Exercise and Hippocampus

The antidepressant effect of running is associated with increased hippocampal cell proliferation

A R T IC L E

International Journal of Neuropsychopharmacology (2005), 8, 357–368. Copyright f 2005 CINP doi :10.1017/S1461145705005122

Astrid Bjørnebekk1, Aleksander A. Mathe´2 and Stefan Brene´1,2 1 2

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Division of Psychiatry, Neurotec Department, Karolinska University Hospital, Huddinge, Stockholm, Sweden

Abstract A common trait of antidepressant drugs, electroconvulsive treatment and physical exercise is that they relieve depression and up-regulate neurotrophic factors as well as cell proliferation and neurogenesis in the hippocampus. In order to identify possible biological underpinnings of depression and the antidepressant effect of running, we analysed cell proliferation, the level of the neurotrophic factor BDNF in hippocampus and dynorphin in striatum/accumbens in ‘depressed’ Flinders Sensitive Line rats (FSL) and Flinders Resistant Line (FRL) rats with and without access to running-wheels. The FRL strain exhibited a higher daily running activity than the FSL strain. Wheel-running had an antidepressant effect in the ‘depressed’ FSL rats, as indicated by the forced swim test. In the hippocampus, cell proliferation was lower in the ‘depressed’ rats compared to the control FRL rats but there was no difference in BDNF or dynorphin levels in striatum/accumbens. After 5 wk of running, cell proliferation increased in FSL but not in FRL rats. BDNF and dynorphin mRNA levels were increased in FRL but not to the same extent in the in FSL rats ; thus, increased BDNF and dynorphin levels were correlated to the running activity but not to the antidepressant effect of running. The only parameter that was associated to basal level of ‘depression’ and to the antidepressant effect was cell proliferation in the hippocampus. Thus, suppression of cell proliferation in the hippocampus could constitute one of the mechanisms that underlie depression, and physical activity might be an efficient antidepressant.

Received 8 June 2004 ; Reviewed 20 October 2004 ; Revised 22 October 2004 ; Accepted 1 November 2004 Key words : Depression, Flinders Sensitive Line rats, hippocampus, neurogenesis, neurotrophic factors.

Introduction Imaging studies have demonstrated structural brain changes associated with early onset depression in the hippocampus, amygdala, striatum and frontal cortex ; areas that are all extensively interconnected. Most consistent are the findings of volume loss in the hippocampal formation (Bremner et al., 2000 ; Sheline et al., 1996, 1999). Factors that might contribute to the hippocampal volume loss in depression are possible suppression of hippocampal neurogenesis or the loss of neurotrophic support. Until recently the dogma was that there was no neurogenesis in the adult mammalian brain even though there were early reports to the contrary (Altman, 1963, 1969 ; Kaplan and Hinds, 1977). Currently it is widely accepted that neurogenesis Address for correspondence : Dr S. Brene´, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden.

Tel. : 46-8-52487451 Fax : 46-8-323742 E-mail : stefan.brene@neuro.ki.se

occurs in the subgranular zone of the dentate gyrus and in the subventricular zone. Although the physiological role of neurogenesis is still controversial, a number of factors shown to regulate neurogenesis in the dentate gyrus have been identified. Interestingly stress, a key factor implicated in the cellular pathology of depression, down-regulates neurogenesis (Gould et al., 1997) and the brain-derived neurotrophic factor (BDNF) (Smith et al., 1995). In contrast, treatments which have an antidepressant effect in patients, e.g. antidepressant drugs, (Malberg et al., 2000), electroconvulsive treatment (Hellsten et al., 2002 ; Nibuya et al., 1995) and physical exercise (Neeper et al., 1996 ; van Praag et al., 1999b) increase both hippocampal neurogenesis and the levels of BDNF. Exercise promotes physical health and has an antidepressant effect in patients similar to conventional antidepressant treatment and psychotherapy.

Follow-up studies of patients reveal that continued exercise is more efficient in preventing depressive

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relapses than antidepressant medication (Babyak et al., 2000 ; Martinsen et al., 1985, 1989 ; Strawbridge et al., 2002). However, the mechanisms by which exercise produces therapeutic effects are still poorly understood.

Depression is a heterogeneous syndrome and it seems likely that several brain regions are involved mediating the diverse symptoms of depression. The mesolimbic dopamine system has a central function for motivational behaviours and pleasure seeking and it is possible that malfunctioning of the brain-reward systems could be an underlying mechanism of the anhedonia experienced in depressive illness (Naranjo et al., 2001 ; Nestler et al., 2002).

In this study we investigated whether running has an effect on immobility in the forced swim test (FST), a test routinely used for screening potential efficacy of antidepressant treatments (Porsolt et al., 1977). Moreover, we analysed an animal model of depression, the Flinders Sensitive Line (FSL) rats and the control Flinders Resistant Line (FRL) rats, thereby making it more likely to identify biological underpinnings of ‘depression ’ as well as effects of running on ‘depression ’. Levels of endogenous opioid peptides in striatum and accumbens, regions suggested playing a role in motivation and anhedonia, and BDNF known to be up-regulated after running and antidepressive treatments in hippocampus were analysed. Finally, we investigated whether hippocampal cell proliferation differed in ‘depressed ’ FSL compared to control FRL rats and if cell proliferation could be one of the possible mechanisms mediating the antidepressive effect of wheel-running.

Materials and methods Wheel-running Male FSL rats (n=16), and their controls, FRL rats (n= 16) were bred at the Karolinska Institute. All animal experiments were approved by the local Ethical Committee for Animal Research in Stockholm. The animals were individually housed with free access (FSL, n=8 ; FRL, n=8) or no access (FSL, n=8 ; FRL, n=8) to running wheels (diameter, 34 cm ; one revolution corresponding to 1.07 m). Running data were sampled 48 times/d using a computer-based data system with customized software. Behavioural testing (see below) was performed after 30 d of running. The total period with access to running-wheels was 35 d. Animals had access to food and water ad libitum, and were subjected to a controlled 12-h light/dark cycle (lights on at 07:00 hours).

FST A modified FST was performed (Caldarone et al., 2003 ; Cryan et al., 2003 ; Hall et al., 2001 ; Nowak et al., 2003 ; Zangen et al., 1999, 2001) after 30 d of running.

All animals were placed one at a time, into a cylinder (24-cm diameter) containing 25-cm deep water (25 xC).

After 15 min the animals were removed from the water, dried with towels and placed in a warmed enclosure before being returned to the home cage.

Swimming vs. floating behaviour (immobility) was recorded with a stopwatch by an observer blind to the treatment condition of the animals. Immobility was defined as a stationary posture and at least three of the rat’s paws had to be immobile.

5-bromo-2-deoxyuridine (BrdU) administration After the FST the rats were returned to their cages containing running-wheels. To evaluate cell proliferation, four injections of BrdU (75 mg/kg i.p., SigmaAldrich, Stockholm, Sweden) with 2-h intervals were administered 4 d after the swim test. The animals were sacrificed approx. 20 h after the last injection.

BrdU immunohistochemistry For the immunohistochemistry, coronal 40 mm sections were collected with a cryostat throughout the hippocampal formation. Antibodies and dilutions used were : mouse a-BrdU (1 : 100 ; DAKO A/S, Glostrup, Denmark), horse a-mouse-biotin (1 : 200 ; Vector, Burlingame, CA, USA). Immunohistochemistry for BrdU was performed as follows : sections were removed from a freezer (x20 xC) and post-fixed for 10 min in 4 % formaldehyde, rinsed in PBS 4r5 min, incubated for 30 min in 2 M HCl at 37 xC, rinsed 3r5 min in PBS, and incubated for 1 h in blocking solution (horse serum 10 %, 0.1 % Tween in PBS) at room temperature. This 1-h incubation was followed by overnight incubation with mouse a-BrdU at 4 xC. On day 2, the samples were rinsed 3r30 min in 0.1 % Tween PBS, incubated with horse a-mouse-biotin for 60 min at room temperature, rinsed again for 90 min in PBS 0.1 % Tween followed by 30 min in PBS only.

The sections were then incubated for 40 min at room temperature with avidin–biotin–peroxidase complex (1 : 100 in PBS, Vectastain Elite ; Vector), then rinsed in PBS for 1 h, followed by peroxidase detection (0.7 mg/ml, DAB dissolved in H2O) (DAB peroxidase substrate, Sigma) for y25 s per section. The sections were rinsed in PBS and stained with a hematoxylin solution (Vector).

Running is antidepressive and cell proliferative Stereology of BrdU-positive cells For quantification of BrdU-positive cells in the dentate gyrus the unbiased optical fractionater counting procedure was performed (West et al., 1991). Coronal 40mm sections were taken throughout the hippocampus and every fifteenth section (600-mm apart) [section sampling fraction (s.s.f.)] was selected for analysis of the right dentate gyrus. An unbiased counting frame with known area was superimposed on the field of view using appropriate software (StereologerTM, SPA Inc. VA, USA). The counting frames were systematically distributed with known x and y steps throughout the marked region from a random starting point. The area of the counting frame relative to the area associated with the x and y steps gives the second fraction [area sampling fraction (a.s.f.)]. The height of the optical dissector relative to the thickness of the section results in the third fraction [height (h)/thickness (h)].

The total number of neurons is given by Ntotal =SQx

1 1 t , s:s:f: a:s:f: h

where SQ is the number of neurons counted in the dissectors. The dentate gyrus was manually outlined using a 10r lens. Cell counts were performed with a 60r lens (numerical aperture=1.4). Positive cells were counted if they were within the dissectors. Cells situated further than two cell-body widths away from the base of the granular cell layer were defined as belonging to the hilus, and thus not counted. Moreover, cells were excluded if they were situated in the uppermost focal plane. To estimate total number of BrdU cells per individual, a representative material of BrdU immunoreactive cells in the dentate gyrus of the left hemispheres was compared to that of the right hemispheres. t tests showed that there were no differences in number of BrdU immunoreactive cells between the two hemispheres, and the total number of cells per individual was calculated.

In situ hybridization Coronal brain sections (14 mm) were cut on a cryostat at x20 xC, and sections were thawed onto glass slides.

The hybridization cocktail contained 50 % formamide, 4rSSC (1rSSC is 0.15 M NaCl, 0.015 M sodium citrate ; pH 7.0), 1rDenhardt’s solution, 1 % Sarcosyl, 0.02 M Na3PO4 ; pH 7.0 ; 10 % dextransulphate, 0.06 M dithiothreitol and 0.1 mg/ml sheared salmon sperm DNA. Single-stranded oligonucleotide 48-mer DNA probes specific for dopamine D1 receptor mRNA (72–121) (Wilkie et al., 1993), dopamine D2 receptor mRNA (772–816) (Guiramand et al., 1995), substance P (20–67)

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(Chiwakata et al., 1991), dynorphin (296–345) (Douglass et al., 1989), enkephalin (235–282) (Zurawski et al., 1986) and BDNF (250–298) (Leibrock et al., 1989) mRNA were used. The probes were 3k-end labelled with [a-33P]dATP (Dupont NEN, Wilmington, DE, USA) using terminal deoxynucleotidyl transferase (Gibco, Ta¨by, Sweden) to a specific activity of approximately 1r109 c.p.m./mg. Hybridization was performed for 18 h in a humidified chamber at 42 xC. Following hybridization, the sections were rinsed 4r20 min each in 1rSSC at 60 xC. Finally, the sections were rinsed in autoclaved water for 10 s, dehydrated in alcohol and air-dried. Thereafter, the slides were exposed to film (Kodak Biomax MR film, Kodak, Rochester, NY, USA) for 5–12 d and developed. Films were scanned and optical density values quantified using appropriate software [NIH image analysis program, version 1.62 (National Institute of Health, USA)]. A 14C step standard (Amersham, Buckinghamshire, UK) was included to calibrate optical density readings and convert measured values into nCi/g.

Statistical procedures Repeated measurements ANOVA was performed to examine differences in running-behaviour between the two strains, and to analyse the effect of treatment on weight or weight differences between the strains.

To analyse the effect of running on immobility behaviour, Mann–Whitney U tests were performed for each of the two strains. A two-way ANOVA, with planned comparison post-hoc test was performed to analyse cell proliferation as well as for the analysis of different mRNA levels. To further investigate the relationship between treatment and outcome variables Pearson’s product-moment correlation was calculated (Statistica ; v. 99, StatSoft, Tulsa, USA).

Results Body weight At the start of the experiment, the average weight of the animals was 360 g and there were no differences between the two strains, or within the strains. Body weight increased during the experiment in all rats (p<0.0001). The average weight gain was higher in controls (44 g) than in runners (39 g) (p<0.05).

Running behaviour Degree of motivation to engage in a goal-oriented reward-seeking behaviour was assessed by measuring the running distance per day. The FSL rats ran distances corresponding to y1600 m, whereas the

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12000 # ##

Running per day (m)

10000

8000 # 6000

4000

2000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Days

FRL rats ran y2500 m during the first 10 d (Figure 1).

Both strains increased their amount of running between the first 10-d period and the second 10-d period (p<0.05). However, the most robust difference in running behaviour was between the first 10 and the last 10 d (p<0.001), thus, during the last 10 d the FSL rats ran on average y2600 m and the FRL rats y5600 m/d (Figure 1). The FSL and the FRL rats displayed similar circadian rhythms of their running pattern indicating that a disturbance in circadian rhythms is not a factor in the differential running behaviour. That is the FRL rats had a more intense running behaviour at the times of activity (data not shown).

The FST after running The animals were subjected to a modified version of the FST in order to investigate the effect of voluntary running during 30 d on a measure of depressive-like behaviour. The modified FST version was chosen to investigate the effect of chronic treatment and also to eliminate the possibility of repeated stress exposure as a confounding factor (Caldarone et al., 2003 ; Cryan et al., 2003 ; Hall et al., 2001 ; Nowak et al., 2003 ; Zangen et al., 1999). Consistent with earlier reports (Bellido et al., 2002 ; Caberlotto et al., 1999 ; Overstreet et al., 1994 ; Schiller et al., 1992), the FSL rats were more immobile than the FRL control strain (p<0.01). Running decreased the immobility time in the FSL rats (p<0.05),

Immobility (s)

Figure 1. Running behaviour in two rat strains. Flinders Sensitive Line (FSL) rats and Flinders Resistant Line (FRL) rats, were individually housed and had free access to running-wheels for 30 d. Values are means¡S.E.M. (n=8 per group). There was a significant difference between the FRL (–&–) and the FSL (–$–) rats in total running (p<0.05). # Indicates an increase in running (distance per 10 d) from one period to another for both strains. # p<0.05, ### p<0.001.

900 800 700 600 500 400 300 200 100 0

∗∗

∗

FSL

FRL

Figure 2. Effect of free wheel-running on immobility in the Forced Swim Test. FSL and FRL rats with and without access to running-wheels for the preceding 30 d were placed in a cylinder (24 cm diameter, depth 25 cm) of water (25 xC) for 15 min, and an observer blind to the experimental condition recorded immobility time. Immobility time is presented as means¡S.E.M. * p<0.05, ** p<0.01. %, Control ; &, Run.

whereas it had no statistically significant effect in the FRL rats. Although, there was a trend to increased immobility in the FRL rats after running (p=0.074) (Figure 2).

Basal levels of dopamine receptors and neuropeptide mRNA in striatal subregions and the effect of chronic running Levels of mRNAs encoding the neuropeptides dynorphin, enkephalin, and substance P, presumably involved in depression or reported to have a putative antidepressant or dysphoric effect (Baamonde et al.,

Running is antidepressive and cell proliferative

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Table 1. Enkephalin and substance P mRNA levels after running NAcSh

LCPu

MCPu

NAcSh

NAcC

Figure 3. Quantitative computerized image analysis was performed over the indicated areas. Analyses were performed approximately at the level of Bregma 1.60 mm.

LCPu, lateral caudate putamen ; MCPu, medial caudate putamen ; NAcSh, accumbens shell ; NAcC, accumbens core.

1992 ; Holsboer, 2003 ; Kramer et al., 1998 ; Pliakas et al., 2001 ; Stengaard-Pedersen and Schou, 1985), were analysed in striatal subregions including nucleus accumbens (NAc) (Figure 3). No difference was found between FSL and FRL in basal levels of mRNAs encoding dynorphin, enkephalin, and substance P in striatal subregions (Table 1). Basal levels of the dopamine D1 and D2 receptor mRNAs also did not differ between the two strains (data not shown).

Dynorphin mRNA was increased in all striatal subregions after chronic running (Figure 4) in the FRL rats (p<0.05–0.01). In contrast, in the FSL rats dynorphin mRNA increased only in the NAc shell region and in the medial caudate putamen (p<0.01 and 0.05).

Running did not affect mRNA levels coding for the D1 and D2 receptor (data not shown), or for the other neuropeptides analysed.

Basal levels of BDNF mRNA in the hippocampus and the effect of chronic running Levels of the neurotrophic factor BDNF are increased in rats after antidepressant treatments and thus, BDNF is suggested to be important in antidepressive treatments (Altar, 1999 ; Dias et al., 2003 ; Nibuya et al., 1995). Therefore, we first analysed whether there were

Enkephalin FRL Controls Runners FSL Controls Runners Substance P FRL Controls Runners FSL Controls Runners

NAcC

79.8¡9.0 83.5¡10.0 83.2¡7.0 87.2¡7.4 76.6¡7.1 85.1¡6.2 79.6¡6.0 86.4¡5.2

MCPu

LCPu

89.2¡8.7 97.1¡8.1

86.6¡8.6 99.6¡7.9

92.4¡4.6 92.6¡3.5 104.7¡6.5 102.0¡8.0

73.2¡7.1 38.9¡3.0 82.8¡6.7 45.9¡5.6

45.0¡1.7 52.0¡5.8

74.3¡3.2 87.8¡4.5

85.7¡4.9 44.2¡7.3 93.2¡7.1 49.3¡10.3

50.7¡5.5 56.3¡6.0

84.5¡6.1 95.6¡8.3

Levels of enkephalin and substance P mRNA in ventral and dorsal striatum (represented as optical densities) were quantified and are listed as mean nCi/g¡S.E.M. FSL and FRL rats that were housed individually with no access to running-wheels (Controls) (n=8 per strain) were compared to animals that had access to running-wheels for 30 d (Runners) (n=8 per strain).

NAcSh, accumbens shell ; NAcC, accumbens core ; MCPu, medial caudate putamen ; LCPu, lateral caudate putamen.

any basal BDNF mRNA level differences in hippocampal subregions when comparing the ‘ depressed’ FSL and non-depressed FRL rats. No differences in those levels were found. Running increased levels of BDNF mRNA in the dorsal and ventral blade of the dentate gyrus in the FRL rats (p<0.01) but not in the FSL rats (Figures 5 and 6). Interestingly, BDNF levels were correlated to the activity in the running-wheels, but not to immobility in the FST. Each week of running was strongly correlated to BDNF mRNA both in the dorsal and in the ventral blade of the dentate gyrus (Figure 6 illustrates the correlation of running distance during the last week and BDNF levels in the ventral blade of the dentate gyrus, r=0.69, p<0.05).

Cell proliferation in the hippocampus and the effect of chronic running Cell proliferation and neurogenesis have recently been demonstrated to have a major role in the antidepressant effect of fluoxetine (Santarelli et al., 2003).

Therefore, to analyse the impact of cell proliferation in ‘depression ’, we first compared the basal level of cell proliferation between the FSL and FRL rat strains.

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90 80

**

FRL Control *

Dynorphin mRNA (nCi/g)

FRL Run 70 60

FSL Control FSL Run **

50 40 ** 30

*

**

20 10 0 LCPu

MCPu

NAcSh

NAcC

Figure 4. Dynorphin mRNA levels after chronic running. Upper panel shows an in-situ autoradiogram of dynorphin mRNA expression in accumbens and caudate putamen at the level of Bregma 1.60 mm. A 48-mer oligonucleotide probe specific for dynorphin mRNA was used. The panel illustrates dynorphin mRNA after free wheel-running for 30 d. Upper left and right : FRL and FSL respectively. Note the overall increase in intensity in the FRL strain in the group of runners compared to controls and the selective increases in intensity in the nucleus accumbens shell and mediate caudate putamen in the FSL strain. Lower panel shows histograms of the levels of dynorphin mRNA in FSL and FRL animals with/without access to running-wheels (n=8 per group). Values are means¡S.E.M. * p<0.05 and ** p<0.01 indicate significantly higher levels of dynorphin mRNA after running. LCPu, lateral caudate putamen ; MCPu, medial caudate putamen ; NAcSh, accumbens shell ; NAcC, accumbens core.

Newly proliferated cells were mainly found in the subgranular layer of the dentate gyrus and appeared in clusters (Figure 7a, b). The cell proliferation level was lower in the FSL strain (p<0.05), FRL rats having four times as many newly proliferating cells than FSL rats. Running increased cell proliferation in FSL rats by y450 % (p<0.05) but running had no effect in FRL rats (Figure 7c). After running, there was no difference in the level of cell proliferation between the two strains.

Discussion Physical exercise is a naturally rewarding behaviour that also has an antidepressant effect in depressed subjects under certain circumstances (Babyak et al., 2000 ; Martinsen, 1990 ; Martinsen et al., 1985 ; Strawbridge et al., 2002). In this study we investigated the effects of a 5-wk free access to running-wheels in ‘depressed ’ FSL and non-depressed FRL rats on behaviour and brain biochemistry. The aim was to

Running is antidepressive and cell proliferative (a)

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similarities to human depression and respond to chronic but not acute treatment with several antidepressants (Overstreet, 1993, 1995 ; Zangen et al., 1997).

‘Depressed ’ rats have a lower level of proliferated cells in dentate gyrus that can be normalized by running

(b)

Figure 5. In-situ hybridization of BDNF mRNA expression in the hippocampus. The autoradiogram illustrates BDNF mRNA in FRL rats after 5 wk of free wheel-running. In panel (b) note the increase in BDNF mRNA in runners in the ventral and dorsal blade of the dentate gyrus, indicated by arrows, compared to control animals (a).

identify neurobiological underpinnings of depression and the mechanisms underlying the antidepressant effect of exercise in humans.

Antidepressant effect of running in FSL rats Recently, an antidepressant property of free wheelrunning was demonstrated in a rodent model of depression, the learned helplessness model. Six weeks of free wheel-running reduced the rats responses to uncontrollable stress (Greenwood et al., 2003). However, another study that combined antidepressive treatment and free wheel-running could not detect an antidepressant potential of running in itself as measured by the FST (Russo-Neustadt et al., 2001). In the present study, free access to a running-wheel over a period of 30 d remarkably decreased immobility time in FSL rats. These results are in line with those obtained on learned-helplessness rats, and support the hypothesis that running can have an antidepressant effect.

Similar to the behaviour of the learned-helplessness animals, we could not demonstrate an antidepressant effect of running in our non-depressed strain. This result is consistent with the fact that antidepressants do not affect mood in persons who are not depressed.

The FSL rats show behavioural and neurochemical

Antidepressant treatments, including exercise, increase neurogenesis in the hippocampus in animal experiments and it has been suggested that neurogenesis is required for the antidepressant effect of drugs such as fluoxetine (Santarelli et al., 2003). Thus, it is conceivable that depressed individuals have a lower neurogenesis and that antidepressant treatments will normalize this.

‘Depressed’ rats had a lower level of proliferated cells in dentate gyrus compared to non-depressed rats and this was normalized following running. Moreover, the increase in cell proliferation was associated with a decrease in immobility in the FST. It is, therefore, possible that one underlying neurobiological cause of ‘depression ’ in the FSL strain is suppression of cell proliferation rate in the subgranular zone of the dentate gyrus. The fact that running had an antidepressant effect and increased cell proliferation further supports the idea that cell proliferation is important for both determining basal affective state and effects of antidepressive treatments. Running did not increase cell proliferation in the FRL rat strain. This result is not in accordance with earlier reports that wheel-running significantly enhances neurogenesis in rodents (Brown et al., 2003 ; Fabel et al., 2003 ; van Praag et al., 1999a).

However, these studies differ from the present study in several aspects. Most studies are performed with mice that are group housed with access to a runningwheel over a relative short period of time (between 1 and 2 wk). We have analysed rats that have been individually housed for more than 1 month. Single housing of rodents is a mild stressor (D’Arbe et al., 2002 ; Plaznik et al., 1993) that might have an impact on running behaviour, the response in the FST and possibly on cell proliferation. Moreover, single housing potentiates preference for addictive drugs such as alcohol and morphine (Hadaway et al., 1979 ; Wolffgramm, 1990). As mentioned previously running in running-wheels can be addictive for rodents and it is likely that the individually housed animals in our study have developed a more extreme, possibly compulsive, behaviour than animals from earlier studies.

The FRL rats that have a more extreme running behaviour do not show any increase in cell proliferation

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160.0

(a) ** **

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140.0

FRL control FRL run

120.0

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80.0 60.0 40.0 20.0 0.0 Ca1

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Ctx

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12000 10000 8000 6000 4000 2000 0 80

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Figure 6. BDNF mRNA levels after chronic running. (a) Histograms illustrate the levels of BDNF mRNA in FSL and FRL animals with/without access to running-wheels. Note the increased level of BDNF mRNA in the dorsal and ventral blade of dentate gyrus in FRL rats after running. Analyses was performed approximately at the level of Bregma x3.30 mm. Ca1–Ca4, fields of Ammon’s horn ; DGdb, dentate gyrus dorsal blade ; DGvb, dentate gyrus ventral blade ; Ctx, cortex ; BLA/BLP, basolateral amygdaloid nucleus anterior/-posterior. ** p<0.01, indicates significantly higher levels of BDNF mRNA after running. (b) Illustrates how BDNF mRNA levels in the dentate gyrus are correlated to running activity in the last week’s experiments (r=0.69, p<0.05).

or any positive effects in the FST after running. The FSL rats that have a lower running activity do have positive effects in the FST and an increase in cell proliferation. Thus, we suggest that running has a beneficial effect on both cell proliferation and in the FST at appropriate dosing.

BDNF levels are correlated to running but not to hippocampal cell proliferation or the antidepressant effect of running Wheel-running and other antidepressive treatments increase both cell proliferation and BDNF in the hippocampus (Malberg et al., 2000 ; Neeper et al., 1996 ;

Nibuya et al., 1995 ; Widenfalk et al., 1999). In fact, it is generally assumed that BDNF signalling plays an important role in regulating adult hippocampal neurogenesis (Lee et al., 2002), and that the effect of antidepressants is contingent on increased activity of trophic factors, such as BDNF (Altar, 1999 ; Duman, 1998). However, mice in which trkB, the receptor for BDNF, are selectively deleted in the hippocampus and the forebrain neocortex do not express more depressive-like or anxious behaviour. Instead these mice have alterations in their locomotor behaviour (Zorner et al., 2003). In our study a correlation between running distance and the level of BDNF mRNA in the dentate gyrus of the hippocampus was demonstrated. Thus,

Running is antidepressive and cell proliferative (a)

(b)

(c) BrdU-immunoreactive cells

4500 4000

# *

3500 3000 2500 2000 1500 1000 500 0

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Figure 7. Cell proliferation in hippocampus in FRL and FSL rats with/without access to running-wheels. (a) Low magnification micrograph of dark BrdU-immunoreactive cells illustrates their localization in the subgranular zone of the dentate gyrus. (b) The high magnification micrograph shows the same dark BrdU-immunoreactive cells that typically were detected in cluster. (c) Histograms illustrate that FSL rats have less proliferated cells than control FRL rats.

Running increases cell proliferation in FSL rats to about the same level as that seen in FRL rats. * p<0.05 indicates a significantly higher level of proliferating cells after running. # p<0.05 indicates a basal difference in cell proliferation level between the two strains (white bars). %, Control ; &, run.

the FRL strain that ran more than the FSL strain had a distinct BDNF mRNA increase in the dentate gyrus while the ‘ depressed’ FSL rats had unchanged BDNF levels. One possible explanation of these findings is that the antidepressant effect of running in our model is not dependent on regulation of BDNF levels in hippocampus. Whether the antidepressant effects of pharmacotherapy and electroconvulsive treatment are contingent on BDNF increase can not be answered in our study. However, mice with a deletion of the CREB gene respond similarly to desipramine and fluoxetine in the FST as wild-type mice. Interestingly, the antidepressant effect of these two antidepressants does not require an increase of BDNF in the hippocampus in CREB knockout animals (Conti et al., 2002). Together these findings support the view that BDNF regulation in the hippocampus is not required in antidepressant treatments. However, we still can not exclude a role of BDNF in other parts of the brain such as the ventral

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tegmental–NAc pathway in antidepressant treatments (Eisch et al., 2003 ; Siuciak et al., 1997). One role of BDNF in the hippocampus after excessive running could be to counteract the negative effects of a possible overtraining phenomenon in the FRL rats. The hippocampal formation is a sensitive brain region vulnerable to stressors. Stress has been demonstrated to cause atrophy of dendrites in the CA3 region (Gould et al., 1997). Exercise and also acute stress is generally beneficial for body and brain. However, cumulative amounts of exercise, as in the case of overtraining, could induce a load on the brain where the brain is at increased risk for damage (McEwen, 2003). It has previously been demonstrated that BDNF is increased in the hippocampus to give neurotrophic support following excitotoxic challenge with kainic acid (Ballarin et al., 1991 ; Zafra et al., 1990) or focal ischaemia (Lindvall et al., 1992). Thus, it is possible that the BDNF increase seen in FRL after running might be a protective mechanism for vulnerable hippocampal neurons.

Neuropeptide mRNA levels in brain-reward pathways after running mRNA levels for neuropeptides with a putative antidepressant effect and important for regulating activity in the mesolimbic brain-reward system and dopamine receptor mRNAs were analysed to search for a possible mechanism underlying anhedonia in depressive illness and the antidepressant effect of exercise. No differences in basal levels of dopamine receptors or these neuropeptides were detected between the FSL and FRL strain in dorsal striatum and accumbens.

Chronic running did not alter levels of substance P or enkephalin. However, running increased the levels of dynorphin mRNA in both strains. Interestingly, the pattern and magnitude of the changes in different striatal and accumbens subregions were different between the FSL and FRL strains.

An increased dynorphin tone is suggested to underlie a negative mood state (Pfeiffer et al., 1986 ; Pliakas et al., 2001) In the FRL rats, dynorphin levels were up-regulated after running in all subregions analysed, confirming previous reports of the effect of running on striatal dynorphin levels (Werme et al., 2000). It is probable that the dynorphin induction seen in both FSL and FRL rats is aversive, and possibly serves as a brake to prevent the rats from initiating a compulsive reinforcing running behaviour. FRL rats with higher activity in the running-wheels are closer to development of compulsive running. Thus compensatory mechanisms might be more pronounced.

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Conclusion We demonstrated that cell proliferation in the subgranular zone of the hippocampus is linked to emotional state. The ‘depressed ’ FSL rat strain’s basal cell proliferation is lower compared to the nondepressed FRL strain. Running has an antidepressant effect in the FSL strain accompanied by increased cell proliferation. No correlations between the levels of BDNF in the hippocampus or dynorphin in striatum/ accumbens and ‘ depression ’ or an antidepressant effect were detected, indicating that cell proliferation might be a better index of the antidepressant effect of running.

Acknowledgements This work was supported by the Swedish Research Council grant nos. 11642 and 10414, Centrum fo¨r idrottsforskning, the G. & Z. Costakis Swedish Foundation for medical research, the National Institute on Drug Abuse, the National Institute on Aging, and Karolinska Insitutet. We thank Dr Patricia Jime´nez Vasquez, Department of Clinical Neuroscience for expert support on FST analysis.

Statement of Interest None.

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