Behavioural Brain Research 299 (2016) 27–31
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Ambient temperature influences the neural benefits of exercise Mark E. Maynard a , Chasity Chung a , Ashley Comer a , Katharine Nelson a , Jamie Tran a , Nadja Werries a , Emily A. Barton a , Michael Spinetta c , J. Leigh Leasure a,b,∗ a b c
Department of Psychology, University of Houston, Houston, TX 77204-5022, United States Department of Biology & Biochemistry, University of Houston, Houston, TX 77204-5022, United States Department of Psychology, Seattle University, Seattle, WA 98122, United States
h i g h l i g h t s • Ambient temperature influences voluntary exercise time, speed and distance. • A limited exercise distance in heat or cold increases hippocampal cell division. • A limited exercise distance in heat or cold increases neuronal differentiation.
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 6 November 2015 Accepted 16 November 2015 Keywords: Temperature Exercise Neurogenesis Hippocampus Brain injury
a b s t r a c t Many of the neural benefits of exercise require weeks to manifest. It would be useful to accelerate onset of exercise-driven plastic changes, such as increased hippocampal neurogenesis. Exercise represents a significant challenge to the brain because it produces heat, but brain temperature does not rise during exercise in the cold. This study tested the hypothesis that exercise in cold ambient temperature would stimulate hippocampal neurogenesis more than exercise in room or hot conditions. Adult female rats had exercise access 2 h per day for 5 days at either room (20 ◦ C), cold (4.5 ◦ C) or hot (37.5 ◦ C) temperature. To label dividing hippocampal precursor cells, animals received daily injections of BrdU. Brains were immunohistochemically processed for dividing cells (Ki67+), surviving cells (BrdU+) and new neurons (doublecortin, DCX) in the hippocampal dentate gyrus. Animals exercising at room temperature ran significantly farther than animals exercising in cold or hot conditions (room 1490 ± 400 m; cold 440 ± 102 m; hot 291 ± 56 m). We therefore analyzed the number of Ki67+, BrdU+ and DCX+ cells normalized for shortest distance run. Contrary to our hypothesis, exercise in either cold or hot conditions generated significantly more Ki67+, BrdU+ and DCX+ cells compared to exercise at room temperature. Thus, a limited amount of running in either cold or hot ambient conditions generates more new cells than a much greater distance run at room temperature. Taken together, our results suggest a simple means by which to augment exercise effects, yet minimize exercise time. © 2015 Elsevier B.V. All rights reserved.
Exercise provides the brain with many benefits, including enhanced hippocampal neurogenesis [1], which has been linked to improved cognition [1,2]. However, these neural benefits take time to emerge. A recent study showed that 2 weeks of exercise was necessary in order to increase neurogenesis and 8 weeks was necessary to increase synaptic efficacy [3]. It would be useful to discover ways to accelerate the neural benefits of exercise. Exercise represents a significant challenge to the brain because it produces heat [4]. Brain functions occur in a relatively narrow
∗ Corresponding author at: Department of Psychology, University of Houston, Houston, TX 77204, United States. Fax: +1 713 743 8588. E-mail address: [email protected] (J.L. Leasure). http://dx.doi.org/10.1016/j.bbr.2015.11.017 0166-4328/© 2015 Elsevier B.V. All rights reserved.
temperature range, which is maintained by balancing heat production and heat loss [4,5]. Regulatory mechanisms protect the brain from being damaged by increased core temperature (for review see Refs. [6,7]), but these are challenged during exercise in the heat [6,8]. In contrast, exercise in cold ambient temperature seems to enhance physical performance [9]—both humans [9] and rats [10] are able to run longer before volitional fatigue in cold ambient temperatures versus hot. Moreover, when animals exercised in thermoneutral conditions (25 ◦ C), brain temperature rose, but when they exercised in the cold (12 ◦ C) the increase in brain temperature was attenuated. The present study was conducted in order to test the hypothesis that exercise in cold ambient temperature (4.5 ◦ C) would more effectively promote neurogenesis in the hippocampal dentate gyrus
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M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31
(DG) than exercise at room temperature (20 ◦ C). Because exercising in the heat represents a considerable challenge to the brain [6,8], we included a hot ambient temperature condition (37.5 ◦ C) in order to test the hypothesis that running in the heat would not increase neurogenesis beyond exercise at room temperature. Forty-nine female Long-Evans rats (Harlan Sprague Dawley, IN, USA), weighing 180–260 g were randomly divided into 6 groups in a 3 × 2 design comparing Temperature (Room, Cold, or Hot) and Activity (Sedentary or Exercise). Each group had 8 animals, with the exception of Cold Exercise, which had 9. Rats were housed in groups in the vivarium (temperature 21.7 ◦ C) on a reversed light/dark cycle (10:00 off/22:00 on) with ad libitum food and water. Vaginal smears were taken once each day between 9:00 and 9:30 A.M. On one occasion (three days prior to the experiment) animals were acclimated to experimental temperature conditions and exercise wheels for 2 h. During the experiment, animals in exercise groups had access to wheels at room (20 ◦ C), cold (4.5 ◦ C) or hot (37.5 ◦ C) ambient temperature, 2 h a day for 5 consecutive days. Sedentary rats were placed in pairs in locked exercise wheels for two hours at their respective group’s temperature. Exercisers had individual access to an exercise wheel, to determine individual running distance, speed and time. After exercise, animals were returned to group-housing. To label cells generated in response to changes in temperature and/or activity, bromodeoxyuridine (BrdU, Sigma, MO, USA, 50 mg/kg, i.p), a thymidine analog, was administered daily prior to wheel access. Fecal samples were collected after exercise on the last day of the experiment. Fecal corticosterone is highly correlated with serum corticosterone and it has been shown that an acute stressor is reflected in elevated fecal corticosterone levels the following day [11]. Thus, if 2 h of exposure to cold or hot temperature elevated corticosterone, we would expect to detect this in fecal samples collected the following day. Also, we could detect any sustained increase in corticosterone caused by repeated exposure to the different temperature conditions. Corticosterone levels were quantified using a commercially available enzyme immunoassay kit (Enzo Life Sciences, New York, USA), according to the manufacturer’s instructions as we have previously described [12]. Following exercise on the fifth day, rats were perfused and brain tissue processed as previously described [12]. For BrdU immunohistochemistry, the primary antibody was sheep anti-Brdu (Exalpha Biologicals, Maynard, MA, USA; 1:400), followed by a biotinylated secondary (donkey anti-sheep, Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:250). To assess cell proliferation and neuronal differentiation, separate one-in-twelve and one-insix series of sections were processed for Ki67 and doublecortin (DCX) respectively. The primary antibodies were rabbit anti-Ki67 (Vector Laboratories, Burlingame, CA; 1:1800) and goat anti-DCX
(Santa Cruz Biotechnology, Santa Cruz, CA; 1:100). The secondary antibodies were biotinylated donkey anti-rabbit and biotinylated donkey anti-goat (both from Jackson ImmunoResearch Laboratories, 1:250). Sections were mounted onto gelatin-coated slides, counterstained with methyl-green, coverslipped and coded. BrdU+ and DCX+ cells in the DG were quantified at 40× and 100× respectively, using the optical fractionator method applied via our automated stereology system (StereoInvestigator, MicroBrightField, VT, USA) as previously described [12]. Cells labeled with Ki67 were not abundant enough in the sedentary groups to be counted stereologically. Instead, each labeled soma in the granule cell layer or subgranular zone was counted in every twelfth section from Bregma −1.88 through Bregma −6.04, using a 40× oil objective. One-way ANOVA’s were conducted to determine the effect of Temperature on exercise distance, time and speed. Unexpectedly, temperature had a profound effect on exercise distance—animals at room temperature ran much further than those in cold or hot conditions. Therefore, we used one-way ANOVA to analyze the number of Ki67+, BrdU+ and DCX+ cells normalized for shortest distance run. We used the following formula for each exercised rat: (290 m/average distance traveled) × (cell count). Because the sedentary animals did not exercise, their cell counts could not be normalized for shortest distance run, therefore, cell count data from the sedentary groups were analyzed with a separate oneway ANOVA. All other data were analyzed with two-way factorial ANOVA with Temperature and Activity as independent variables. For all statistical analyses, an alpha level of 0.05 was set to determine significance. Bonferroni and Games Howell corrected post hoc analysis were conducted where appropriate. To determine whether the stage of estrus affected neurogenesis, a factorial ANOVA using the variables Temperature, Activity, and Day 1–5 of the experiment were analyzed. Because stage of estrus is a categorical variable, it was necessary to dummy code by assigning a numerical value for stage of estrus (Diestrus = 0, Proestrus = 1, Estrus = 2, Metestrus = 3) for each of the five days of the experiment. There was a significant main effect of Temperature [F(2,22) = 7.71, p < .005] on distance traveled (Fig. 1). Rats at room temperature ran significantly further than rats in cold (p < .05) and hot (p < .005) conditions, with no difference between the cold and hot conditions (p = .891). Analysis of average speed revealed a significant main effect of Temperature [F(2,22) = 4.832, p < .05]. Post hoc analysis showed that animals running at room temperature ran faster than animals in the hot (p < .05) condition. There was no difference between the cold and hot conditions (p = .582). Time spent running was significantly affected by Temperature [F(2,22) = 6.737, p < .005]. Post hoc analysis revealed that animals running in room temperature conditions spent more
Fig. 1. Rats in the cold and hot ambient temperature conditions ran shorter distances (A) at slower average speeds (B) and spent less time running (C) than rats running at room temperature. *p < 0.05 significantly different from room temperature control.
M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31
time running than animals in both cold (p < .05) and hot (p < .05) conditions, with no difference between cold and hot conditions (p = .749). The number of fecal pellets deposited by each animal during each day’s exercise period were counted in order to obtain a measure of intestinal motility. For paired sedentary animals, counts were halved. There was a significant main effect of Temperature [F(2,35) = 21.61, p < .005] and of Activity [F(1,35) = 13.15, p < .005], but no interaction [F(2,35) = .65, p = .529]. As shown in Table 1, animals that exercised defecated more than sedentary animals, regardless of temperature. Sedentary and/or exercised animals in the cold defecated more than animals in the room or hot conditions. For total fecal corticosterone, there were no significant main effects of Temperature [F(2,21) = 2.304, p = .125] or Activity [F(1,21) = 1.814, p = .192], and no significant interaction [F(2,21) = .073, p = .93] (see Table 1). Stage of estrus for any day of the experiment did not influence DCX (D1: p = .939; D2: p = .194; D3: p = .184; D4: p = .207; D5: p = .807), Ki67 (D1: p = .879; D2: p = .598; D3: p = .634; D4: p = .833; D5: p = .774), or BrdU (D1: p = .382; D2: p = .654; D3: p = .125; D4: p = .419; D5: p = .643). In sedentary rats, there was no main effect of Temperature for Ki67+ (proliferating) cells [F(2,22) = 2.37, p = .119], BrdU+ (surviving) cells [F(2,23) = 1.87, p = .179], or DCX+ cells (newly generated neurons) [F(2,23) = 1.21, p = .318]. Data (collapsed across groups) are shown in Fig. 2. In exercised rats, for Ki67 there was a significant effect of Temperature [F(2,24) = 7.862, p < .003]. Post hoc Games–Howell corrected analysis showed that exercised rats in the cold and hot groups had more Ki67+ cells than rats that ran at room temperature (p < .02 for both comparisons), but no difference between the cold and hot groups (Fig. 2A). For BrdU, there was a significant effect of Temperature [F(2,24) = 12.906, p < .001]. Post hoc Games–Howell corrected analysis showed that rats that ran in either the cold or hot group had more BrdU+ cells compared to rats that ran at room temperature (p < .05 for both comparisons). However, there were no differences in BrdU+ cells between the cold and hot conditions (Fig. 2B). The pattern of results was the same for DCX+ cells, with a significant effect of Temperature [F(2,24) = 15.310, p < .001]. Post hoc analysis again showed that rats that ran in the cold or hot conditions had significantly more DCX+ cells than rats that ran at room temperature (p < .05), but no difference between the cold and hot conditions (Figs. 2 and 3C, ). Exercise powerfully promotes neuroplasticity, however, many of these changes take a great deal of time to emerge. For example, a recent study showed that 2 weeks of exercise was necessary in order to increase neurogenesis and 8 weeks was necessary to increase synaptic efficacy [3]. We conducted the present experiment in order to determine whether the neurogenic effect of exercise would be accelerated if exercise occurred in the cold. Exercise produces heat, and invokes regulatory mechanisms that function to protect the brain from heat-induced injury [6,7]. Exercise therefore presents the brain with both a benefit and a challenge. We reasoned that if the challenge could be removed, the benefits might emerge sooner. Exercise in the cold removes the challenge, because while brain and body temperatures rise when sitting or exercising in hot conditions (38 ◦ C) [13], they did not change in rats exercising at 12 ◦ C [10]. We therefore hypothesized that exercise in cold conditions would accelerate brain benefits
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Fig. 2. Cell counts (non-normalized data) are shown as a function of distance run for each subject. Rats in either hot or cold ambient conditions ran very little, yet had more Ki67+ (A), BrdU+ (B) and DCX+ (C) cells compared to animals that ran at room temperature. The dotted line represents the mean number of cells for sedentary animals.
compared to exercise in thermoneutral conditions. We also hypothesized that exercise in the heat would delay exercise benefits, due to rising temperature. Consistent with prior findings [14], both cold and hot ambient temperatures had a marked effect on exercise distance, speed and time. The primary finding of this study is that a limited amount of running (in terms of distance, speed and time) in cold or hot ambient conditions generated more new cells and newly differentiated neurons than a much longer distance at room temperature (see Fig. 2). Sedentary exposure to cold or hot ambient temperatures is not sufficient to alter hippocampal precursor cells, as there were no differences between sedentary groups. Thus, activity (but not being sedentary) in the heat or the cold had a stimulating effect on hippocampal precursor cells. Previous reports have shown that 10–14 days of exercise under normal room temperature conditions is typically necessary for an increase in the number of immature neurons [3,15]. However, in the present study, exercising less than 500 m
Table 1 Average number of fecal pellets and fecal corticosterone values (±SEM). Room sedentary Fecal pellets Fecal corticosterone (pg/ml)
1.6 ± 0.4 1771 ± 396
Room exercise
Cold sedentary
Cold exercise
Hot sedentary
Hot exercise
3.2 ± 0.6 2534 ± 602
5.5 ± 0.5 1333 ± 208
6.5 ± 0.5 1719 ± 409
2.8 ± 0.3 2357 ± 386
5.1 ± 0.9 2939 ± 429
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M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31
effects of cold temperature on exercise-driven neurogenesis. We also reasoned that neurogenesis might be influenced by metabolic increases due to exercise and/or exposure to cold or heat. While we did not measure metabolism directly, running increases intestinal motility [17]. Therefore, we counted fecal pellets as a proxy measure of metabolic activation. Animals in the cold defecated most, followed by hot exercise animals. There was increased proliferation, survival and neuronal differentiation in the cold exercise and hot exercise groups, but not the cold sedentary group, thus, there may be involvement of increased metabolism, but other factors are also likely important. Future studies will investigate inflammation and trophic factor expression as potential mechanisms underlying the accelerating effect of temperature on exercise brain benefits. Enhanced neurogenesis has been linked to enhanced cognition [1,2]. In contrast, decreased neurogenesis, such as happens with brain radiation (for cancer treatment) or brain aging, is associated with cognitive impairments [18–20]. Exercise has been shown to be an effective means by which to enhance hippocampal neurogenesis in animal models of these conditions [21,22]. However, both the aged and cancer survivors may have mobility limitations that decrease their capacity to reap the brain benefits of exercise. The same is true for traumatic brain injury or stroke, where exercise would be a useful component of rehabilitation efforts, but motor impairments and deconditioning may limit exercise capacity. Additionally, spaceflight is a situation in which exercise countermeasures are essential for maintaining health, yet difficult, due to space and time constraints [23]. Thus, identifying simple means by which the neurogenic effects of exercise can be augmented could improve exercise-based treatment interventions. In summary, the idea that exercise could augment the effects of other therapies is widely acknowledged. Ironically, the brains that would benefit most from the neurogenic effects of exercise are often paired with bodies that can withstand only minimal physical activity. Our results suggest a simple means by which to maximize exercise benefits while minimizing exercise time. Conflict of interest None. Acknowledgements This study was supported by a Grant-in-Aid to JLL from the College of Liberal Arts and Social Sciences, University of Houston. We thank Dr. Shaefali Rodgers for helpful comments on the manuscript. References Fig. 3. Pictured are sections stained for doublecortin at 20× magnification. Panel A depicts a control animal that ran a high average distance. Animals in the cold (B) and hot (C) ambient temperature conditions had more DCX+ cells, yet ran a much shorter average distance. This pattern of staining (few processes) is typical of LongEvans rats, which show less complex branching than other rat strains [24]. Scale bar = 100 m.
per day in cold or hot conditions increased the number of immature neurons (see Fig. 2). Viewed within this context, our results suggest that either cold or hot ambient temperature may accelerate the brain benefits of exercise. There are several potential mechanisms by which temperature could interact with exercise to alter its neural effects. One possibility is circulating corticosterone. Stress is known to decrease neurogenesis [16] and exposure to both cold and hot temperatures has been shown to be stressful [4]. However, we found no significant effects of either Activity or Temperature, indicating that corticosterone levels were not directly involved in the
[1] H. van Praag, B.R. Christie, T.J. Sejnowski, F.H. Gage, Running enhances neurogenesis, learning, and long-term potentiation in mice, Proc. Natl. Acad. Sci. U. S. A. 96 (23) (1999) 13427–13431. [2] T.J. Shors, M.L. Anderson, D.M. Curlik 2nd, M.S. Nokia, Use it or lose it: how neurogenesis keeps the brain fit for learning, Behav. Brain Res. 227 (2) (2012) 450–458. [3] A.R. Patten, H. Sickmann, B.N. Hryciw, T. Kucharsky, R. Parton, A. Kernick, et al., Long-term exercise is needed to enhance synaptic plasticity in the hippocampus, Learn. Mem. 20 (11) (2013) 642–647. [4] J. Terrien, M. Perret, F. Aujard, Behavioral thermoregulation in mammals: a review, Front. Biosci. 16 (2011) 1428–1444. [5] H. Wang, B. Wang, K.P. Normoyle, K. Jackson, K. Spitler, M.F. Sharrock, et al., Brain temperature and its fundamental properties: a review for clinical neuroscientists, Front. Neurosci. 8 (2014) 307. [6] L. Nybo, N.H. Secher, Cerebral perturbations provoked by prolonged exercise, Prog. Neurobiol. 72 (4) (2004) 223–261. [7] F.E. Marino, The critical limiting temperature and selective brain cooling: neuroprotection during exercise? Int. J. Hyperthermia 27 (6) (2011) 582–590. [8] B. Nielsen, L. Nybo, Cerebral changes during exercise in the heat, Sports Med. 33 (1) (2003) 1–11. [9] S.D. Galloway, R.J. Maughan, Effects of ambient temperature on the capacity to perform prolonged cycle exercise in man, Med. Sci. Sports Exerc. 29 (9) (1997) 1240–1249.
M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31 [10] C.G. Fonseca, W. Pires, M.R. Lima, J.B. Guimaraes, N.R. Lima, S.P. Wanner, Hypothalamic temperature of rats subjected to treadmill running in a cold environment, PLoS One 9 (11) (2014) e111501. [11] P.K. Thanos, S.A. Cavigelli, M. Michaelides, D.M. Olvet, U. Patel, M.N. Diep, et al., A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm, Physiol. Res. 58 (2) (2009) 219–228. [12] D.F. Hawley, K. Morch, B.R. Christie, J.L. Leasure, Differential response of hippocampal subregions to stress and learning, PLoS One 7 (12) (2012) e53126. [13] A. Fuller, R.N. Carter, D. Mitchell, Brain and abdominal temperatures at fatigue in rats exercising in the heat, J. Appl. Physiol. 84 (3) (1998) 877–883. [14] F.W. Finger, Relation of general activity in rats to environmental temperature, Percept. Motor Skills 43 (1976) 875–890, 3 pt. 1. [15] G. Kronenberg, A. Bick-Sander, E. Bunk, C. Wolf, D. Ehninger, G. Kempermann, Physical exercise prevents age-related decline in precursor cell activity in the mouse dentate gyrus, Neurobiol. Aging 27 (10) (2006) 1505–1513. [16] E. Gould, P. Tanapat, Stress and hippocampal neurogenesis, Biol. Psychiatry 46 (11) (1999) 1472–1479. [17] F.M. Moses, The effect of exercise on the gastrointestinal tract, Sports Med. 9 (3) (1990) 159–172.
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[18] M.L. Monje, T. Palmer, Radiation injury and neurogenesis, Curr. Opin. Neurol. 16 (2) (2003) 129–134. [19] M. Monje, J. Dietrich, Cognitive side effects of cancer therapy demonstrate a functional role for adult neurogenesis, Behav. Brain Res. 227 (2) (2012) 376–379. [20] H.G. Kuhn, H. Dickinson-Anson, F.H. Gage, Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation, J. Neurosci. 16 (6) (1996) 2027–2033. [21] H. van Praag, T. Shubert, C. Zhao, F.H. Gage, Exercise enhances learning and hippocampal neurogenesis in aged mice, J. Neurosci. 25 (38) (2005) 8680–8685. [22] S.J. Wong-Goodrich, M.L. Pfau, C.T. Flores, J.A. Fraser, C.L. Williams, L.W. Jones, Voluntary running prevents progressive memory decline and increases adult hippocampal neurogenesis and growth factor expression after whole-brain irradiation, Cancer Res. 70 (22) (2010) 9329–9338. [23] A.R. Hargens, R. Bhattacharya, S.M. Schneider, Space physiology VI: exercise, artificial gravity, and countermeasure development for prolonged space flight, Eur. J. Appl. Physiol. 113 (9) (2013) 2183–2192. [24] J.R. Epp, N.A. Scott, L.A. Galea, Strain differences in neurogenesis and activation of new neurons in the dentate gyrus in response to spatial learning, Neuroscience 172 (2011) 342–354.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Ambient temperature influences the neural benefits of exercise Mark E. Maynard a , Chasity Chung a , Ashley Comer a , Katharine Nelson a , Jamie Tran a , Nadja Werries a , Emily A. Barton a , Michael Spinetta c , J. Leigh Leasure a,b,∗ a b c
Department of Psychology, University of Houston, Houston, TX 77204-5022, United States Department of Biology & Biochemistry, University of Houston, Houston, TX 77204-5022, United States Department of Psychology, Seattle University, Seattle, WA 98122, United States
h i g h l i g h t s • Ambient temperature influences voluntary exercise time, speed and distance. • A limited exercise distance in heat or cold increases hippocampal cell division. • A limited exercise distance in heat or cold increases neuronal differentiation.
a r t i c l e
i n f o
Article history: Received 7 May 2015 Received in revised form 6 November 2015 Accepted 16 November 2015 Keywords: Temperature Exercise Neurogenesis Hippocampus Brain injury
a b s t r a c t Many of the neural benefits of exercise require weeks to manifest. It would be useful to accelerate onset of exercise-driven plastic changes, such as increased hippocampal neurogenesis. Exercise represents a significant challenge to the brain because it produces heat, but brain temperature does not rise during exercise in the cold. This study tested the hypothesis that exercise in cold ambient temperature would stimulate hippocampal neurogenesis more than exercise in room or hot conditions. Adult female rats had exercise access 2 h per day for 5 days at either room (20 ◦ C), cold (4.5 ◦ C) or hot (37.5 ◦ C) temperature. To label dividing hippocampal precursor cells, animals received daily injections of BrdU. Brains were immunohistochemically processed for dividing cells (Ki67+), surviving cells (BrdU+) and new neurons (doublecortin, DCX) in the hippocampal dentate gyrus. Animals exercising at room temperature ran significantly farther than animals exercising in cold or hot conditions (room 1490 ± 400 m; cold 440 ± 102 m; hot 291 ± 56 m). We therefore analyzed the number of Ki67+, BrdU+ and DCX+ cells normalized for shortest distance run. Contrary to our hypothesis, exercise in either cold or hot conditions generated significantly more Ki67+, BrdU+ and DCX+ cells compared to exercise at room temperature. Thus, a limited amount of running in either cold or hot ambient conditions generates more new cells than a much greater distance run at room temperature. Taken together, our results suggest a simple means by which to augment exercise effects, yet minimize exercise time. © 2015 Elsevier B.V. All rights reserved.
Exercise provides the brain with many benefits, including enhanced hippocampal neurogenesis [1], which has been linked to improved cognition [1,2]. However, these neural benefits take time to emerge. A recent study showed that 2 weeks of exercise was necessary in order to increase neurogenesis and 8 weeks was necessary to increase synaptic efficacy [3]. It would be useful to discover ways to accelerate the neural benefits of exercise. Exercise represents a significant challenge to the brain because it produces heat [4]. Brain functions occur in a relatively narrow
∗ Corresponding author at: Department of Psychology, University of Houston, Houston, TX 77204, United States. Fax: +1 713 743 8588. E-mail address: [email protected] (J.L. Leasure). http://dx.doi.org/10.1016/j.bbr.2015.11.017 0166-4328/© 2015 Elsevier B.V. All rights reserved.
temperature range, which is maintained by balancing heat production and heat loss [4,5]. Regulatory mechanisms protect the brain from being damaged by increased core temperature (for review see Refs. [6,7]), but these are challenged during exercise in the heat [6,8]. In contrast, exercise in cold ambient temperature seems to enhance physical performance [9]—both humans [9] and rats [10] are able to run longer before volitional fatigue in cold ambient temperatures versus hot. Moreover, when animals exercised in thermoneutral conditions (25 ◦ C), brain temperature rose, but when they exercised in the cold (12 ◦ C) the increase in brain temperature was attenuated. The present study was conducted in order to test the hypothesis that exercise in cold ambient temperature (4.5 ◦ C) would more effectively promote neurogenesis in the hippocampal dentate gyrus
28
M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31
(DG) than exercise at room temperature (20 ◦ C). Because exercising in the heat represents a considerable challenge to the brain [6,8], we included a hot ambient temperature condition (37.5 ◦ C) in order to test the hypothesis that running in the heat would not increase neurogenesis beyond exercise at room temperature. Forty-nine female Long-Evans rats (Harlan Sprague Dawley, IN, USA), weighing 180–260 g were randomly divided into 6 groups in a 3 × 2 design comparing Temperature (Room, Cold, or Hot) and Activity (Sedentary or Exercise). Each group had 8 animals, with the exception of Cold Exercise, which had 9. Rats were housed in groups in the vivarium (temperature 21.7 ◦ C) on a reversed light/dark cycle (10:00 off/22:00 on) with ad libitum food and water. Vaginal smears were taken once each day between 9:00 and 9:30 A.M. On one occasion (three days prior to the experiment) animals were acclimated to experimental temperature conditions and exercise wheels for 2 h. During the experiment, animals in exercise groups had access to wheels at room (20 ◦ C), cold (4.5 ◦ C) or hot (37.5 ◦ C) ambient temperature, 2 h a day for 5 consecutive days. Sedentary rats were placed in pairs in locked exercise wheels for two hours at their respective group’s temperature. Exercisers had individual access to an exercise wheel, to determine individual running distance, speed and time. After exercise, animals were returned to group-housing. To label cells generated in response to changes in temperature and/or activity, bromodeoxyuridine (BrdU, Sigma, MO, USA, 50 mg/kg, i.p), a thymidine analog, was administered daily prior to wheel access. Fecal samples were collected after exercise on the last day of the experiment. Fecal corticosterone is highly correlated with serum corticosterone and it has been shown that an acute stressor is reflected in elevated fecal corticosterone levels the following day [11]. Thus, if 2 h of exposure to cold or hot temperature elevated corticosterone, we would expect to detect this in fecal samples collected the following day. Also, we could detect any sustained increase in corticosterone caused by repeated exposure to the different temperature conditions. Corticosterone levels were quantified using a commercially available enzyme immunoassay kit (Enzo Life Sciences, New York, USA), according to the manufacturer’s instructions as we have previously described [12]. Following exercise on the fifth day, rats were perfused and brain tissue processed as previously described [12]. For BrdU immunohistochemistry, the primary antibody was sheep anti-Brdu (Exalpha Biologicals, Maynard, MA, USA; 1:400), followed by a biotinylated secondary (donkey anti-sheep, Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:250). To assess cell proliferation and neuronal differentiation, separate one-in-twelve and one-insix series of sections were processed for Ki67 and doublecortin (DCX) respectively. The primary antibodies were rabbit anti-Ki67 (Vector Laboratories, Burlingame, CA; 1:1800) and goat anti-DCX
(Santa Cruz Biotechnology, Santa Cruz, CA; 1:100). The secondary antibodies were biotinylated donkey anti-rabbit and biotinylated donkey anti-goat (both from Jackson ImmunoResearch Laboratories, 1:250). Sections were mounted onto gelatin-coated slides, counterstained with methyl-green, coverslipped and coded. BrdU+ and DCX+ cells in the DG were quantified at 40× and 100× respectively, using the optical fractionator method applied via our automated stereology system (StereoInvestigator, MicroBrightField, VT, USA) as previously described [12]. Cells labeled with Ki67 were not abundant enough in the sedentary groups to be counted stereologically. Instead, each labeled soma in the granule cell layer or subgranular zone was counted in every twelfth section from Bregma −1.88 through Bregma −6.04, using a 40× oil objective. One-way ANOVA’s were conducted to determine the effect of Temperature on exercise distance, time and speed. Unexpectedly, temperature had a profound effect on exercise distance—animals at room temperature ran much further than those in cold or hot conditions. Therefore, we used one-way ANOVA to analyze the number of Ki67+, BrdU+ and DCX+ cells normalized for shortest distance run. We used the following formula for each exercised rat: (290 m/average distance traveled) × (cell count). Because the sedentary animals did not exercise, their cell counts could not be normalized for shortest distance run, therefore, cell count data from the sedentary groups were analyzed with a separate oneway ANOVA. All other data were analyzed with two-way factorial ANOVA with Temperature and Activity as independent variables. For all statistical analyses, an alpha level of 0.05 was set to determine significance. Bonferroni and Games Howell corrected post hoc analysis were conducted where appropriate. To determine whether the stage of estrus affected neurogenesis, a factorial ANOVA using the variables Temperature, Activity, and Day 1–5 of the experiment were analyzed. Because stage of estrus is a categorical variable, it was necessary to dummy code by assigning a numerical value for stage of estrus (Diestrus = 0, Proestrus = 1, Estrus = 2, Metestrus = 3) for each of the five days of the experiment. There was a significant main effect of Temperature [F(2,22) = 7.71, p < .005] on distance traveled (Fig. 1). Rats at room temperature ran significantly further than rats in cold (p < .05) and hot (p < .005) conditions, with no difference between the cold and hot conditions (p = .891). Analysis of average speed revealed a significant main effect of Temperature [F(2,22) = 4.832, p < .05]. Post hoc analysis showed that animals running at room temperature ran faster than animals in the hot (p < .05) condition. There was no difference between the cold and hot conditions (p = .582). Time spent running was significantly affected by Temperature [F(2,22) = 6.737, p < .005]. Post hoc analysis revealed that animals running in room temperature conditions spent more
Fig. 1. Rats in the cold and hot ambient temperature conditions ran shorter distances (A) at slower average speeds (B) and spent less time running (C) than rats running at room temperature. *p < 0.05 significantly different from room temperature control.
M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31
time running than animals in both cold (p < .05) and hot (p < .05) conditions, with no difference between cold and hot conditions (p = .749). The number of fecal pellets deposited by each animal during each day’s exercise period were counted in order to obtain a measure of intestinal motility. For paired sedentary animals, counts were halved. There was a significant main effect of Temperature [F(2,35) = 21.61, p < .005] and of Activity [F(1,35) = 13.15, p < .005], but no interaction [F(2,35) = .65, p = .529]. As shown in Table 1, animals that exercised defecated more than sedentary animals, regardless of temperature. Sedentary and/or exercised animals in the cold defecated more than animals in the room or hot conditions. For total fecal corticosterone, there were no significant main effects of Temperature [F(2,21) = 2.304, p = .125] or Activity [F(1,21) = 1.814, p = .192], and no significant interaction [F(2,21) = .073, p = .93] (see Table 1). Stage of estrus for any day of the experiment did not influence DCX (D1: p = .939; D2: p = .194; D3: p = .184; D4: p = .207; D5: p = .807), Ki67 (D1: p = .879; D2: p = .598; D3: p = .634; D4: p = .833; D5: p = .774), or BrdU (D1: p = .382; D2: p = .654; D3: p = .125; D4: p = .419; D5: p = .643). In sedentary rats, there was no main effect of Temperature for Ki67+ (proliferating) cells [F(2,22) = 2.37, p = .119], BrdU+ (surviving) cells [F(2,23) = 1.87, p = .179], or DCX+ cells (newly generated neurons) [F(2,23) = 1.21, p = .318]. Data (collapsed across groups) are shown in Fig. 2. In exercised rats, for Ki67 there was a significant effect of Temperature [F(2,24) = 7.862, p < .003]. Post hoc Games–Howell corrected analysis showed that exercised rats in the cold and hot groups had more Ki67+ cells than rats that ran at room temperature (p < .02 for both comparisons), but no difference between the cold and hot groups (Fig. 2A). For BrdU, there was a significant effect of Temperature [F(2,24) = 12.906, p < .001]. Post hoc Games–Howell corrected analysis showed that rats that ran in either the cold or hot group had more BrdU+ cells compared to rats that ran at room temperature (p < .05 for both comparisons). However, there were no differences in BrdU+ cells between the cold and hot conditions (Fig. 2B). The pattern of results was the same for DCX+ cells, with a significant effect of Temperature [F(2,24) = 15.310, p < .001]. Post hoc analysis again showed that rats that ran in the cold or hot conditions had significantly more DCX+ cells than rats that ran at room temperature (p < .05), but no difference between the cold and hot conditions (Figs. 2 and 3C, ). Exercise powerfully promotes neuroplasticity, however, many of these changes take a great deal of time to emerge. For example, a recent study showed that 2 weeks of exercise was necessary in order to increase neurogenesis and 8 weeks was necessary to increase synaptic efficacy [3]. We conducted the present experiment in order to determine whether the neurogenic effect of exercise would be accelerated if exercise occurred in the cold. Exercise produces heat, and invokes regulatory mechanisms that function to protect the brain from heat-induced injury [6,7]. Exercise therefore presents the brain with both a benefit and a challenge. We reasoned that if the challenge could be removed, the benefits might emerge sooner. Exercise in the cold removes the challenge, because while brain and body temperatures rise when sitting or exercising in hot conditions (38 ◦ C) [13], they did not change in rats exercising at 12 ◦ C [10]. We therefore hypothesized that exercise in cold conditions would accelerate brain benefits
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Fig. 2. Cell counts (non-normalized data) are shown as a function of distance run for each subject. Rats in either hot or cold ambient conditions ran very little, yet had more Ki67+ (A), BrdU+ (B) and DCX+ (C) cells compared to animals that ran at room temperature. The dotted line represents the mean number of cells for sedentary animals.
compared to exercise in thermoneutral conditions. We also hypothesized that exercise in the heat would delay exercise benefits, due to rising temperature. Consistent with prior findings [14], both cold and hot ambient temperatures had a marked effect on exercise distance, speed and time. The primary finding of this study is that a limited amount of running (in terms of distance, speed and time) in cold or hot ambient conditions generated more new cells and newly differentiated neurons than a much longer distance at room temperature (see Fig. 2). Sedentary exposure to cold or hot ambient temperatures is not sufficient to alter hippocampal precursor cells, as there were no differences between sedentary groups. Thus, activity (but not being sedentary) in the heat or the cold had a stimulating effect on hippocampal precursor cells. Previous reports have shown that 10–14 days of exercise under normal room temperature conditions is typically necessary for an increase in the number of immature neurons [3,15]. However, in the present study, exercising less than 500 m
Table 1 Average number of fecal pellets and fecal corticosterone values (±SEM). Room sedentary Fecal pellets Fecal corticosterone (pg/ml)
1.6 ± 0.4 1771 ± 396
Room exercise
Cold sedentary
Cold exercise
Hot sedentary
Hot exercise
3.2 ± 0.6 2534 ± 602
5.5 ± 0.5 1333 ± 208
6.5 ± 0.5 1719 ± 409
2.8 ± 0.3 2357 ± 386
5.1 ± 0.9 2939 ± 429
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effects of cold temperature on exercise-driven neurogenesis. We also reasoned that neurogenesis might be influenced by metabolic increases due to exercise and/or exposure to cold or heat. While we did not measure metabolism directly, running increases intestinal motility [17]. Therefore, we counted fecal pellets as a proxy measure of metabolic activation. Animals in the cold defecated most, followed by hot exercise animals. There was increased proliferation, survival and neuronal differentiation in the cold exercise and hot exercise groups, but not the cold sedentary group, thus, there may be involvement of increased metabolism, but other factors are also likely important. Future studies will investigate inflammation and trophic factor expression as potential mechanisms underlying the accelerating effect of temperature on exercise brain benefits. Enhanced neurogenesis has been linked to enhanced cognition [1,2]. In contrast, decreased neurogenesis, such as happens with brain radiation (for cancer treatment) or brain aging, is associated with cognitive impairments [18–20]. Exercise has been shown to be an effective means by which to enhance hippocampal neurogenesis in animal models of these conditions [21,22]. However, both the aged and cancer survivors may have mobility limitations that decrease their capacity to reap the brain benefits of exercise. The same is true for traumatic brain injury or stroke, where exercise would be a useful component of rehabilitation efforts, but motor impairments and deconditioning may limit exercise capacity. Additionally, spaceflight is a situation in which exercise countermeasures are essential for maintaining health, yet difficult, due to space and time constraints [23]. Thus, identifying simple means by which the neurogenic effects of exercise can be augmented could improve exercise-based treatment interventions. In summary, the idea that exercise could augment the effects of other therapies is widely acknowledged. Ironically, the brains that would benefit most from the neurogenic effects of exercise are often paired with bodies that can withstand only minimal physical activity. Our results suggest a simple means by which to maximize exercise benefits while minimizing exercise time. Conflict of interest None. Acknowledgements This study was supported by a Grant-in-Aid to JLL from the College of Liberal Arts and Social Sciences, University of Houston. We thank Dr. Shaefali Rodgers for helpful comments on the manuscript. References Fig. 3. Pictured are sections stained for doublecortin at 20× magnification. Panel A depicts a control animal that ran a high average distance. Animals in the cold (B) and hot (C) ambient temperature conditions had more DCX+ cells, yet ran a much shorter average distance. This pattern of staining (few processes) is typical of LongEvans rats, which show less complex branching than other rat strains [24]. Scale bar = 100 m.
per day in cold or hot conditions increased the number of immature neurons (see Fig. 2). Viewed within this context, our results suggest that either cold or hot ambient temperature may accelerate the brain benefits of exercise. There are several potential mechanisms by which temperature could interact with exercise to alter its neural effects. One possibility is circulating corticosterone. Stress is known to decrease neurogenesis [16] and exposure to both cold and hot temperatures has been shown to be stressful [4]. However, we found no significant effects of either Activity or Temperature, indicating that corticosterone levels were not directly involved in the
[1] H. van Praag, B.R. Christie, T.J. Sejnowski, F.H. Gage, Running enhances neurogenesis, learning, and long-term potentiation in mice, Proc. Natl. Acad. Sci. U. S. A. 96 (23) (1999) 13427–13431. [2] T.J. Shors, M.L. Anderson, D.M. Curlik 2nd, M.S. Nokia, Use it or lose it: how neurogenesis keeps the brain fit for learning, Behav. Brain Res. 227 (2) (2012) 450–458. [3] A.R. Patten, H. Sickmann, B.N. Hryciw, T. Kucharsky, R. Parton, A. Kernick, et al., Long-term exercise is needed to enhance synaptic plasticity in the hippocampus, Learn. Mem. 20 (11) (2013) 642–647. [4] J. Terrien, M. Perret, F. Aujard, Behavioral thermoregulation in mammals: a review, Front. Biosci. 16 (2011) 1428–1444. [5] H. Wang, B. Wang, K.P. Normoyle, K. Jackson, K. Spitler, M.F. Sharrock, et al., Brain temperature and its fundamental properties: a review for clinical neuroscientists, Front. Neurosci. 8 (2014) 307. [6] L. Nybo, N.H. Secher, Cerebral perturbations provoked by prolonged exercise, Prog. Neurobiol. 72 (4) (2004) 223–261. [7] F.E. Marino, The critical limiting temperature and selective brain cooling: neuroprotection during exercise? Int. J. Hyperthermia 27 (6) (2011) 582–590. [8] B. Nielsen, L. Nybo, Cerebral changes during exercise in the heat, Sports Med. 33 (1) (2003) 1–11. [9] S.D. Galloway, R.J. Maughan, Effects of ambient temperature on the capacity to perform prolonged cycle exercise in man, Med. Sci. Sports Exerc. 29 (9) (1997) 1240–1249.
M.E. Maynard et al. / Behavioural Brain Research 299 (2016) 27–31 [10] C.G. Fonseca, W. Pires, M.R. Lima, J.B. Guimaraes, N.R. Lima, S.P. Wanner, Hypothalamic temperature of rats subjected to treadmill running in a cold environment, PLoS One 9 (11) (2014) e111501. [11] P.K. Thanos, S.A. Cavigelli, M. Michaelides, D.M. Olvet, U. Patel, M.N. Diep, et al., A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm, Physiol. Res. 58 (2) (2009) 219–228. [12] D.F. Hawley, K. Morch, B.R. Christie, J.L. Leasure, Differential response of hippocampal subregions to stress and learning, PLoS One 7 (12) (2012) e53126. [13] A. Fuller, R.N. Carter, D. Mitchell, Brain and abdominal temperatures at fatigue in rats exercising in the heat, J. Appl. Physiol. 84 (3) (1998) 877–883. [14] F.W. Finger, Relation of general activity in rats to environmental temperature, Percept. Motor Skills 43 (1976) 875–890, 3 pt. 1. [15] G. Kronenberg, A. Bick-Sander, E. Bunk, C. Wolf, D. Ehninger, G. Kempermann, Physical exercise prevents age-related decline in precursor cell activity in the mouse dentate gyrus, Neurobiol. Aging 27 (10) (2006) 1505–1513. [16] E. Gould, P. Tanapat, Stress and hippocampal neurogenesis, Biol. Psychiatry 46 (11) (1999) 1472–1479. [17] F.M. Moses, The effect of exercise on the gastrointestinal tract, Sports Med. 9 (3) (1990) 159–172.
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[18] M.L. Monje, T. Palmer, Radiation injury and neurogenesis, Curr. Opin. Neurol. 16 (2) (2003) 129–134. [19] M. Monje, J. Dietrich, Cognitive side effects of cancer therapy demonstrate a functional role for adult neurogenesis, Behav. Brain Res. 227 (2) (2012) 376–379. [20] H.G. Kuhn, H. Dickinson-Anson, F.H. Gage, Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation, J. Neurosci. 16 (6) (1996) 2027–2033. [21] H. van Praag, T. Shubert, C. Zhao, F.H. Gage, Exercise enhances learning and hippocampal neurogenesis in aged mice, J. Neurosci. 25 (38) (2005) 8680–8685. [22] S.J. Wong-Goodrich, M.L. Pfau, C.T. Flores, J.A. Fraser, C.L. Williams, L.W. Jones, Voluntary running prevents progressive memory decline and increases adult hippocampal neurogenesis and growth factor expression after whole-brain irradiation, Cancer Res. 70 (22) (2010) 9329–9338. [23] A.R. Hargens, R. Bhattacharya, S.M. Schneider, Space physiology VI: exercise, artificial gravity, and countermeasure development for prolonged space flight, Eur. J. Appl. Physiol. 113 (9) (2013) 2183–2192. [24] J.R. Epp, N.A. Scott, L.A. Galea, Strain differences in neurogenesis and activation of new neurons in the dentate gyrus in response to spatial learning, Neuroscience 172 (2011) 342–354.
