Part 3:A Unique Mouse Model Of Early Life Exercise Enables Hippocampal Memory And Synaptic Plasticity

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Materials and Methods

Animals. Wild-type mice were the progeny of C57Bl6J dams, originally obtained from Jackson Laboratories and further bred in our animal facilities. Mice had free access to food and water and lights were maintained on a 12 h light/dark cycle. Upon weaning on a postnatal day (P) 21, mice were housed in either standard bedding cages or cages equipped with a running wheel. Both male and female mice were used for all exercise and behavior experiments. Only male mice were used for electrophysiological studies. All behavioral testing was performed during the light cycle. Experiments were conducted according to US National Institutes of Health guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

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Running wheel paradigm. Upon weaning on P21, mice were pair-housed in either standard cages without a wheel or equipped with either a mobile or stationary stainless-steel running wheel (diameter 4.5 cm, 112 grams, Starr Life Sciences). Animals housed with running wheels had free access for predetermined durations: either one week during the juvenile period (P21–27; juv ELE), one week during adolescence (P35–41; Adol ELE), or three consecutive weeks of access (P21–41; juv-adol ELE). An additional group of mice was housed with a stationary wheel during P21–41 (sta) to control for environmental enrichment from the presence of a wheel in the cage. Each wheel was fitted with a lightweight polyurethane mesh to prevent the small limbs of weanling mice from slipping through the rungs of the wheels. Running activity was tracked via digitally monitored sensors attached to a data port and computer for real-time data acquisition (Vital View Sofware) via magnetic detection of the wheel revolutions. Each digitally monitored wheel tracked the running distance for the cage (two mice housed per cage, thus the distance traveled per cage represents the total distance ran between two mice). Revolutions were quantified by the minute daily for the duration of running and converted to distance ran per cage (km).

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Object location memory (OLM) protocol. The object location memory protocol used in this study has been adapted and modified from previous protocols44. On a postnatal day 36, mice were brought into a testing room with reduced room brightness. Mice were handled for approximately 2 min each, for a total of 5 consecutive days before the OLM training session (twice a day for the first 2 days followed by once a day for the next 3 days). Habituation sessions were 5 min, twice per day for 3 days (P38-P40) and occurred within chambers containing four unique spatial cues on each wall of the chamber (horizontal lines, black X, vertical strip, and blank wall). Habituation overlapped with the last 2 days of handling. During the training phase (P41), mice were placed into the same chambers with two identical objects and exposed to either a subthreshold (3 min) or threshold (10 min) training period; a study design informed by prior work demonstrating that 3 min of training is typically insufficient for long-term memory formation45. For mice in exercise cages, running wheels were locked immediately after OLM training to eliminate any acute effects of exercise on memory consolidation. On the testing day (P42; 24 hours afer training), one of the objects was moved to a novel location inside the chamber, and mice were then allowed to explore the objects for 5 minutes. Te times spent with each object was determined via blind analysis by hand-scoring, and values obtained on both training and testing days were converted into a Discrimination Index (DI), based on the novel location of the object during testing: [(time spent exploring novel object – time spent exploring familiar object)/(total time exploring both objects) × 100%]. There were no significant differences in memory performance between sedentary animals and those housed with a stationary wheel, so these groups were combined and represented as “no ELE” in all OLM analyses. Habituation trials were analyzed for distance traveled across sessions using ANY-maze behavioral analysis software (Stoelting Co). Mice with uneven exploration times between the two objects during the training phase (DI > 20) or who showed no decrease in total distance traveled across habituation sessions were excluded from the analysis.

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electrophysiology studies. Electrophysiology experiments were performed as previously described, and the source of these methods has been published elsewhere66. Only male mice were used for electrophysiological experiments. Briefly, male mice from all ELE groups were sacrificed between P42–51, and slices were collected from the rostral hippocampus. Coronal slices were sectioned (Leica VT1000 S) at 320um thickness and placed in an interface recording chamber with preheated (31 ± 1 °C) artificial cerebrospinal fluid (124 mM NaCl, 3 mM KCl, 1.25 mM KH2PO4, 1.5 mM MgSO4, 2.5 mM CaCl2, 26 mM NaHCO3, and 10 mM d-glucose). Slices were continuously perfused at a rate of 1.75–2 ml per min while the surface of the slices was exposed to warm, humidified 95% O2/5% CO2. Recordings began afer at least 2 h of incubation. Field excitatory postsynaptic potentials (fEPSPs) were recorded from CA1b stratum radiatum using a single glass pipette (2–3 MΩ) flled with 2 M NaCl. Stimulation pulses (0.05 Hz) were delivered to Schafer collateral-commissural projections using a bipolar stimulating electrode (twisted nichrome wire, 65 μm) positioned in CA1c. The current intensity was adjusted to obtain 50% of maximal fEPSP response. After a stable baseline was established, LTP was induced with a single train of fve theta bursts, in which each burst (four pulses at 100 Hz) was delivered 200 ms apart (i.e., at theta frequency). The stimulation intensity was not increased during TBS. Data were collected and digitized by NAC 2.0 Neurodata Acquisition System (Teta Burst). Data in the LTP figure were normalized to the last 10 min of baseline and presented as mean ± SE. Baseline measures on I/O curves, paired-pulse facilitation and LTP were analyzed using a two-way ANOVA and linear regression analysis (Graphpad Prism 8 software). Cistanche can help improve memory.

Statistical analyses. One-way ANOVAs were used to analyze the effect of the timing of early-life exercise on OLM Acquisition DIs (Fig. 2d, g), OLM Testing DIs (Fig. 2e, h) and OLM Testing total exploration times (Fig. 2f, i) within each sex. Dunnett’s post-hoc multiple comparisons were used to make specific comparisons to the “no ELE” (sedentary) group when significant ANOVA results were observed. Two-way ANOVA was used to analyze mouse weight gained during the ELE period (Fig. 1b, factors of ELE group and sex). Two-way repeated measures (RM)-ANOVAs were used to analyze daily running distance (Fig. 1c–e, factors of postnatal day and sex), habituation sessions (Fig. 2b, c) and to compare OLM acquisition DI vs testing DI for each mouse for within-group comparisons. Sidak’s post hoc tests were used to make specific comparisons when significant interactions or main effects of a running group or OLM session were observed. Additionally, a three-way ANOVA was performed for OLM Testing DIs to determine any significant interactions between three nominal variables (OLM training duration, ELE group, and sex). For rare instances that 24 h running data for a particular day were not recorded, values were imputed based on taking the average distance run of the prior three days, to perform statistical analyses. Unpaired Student’s t-tests were used to analyze total running distances for each ELE group (Fig. 1f–h), as well as comparing OLM testing DIs between 3-min and 10-min trained sedentary mice. Cumulative running distances are expressed as Means ± SD. Main effects and interactions for all ANOVAs are described in the text and figure legends, along with the specific number of animals of each sex used in each experiment. All analyses were two-tailed and required an α value of 0.05 for significance. Error bars in all figures represent SEM. For all experiments, values ±2 standard deviations from the group mean were considered outliers and were removed from analyses. All statistics were performed with GraphPad Prism 8 software. Total number of mice used in behavior experiments = 152.

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Received: 16 January 2020; Accepted: 13 May 2020;

Published: xx xx xxxx

Acknowledgments

This research was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health, under award number K12NS098482 (awarded to A.S.I.) and the UC Irvine School of Medicine, Department of Pediatrics. We would also like to thank Dr. Marcelo Wood for their scientific discussions and critical input on the project.

Author contributions

A.S.I., T.Y., S.P., F.S., and T.V. performed exercise and behavior experiments and analyzed data; E.K. performed all electrophysiology experiments, analyzed the data, and edited the manuscript; A.S.I. designed study, analyzed data, interpreted results, and wrote the manuscript.

competing interests

The authors declare no competing interests.