1	group2	n1	n2	statistic	df	p	p.adj	p.adj.signif
Saline	value	Neutral side	TMT side	19	19	4.6050714	18	0.00022	0.00022	***
Nor-BNI	value	Neutral side	TMT side	21	21	0.6281951	20	0.53700	0.53700	ns

Additionally, the change in freezing behaviour between the two sessions was significantly greater for Saline-treated mice than for NorBNI (t(38) = 2.71, p = 0.01)

## 
##  Welch Two Sample t-test
## 
## data:  change by Drug
## t = -2.7084, df = 38, p-value = 0.01008
## alternative hypothesis: true difference in means between group Saline and group Nor-BNI is not equal to 0
## 95 percent confidence interval:
##  -25.186925  -3.640142
## sample estimates:
##  mean in group Saline mean in group Nor-BNI 
##            -16.842105             -2.428571

5.1.3 Average Duration of Freeze

Repeated measures ANOVA on the average duration of freezing episode indicated a main effect of drug only (F (1,38) = 15.71, p < 0.001).

Effect	DFn	DFd	F	p	p<.05	ges
Drug	1	38	15.713	0.000314	*	0.252
variable	1	38	1.561	0.219000		0.007
Drug:variable	1	38	1.716	0.198000		0.008

Nor-BNI treated mice exhibited a significantly higher average duration of freezing episode than did saline-injected mice on both the neutral (p = 0.001) and the TMT-paired (p < 0.001) sides of the CPA apparatus.

variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
Neutral side	value	Saline	Nor-BNI	19	21	0.00107	**	0.00107	**
TMT side	value	Saline	Nor-BNI	19	21	0.00074	***	0.00074	***

The within-subjects change in the average duration of freezing episode between the two sessions was not significant for either NorBNI-injected (p = 0.13) or saline-injected (p = 0.96) mice.

Drug	.y.	group1	group2	n1	n2	statistic	df	p	p.adj	p.adj.signif
Saline	value	Neutral side	TMT side	19	19	0.055716	18	0.956	0.956	ns
Nor-BNI	value	Neutral side	TMT side	21	21	-1.572287	20	0.132	0.132	ns