Experiment 1: Effect of 30-min of exercise 2-h before cued

on extinction acquisition and subsequent memory

The aim of experiment 1 was to test the effect of a single 30-min bout of voluntary wheel running 2-h before extinction training on extinction acquisition and subsequent memory tests. We hypothesized that an acute bout of exercise 2-h prior to extinction would enhance extinction memories as indicated by reduced levels of freezing

at long-term memory (LTM) and/or reinstatement tests. To test these effects, we employed a 2x2 design in which rats underwent one of two habituation conditions in which there was a freely moving wheel available (Habituation) or an empty cage (No Habituation). This was done to reduce neophobia and allow rats to gain dexterity for wheel running, potentially enhancing the amount of voluntary exercise displayed during the exercise treatment prior to extinction. Subsequently rats were then divided into two groups in which prior to extinction training there was wheel access for exercising (Exercise) or a group that remained sedentary with no access to a wheel (Sedentary) (see Figure 3.1 for a timeline of experimental procedures). This resulted in four groups: No Habituation-Sedentary (SED (no hab); n = 8), Habituation-Sedentary (SED (hab); n = 8), No Habituation-Exercise (EX (no hab); n = 8), Habituation-Exercise (EX (hab); n = 10).

3.6.1.1 Methods

Subjects. Thirty-four male Sprague-Dawley albino rats (Harlan

Laboratories) were ordered at 275-300 g and housed in pairs throughout the entirety of the experiment. Rats were allowed one week to acclimate to the colony. All rats were housed in 42x20x20 cm polycarbonate cages. Housing rooms were maintained at a constant temperature (21 ± 1°C) and a 12-12 light-dark cycle (lights on at 6:00 and off at 18:00). Food and water were provided ad libitum throughout the entire experiment.

Equipment. All test procedures were conducted in a Habitest Modular System

conditioning chamber (Coulbourn Instruments) equipped with metal rod flooring connected to a shock generator and a speaker connected to a tone generator, and enclosed in a sound attenuating chamber. All behavioral testing occurred in the same context across all procedures. Chambers were illuminated with a red light and wiped with disinfectant cleaner between sessions. All videos were recorded from overhead cameras

and output to AVI files, to be scored by a researcher blinded to experimental conditions. Graphic state software controlled video recording and stimulus presentations.

For both the wheel Habituation and/or Exercise treatment sessions, all rats were exposed alone to polycarbonate cages exactly like the ones in which they were housed but with an attached 35.6 cm diameter running wheel (Harvard Apparatus) on one-half of the cage. Wheels were fitted with magnetic counters, which recorded every quarter turn of the wheel in either direction. In No Habituation and/or Sedentary sessions, rats were exposed to cages alone without a locked wheel. This was done to reduce the likelihood that they would climb on the wheel and engage in physical activity nullifying the comparison to freely moving wheels (Koteja, Garland, Sax, Swallow, & Carter, 1999).

Procedures.

Wheel habituation. To reduce novelty to the exercise wheels and to allow rats to gain dexterity for running on the wheels, the rats in the EX (hab) and SED (hab) groups were allowed 10-min of individual access to the exercise wheel three mornings preceding behavioral testing. Rats in the EX (no hab) and SED (no hab) were placed individually in an empty cage for the 10-min. Habituation or No Habituation procedures were continued throughout the experiment to reduce any potential confounds of stress related to removal of wheel access (Greenwood et al., 2012; Nishijima et al., 2013). All habituation procedures occurred in the last hour of the rat’s dark cycle (5:00-6:00), with at least 6-h between behavioral testing and habituation procedures. Between sessions, cages were wiped with 70% ethanol and bedding was replaced.

Fear acquisition. All rats were allowed 10-min of habituation to the conditioning chamber before receiving three conditioning trials of a tone (5 kHz for 20-s) co- terminating with a foot shock (0.7 mA for 0.5 s) separated by a variable intertrial interval (ITI) 3-min on average.

Treatment. Previous findings from Soya et al. (Soya et al., 2007) indicate that after a single 30-min bout of low intensity forced exercise there is a maximal 1.5- 2 fold increase of BNDF in the CA1, CA3, and dentate gyrus within the hippocampus 2-h after the start of exercise. This is further supported by Huang et al. (Huang et al., 2006) who also show an up-regulation of BDNF at a 2-h time point after forced exercise. Based on these findings we provided a single bout of voluntary exercise 2-h before the extinction training session expecting that upregulation of BDNF will occur as the extinction session begins to occur (i.e. during extinction acquisition). The use of voluntary exercise rather then forced exercise was chosen based on previous findings suggesting that forced exercise can be stressful, and even an acute stressor such as this has the potential to negatively impact fear extinction (Chauveau et al., 2012; Greenwood et al., 2003; Maroun et al., 2013); thus, by using voluntary wheel running we aimed to reduce exercise type as a potential confound. To measure voluntary running behavior the total distance run by each rat was measured. All wheels were equipped with magnetic counters to track every quarter turn of the exercise wheel, this data was recorded after each exercise session and converted into distance measurements.

Extinction. An extinction training session consisted of 19 presentations of the tone alone separated by a variable intertrial interval (ITI), 3-min on average.

Long-term memory test. 24-h after the extinction session, rats were exposed to three nonreinforced tone presentations separated by a variable intertrial interval (ITI) 3- min on average.

Reinstatement. 24-h after LTM rats were exposed to five unsignaled foot shocks with a fixed intertrial interval (ITI) of 1-min. All foot shocks were at the same (0.7 mA for 0.5-s) level used at the initial fear acquisition.

Reinstatement test. To test the reinstatement of freezing, 24-h after exposer to the unsignaled foot shocks three nonreinforced tone presentations were given, each separated by a variable intertrial interval (ITI) 3-min on average.

Measures.

Freezing behavior. Across all behavioral tests freezing was measured as a total number of seconds and expressed as a percentage of time during each 20-s cue presentation. Freezing was assessed for each individual trial for fear acquisition and extinction, whereas on memory tests the percentage of freezing was averaged across trials.

Analysis. A type III sums of squares Analysis of Variance (ANOVA) was used to assess group differences and included 2-factors, habituation condition (Habituation or No Habituation) and treatment (Exercise or Sedentary) across all outcomes. Additional linear models were run to test the relationship between average distance run across treatment session (in groups receiving wheel access) and freezing during long-term memory and reinstatement tests. Data were analyzed using RStudio (Version 0.99.902) using R (Version 3.3.0).

3.6.1.2 Results

Freezing behavior. For individual findings across all behavioral tests see Table

3.7. During fear acquisition a repeated measures ANOVA indicated a significant effect by trial indicating within session acquisition of freezing by CS3 (Figure 3.3A). Following the treatment session rats showed an overall main effect of trial indicating within session extinction occurred across groups (Figure 3.4A) and a significant habituation x treatment x trial interaction, such that there was no difference in extinction behavior until trials 12 to 15 and 17 for which an effect of exercise exists only in rats exposed to the habituation

condition. The next day rats were tested for LTM for which no significant differences in freezing between groups (Figure 3.4B). Finally, after reinstatement procedures a repeated measures ANOVA indicated significant reinstatement of freezing from LTM to reinstatement test across groups but no significant differences between groups in freezing behavior during the reinstatement test (Figure 3.4C).

Table 3.7 Primary results rat cued extinction experiments.

Experiment 1 Experiment 2 Experiment 3

df F p df F p df F p

Within Session Acquisition

habituation 1, 30 0.142 0.708 1, 28 7.416 0.011 treatment 1, 30 2.184 0.150 1, 28 0.434 0.515 2, 33 0.246 0.783 habituation x treatment 1, 30 1.521 0.227 1, 28 0.805 0.377 trial 2, 60 297.335 < 0.001 2, 56 181 < 0.001 2, 66 603.53 < 0.001 habituation x trial 2, 60 0.224 0.800 2, 56 2.428 0.097 treatment x trial 2, 60 0.766 0.469 2, 56 0.061 0.94 4, 66 0.593 0.669 habituation x treatment x trial 2, 60 1.229 0.300 2, 56 0.511 0.603

Within Session 1 Extinction

habituation 1, 30 0.702 0.409 1, 28 2.641 0.115 treatment 1, 30 0.062 0.805 1, 28 0.326 0.573 2, 33 0.219 0.805 habituation x treatment 1, 30 0.076 0.785 1, 28 0.569 0.457 trial 18, 540 8.524 < 0.001 18, 504 10.482 < 0.001 18, 594 3.625 < 0.001 habituation x trial 18, 540 1.528 0.075 18, 504 1.295 0.185 treatment x trial 18, 540 1.045 0.407 18, 504 0.636 0.872 36, 594 0.699 0.907 habituation x treatment x trial 18, 540 1.849 0.018 18, 504 0.725 0.786

Within Session 2 Extinction

habituation 1, 28 0.810 0.376 treatment 1, 28 0.962 0.335 2, 33 1.143 0.331 habituation x treatment 1, 28 1.903 0.179 trial 18, 504 25.052 < 0.001 18, 594 11.5 < 0.001 habituation x trial 18, 504 0.602 0.899 treatment x trial 18, 504 0.994 0.465 36, 594 0.727 0.88

Note. Empty units are data not applicable to that individual experiment Table 3.7 cont. Primary results rat cue based extinction experiment

Experiment 1 Experiment 2 Experiment 3

df F p df F p df F p

Long-term Memory Session 1

habituation 1,30 0.985 0.329 1,28 0.172 0.682

treatment 1,30 0.005 0.947 1,28 0.011 0.916 2,33 0.699 0.504

habituation x treatment 1,30 0.883 0.355 1,28 0.429 0.518

Long-term Memory Session 2

treatment 2,33 0.311 0.735

Reinstatement Test

habituation 1,30 0.039 0.845 1,28 0.134 0.717

treatment 1,30 0.008 0.928 1,28 0.778 0.385 2,33 1.04 0.365

habituation x treatment 1,30 0.267 0.609 1,28 0.198 0.66

Long-term Memory Test to Reinstatement Test

habituation 1,30 0.008 0.930 1,28 0.19 0.666 treatment 1,30 0.417 0.523 1,28 0.257 0.617 2,33 0.803 0.456 habituation x treatment 1,30 0.042 0.840 1,28 0.387 0.539 test 1,30 16.000 < .001 1,28 35 < .001 1,33 14.336 0.001 habituation x test 1,30 0.001 0.974 1,28 0.019 0.89 treatment x test 1,30 0.710 0.406 1,28 0.585 0.451 2,33 0.262 0.771

Figure 3.3 Fear acquisition. (A-C) Rats in all groups show within session fear acquisition, p’s < 0.001. (B) Data indicate a main effect of habituation condition, p = 0.01. Data are expressed as mean ± CI (n = 8–12 per group).

Figure 3.4 Experiment 1. (A) within session one extinction, (B) long-term memory and (C) reinstatement data used in cumulative analysis. (A) All rats show within session extinction, p < 0.001. (A) Data indicate a significant habituation by treatment by cue interaction, p = 0.02. Data are expressed as mean ± CI (n = 8–10 per group).

Voluntary running behavior. On average, both exercise groups ran similar

amounts (EX (no hab) M = 55.25, SD = 26.92; EX (hab) M = 68.70, SD = 33.65; t(16) = 0.94, p = 0.36). For average distances run across both groups during the intervention period, see Table 3.8. Data indicated a non-significant relationship between the average

distance run and later freezing during the LTM test (r(17) = -0.16, p = 0.52, Figure 3.5A)2_{. However, there was a significant, negative relationship between average distance}

run and later freezing during the reinstatement test (r(17) = -0.59, p = 0.01, figure 3.6A). It is important to note that this significant relationship appears to be driven by one outlier who ran a distance 2.67 standard deviations above the mean because when this outlier is removed the negative relationship observed is no longer significant. However, since the purpose of this study was to examine voluntary exercise the outlier was retained for all analyses.

Average Distance (meters) SD

30-min of access Experiment 1 62.72 30.74 Experiment 2 64.31 22.07 3-h of access Experiment 3 126.58 99.69 Experiment 4 148.08 128.95

Table 3.8 Average distance in meters run across each experiment.

2_{Initial analysis found no differences between the EX (no hab) and EX (hab) groups in}

the relationship between average distances run prior to the extinction sessions and later freezing behavior during the LTM test or the reinstatement test. Therefore, data from the EX (no hab) and EX (hab) were combined.

Figure 3.5 Linear relationship between average distance run before extinction

session(s) and freezing during the long-term memory test. (A, B, D) and

the second (final) long-term memory test (C) after extinction. No significant relationships were observed across all experiments. (A-B) Geometric shapes indicate habituation condition; there were statistically no differences

between groups so data was combined. Note: experiments 1 and 2 rats were allowed 30-min of wheel access whereas experiments 3 and 4 there was 3-h of access.

Figure 3.6 Linear relationship between average distance run before extinction

session(s) and freezing during relapse tests. Reinstatement (A-C) or

spontaneous recovery (D). (A) Data indicate a significant negative

relationship between distance run and freezing, R2 _{= .3486 p = .01, however} data are driven by one rat who ran a significantly greater distance, see result section for greater details. (B, C, D) No significant relationships were observed. (A-B) Geometric shapes indicate habituation condition; there were statistically no differences between groups so data was combined. Note: experiments 1 and 2 rats were allowed 30-min of wheel access whereas experiments 3 and 4 there was 3-h of access.