JOURNAL OF NEUROCHEMISTRY

| 2014

doi: 10.1111/jnc.12976

,

,

*Departamento de Fisiologia, Universidade Federal de S~ ao Paulo/Escola Paulista de Medicina (UNIFESP/EPM), S~ao Paulo, SP, Brazil †Hospital Israelita Albert Einstein. Instituto do Cerebro, S~ ao Paulo, SP, Brazil ‡Disciplina de Neurologia Experimental, Universidade Federal de S~ ao Paulo/Escola Paulista de Medicina (UNIFESP/EPM), S~ ao Paulo, SP, Brazil §Department of Pharmacobiology, Center of Research and Advanced Studies, Mexico City, Mexico ¶Psychiatric Hospital of the University of Basel, Center for Affective-, Stress- and Sleep Disorders, Basel, Switzerland **Institute of Exercise and Health Sciences, University of Basel, Basel, Switzerland

Abstract Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or longterm effects (chronic) of exercise, overall, higher opioid

receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [35S]GTPcS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. Keywords: brain, DOR, exercise, KOR, MOR, opioid receptor. J. Neurochem. (2014) 10.1111/jnc.12976

In humans, physical exercise induces psychological benefits such as positive mood changes and decreased levels of anxiety (Rosch 1985; Wildmann et al. 1986; Bender et al. 2007). Although many physiological hypotheses have also been suggested to explain the anxiolytic and anti-depressive effects of exercise (Landers and Arent 2001) (monoamine, endorphin, and thermogenic hypothesis), these effects have been somewhat mediated by the opioid system (Jarvekulg and Viru 2002), suggesting a relationship between exerciseinduced effects in the brain and the opioid receptors.

Received July 21, 2014; revised manuscript received September 6, 2014; accepted October 6, 2014. Address correspondence and reprint requests to Dr Ricardo M. Arida, Rua Botucatu 862, Ed. Ci^encias Biom
edicas 5 andar. 04023-900, S~ao Paulo, Brazil. E-mail: [email protected] Abbreviations used: BL, basolateral amygdala; CA1, cornu ammonis 1; CA2, cornu ammonis 2; CA3, cornu ammonis 3; CD, central amygdala; DAMGO, [D-ala2-N-methyl-phe-glycol5][tyrosyl-3-4-3H] enkephalin; DG, dentate gyrus; DOR, delta opioid receptor; DPDPE, D-penicillamine2,5 enkephalin; DS, dorsal; Hypo, hypothalamus; KOR, kappa opioid receptor; ML, medial amygdala; MOR, mu opioid receptor; Pirf, piriform cortex; Sen, sensorimotor cortex; VT, ventral.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

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R. M. Arida et al.

Endogenous opioids (endorphins, enkephalins, and dynorphins) act by binding to mu opioid receptor (MOR), kappa opioid receptor (KOR) or delta opioid receptor (DOR) and there is a widespread distribution of these receptors throughout the peripheral nervous system, spinal cord, and brain (Goodman et al. 1988). They modulate mood, appetite and pain, by activating particular G protein-coupled receptors. To this extent, alterations in opioid receptors after physical exercise have been investigated. For instance, some reports demonstrated an increase in opioid receptor occupancy in the brain after running (Pert and Bowie 1979) or swimming (Sforzo et al. 1986). On the other hand, after longer periods of time (chronic exercise), the sensitivity to the effects of exogenously administered opioids is reduced (Kanarek et al. 1998; Mathes and Kanarek 2001). Thus, rats submitted to voluntary running present fewer beta-endorphin binding sites than control rats, an effect that could suggest a compensatory down-regulation of opioid receptors during exercise (Houghten et al. 1986). In line with the above observations, we have recently verified whether acute and chronic exercise could modify the expression of MOR in rat hippocampal formation. Significant enhance of MOR expression in the hippocampal formation was observed after acute exercise (seven consecutive days) compared to control animals. Conversely, a significant reduction in MOR expression was noted after chronic exercise (45 consecutive days) compared to the acute-exercise animals (de Oliveira et al. 2010). Although these results indicate higher expression of MOR in hippocampus, it is important to consider that the receptor expression sometimes does not match the changes in binding and/or G-protein activation (Rocha et al. 2009). Behaviors associated to exercise such as reward, nociception, changes in mood, anxiety and performance are believed to be in part related to altered endogenous opioid peptides or their receptors sensitivity (Rosch 1985; Wildmann et al. 1986; Boecker et al. 2008). Although there are data that relate exercise to neuropeptide expression in the brain, the effects of exercise-related stress condition on opioid receptor binding and signal transduction mechanisms downstream of these receptors still remain unknown. Because of the lack of substantial evidence as to the receptor subtype playing the predominant role in eliciting these effects, we are interested in MOR, KOR and DOR, since activation of these receptors by the endogenous ligands can activate all the Gi/Go with equal potency (Kenakin 1993). When opioids bind to the G protein-coupled receptors, they induce an activation of the associated G protein by exchanging its bound GDP for a GTP. The G protein a subunit, with the bound GTP, can then dissociate from the b and c subunits to further affect intracellular signaling proteins (Kenakin 1993). Therefore, quantification of [35S]GTPcS binding (a radiolabeled GTP analog) represents an excellent opportunity to determine exercise-induced changes in the ability of opioid receptors to activate G proteins and subsequent transduction (Harrison

and Traynor 2003). In this study, we characterized the binding and G protein activation of MOR, KOR, and DOR in several brain regions such as cortex, amygdala, hippocampus, and hypothalamus following acute and chronic exercise at low intensity and long duration.

Material and methods Physical exercise procedure Eighteen adult male Wistar rats (outbred, raised, and maintained in the Centre for Development of Experimental Models in Medicine and Biology of Universidade Federal de S~ao Paulo) weighing 200– 280 g were used in this study. The colony room was maintained at 22
2°C with a 12 h light/dark schedule (light: 7 a.m. until 7 p.m.), and food and water were provided ad libitum throughout the experimental period. The rats were divided into three groups: acute exercise, chronic exercise, and control (n = 6 for each group). Animals assigned to the exercise groups were familiarized with the apparatus for 2 days by placing them on a treadmill (Columbus instruments) for 5 min/day at a speed of 8 m/min at a 0% degree incline. Considering that animals were submitted to forced exercise with a motivational stressor, i.e., foot shock, we rated each animal’s treadmill performance (as measure of trainability) on a scale of 1–5 according to previous study (Dishman et al. 1988). Animals with a mean rating of 3 or higher were included in the exercise groups. This procedure was used to exclude possible different levels of stress between animals (Arida et al. 2011). Subsequently they were submitted to an exercise program of 7 (acute exercise group) or 30 (chronic exercise group) consecutive sessions on a treadmill at low intensity and long duration. If eventually animals reduced their speed of running and contacted the aversive shock grid at the end of the treadmill, the intensity was decreased. Both acute and chronic exercise groups ran during 60 min/day at speed of 10 m/ min. Training period occurred between 9:00 and 10:00 h a.m. Animals from the control group were transferred to the experimental room and handled in the same way as animals in the exercise group (privation of water and food during treadmill exercise for 7 or 30 days). Animals from the exercise groups were killed 1 h after the last training session. The control group was killed in the same time from other groups. The brain was quickly removed, frozen in pulverized dry ice, and stored at 70°C. All procedures followed the guidelines under European Community Council (86/609/EEC) and experimental protocols were approved by the local ethics committee of the Universidade Federal de S~ao Paulo (#0607/09). Autoradiography experiments Preparation of tissue sections Frozen coronal sections of 20 lm were cut in a cryostat, thawmounted on gelatin-coated slides, and stored again at 70°C. Serial and parallel sections were obtained from each sample for quantitative and functional autoradiography from the sensorimotor (Sen) (Hasbi et al. 2007) and piriform (Pirf) cortex, basolateral (BL), medial (ML) and central amygdala (CD) (Dietrich and McDaniel 2004), dentate gyrus (DG) (Greenwood et al. 2011) and Ammon’s horn [subregions: cornu ammonis (CA)1, CA2 and CA3], dorsal (DS) and ventral (VT) hypothalamus (Hypo).

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

Differential effects of exercise on brain opioid receptor

Quantitative autoradiography Experiments were performed on brain sections according to the procedures described previously (Rocha et al. 1991; Gartshore et al. 1996; Clarke et al. 2001, see Table 1). Briefly, brain sections were initially washed in tris buffer during 30 min, and then for each of the receptors evaluated, slices were incubated in a solution containing a specific [3H]ligand at a concentration equivalent to its Kd value. Binding obtained in presence of a non-labeled ligand was considered to be non-specific. Values of specific binding reported in this study were obtained from the difference between non-specific and total binding. Finally, incubation was completed with two consecutive washes in buffer and a distilled water rinse (2 s) at 4°C. Sections were quickly dried under a gentle stream of cold air, and then they were arrayed in X-ray cassettes and opposed to tritiumsensitive film (Kodak-MR) together with tritium standards (American Radiolabeled Chemicals, Inc., 101 ARC Drive. Saint Louis, MO 63146 USA.) at 22°C, from 8 to 16 weeks, depending on the [3H]ligand (see Table 1). Thereafter, they were developed using standard Kodak developer (D-11) and fast fixer at 22°C. Optical densities of specific brain areas were determined using a videocomputer enhancement program (JAVA Jandel Video Analysis Software. Jandel Scientific. 65 Koch Road. Corte Madera, CA 94925. USA). For each structure, ten optical density readings were taken from at least three sections over the entire region resected, and average was obtained. The optical density readings of the standards were used to construct a standard curve to determine tissue radioactivity values for the accompanying tissue sections (dpm/ mm2). The tritium standards were previously calibrated to brain homogenates with known protein concentrations to allow a transformation of gray values into total binding. Then, dpm/mm2 values were converted to fmol/mg protein based on the specific activity of each [3H]ligand and tissue thickness (20 lm). Functional autoradiography Agonist-stimulated [35S]GTPcS autoradiography was performed as described previously (Sim et al. 1995; Sim and Childers 1997). Sections were soaked for 10 min at 25°C in 50 mM Tris-HCl buffer (pH 7.4) to remove endogenous ligands. They were then preincubated for 15 min in the same buffer, to which 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, and 2 mM GDP were added. Forty pM [35S]GTPcS were then added to this solution, for a 2 h incubation at 25°C to determine basal binding, i.e. the labeling of G protein in the absence of agonists. Opioid receptor-induced G protein activation was evaluated using the solution containing [35S] GTPcS in presence of 3 lM of [D-ala2-N-methyl-phe-glycol5][tyrosyl-3-4-3H]enkephalin for MOR, 10 lM of D-penicillamine2,5

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enkephalin for DOR, or 1 lM of U-50488 for KOR, with and without antagonists (1 lM naloxone for MOR and DOR, or 1 lM norbinaltorphimine, for KOR). Slides were then rinsed twice for 2 min each in 50 mM Tris-HCl buffer, at 4°C, pH 7.4, and once in deionized H2O at 4°C. Slides were dried overnight and exposed to film (Kodak-MR) for 5 days in film cassettes containing [14C] microscales (American Radiolabeled Chemicals, Inc.). Densitometric analysis of the different brain areas was performed as described above for tritium autoradiography. Values were converted into fmol/ mg of protein based on the specific activity of [35S]GTPcS and tissue thickness (20 m). For each animal, net agonist-stimulated [35S]GTPcS binding in specific brain areas was calculated by subtracting values obtained under basal conditions from those obtained under agonist-stimulated [35S]GTPcS incorporation. Data analysis Results obtained from quantitative and functional autoradiography were examined statistically by Kruskal–Wallis non-parametric test followed by repeated Mann–Whitney tests. Bonferroni correction for the p value was adopted (p value/number of comparisons; 0.05/ 3 = 0.017). Results are presented as median and inferior and superior quartiles (M and Q1 and Q3). All analyses were performed using IBM SPSS version 20.0 software (SPSS Inc., IBM Company, Armonk, NY, USA).

Results MOR binding and functional coupling to G proteins A significant reduction in MOR binding was found in the Sen cortex of rats from chronic exercise group in relation to rats from control (p = 0.004) and acute exercise (p = 0.006) groups (Table 2). No significant change in this brain area was noted to [35S]GTPcS incorporation as a consequence of MOR activation among groups. In the Pirf cortex, MOR binding as well as MOR-induced G protein activation were significantly higher in acute and chronic exercise groups (p = 0.006) than in the control group. In the amygdala, a significant increase in MOR binding in the BL and ML nuclei was observed in acute exercise group when compared to control and chronic exercise groups (p = 0.004 in both groups). Concerning MOR-mediated G protein activation, an increase in the ML amygdala nucleus of chronic exercise group and a reduction in the CD amygdala nucleus of acute exercise group compared to control group (p = 0.006 in both

Table 1 Conditions for quantitative autoradiography experiments

Binding Mu Delta Kappa

Ligand-(nM) specific activity [3H]DAMGO (2 nM) 61 Ci/mmol [3H]DPDPE (10 nM) 40.7 Ci/mmol [3H]U-69593 (3 nM) 63 Ci/mmol

Buffer (pH 7.4)

Incubation 22°C

Exposition 22°C

Non-labeled ligand

Tris HCl (50 mM)

60 min

8 weeks

Naloxone (1 lM)

Tris HCl (50 mM)

60 min

16 weeks

DPDPE (1 lM)

Tris HCl (50 mM)

90 min

12 weeks

U-68593 (1 lM)

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

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R. M. Arida et al.

Table 2 Quantitative and functional autoradiography of MOR in the sensorimotor (Sen) and piriform (Pirf) cortex, basolateral (BL), medial (ML), and central (CD) (Dietrich and McDaniel) amygdala, hippocampal regions of dentate gyrus and Ammon’s horn (subregions: CA1, CA2 and CA3), and dorsal (DS) and ventral (VT) hypothalamus of rats from control, acute exercise (7 days) and chronic exercise (30 days) groups Control Quantitative autoradiography Cortex Sen 29.65 Pirif 4.37 Amygdala BL 47.40 ML 7.20 CD 3.31 Hippocampus DG 21.56 CA1 5.09 CA2 4.87 CA3 4.60 Hypothalamus DS # VT # Functional autoradiography Cortex Sen 251.37 Pirif 56.10 Amygdala BL 588.33 ML 68.45 CD 471.16 Hippocampus DG 369.21 CA1 69.60 CA2 238.09 CA3 150.46 Hypothalamus DS 444.00 VT 510.00

Acute exercise

(22.37–39.03) (3.33–6.46)

Chronic exercise

6.69 (3.82–9.51)a,b 32.46 (20.96–49.38)a

28.32 (13.60–36.77) 26.26 (21.22–34.97)a 157.73 (129.85–257.22)a,c 35.54 (26.38–45.58)a,c 3.92 (3.06–5.91)

(34.41–61.60) (5.23–8.17) (2.90–4.89) (14.27–53.73) (3.16–6.58) (3.21–5.14) (3.34–5.13)

21.65 6.16 6.37 25.25

54.57 (31.74–69.57) 6.54 (4.61–8.76) 4.41 (3.33–5.29)

(14.64–59.16) (3.45–9.23) (4.37–9.50) (14.03–40.86)a,c

5.50 5.52 6.36 7.09

(4.54–8.25)a,b (4.42–8.43) (5.03–7.78) (6.06–8.26)a

# #

# #

(242.22–439.12) (39.90–98.71)

253.87 (185.78–258.02)a 225.25 (179.59–335.85)

363.32 (265.20–475.60)a 346.77 (294.37–438.29)

(483.80–896.93) (52.38–95.15) (339.88–503.06)

502.02 (401.18–738.70) 118.95 (99.47–134.00)a 227.11 (133.88–297.06)a

533.46 (483.96–683.80) 159.88 (120.11–226.37) 203.87 (146.78–381.53)

(333.04–518.83) (38.86–186.83) (216.02–371.08) (125.01–293.53) (370.5–570.25) (340–632.25)

158.04 (143.50–202.83)a

#

#

#

341.09 (251.71–425.33) 288.26 (156.34–355.13)

#

733.50 (535.75–958.75) 953.50 (586.25–1068)

330.00 (179.5–609.5) 299.00 (132–563.5)

159.67 (122.29–185.88)

Values are expressed in fmol/mg tissue and presented as median and quartiles 1 and 3 of six animals. Statistical analysis was conducted by Kruskal–Wallis non-parametric test followed by repeated Mann–Whitney tests. Values were considered significant when p < 0.017 (after Bonferroni correction): a significant difference to the control group; b significant difference to the acute exercise group; c significant difference to the chronic exercise group. Values presented as #indicates results below detectable limits.

groups) were found. In the hippocampal formation, MOR binding was significantly lower in DG of rats from exercise chronic group than in rats from control (p = 0.011) and acute exercise (p = 0.004) groups. No significant changes were noted to MOR binding in the CA1 and CA2 regions. In CA3 region, a significant increase in MOR binding was found in exercise groups (acute and chronic), relative to the control group (p = 0.004 and p = 0.005, respectively). Thus, a significant increase in MOR binding was noted in the CA3 of rats of the acute exercise group when compared to those of the chronic exercise group (p = 0.004). The MOR-mediated G protein activation in DG was significantly reduced in rats from acute group in relation to rats from control group (p = 0.006). No MOR-induced [35S]GTPcS incorporation

was detected in CA1 for acute exercise group and in DG, CA1, and CA2 for the chronic exercise group. MOR receptor binding in DS and VT Hypo was lower and unable to be evaluated in the different experimental groups under our experimental conditions, whereas MOR-induced [35S]GTPcS incorporation did not show significant changes in these brain areas of animals from acute and chronic exercise (Table 2). KOR binding and functional coupling to G proteins Quantitative autoradiography revealed a significant increase in KOR binding in the Sen cortex of rats of the acute exercise group compared to control and chronic exercise groups (p = 0.004 in both groups) (Table 3). In contrast, a significant reduction in KOR-induced [35S]GTPcS incorporation

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

Differential effects of exercise on brain opioid receptor

was found in this brain area of acute exercise group when compared to the control group (p = 0.006). KOR binding in the Pirf cortex showed a significant reduction in the chronic exercise group in relation to control and acute exercise groups (p = 0.004 in both groups). No KOR-mediated G protein activation was detected in Pirf cortex of rats from exercise groups (acute and chronic). In amygdala, there was no KOR-mediated G protein activation greater than basal levels in the BL nucleus of the chronic exercise group when compared with control and acute exercise groups (p = 0.006 in both groups). No significant changes were noted to KOR binding in ML and CD nuclei among groups. BL and CD nuclei demonstrated significant increase in KOR-mediated G

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protein activation of the acute exercise group when compared to control group (p = 0.004 and p = 0.006, respectively). KOR-mediated G protein activation was absent in ML nucleus for the acute exercise group and in BL, ML and CD nuclei for the chronic exercise group. In hippocampal formation, a significant increase in CA1 KOR binding was observed in acute exercise group in relation to control and chronic exercise groups (p = 0.006 and p = 0.009, respectively). On the other hand, KOR binding was reduced in hippocampal regions of the DG, CA2 and CA3 of chronic exercise group compared to control (p = 0.004, p = 0.006 and p = 0.006, respectively) and acute exercise (p = 0.009 for all) groups. Concerning KOR-mediated G protein

Table 3 Quantitative and functional autoradiography of KOR in the sensorimotor (Sen) and piriform (Pirf) cortex, basolateral (BL), medial (ML) and central (CD) (Dietrich and McDaniel) amygdala, hippocampal regions of dentate gyrus and Ammon’s horn (subregions: CA1, CA2 and CA3), and dorsal (DS) and ventral (VT) hypothalamus of rats from control, acute exercise (7 days) and chronic exercise (30 days) groups Control Quantitative autoradiography Cortex Sen 4.96 Pirif 19.75 Amygdala BL 21.08 ML 41.05 CD 9.09 Hippocampus DG 17.92 CA1 6.94 CA2 21.03 CA3 20.25 Hypothalamus DS 25.5 VT 24 Functional autoradiography Cortex Sens 198.73 Pirif 316.11 Amygdala BL 200.06 ML 134.57 CD 162.91 Hippocampus DG 746.03 CA1 374.52 CA2 347.42 CA3 174.44 Hypothalamus DS 688.5 VT 403.00

Acute exercise

(4.45–8.54) (17.58–22.53)

18.33 (15.99–19.74)a,c 17.89 (13.96–20.64)

(19.79–28.18) (29.16–55.64) (8.58–10.31)

20.96 (17.22–25.43) 27.67 (18.54–32.26) 7.59 (4.14–9.94)

(14.50–20.42) (5.03–7.76) (17.53–24.79) (18.15–22.26)

26.07 25.47 21.15 20.59

(24.00–32.00) (22.00–29.75)

24.00 (23.00–26.25) 24.5 (21.75–28.00)

(178.90–263.82) (217.31–457.80) (160.43–339.96) (106.04–192.12) (147.03–205.77) (695.21–773.92) (265.30–465.79) (308.66–438.18) (137.64–242.21) (527.75–881.25) (269.25–666.5)

Chronic exercise

5.65 (2.59–8.12) 7.10 (5.66–8.73)a,b 8.26 (3.21–9.05)a,b 26.31 (20.96–32.81) 4.48 (2.95–6.41)

(18.28–38.28) (20.09–28.38)a,c (15.32–25.62) (18.66–22.79)

3.53 3.31 3.84 3.30

(2.55–4.20)a,b (2.82–4.54) (2.64–6.51)a,b (2.85–5.17)a,b

23.00 (23.00–24.00)a,b 24.00 (22.00–26.00)

126.14(101.13–153.18)a

225.67 (149.18–314.53)

#

#

949.79 (752.77–1020.39)a

#

#

#

296.34 (237.28–353.66)a

#

589.07 (531.39–681.13) 132.74 (115.62–137.66)a #

333.47 (281.21–355.08) 660.5 (516.00–930.25) 527.00 (212.75–609.25)

# # #

a

#

45.00 (0–592.5)b 15.00 (7.00–554.00)

Values are expressed in fmol/mg tissue and presented as median and quartiles 1 and 3 of six animals. Statistical analysis was conducted by Kruskal–Wallis non-parametric test followed by repeated Mann–Whitney tests. Values were considered significant when p < 0.017 (after Bonferroni correction): a significant difference to the control group; b significant difference to the acute exercise group; c significant difference to the chronic exercise group. Values presented as #indicates results below detectable limits.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

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R. M. Arida et al.

DOR binding and functional coupling to G proteins A significant increase in DOR binding was detected in the Sen cortex of the acute exercise group compared to control and chronic exercise groups (p = 0.004 in both groups) (Table 4). In the Pirf cortex, a significant DOR binding reduction was observed in the chronic exercise group when compared to control and acute exercise groups (p = 0.004 in both groups). The analysis [35S]GTPcS incorporation as a consequence of DOR activation in this brain area showed a significant increase in acute and chronic exercise groups in relation to control group (p = 0.006 and p = 0.004, respectively). In ML amygdala, decreased DOR binding was found in chronic exercise animals compared to control acute exercise groups (p = 0.004 in both groups). DOR-mediated

activation, a reduction in CA1 and an increase in CA3 of rats of the acute exercise group in relation to those of the control group were observed (p = 0.004 and p = 0.006, respectively). No KOR-mediated G protein activation was detected in CA2 for acute exercise group in DG, CA1, CA2, and CA3 for chronic exercise group. KOR receptor binding in VE Hypo of the acute and chronic groups did not demonstrate significant changes. In contrast, animals from the chronic group demonstrated low KOR induced [35S]GTPcS incorporation in the DS Hypo compared to control (p = 0.003) and acute (p = 0.016) groups. Furthermore, animals from the chronic group demonstrated low KOR-induced [35S]GTPcS incorporation in the DS Hypo compared to acute (p = 0.014) group (Table 3).

Table 4 Quantitative and functional autoradiography of DOR in the sensorimotor (Sen) and piriform (Pirf) cortex, basolateral (BL), medial (ML) and central (CD) (Dietrich and McDaniel) amygdala, hippocampal regions of dentate gyrus and Ammon’s horn (subregions: CA1, CA2 and CA3), and dorsal (DS) and ventral (VT) hypothalamus of rats from control, acute exercise (7 days) and chronic exercise (30 days) groups Control Quantitative autoradiography Cortex Sens 59.09 Pirif 299.65 Amygdala BL 327.02 ML 195.40 CD 34.98 Hippocampus DG 483.18 CA1 130.37 CA2 71.78 CA3 65.55 Hypothalamus DS 6.5 VT 7.00 Functional autoradiography Cortex Sen 265.52 Pirif 90.35 Amygdala BL 442.49 ML 56.67 CD 458.74 Hippocampus DG 195.92 CA1 114.10 CA2 53.42 CA3 41.59 Hypothalamus DS 162.5 VT 247.5

Acute exercise

(53.74–78.35) (145.29–331.91)

181.68 (115.67–310.04)a,c 266.82 (129.44–362.56)

(189.64–446.38) (156.45–327.27) (24.63–45.60)

361.56 (229.89–540.04) 161.17 (107.91–573.30) 53.54 (30.72–79.57)

(327.70–616.45) (110.42–150.03) (48.38–96.94) (41.23–86.83)

229.75 80.33 186.77 238.74

(4.75–7.28) (5.00–8.05)

(183.85–356.20) (40.50–102.18) (270.24–654.76) (20.70–74.24) (309.59–618.62)

Chronic exercise

52.33 (26.82–79.34) 61.25 (46.54–71.56)a,b 409.93 (230.76–558.25) 68.86 (34.17–90.58)a,b 46.60 (22.85–56.71)

(133.05–611.12) (69.27–97.09)a,c (121.74–400.00)a,c (131.66–621.57)a,c

63.71 40.21 40.36 29.52

6.75 (5.90–7.90) 7.40 (6.65–8.93)

322.54 (232.96–411.35) 303.22 (223.32–316.54)a

(39.25–78.01)a,b (20.59–68.57)a (23.54–62.72) (14.92–44.50)

6.1 (6.00–8.10) 7.00 (6.00–9.10)

274.17 (155.49–451.80) 234.41 (160.49–276.81)a

365.02 (275.63–480.06)

335.70 (202.31–433.65)

#

#

154.61 (121.24–175.50)

#

(122.25–357.77) (63.95–189.02) (32.28–72.16) (17.57–58.15)

#

#

#

#

#

#

#

#

(136.75–216.25) (216.75–347.00)

195.00 (166.5–426.25) 366.5 (265.50–572.75)

234.00 (24.00–374.00) 57.00 (0–551.50)

Values are expressed in fmol/mg tissue and presented as median and quartiles 1 and 3 of six animals. Statistical analysis was conducted by Kruskal–Wallis non-parametric test followed by repeated Mann–Whitney tests. Values were considered significant when p < 0.017 (after Bonferroni correction): a significant difference to the control group; b significant difference to the acute exercise group; c significant difference to the chronic exercise group. Values presented as #indicates results below detectable limits.

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

Differential effects of exercise on brain opioid receptor

G protein activation was absent in ML amygdala nucleus for the acute and chronic exercise groups and in CD nucleus for the chronic exercise group. In hippocampal region, DOR binding was reduced in DG and CA1 of rats of the chronic exercise group compared to those of the control group (p = 0.004 in both regions). Thus, a significant reduction in DOR binding was noted in DG of the chronic exercise group in relation to the acute exercise group (p = 0.004). In CA1, DOR binding of the acute exercise group was lower than of the control group (p = 0.004) and higher than of the chronic exercise group (p = 0.016). In CA2 and CA3 regions, a significant increase in DOR binding was observed in the acute exercise group when compared to control (p = 0.004 in both regions) and chronic exercise (p = 0.004 in both regions) groups. Interestingly, no DOR-mediated G protein activation was detected in hippocampal region (DG, CA1, CA2, and CA3) of acute and chronic exercise groups. DOR receptor binding as well as DOR induced [35S]GTPcS incorporation in DS and VT Hypo of the acute and chronic groups did not show significant alterations (Table 4). Table 5 summarizes the quantitative and functional autoradiography findings of opioid receptors in the brain areas studied of exercised animals compared to control animals.

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Pseudo-color autoradiograms showing the distribution of the [35S]GTPcS incorporation under basal conditions (Fig. 1). Analysis of basal G -protein activation According to the previous results, we found that the different opioid agonists were unable to induce G protein activation in specific brain areas. The low efficacy of an agonist to induce GDP/GTP exchange during the functional autoradiography can be explained because the G protein-coupled receptors are in an active state. This situation that can be identified with increased labeling of the active G protein with [35S]GTPcS under basal conditions (absence of agonists). Therefore, we analyzed if the different experimental conditions augment the levels of basal G protein activation in the brain areas evaluated. In relation to control and acute groups, increased values of basal activation were noted in sensorimotor cortex and the different areas of hippocampus in the chronic exercise group (p < 0.017) (Table 6). Pseudo-color autoradiograms showing the distribution of the [35S]GTPcS incorporation under basal conditions in coronal sections of an animal of the control group, and rats submitted to acute and chronic exercise (Fig. 2).

Table 5 Summary of quantitative and functional autoradiography findings of opioid receptors in the cortex, amygdala, hippocampus, and hypothalamus of rats submitted to acute and chronic exercise compared to control animals Binding

Activation

Cortex

Acute

Chronic

Acute

Chronic

MOR KOR DOR

Increased Increased Increased/no difference

Reduced/increased Reduced/no difference Reduced/no difference

Increased Reduced Increased/no difference

Increased/no difference Reduced/no difference Increased/no difference

Binding

Activation

Amygdala

Acute

Chronic

Acute

Chronic

MOR KOR DOR

Increased/no difference No difference No difference

No difference Reduced/no difference Reduced/no difference

Reduced/no difference Reduced/increased Reduced/no difference

Increased/no difference Reduced Reduced/no difference

Binding

Activation

Hippocampus

Acute

Chronic

Acute

Chronic

MOR KOR DOR

Increased/no difference Increased Increased

Reduced/increased/no difference Reduced/no difference Reduced/no difference

Reduced/no difference Reduced/increased/no difference Reduced

Reduced/no difference Reduced Increased/no difference

Binding

Activation

Hypothalamus

Acute

Chronic

Acute

Chronic

MOR KOR DOR

# No difference No difference

# No difference/reduced No difference

No difference No difference No difference

No difference Reduced No difference

Values presented as # indicates results below detectable limits.

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R. M. Arida et al.

Fig. 1 Pseudo-color autoradiograms showing the distribution of MOR, DOR and KOR receptor binding labeled with [3H]DAMGO, [3H]DPDPE and [3H]U-69593, respectively, in coronal sections of a control animal (Control) and a rat submitted to chronic exercise (CE). High receptor binding appears as red and yellow areas, whereas green and blue areas indicate regions with low receptor binding.

Discussion Studies have investigated the involvement of opioid system and exercise in several conditions such as memory induced by stressful situations, rewarding (Greenwood et al. 2011), dependence (Smith and Yancey 2003) and anxiety-like behaviors (Randall-Thompson et al. 2010). Although there are diverse effects of endogenous opioid on central nervous system (Froehlich 1997), we were especially interested in the effects of exercise-related stress condition on opioid receptor binding and G protein activation. Exercise represents a physical stress that challenges homeostasis and stress associated with endogenous opioid peptides (Radak et al. 2008; Van’t Veer and Carlezon 2013). Moreover, there is a positive correlation between adrenocorticotropic hormone and beta-endorphin, suggesting the role of stress in increasing beta-endorphin production (Bender et al. 2007). Since different exercise stimuli may affect the brain opioid system, the influence of exercise on opioid receptor binding and activation was analyzed in animals submitted to acute or chronic exercise at low intensity and long duration. The effect of exercise duration on opioid response remains controversial, with most of our understanding being confined to the peripheral effects induced by exercise. In initial human studies, higher blood endorphins were observed in exercise of longer duration (Thompson et al. 1985), that is, a minimal duration of exercise appears to be necessary to stimulate

beta-endorphin release (McMurray et al. 1987; Goldfarb et al. 1990). However, there is no consistent information of central opioid system induced by exercise, and limited evidence has been proposed based on animal studies (Hoffmann et al. 1990; Tendzegolskis et al. 1991; Aravich et al. 1993). In animal studies, there is a lack of specific exercise protocols to evaluate precisely the effect of different exercise stimuli as found in human reports. From the few studies on this topic, Sforzo et al. (1986) observed an increase in opioid receptor binding in several brain areas after 2-h but not after 1-h swim. Another investigation reported altered beta-endorphin in nucleus accumbens in trained rats following 2-h of treadmill running (Blake et al. 1984). Our findings are in accordance with previous works indicating changes in opioid receptors binding in several brain areas of the acute group submitted to aerobic exercise of long duration (60 min). Concerning short- (acute) or long-term effects (chronic) of exercise on opioid receptors, overall, a higher MOR, KOR, and DOR binding in acute-exercise animals was observed, and the opposite was found in chronic-exercise animals. Houghten et al. (1986) noted that rats exercising for 5 months presented fewer beta-endorphin binding sites than sedentary rats. Similarly, chronic treatment with exogenous opioids reduced the number of opioid receptors (Yoburn et al. 1993; Diaz et al. 1995). It is suggested that if exercise is applied for long periods of time, the effects of exogenously opioids are lower as a consequence of reduced opioid receptor binding. Smith and Yancey (2003) investigated the effects of chronic exercise on MOR sensitivity and verified whether these effects might be attributed to the development of opioid dependence and tolerance. Interestingly, the sensitivity to buprenorphine (MOR agonist) was decreased in control animals that were transferred to cages provided with wheel running, and increased in trained animals that were transferred to control housing conditions. We have previously reported significant enhancement of MOR expression in hippocampal formation after acute exercise and significant reduction after chronic exercise (de Oliveira et al. 2010), suggesting a down-regulation of MOR after long-term exercise. Another point to be considered is related to the intensity of exercise. Previous investigations have demonstrated that the intensity of effort is correlated with a beta-endorphin release (Mougin et al. 1988; Goldfarb et al. 1990; Mehl et al. 2000). Earlier exercise studies in humans have indicated that beta-endorphin release depends more on the intensity of exercise (Langenfeld et al. 1987). It has also been suggested that the blood beta-endorphin levels do not increase until the anaerobic threshold is exceeded (Viswanathan et al. 1987; Brooks et al. 1988). McMurray et al. (1987) reported enhanced circulating beta-endorphins following high-intensity exercise (80% VO2max) but not after low or moderate intensities (40% and 60% VO2max). Despite the association

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

Differential effects of exercise on brain opioid receptor

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Table 6 Basal G protein activation previous to MOR, KOR, and DOR stimulation in sensitive (Sen) and piriform (Pirf) cortex, basolateral (BL), medial (ML) and central (CD) amygdala, dentate gyrus (DG) and Ammon’s horn (subregions: CA1, CA2 and CA3), and dorsal (DS) and ventral (VT) hypothalamus

Cortex Sen Pirif Amygdala BL ML CD Hippocampus DG CA1 CA2 CA3 Hypothalamus DS VT

Control

Acute exercise

Chronic exercise

665.54 (544.98–747.98) 1511.31 (1072.7–1781.28)

674.43 (469.39–959.23) 1592.43 (1388.6–1755.97)

876.72 (683.14–1234.48)a,b 1476.24 (1245.43–1650.29)b

1381.16 (1141.24–1592.3) 2045.14 (1945.73–2059.59) 1551 (1340.54–1708.40)

1483.75 (1196.70–1715.06) 2061.44 (1962.42–2094.44) 1651.21 (1413.50–1791.44)

1529.91 (1300.94–1688.15) 2012.56 (1894.26–2058.91)b 1541.45 (1042.27–1800.35)

993.18 1041.47 1048.63 1106.85

1391.29 1284.95 1242.87 1455.76

(828.90–1212.36) (953.74–1361.20) (901.72–1215.86) (834.77–1513.98)

1237.5 (1016.00–1432.00) 1278 (1150.25–1442.50)

(1145.02–1610.72)a (1011.57–1501.54) (1071.73–1515.98) (973.69–1630.48)

1206.5 (1002.00–1390.25) 1267 (959.00–1384.25)

1624.33 1542.17 1556.05 1642.32

(1500.07–1752.05)a,b (1292.36–1650.09)a,b (1368.93–1682.47)a,b (1429.29–1758.94)a,b

932 (790.00–1262.00) 842 (745.00–1232.00)a

Values are expressed in fmol/mg tissue and presented as median and quartiles 1 and 3. Statistical analysis was conducted by Kruskal–Wallis nonparametric test followed by repeated Mann–Whitney tests. Values were considered significant when p < 0.017. a significant difference of control group. b significant difference of acute group; c significant difference of chronic group.

Fig. 2 Pseudo-color autoradiograms showing the distribution of the [35S]GTPcS incorporation under basal conditions in coronal sections of an animal of the control group, and rats submitted to acute and chronic.

between exercise-induced effects in brain and the opioid system, we have to bear in mind that the beneficial effect of exercise on mood, anxiety (Rosch 1985; Wildmann et al. 1986; Bender et al. 2007) and depression (Landers and Arent 2001) is commonly noted at low exercise intensity (Jarvekulg

and Viru 2002). Indeed, there is no consistent information to suggest that these effects in the brain are similar to those found in the periphery. We therefore proposed to evaluate the effect of low-intensity exercise on the brain opioid system. We noted differential binding of opioid receptors in brain regions at speed of 10 m/min. One of the few studies that addressed this issue evaluated the central opioidergic mechanisms of the runner’s high imaging in the human brain (Boecker et al. 2008). The release of endogenous opioids occurred in frontolimbic brain regions after 2-h running, i.e., moderate intensity over an extended distance or duration. As observed above, the majority of human and animal investigations have focused on the influence of exercise on endorphin and its MOR receptor. Indeed, limited investigation on KOR and DOR involvement during exercise has been reported in the literature. Christie and Chesher (1983) found an increase in KOR binding after chronic swimming in rats. Thus, chronic exercise (7 weeks of treadmill running) induced an increase in KOR tissue content in caudateputamen and DOR in paraventricular hypothalamic nucleus (Chen et al. 2007). In accordance, Blake et al. (1984) found enhanced DOR content in the ventral tegmentum after 8 weeks of treadmill training. Although our study demonstrated increased or unaltered KOR and DOR binding in most structures analyzed for acute exercise, reduced binding was observed in chronic exercise. Particularly, we observed an increase in KOR binding only in the CA1 area after acute exercise and a reduction in KOR binding in DG, CA2, CA3 and BL amygdala after chronic exercise. The few brain alterations noted in our investigation could be due to a

© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12976

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R. M. Arida et al.

relatively low level of physical stress (low exercise intensity) imposed on our animals. Dynorphin/KOR system has an important contribution to stress response. Behavioral stress in animals induces an increase in dynorphin secretion and KOR activation in stress-related brain areas (Shirayama et al. 2004). Indeed, BL amygdala and CA1 region have been suggested as critical sites in mediating the effects of stress hormones on memory formation (McGaugh et al. 1996). Preclinical findings support the role of DOR in emotional processes and drug reward and addiction (for review see Chu Sin Chung and Kieffer 2013). Pharmacological blockade of DOR increases anxiety (Perrine et al. 2006) and activation of DOR, either endogenously or by agonist treatment, can improve emotional responses and treatment of anxiety and depression (Pradhan et al. 2011). The increased (acute exercise) DOR binding in some hippocampal regions in our study could in part explain the anxiolytic effects of exercise. Disparate results were observed for MOR, KOR and DOR receptor activation in our study. Investigations have reported that chronic opiate treatment decreases the MOR-mediated G protein activation in specific brain regions (Noble and Cox 1996; Celver et al. 2004). In line with this, decreased MORactivated G proteins found in several brain regions in our study could be associated with receptor desensitization similar to those observed after chronic opiate administration (Zhu et al. 1998; Maher et al. 2005; Bourova et al. 2010). Future studies should be focused on elucidating the formation of opioid receptor hetero-oligomers as a consequence of exercise and their role in mood and neurological disorders (Lee et al. 2009; Pei et al. 2010). It is import to bear in mind that no meaningful comparisons can yet be made considering different exercise protocols from the above studies. Undoubtedly, the opioid system has complex and divergent functions in the brain, and different stimuli such as type, duration and intensity of exercise can mediate the diverse effects of endogenous opioid and their receptors which can potentially confound the interpretation of the results. These effects can comprise the influence on cell proliferation (Ra et al. 2002; Lee et al. 2003; Koehl et al. 2008) or emotional learning and memory (Ukai et al. 2000; Katzen-Perez et al. 2001). The discrepancy of some results might also be attributed to interactions of opioids with other neurotransmitter systems in the central nervous system. An important finding from this study was that animals with chronic exercise showed lower or absence of [35S]GTPcS incorporation in the presence of agonists into MOR, DOR and KOR in different brain areas. In contrast, the binding of [35S] GTPcS under basal conditions (absence of agonists) was mainly elevated in sensorimotor cortex and hippocampus, a situation more evident after chronic exercise. This situation suggests an increased constitutive activity of receptors coupled to G proteins, such as opioid receptors. There are many possibilities that could lead to changes in basal [35S]GTPcS

binding. For example, exercise has been shown to regulate the expression of G protein subunits (Esmaeili-Mahani et al. 2013). MOR constitutive activity has been described to augment as a consequence of chronic exposure to endogenous and exogenous opiate agonists (Liu and Prather 2002; Shoblock and Maidment 2006, 2007; Meye et al. 2012), a situation associated with opiate dependence (Cruz et al. 1996; Wang et al. 2004). The up-regulation of constitutively active MORs may activate downstream counter adaptive processes that ultimately produce cellular and physical dependence (Corder et al. 2013). Future experiments should seek to determine if the augmented binding of [35S]GTPcS under basal conditions depends on increased opioid receptor constitutive activity as a consequence of chronic exercise, a condition that may be associated with opioid release during chronic exercise. How exercise stress could activate the central opioid system has yet to be properly defined. This study provides evidence that binding and activation of opioid receptors occur differentially in brain regions in response to diverse exercise stimuli. In this context, it is important to point out that opioid receptors-induced G protein activation is decreased in several brain areas of the chronic group in spite of increased or absence of changes in receptor binding. This finding is relevant because it indicates lower signal transductional mechanisms that previously have not been described using other procedures such as western blot or in vitro autoradiography. However, in a more general view, changes of opioid receptors in different areas of the rat brain are ‘bidirectional’, showing decreases in some areas and increases in others. This is of particular interest as different opioid receptor types have complex and somewhat opposing functions in nociception, modulation of mood states, reward, and specific role in the generation of the runner’s high sensation. Thus, the opioid adjustment to exercise is complex and the involvement of the central opioid may be masked by actions of neurotransmitters, hormones and metabolic byproducts of exercise. In conclusion, our findings indicate that depending on exercise stimulus, specific areas of brain can be activated or adjusted to modulate the factors cited above.

Acknowledgments and conflict of interest disclosure Research supported by CAPES, FAPESP, CNPq, Instituto Nacional de Neuroci^encia Translacional (INNT) and the National Council for Sciences and Technology of Mexico (grant 98386). The authors declare no conflicts of interest. All experiments were conducted in compliance with the ARRIVE guidelines.

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