MEDICION DEL PODER CALORIFICO

In addition to primary group differences, we wanted to explore the possibility that the voluntary nature of the exercise used may have an influence on either memory

retention or adult conditioning, as distances run were variable among rats. First a model with average distance run across all 21 days and early life condition was tested against freezing during cue one prior to adult conditioning/re-conditioning, for which a

significant interaction was found (B= -0.0687 p= .0467) indicating a difference in the slopes between rats conditioned at P17 and those that were not, see Figure 2.9. This difference in slopes can be explained by the significant negative correlation seen in P17

conditioned rats (R²= 0.5517; F(1,12) = 44.5801; p = 0.03477) and the non-significant positive correlation in rats that remained naïve at P17 (R²= 0.00196; F(1,12) = 0.0118; p = 0.917). This effect was then tested in rats of the P25 experiment for which there was no significant interaction (B= 0.7319 p = .40667; figure not shown).

Figure 2.9 Linear relationships between average distance run across the 21-day exercise intervention and freezing during the memory retention test (cue 1) in adulthood in rats conditioned or not at P17. Data indicate an interaction between average distance run and early life condition. Data indicate an interaction between average distance run and early life condition (p = 0.047). Rats previously conditioned at P17 show a significant negative correlation (R²= 0.5517; p = 0.03477), with no correlation in rats that remained naïve at P17 (R²= 0.00196; p = 0.917).

Exercise could also be influencing adult outcomes through its effects on adult conditioning. This was tested using a model that included early life experience, average distance run during late adolescence and adult conditioning, for which the P17

experiment indicates no significant interactions in the model, p’s > 0.05 (figure not shown). Next, data from the P25 experiment were tested, again no significant interactions were found in the model, p’s > 0.05. However, after visual inspection a similar pattern was seen in the slopes based on adult conditions (FC vs Ctl) such that general linear hypotheses testing was used to test the differences in slopes. Results found a

non-significant difference in adult control rats (p= 0.932) and adult conditioned (p= 0.932), so data were collapse, Figure 2.10. The model was then re-run including average distance run against freezing during long-term memory based on adult condition, data indicate a trend for a significant interaction (B= 0.0361 p= .0773). When broken down a significant negative correlation was seen in adult conditioned rats (R²= 0.3134; F(1,16) = 7.303; p = 0.01569) with a non-significant positive correlation in rats that served as adult controls (R²= 0.02287; F(1,16) = 0.3745; p = 0.5492).

Figure 2.10 Linear relationships between average distance run across the 21-day intervention period and freezing during LTM in rats conditioned or not in adulthood. Analysis was initially run across all four groups receiving exercise during late adolescence, for which no effects were seen. However, no differences in slopes were seen for adult conditioned groups (p= 0.932) or no adult conditioning (control) groups (p= 0.932), data were collapsed and are presented in the figure above. A trend for a significant interaction between average distance and adult condition was seen (p= .077). Indicating rats conditioned in adulthood show a significant negative correlation (R²= 0.3134; p = 0.0157), with no correlation in rats that served as adult controls (R²= 0.0229; p = 0.5492).

2.6.4 Discussion

Understanding how fear learning and memory develop over time is critical to understand how fear and anxiety-related disorders manifest and persist during a lifetime.

The goal of this work was to determine if an intervention of chronic exercise during late adolescence could serve as an indirect approach to reduce persistence of fear. Our results reveal that early predictable aversive learning at P17 or P25 differentially modulate fear retention in adulthood and that these memories and the acquisition of fear in adulthood could be differentially modulated by exercise during late adolescence. Specifically, in the P17 experiment data again suggest no fear memory retention in adulthood, as indicated by no difference in freezing compared to naïve controls. However even without an initial difference in overall fear responding, an increase in average distance run during

adolescence predicted lower levels of freezing during this retention test. Conversely, in the P25 experiment data indicate a trend for an interaction between the relationship of distance run and freezing between those rats conditioned in adulthood versus those that were not. However, rats that were conditioned in adulthood exhibit lower levels of freezing at long-term memory, irrespective of early life condition, as levels of previous exercise experience increase. This work replicates the primary findings from chapter 2, as well as a large breath of work which indicates that, when acquired around P17 in rats, fear memories are no longer explicitly expressed two or more days after conditioning (Akers et al., 2012; Campbell & Campbell, 1962; Coulter et al., 1976; Jones & Monfils, 2016; Li & Richardson, 2013; Travaglia, Bisaz, Cruz, et al., 2016; Travaglia, Bisaz, Sweet, et al., 2016). It also contributes to the literature by showing that while rats

conditioned at P17 do not freeze significantly differently from rats not previously conditioned, being exposed to an adolescent intervention of chronic exercise produces a reduction in freezing during the retention test. Of particular interest is the fact that when children who developed PTSD in early life are exposed to an exercise regimen three times a week for 8-weeks (24 days), they show a reduction in PTSD symptoms, as well as a reduction on measures of depression and anxiety (Newman & Motta, 2007).

Additional work in non-human animal models has also shown that exercise can serve to produce stress resistance (Greenwood et al., 2003), as well as reduce levels of anxiety (Greenwood, Strong, Brooks, & Fleshner, 2008; Motaghinejad, Fatima, Karimian, &

Ganji, 2016; Pietrelli, Lopez-Costa, Goñi, Brusco, & Basso, 2012; Salam et al., 2009).

The increased levels of exercise, thus, may be working not on direct fear memories, but to reduce any generalized anxiety response (e.g., increased startle responses; Bazak et al., 2009; Jovanovic et al., 2009, or decreased locomotor behavior Ishikawa, Nishimura,

& Ishikawa, 2015), that may have developed as a result of early life trauma. Interestingly, maternal separation, a form of early life stressor, that promotes adult-like potentiation of fear responses rather than persistent reductions of fear,(Callaghan & Richardson, 2011;

Kim & Richardson, 2007a, 2007b), also promotes fear memories to persist into adulthood after extinction in P17 rats (Callaghan & Richardson, 2012). Future work should determine if the initial potentiation and persistence of fear through stress exposure can be reduced through the application of chronic exercise.

Interestingly, within these supplemental studies presented here, looking at exercise as a possible intervention to reduce the potentiation of fear after reconditioning

in adulthood, neither P17 nor P25 conditions showed a potentiation of fear as was previously seen in primary analysis, in which both P17 and P25 rats conditioned using a predictable, paired, procedure in early life and in adulthood resulted in a potentiated fear response in adulthood. One reason for these differences may arise from differences in methodology. For example, there was a difference in the cue duration. Previous research has found that not only will a 3-second tone cue produce a significant increase in the freezing response above and beyond that of a 20-second cue, but the freezing response is more resistant to extinction (Kiyokawa et al., 2015). The current findings are also

consistent with other work from our lab in which a the use of a 20-second cue during paired conditioning in early life and again during paired reconditioning did not potentiate freezing (Jones & Monfils, 2016). However, rats that exercised in adolescence within the P25 experiment did show an influence on conditioning in adulthood, such that there was a negative correlation between average distance run across the 21-days of exercise access and freezing during the long-term memory test irrespective of early life conditioning or not. This effect, however, was confounded by the fact that there was only a trend for a difference in this effect compared to the relationship seen in adult control rats.

Nonetheless, this finding can be supported by the fact that chronic exercise has been shown to produce stress resistance effects, that counter the negative consequences stress has on enhancing fear responses (Greenwood & Fleshner, 2011). While rats within the current set of experiments were not exposed to stress, this effect may indicate exercise acts as a buffer for future fear responding, where the conditioning procedure itself may serve as a stress-inducing event. Yet, this relationship may not significantly differ from

rats not exposed to conditioning in adulthood, as a serious detriment needs to be in place (such as prior uncontrollable stress exposure) for exercise to produce a robust enough effect. It is also important to note that there were no main effects of exercise over sedentary conditions on freezing during this long-term memory test, nor did any level of exercise result in a complete lack of a freezing response, indicating that exercise my not be eliminating the fear response all together but curbing the overall effect. Additionally, previous literature has shown that contextual fear conditioning (Baruch, Swain, &

Helmstetter, 2004; Greenwood, Strong, Foley, & Fleshner, 2009; Kohman et al., 2012) cued fear conditioning (Falls, Fox, & MacAulay, 2010) can be enhanced by chronic voluntary exercise. However, less is known about the relationship between distance run and fear responding, but work by Greenwood and colleagues found a correlation similar to ours between fear responding and exercise after stress exposure (Greenwood, Foley, Burhans, Maier, & Fleshner, 2005). Specifically, 6-weeks but not 3-weeks of voluntary wheel access was sufficient to reduce freezing levels to that of non-stressed controls.

The current findings add support to the knowledge that pre-weaning aversive experience produces more generalized neurobehavioral consequences that can be influenced by an indirect approach, such as chronic exercise. This is distinct from findings that aversive experiences acquired after weaning are not influenced in the same way. Future studies should examine the neural mechanisms that underlie the different behavioral effects of early life fear experience and delineate how chronic exercise may be influencing the similar negative correlations presented in this work. The present work highlights the fact that chronic exercise may have the potential to influence fear

responses after adult conditioning, and may serve to buffer, but not eliminate, the fear response.

Chapter 3: Effects of Acute Exercise on Fear Extinction in Rats and