www.elsevier.com/locate/brainres

Brain Research 1065

Research Report

Exercise affects glutamate receptors in postsynaptic densities

from cortical mice brain

Marcelo O. Dietrich a, Carlos E. Mantese a, Lisiane O. Porciuncula a, Gabriele Ghisleni a,

Lucia Vinade a,b, Diogo O. Souza a, Luis V. Portela a,*

aDepartamento de Bioquımica, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul,

Rua Ramiro Barcelos, 2600-Anexo 90035-003, Porto Alegre/RS, BrazilbLaboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA

Accepted 27 September 2005

Available online 18 November 2005

Abstract

Physical activity has been proposed as a behavior intervention that promotes mental health and some of the benefits induced by exercise

have been related to the glutamatergic system. Indeed, glutamate is the most abundant excitatory neurotransmitter in brain. Thus, we

evaluated if voluntary exercise in mice could modulate glutamatergic synapses at level of postsynaptic density (PSD). Through Western blot,

we found that exercise during 1 month increased glutamatergic-related protein content in PSD from cortex of mice. Exercise increased the

immunocontent of GluR1 (129%), SAP-97 (179%), GRIP-1 (129%), and in less extent, GluR2/3 (118%) and PSD-95 (112%) proteins. The

overall content of NMDA subunits R1, R2A and R2B were not altered in mice that had exercised, however, the phosphorylated NMDA

subunits, phospho-NMDAR1 (150%), and phospho-NMDAR2B (183%) showed a strong increase. Because exercise increased the content of

phosphorylated forms of NMDA receptors, we evaluated the binding of MK-801, a specific ligand that binds to open NMDA channel.

Exercise increased the binding of MK-801 in cortical cellular membranes in 51%. Altogether, our results point to a modulation of

glutamatergic synapses by exercise with likely implications in the exercise-induced mental health.

D 2005 Elsevier B.V. All rights reserved.

Theme: Excitable membranes and synaptic transmission

Topic: Postsynaptic mechanisms

Keywords: Voluntary exercise; Brain; Glutamate receptor; NMDA; Phosphorylation

1. Introduction

Glutamate is the most abundant neurotransmitter in the

mammalian central nervous system (CNS), acting on iono-

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.09.038

Abbreviations: AMPA, a-amino-3-hydroxi-5-methyl-4-isoxazol pro-

pionic acid; GluR1, GluR2/3, GluR6/7, glutamate receptor subunit type

1, 2/3 and 6/7, respectively; GRIP-1, glutamate receptor interacting protein

1; MAGUK, membrane-associated guanylyl kinase; NMDA, N-methyl-d-

aspartate; p-NR1 and p-NR2B, phosphorylated NMDA receptor subunit

type 1 and type 2B, respectively; PSD, postsynaptic density; PSD-95,

postsynaptic density protein of 95 kDa; SAP-97, synapse associated protein

97

* Corresponding author. Fax: +55 51 3316 5540.

E-mail address: roskaportela@gmail.com (L.V. Portela).

tropic N-methyl-d-aspartate (NMDA), a-amino-3-hydroxi-

5-methyl-4-isoxazol propionic acid (AMPA) and kainate

(KA) receptors, and on metabotropic G protein-coupled

receptors, all of which are essential receptors for normal brain

functions [35]. However, as overstimulation of glutamatergic

receptors triggers neuronal damage involved in acute and

chronic brain diseases [28,31,35,46], a fine modulation of

glutamatergic synapses activity is pivotal for CNS function.

Regular physical activity, an essential component of a

healthy lifestyle, recently has been shown to mediate central

nervous system (CNS) adaptations, up to now more

extensively reported in hippocampus [1,6]. Animal studies

have demonstrated that exercise protects neurons from

various brain insults [4,18,47,54], activates neuronal cells

(2005) 20 – 25

Fig. 2. Effect of 4 weeks voluntary exercise on the binding of [3H]MK-

801, a specific ligand to the MK-801 binding site in the inner portion of

the NMDA channel. Exercise increased the binding of 4 nM [3H]MK-801

M.O. Dietrich et al. / Brain Research 1065 (2005) 20–25 21

[7], promotes neurogenesis [48] and improves the perform-

ance in learning tasks [49].

The postsynaptic density (PSD) is a protein complex that

lays postsynaptic membranes at the synaptic contact zone.

PSD contains key elements involved in neuronal postsy-

naptic activity, such as ionic channels, neurotransmitter

receptors, anchoring proteins and membrane signal trans-

duction enzymes [34,44]. A subcellular preparation enriched

in PSD maintains much of the molecular organization and

activities observed in situ, thus, it becomes an excellent tool

to investigate plastic synaptic characteristics of excitatory

glutamatergic synapses [38,42,51]. In the present work, we

have evaluated the effects of voluntary exercise on

glutamatergic ionotropic receptors in PSD preparation

obtained from mice cerebral cortex.

in 51 T 11% ( P < 0.05), which mostly indicates the state of opening/

activity of the NMDA channel (absolute binding value of 100%: 0.33 T

0.04 pmol/mg protein).

2. Results

Voluntary exercise triggered an increase in the immuno-

content of AMPA (GluR1, GluR2/3; P < 0.05), but not KA

(GluR6/7, P > 0.05) and nor NMDA (NR1, NR2A, NR2B)

receptor subunits (Fig. 1) in PSD from cerebral cortex, a

brain area engaged in physical activity.

The glutamatergic synaptic strength depends on the

number and activity of glutamate receptors, and their activity

may be modulated by many factors, as their phosphorylated/

Fig. 1. Effect of 4 weeks of voluntary exercise on the immunocontent of

specific proteins in cortical PSD fraction of adult mice. Representative paired

Western blot lanes are shown. Histograms represent percentage of increase in

relation to sedentary controls through densitometric quantification of

immunoblots, from above to below: NMDAR1 (108 T 7%); NMDAR2A

(111 T 7%); NMDAR2B (102 T 1%); GluR1 (129 T 4%); GluR2/3 (118 T

3%); GluR6/7 (131 T 10%); PSD-95 (112 T 2%); SAP-97 (179 T 26%);

GRIP-1 (129 T 6%); p-NMDAR1 (150 T 7%); and p-NMDA2B (183 T 28%).

Results are mean T standard error of the mean. *P < 0.05 vs. sedentary

control. n = 4–5 PSD preparations for all experiments. S, sedentary group;

Ex, voluntary exercise group.

dephosphorylated state. Since the phosphorylation of NMDA

receptor subunits affects their activities [8,21,22,25,45], the

immunocontent of phosphorylated NMDA subunits in PSD

fraction was assessed with phospho-specific antibodies,

aiming to better characterize the effects of exercise on these

subunits, which represent most of glutamate receptors in the

brain [35]. There was an increase in phosphorylated subunits,

phospho-Ser896-NR1 (p-NMDAR1, P < 0.05) and phospho-

Ser1303-NR2B (p-NMDAR2B, P < 0.05), triggered by

exercise (Fig. 1). Since phosphorylation of NMDA receptors

increases the activity/opening of the channels, we used a

second approach to support our data. Using a binding assay

with a specific NMDA ligand, MK-801, which binds in the

open state of NMDA receptor, we were able to better charac-

terize the opening/activity of NMDA receptor. Notably, as

present in Fig. 2, exercise increased the binding of [3H] MK-

801 in cortical cellular membranes preparation (P < 0.05).

In addition to postsynaptic receptors, PSD also contains

signal- and scaffolding-proteins that organize the functional

postsynaptic structure [15]. Thus, we also quantified PSD

proteins such as PSD-95, SAP-97 and GRIP-1, which

anchor glutamate receptors. Exercise promoted a slight but

statistically significant increase in PSD-95 (P < 0.05) and,

more prominently in SAP-97 (P < 0.05) and GRIP-1 (P <

0.05) (Fig. 1). Altogether, these results point to an

adaptation of postsynaptic glutamatergic arrangement to

voluntary exercise.

3. Discussion

It has been shown that glutamatergic system modulation

is involved in various brain processes. The present results

indicate that voluntary exercise affected cortical postsynap-

tic proteins related to the glutamatergic system, an effect

that could be involved in the benefits caused by exercise on

the CNS.

M.O. Dietrich et al. / Brain Research 1065 (2005) 20–2522

AMPA receptors are involved in fast synaptic trans-

mission at glutamatergic synapses with consequent depola-

rization of the cellular membrane, promoting the opening of

NMDA receptors, a mechanism that has been related to

long-term potentiation (LTP) [17], neuroprotection [14], and

neurotrophic processes [24]. Additionally, AMPA receptors

have a particular and complex system of trafficking,

involved in the synaptic plasticity [2,27,36,43]. Although

KA and AMPA receptors have been difficult to be differ-

entiated, it has been shown that at postsynaptic membranes

KA receptors modulate the glutamatergic neurotransmission

[20]. Recently, it has been evidenced that specific membrane

proteins are able to connect KA to AMPA receptors,

mediating synaptic transmission in some neuronal networks

[5]. Our results suggest that regular physical exercise

promotes synapse plasticity by increasing the content of

AMPA in the PSD (Fig. 1). However, the importance of

increased AMPA receptors content in postsynaptic sites

needs to be further evaluated to access their function under

physiological adaptations.

Some benefits induced by exercise seem to involve

NMDA receptors, as the increase in neurogenesis [16] and

improvement in long-term potentiation [49]. In addition,

molecular approaches demonstrated that induction by

exercise of striatal Fos expression is dependent of NMDA

receptor activity [26]. Recently, it has been suggested that

NMDA receptors activity is an essential step to exercise-

induced synaptic plasticity in hippocampus [50]. NMDA

receptors, mostly studied due to their wide-ranging involve-

ment in many brain functions, have been associated with

many physiological and pathological brain conditions.

NMDA receptors exert a key role on neuronal excitability

and synaptic signaling through a fine control of its channel

activity. Accordingly, phosphorylation of NMDA receptors

increases the ion current through the receptor, whereas their

activity decreases by action of protein phosphatases

[23,41,53]. Moreover, phosphorylation of NMDA receptors

modulates their trafficking [39]. The present study gives

evidences of NMDA receptor modulation by exercise

through an increase in the phosphorylation of the subunits

NR1 and NR2B, and an increase in the binding of a channel

ligand (MK-801), which binds to the open NMDA channel,

with likely implications in brain physiology. Both data

together should indicate that our protocol of exercise

induced an opening of NMDA receptors and consequently

should increase their activities.

In addition to the evaluation of glutamate receptors, we

also studied the content of glutamate-related proteins, which

anchor receptors, maintaining the architecture and function-

ality of postsynaptic densities. Accordingly, the content of

anchoring proteins (PSD-95, SAP-97 and GRIP-1) accom-

panied the increase of the glutamate receptors subunits.

PSD-95 and SAP-97 are members of the MAGUK

(membrane-associated guanylyl kinase) proteins. PSD-95

has been described to anchor NMDA receptors (through

NR2 subunits) [42] and kainate receptors (through GluR6

subunits) [10]. SAP-97 anchors both AMPA (through

GluR1 subunit) [19] and kainate receptors (GluR6 subunit)

[30]. Similarly, GRIP-1 anchors the GluR2/3 subunits of

AMPA receptors [9] and the GluR5 subunit of kainate

receptors, as well [12]. These complex interactions among

anchoring proteins and glutamatergic receptors are involved

in the synaptic plasticity. Therefore, our results suggest that

voluntary exercise modulate these interactions.

Even though the majority of molecular and neuro-

chemical alterations were reported in the hippocampus, we

cannot exclude the importance of the cortical region in the

exercise-mediated effects. In fact, monitoring of local

cerebral glucose utilization showed that running wheel

exercise increase this index in some cortical regions at the

same magnitude as in the hippocampus [52]. Moreover,

exercise promotes increase in the cortical mRNA BDNF, the

molecule proposed as responsible for the exercise benefits

[32]. Recently, the levels of proteins involving in the

intracellular signaling including the presynaptic protein

synapsin I were found to be increase in cortical regions

from exercised animals [11]. Despite these observations,

studies exploring the effects of physical activity in other

brain regions and in different synaptic compartments are

very scarce. Thus, our study highlights some alterations in

synaptic proteins in a very specialized region of the synapse,

which could reflect on the brain function.

Summarizing, our results demonstrate that voluntary

physical exercise affected the immunocontent of glutamate

receptors type AMPA and related-proteins, and the opening/

activity of NMDA (through the modulation of the phos-

phorylation/dephosphorylation state), suggesting that the

glutamatergic system may be a putative target for modu-

lation by regular exercise activity.

It has been shown that the activation of the glutamatergic

neurotransmission may be beneficial or detrimental, depend-

ing among other factors on the degree of activation. Slight

activation may beneficial, as observed in memory studies

(see [13]), while over stimulation may be detrimental, as

observed in various acute and chronic brain injuries (see

[28,29]). Concerning the involvement of the glutamatergic

neurotransmission on brain effects of exercise, this duality is

also present, the exercise potentially being beneficial [1] or

detrimental [40].

Thus, the functional relevance of the effects of exercise

on the glutamatergic receptors presented in this study is

opened and under investigation by our group.

4. Experimental procedures

4.1. Animals

Male albino CF1 mice, 2 months old (from Fundacao

Estadual de Producao e Pesquisa do Rio Grande do Sul,

Porto Alegre, Brazil), were housed in standard cages (48 �26 cm), 3 to 4 animals per cage. Animals were allocated in a

M.O. Dietrich et al. / Brain Research 1065 (2005) 20–25 23

room with controlled temperature (22 T 1 -C), under a 12-hlight/12-h dark cycle. Mice were divided in two groups:

sedentary (S) and voluntarily physical active (exercise, Ex)

groups. Animals from Ex group had free access, for 1 month,

to a running wheel connected to an electronic counter, which

was located within the living cage; S group stayed for the

same time in similar conditions without running wheel. The

animals were controlled for a distance. After 4 weeks of

running wheel, the average distance was 3500 m. All

experiments were in agreement with Committee on Care

and Use of Experimental Animal Resources, UFRGS, Brazil.

4.2. Materials

Antibodies against GRIP-1 (glutamate receptor interact-

ing protein 1), GluR1, GluR2/3, GluR6/7 (glutamate

receptor subunit type 1, 2/3 and 6/7, respectively),

phospho-NR1ser896 and phospho-NR2Bser1303 (phosphory-

lated subunits of NMDA receptor type 1 and type 2B,

respectively) were obtained from Upstate (Lake Placid, NY,

USA). Antibodies against PSD-95 (postsynaptic density

protein of 95 kDa) and SAP-97 (synapse associated protein

97) were obtained from ABR (Golden, CO, USA). Anti-

bodies against NR1, NR2A and NR2B (NMDA receptor

subunits type 1, 2A, and 2B respectively) were purchased

from Chemicon Int. (Temecula, CA, USA). [3H] MK-801

(24.2Ci/mmol) was purchased from Perkin-Elmer Life

Sciences, Ltd., USA.

4.3. Postsynaptic density (PSD) preparation

The PSD fraction was prepared as previously described

[3] with some modifications. Cerebral cortices from Wistar

rats were dissected at 0–4 -C, manually homogenized in

0.32 M sucrose, 1 mM MgCl2 and 1 Ag/ml leupeptin in 1

mM of HEPES (pH 7.4) (1 g of tissue/4 ml) and

synaptosomes were obtained by differential and density-

gradient centrifugations as follows: sucrose gradients

containing 8 ml of the resuspended brain preparation and

gradients containing 10 ml each of 1.25 M, 1.0 M and

0.85 M sucrose solutions were centrifuged for 2 h at

80,000 � g using SW 28 rotor (Beckman Instruments,

Spinco Division, Palo Alto, CA, USA). The synaptosomal

fraction was collected from the interface of 1.25 M and 1.0

M sucrose and diluted with 1 mM of HEPES, pH 7.4, to

11 ml/g of original tissue. An equal volume of 1% Triton

X-100 in 0.32 M sucrose was added and stirred at 4 -C for

15 min. This suspension was centrifuged at 36,000 � g for

30 min and the pellets were resuspended into 0.32 M sucrose

(0.4 ml/g of original tissue). Two milliliters of this material

layered onto gradients in polyallomer tubes containing 4 ml

of 2.1 M sucrose and 3 ml each of 1.5 M and 1.0 M sucrose.

The gradients were centrifuged for 2 h at 200,000 � g in a

SW 40 rotor (Beckman Instruments). The postsynaptic

fraction was collected from the 2.1–1.5 M interface with a

siliconized plastic pipette and diluted with 20 mM of HEPES

and an equal volume of 1% Triton X-100 in 150 mM KCl to

obtain 0.5 ml/g of original tissue. The PSD fraction was

collected on the 2.1 M sucrose cushion by centrifugation at

20,0000 � g for 40 min. This interface was then

resuspended in 20 mM HEPES, pH 7.4, and again collected

on the 2.1 M sucrose by centrifugation at 200,000 � g for 30

min. The PSD fraction from the interface was resuspended

by stirring in 40% glycerol and stored at �20 -C.

4.4. Synaptic membrane preparation

Synaptic membrane preparation was obtained from the

cerebral cortices, as described by Jones and Matus [15], and

stored frozen at �70 -C for up to 2 months. On the day of

assay, membranes were thawed at 37 -C, for 30 min, washed

3 times with 5 mM Tris–HCl, pH 7.4 at 27,000 � g for 15

min. The final pellet was resuspended in the same buffer to

yield a protein concentration of 0.5–2 mg/ml and used for the

[3H]MK-801 binding assay.

4.5. Protein measurement

The protein content of PSD fraction and of synaptic

membrane preparation was estimated by the method of

Peterson [37].

4.6. [3H] MK-801 binding assay

Incubations were started by adding 50 Ag of membrane

protein to a medium containing 5 mM Tris–HCl, pH 7.4

and 4 nM [3H] MK-801. Following 60 min of incubation at

37 -C, bound and free [3H] MK-801 were separated by

filtration through GF/B Whatman filters. Radioactivity was

determined using a Wallac liquid scintillation counter.

Specific binding was calculated as the difference between

total binding and non-specific binding, which was measured

in the presence of 4AM MK-801. Non-specific binding

typically amounted to 20–30% of the total binding [33].

4.7. Electrophoresis and Western blot analysis

Proteins from PSD preparations were separated by 7.5%

SDS-PAGE mini-gels and transferred to nitrocellulose

membrane using a Trans-Blot system (Bio-Rad, Hercules

CA). Membranes were processed as follow: (1) blocking

with 2% bovine serum albumin for 2 h; (2) incubation with

primary antibody for 1 h: 1:5000 anti-PSD-95; 1:1000, anti-

SAP-97; 1:1000 anti-GRIP-1; 1:1000 anti-GluR1; 1:1000

anti-GluR2/3; 1:1000 anti-GluR 6/7; 1:5000 anti-NR1;

1:1000 anti-p-NR1; 1:100 anti-NR2B; 1:1000 anti-p-

NR2B; 1:1000 anti-NR2A; (3) incubation with alkaline

phosphatase-conjugated secondary antibody (1:5000 anti-

mouse or 1:10,000 anti-rabbit) for 1 h; (4) developing with

alkaline phosphatase substrate. Membranes were then dried

and scanned. Measurement of bands intensity through total

area of the peaks was performed using the public domain

M.O. Dietrich et al. / Brain Research 1065 (2005) 20–2524

NIH Image program (Developed at the U.S. National

Institutes of Health and available on the Internet at http://

rsb.info.nih.gov/nih-image/). An internal control in PSD is

unknown, i.e., a protein which content does not vary with

exercise; thus it was performed additional examination of

the total amount of protein loaded on the gel. We used

Coomassie blue stain in gels, analyzed by NIH Image, and

Ponceau S red stain in membranes to be sure the same

quantity of protein was loaded in each lane (5 Ag/lane).When protein stained among lanes differed more than 5%

we did not develop the membrane.

4.8. Statistical analysis

Optical densities (OD) and binding values were com-

pared through paired t test. It was necessary 8 animals for

each PSD preparation. We performed 4 PSD preparations in

each experiment (two exercise and two sedentary). Thus, we

used unpaired t test (two-tailed) comparing samples

prepared in the same day to avoid bias due to PSD

preparation. Additionally, these pairs of samples were run

in the same gel in paired lanes. The results are presented as

percent average T standard error of the mean (SEM),

considering the S group as 100%. P < 0.05 was considered

statistically significant.

Acknowledgments

This work was support by Brazilian agencies, CNPq,

CAPES and FAPERGS (BPV 04/60002.2 for LV) and USA/

NIH intramural program.

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