Chen et al 2007

1. Treadmill exercise enhances passive avoidance learning in rats: The role of down-regulated serotonin system in the limbic system Chen et al 2007

2. Introduction • Treadmill running and running wheel exercise • Voluntary wheel running could not rule out possible confounding effects of non-exercise training factors • Genetic differences between good runners and poor runners • The effects of environment enrichment • The temporal relationship between the end of running wheel exercise and the effect measurement is poor-defined (Huang et al 2006)

3. Treadmill running: a stressor to rodents? • Moderate exercise training with careful animal handling and previous familiarization can minimize the possible stress: acutely upregulate rat hippocampal BDNF gene expression and improve learning. • Strenuous exercise without familiarization reduces the BDNF mRNA expression, similar to that exposed to the immobilization stress. (Huang et al 2006) • The treadmill-running: a mandatory exercise paradigm with defined exercise intensity and duration. • improves passive avoidance (PA) retention and spatial learning.

4. Two avoidance tests • Passive avoidance: the animal has to refrain from executing a previous response, such as touching food or water, stepping down or stepping through. (Myhrer, 2003) • Active avoidance: the animal has to emit a specific response in order to escape a noxious stimulus. (Cimadevilla et al., 2000) • Passive avoidance: the resulting increase in response latency reflects the strength of the memory trace. (Cimadevilla et al., 2000)

5. Serotonin (5-HT) influences a wide range of physiological systems and behavioral functions. • 5-HT modulates cognitive processes: fear learning and memory • 5-HT1A receptors: 5-HT may impair learning and memory performance via activating 5-HT1A receptors.

6. Mainly 5-HT projections are showed in brain areas important for memory (Meneses and Perez-Garcia, 2007)

7. Regular exercise improves learning and memory capabilities. • Exercise alters the 5-HT levels in different brain regions. • Are the alterations in 5-HT metabolism or its receptors in various brain regions involved in chronic exercise-enhanced learning and memory?

8. Materials and methods • Animals: male Sprague-Dawley rats • Treadmill exercise protocol • Familiarization: 9m/min for 10 min each day for 5 days • Control and exercise groups • Exercise: a leveled treadmill at an intensity of 70% of maximal oxygen consumption for 60 min/d, 5d/week for 4 weeks, 12m/min – 15m/min • Control: placed in the treadmill without running for 10 min/d • No electric shock was applied

9. Passive avoidance apparatus • A sliding door between two compartments • A stainless-steel floor • An illuminating bulb

10. Behavioral tests • One-trail step-through passive avoidance test • Exploration for 60s each without electric shock • The rat was placed in the lighted compartment facing away from the closed door  it turned around  the door was opened  once the rat had entered the dark compartment, the door was closed and an electrical current was delivered • Latency to cross into the dark compartment was recorded. (pre-shock latency) • Retention of PA performance was tested 24h and 72h afterwards. (testing latency) • The role of 5-HT1A receptor: sc injection of 8-OH-DPAT (0.1mg/kg) or vehicle 15 min prior to PA training, PA retention latencies were tested 24 h after training.

11. Pain sensitivity test • Rule out the effect of drug-induced alteration in sensory input • Test was performed 15 min after sc. injection • Rats were individually placed on a hot plate • The latency to the first hind paw withdraw was recorded as in index of nociceptive threshold to thermal stimulation

12. Biochemical studies • Brain tissue dissection: prefrontal cortex, hippocampus, amygdala • Determination of citrate synthase activity in soleus muscles • Confirm the exercise training effect • Measurement of 5-HT and 5-HIAA by HPLC • Measurement pf 5-HT1A receptor and 5-HT2A receptor and 5-HT transporter SERT by Western blotting • Statistical analysis

13. Results • The exercise training effect • the citrate synthase enzyme activity was significantly increased: 1.96±0.01 vs. 1.77±0.02 µmol/min/g wet weight for exercise and control groups.

14. Passive avoidance performance • Pre-shock latency was not different between control and exercise groups. • 24-h testing retention latency was significantly increased in the exercise group compared to the control group.

15. No significant differences in pre-shock training latencies • Exercise significantly enhanced 24-h testing retention latencies • The exercise effect was abolished by the administration of 8-OH-DPAT • 8-OH-DPAT significantly suppressed the PA performance in both exercise and control groups. • Rule out the drug influence to the sensory input: 8-OH-DPAT did not significantly affect their pain sensitivity • The paw withdraw latency: • Saline group: 5.5±0.3s • 8-OH-DPAT group: 6.1±0.7s

16. Levels of 5-HT and 5-HIAA in different brain regions • The 5-HT level in the hippocampus was significantly reduced in the exercise group at various points after exercise. • The 5-HIAA levels in these three brain areas were not significantly affected by exercising training.

17. Protein expression in different brain regions • 5-HT receptors • The protein expression level of 5-HT1A receptor in the amygdala was significantly reduced in the exercise group at all studied time points

18. Treadmill exercise training significantly down-regulated 5-HT2A receptor in rat amygdala, but not in hippocampus or frontal cortex.

19. 5-HT transporter SERT • Exercise did not significantly alter the protein expression level of SERT in these three regions.

20. Discussion • Exercise Training can enhance aversive learning and memory, possibly due to blunted serotonergic inhibition in the brain limbic system. • The 5-HT system influenced by the exercise may modulate multiple neurotransmitter systems: cholinergic and glutamatergic etc. • Other 5-HT receptors may also modulate the cognitive function: 5-HT1B, 5-HT2. • The exercise-decreased hippocampal 5-HT levels were unlikely to be caused by changes in 5-HT uptake or degradation.

21. The impact of stressful experiences? • Unlikely • Due to the separate groups of rats for PA performance test or monitoring the 5-HT-related parameters, the treadmill familiarization, moderate exercise intensity and the absence of electric shocks.

22. References • 1 Chen et al. (2007) Treadmill exercise enhances passive avoidance learning in rats: The role of down-regulated serotonin system in the limbic system. Neurobiology of Learning and Memory. Doi:10.1016/j.nlm.2007.08.004 • 2 Huang et al. (2006) Compulsive exercise acutely upregulates rat hippocampal brain-derived neurotrophic factor. Journal of Neural Transmission. 113,803-811 • 3 Myhrer, T. (2003) Neurotransmitter systems involved in learning and memory in the rat: a meta-analysis based on studies of four behavioral tasks. Brain research reviews. 41, 268-287 • 4 Cimadevilla et al. (2000) Passive and active place avoidance as a tool of spatial memory research in rats. Journal of neuroscience methods. 192, 155-164 • 5 Meneses, A. and Perez-Garcia, G. (2007) 5-HT1A receptors and memory. Neuroscience and biobehavioral reviews . 31, 705-727