Short-term, moderate exercise is capable of inducing ... · Research Report Short-term, moderate exercise is capable of inducing structural, bdnf-independent hippocampal plasticity

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Research Report

Short-term, moderate exercise is capable of inducingstructural, bdnf-independent hippocampal plasticity

Ana F.B. Ferreiraa,⁎, Caroline C. Reala, Alice C. Rodriguesb,Adilson S. Alvesa, Luiz R.G. Brittoa

aDepartment of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, BrazilbDepartment of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil

A R T I C L E I N F O

⁎ Corresponding author at: Laboratory of Cellulaof São Paulo, Av. Prof. Lineu Prestes, 1524 São

E-mail address: [email protected]

Please cite this article as: Ferreira, A.Findependent hippocampal plasticity, Bra

0006-8993/$ – see front matter © 2011 Elseviedoi:10.1016/j.brainres.2011.10.004

A B S T R A C T

Article history:Accepted 2 October 2011

Exercise is known to improve cognitive functions and to induce neuroprotection. In this studywe used a short-term, moderate intensity treadmill exercise protocol to investigate the effectsof exercise onusualmarkers of hippocampal synaptic and structural plasticity, such as synapsinI (SYN), synaptophysin (SYP), neurofilaments (NF),microtubule-associated protein 2 (MAP2), glu-tamate receptor subunits GluR1 and GluR2/3, brain-derived neurotrophic factor (BDNF) and glialfibrillary acidic protein (GFAP). Immunohistochemistry, Western blotting and real-time PCRwere used. We also evaluated the number of cells positive for the proliferation marker 5-bromo-2-deoxyuridine (BrdU), the neurogenesismarker doublecortin (DCX) and the plasma cor-ticosterone levels. AdultmaleWistar rats were adapted to a treadmill and divided into 4 groups:sedentary (SED), 3-day exercise (EX3), 7-day exercise (EX7) and 15-day exercise (EX15). The pro-tein changes detected were increased levels of NF68 and MAP2 at EX3, of SYN at EX7 and ofGFAPat EX15, accompaniedbyadecreased level of GluR1at EX3. Immunohistochemical findingsrevealed a similar pattern of changes. The real-time PCR analysis disclosed only an increase ofMAP2 mRNA at EX7. We also observed an increased number of BrdU-positive cells and DCX-positive cells in the subgranular zone of the dentate gyrus at all time points and increased corti-costerone levels at EX3 and EX7. These results reveal a positive effect of short-term, moderatetreadmill exercise on hippocampal plasticity. This effect was in general independent of tran-scriptional processes and of BDNF upregulation, and occurred even in the presence of increasedcorticosterone levels.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Physical exerciseSynaptic plasticityStructural plasticityNeurogenesisDoublecortinBDNFCorticosterone

1. Introduction

Physical exercise has beneficial effects on brain health and cog-nition. It uses the processes of energymetabolism and synapticplasticity to promote brain health, upregulating proteins relatedto cognitive (Ding et al., 2006) and mitochondrial function(Kirchner et al., 2008). Exercise hasalso protective effects against

r Neurobiology, DepartmentPaulo, SP 05508-900, Brazr (A.F.B. Ferreira).

.B., et al., Short-term,in Res. (2011), doi:10.101

r B.V. All rights reserved.

several neurological diseases including Parkinson's disease(Smith and Zigmond, 2003), Alzheimer's disease (Mirochnic etal., 2009), and ischemic stroke (Stummer et al., 1994). In addition,exercisehas beenassociatedwith a reduced risk of cognitive im-pairment and dementia with age (Laurin et al., 2001).

Both acute and chronic exercises increase hippocampal ac-tivity (Holschneider et al., 2003, 2007). Exercise induces hippo-

of Physiology and Biophysics, Institute of Biomedical Sciences, Universityil. Fax: +55 11 3091 7426.

moderate exercise is capable of inducing structural, bdnf-6/j.brainres.2011.10.004

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campal synaptic plasticity mainly by enhancing synaptic effi-cacy and the expression of molecules involved in learning andmemory (Farmer et al., 2004; Vaynman et al. 2003, 2004). In-crease of neurotrophic factors, such as brain-derived neuro-trophic factor (BDNF), nerve growth factor (NGF) andfibroblast growth factor (FGF), and their mRNAs have beenwidely reported after exercise (Berchtold et al., 2010; Gomez-Pinilla et al., 1997; Neeper et al., 1996). Components of the pre-synaptic vesicle membrane, such as synapsin I (SYN) andsynaptophysin (SYP) are also found to be increased after exer-cise training (Vaynman et al., 2006). Exercise induces long-term potentiation (LTP) (van Praag et al., 1999a) and increasedglutamatergic activity (Leung et al., 2006), but the simulta-neous increase of the expression of neurotrophic factors,such as BDNF, promotes neuroprotection against the excito-toxic effects of glutamate in cell cultures (Jiang et al., 2005).There is evidence that treadmill exercise increases the num-ber of astrocytes and the level of glial fibrillary acidic protein(GFAP) in the frontoparietal cortex and dorsolateral striatumof exercised rats (Li et al., 2005) and stimulates the prolifera-tion of astrocytes in the subgranular zone (SGZ) (Uda et al.,2006). Voluntary physical activity is also known to induceadult hippocampal neurogenesis, increasing cell proliferationand survival (Ehninger and Kempermann, 2003; van Praag etal., 1999a, 1999b). In addition, structural neuronal proteinsmay also be affected by exercise due to their plastic character-istics in face of various stimuli (for reviews, see Sánchez et al.,2000; Julien, 1999).

Considering that for humans the recommended guidelinefor the practice of physical activity states at least 30 min ofmoderate-intensity activity on most days of the week (Hill-man et al., 2008), here we used an animal model of short-term, moderate intensity treadmill exercise protocol (Ferreiraet al., 2010) to investigate if this would be enough to induceplastic changes of synaptic and structural elements of therat hippocampus. We chose to study synaptic densitymarkers, such as SYN and SYP; structural neuronal proteinsto predict axonal and dendritic growth or remodeling, suchas NFs and MAP2; the neurotrophic factor BDNF, which hasbeen repeatedly associated to exercise-induced plasticity(Berchtold et al., 2010; Ding et al., 2006; Griesbach et al., 2004;Vaynman et al., 2003, 2004, 2006); glutamate receptor sub-units, such as GluR1 and GluR2/3, which are the predominantsubunits expressed in granular and pyramidal cells in the hip-pocampus (Petralia and Wenthold, 1992) and are related toexercise-induced increases of LTP (van Praag et al., 1999a);and the astrocytic marker GFAP to predict growth or remodel-ing of astrocytic processes, which are critical for neurovascu-lar coupling (Zonta et al., 2003) and energy metabolismespecially during exercise (Magistretti and Pellerin, 1996).Since the increase of adult hippocampal neurogenesis due tovarious exercise protocols has been widely reported (Ehningerand Kempermann, 2003; Kim et al., 2010; Uda et al., 2006; vanPraag et al., 1999a, 1999b), we studied the effect of this proto-col on cell proliferation and neurogenesis by evaluating, re-spectively, the number of 5-bromo-2-deoxyuridine (BrdU)-positive cells and doublecortin (DCX)-positive cells in theSGZ. In addition, due to the stressful nature of exercise, plas-ma corticosterone was measured to predict stress levels in-duced by the present treadmill protocol.

Please cite this article as: Ferreira, A.F.B., et al., Short-term,independent hippocampal plasticity, Brain Res. (2011), doi:10.101

2. Results

Our immunohistochemical data revealed a puntiform-granular pattern of hippocampal staining for anti-SYN andanti-SYP. Anti-SYN intensely stained the hilus (polymorphiclayer), whereas anti-SYP generated a less dense pattern withonly a few perikarya stained in the polymorphic layer. We ob-served an increased staining for SYN at EX7 (p<0.05) [F(3,28)=3.526, p=0.0276], accompanied by increased protein levels(p<0.05) [F(3,28)=5.343, p=0.0049] (Fig. 1). SYP immunoreac-tivity [F(3,28)=0.090, p=0.965] and protein levels [F(3,28)=0.535, p=0.662] were unaltered with the present exercise pro-tocol (Fig. 1).

For anti-NF, which stains all 3 polypeptides that constitutethe neuronal NF, we observed a staining pattern along axonsmainly in the polymorphic layer which increased at EX3(p<0.05) [F(3,28)=8.170, p=0.0005] with some staining in themolecular layer, which remained unchanged after exercise.The protein levels of only NF68 were increased at EX3 (p<0.05)[F(3,28)=5.335, p=0.0049], whereas the levels ofNF160 remainedunchanged [F(3,28)=1.162, p=0.3418] and NF200 was notdetected (Fig. 2). Anti-MAP2 stained neuropil in all regions ofthe DG and we could observe increased staining in the hilar re-gion for all exercise groups (p<0.001) [F(3,28)=16.39, p<0.0001],whereas protein levels were significantly increased only atEX3 (p<0.05) [F(3,28)=4.349, p=0.0123] (Fig. 2). Anti-GFAP alsoproduced a very diffuse staining of astrocytic processesthroughout the DG and we could observe increased staining inthe hilar region at EX3 (p<0.05) and EX15 (p<0.001) [F(3,28)=14.64, p<0.0001], accompanied by increased protein levels onlyat EX15 (p<0.01) [F(3,28)=7.019, p=0.0012] (Fig. 2).

Anti-GluR1 and anti-GluR2/3 stained perikarya and neuropilin the polymorphic layer, whereas some cells in the granularcell layer stained only for GluR1.We observed a decreased stain-ing for GluR1 in the hilar region at all exercise periods (p<0.001)with a more pronounced decrease at EX3 [F(3,28)=39.11,p<0.0001], which was the only group that presented decreasedprotein levels (p<0.05) [F(3,28)=4.046, p=0.0165] (Fig. 3). As forGluR2/3, whereas protein changes were not detected [F(3,28)=2.833, p=0.0563], the staining pattern in the hilar region sug-gested increases at EX7 and EX15 (p<0.05) [F(3,28)=5.612,p=0.0038] (Fig. 3). Anti-BDNF, on the other hand, stained mostlyperikarya in the polymorphic and granular cell layers and, inter-estingly, neither the staining for BDNF [F(3,28)=1.445, p=0.2509]nor its protein levels [F(3,28)=2.527, p=0.0777] showed changesafter the exercise protocol used here (Fig. 4).

As for the real-time PCR analysis, we only observed an in-crease of MAP2 mRNA expression at EX7 (p<0.05) [F(3,28)=4.788, p=0.0081]. No changes were observed for any of theother gene transcripts, and the expression of the GluR3mRNA could not be detected in the hippocampus in our condi-tions. The results for the mRNA analysis are summarized inTable 1.

In regard to cell proliferation and neurogenesis, we ob-served an increased number of BrdU-positive cells [F(3,20)=25.39, p<0.0001] and of DCX-positive cells [F(3,20)=24.99,p<0.0001] in the SGZ at all exercise periods. The number ofBrdU-positive cells reached a ca. 2-fold increase at EX3, washighest at EX7 and was still increased at EX15 (p<0.001),



Fig. 1 – Effects of treadmill running on SYN and SYP in the hippocampus. (A) Digital images of coronal sections of the dentategyrus stained for SYN and SYP. (B) Mean ratio of SYN and SYP integrated density data in the hilus in relation to the backgroundoptical density (N=8). (C) Mean ratio of SYN/β-actin and SYP/β-actin densitometry (N=8). Density data relative to the sedentarygroup (normalized) and typical immunoblots in each condition. SYN: synapsin I; SYP: synaptophysin; SED: sedentary; EX3:exercise training for 3 days; EX7: exercise training for 7 days; EX15: exercise training for 15 days (*p<0.05).

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although not as high as at EX3 and EX7 (Fig. 5). The stainingfor the neurogenesis marker DCX appeared to increase pro-gressively with exercise exposure and was found to be in-creased at all exercise periods (p<0.001) (Fig. 5). We also

Fig. 2 – Effects of treadmill running on NF, MAP2 and GFAP in thdentate gyrus stained for NF, MAP2 and GFAP. (B) Mean ratio of Nrelation to the background optical density (N=8). (C) Mean ratio o(N=8). Density data relative to the sedentary group (normalized)ments; NF68: 68 kDa neurofilament; NF160: 160 kDa neurofilamefibrillary acidic protein; SED: sedentary; EX3: exercise training fotraining for 15 days (*p<0.05; **p<0.01; ***p<0.001).


verified the occurrence of SGZ neurons co-localizing BrdUand DCX (Fig. 6), as expected.

The results of the corticosterone measurements revealed anincrease of plasma levels at EX3 (ca. 73%, 3742±431 pg/ml,

e hippocampus. (A) Digital images of coronal sections of theF, MAP2 and GFAP integrated density data in the hilus inf NF/β-actin, MAP2/β-actin and GFAP/β-actin densitometryand typical immunoblots in each condition. NF: neurofila-nt; MAP2: microtubule-associated protein 2; GFAP: glialr 3 days; EX7: exercise training for 7 days; EX15: exercise


image of Fig.�1

image of Fig.�2


Fig. 3 – Effects of treadmill running on GluR1 and GluR2/3 in the hippocampus. (A) Digital images of coronal sections of thedentate gyrus stained for GluR1 and GluR2/3. (B) Mean number of labeled cells/mm2 for GluR1 and GluR2/3 in the hilus (N=8). (C)Mean ratio of GluR1/β-actin and GluR2/3/β-actin densitometry (N=8). Density data relative to the sedentary group (normalized)and typical immunoblots in each condition. GluR1: glutamate receptor subunit 1; GluR2/3: glutamate receptor subunit 2/3; SED:sedentary; EX3: exercise training for 3 days; EX7: exercise training for 7 days; EX15: exercise training for 15 days (*p<0.05;**p<0.01; ***p<0.001).


p<0.05) andat EX7 (ca. 174%, 5901±721 pg/ml, p<0.001),whereasthe levels for EX15 (2678±313 pg/ml) were similar to the levelsdetected in the sedentary group (2151±276 pg/ml) [F(3,31)=13.69, p<0.0001], as previously reported (Real et al., 2010).

3. Discussion

The short-term exercise protocol used here appeared to in-duce increases of SYN, NF68, MAP2 and GFAP, accompaniedby a decrease of GluR1. The only transcriptional effectdetected was an increase of the mRNA coding for MAP2. Theother proteins and mRNAs studied remained unchanged,whereas BrDU- and DCX-positive cells increased.

The effects of exercise may depend on factors such as du-ration of exercise exposure, type of exercise performed andpossibly other, still uncharacterized variables (Berchtold etal., 2010); therefore, it is important to discuss the exercise pro-tocol used in this study for comparative purposes. Since ro-dents normally exhibit intense physical activity during thedark (active) period (Holmes et al., 2004), we conducted our

Fig. 4 – Effects of treadmill running on BDNF in the hippocampusstained for BDNF. (B) Mean ratio of BDNF integrated density data in(C) Mean ratio of BDNF/β-actin densitometry (N=8). Density datamunoblots in each condition, including a recombinant human Brotrophic factor; SED: sedentary; EX3: exercise training for 3 days15 days.


exercise training during their active cycle. In order to mini-mize stress, our protocol started with an adaptation periodallowing the animals to become familiarized to the treadmill,and we used a moderate intensity protocol (Ferreira et al.,2010). Treadmill exercise is a key component of many neuro-logical rehabilitation programs (Holschneider et al., 2007).However, it is considered to be a forced type of exercise(Arida et al., 2004). In fact, when submitted to stressful tread-mill exercise protocols, rats show activation of the amygdala(Vissing et al., 1996), although, rats exercised on runningwheels may also display increased anxiety-like behaviors(Grace et al., 2009).

Whereas high-intensity exercise protocols may increasecorticosterone levels, which inhibit the beneficial effects ofBDNF (Cosi et al., 1993) and neurogenesis (Gould et al., 1992),basal levels of glucocorticoids are necessary to maintain neu-rogenesis (Sloviter et al., 1993). We have measured corticoste-rone levels from our animals and found them to be increasedonly at EX3 and EX7, in agreement with earlier data (Tharpand Buuck, 1974). Another factor that should be taken intoconsideration is the novelty of the exercise experience during

. (A) Digital images of coronal sections of the dentate gyrusthe hilus in relation to the background optical density (N=8).relative to the sedentary group (normalized) and typical im-DNF sample (rhBDNF) as a control. BDNF: brain-derived neu-; EX7: exercise training for 7 days; EX15: exercise training for


image of Fig.�3

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Table 1 –mRNA expression of synaptic and structural elements after different periods of moderate exercise.

mRNAs Real-time PCR

SEDMean (±SEM)

EX3Mean (±SEM)

EX7Mean (±SEM)

EX15Mean (±SEM)

F values and degrees of freedom

SYN 1 2.27 2.49 1.52 [F(3,28)=1.055, p=0.384](±0.79) (±0.83) (±0.52) (±0.47)

SYP 1 5.62 4.59 2.76 [F(3,28)=1.251, p=0.310](±1.88) (±2.68) (±1.14) (±1.15)

BDNF 1 1.1 1.16 1.12 [F(3,28)=0.493, p=0.690](±0.07) (±0.11) (±0.10) (±0.10)

GLUR1 1 4.08 2.76 1.54 [F(3,28)=2.309, p=0.098](±0.53) (±1.46) (±0.71) (±0.58)

GLUR2 1 0.91 0.9 1.63 [F(3,28)=0.031, p=0.824](±0.41) (±0.68) (±0.82) (±0.56)

GLUR3 nd nd nd nd –NF68 1 1 1.29 0.98 [F(3,28)=0.712, p=0.553]

(±0.20) (±0.24) (±0.11) (±0.12)NF160 1 1.13 1.11 1.14 [F(3,28)=0.539, p=0.660]

(±0.09) (±0.08) (±0.09) (±0.09)NF200 1 0.93 1 0.88 [F(3,28)=0.189, p=0.903]

(±0.16) (±0.15) (±0.09) (±0.13)MAP2 1 1.41 4.04 1.18 [F(3,28)=4.788, p=0.008]

(±0.33) (±0.38) (±1.19) * (±0.21)GFAP 1 2.68 3.44 2.77 [F(3,28)=1.233, p=0.316]

(±1.02) (±1.05) (±0.58) (±1.01)

SED: sedentary; EX3: exercise training for 3 days; EX7: exercise training for 7 days; EX15: exercise training for 15 days; SYN: synapsin I; SYP:synaptophysin; BDNF: brain-derived neurotrophic factor; GLUR1.2 and 3: glutamate receptors types 1. 2 and 3; NF68: neurofilament 68 kDa;NF160: neurofilament 160 kDa; NF200: neurofilament 200 kDa; MAP2: microtubule associated protein 2; GFAP: glial fibrilary associated protein;nd: not detected; (*p<0.05 vs. sedentary groups).


the first few days. Therefore, the changes discussed here maybe at least in part the result of environmental stimuli and notonly of the exercise protocol, whichmight account for some ofthe differential changes that occurred at different time points.

The neurotrophin BDNF has been shown to increasesynaptogenesis (Mattson, 2008) and neurogenesis (Lee andSon, 2009; Zigova et al., 1998), to modulate synaptic plasticityin the adult brain (Vaynman et al., 2003, 2004), change themorphology of cells and dendrites (Tolwani et al., 2002), and

Fig. 5 – Effects of treadmill running on cell proliferation and neurosections of the dentate gyrus stained for BrdU and DCX. (B) Meancells/mm2 in the subgranular zone (N=6). BrdU: 5-bromo-2-deoxtraining for 3 days; EX7: exercise training for 7 days; EX15: exerc


to modify synaptic function in the hippocampus by modulat-ing the efficacy of neurotransmitter release (Kang and Schu-man, 1995). Even though BDNF is widely reported to beincreased after various exercise protocols (Ding et al., 2006;Griesbach et al., 2004; Kim et al., 2010; Vaynman et al., 2003,2004), we did not observe any changes of BDNF protein andmRNA levels after the exercise protocol used here. This sug-gests that the changes observed for the synaptic and structur-al proteins, some of which are regulated by BDNF, might be

genesis in the rat hippocampus. (A) Digital images of coronalnumber of BrdU-positive cells/mm2 and DCX-positive

yuridine; DCX: doublecortin; SED: sedentary; EX3: exerciseise training for 15 days (**p<0.01; ***p<0.001).


image of Fig.�5


Fig. 6 – Confocal images of coronal sections of the dentate gyrus double-stained for BrdU and DCX. Double-stained cells areclearly visible (arrows) throughout the SGZ. BrdU: 5-bromo-2-deoxyuridine; DCX: doublecortin.


regulated in the present conditions by neurotrophic factorsother than BDNF. As mentioned earlier, FGF-2 also increasesafter exercise and may be critical to mediate exercise-induced changes in the brain (Gomez-Pinilla et al., 1997). FGFis angiogenic (Folkman and Klagsburn, 1987), stimulates pro-liferation of astrocytes (Gomez-Pinilla et al., 1995) and pro-motes survival and growth of neurons (Anderson et al.,1988), all of which are increased by exercise (Black et al.,1990; Li et al., 2005; van Praag et al., 1999b). Another candidateto participate in the regulation of the above mentioned plasticmechanisms is the epidermal growth factor (EGF). AlthoughEGF has been shown to promote survival and differentiationof postmitotic neurons (Morrison et al., 1987) and to increasethe density of newborn cells in the subventricular zone, it ap-pears to shift the ratio of differentiation of those cells towardsa glial lineage in the SGZ (Kuhn et al., 1997), whereas we ob-served a shift towards the neuronal fate based on the increaseof DCX-positive cells. On the other hand, EGF might haveplayed some role in exercise-induced hippocampal plasticityobserved here, as we detected increased GFAP levels, andEGF is known to induce proliferation of astrocytes (Kornblumet al., 1998). Alternatively, it is possible that BDNF signalingis increased by the present protocol through receptor sensiti-zation/upregulation, which remains to be evaluated.

BDNF is involved in the synthesis (Vaynman et al., 2006)and phosphorylation of SYN (Jovanovic et al., 1996, 2000).Even though we found BDNF levels to be unchanged afterthe present protocol of exercise, we observed increased levelsof SYN at EX7. SYN is involved in vesicle clustering, neuro-transmitter release, axonal elongation and maintenance ofsynaptic contacts (Fornasiero et al., 2010; Greengard et al.,1993; Jovanovic et al., 1996). This synaptic protein is frequentlyused as a predictor of synaptic density (De Camilli et al., 1983;Fornasiero et al., 2010) and is increased by exercise (Molteni etal., 2002; Vaynman et al., 2006). These studies, as well asmanyothers, support our findings of increased SYN after exercise.We did not notice, however, changes of SYP, the secondnerve terminal protein studied here, even though this proteinhas been noted to change in the same proportion as SYN aftersome exercise protocols (Vaynman et al., 2006).

As mentioned earlier, exercise increases glutamatergic ac-tivity (Leung et al., 2006). Glutamate is definitely involved inthe mechanisms that promote learning and memory, andthe activation of glutamate receptors has a role in the genera-tion of LTP as a response to exercise (van Praag et al., 1999a).


GluR1 and GluR2 are clearly related to LTP mechanisms anddo undergo plastic changes after exercise (Dietrich et al.,2005; Real et al., 2010). Since it has been shown that short-term exercise promotes an increase of glutamate (Leung etal., 2006), the decreased levels of GluR1 at EX3 observed inour study could represent a protective strategy to preventover-excitation and neurotoxicity by glutamate. The concen-tration of glutamate appears to decrease after 7 days of exer-cise (Leung et al., 2006), when we found the protein levels ofGluR1 to have returned to control levels. There is evidence,however, of increased GluR1 mRNA expression after 3 and7 days of voluntary exercise (Molteni et al., 2002). Nonetheless,Chen et al. (2007) have demonstrated that voluntary andforced exercise may activate distinct signaling pathways,which could explain the different findings between voluntaryexercise (Molteni et al., 2002) and the present protocol.

During development, GluR1 and GluR2 are related to in-creases of length and complexity of dendritic arborizations(Chen et al., 2009). This doesn't appear to be a mechanism in-volved in the changes that occur in the adult brain and theones observed here, as we noticed increases of MAP2 and NF68after exercise despite the decreased levels of GluR1 and unal-tered levels of GluR2/3. MAP2 is an early and sensitive markerof neuronal damage following traumatic brain injury (Huh etal., 2003), and has not yet been associated with exercise-dependent plasticity. Increased levels of MAP2 mRNA in thegranule cell dendrites have been associated with the inductionof LTP in hippocampal perforant path/granule cell synapses inrats (Roberts et al., 1998) and with some forms of hippocampus-mediated memory processes (Fanara et al., 2010). On the otherhand, decreased levels of MAP2 and NFs have been associatedwith hypercortisolism (Cereseto et al., 2006). Our findingsrevealed increases ofMAP2 protein andmRNA, together with in-creased immunoreactivity and levels of NF68. To the best of ourknowledge, this is the first evidence of changes of protein levelsof NFs and MAP2 in response to exercise, despite reports of in-creased dendritic length (Stranahan et al., 2007) and complexity(Eadie et al., 2005). Together with previous literature, the presentdata can be interpreted as a beneficial plastic effect. In fact, in-creased perikaryal levels of NF proteins are thought to be neuro-protective in diseases such as amyotrophic lateral sclerosis, dueto NF association to calcium-binding proteins (for a review, seeJulien, 1999). It is noteworthy, however, that the increase ofMAP2 preceded the increase of MAP2 mRNA, whereas no NFmRNA has changed after exercise. Changes of protein levels in


image of Fig.�6



the absenceofmRNAchangesmaybe explainedbyprotein accu-mulationdue to increasedprotein stability and/or decreasedpro-tein degradation, which also applies to SYN and GFAP data.

Exercise-induced astrocytic changes have also been previ-ously reported. It was observed that astrocytic density andGFAP levels increase in the cortex and striatum after 3 and6 weeks of treadmill exercise (Li et al., 2005). In the SGZ,GFAP-expressing cells increase after 7 days of wheel running(Komitova et al., 2005). The association of exercise-inducedastrocytosis and angiogenesis is believed to strengthen theneurovascular unit and consequently the blood–brain barrier,which provides protective functions, especially after brain in-jury (Li et al., 2005). Considering the two studies mentionedabove, the present report appears to be one of the few to ob-serve astrocytic plasticity after exercise, as we demonstratedincreases of GFAP after 3 and 15 days of moderate exercise.

We also detected an exercise-induced increase of cell prolif-eration andneurogenesis in the SGZ after all periods of exercise,and demonstrated that 3 days of moderate intensity treadmillexercisewere sufficient to induce these changes. The immunos-taining for DCX, a protein that promotes microtubule polymeri-zation and is present in migrating neuroblasts and youngneurons (von Bohlen Und Halbach, 2007), revealed increasesthat progressed with exercise exposure. This finding suggeststhat even though the levels of cell proliferation remained stablethrough time, the ratio of cell differentiation possibly shifted to-wards the neuronal fate. Voluntary exercise has been shown toenhance neurogenesis and improve cognitive performance(Fabel and Kempermann, 2008; van Praag, 2008). Other authorshave also reported increased neurogenesis after 3 days of exer-cise, but they do not comment on the distance their mice ranper night, therefore limiting possible comparisons to our resultswith rats (Kronenberg et al., 2006).

In conclusion, the changes of SYN, NF68, GluR1, MAP2 andGFAP as a result of different periods of treadmill runningreported here suggest a positive effect of short-term, moderateintensity treadmill exercise on hippocampal plasticity, whichwas in general independent of transcription regulation and ofBDNF upregulation. In addition, the present protocol appearedto be sufficient to increase hippocampal neurogenesis as earlyas 3 days after exercise training. These changes might be subja-cent to anatomical and functional plasticity of the hippocampalarea generated by physical exercise. Furthermore, the increasingbody of information on exercise-induced changesmay be usefulto develop strategies to prevent or treat functional decline fol-lowing aging, neurological disorders and trauma.

4. Experimental procedures

4.1. Animals

Male 2month-oldWistar rats weighing ca. 250 g (obtained fromtheAnimal Facility of the Institute of Biomedical Sciences of theUniversity of São Paulo) were housed in groups in standardpolyethylene cages with food and water ad libitum, room tem-perature of 23 °C and a 12/12 h light–dark inverted cycle(Holmes et al., 2004). All protocols were approved by the EthicsCommittee for Animal Research of the University of São Pauloand experimental procedures were performed in accordance


with the guidelines of the Brazilian College for Animal Experi-mentation (COBEA) and the animal care guidelines of the Na-tional Institutes of Health (NIH/USA).

4.2. Exercise protocol

All animals went through a two-day adaptation period to atreadmill (KT 3000— IMBRAMED, Brazil, adapted for rats) dur-ing which they were allowed to explore the equipment andthe treadmill was turned on for only 15 min at low speeds(0.3 to 0.5 km/h). This procedure has the purpose of excludinganimals which are intolerant to the treadmill and refuse torun, providing a homogenous group of rats that will then berandomly divided into 4 groups: sedentary (SED) and exercisefor 3 days (EX3), 7 days (EX7) and 15 days (EX15). Each of theexperimental groups exercised for 40 min a day at 10 m/min(0.6 km/h) in the middle of the active cycle (between 11 amand 1 pm), whereas the sedentary group remained in thecages near the treadmill. The inverted cycle and this periodof training were used to avoid the development of internaldesynchronization, similar to the effect observed in night-shift workers, which was previously detected in rats that exer-cised during their light cycle (Salgado-Delgado et al., 2008).The animals which presented problems adapting to the tread-mill or refused to run were excluded. Different groups of ratswere used for immunohistochemistry, immunoblotting andreal-time PCR assays.

4.3. Immunohistochemistry

4.3.1. Tissue processingAfter the exercise period, the animals (8 animals per group)were deeply anesthetized (ketamine, 20 mg/100 g of bodyweight; xylazine, 2 mg/100 g, i.m.) and perfused transcardiallywith 300 mL of 0.1 M phosphate buffered saline (PBS) followedby 300 mL of 2% paraformaldehyde in 0.1 M sodium phos-phate buffer (PB), pH 7.4. The brains were then removed andpost-fixed for 4 h in the same fixative at 4 °C and cryopro-tected with a 30% sucrose solution (in PB) for 48 h at 4 °C. Cor-onal sections (30 μm) were cut on dry ice using a slidingmicrotome (Leica SM 2000R—Heidelberger, Nussloch, Germa-ny). Sections were stored in PB at 4 °C until use.

4.3.2. ImmunostainingFree-floating sections were stained with a series of antibodies,namely rabbit polyclonal anti-SYN (1:1000) (Chemicon, Temecula,USA), rabbit polyclonal anti-SYP (1:250) (DakoCytomation,Glostrup, Denmark),mousemonoclonal anti-NFs (PAN, recogniz-ing 68 kDa, 160 kDa and 200 kDa neurofilaments) (1:2000) (ZymedLaboratories, San Francisco, CA, USA), rabbit polyclonal anti-BDNF (1:500) (Chemicon, Temecula, USA), mouse monoclonalanti-MAP2 (1:1000) (Chemicon, Temecula, USA),mousemonoclo-nal anti-GFAP (1:1000) (Immunon, Pittsburgh, PA, USA), rabbitpolyclonal anti-GluR1 and anti-GluR2/3 (1:250) (Chemicon, Te-mecula, CA, USA). The antiserum against GluR2/3 recognizes anepitope common to theGluR2 andGluR3 subunits. As the expres-sion of GluR3 in the hippocampus is very lowwhen compared tothe expression of GluR2, it is generally assumed that the widelyusedGluR2/3 antibody provides a goodpicture of theGluR2distri-bution in the brain (Petralia and Wenthold, 1992). All antibodies




are routinely used by several laboratories. The secondary anti-bodies were biotinylated goat anti-rabbit antisera for SYN andBDNF, donkey anti-rabbit antisera for SYP, GluR1 and GluR2/3,donkey anti-mouse antisera forMAP2 andGFAP (all from JacksonImmuno Research Lab., West Grove, Pennsylvania, USA) and agoat anti-mouse antiserum for NFs (Vector, Burlingame, CA,USA). The primary antibodies were diluted in PBwith 0.3% TritonX-100 and 5% normal goat serum (for anti-SYN, anti-BDNF andanti-NFs) or normal donkey serum (for anti-SYP, anti GluR1,anti-GluR2/3, anti-MAP2andanti-GFAP) and the sectionswere in-cubated overnight (14–20 h) at room temperature (ca. 24 °C). Afterwashing the sections with PB (3×10min), they were incubatedwith the corresponding secondary antibodies, which were all di-luted 1:200 in PB with 0.3% Triton X-100 for 2 h at room tempera-ture. Following additional washes (3×10min), the sections wereincubated with the avidin–biotin-peroxidase complex (ABC Elitekit, Vector Labs., Burlingame, CA, USA) for 2 h at room tempera-ture. Labelingwas developedwith 0.05% diaminobenzidine tetra-hydrochloride (DAB) and 0.03% (final concentration) hydrogenperoxide in PB. To confirm the specificity of the antibodies, a sep-arate set of sections fromeach groupwas incubatedonlywith thesecondary antibodies, a condition in which no staining waspresent.

After the staining procedure, the sections were mountedon glass slides and the staining was intensified with 0.05% os-mium tetroxide in water. They were then dehydrated and cov-erslipped using Permount (Fisher, Pittsburg, PA, USA). Theregion of interest was identified based on a stereotaxic atlas(Paxinos and Watson, 2005) using a 20× objective on a NikonE1000 microscope (Melville, NY, USA). Images were capturedusing a Nikon DMX1200 digital camera, encompassing anarea of 54,000 μm2 of the dorsal hippocampus, between 3and 4 mm behind the bregma (5–7 sections/brain) (Image J,NIH/USA).

4.4. Western blotting

The animals (8 animals per group) were decapitated and theirhippocampi quickly collected, frozen in liquid nitrogen andstored at −70 °C until use. The tissue was then homogenizedat 4 °C in extraction buffer (Tris, pH 7.4, 100 mM; EDTA10 mM; PMSF 2 mM; aprotinin 0.01 mg/ml). The homogenateswere centrifuged at 12,000 rpm (15294 g) (Eppendorf Centri-fuge 5804R — Westbury, NY, USA) at 4 °C for 20 min, and theprotein concentration of the supernatant was determinedusing a protein assay kit (Bio-Rad, Hercules, CA, USA) (Brad-ford, 1976). The material was stored in a sample buffer(Tris/HCl 125 mM, pH 6.8; 2.5% (p/v) SDS; 2.5% 2-mercaptoethanol, 4 mM EDTA and 0.05% bromofenol blue)(Laemmli, 1970) at −70 °C until starting the assays. Samplescontaining 75–100 μg of total proteins in Laemmli bufferwere boiled for 5 min and separated by 6.5%, 8% and 12% ac-rylamide SDS gels (Bio-Rad, Hercules, CA, USA) at 25 mA(Laemmli, 1970) and electrophoretically transferred to nitro-cellulose membranes (Millipore, Temecula, CA, USA) at 100 Vfor 80 min using a Trans-Blot cell system (Bio-Rad, Hercules,CA, USA). A sample of 800 ng of recombinant human BDNF(rhBDNF) (Sigma, St. Louis, MO, USA) reconstituted with0.2 μm-filtered PBS/0.1% BSA to a concentration of 50 mg/mlwas also applied to the 12% gels as a control for BDNF (Das


et al., 2001). The membranes were then blocked for 2 h atroom temperature with PBS containing 0.05% Tween-20(TTBS) and 5% non-fat milk, and incubated overnight at 4 °Cwith the same primary antibodies used for immunohisto-chemistry at a concentration of 1:1000. The membranes werethen incubated for 2 h with anti-rabbit-HRP IgG for SYN, SYP,BDNF, GluR1 and GluR2/3 and anti-mouse-HRP IgG for NFs,MAP2 and GFAP (Amersham, Little Chalfont, Buckingham-shire, UK) diluted 1:10,000 in TTBS with 1% non-fat milk. Theprobed proteins were developed by using a chemiluminescentkit (ECL, Amersham Biosciences, NJ, USA). Themembrane wasthen incubated for 30 min at room temperature with strippingbuffer and an anti-β-actin antibody (Sigma, St. Louis, MO,USA) was used to quantify β-actin as a loading control. Thebound antibodies were visualized using radiographic filmswhich were placed in contact with the membranes, then de-veloped and fixed. The quantification of band intensity wasperformed with Scion Image 4.0.2 (Scion Corporation, Freder-ick, MD, USA).

4.5. RNA isolation, cDNA synthesis and real-time PCR

The hippocampi were collected (8 animals per group) and im-mediately homogenized in 1 mL TRIzol (Invitrogen, Carlsbad,CA, USA) with a homogenizer and total RNA was isolated fol-lowing themanufacturer's suggested protocol. Briefly, followingone chloroform extraction step, RNA was precipitated with iso-propanol and the pellet washed once in 70% ethanol. After air-drying, RNA was resuspended in DEPC-treated water and theconcentration of each sample was obtained from A260/A280nm measurements. Residual DNA was removed using DNase I(Invitrogen) by following the manufacturer's protocol. For each20 μL reverse transcription reaction, 4 μg total RNA was mixedwith 1 μL oligodT primer (0.5 μg/μL; Invitrogen) and incubatedfor 10min at 65 °C. After cooling on ice the solution was mixedwith 4 μL 5× first strand buffer, 2 μL of 0.1 M DTT, 1 μL of dATP,dTTP, dCTP and dGTP (10 mM each), and 1 μL SuperScript III re-verse transcriptase (200 U/μL; Invitrogen) and incubated for60 min at 50 °C. Reaction was inactivated by heating at 70 °Cfor 15 min, and the samples were diluted four times.

The real-time PCR reaction system included the following:200 to 400 nM primers, 5 ng cDNA samples, and 1× SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, CA,USA). Using the Rotor-Gene 3000 Real-time PCR detection sys-tem (Corbett Research, Mortlake, NSW, Australia), cycling con-ditions were set as follows: after initial activation at 50 °C for2 min and 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °Cfor 1 min, then melt curve analysis was performed by heatingsamples from65 °C to 99 °C (1 °C increment changes at 5 s inter-vals), in order to evaluate primer specificity. All sample mea-surements were performed in duplicate. Primers used for thehousekeeping genes, hydroxymethylbilane synthase (HMBS)and hypoxanthine phosphoribosyltransferase 1 (HPRT1), weredescribed by Depreter et al. (2002) and the primers for thegenes of interest were designed using software primer expressv3.0 (Applied Biosystems) (Depreter et al., 2002). The efficiencyof the primers used ranged from 98 to 118% (106±7%) andtheir properties are described in Table 2.

Relative quantification of target gene expression was per-formed using the comparative CT method, as described in detail



Table 2 – Description of primers used to study the genesof interest and the housekeeping genes*.

Gene Refseq Primers sequence(5′ to 3′)

Ampliconlength (bp)

NF68 NM_031783.1 FW: AGA CAT CAG CGCCAT GCA

60

RV:TTC GTG CTT CGCAGC TCA T

NF160 NM_017029.1 FW: GGC TCC AGA CATTGT ATT TTC CTT

63

RV: GGC ACC CTG AGCTTG CAT

NF200 NM_012607.2 FW: AGA GGA GTG GTTCCG AGT GAG A

70

RV: GCG CAT AGC ATCCGT GTT C

SYN NM_001110782.1and NM_019137.1

FW: TTC AGC ATG GCACGT AAT GG

59

RV: CCA GCA TAC TGCAGC CCA AT

SYP NM_012664.1 FW: CCC TTC AGG CTGCAC CAA

62

RV: TTG GTA GTG CCCCCT TTG AC

BDNF NM_007540.4 FW: CAC TTT TGA GCACGT GAT CGA

59

RV: CGT TGG GCC GAACCT TCT

MAP2 NM_013066.1 FW: AGA TCA GAAAGA CTG GTT CAT CGA

65

RV: CAG CTA AAC CCCATT CAT CCT T

GFAP NM_017009.2 FW: CCT TGA CCT GCGACC TTG AG

62

RV: GCG CAT TTG CCTCTC CAA

GLUR1 NM_031608.1 FW: ATG CGG CTT TTAGGT TTG CTT

115

RV: TGG GAA CAG AAACGG TAA GTC AT

GLUR2 NM_017261.2 FW: ATG GTT CAG TTTTCC ACT TCG G

120

RV: TGC GTA GAC TCCTCT TGA AAA CTG

GLUR3 NM_032990.2 FW: ACG GGC AGA GTCCAA ACG

192

RV: GCA GCC ACG TTTTCA GTG ATT

HMBS* NM_013168 FW: TCT AGA TGG CTCAGA TAG CAT GCA

76

RV: TGG ACC ATC TTCTTG CTG AAC A

HPRT1* X62085 FW: GCG AAA GTG GAAAAG CCA AGT

76

RV: GCC ACA TCA ACAGGA CTC TTG TAG

NF68: neurofilament 68 kDa; NF160: neurofilament 160 kDa; NF200:neurofilament 200 kDa; SYN: synapsin I; SYP: synaptophysin;BDNF: brain-derived neurotrophic factor; MAP2: microtubule asso-ciated protein 2; GFAP: glial fibrillary associated protein; GLUR1, 2and 3: glutamate receptors types 1, 2 and 3; HMBS: hydroxymethyl-bilane synthase; HPRT1: hypoxanthine.


elsewhere (Medhurst et al., 2000). The ΔCT valuewas determinedby subtracting the target CT of each sample from the respectivehousekeeping genes mean values. Calculation of ΔΔCT involved


the sedentary group mean ΔCT value as an arbitrary constant tosubtract from all other ΔCT mean values. Fold-changes in geneexpression of the target gene are equivalent to 2−ΔΔCT.

4.6. Detection of 5-bromo-2-deoxyuridine and doublecortin

BrdU (AmershamCell Proliferation Kit, Little Chalfont, Bucking-hamshire, UK) was dissolved in dH2O. Each rat received a singleinjection of 50mg/kg of body weight at a concentration of50 mg/mL at the end of the training period. The animals weretranscardially perfused 3 h after the injection of BrdU and im-munohistochemistry for BrdUwas performed. The transcardiacperfusion and tissue preparation for immunohistochemistrywere both performed as described in the tissue processing sec-tion. Free-floating 30 μm-sections were washed (3×10 min)with PBST (PBS+0,1% Triton X-100), pretreated/denaturatedwith 2 N HCl for 1 h, washed again (3×10min), fixed with0.1 M Na2B4O7 at 4 °C for 10min and washed again (3×10min).After the pretreatment, the sections were incubated overnightat room temperature with amouse monoclonal anti-BrdU anti-body (1:1000) (Amersham, Little Chalfont, Buckinghamshire,UK) and 5%normal donkey serum. The sectionswere then incu-bated for 2 h with a biotinylated donkey anti-mouse secondaryantibody (1:200) (Jackson Immuno Research Lab., West Grove,Pennsylvania, USA). For the staining of DCX, the sections werepre-incubated in 10% normal donkey serum and incubated for48 h at room temperature with a goat polyclonal anti-DCX anti-body (1:100) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA) and 10%normal donkey serum.The sectionswere then in-cubated for 2 h with a biotinylated donkey anti-goat secondaryantibody (1:200) (Jackson Immuno Research Lab., West Grove,Pennsylvania, USA). Immunostaining for both sets of tissuewas performed as described above. Digital images were cap-tured using a 20× objective on a Nikon microscope (NikonE1000, Melville, NY, USA) and camera (Nikon DMX1200). BrdU-and DCX-positive cells in the SGZ were counted (Image J,NIH/USA) in areas of 54,000 μm2 from 5 to 7 sections per animal(N=6), between 3 and 4mm behind the bregma (Paxinos andWatson, 2005).

To verify possible co-localization of these markers, the sec-tions were pre-incubated in 10% normal donkey serum for 1 h,incubated with Alexa Fluor 488-conjugated mouse monoclonalanti-BrdU antibody (1:500) (Caltag Laboratories, Invitrogen Cor-poration, Carlsbad, CA, USA) overnight at room temperatureand with the goat polyclonal anti-DCX antibody (1:100) (SantaCruz Biotechnology, Inc., Santa Cruz, CA, USA) for 48 h at roomtemperature followed by TRITC-conjugated IgG donkey anti-goat (1:50) (Jackson Immuno Research Lab., West Grove, Penn-sylvania, USA). Confocal images were acquired using a 40× oilobjective on an inverted confocal microscope (Zeiss AxiovertLSM510 — Carl Zeiss, Germany) and Z-series was conductedfrom a total of 20 μm (1 μm interval).

4.7. Corticosterone determination

For corticosterone determination, the rats (7–10 animals pergroup)weredecapitated, and the bloodwas collected invacutai-ners containing sodium heparine. Samples were then spun at1,000×g for 15min at 4 °C to obtain plasma. The plasma sam-ples were stored at −70 °C until dosage of the hormone with




an ELISA kit (Cayman Chemical Co., Ann Arbor, MI,USA —500651 Corticosterone EIA Kit). All the samples were processedin duplicate at a dilution of 1:10. The method used here hasbeen described elsewhere (Pradelles et al., 1985). Briefly, the di-luted samples were incubated for 2 h at room temperature incorticosterone conjugated with acetylcholinesterase and a spe-cific antiserum in a 96-well plate pre-covered with rabbit anti-IgG antibody. After the incubation, the plates were washedwith the provided wash buffer (1:400) and Tween 20 (1:2000) di-luted in ddH2O and the enzymatic substrate was added (Ellmanreagent). The optic density of the sampleswas determined after1 h using the ELISA reader (412 nm) and the concentration ofcorticosterone was calculated using a standard curve.

4.8. Statistical analysis

Data are expressed as the mean±SEM. Statistical analyseswere performed using one-way ANOVA with Tukey post-hoctest for immunohistochemistry, Western blotting and mRNAexpression data and one-way ANOVA with the Bonferronipost-hoc test for plasma corticosterone.

Acknowledgments

This study was supported by FAPESP and CNPq (Brazil). Theauthors would like to thank Drs. Andréa S. Torrão, Rui Curiand Mauro Leonelli for their helpful suggestions and support,and Daniel O. Martins for helping with some experimentalprotocols. A.F.B.F., C.C.R. and A.C.R. are the recipients of fel-lowships from FAPESP.

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Short-term, moderate exercise is capable of inducing ... · Research Report Short-term, moderate exercise is capable of inducing structural, bdnf-independent hippocampal plasticity