Voluntary exercise improves both learning and consolidation of cued conditioned fear in C57 mice

Behavioural Brain Research 207 (2010) 321–331

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Research report

Voluntary exercise improves both learning and consolidation of cued conditioned fear in C57 mice William A. Falls ∗ , James H. Fox, Christina M. MacAulay Department of Psychology, University of Vermont, John Dewey Hall, 2 Colchester Ave., Burlington, VT 05405, USA

a r t i c l e

i n f o

Article history: Received 4 August 2009 Received in revised form 5 October 2009 Accepted 10 October 2009 Available online 29 October 2009 Keywords: Exercise Learning Memory Consolidation Conditioned fear Anxiety Acoustic startle

a b s t r a c t Exercise is associated with improved cognitive function in humans as well as improved learning across a range of tasks in rodents. Although these studies provide a strong link between exercise and learning, to date studies have largely focused on tasks that principally involve the hippocampus. However, exercise has been shown to produce alterations in other brain areas suggesting that the cognitive enhancing effects of exercise may be more general. Therefore we set out to examine the effects of voluntary exercise on cued Pavlovian fear conditioning, a form of learning that is critically dependent on the amygdala. In Experiment 1 we showed that mice given 2 weeks of access to a running wheel prior to tone and foot shock fear conditioning showed enhanced conditioned fear as measured by fear-potentiated startle. This effect was not the result of altered shock reactivity nor was it to due to reduced baseline startle amplitude in exercising mice. In subsequent experiments we sought to examine whether the enhanced cued conditioned fear was the result of an improvement in learning, consolidation or retrieval of conditioned fear. In separate groups of mice, two weeks of access to a running wheel was begun either prior to fear conditioning, immediately after fear conditioning (consolidation period) or 2 weeks after fear conditioning. Compared to sedentary mice, mice that exercised either prior to fear conditioning, or immediately after fear conditioning, showed enhanced cued conditioned fear. Fear conditioning was not enhanced in mice that began exercising 2 weeks after fear conditioning. Taken together these results suggest that voluntary exercise improves the learning and consolidation of cued conditioned fear but does not improve the retrieval or performance of conditioned fear. Because a great deal is known about the neural circuit for cued conditioned fear, it is now possible to examine the cellular, molecular and pharmacological changes associated with exercise in this well-understood neural circuit. © 2009 Elsevier B.V. All rights reserved.

1. Introduction It has been known for some time that physical activity (i.e., exercise) improves physical health by benefiting the cardiovascular system. In recent years it has been shown that physical activity also affects the brain [10,11]. Exercise in animals is associated with neurogenesis and increased neuronal survival in the hippocampus [5,24,92–95], capillary growth and increased vascular flow in cerebellum and motor cortex [4,45,86], increased expression of neurotrophins in several brain areas including the hippocampus and frontal cortex [34,67,68,96,97], changes in the expression of genes [88] and signaling molecules in the hippocampus [84] and enhancement of hippocampal long-term potentiation [24,92]. These alterations in the brain are thought to promote brain health and improve the long-term functioning of the brain. In humans, exercise is associated with improved cognitive function [40]. Similarly, exercise in rodents is associated with

∗ Corresponding author. Tel.: +1 802 656 5748; fax: +1 802 656 8783. E-mail address: [email protected] (W.A. Falls). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.10.016

improved learning and memory in several hippocampal-dependent tasks. For example, exercise improves learning in water maze [27–29,93], radial-arm maze [1], passive avoidance [71,80] and context-dependent freezing [2]. These data, together with the clear effects of exercise on hippocampal cell morphology, gene expression and neuropharmacology, suggest that a primary effect of exercise is to improve hippocampal function. Although studies have provided a link between exercise and learning, to date studies have largely focused on tasks that principally involve the hippocampus [2]. However, exercise has been shown to produce alterations in other brain areas including the cortex [68,86], cerebellum [4,45], raphe nucleus [35,37] and the amygdala [38] suggesting that exercise may have effects on learning that involve structures in addition to the hippocampus. The goal of the present study is to examine the effects of voluntary exercise on cued Pavlovian fear conditioning, a form of learning that is not critically dependent on the hippocampus [22,46,69]. In cued Pavlovian fear conditioning, a neutral stimulus, such as a tone, is paired with mild foot shock. Following this pairing, the tone acquires the ability to elicit a variety of behaviors indicative of a state of fear [59], such as freezing [20] and potentia-

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tion of the acoustic startle reflex [14]. Many studies have shown that the amygdala plays a critical role in cued fear conditioning (e.g., [41,46,48,51,54,56,65,66,70,74,82]) and is central to a well-understood neural circuit for conditioned fear [13,23,25,50]. Therefore, examining the effect of exercise on cued fear conditioning will not only test the generality of the beneficial effects of exercise on learning, but it will also provide the opportunity to examine the cellular, molecular and pharmacological changes associated with improved learning across this well understood neural circuit. In Experiment 1 we examined whether two weeks of voluntary exercise would affect cued conditioned fear. Male C57BL/6J mice were given access to a running wheel in their home cage. For one group of mice the wheels were functional and for the other group, the wheels were locked preventing running. After 14 days of access to the running wheel, mice underwent Pavlovian fear conditioning in which a tone was paired with foot shock. Fear conditioning was assessed using the fear-potentiated startle procedure [14] in which conditioned fear is operationally defined as elevated startle amplitude in the presence versus absence of the cue that was previously paired with shock. The results of Experiment 1 indicate that two weeks of exercise enhanced conditioned fear to a tone previously paired with shock. However, in this experiment, as in most examining the effects of exercise, exercise was initiated prior to learning (e.g., conditioned fear) and was continued through to testing. Thus, it is difficult to determine whether voluntary exercise improves learning, consolidation or retrieval of Pavlovian conditioned fear. In an effort to isolate which of these processes are affected by voluntary exercise, we ran additional experiments in which exercise was restricted to time periods that would most likely affect learning, consolidation, or retrieval of conditioned fear. 2. Experiment 1: voluntary exercise enhances cued conditioned fear 2.1. Method 2.1.1. Animals Eight weeks old, male C57BL6/J mice were obtained from Jackson Laboratories in Bar Harbor, Maine. Mice were housed in groups of two–four in standard acrylic cages (24 cm (W) × 45 cm (D) × 20 cm (H)) located in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved conventional animal facility. Mice were maintained on a 12-h light/dark cycle (lights on at 07:00 h) with food and water available at all times. Fear conditioning and testing were carried out between 09:00 and 14:00 h. A 7-day acclimation period was given to mice before introduction of the running wheels. All procedures were approved by the University of Vermont Animal Care and Use Committee. 2.1.2. Voluntary wheel running Mice were given ad lib access to a running wheel (Superpet mini run-a-round, measuring 11.4 cm in diameter) for two weeks prior to the start of behavioral testing. For half of the cages, the wheels were locked preventing running (sedentary control) and for the remaining cages the wheels were functional. 2.1.3. Apparatus Fear conditioning and fear-potentiated startle testing was carried out in eight sound attenuating cubicles measuring 58 cm (W) × 32 cm (D) × 55 cm (H). Each cubicle was lined with black, sound absorbing foam with no internal source of light. Each cubicle contained a stabilimeter device consisting of a load cell platform onto which the behavioral chamber was mounted. The chamber was constructed of clear acrylic, cylindrical in shape, 12.5 cm in

length, with an inner diameter of 5 cm. The floor of the chamber consisted of a removable shock grid composed of six steel rods 3.2 mm in diameter, and spaced 6.4 mm apart (Med-Associates, Georgia, VT) through which shock could be administered. Startle responses were transduced by the load cell, amplified, and digitized over a range of 0–4096 units. Startle stimuli and the tone conditioned stimulus (CS) were provided through a Radio Shack Supertweeter located 10 cm behind the behavioral chamber. Data collection and the control and sequencing of all stimuli were controlled by Med-Associates startle reflex hardware and software (Georgia, Vermont). Startle amplitude was defined as largest peak-to-peak value within 100 ms after the onset of the startle stimulus. 2.1.4. Procedure Mice were randomly assigned to sedentary and exercising conditions. Experimental procedures began 14 days after introduction of the running wheels. We have previously shown that mice gradually increase their running distance over the first 7 days of wheel access and by day 14 run an average of 18.3 km (±0.7 km), 17.6 km during the dark cycle alone [79]. The amount of time mice run is also stable over the 14 days averaging 578 min (±19.9 min) during the dark cycle and 58.7 min (±10.6 min) during the light cycle. We have also shown, using near-infrared video monitoring during the dark cycle, that, on average, all 4 mice in a group cage run on the wheel an equivalent proportion of the total time. Prior to fear conditioning, mice were given startle stimulus alone test sessions in order to assess the effects of voluntary exercise on startle amplitude and to acclimate the mice to handling and the behavioral chambers prior to fear conditioning [19]. For these tests, mice were placed in the startle apparatus and presented with startle stimulus alone trials. A 5-min acclimation period preceded presentation of any stimuli. The startle stimuli were 20-ms white noise bursts with a rise-decay time of 1 ms. Thirty startle stimuli were presented in a pseudorandom order. Ten stimuli of each intensity level (95, 100, 105 dB) were presented with an inter-trial interval (ITI) of 60 s. A total of three startle stimulus alone test sessions were given, one session per day for three consecutive days. These data were not used. Following startle stimulus alone test sessions, mice were returned to the startle chambers and given a pre-conditioning potentiated startle test in order to assess the unconditioned effect of the tone conditioned stimulus on startle amplitude before fear conditioning [19,39]. This test consisted of nine startle stimulus alone trials (three each at 95, 100 and 105 dB) followed by nine tone + startle stimulus trials (three each at 95, 100 and 105 dB) intermixed with an additional nine startle stimulus alone trials (three each at 95, 100 and 105 dB). The interval between successive startle stimuli was 60 s. Trials were presented in a pseudorandom order with the constraint that one of each intensity was presented in each of three blocks. The tone CS was a 30-s, 12-kHz 70-dB pure tone. The startle stimulus occurred 29.75 s after the onset of the tone conditioned stimulus. Conditioning was conducted 72 h following the pre-training test for fear-potentiated startle. Conditioning consisted of 5 tone + shock trials in which the 30-s tone coterminated with a 0.25s, 0.4-mA foot shock. The average ITI for conditioning was 2 min. Reactivity to the foot shock was measured during each fear conditioning trial by integrating the output of the stabilimeter over the 250 ms shock period. This allowed us to assess whether shock reactivity differed between the groups over the course of fear conditioning. The day following fear conditioning, mice were returned to the startle chamber for a post-conditioning potentiated startle test session. This test session was identical to that given prior to training.

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Table 1 Startle amplitude for the three pre-conditioning startle stimulus alone test sessions in Experiment 1. Test #1

Test #2

Test #3

95 db

100 db

105 db

95 db

100 db

105 db

95 db

100 db

105 db

Sedentary sem

889.1 91.9

1139.8 75.7

1335.9 93.7

801.4 87.3

1242.6 110.2

1364.8 108.6

778.3 78.0

1197.0 95.6

1173.0 88.0

Exercising sem

497.3 54.7

683.7 64.6

554.3 61.0

657.1 75.4

791.3 67.5

507.3 50.6

616.9 49.8

637.8 48.6

764.5 61.3

Numbers in italics represent 1 sem. Mice in the exercising groups had lower startle amplitude across the three test sessions (p < 0.001).

Conditioned fear was defined as an increase in potentiated startle to the tone following fear conditioning. 2.1.5. Data reduction and statistics For the startle stimulus alone tests, mean startle amplitudes were computed for each mouse at each stimulus intensity. For the tests of potentiated startle (pre-conditioning, post-conditioning), mean startle amplitudes were computed for startle stimulus alone (SS) and tone plus startle stimulus trials (CS). Following the recommendations of Walker and Davis [100], percent potentiated startle was computed for each mouse using the formula ([CS − SS]/SS × 100). Larger percentages represent greater potentiated startle. The mean startle amplitudes for startle stimulus alone trials during the potentiated startle tests were analyzed separately. All data were analyzed using a mixed model ANOVA with group (sedentary versus exercising) as a between subjects factor and test and startle stimulus intensity as within subjects factors. Startle amplitude in the absence of the tone was analyzed separately (see Table 2). Significant interactions were followed by lower order ANOVA’s and t-tests. The alpha level for all tests was set as 0.05. 2.2. Results 2.2.1. Startle stimulus alone tests prior to fear conditioning Mice given free access to a functioning running wheel had lower startle amplitude across all three test sessions (F(1,30) = 35.6; Table 1). Startle amplitude increased with increasing startle stimulus intensity (F(2,120) = 53.6). No other main effects or interactions were statistically reliable. 2.2.2. Shock reactivity during fear conditioning Shock reactivity was measured for each mouse during fear conditioning by integrating the output of the stabilimeter over the 250 ms shock period. Fig. 2 shows that shock reactivity did not differ between the exercise groups (F < 1) nor did it change over the course of the conditioning session (F < 1). Thus, any differences in fear-potentiated startle between the exercise groups following fear conditioning cannot be easily attributed to differences in the response to foot shock. 2.2.3. Fear-potentiated startle Exercising mice showed greater fear-potentiated startle (group by test interaction F(1,30) = 4.62; Fig. 1). Startle potentiation in the pre-conditioning potentiated startle test did not differ between the groups (F < 1) suggesting that the unconditioned effects of the tone were not affected by exercise. However, following fear conditioning the tone produced a greater increase in potentiated startle in exercising mice (F(1,30) = 8.24). These data suggest that voluntary exercise prior to fear conditioning results in an enhancement in cued conditioned fear. 2.2.4. Startle amplitude in the absence of the tone CS Exercising mice had lower startle amplitude in the absence of the tone both before and after conditioning (Table 2.

Fig. 1. Two weeks of voluntary exercise prior to tone and foot shock fear conditioning resulted in enhanced conditioned fear. Mice were given free access to a running wheel for two weeks following which they were given 5 tone and foot shock fear conditioning trials. Exercising and sedentary mice did not differ in fear-potentiated startle prior to fear conditioning (F < 1) suggesting that exercise does not alter the unconditioned effect of a tone on startle. However, after fear conditioning, exercise mice showed greater fear-potentiated startle (F(1,30) = 8.24) suggesting that exercise enhanced cued conditioned fear.

F(1,30) = 18.04). Sedentary, but not exercising, mice showed an increase in startle amplitude following fear conditioning (F(1,30) = 9.09; before conditioning versus after conditioning t-test, t(15) = 3.63 and t(15) = 0.47 for sedentary and exercising groups respectively).

Table 2 Startle amplitude for startle stimulus alone trials Experiments 1 and 2. Group

Experiment 1: potentiated startle tests

Experiment 2: shock sensitization

Before conditioning

After conditioning

Before shock

Sedentary

687.4 73.1

984.8 90.7

823.5 93.7

Exercising

444.5 58.9

479.7 67.4

366.1 46.4

Numbers in italics represent 1 SEM. Mice in the exercising groups had lower startle amplitude (Experiment 1: before conditioning, p = . 015; after conditioning: p < 0.001; Experiment 2 before shock p < 0.001). In Experiment 1, sedentary, but not exercising, mice showed an increase in startle amplitude following fear conditioning (p = 0.002 and p = 0.65 for sedentary and exercising groups respectively).

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2.3. Discussion Several experiments have demonstrated that voluntary exercise enhances learning and memory in rodents [1,2,6,27–29,91,93]. It is noteworthy that Baruch et al. [2] were the first to show that conditioned fear is enhanced by prior voluntary exercise (see also [6,38,91]). However, in their experiment the beneficial effect of exercise was limited to contextual fear, cued fear was not enhanced by prior exercise. Although contextual fear has been measured with the fear-potentiated startle procedure [31,61], we did not arrange to explicitly measure contextual fear. However, we did observe and increase in startle amplitude on startle stimulus alone trials following fear conditioning which may be interpreted as contextual fear. However, the increase in startle amplitude was only observed in sedentary mice. If this was contextual fear we would have expected to see greater contextual potentiation in exercising mice consistent with studies showing increased contextual fear following exercise [2,6,38,91]. While any number of procedural differences can account for the fact that we observe enhanced cued fear (species, fear conditioning parameters, measure of conditioned fear, etc.) one deserves particular attention. We have previously shown that voluntary exercise reduces startle amplitude in mice and that this effect is associated with a reduction in anxiety-like behaviors across a number of tasks [79]. However, this raises the possibility that the greater fear-potentiated startle in the exercising group is a consequence of lower startle amplitude rather than improved fear conditioning. Although mice in the sedentary group were not at a measurement ceiling (the average startle amplitude on tone and startle stimulus trials was 1066 out of a possible 4095), mice in the exercising group had a larger range in which to show fear-potentiated startle. It is precisely because of the differences in startle amplitude between the exercise groups that we chose to evaluate fear-potentiated startle using a proportional measure. Walker and Davis [100] have shown that, across groups with different startle baselines, conditioned fear is most accurately reflected in the proportional increase in fear-potentiated startle. In their experiment, manipulations that produce changes in baseline startle without affecting fear did not produce changes in fear-potentiated startle as measured by proportional increases in startle. Thus, proportional measures of fear-potentiated startle seem to reflect the amount of conditioned fear independent of startle amplitude. In the present experiment, mice given free access to the running wheel showed a larger proportional increase in fear-potentiated startle (Fig. 1) suggesting greater conditioned fear. Moreover, exercising and sedentary mice showed similar unconditioned potentiation of startle by the tone in the preconditioning potentiated startle test (Fig. 1; before conditioning). The unconditioned effects of a tone on startle amplitude are thought to reflect an interaction between the tone and the startle stimulus within the auditory system. For example, the tone may decrease the threshold for recruitment of neurons in the startle pathway [7]. The fact that this ‘potentiation’ of startle was not enhanced in exercising mice suggests that their lower startle amplitude is not simply more susceptible to potentiation. However, in order to attempt to evaluate the possibility that mice in the exercise group show greater fear-potentiated startle because they have lower startle amplitudes, we conducted a second experiment in which we evaluated shock sensitization of startle in mice given voluntary access to a running wheel. Shock sensitization of startle is thought an unconditioned response to foot shock or a rapid form of contextual fear [12,75]. We have already shown in the present experiment that mice given voluntary access to a running wheel do not show altered shock reactivity (Fig. 2). If mice in the exercise group have a greater likelihood of startle potentiation owing to their lower startle amplitudes, they may also show greater shock sensitization of startle. If, on the other hand, mice in

Fig. 2. Shock reactivity during tone and foot shock fear conditioning in Experiment 1. Shock reactivity was similar in exercising and sedentary mice (F < 1) suggesting that the difference in cued conditioned fear (Fig. 1) is not directly attributable to differences in shock reactivity.

the sedentary group show greater fear-potentiated startle because of greater conditioned fear alone, they may be expected to not show greater shock sensitization of startle as it reflects an unconditioned response to foot shock. 3. Experiment 2—voluntary exercise does not alter shock sensitization of startle 3.1. Method 3.1.1. Subjects Eight weeks old, male C57BL6/J (n = 32) mice were obtained from Jackson Laboratories in Bar Harbor, Maine. Mice were housed in Experiment 1. One mouse from the exercise group was euthanized prior to the start of behavioral testing because of hydrocephalus. 3.1.2. Apparatus The apparatus was identical to that used in Experiment 1. 3.1.3. Procedure As in Experiment 1, mice were given 1 week of acclimatization to the colony prior to introduction of the running wheels. Mice were randomly assigned to sedentary (n = 16) and exercising (n = 15) running conditions. Experimental procedures began 14 days after introduction of the running wheels. Prior to shock sensitization testing, mice were given startle stimulus alone test sessions as described in Experiment 1. Briefly, mice were placed in the startle apparatus and, after a 5-min acclimation period, were given the first of 30 startle stimulus alone trials. Ten stimuli of each intensity level (95, 100, 105 dB) were presented with a mean ITI of 60 s. A total of three startle stimulus alone test sessions were given, one session per day for three consecutive days. Twenty-four hours following the last startle stimulus alone test session, mice were returned to the startle chambers for shock sensitization testing. Following a 5 min acclimation period, mice were presented with the first of 30 startle stimulus alone trials (10 each of 95, 100, 105 dB) at an ITI of 60 s. One minute after trial 30, the mice were presented with a single train of 10 foot shocks given

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3.3. Discussion Voluntary exercise does not affect shock sensitization of startle. Shock sensitization may be associated with acute stress-like responses or rapid acquisition of contextual fear [75,76]. Importantly for the present experiment, the fact that the exercise group did not show greater shock sensitization than the sedentary group, suggests that the lower startle amplitude in the exercising group is not necessarily more apt to be potentiated. Thus, the idea that fearpotentiated startle is exaggerated in the exercising group simply because of lower startle amplitude is not supported (also see Experiment 4). It should be noted that Richardson [75,76] has argued that shock sensitization reflects a form or rapid context conditioning. As such, our data would suggest that such rapidly acquired contextual fear is not affected by prior voluntary exercise, an effect also reported by Greenwood et al. [38]. 4. Experiment 3: voluntary exercise initiated before or after fear conditioning enhances cued conditioned fear

Fig. 3. Two weeks of voluntary exercise did not affect shock sensitization of acoustic startle. Mice were given free access to a running wheel for two weeks following which they tested for acoustic startle before and after 10 unsignaled foot shocks. There were no differences between exercising and sedentary mice in the ability of foot shock to sensitize startle (F < 1). Thus, enhanced fear-potentiated startle (Fig. 1) is not the direct consequence of lower startle amplitude in exercising mice (see Table 2).

with a 10-s inter-stimulus interval. Each foot shock was 250-ms in duration and was 0.4 mA. One minute after the final foot shock the mice were given the first of 30 startle stimulus alone trials identical to those given prior to shock.

3.1.4. Data reduction and statistics For the startle stimulus alone tests, mean startle amplitudes were computed for each mouse at each stimulus intensity. For the shock sensitization test mean startle amplitudes were computed for startle stimulus trials given before foot shock and those given after foot shock. Percent shock sensitization of startle was computed for each mouse using the formula ([post-shock − pre shock]/pre shock × 100). Larger percentages represent greater shock sensitization. All data were analyzed using a mixed model ANOVA with group (sedentary versus exercise) as a between subjects factor and startle stimulus intensity as a within subjects factor. The alpha level for all tests was set as 0.05.

3.2. Results 3.2.1. Startle stimulus alone tests prior to shock sensitization Exercising mice had lower startle amplitude (F(1,29) = 21.28; Table 2). Startle amplitude increased with increasing startle stimulus intensity (F(2,116) = 25.93; data not shown) with the increase in startle amplitude being pronounced in the sedentary group (F(2,116) = 10.39, data not shown).

3.2.2. Shock sensitization Both the sedentary and exercising groups showed shock sensitization of startle (t(15) = 2.59 and t(14) = 4.04 respectively) but there were no differences between the groups (Fig. 3; F < 1). The main effect of startle stimulus intensity and the group by intensity interaction were not significant.

The goal of Experiment 3 is to determine whether voluntary exercise initiated immediately after fear conditioning and before testing would also improve cued fear conditioning. In this experiment mice were given access to a running wheel either for the two weeks before fear conditioning, or the two weeks after fear conditioning. If the benefit of voluntary exercise is limited to learning, then only mice exercising prior to fear conditioning should show enhanced fear-potentiated startle. If, on the other hand, exercise can affect post acquisition processes, such as consolidation or memory retrieval, then mice that exercise after fear conditioning should also show enhanced fear-potentiated startle. 4.1. Method 4.1.1. Subjects Eight weeks old, male C57BL6/J (n = 88) mice were obtained from Jackson Laboratories in Bar Harbor, Maine. Mice were housed as described in Experiment 1. 4.1.2. Apparatus The apparatus was identical to that used in Experiment 1. 4.1.3. Procedure Mice were given access to functioning or locked running wheels for 14 days. Mice were then given a pre-conditioning test of potentiated startle followed by fear conditioning as described in Experiment 1. Immediately after fear conditioning half of the functioning running wheels were locked and half of the locked running wheels were unlocked. The post-conditioning test for potentiated startle occurred two weeks later. Thus, there were four groups: mice that were sedentary for the two weeks before and the two weeks after fear conditioning (group Sedentary/Sedentary, n = 24), mice that exercised before and after fear conditioning, group Exercise/Exercise n = 24) and mice that either exercised before (group Exercise/Sedentary n = 20) or after fear conditioning (Sedentary/Exercise n = 20). 4.1.4. Data reduction and statistics Percent potentiated startle was computed for each mouse and data were analyzed using a mixed model ANOVA with group (sedentary/sedentary, exercising/sedentary, sedentary/exercising, and exercising/exercising) as a between subjects factor and test (before conditioning and after conditioning) as a within subjects factor. Startle amplitude in the absence of the tone CS was analyzed separately (see Table 3).

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Table 3 Startle amplitude for startle stimulus alone trials Experiment 3. Group

Potentiated start tests Before conditioning

After conditioning

(1) Sedentary/sedentary

950.0 88.1

1048.9 115.0

(2) Sedentary/exercising

822.9 67.1

689.0 88.6

(3) Exercising/sedentary

668.1 79.9

606.1 55.1

(4) Exercising/exercising

662.5 38.4

613.5 62.8

Numbers in italics represent 1 SEM. Mice that had exercised have lower startle amplitude (before conditioning: 1 and 2 versus 3 and 4, p = 0.031; after conditioning: 1 versus 2–4, p < 0.001). Unlike Experiment 1, sedentary/sedentary mice did not show an increase in startle amplitude following fear conditioning (p = 0.373).

4.2. Results 4.2.1. Fear-potentiated startle Mice that exercised either before fear conditioning, after fear conditioning, or throughout the experiment, showed greater fearpotentiated startle than mice who did not exercise (Fig. 4; group by test interaction; F(3,84) = 2.75). There were no group differences in potentiated startle before conditioning (F < 1). However, after fear conditioning, all exercising groups showed greater potentiated startle than the sedentary group (pairwise comparisons (Fisher’s LSD, ps < 0.05). All groups showed fear-potentiated startle (before conditioning versus after conditioning, ps < 0.05). 4.2.2. Startle amplitude in the absence of the tone CS Startle amplitude was lower in exercising mice than in sedentary mice prior to fear conditioning (planned contrast comparing mice that exercised (exercising/sedentary and exercising/exercising) to those that did not (sedentary/sedentary and sedentary/exercising; F(1,88) = 4.82; Table 3). After fear condition-

ing, startle amplitude was lower in all exercising groups than in the sedentary group, even for the group that exercised before conditioning only (pairwise comparisons (Fisher’s LSD, ps < 0.03; Table 3). Thus, the startle reducing effect of exercise persists for at least two weeks after wheel running is stopped. There were no differences in startle amplitude among the groups that had an opportunity to exercise. Unlike in Experiment 1, startle amplitude did not change from the pre-conditioning to the post-conditioning in sedentary mice (t(23) = 0.91). 4.3. Discussion As in Experiment 1, exercise prior to fear conditioning improves fear-potentiated startle. The improvement is evident even in mice that became sedentary immediately after fear conditioning suggesting that exercise after fear conditioning or before testing is not required to show improvement in conditioned fear. However, the most significant finding of this experiment is that voluntary exercise following fear conditioning also improves conditioned fear. This suggests that, in addition to affecting learning, voluntary exercise affects post acquisition processes, such as memory consolidation or memory retrieval. However, because mice exercised after fear conditioning and immediately before testing, it is not possible to determine whether exercise is affecting consolidation or retrieval. In an effort to tease these apart, we carried out Experiment 4 in which we attempted to isolate the effect of exercise to either the period after fear conditioning or the period before testing. 5. Experiment 4—voluntary exercise following fear conditioning, but not before potentiated startle testing, is associated with an enhancement in cued Pavlovian fear conditioning The goal of Experiment 4 is to determine whether exercise immediately after fear conditioning (i.e., during consolidation) or immediately before testing (prior to retrieval) affects cued conditioned fear. Mice were given fear conditioning and then tested for potentiated startle four weeks later. Separate groups of mice were given access to running wheels for the two-week period immediately after fear conditioning or the two-week period immediately before testing. If exercise effects memory consolidation, then mice given access to the running wheel after fear conditioning should show improved fear-potentiated startle. On the other hand, if exercise improves memory retrieval, then mice given access to the running wheel prior to testing should show improved fearpotentiated startle. 5.1. Method 5.1.1. Subjects Eight weeks old, male C57BL6/J (n = 66) mice were obtained from Jackson Laboratories in Bar Harbor, Maine and were housed as described in Experiment 1. 5.1.2. Apparatus The apparatus was as described in Experiment 1.

Fig. 4. Two weeks of voluntary exercise prior to, or immediately after, tone and foot shock fear conditioning resulted in enhanced conditioned fear. Mice were given two weeks free access to a running wheel prior to tone and foot shock fear conditioning (exercising/sedentary), after tone and foot shock fear conditioning (sedentary/exercising) or before and after fear conditioning (exercising/exercising). There were no group differences in fear-potentiated startle before conditioning (F < 1). However, after fear conditioning, all exercising groups showed greater fearpotentiated startle than the sedentary group (pairwise comparisons (Fisher’s LSD, ps < 0.05) suggesting that exercise effects learning ass well as the consolidation or retrieval of cued conditioned fear.

5.1.3. Procedure Mice were given access to locked running wheels for 14 days. Mice were then given a test of potentiated startle before fear conditioning followed by fear conditioning as described in Experiment 1. Wheels in group Sedentary remain locked for the entire four-week fear conditioning to test interval. In group Post-Conditioning Exercise, the wheels were unlocked immediately after fear conditioning

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Table 4 Startle amplitude for startle stimulus alone trials Experiment 4. Group

Potentiated start tests Before conditioning

After conditioning

(1) Sedentary

753.4 71.4

877.5 97.3

(2) Post-conditioning exercise

842.7 111.8

742.5 108.1

(3) Pre-test exercise

785.3 82.1

591.4 64.4

Numbers in italics represent 1. SEM. There were no group differences in startle amplitude before fear conditioning. However, mice that exercised the two weeks immediately before testing (Group 3 above) had lower startle amplitude than nonexercising mice after conditioning (p = 0.032). Sedentary mice did not show an increase in startle amplitude following fear conditioning (p = 0.215).

and remained unlocked for two weeks. The wheels in group PostConditioning Exercise were then locked and the wheels in group Pre-Test Exercise were unlocked. Two weeks later all groups were tested for potentiated startle as described in Experiment 1. Thus, there were three groups: mice that were sedentary for the four weeks between fear conditioning and testing (group Sedentary, n = 22), mice that exercised for the two weeks after fear conditioning and were sedentary for the two weeks before testing (group Post-conditioning Exercise; n = 22) and mice that were sedentary for the two weeks after fear conditioning and exercised the two weeks before testing (group Pre-test Exercise; n = 22). 5.1.4. Data reduction Percent potentiated startle was computed for each mouse and data were analyzed using a mixed model ANOVA with group (Post-conditioning exercise, Pre-test Exercise and Sedentary) as a between subjects factor and test (before conditioning and after conditioning) as a within subjects factor. Startle amplitude in the absence of the tone CS was analyzed separately (see Table 4). 5.2. Results 5.2.1. Fear-potentiated startle Mice that exercised after fear conditioning, but not those that exercised before testing, showed improved fear-potentiated startle (Fig. 5; group by test interaction; F(2,63) = 3.46). There were no group differences in potentiated startle before fear conditioning (F < 1). After fear conditioning, group Post-conditioning showed greater potentiated startle than either group Pre-Test or group Sedentary (ps < 0.03). All groups showed fear-potentiated startle (before conditioning versus after conditioning, ps < 0.05). 5.2.2. Startle amplitude in the absence of the tone CS There were no group differences in startle amplitude in the absence of the tone CS prior to fear conditioning (F < 1). However, at the post-conditioning potentiated startle test, group Pre-test Exercise had lower startle amplitude than group Sedentary (pairwise comparisons (Fisher’s LSD, ps < 0.03; Table 4). Group Postconditioning Exercise did not differ from either group Sedentary or group Pre-test Exercise. This suggests that the reduction in startle amplitude produced by voluntary exercise may not always last once wheels are locked (see Experiment 3 above), and more importantly, that improved fear-potentiated startle does not require an exerciseassociated reduction in startle amplitude (group Post-conditioning Exercise), and likewise, a reduction in startle amplitude does not result in improved fear-potentiated startle (group Pre-test Exercise). Finally, there was no change in startle amplitude from the pre-conditioning to post-conditioning test in sedentary mice (t(21) = 1.27).

Fig. 5. Two weeks of voluntary exercise immediately after tone and foot shock fear conditioning but not the two weeks before fear-potentiated startle testing, resulted in enhanced conditioned fear. Mice were given two weeks free access to a running wheel beginning immediately after tone and foot shock fear conditioning (post-conditioning exercise) or beginning two weeks after fear conditioning (pre-test exercise). All mice were tested for fear-potentiated startle 4 weeks after fear conditioning. There were no group differences in fear-potentiated startle before fear conditioning (F < 1). Mice that exercised after fear conditioning, but not those that exercised before fear-potentiated startle testing, showed improved fearpotentiated startle. After fear conditioning, group post-conditioning showed great fear-potentiated startle than either group pre-test or group Sedentary (ps < 0.03).

6. Discussion Our results show that two weeks of voluntary exercise in mice occurring either before (Experiments 1 and 3) or immediately after tone and foot shock pairings (Experiments 2 and 4) improves conditioned fear as measured with the fear-potentiated startle effect. The improvement in fear conditioning was not attributable to altered shock reactivity during fear conditioning (Experiment 1) nor was it a direct consequence of the reduction in acoustic startle amplitude that accompanies voluntary exercise (Experiments 2 and 4). Voluntary exercise did not affect the retrieval or performance of conditioned fear as mice that began to exercise two weeks after tone and foot shock fear conditioning showed fear-potentiated startle that was similar to sedentary mice. These results suggest that voluntary exercise improves learning and consolidation of cued conditioned fear. Several recent studies have shown that voluntary exercise improves Pavlovian contextual conditioned fear [2,6,8,38,91]. Although contextual fear has been measured with the fearpotentiated startle procedure [31,61], we did not arrange to explicitly measure contextual fear. Nevertheless, we did observe an increase in startle amplitude in the absence of the tone CS following fear conditioning (see Tables 2–4). While this may be interpreted as evidence of contextual fear, it was only observed in sedentary mice and was only statistically reliable in Experiment 1 (it is worth noting that Experiment 1 is the only experiment in the series where post-conditioning potentiated startle testing occurred 24 h after fear conditioning. Perhaps this increase in startle amplitude in the absence of the CS weakens over time). Nevertheless, if the increase in startle amplitude in the absence of the CS were contextual fear, this result would be in contrast to the several reports that have shown that exercise improves contextual fear. However, this result is very difficult to interpret because startle in the absence of the tone CS may be influenced by prior presentations of the tone and may not reflect fear to contextual cues alone. Moreover, in Experi-

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ments 3 and 4 any between group comparison of contextual fear is confounded by the exercise-associated reduction in startle amplitude. Thus the only adequate measure of contextual fear would be with a within group comparison in which startle is measured in both the training context and a neutral context. Of the several studies that have reported increases in contextual fear following voluntary exercise, only Baruch et al. [2] evaluated whether exercise also affected cued conditioned fear. Unlike our results, they reported no effect of exercise on cued conditioned fear. While any number of procedural differences can account for these different outcomes (species, fear conditioning parameters, measure of conditioned fear, etc.), one possible explanation is that in the Baruch et al. study both the sedentary and exercise groups showed robust cued conditioned fear that was much greater than fear measured to the context alone. As Baruch et al. point out, this may have made contextual fear more sensitive to the effects of exercise. Said another way, lack of an effect of exercise on cued conditioned fear may have been the result of a “ceiling effect”. In our experiments sedentary mice showed modest levels of cued conditioned fear as is typical in our experiments [19,39,98] which may make our procedure more sensitive to the effects of exercise on cued conditioned fear. The hippocampus plays an important role in contextual fear conditioning [2,6,8,38,91]. Given the well-documented affects of voluntary exercise on hippocampal function [4,5,24,32–34,45,67,68,84,86,88,92–95,97] it is very likely that these or similar changes in hippocampal function mediate the improvements in contextual fear conditioning following voluntary exercise [2,6,8,38,91]. Consistent with this, Greenwood et al. [38] have shown that voluntary exercise reduces the amount of context pre-exposure time that is required to show contextual fear conditioning and improves context discrimination learning two effects that are also dependent on the hippocampus. However, unlike contextual fear, the hippocampus is not required for cued fear conditioning (however see [57]). Many studies have shown that the amygdala plays a critical role in both cued and contextual conditioned fear [15,22,23,25,49,58]. For example, lesions of the amygdala performed either before [51,53] or after [39,55,81] cued fear conditioning prevent the acquisition and expression of conditioned fear. Similarly, drugs known to prevent synaptic plasticity in vitro prevent the acquisition of conditioned fear when infused into the amygdala [56,65]. The amygdala is central to a well-understood neural circuit for cued fear conditioning that includes cortical and thalamic inputs to the lateral nucleus of the amygdala [51], projections from the central nucleus of the amygdala to midbrain and brain stem areas known to be critical for many of the behaviors indicative of fear [21,42,62,102–104] as well as several areas known to contribute to the expression of conditioned fear including the deep layers of the superior colliculus/deep mesencephalic nucleus [62], periacquductal gray [73], bed nucleus of the stria terminalis [63], ventromedial hypothalamus [105], dorsal striatum [26] and medial prefrontal cortex [64]. Despite the apparent complexity of this circuit, the data strongly suggest that plasticity within the amygdala mediates learning and consolidation of conditioned fear (e.g., [65,77,82,83]). Given that exercise improves cued conditioned fear, it is likely that exercise-induced changes either in amygdala itself, or in areas of the circuit that directly influence amygdala plasticity, contribute to improved cued conditioned fear following exercise. To date, very few studies have reported attempts to examine exercise-induced changes in amygdala function (see [38,52]). Burghardt et al. [6] have shown that exercise increases the expression of the immediately early gene fos in the central nucleus of the amygdala following fear conditioning. More recently, Greenwood et al. [38] reported that six weeks of voluntary exercise increased BDNF mRNA in the basolateral amygdala. BDNF in the

amygdala has been implicated in the plasticity underlying cued fear conditioning [72] and may very well contribute to the improved cued conditioned fear following voluntary exercise. However, it is important to note that despite this strong evidence for exerciseinduced changes in amygdala function, Greenwood et al. [38] reported no effect of exercise on immediate post-shock freezing (see also [6,91]), a form of fear conditioning that, like cued fear conditioning, is dependent on the amygdala [47]. It is not clear why we observe improved cued fear conditioning whereas Greenwood et al. did not observe improved post-shock freezing. One possibility is that the strength of conditioning or the measure of conditioned fear (freezing versus fear-potentiated startle) is a critical determinant of whether one observes improved amygdaladependent fear. Another possibility is that exercise may affect plasticity in subregions of the amygdala (e.g., lateral and basal nuclei) that are important for cued fear conditioning and not in those regions that are more important for amygdala-dependent post-shock freezing. Interestingly, exercise did not affect shock sensitization of startle which, like post-shock freezing, is thought to reflect a form of rapid context conditioning [75,76]. Thus, our data suggest that such rapidly acquired contextual fear is not affected by prior voluntary exercise [38]. Alternatively, despite evidence for exercise-induced increase in amygdala BDNF, exercise-induced changes in the amygdala may not underlie the improved cued fear conditioning. The improved cued fear conditioning may be mediated by exercise-induced plasticity elsewhere in the neural circuits for cued conditioned fear. For example, Dishman and colleagues [43,85] have shown that voluntary exercise is associated with changes in the locus coeruleus and Greenwood et al. [36] have shown that exercise alters the function of the dorsal raphe nucleus. Both of these areas project to and modulate the amygdala and may play important roles in exercise-induced improvement of cued conditioned fear. Exercise not only improves learning, but it also produces stressresilience [16,17,35,37]. Consistent with this, we have shown that two weeks of voluntary exercise reduce anxiety across several different measures including acoustic startle [79]. The fact that voluntary exercise is anxiolytic, yet also improves conditioned fear, is somewhat surprising given that they are thought to be highly correlated emotions. However, conditioned fear is, by definition, a form of learning and there is substantial evidence that exercise improves learning. Moreover, there is growing evidence that fear and anxiety are mediated by separate, but overlapping, neural circuits [99]. Interestingly, it has been suggested that the memory-enhancing effects of exercise may be directly related to its stress-reducing and anxiolytic effects [9,89]. That is, exercise may reduce anxiety and in turn enhance conditioned fear. Although that hypothesis has not been directly evaluated, acute pre-conditioning stress has been shown to enhance anxiety and interfere with cued fear conditioning [87]. Perhaps anxiety, which has been thought of as an overgeneralized or more persistent form of fear, interferes with fear conditioning by blocking conditioning to discrete cues. Exercise may improve conditioned fear by reducing this overgeneralization. Interestingly, we have preliminary data indicating that exercise may reduce anxiety by altering the responsiveness of the bed nucleus of the stria terminalis (BNST). The BNST plays a role in mediating several anxiety related behaviors [101] and has been shown to play a role in modulating conditioned fear [63]. Perhaps a reduction in the excitability of the BNST following exercise reduces anxiety and its overgeneralization during fear conditioning. Consistent with this, Duvarci et al. [18] have reported that lesions of the BNST improve discriminative cued fear conditioning by reducing the generalization of fear from the CS+ to the CS-. Studies are underway to determine whether exercise-induced changes in the BNST contribute to improved cued conditioned fear.

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One important finding of our study is that voluntary exercise not only affects learning, but it also affects consolidation of cued conditioned fear. Mice that exercised for the two weeks immediately after fear conditioning (Experiments 3 and 4) showed improved cued conditioned fear. Importantly, mice that began exercising two weeks after fear conditioning did not show enhanced memory suggesting that there is a time window over which post-training exercise can improve consolidation and that exercise does not affect memory retrieval per se. Very few studies have evaluated the effects of post-conditioning exercise. In one study, two weeks of exercise following maze acquisition improved subsequent maze performance [90] suggesting that exercise improved memory consolidation. However, in contextual fear conditioning, six weeks of exercise following fear conditioning did not affect contextual fear memory despite the fact six weeks of exercise increased BDNF mRNA in both the hippocampus and the amygdala [38]. These later results suggest that for contextual fear conditioning, exerciseinduced changes in the brain must be in place prior to learning. Again, the difference between our results and those for contextual fear conditioning may reflect differences in the neural structures supporting the two types of fear conditioning or in the different measures of conditioned fear. Once again, more work is needed to examine these differences and the apparent differential sensitively to the effects of exercise. On some reflection, the fact that post-conditioning exercise improves consolidation may seem a bit surprising given that the exercise-induced behavioral and molecular changes [35], including those in the hippocampus [3], develop only after several weeks of exercise. Given this time frame it is difficult to understand how memory consolidation that is thought to occur in the hours or days after fear conditioning could be affected by changes that require 1–2 weeks to develop. Thus, there must be physiological effects of exercise that appear more acutely to affect consolidation. Interestingly, studies have shown that acute exercise may be stressful. For example, acute exercise supports conditioned taste aversion to foods consumed before running [30]. Moreover, we have shown that following 3 days of voluntary exercise acoustic startle amplitude is increased (unpublished observations) suggesting increased anxiety. A large number of studies have shown that post-conditioning administration of stress hormones, or exposure to acute stress, enhances memory consolidation through amygdala-dependent mechanisms [60,78]. For example, Hui et al. [44] showed that acute handling, or a vehicle injection, immediately following Pavlovian fear conditioning resulted in enhanced cued fear conditioning tested 24 h later. Therefore, in contrast to pre-conditioning exercise which may enhance cued fear conditioning in part by reducing stress and anxiety, post-conditioning exercise may enhance fear consolidation because it is initially stressful. If true, then post-conditioning exercise for several hours to a few days immediately after fear conditioning, but not several days later, should also improve consolidation of conditioned fear. Although acute exercise stress could possibly underlie the enhanced consolidation of cued conditioned fear, it is important to again note that Greenwood et al. [38] did not observe an effect of post-conditioning exercise on consolidation of contextual fear. Thus, with this hypothesis we would have to argue that acute exercise stress is sufficient to facilitate cued fear but is not sufficient to facilitate consolidation of contextual memory. This may further suggest that acute exercise stress may differentially affect amygdala and hippocampus-dependent plasticity.

Acknowledgements This work was supported by the Department of Psychology, University of Vermont. The authors wish to thank Jom Hammack

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and John Green for the many insightful discussions concerning this project.

References [1] Anderson BJ, Rapp DN, Baek DH, McCloskey DP, Coburn-Litvak PS, Robinson JK. Exercise influences spatial learning in the radial arm maze. Physiology & Behavior 2000;70(5):425–9. [2] Baruch DE, Swain RA, Helmstetter FJ. Effects of exercise on Pavlovian fear conditioning. Behavioral Neuroscience 2004;118(5):1123–7. [3] Berchtold NC, Chinn G, Chou M, Kesslak JP, Cotman CW. Exercise primes a molecular memory for brain-derived neurotrophic factor protein induction in the rat hippocampus. Neuroscience 2005;133(3):853–61. [4] Black JE, Isaacs KR, Anderson BJ, Alcantara AA, Greenough WT. Learning causes synaptogenesis, whereas motor activity causes angiogenesis, in cerebellar cortex of adult rats. Proceedings of the National Academy of Sciences of the United States of America 1990;87(14):5568–72. [5] Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler J, Gage FH, et al. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. European Journal of Neuroscience 2003;17(10):2042–6. [6] Burghardt PR, Pasumarthi RK, Wilson MA, Fadel J. Alterations in fear conditioning and amygdalar activation following chronic wheel running rats. Pharmacology, Biochemistry & Behavior 2006;84:306–12. [7] Campeau S, Davis M. Fear-potentiation of the acoustic startle reflex in rats using noises of various spectral frequencies. Animal Learning and Behavior 1992;20(2):177–86. [8] Clark P, Brzezinska W, Thomas M, Ryzhenko N, Toshkov S, Rhodes J. Intact neurogenesis is required for benefits of exercise on spatial memory but not motor performance or contextual fear conditioning in C57BL/6J mice. Neuroscience 2008;155:1048–58. [9] Collins A, Hill L, Chandramohan Y, Whitcomb D, Droste S, Reul J. Exercise improves cognitive responses to psychological stress through enhancement of epigenetic mechanisms and gene expression in the dentate gyrus. PLoS One 2009;4:e4330. [10] Cotman CW, Berchtold NC. Exercise: a behavioral intervention to enhance brain health and plasticity. Trends in Neurosciences 2002;25(6):295–301. [11] Cotman CW, Engesser-Cesar C. Exercise enhances and protects brain function. Exercise & Sport Sciences Reviews 2002;30(2):75–9. [12] Davis M. Sensitization of the acoustic startle reflex by footshock. Behavioral Neuroscience 1989;103:495–503. [13] Davis M. Neural systems involved in fear and anxiety measured with fearpotentiated startle. American Psychologist 2006;61(8):741–56. [14] Davis M, Astrachan DI. Conditioned fear and startle magnitude: effects of different footshock or backshock intensities used in training. Journal of Experimental Psychology: Animal Behavior Processes 1978;4:95–103. [15] Davis M, Whalen PJ. The amygdala: vigilance and emotion. Molecular Psychiatry 2001;6(1):13–34. [16] Dishman RK. Brain monoamines, excercise, and behavioral stress: animal models. Medicine and Science in Sports and Exercise 1997;29(1):63–74. [17] Droste SK, Gesing A, Linthorst ACE, Muller MB, Reul JMHM, S. U. Effects of longterm voluntary exercise on the mouse hypothalamic-pituitary-adrenocortical axis. Endocrinology 2003;144(7):3012–23. [18] Duvarci S, Bauer EP, Pare D. The bed nucleus of the stria terminalis mediates inter-individual variations in anxiety and fear. The Journal of Neuroscience 2009;29(33):10357–61. [19] Falls WA, Carlson S, Turner J, Willott JE. Fear-potentiated startle in two strains of inbred mice. Behavioral Neuroscience 1997;111(4):855–61. [20] Fanselow MS. Conditioned and uncondiitoned components of post-shock freezing. Pavlovian Journal of Biological Science 1980;15(4):177–82. [21] Fanselow MS. Neural organization of the defensive behavior systems responsible for fear. Psychonomic Bulletin & Review 1994;1(4):429–38. [22] Fanselow MS. Modality-specific memory of fear: differential involvement of the amygdala and hippocampal formation in Pavlovian fear conditioning. In: Taketoshi O, McNaughton BL, Molotchnikoff S, editors. Perception, memory and emotion: frontiers in neuroscience. NewYork: Pergamon; 1996. p. 499–511. [23] Fanselow MS, Gale GD. The amygdala, fear, and memory. Annals of the New York Academy of Sciences 2003;985:125–34. [24] Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR. Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague-Dawley rats in vivo. Neuroscience 2004;124(1):71–9. [25] Fendt M, Fanselow MS. The neuroanatomical and neurochemical basis of conditioned fear. Neuroscience & Biobehavioral Reviews 1999;23(5):743–60. [26] Ferreira T, Shammah-Lagnado S, Bueno O, Moreira K, Fornari R, Oliveira M. The indirect amygdala-dorsal striatum pathway mediates conditioned freezing: insights on emotional memory networks. Neuroscience 2008;153:84–94. [27] Fordyce DE, Farrar RP. Enhancement of spatial learning in F344 rats by physical activity and related learning-associated alterations in hippocampal and cortical cholinergic functioning. Behavioural Brain Research 1991;46(2):123–33. [28] Fordyce DE, Starnes JW, Farrar RP. Compensation of the age-related decline in hippocampal muscarinic receptor density through daily exercise or underfeeding. Journal of Gerontology 1991;46(6):B245–8.

330

W.A. Falls et al. / Behavioural Brain Research 207 (2010) 321–331

[29] Fordyce DE, Wehner JM. Physical activity enhances spatial learning performance with an associted alteration in hippocampal protein kinase C activity in C57BL/6 and DBA/2 mice. Brain Research 1993;619:111–9. [30] Forristall J, Hookey B, Grant V. Conditioned taste avoidance induced by forced and voluntary wheel running in rats. Behavior Processes 2007;74:326–33. [31] Gewirtz JC, McNish KA, Davis M. Is the hippocmapus necessary for contextual fear conditioning? Behavioural Brain Research 2000;110:83–95. [32] Gomez-Pinilla F, Vaynman S, Ying Z. Exercise induces BDNF and synapsin I to specific hippocampal subfields. Journal of Neuroscience Research 2004;76:356–62. [33] Gomez-Pinilla F, Vaynman SS, Ying Z. Exercise differentially regulates synaptic proteins associated to the function of BDNF. Brain Research 2005;1070:124–30. [34] Gomez-Pinilla F, Ying Z, Roy RR, Molteni R, Edgerton VR. Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity. Journal of Neurophysiology 2002;88(5):2187–95. [35] Greenwood BN, Foley TE, Burhans D, Maier SF, Fleshner M. The consequences of uncontrollable stress are sensitive to duration of prior wheel running. Brain Research 2005;1033(2):164–78. [36] Greenwood BN, Foley TE, Day H, Burhans D, Brooks L, Campaeu S, et al. Wheel running alters serotonin (5-HT) transporter, 5-HT(1a), 5-HT(1b), and alpha(1b)-adrenergic receptor mRNA in the rat raphe nuclei. Biological Psychiatry 2004;57(5):559–68. [37] Greenwood BN, Foley TE, Day HE, Campisi J, Hammack SH, Campeau S, et al. Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. Journal of Neuroscience 2003;23(7):2889–98. [38] Greenwood BN, Strong PV, Foley TE, Fleshner M. A behavioral analysis of the impact of voluntary physical activity on hippocampus-dependent contextual conditioning. Hippocampus 2009;19(10):988–1001. [39] Heldt SA, Sundin V, Willott JF, Falls WA. Post-training lesions of the amygdala interfere with fear-potentiated startle to both visual and auditory conditioned stimuli in C57BL/6J mice. Behavioral Neuroscience 2000;114(4):749–59. [40] Hillman CH, Belopolsky AV, Snook EM, Kramer AF, McAuley E. Physical activity and executive control: implications for increased cognitive health during older adulthood. Research Quarterly for Exercise & Sport 2004;75(2):176–85. [41] Hitchcock JM, Davis M. Lesions of the amygdala, but not of the cerebellum or red nucleus, block conditioned fear as measured with the potentiated startle paradigm. Behavioral Neuroscience 1986;100:11–22. [42] Hitchcock JM, Davis M. The efferent pathway of the amygdala involved in conditioned fear as measured with the fear-potentiated startle paradigm. Behavioral Neuroscience 1991;105:826–42. [43] Holmes P, Yoo H, Dishman R. Voluntary exercise and clomipramine treatment elevate prepro-galanin mRNA levels in the locus coeruleus in rats. Neuroscience Letters 2006;408:1–4. [44] Hui I, Hui G, Roozendaal B, McGaugh J, Weinberger N. Posttraining handling facilitates memory for auditory-cue fear conditioning in rats. Neurobiology of Learning and Memory 2006;86:160–3. [45] Isaacs KR, Anderson BJ, Alcantara AA, Black JE, Greenough WT. Exercise and the brain: angiogenesis in the adult rat cerebellum after vigorous physical activity and motor skill learning. Journal of Cerebral Blood Flow & Metabolism 1992;12(1):110–9. [46] Kim JJ, Fanselow MS. Modality specific retrograde amnesia of fear. Science 1992;256:675–7. [47] Kim JJ, Fanselow MS. Effects of amygdala, hppocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behavioral Neuroscience 1993;107(6):1093–8. [48] Kim JJ, Rison RA, Fanselow MS. Effects of amygdala, hippocampus, and periaqueductal gray lesions on short- and long-term contextual fear. Behavioral Neuroscience 1993;107(6):1093–8. [49] LeDoux JE. Emotion circuits in the brain. Annual Review of Neuroscience 2000;23:155–84. [50] LeDoux JE. The emotional brain, fear, and the amygdala. Cellular and Molecular Neurobiology 2003;23(4–5):727–38. [51] LeDoux JE, Cicchetti P, Xagoraris A, Romanski LM. The lateral amygdaloid nucleus: Sensory interface of the amygdala in fear conditioning. Journal of Neuroscience 1990;10(4):1062–9. [52] Liu Y, Chen H, Wu C, Kuo Y, Yu L, Huang A, et al. Differential effects of treadmill running and wheel running on spatial or aversive learning and memory: roles of amygdalar brain-derived neurotrophic factor and synaptotagmin I. Journal of Physiology 2009;587:3221–31. [53] Maren S. Neurotoxic basolateral amygdala lesions impair learning and memory but not the performance of conditioned fear in rats. The Journal of Neuroscience 1999;19(19):8696–703. [54] Maren S. Synaptic mechanisms of associative memory in the amygdala. Neuron 2005;47(6):783–6. [55] Maren S, Aharonov G, Fanselow MS. Retrograde abolition of conditioned fear after excitotoxic lesions in the basolateral amygdala of rats: absence of a temporal gradient. Behavioral Neuroscience 1996;110(4):718–26. [56] Maren S, Aharonov G, Stote DL, Fanselow MS. N-methyl-D-aspartate receptors in the basolateral amygdala are required for both acquisition and expression of conditioned fear in rats. Behavioral Neuroscience 1996;110(6):1365–74. [57] Maren S, Holt W. Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus. Behavioral Neuroscience 2004;118:97–110.

[58] Maren S, Quirk GJ. Neuronal signaling of fear memory. Nature Reviews Neuroscience 2004;5:844–52. [59] McAllister WR, McAllister DE. Behavioral measurements of conditioned Fear. In: Brush FR, editor. Aversive Conditioning and Learning. New York: Academic Press; 1971. p. 105–79. [60] McGaugh JL. Memory consolidaiton and the amygdala: a systems perspective. Trends in Neurosciences 2002;25(9):456–61. [61] McNish KA, Gewirtz JC, Davis M. Evidence of contextual fear after lesions of the hippocampus: a disruption of freezing but not fear-potentiated startle. The Journal of Neuroscience 1997;17(23):9353–60. [62] Meloni EG, Davis M. Muscimol in the deep layers of the superior colliculus/mesencephalic reticular formation blocks expression but not acquisition of fear-potentiated startle. Behavioral Neuroscience 1999;113(6):1152– 60. [63] Meloni EG, Jackson A, Gerety LP, Cohen BM, Carlezon Jr WA. Role of the bed nucleus of the stria terminalis (BST) in the expression of conditioned fear. Annals of the New York Academy of Sciences 2006;1071:538–41. [64] Milad M, Quirk GJ. Neurons in medial prefrontal cortex signal memory for fear extinction. Nature 2002;420:70–4. [65] Miserendino MJD, Sananes CB, Melia KR, Davis M. Blocking of acquisition but not expression of conditioned fear-potentiated startle by NMDA antagonists in the amygdala. Nature 1990;345(6277):716–8. [66] Nader K, LeDoux JE. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature 2000;406:722–6. [67] Neeper SA, Gomez-Pinilla F, Choi J, Cotman C. Exercise and brain neurotrophins. Nature 1995;373(6510):109. [68] Neeper SA, Gomez-Pinilla F, Choi J, Cotman CW. Physical activity increases mRNA for brain-derived neurotrophic factor and nerve growth factor in rat brain. Brain Research 1996;726(1–2):49–56. [69] Phillips RG, LeDoux JE. Differential contributions of amygdala and hippocampus to cued and contextual fear conditioning. Behavioral Neuroscience 1992;106(2):274–85. [70] Quirk GJ, Repa JC, LeDoux JE. Fear conditioning enhances short-latency auditory responses of lateral amygdala neurons: Parallel recordings in the freely behaving rat. Neuron 1995;15(5):1029–39. [71] Radak Z, Kaneko T, Tahara S, Nakamoto H, Pucsok J, Sasvari M, et al. Regular exercise improves cognitive function and decreases oxidative damage in rat brain. Neurochemistry International 2001;38(1):17–23. [72] Rattiner LM, Davis MJ, French CT, Ressler KJ. Brain-derived neurotrophic factor and tyrosine kinase receptor B involvement in amygdala-dependent fear conditioning. Journal of Neuroscience 2004;24(20):4796–806. [73] Reimer A, Oliveira A, Brandão M. Selective involvement of GABAergic mechanisms of the dorsal periaqueductal gray and inferior colliculus on the memory of the contextual fear as assessed by the fear potentiated startle test. Brain Research Bulletin 2008;76:545–50. [74] Ressler KJ, Paschall G, Zhou X-l, Davis M. Regulation of synaptic plasticity genes during consolidation of fear conditioning. Journal of Neuroscience 2002;22(18):7892–902. [75] Richardson R. Shock sensitization of startle: learned or unlearned fear? Behavioural Brain Research 2000;110:109–17. [76] Richardson R, Elsayed H. Shock sensitization of startle in rats: the role of contextual conditioning. Behavioral Neuroscience 1998;112(5):1136–41. [77] Rogan MT, Staubli U, LeDoux JE. Fear-conditioning induces associative longterm potentiation in the amygdala. Nature 1997;390:604–7. [78] Roozendaal B, McEwen B, Chattarji S. Stress, memory and the amygdala. Nature Reviews Neuroscience 2009;10:423–33. [79] Salam JN, Fox JH, DeTroy EM, Guignon MH, Wohl DF, Falls WA. Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety. Behavioural Brain Research 2009;197(1):31–40. [80] Samorajski T, Delaney C, Durham L, Ordy JM, Johnson JA, Dunlap WP. Effect of exercise on longevity, body weight, locomotor performance, and passive-avoidance memory of C57BL/6J mice. Neurobiology of Aging 1985;6(1):17–24. [81] Sananes CB, Davis M. N-methyl-D-asparate lesions of the lateral and basolateral nuclei of the amygdala block fear-potentiated startle and shock sensitization of startle. Behavioral Neuroscience 1992;106:72–80. [82] Schafe GE, Atkins CM, Swank MW, Bauer EP, Sweatt JD, LeDoux JE. Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of Pavlovian fear conditioning. The Journal of Neuroscience 2000;20(21):8177–87. [83] Schafe GE, LeDoux JE. Memory consolidation of auditory Pavlovian fear conditioning requires protein synthesis and protein kinase A in the amygdala. The Journal of Neuroscience 2000;20(RC96):1–5. [84] Shen H, Tong L, Balazs R, Cotman CW. Physical activity elicits sustained activation of the cyclic AMP response element-binding protein and mitogen-activated protein kinase in the rat hippocampus. Neuroscience 2001;107(2):219–29. [85] Soares J, Holmes PV, Renner KJ, Edwards GL, Bunnell BN, Dishman RK. Brain noradrenergic responses to footshock after chronic activity wheel running. Behavioral Neurosciecne 1999;113(3):558–66. [86] Swain RA, et al. Prolonged exercise induces angiogenesis and increases cerebral blood volume in primary motor cortex of the rat. Neuroscience 2003;117(4):1037–46. [87] Todorovic C, Radulovic J, Jahn O, Radulovic M, Sherrin T, Hippel C, et al. Differential activation of CRF receptor subtypes removes stress-induced memory deficit and anxiety. European Journal of Neuroscience 2007;25:3385–97.

W.A. Falls et al. / Behavioural Brain Research 207 (2010) 321–331 [88] Tong L, Shen H, Perreau VM, Balazs R, Cotman CW. Effects of exercise on gene-expression profile in the rat hippocampus. Neurobiology of Disease 2001;8(6):1046–56. [89] Trejo JL, LLorens-Martín MV, Torres-Alemán I. The effects of exercise on spatial learning and anxiety-like behavior are mediated by an IGF-I-dependent mechanism related to hippocampal neurogenesis. Molecular and Cellular Neuroscience 2008;37:402–11. [90] Van der Borght K, Havekes R, Bos T, Eggen BJL, Van der Zee EA. Exercise improves memory acquisition and retrieval in the Y-maze task: relationship with hippocampal neurogenesis. Behavioral Neuroscience 2007;121:324–34. [91] Van Hoomissen JD, Holmes PV, Zellner AS, Poudevigne AM, Dishman RK. Effects of beta-adrenoreceptor blockade during chronic exercise on contextual fear conditioning and mRNA for galanin and brain-derived neurotrophic factor. Behavioural Neuroscience 2004;118(6):1378–90. [92] van Praag H, Christie BR, Sejnowski TJ, Gage FH. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proceedings of the National Academy of Sciences of the United States of America 1999;96(23):13427–31. [93] van Praag H, Chunm TS, Gage FH. Exercise enhances learning and hippocampal neurogenesis in aged mice. The Journal of Neuroscience 2005;25(38): 8680–6. [94] van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature Neuroscience 1999;2(3):266–70. [95] van Praag H, Kempermann G, Gage FH. Neural consequences of environmental enrichment. Nature Reviews Neuroscience 2000;1(3):191–8. [96] Vaynman S, Ying Z, Gomez-Pinilla F. Exercise induces BDNF and synapsin I to specific hippocampal subfields. Journal of Neuroscience Research 2004;76(3):356–62.

331

[97] Vaynman S, Ying Z, Gomez-Pinilla F. Hippocampal BDNF mediates the efficacy of exercise on synaptic plasticity and cognition. European Journal of Neuroscience 2004;20(10):2580–90. [98] Waddell J, Dunnet C, Falls WA. C57BL/6J and DBA/2J mice differ in extinction and renewal of extinguished conditioned fear. Behavioural Brain Research 2004;154:567–76. [99] Walker DL, Davis M. Double dissociation between the involvement of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear. The Journal of Neuroscience 1997;17(23):9375–83. [100] Walker DL, Davis M. Quantifying fear potentiated startle using absolute versus proportional increase scoring methods: implications for the neurocircuitry of fear and anxiety. Psychopharmacology 2002;164(3):318–28. [101] Walker DL, Toufexis DJ, Davis M. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. European Journal of Pharmacology 2003;463:199–216. [102] Yeomans JS, Frankland PW. The acoustic startle reflex: neurons and connections. Brain Research Reviews 1996;21:301–14. [103] Yeomans JS, Polard BA. Amygdala efferents mediating electrically evoked startle-like responses and fear potentiation of acoustic startle. Behavioral Neuroscience 1993;107:596–610. [104] Zhao Z, Davis M. Fear-potentiated startle in rats is mediated by neurons in the deep layers of the superior colliculus/deep mesencephalic nucleus of the rostral midbrain through the glutamate non-NMDA receptors. The Journal of Neuroscience 2004;24(46):10326–34. [105] Zhao Z, Yang Y, Walker D, Davis M. Effects of substance P in the amygdala, ventromedial hypothalamus, and periaqueductal gray on fear-potentiated startle. Neuropsychopharmacology 2009;34:331–40.