| Objective:Human intelligence is a complex genetic trait that rests on the interplay between a multitude of genes and certain environmental factors. Environmental factors (e.g., drugs, diet, and stress) experienced by parents can alter the development of future generations. Regular physical exercise has been demonstrated to beneficially effect neural health and function and can protect neurons from various brain insults. It has been recently reported that regular physical activity during pregnancy is beneficial to both mother and developing fetus. Studies in humans have shown that children of the mothers who were active during their pregnancy scored higher on the orientation and state regulation subscales of the Brazelton Neonatal Behavioral Assessment Scales.Maternal behavior and associated brain plasticity has been well characterized. However, little information exists about the effects of paternal exercise on cognitive brain function in offspring. It is therefore of great interest to define the transgenerational effects of paternal environmental conditions on mammals and the underlying mechanisms that may mediate these effects. We investigated whether paternal physical exercise positively influences hippocampal neurogenesis as well as memory and learning abilities of the fetal brain. Thus, we studied the effects of paternal treadmill exercise on spatial learning and memory by assessing performance in the open field and MWM tests and neuron numbers in the hippocampus of pups.Methods:(1) Animal model:Sibling males were divided into two groups:the control (C) and the exercise group (E). The mice in the running group were exercised on a motor-driven rodent treadmill for5days a week for a total of6weeks. After6weeks of exercise, one male and one female were mated. One to two days later, male mice were removed, and pregnant females were left alone. After weaning, male pups underwent behavioral assessment.(2) Training protocol:Mice randomized to CM and EM group were given exercise training on motorized treadmill at12m/min (75%VO2max) on a0%grade, training took place5days per week,60min per day, maintained for6weeks.(3) Open field test:The open field test was used to evaluate the learning and memory ability of the male pups22days after birth. The OFT is an experiment used to assay general locomotor activity levels and anxiety in rodents in scientific research. Each rat was placed in the center of the field while we recorded its behavior (moving or staying in the same area) for5min. Crossing and rearing are typical exploratory behaviors in mice.(4) Morris water maze test:The Morris water maze is one of the most widely used tasks in behavioral neuroscience for studying the psychological processes and neural mechanisms of spatial learning and memory by observing and recording of escape latency, distance moved and velocity during the time spend in the MWM water tank.(5) Nissl staining:The Nissl staining is a classic nucleic acid staining method traditionally used on nervous tissue sections. All animals were killed the morning following the last treatment day. Brain sections were dried, rehydrated, and immersed in0.1%cresyl violet for20-30minutes. After washing in water, the sections were dehydrated in graded alcohols, cleared in xylenes and cover-slipped with permount. The number of neurons was counted in hippocampal CA1, CA2, CA3, and DG regions.(6) Immunohistochemistry:IHC is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Images of positive staining in the CA1, CA2, CA3, and DG region of hippocampus were captured by an Olympus BX50microscope (Olympus, Tokyo, Japan) and the staining density was measured in integrated optical density (IOD) by Image-Pro Plus6.0.(7) Real-time PCR was used to detect BDNF and reelin mRNA level in hippcampal.(8) Western blot was used to detect BDNF and reelin protein level in hippcampal.Results:(1) Open field test:The results of the open field test showed that the number of crossing and rearing in exercise male pups were increased by31.73%(P＜0.05) and45.51%(P＜0.05) respectively comparing to CM group.(2) Morris water maze test: There were no significant differences in latency between CM and EM group on days1and2, but exercise significantly (p<0.05) decreased the latency to locate the platform by day3as compared to control male pups and remained significant on days4and5. Specifically, we found that exercise male pups had significantly shorter escape latencies on day3(44.34±2.61s, p＜0.05),4(42.97±2.64s, p<0.05), and5(32.14±2.59s, p<0.05) of MWM training as compared to control male pups (52.87±1.77s,49.78±2.01s, and45.02±2.46s, respectively). During probe trials, we found that the exercise male pups showed a clear preference for the3quadrant over control male pups, as they spent a significantly (p<0.05) greater percentage of time and path length in quadrant3(32.4±1.4%,33.2±1.3%) than control male pups (18.0±5.6%,22.8±8.7%, respectively).(3) Nissl staining demonstrated the presence of intact cells in the hippocampus of pups. The number of neurons increased CA1(47.37%, P<0.05), CA2(29.6%, P<0.05), CA3(32.19%, P＜0.05), and GD (13.49%, P＜0.05) in male pups of rats that exercised.(4) Immunohistochemistry:The paternal exercise significantly increased BDNF and reelin protein levels in the four regions (CA1, CA2, CA3, DG) of pups hippocampus (P＜0.05vs CM), which was shown as increased density.(5) BDNF mRNA and protein expression in hippocampus:There were significant increases in BDNF mRNA (42.34%, P<0.05) and protein (35.7%, P＜0.05) expression in EM group, compared with CM group.(6) reelin mRNA and protein expression in hippocampus:Expression of reelin mRNA and protein in mice of EM group were significantly increased by295.17%(p＜0.05) and124.46%(P＜0.05) respectively comparing to CM group.Conclusions:We proved the idea that paternal treadmill exercise increases hippocampal cell proliferation in the hippocampus, thereby resulting in improved spatial memory capability of male pups. This is the first study examining the effects of paternal regular aerobic exercise on the learning and memory of offspring. We also have investigated the possibility that this positive effect of paternal treadmill exercise on spatial learning and memory of male pups might be partially mediated by exercise-induced BDNF and reelin expression. The present study proved that paternal treadmill exercise may enhance the brain functions of the offspring, but further studies are needed to elucidate the underlying mechanisms by which BDNF and reelin mediated the paternal exercise-induced improvement of hippocampus functions of male pups. |
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