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Behavioural Brain Research 323 (2017) 56–67

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

xercise increases mTOR signaling in brain regions involved inognition and emotional behavior

rian A. Lloyd a, Holly S. Hake a, Takayuki Ishiwata b, Caroline E. Farmer a,steban C. Loetz a, Monika Fleshner c, Sondra T. Bland a, Benjamin N. Greenwood a,∗

Department of Psychology, University of Colorado Denver, United StatesDepartment of Sport and Wellness, Rikkyo University, Saitama, JapanDepartment of Integrative Physiology and Center for Neuroscience, University of Colorado Boulder, United States

r t i c l e i n f o

rticle history:eceived 29 November 2016eceived in revised form 11 January 2017ccepted 18 January 2017vailable online 24 January 2017

a b s t r a c t

Exercise can enhance learning and memory and produce resistance against stress-related psychiatricdisorders such as depression and anxiety. In rats, these beneficial effects of exercise occur regardless ofexercise controllability: both voluntary and forced wheel running produce stress-protective effects. Themechanisms underlying these beneficial effects of exercise remain unknown. The mammalian target ofrapamycin (mTOR) is a translation regulator important for cell growth, proliferation, and survival. mTORhas been implicated in enhancing learning and memory as well as antidepressant effects. Moreover,mTOR is sensitive to exercise signals such as metabolic factors. The effects of exercise on mTOR signaling,however, remain unknown. The goal of the present study was to test the hypothesis that exercise, regard-less of controllability, increases levels of phosphorylated mTOR (p-mTOR) in brain regions important forlearning and emotional behavior. Rats were exposed to 6 weeks of either sedentary (locked wheel),voluntary, or forced wheel running conditions. At 6 weeks, rats were sacrificed during peak runningand levels of p-mTOR were measured using immunohistochemistry. Overall, both voluntary and forcedexercise increased p-mTOR-positive neurons in the medial prefrontal cortex, striatum, hippocampus,hypothalamus, and amygdala compared to locked wheel controls. Exercise, regardless of controllability,also increased numbers of p-mTOR-positive glia in the striatum, hippocampus, and amygdala. For bothneurons and glia, the largest increase in p-mTOR positive cells was observed after voluntary running,

with forced exercise causing a more modest increase. Interestingly, voluntary exercise preferentiallyincreased p-mTOR in astrocytes (GFAP+), while forced running increased p-mTOR in microglia (CD11+)in the inferior dentate gyrus. Results suggest that mTOR signaling is sensitive to exercise, but subtle dif-ferences exist depending on exercise controllability. Increases in mTOR signaling could contribute to thebeneficial effects of exercise on cognitive function and mental health.

© 2017 Elsevier B.V. All rights reserved.

. Introduction

Exercise can enhance learning and memory [1–4] and reducehe incidence of stress-related psychiatric disorders such as depres-ion [5–11] and anxiety [12–14]. In rats, these beneficial effects ofxercise occur regardless of exercise controllability. Both volun-ary wheel running [15–18] and forced treadmill training [19–21]

mprove aspects of cognition, and both voluntary and forced wheelunning produce protective effects against the development oftress-induced anxiety- and depression-like behavior [22]. Iden-

∗ Corresponding author.E-mail address: benjamin.greenwood@ucdenver.edu (B.N. Greenwood).

ttp://dx.doi.org/10.1016/j.bbr.2017.01.033166-4328/© 2017 Elsevier B.V. All rights reserved.

tification of the mechanisms underlying these beneficial effects ofexercise could lead to novel therapeutic approaches.

The mammalian target of rapamycin (mTOR), a serine/threoninekinase important for cell growth, proliferation, and survival[23], has been increasingly implicated in cognitive function[24–27]. For example, learning transiently increases p-mTOR inthe hippocampus [28,29] and blockade of mTOR signaling withRapamycin impairs hippocampus-dependent learning in tasks suchas inhibitory avoidance [30], and both voluntary [31,32] and forced[33] exercise enhance learning in this same task. Considering thatmTOR activates proteins involved in synaptic protein synthesis

such as ribosomal S6 kinase 1 (RS6K1) and eukaryotic translationinitiation factor 4E-binding protein 1 (4E-BP1) [34,35], enhancedsynaptic plasticity could contribute to the beneficial effects of

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TOR on cognitive function. Indeed, mTOR can increase dendriticrborization in the hippocampus via calmodulin-dependent pro-ein kinase II� (CaMKII�) [27] and downstream activation of RS6K1as been reported to enhance dendritic arborization in the PFC [36].

nterestingly, Rapamycin has been recently reported to block theacilitation of hippocampal long-term potentiation provided by annriched environment [37], suggesting that mTOR signaling coulde critical for beneficial effects of environmental manipulations onognitive function.

In addition to improving cognitive function, mTOR has alsoeen implicated in providing antidepressant effects [36,38–40]. Forxample, inhibition of mTOR signaling in the prefrontal cortex (PFC)ith Rapamycin can block the antidepressant effects of low-dose

etamine [40]. Moreover, inactivation of RS6K1, a protein that canncrease dendritic arborization, in the PFC can cause depressive-likeehavior in the absence of external stressors [36].

It may not be a coincidence that the beneficial effects of mTORignaling seem to parallel the broad benefits of exercise. In fact,TOR is a compelling candidate for mediating the cognitive ben-

fits and anti-depressant effects of exercise. mTOR signaling isensitive to metabolic signals such as glucose [41] and amino acids42–44]; availability of which are increased in circulation and therain during exercise [45–47]. Additionally, mTOR signaling is alsotimulated by factors such as glutamate, tumor necrosis factor�TNF�) and receptor activator of nuclear factor kappa-B ligandRANKL) [46,48,49], as well as by growth factors such as brain-erived neurotrophic factor (BDNF) [50] and insulin-like growth

actor-1 (IGF-1) [51,52]. The growth factors BDNF and IGF-1 arencreased by exercise [53,54] and are thought to be important forhe enhanced plasticity [53], cognitive benefits [55] and antidepres-ant effects [56] conferred by exercise. BDNF signaling, for example,an lead to phosphorylation of mTOR through the PI3K/AKT intra-ellular cascade [48,57–59]. Thus, it is possible that mTOR signalingepresents a common downstream target of exercise signals impor-ant for beneficial effects of exercise [60].

Although it has been previously reported that forced treadmillraining increases mTOR signaling in the brain [61–63], these priortudies focused principally on the hippocampus. This attention tohe hippocampus is logical due to the consistent beneficial effects ofreadmill training on hippocampal-dependent memory [33,64,65].owever, forced treadmill training has produced inconsistent anx-

olytic and antidepressant effects in rodents (for a review, [66]).oluntary wheel running, on the other hand, produces well knownntidepressant and anxiolytic effects [6,13,66–69], and both vol-ntary and forced wheel running increase resistance against theepression- and anxiety-like behavioral effects of stress [22]. Theffects of exercise on mTOR signaling in brain regions involved inmotional regulation beyond the hippocampus; however, remainnknown.

The goal of the current experiment was to test the hypothesishat both voluntary and forced wheel running increase phosphory-ation of mTOR in brain regions implicated in cognitive function andmotional behavior. Forced exercise was controlled by a motorizedheel driven by software designed to rotate the wheel in a pattern

hat closely resembles natural running behavior [22]. Immunohis-ochemistry was utilized to allow quantification of both neuronsnd glia in specific brain subregions, as p-mTOR in these cell typesould have different effects on neural circuit function and behavior.

. Materials and methods

.1. Subjects

A total of 23, young adult (between P42-P49 upon arrival),ale Fischer 344 rats (Harlan SPF, Indianapolis, IN., USA) were pair

Research 323 (2017) 56–67 57

housed in Nalgene Plexiglas cages (45 cm × 25.2 cm × 14.7 cm) ina humidity-controlled environment at a temperature of 22 ◦C. Therats were maintained on a 12:12 h light:dark cycle with the lights on07:00–19:00 h. All rats had ad libitum access to water and food (labchow). Rats were weighed weekly and acclimated to these hous-ing conditions for 7 days prior to any experimental manipulation.Precautions were taken to minimize animal discomfort during allprocedures. The University of Colorado Boulder Animal Care andUse Committee approved all experimental protocols.

2.2. Exercise protocols

Rats were randomly assigned to one of three conditions: lockedwheel (n = 8), voluntary running wheel (n = 8), or forced run-ning wheel (n = 7). Voluntary and forced running protocols werecarried out as previously described [22,70]. To minimize non-running behaviors in the forced wheels (e.g. tumbling, hangingonto wheel rungs), experience with voluntary wheel running isrequired. Motorized wheels belonging to rats in the forced exer-cise group were therefore removed from the motor and all rats inthe voluntary and forced wheel running conditions were allowedvoluntary access to their individual wheels for 5 consecutive active(dark) cycles prior to the start of differential group treatment.Wheels belonging to rats in the forced wheel running group werere-connected to motors following these 5 nights of voluntary run-ning, and all subsequent running by the forced exercise group wascontrolled by the motor. Rats were transported 5 nights a weekfrom their home cages to their assigned locked, freely mobile ormotorized running wheels (1.1 m circumference; Lafayette Instru-ments, Lafayette, IN., USA). The forced wheels were driven by amotor controlled by the Activity Wheel Monitor software (LafayetteInstruments, Lafayette, IN., USA) according to a protocol pre-programmed by the experimenters based on previous analysis ofF344 rats’ natural voluntary running behavior [22]. In order to min-imize tumbling in the forced wheel, the motorized wheels slowlyincreased in speed during the first few days of forced running, andrunning speeds in the forced wheel running group were kept below17 m/min for the duration of the experiment. Due to the slowerspeeds run by the forced exercise rats, the average distance run perday of rats in the forced wheel running condition was less than thatof the voluntary wheel condition [70]. Nevertheless, the pattern ofexercise, namely the duration of rest periods (range 0.33–30 min)and running bout length (average 2.04 ± 1.95 min), were carefullymatched between voluntary and forced wheel running conditions[22,70]. Stress-protective [22] and rewarding [70] effects of thisforced running pattern have been previously described. Rats in thelocked, freely-mobile, and motorized groups were confined to theirwheels for their assigned running periods, which was the entire12 h duration of the active cycle. We have previously observed thatrats eat and drink equal amounts regardless of exercise condition[22]. A food tray and water bottle mounted on the side of the wheelallowed access to food and water while rats were confined to thewheel. Locked, freely-mobile, and motorized wheel conditions con-tinued for 5 nights/week for 6 weeks following the initial 5 nights ofvoluntary running. The Lafayette Activity Wheel Monitor softwareautomatically recorded wheel revolutions. The analyses of volun-tary and forced running patterns of these rats in addition to bodyweight data have been previously published [70].

2.3. Single p-mTOR immunohistochemistry

On the last night of the experiment, rats were transported

to their locked, freely-mobile or motorized wheel as usual andwere allowed to run at least 2 h prior to being removed from thewheels and deeply anesthetized with sodium pentobarbital. Twohours was required to perfuse all the rats, thus rats remained in

5 l Brain Research 323 (2017) 56–67

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heir wheels between 2 and 4 h prior to sacrifice. Times prior toacrifice were counterbalanced between groups. Rats were per-used transcardially with cold saline, followed by 300–400 ml of% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Brainsere extracted, post-fixed in PFA overnight, and then transferred

o 30% sucrose solution for 3 days. Brains were then flash frozenn isopentane with dry ice and stored at −70 ◦C. Brains were slicedn a cryostat at 35 �m in coronal sections. Brain slices were stored

n cryoprotectant at −20 ◦C prior to staining. All tissue from eachrain region was processed in 25-well staining dishes so that directroup comparisons could be made with three tissue sections per rat.he number of rats per group differed slightly (from n = 6 to n = 8)ithin and between brain regions due to tissue damage incurred

uring slicing or IHC.Immunohistochemistry (IHC) was performed as previously

escribed [6,70–72] on brain sections containing (from rostralo caudal) PFC (3.7 mm–1.70 mm rostral from Bregma), striatum1.6 mm–0.2 mm rostral from Bregma), hippocampus/amygdala−2.12 mm to − 4.52 mm caudal from Bregma) and hypothalamus−0.20 mm to −3.30 mm caudal from Bregma). Sections were rinsedor 10 min 3 times in 0.01 M tris-buffered saline (TBS) followed by

40 min incubation in a solution of 0.3% hydrogen peroxide, 3.75%riton-X in 0.01 M TBS. Sections were incubated for 48 h at 4 ◦C inlocking solution containing 2.5% Triton X, 5% normal goat serumNGS), and rabbit anti-p-S2448-mTOR (catalog number 49F9, Cellignaling, Danvers, MA) at 1:1000. Our study focused on phospho-ylated S2448 mTOR (p-mTOR), as this is the phosphorylation sitehat has been shown to activate proteins downstream of mTOR suchs RS6K1 [40,73,74]. The specificity of the chosen antibody has beenemonstrated by western blot [75,76]. This antibody has also beensed by others to quantify p-mTOR in brain [77,78], including afterreadmill training [62].

Incubation in primary antisera was followed by another seriesf washes for 10 min 3 times in 0.01 M phosphate buffed salinePBS) with 0.75% Triton-X and 5% NGS. The sections were incu-ated at room temperature (RT) for 120 min in blocking solutionontaining a 1:200 dilution of biotinylated goat anti-rabbit IgGJackson Immunoresearch, West Grove, PA). The brain sectionsere rinsed for 10 min 3 times in 0.01 M PBS. Sections were then

ncubated with avidin-biotin-horseradish peroxidase complexesABC; Vecastain Elite ABC kit, Vector Laboratories, Burlingame, CA)n PBS containing 0.5% Triton X for 2 h. After 3 washes for 10 min

ith 0.1 M PB, sections were placed in a solution containing 3,3′-iaminobenzidene tetrahydrochloride (DAB) and glucose oxidase

n PBS for 10 min. The peroxidase reaction was started by additionf glucose solution and reacted for ∼15 min, yielding a dark browneaction product. The reaction was stopped by 3 rinses for 10 minn PBS. Stained sections were mounted onto gelatin coated, glasslides and air-dried overnight. Slide-mounted sections were dehy-rated in a series of alcohols, rinsed in Histoclear, and cover-slippedith Permount.

.4. Double fluorescent IHC

Tissue slices from the hippocampus and amygdala from a sub-et of rats with numbers of p-mTOR-positive glia closest to theirroup’s mean were used to identify the type of neuroglia expressing-mTOR in these regions (n = 2/group), because the highest numberf p-mTOR-positive glia were observed in these regions. The brainections were washed in 0.01 M phosphate buffer saline (PBS) for0 min 3 times, followed by a 1 h incubation in a blocking solutionf 5% normal goat serum (NGS) in PBS. Sections were incubated

or 48 h at 4 ◦C in blocking solution containing rabbit anti-p-mTORcatalog number 49F9, Cell Signaling, Danvers, MA) at 1:500 andhen a solution containing rat monoclonal GFAP (marker of astro-ytes; catalog number G3893, Sigma-Aldrich, St Louis, MO) or rat

Fig. 1. Quantification parameters of neurons and glia. (A) Representative photomi-crogram of a p-mTOR-positive neuron. (B) Representative photomicrogram of ap-mTOR-positive glia.

monoclonal CD11 (OX42) (marker of microglia; catalog number554859, BD Pharmingen, San Jose, CA) at 1:500. The next stepsof the procedure were completed under low light. The brain sec-tions were washed again in 0.01 M phosphate buffer saline (PBS)for 10 min 3 times, followed by a 2 h incubation at RT in a solutionof goat anti-mouse Alexa Fluor 488 IgG (catalog number A11001,BD Pharmingen, San Jose, CA) at 1:400. Then, a 2 h incubation at RTin a solution of goat anti-rabbit Alexa Fluor 594 IgG at 1:400 (cat-alog number A11007, BD Pharmingen, San Jose, CA). The reactionwas stopped by rinses in PBS. The coverslips were mounted usingProlongGold and sealed with clear nail polish.

2.5. Quantification of immunohistochemistry

Images of the prelimbic (PL) and infralimbic (IL) PFC, dorsal lat-eral (DLS) and dorsal medial (DMS) striatum, nucleus accumbenscore (NAcC) and nucleus accumbens shell (NAcS), central (CeA) andbasolateral (BLA) amygdala, superior and inferior dentate gyrus(DG), cornu ammonis (CA1, CA2, CA3), median preoptic nucleus(MnPO), medial preoptic area (MPO), dorsomedial hypothalamus(DMH), ventromedial hypothalamus (VMH), and paraventricularnucleus (PVN) were captured digitally at 40X on an Olympus BX51microscope. Quantification of neurons, glia, and unbiased densit-ometry was done with VisioPharm Integrator System software bymultiple experimenters blind to treatment condition of the rats.Pearson’s correlation resulted in an inter-rater reliability of 0.8(p < 0.0001).

Cell counts and densitometry values were collected from 3tissue sections and averaged for each region. The number of hemi-spheres quantified in each rat differed slightly due to damageincurred during slicing or IHC. Only neurons and glia staining darkbrown were counted as p-mTOR-positive cells and cells that didnot lie completely within the region of interest or were out offocus were not counted. Neurons and glia were distinguished basedon criteria including number of processes and clarity of nucleus.Representative photomicrograms of a neuron and glia are pro-vided in Fig. 1A and 1B, respectively. Previous studies have alsodocumented p-mTOR-positive glia based on morphology [79]. Thecell counts based on morphology were confirmed by the double-immunofluorescence for p-mTOR and glial markers GFAP and CD11,which yielded p-mTOR-positive glial counts that did not differ fromthe total number of p-mTOR-positive glia observed using morpho-logical identification (p > 0.05 in the DG and BLA). Densitometry

values collected with Visiopharm measure the average intensity ofall pixels in the region of interest and serves as an unbiased mea-sure because it cannot be influenced by the observer. A 450 �mby 450 �m square was drawn around the region of interest and

B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67 59

Fig. 2. Effects of exercise on p-mTOR in the prefrontal cortex (PFC). (A) Brain atlas diagram modified from Paxinos and Watson (2006) and representative photomicrogramsdepicting p-mTOR staining in the prelimbic (PL) of sedentary (left), voluntary run (middle) and forced run (right) groups. White arrows indicate examples of p-mTOR-positiveneurons. (B) Exercise does not alter the number of p-mTOR-positive neurons in either the PL or the infralimbic (IL) cortices. (C) Voluntary exercise increases the intensity ofstain in the PL and IL compared to sedentary rats. *p < 0.01 relative to sedentary rats. Bars represent group means ± SEM.

Fig. 3. Effect of exercise on p-mTOR in the dorsal striatum. (A) Brain atlas diagram modified from Paxinos and Watson (2006) and representative photomicrograms depictingp-mTOR staining in the dorsal lateral striatum (DLS) of sedentary (left), voluntary run (middle) and forced run (right) groups. White arrows indicate examples of p-mTOR-p DLS.s * p < 0g

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ositive neurons. (B) Voluntary exercise increases p-mTOR-positive neurons in theedentary and forced exercise groups in the dorsal medial striatum (DMS) and DLS.roup means ± SEM.

uantification of single p-mTOR took place within that area. Alluantification was performed with the same size region of inter-st and converting quantification to cells per unit area did notmpact the results. A Zeiss 700 laser scanning confocal microscopeoupled to Axiovision BIO software was used to capture double-uorescent images of the inferior DG and BLA. The number ofingle-labeled astrocytes (GFAP+) and microglia (CD11+), as well asouble-labeled GFAP/p-mTOR and CD11/p-mTOR glia were quan-ified with Zeiss software.

.6. Statistical analyses

One-way ANOVA was used to analyze group differences inxpression of p-mTOR in neuron and glial cells, unbiased den-itometry, and numbers of CD11, GFAP, and double-labeled cells.

(C) Voluntary exercise increases the intensity of p-mTOR stain compared to both.05 relative to locked group; � p < 0.05 relative to forced run group. Bars represent

Analyses were followed by Fisher’s protected least significantdifference (PLSD) post hoc tests when appropriate. Regression anal-yses were completed in each region test the hypothesis that averageweekly running distance or running distance on the last night ofrunning prior to sacrifice are related to levels of p-mTOR. Groupdifferences were considered different when p ≤ 0.05.

3. Results

3.1. Voluntary exercise had minimal effects on mTOR signaling inthe PFC

3.1.1. NeuronsRegions where p-mTOR was assessed in the PFC, as well as

representative photomicrographs, are shown in Fig. 2A. Neither

60 B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67

Fig. 4. Effects of exercise on p-mTOR in the nucleus accumbens (NAc) (A) Brain atlas diagram modified from Paxinos and Watson (2006) and representative photomicrogramsdepicting p-mTOR staining in the nucleus accumbens core (NAcC) of sedentary (left), voluntary run (middle) and forced run (right) groups. White arrows indicate examplesof p-mTOR-positive neurons; black arrows indicate examples of p-mTOR-positive glia. (B) Voluntary, but not forced exercise increases p-mTOR-positive neurons comparedto sedentary and forced run groups in the NAcC and the nucleus accumbens shell (NAcS). (C) Both voluntary and forced exercise increase p-mTOR-positive glia compared tosedentary controls in the NAcC and NAcS. In the NAcC, voluntary exercise also increases the number of p-mTOR-positive glia compared to the forced exercise condition. (D)Densitometry results show that only voluntary exercise increases the intensity of stain compared to sedentary and forced wheel running conditions in the NAcC and NAcS. *r d run

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epresents p < 0.05 relative to sedentary rats; � represents p < 0.05 relative to force

oluntary nor forced exercise increased the number of p-mTOR-ositive neurons in the PL (F(2,20) = 0.090; p = 0.914; Fig. 2B) or ILF(2,20) = 0.033; p = 0.967; Fig. 2B). Average weekly distance ran byhe voluntary running group negatively correlated with the numberf p-mTOR-positive neurons in the PL (R = −0.720; F(1,6) = 6.468;

= 0.0439) and the IL (R = −0.845; F(1,6) = 15.007; p = 0.0082; dataot shown).

.1.2. DensitometryDespite the lack of significant differences in p-mTOR-positive

ell counts, voluntary exercise significantly increased the inten-ity of p-mTOR stain as measured by densitometry in both theL (F(2,20) = 4.715; p = 0.0226) and IL (F(2,20) = 4.920; p = 0.0197;ee Fig. 2C for post-hoc analyses). p-mTOR-positive glia were notbserved in the PFC. No significant correlations between runningistance and intensity of p-mTOR stain in the PFC were found.

.2. Exercise increased numbers of p-mTOR-positive neurons andlia in the striatum

.2.1. NeuronsRegions where p-mTOR was assessed in the dorsal and ven-

ral striatum, as well as representative photomicrographs, arehown in Figs. 3A and 4A . Group differences in the number of p-TOR-positive neurons were observed in the DLS (F(2,19) = 0.917;

= 0.04168; Fig. 3B), NAcC (F(2,19) = 15.191; p = 0.0001; Fig. 4B) andAcS (F(2,19) = 8.488; p = 0.0023; Fig. 4B). Post-hoc analyses reveal

hat only voluntary exercise increased the number of p-mTOR-ositive neurons in these areas compared to the sedentary groupp < 0.05; Figs. 3B & 4B). The numbers of p-mTOR-positive neurons

n the forced wheel running group differed from neither the vol-ntary nor locked wheel conditions in the DMS. Average weeklyoluntary running distance positively correlated with the numberf p-mTOR-positive neurons in the DLS (R = 0.718; F(1,6) = 6.369;

group. Bars represent group means ± SEM.

p = 0.0451), but negatively correlated with neuron counts in theNAcC (R = −0.950; F(1,6) = 56.034; p = 0.0003). The distance runon the night prior to sacrifice predicted the number of p-mTOR-positive neurons in the DMS (R = 0.772; F(1,6) = 8.832; p = 0.0249)in the voluntary running group, but not the forced running group.

3.2.2. GliaVery few p-mTOR-positive glia were observed in the dorsal

striatum. In the ventral striatum, both voluntary and forced exer-cise increased p-mTOR-positive glia in the NAcC (F(2,18) = 16.281;p < 0.0001; Fig. 4C) and NAcS (F(2,18) = 8.153; p = 0.0030; Fig. 4C),with voluntary exercise providing the largest increase in theNAcC (p < 0.05 compared to locked and forced exercise conditions;Fig. 4C). Average weekly voluntary running distance negativelycorrelated with numbers of p-mTOR-positive glia in the NAcC(R = −0.829; F(1,6) = 13.195; p = 0.0109) and NAcS (R = −0.829;F(1,6) = 13.195; p = 0.0109).

3.2.3. DensitometryDensitometry, which does not discriminate between neu-

rons and glia, revealed group differences in intensity ofp-mTOR staining in the DMS (F(2,19) = 8.047; p = 0.0029; Fig. 3C),DLS (F(2,19) = 7.017; p = 0.0050; Fig. 3C), NAcC (F(2,18) = 5.220;p = 0.0163; Fig. 4D) and NacS (F(2,18) = 5.353; p = 0.0150; Fig. 4D).Voluntary exercise increased the intensity of p-mTOR stain inthese regions above both sedentary and forced exercise condi-

tions (p < 0.05; Figs. 3C & 4D). Average weekly distance run bythe voluntary running group predicted intensity of stain in boththe DMS (R = 0.715; F(1,6) = 6.259; p = 0.0464) and DLS (R = 0.720;F(1,6) = 6.466; p = 0.0439).

B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67 61

Fig. 5. Effects of exercise on p-mTOR in the hippocampus and amygdala. (A) Brain atlas diagram modified from Paxinos and Watson (2006) and representative photomi-crograms depicting p-mTOR staining in the dentate gyrus (DG) of sedentary (left), voluntary run (middle) and forced run (right) groups. White arrows indicate examples ofp-mTOR-positive neurons; black arrows indicate examples of p-mTOR-positive glia. (B) Both voluntary and forced exercise increase p-mTOR-positive neurons compared tosedentary rats in the cornu ammonis (CA1, CA2, CA3) and DG regions of the hippocampus. Voluntary exercise also increases p-mTOR-positive neurons in the DG comparedto the forced wheel running condition. (C) Both voluntary and forced exercise increase p-mTOR-positive glia compared to sedentary controls in the CA2 and CA3. Forcedexercise increases counts of p-mTOR-positive glia in the DG compared to sedentary rats. (D) Voluntary exercise increases the intensity of p-mTOR stain compared to sedentaryand forced exercise conditions in the CA1 and CA2. Both voluntary and forced exercise increase the intensity of stain compared to sedentary controls in the CA3 and DG.(E) Voluntary exercise increases p-mTOR-positive neurons in the basolateral amygdala (BLA) compared to sedentary rats. (F) Both voluntary and forced exercise increasep-mTOR-positive glia in the BLA and central amygdala (CeA) compared to sedentary controls. (G) Both voluntary and forced exercise increase the intensity of stain in the BLAand CeA relative to sedentary controls, with voluntary providing a larger increase compared to the forced wheel running condition. * represents p < 0.05 relative to sedentaryr s ± SEM

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ats; � represents p < 0.05 relative to forced run group. Bars represent group mean

.3. Exercise increased p-mTOR in the hippocampus andmygdala

.3.1. NeuronsRegions where p-mTOR was assessed in the hippocampus and

mygdala, as well as representative photomicrographs, are shownn Fig. 5A. Both voluntary and forced exercise increased p-mTOR-ositive neurons in the CA1 (F(2,20) = 18.257; p < 0.0001; Fig. 5B),A2 (F(2,20) = 12.442; p = 0.0003; Fig. 5B), CA3 (F(2,20) = 9.689;

= 0.0011; Fig. 5B) and DG (F(2,20) = 44.085, p < 0.0001; Fig. 5B).oluntary exercise elicited the largest increase in p-mTOR-positiveeurons in the DG (p < 0.05 compared to sedentary and forcedxercise conditions; Fig. 5B). Group differences in p-mTOR-positiveeurons were also observed in the BLA (F(2,20) = 4.975; p = 0.0176;ig. 5E), but not in CeA (F(2,20) = 1.849; p = 0.1833; Fig. 5E). In theLA, voluntary running increased p-mTOR-positive neuron counts

ompared to the locked wheel locked wheel condition (p < 0.05;ig. 5E).

.

3.3.2. GliaQuantification of the p-mTOR-positive glia revealed a trend

for group differences in the CA1 (F(2,20) = 3.183; p = 0.0631;Fig. 5C), and significant differences in p-mTOR-positive glia inCA2 (F(2,20) = 4.060, p = 0.0331; Fig. 5C), CA3 (F(2,20) = 4.371;p = 0.0226; Fig. 5C) and DG (F(2,20) = 3.875; p = 0.0378; Fig. 5C).Post-hoc analyses revealed that both voluntary and forced exer-cise significantly increased the number of p-mTOR-positive glia inCA2 and CA3 compared to locked wheel controls (p < 0.05; Fig. 5C),while only forced exercise increased p-mTOR-positive glia in DGrelative to the sedentary condition (p < 0.05; Fig. 5C). Both vol-untary and forced exercise increased p-mTOR-positive glia in theBLA (F(2,20) = 7.280; p = 0.0042; Fig. 5F) and CeA (F(2,20) = 3.612;p = 0.0458; Fig. 5F).

Double immunofluorescence was completed to assess the rel-ative expression of p-mTOR in microglia (CD11-positive) and

astrocytes (GFAP-positive). There was a significant effect of exer-cise on percent glia double-labeled with CD11 and p-mTOR in theDG (F(2,3) = 52.678; p = 0.0046; Fig. 6A), with a significant increase

62 B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67

Fig. 6. Effects of exercise on glial expression of p-mTOR in the hippocampus and amygdala (A) Forced exercise increases the percent of CD11-positive cells expressing p-mTOR compared to sedentary and voluntary exercise conditions in the dentate gyrus (DG). (B) Only voluntary exercise increases the percent of GFAP-positive cells expressingp-mTOR compared to sedentary and forced wheel running conditions in the inferior dentate gyrus. Both voluntary and forced decrease GFAP-positive cells compared tos ion of* ed runm e rea

ilameeabvgwcmaG

3

dipDwos(e

edentary controls in the DG. (C,D) There were no significant changes in the proport represents p < 0.05 relative to sedentary rats; � represents p < 0.05 relative to forc

eans ± SEM. (For interpretation of the references to colour in this figure legend, th

n the forced exercise condition compared to both voluntary andocked wheel groups (p < 0.05; Fig. 6A). Exercise also significantlyffected the percentage of glia double-labeled with GFAP and p-TOR in the DG (F(2,3) = 12.929; p = 0.0335; Fig. 6B), with voluntary

xercise providing the largest increase compared to both forcedxercise and locked wheel conditions (p < 0.05; Fig. 6B). The DG waslso remarkable for a significant effect of exercise on the total num-er of GFAP-positive glia (F(2,3) = 9.945; p = 0.0475; Fig. 6B). Botholuntary and forced running reduced the number of GFAP-positivelia (p < 0.05; Fig. 6B). In the BLA, both microglia and astrocytesere observed to express p-mTOR. Exercise did not significantly

hange counts of GFAP-positive, CD11-positive, or the percent of p-TOR-positive/glia-positive double-labeled cells in the BLA (Fig. 6C

nd D). Representative confocal images of CD11-positive glia andFAP-positive glia are shown in Figs. 7 and 8, respectively.

.3.3. DensitometryUnbiased densitometry was consistent with neuron and glia

ata. Voluntary and forced wheel running increase intensity of stainn CA1 (F(2,20) = 9.737; p = 0.0011; Fig. 5D), CA2 (F(2,20) = 9.811;

= 0.0011; Fig. 5D), CA3 (F(2,20) = 9.256; p = 0.0015; Fig. 5D), andG (F(2,20) = 15.331; p < 0.0001; Fig. 5D) compared to lockedheel controls. The distance run by the forced running group

n the last day negatively correlated to the intensity of p-mTORtain in CA1 (R = −0.850; F(1,5) = 13.044; p = 0.0154) and CA2R = −0.758; F(1,5) = 6.751; p = 0.0483). Both voluntary and forcedxercise increase the intensity of stain in the BLA (F(2,20) = 15.733;

CD11 or GFAP-positive cells expressing p-mTOR in the basolateral amygdala (BLA). group; � represents p < 0.05 relative to voluntary run group. Bars represent group

der is referred to the web version of this article.)

p < 0.0001; Fig. 5G) and CeA (F(2,20) = 14.385; p = 0.0001; Fig. 5D).Voluntary exercise caused the largest increase in densitometry inthe CeA (p < 0.05 compared to sedentary and forced exercise con-ditions; Fig. 5G). No significant correlations were found in theamygdala.

3.4. Both voluntary and forced exercise increasedp-mTOR-positive neurons in the hypothalamus

3.4.1. NeuronsRegions where p-mTOR was assessed in the hypothalamus, as

well as representative photomicrographs, are shown in Fig. 9A. Bothvoluntary and forced wheel running increase p-mTOR-positiveneurons in the MnPO (F(2,19) = 17.306; p < 0.0001; Fig. 7B), MPO(F(2,19) = 32.023; p < 0.0001; Fig. 9B), and DMH (F(2,19) = 36.373;p < 0.0001; Fig. 9B). Group differences were observed in the VMH(F(2,20) = 3.649; p = 0.0446; Fig. 9B), where forced exercise reducesthe number of p-mTOR-positive neurons compared to the seden-tary group (p < 0.05; Fig. 9B). No group differences were observedin the PVN (F(2,20) = 0.227; p = 0.7987). Average weekly voluntaryrunning distance predicts number of p-mTOR-positive neurons inthe MnPO (R = 0.725; F = 7.047; p = 0.0378).

3.4.2. DensitometryGroup differences in intensity of staining were also

observed in the MnPO (F(2,20) = 4.381; p = 0.0273; Fig. 9C),MPO (F(2,20) = 4.602; p = 0.0235; Fig. 9C), VMH (F(2,20) = 6.135;

B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67 63

Fig. 7. Representative confocal images of CD11 (green)/p-mTOR (red) double immunofluorescence in the dentate gyrus of sedentary (top), voluntary run (middle) and forcedrun (bottom) groups. Bar A: Overlay. Bar B: CD11 only. Bar C: p-mTOR only. Bar D: co-localized pixels only. White arrows indicate cells that were counted as double labels.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

F nofluor co-loc( erred

pp

4

m

ig. 8. Representative confocal images of GFAP (green)/p-mTOR (red) double immuun (bottom) groups. Bar A: Overlay. Bar B: CD11 only. Bar C: p-mTOR only. Bar D:

For interpretation of the references to colour in this figure legend, the reader is ref

= 0.0088; Fig. 9C), and DMH (F(2,19) = 6.955; p = 0.0054; Fig. 9C).-mTOR-positive glia were not observed in the hypothalamus.

. Discussion

Here we report the novel findings that wheel running increasesTOR signaling in brain regions involved in cognition and emo-

rescence in the dentate gyrus of sedentary (top), voluntary run (middle) and forcedalized pixels only. White arrows indicate cells that were counted as double labels.to the web version of this article.)

tional behavior. Increases in p-mTOR immunoreactivity followingexercise were observed in almost every brain region investi-gated. Combined with prior reports of increased mTOR signaling

in peripheral tissues following exercise [80,81], these data suggestthat mTOR signaling could be a fundamental and ubiquitous cellu-lar target of physical activity. Given the emerging data supportinga role for mTOR in neural plasticity [27,82], cognitive function

64 B.A. Lloyd et al. / Behavioural Brain Research 323 (2017) 56–67

Fig. 9. Effects of exercise on p-mTOR in the hypothalamus. (A) Brain atlas diagram modified from Paxinos and Watson (2006) and representative photomicrograms depicting p-mTOR staining in the medial preoptic area (MPO) of sedentary (left), voluntary run (middle) and forced run (right) groups. White arrows indicate examples of p-mTOR-positiveneurons. (B) Both voluntary and forced exercise increase the number of p-mTOR-positive neurons compared to locked wheel controls in the dorsomedial hypothalamus( p-mTc el conc roup

[nii

ercBltiDamt[

DMH), MPO, and median preoptic nucleus (MnPO). Forced wheel running decreasesontrols. (C) Voluntary exercise increases intensity of stain compared to locked wheondition (p > 0.05). * represents p < 0.01 relative to sedentary rats. Bars represent g

24–26] and emotional regulation [36,40], increases in mTOR sig-aling following exercise could contribute to the broad beneficial

mpact of exercise on brain health, cognition, and emotional behav-or.

It is not necessarily surprising that both voluntary and forcedxercise increase mTOR signaling. Signals that lead to the phospho-ylation of mTOR would not be expected to be sensitive to exerciseontrollability. Indeed, both voluntary and forced exercise increaseDNF [83–85] and nutrient availability [41–44], which can each

ead to mTOR phosphorylation [45–47,50]. Despite the similari-ies in signaling mechanisms, voluntary running elicited a biggerncrease in mTOR signaling in many regions than forced exercise.ifferences in running distance or stress between the voluntary

nd forced running groups could account for these differences inTOR signaling. Voluntary running rats ran a greater distance over

he course of the study compared to forced wheel running rats70]. However, average weekly distance run was only positively

OR-positive cells in the ventromedial hypothalamus (VMH) compared to sedentarytrols in the DMH, VMH, MPO and MnPO. Forced exercise does not differ from eithermeans ± SEM.

correlated with p-mTOR in the dorsal striatum and MnPO. In fact,running distance was negatively correlated with p-mTOR in otherregions, such as the IL, NAc, CA1 and CA2. These regions mightbe expected to be particularly sensitive to stress; therefore, stresscould contribute to the negative correlations and the differencesbetween voluntary and forced running in these areas. In supportof this, prior work indicates that excessive voluntary running canelicit signs of chronic stress [86], such as gastric ulceration and mor-phologic changes in the hippocampus [87]. We have observed thatforced wheel running can also elicit signs of chronic stress [22],despite its rewarding [70] and stress-protective [22] effects. Sincereductions in mTOR have been reported following stressor expo-sure [88,89], it is possible that the negative correlations between

running and p-mTOR observed in some brain regions, as well asthe more modest effect of forced wheel running on p-mTOR, aredue to the potential stress effects of excessive or forced exercise.In these regions, then, overall levels of p-mTOR would reflect a

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ompetition between factors signaling an increase in p-mTOR andhe potentially inhibitory influence of stress. Consistent with thisnterpretation is the observation that in brain regions in which-mTOR was negatively correlated to voluntary running distance,

orced wheel running either did not increase p-mTOR or elicited more modest increase. However, the possibility that the 5 daysf voluntary wheel access prior to beginning forced wheel runningould have influenced the results in the forced wheel running groupannot be ruled out.

Another remaining question is whether the increase in p-mTORbserved in the brains of exercising rats represents levels of p-TOR elicited by the acute running bout, or an accumulation of

-mTOR or mTOR due to the history of repeated nightly exercise.ime-course data for the activation of p-mTOR in response to exer-ise does not exist at this time; however, previous data acquiredfter acute activation of mTOR signaling with inhibitory avoid-nce training show that the highest levels of phosphorylation occur–6 h after acute manipulation and return to baseline levels within

h [28]. Moreover, previous work in muscle indicates an equivalentncrease in p-mTOR in response to an acute exercise bout followingither 10 or 20 days of prior exercise training which suggests a lackf compounding effects of exercise history on tissue-p-mTOR [90].he levels of p-mTOR observed in the current study therefore most

ikely represent an increase in mTOR activity elicited by the acutexercise bout on the night of sacrifice. Levels of p-mTOR in regionsn which weekly running distance was positively correlated with-mTOR; however, could have been influenced by an increase inTOR protein availability prior to acute exercise. In other regions,

t remains unknown whether repeated nightly exercise is requiredo enable the increase in p-mTOR elicited by an acute exercise bout.

mTOR signaling in the PFC has been especially implicatedn antidepressant effects. The rapid antidepressant effects ofetamine, for example, are dependent on mTOR signaling in theedial PFC [40]. Exercise can reduce the incidence of depression

91] and can be used on its own [92–95] or as an adjunct toypical antidepressants [96,97] for the treatment of depression.imilarly, both voluntary and forced wheel running result in anntidepressant-like effect in the shuttle box escape task [22]. Inhe PFC; however, neither voluntary nor forced wheel runningncreased the number of p-mTOR-positive neurons and only vol-ntary wheel running increased p-mTOR staining intensity. The

act that mTOR signaling in the PFC is minimally sensitive to exer-ise is consistent with prior data suggesting that mechanismsutside of the PFC are involved in the antidepressant effects ofxercise [22,98]. In fact, negative correlations in the voluntary run-ing group were found between average weekly running distancend p-mTOR-positive neurons in the PFC. Interestingly, rats in theoluntary wheel running group that ran more than 5000 m pereek had lower levels of p-mTOR-positive neurons than lockedheel controls. Increases in mTOR signaling in areas outside of the

FC, such as the amygdala, hippocampus, or striatum, could leado neuroplastic adaptations in circuits contributing to the stress-rotective, antidepressant, and anxiolytic effects of exercise.

Increased mTOR signaling in the dorsal and ventral striatumould contribute to the adaptations in locomotor behavior [99]nd reward pathway sensitivity [100–102] known to occur fol-owing habitual physical activity. It is somewhat surprising thatxercise did not increase p-mTOR in the DMS, especially consider-ng that exercise elicits other adaptations in this region includinghanges in mRNA levels of dopamine and opioid receptors [72,103],-HT2C receptors [104], and increases in �FosB [70]. However,here were positive correlations between distance run and num-

ers of p-mTOR-positive neurons in the DMS and DLS of voluntarilyunning rats.

Both voluntary and forced wheel running resulted in largencreases in p-mTOR in the hippocampus. Interestingly, BDNF,

Research 323 (2017) 56–67 65

which is increased in the hippocampus following both voluntary[53,54,105] and forced [84,85] exercise, can increase mTOR signal-ing through trkB receptors [84]. The exercise-induced increase inBDNF in the hippocampus could, therefore, contribute to the par-ticularly robust increase in mTOR signaling observed in this region.These data are also consistent with mTOR being important for thebeneficial effects of exercise on hippocampal learning and memory.For example, mTOR signaling has been reported to be necessary forlearning of the inhibitory avoidance hippocampal-dependent task[30], and both voluntary [31,32] and forced [33] exercise enhanceperformance in this same task. Additionally, blockade of mTOR withRapamycin prevents the acquisition of memory necessary for novelobject recognition [30] which is another hippocampal-dependenttask in which performance is enhanced by voluntary [106] andforced [107] exercise.

Large increases in p-mTOR were observed in the hypothalamus,where both forced and voluntary exercise increased p-mTOR-positive neurons in the MPO, MnPO, and DMH. The increase inp-mTOR in hypothalamic regions could be related to these regions’role in thermoregulation [108,109]. Indeed, mTOR signaling in thehypothalamus has been shown modulate thermoregulation in rats[110] and exercise has been shown to both increase basal temper-ature and alter thermoregulatory response to acute stimuli in rats[111–113]. The positive correlation between running distance andp-mTOR-positive cells in the MnPO could be a result of increaseddemands on the thermoregulatory system with increasing runningdistance.

The majority of p-mTOR was observed in neurons. However, thehippocampus and amygdala contained an appreciable number ofp-mTOR-positive glia, overall numbers of which in the hippocam-pus were sensitive to exercise. Double fluorescent IHC revealedthat the p-mTOR-positive glia observed in the hippocampus andamygdala included a mix of both astrocytes (GFAP+) and microglia(CD11+), with a tendency toward a greater number of p-mTOR-positive astrocytes. Interestingly, however, while voluntary wheelrunning increased the percentage of astrocytes expressing p-mTORin the DG, forced wheel running increased the percentage of p-mTOR-positive microglia. These data suggest that, while both formsof exercise increase mTOR signaling in neurons and glia, the typeof glia sensitive to exercise may differ depending on whetherexercise is voluntary or forced. This is important, as mTOR couldsupport very different functions depending on type of glial cell.For example, astrocytes (GFAP+) release glutamate [114] as wellas BDNF [115]. Proliferation of astrocytes due to mTOR signalingcould thus further activate mTOR in neurons providing enhancedsynaptic plasticity and dendritic arborization [23,27,114]. mTORsignaling in astrocytes also leads to upregulation of glutamatetransporter 1 and can reduce glutamate neurotoxicity [116], there-fore potentially increasing the longevity of neurons. In microglia(CD11/OX42+), mTOR signaling is increased in response to oxida-tive stress, hypoxia, and cytokines including TNF� [49] and IL-1�,and modulates the production of nitric oxide [117]. mTOR signal-ing could also contribute to activation of microglia, as blockade ofmTOR with rapamycin decreases numbers of activated microglia[118,119]. Therefore, increases in p-mTOR-positive astrocytes inthe voluntary exercise condition could contribute to the benefi-cial effects of exercise on neuronal health and plasticity, whereasan increase of p-mTOR-positive microglia following forced wheelrunning could be a consequence of, or contributor to, cellular stressand inflammation. This difference could be driven by the greaterstress response elicited by forced, relative to voluntary, exercise[22]. Additional experiments are required for further speculation

on the effects of voluntary and forced exercise on mTOR signalingin glia.

Exercise activates mTOR in brain regions involved in cognitionand emotion. mTOR signaling in these regions could contribute to

6 l Brain

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6 B.A. Lloyd et al. / Behavioura

he beneficial effects of exercise on cognitive function, emotionalehavior, and brain health. Whether mTOR activation is requiredor the cognitive or stress-buffering effects of exercise remainsnknown. Future experiments utilizing mTOR inhibitors are war-anted to uncover whether mTOR signaling is required for the broadeneficial effects of exercise.

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