Effect of Exercise on Succinate Dehydrogenase Levels in the Brain

Sunjay Koshy1, under the supervision of Prof. Donna Korol2,3, in collaboration with Katherine Mitterling3
1University

Laboratory High School, 2Department of Psychology, 3Neuroscience Program, College of Liberal Arts and Sciences, University of Illinois at Urbana-Champaign

Introduction
Exercise enhances learning and memory in a variety of behavioral tasks in both human and non-human animals. Improvement on behavioral tasks by exercise has been observed; however, the molecular mechanism for this enhancement is unclear. Previous work from the Korol and Gold lab and others has shown that glucose can enhance learning and memory in humans and rodents. Exercise enhances metabolism in the periphery, suggesting that the effects of exercise on learning and memory may be due to increased metabolic efficiency. While exercise has been shown to affect metabolism in the periphery, its effects in the brain are unclear. One possible mechanism is that exercise enhances the activity of the enzymes in the mitochondrial electron transport chain, a series of proteins critical for the production of ATP. mitochondrion

Methods
Animals To determine the correlation between exercise and memory, twelve young adult male rats were used. The voluntary exercise group (exercise) had free access to running wheels for 3 weeks (n=6). The sedentary control group (sedentary) were kept in the normal home cages for the same amount of time (n=6). Tissue prep For tissue collection, the rats were perfused. First the rats were given an overdose injection of sodium pentobarbital. When they no longer had a corneal reflex, a series of incisions on the rat's underside were made and the rib cage was lifted off. A solution of 0.5% paraformaldehyde (PFA) and 10% glycerol was then pumped into the heart. The solution was pumped in for 5 minutes. The brain was then extracted and flash frozen in heptane on dry ice. The brain was stored on dry ice until the tissue was cut. Slices (40 m thick) were taken through the region of interest and mounted on slides. SDH Activity Assay The slides were warmed on a 37 degrees Celsius slide warmer and stained with a sodium succinate solution (0.55mM NBT, 0.05M Na+ succinate in 0.1M phosphate buffer). The slides were post-fixed for 3 to 4 hours in a 4% PFA solution. This staining shows levels of succinate dehydrogenase (SDH) activity in the brain slices, depending on the intensity of the staining. SDH helps in the production of ATP in the brain, and is therefore probably linked to brain activity. The slices were coverslipped and photos were taken of them. Image Analysis The gray-scale images of brain slices were analyzed using ImageJ. For each rat, two brain slices from four regions of the hippocampus (dentate gyrus hilus, corpus callosum, CA1, and CA3) were analyzed. A right and left image were taken of each slice. Using ImageJ, the area (in pixels), standard deviation of color, and mean gray value were calculated. The optical density of the slice was then calculated by using the formula: -log(transmission), where transmission equals (255-mean gray value)/255. For each rat, the optical density values for each brain region was averaged. Then the average optical density value was taken for a specific section of the brain for control group and experimental group separately. The average optical density for each hippocampal region was graphed.

Images

SDH Activity Graphs

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Results
From the graphs, it is evident that SDH activity, and therefore brain activity, in the dentate gyrus, CA1 and CA3 may be higher in exercising rats than in sedentary rats. In the corpus callosum graph , the optical density (and therefore the level of SDH activity) seem to be roughly equivalent in both the sedentary and exercising rats. The lack of difference between exercise and sedentary rats in the corpus callosum suggests that exercise does not change SDH activity in myelinated brain regions.

Dentate gyrus

Optical Density (Mean +/- SEM)

0.30 0.20 0.10 0.00 Sedentary Exercise

Conclusions
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Optical Density (Mean +/- SEM)

0.40 0.30 0.20 0.10 0.00 Sedentary Exercise

From this experiment I have found that exercise does improve levels of succinate dehydrogenase activity, which impact memory and learning capabilities.

CA3

Future Directions
Examine the interaction between estradiol and exercise on learning and SDH activity in young adult female rats Examine the effects of exercise and/or estradiol in aged rats Examine the effects of exercise and/or estradiol on other mitochondria electron transport chain enzymes

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Optical Density (Mean +/- SEM)

0.40 0.30 0.20 0.10 0.00 Sedentary Exercise

CA1

Mitochondrial electron transport chain

Acknowledgments
I would like to thank the I-STEM organizers, including Prof. Liz Destefano, my faculty supervisor Pat Morris, and the other science and math faculty at Uni High. I am grateful to Prof. Korol, Katherine Mitterling, and everyone at the Korol and Gold Labs for their excellent mentorship and guidance.

Aim
This was a two-part study on the effects of exercise and estrogen on memory. The first part was to determine the relationship between exercise and memory in young adult male Long-Evans rats. To examine the effects of exercise on learning and brain function, rats were trained on a behavioral task and the activity of succinate dehydrogenase (SDH; enzyme II in the electron transport chain) was measured. The second part of the study will determine the relationship between estradiol and learning in young adult female Long-Evans rats. The results of the study could be used to further our understanding of the human brain and how we can keep our memory from degrading.

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Corpus callosum

Optical Density (Mean +/- SEM)

0.06 0.04 0.02 0.00 Sedentary Exercise