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Hippocampus-dependent learning and memory were assessed using an Object Location Memory (OLM) task, a non-aversive spatial memory task that relies on the rodent’s innate preference for exploring the novel placement of objects44. All mice were tested in OLM during adolescence (P41–42). Sedentary and stationary wheel-housed mice had no significant differences in their OLM performances, so these groups were combined (no ELE). Mice were exposed to two identical objects for either 3-min or 10-min during the OLM acquisition phase, followed by a 5-min OLM retention test performed 24 hours later (Fig. 2a). During OLM testing one of the objects was placed in a novel location and times spent exploring familiar and novel object locations were quantified and expressed as a discrimination index (DI). All mice were habituated to the OLM chambers for three consecutive days, twice per day, and demonstrated habituation by signifcantly reducing the distance traveled across trials regardless of exercise group (Fig. 2b,c; males: main effect of habituation trial: F(3.6, 146.2) = 75.76, p < 0.0001; no effect of exercise group: F(7, 83) = 1.48, p = 0.19; group x trial interaction: F(15,203) = 2.63, p = 0.001. females: main effect of habituation trial: F(1.201, 45.38) = 8.93, p = 0.003; no effect of exercise group: F(3,40) = 0.55, p = 0.65; no group x trial interaction: F(15,189) = 0.81, p = 0.67).

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In the sub-threshold OLM design used for this study, the 3-min training exposure (acquisition phase) is a duration previously established as sub-threshold for supporting long-term memory formation in adult mice, whereas the 10-min training exposure is typically sufcient45 . Our frst experiments tested whether 10-min of training during OLM acquisition was also sufcient for long-term memory formation during adolescence. No object preference was observed in any of the groups, as indicated by average DIs close to 0 (Fig. 2d; males: F(3, 27) = 0.47, p = 0.70; females: F(3,27) = 1.93, p = 0.15). During OLM testing 24 h later, all 10-min trained groups exhibited strong preference for novel-placed objects, as demonstrated by a signifcant increase in testing DI when compared to acquisition DIs (Fig. 2e; males: main efect of OLM session: F(1,27) = 143.5, p < 0.0001; no efect of running group: F(3,27) = 2.06, p = 0.12; and no interaction: F(3,27) = 0.73, p = 0.54. females: main efect of OLM ses- sion: F(1,26) = 66.4, p < 0.0001; no efect of running group: F(3,26) = 0.78, p = 0.52; and no interaction: F(3,26) = 0.80, p = 0.50). Running exposure did not signifcantly improve memory performance during OLM testing, suggesting a possible plateau of learning reached by all groups afer being trained for 10-min the day prior (males: F(3,27) = 1.71, p = 0.19; females: F(3,26) = 0.23, p = 0.87). Further, there were no diferences in total object exploration times during OLM testing (Fig. 2f; males: F(3,27) = 0.56, p = 0.65; females: F(3,26) = 1.65, p = 0.20). Tese data suggest that performance in the OLM task can be used as a robust measure of long-term memory formation in adolescent male and female mice.

3-min of OLM training has been demonstrated to be insufficient for long-term memory forma - tion in non-exercised adults but can become sufficient if immediately preceded by exercise42,43 . Given the exercise-induced improvements in long-term spatial memory in adult mice, we sought to determine whether this phenomenon also holds true during adolescence. All groups trained for 3-min did not exhibit signifcant object preference during OLM acquisition (Fig. 2g, males: F(3, 27) = 1.17, p = 0.34; females: F(3,24) = 0.03, p = 0.99). Sedentary male and female mice had signifcantly lower DIs than 10-min trained sedentary mice, suggesting that 3-min trained, non-exercised adolescent (P42) mice do not form strong long-term memory for object location (unpaired t-tests, males: t(14) = 4.73, p = 0.0003; females: t(16) = 2.90, p = 0.01). Comparisons of corresponding OLM testing DI to acquisition DI revealed that any exposure to early-life exercise in male mice leads to signif- icant novel object location preference during testing, whereas in female mice, only the juv ELE and juv-adol ELE groups had signifcant object preference during testing when compared to training (Fig. 2h; males: no ELE: p = 0.18; juv-adol ELE: p = 0.0002; juv ELE: p < 0.0001; adol ELE: p = 0.03; females: no ELE: p = 0.15; juv-adol ELE: p = 0.002; juv ELE: p < 0.0001; adol ELE: p = 0.47). When comparing each exercise group to sedentary con- trols, male mice that underwent exercise during the juvenile period (juv ELE and juv-adol ELE) showed strong preference for novel object location when trained for 3-min (Fig. 2h; F(3, 27) = 7.78, p = 0.0007; no ELE vs juv-adol ELE: p = 0.009; no ELE vs juv ELE: p = 0.001) whereas adol ELE mice did not (no ELE vs adol ELE: p = 0.84). In females, only the juv ELE group had signifcantly greater preference for novel object location when compared to sedentary controls (Fig. 2h; F(3,22) = 5.23, p = 0.007; no ELE vs juv ELE: p = 0.01; no ELE vs juv-adol ELE: p = 0.38; no ELE vs adol ELE: p = 0.92). All groups of 3-min trained mice spent similar times exploring objects during OLM testing (Fig. 2i; males: F(3,27) = 0.16, p = 0.92; females: F(3,22) = 0.39, p = 0.76).

Finally, a three-way ANOVA was performed to determine the impact of and interactions between all nominal variables on the DIs of each group, including sex as a biological variable (total of three nominal variables: OLM training duration, ELE group, and sex; measurable variable: DI). Each individual variable was a significant source of variation (OLM training duration: F(1,102) = 13.23, p = 0.0004; sex: F(1,102) = 4.74, p = 0.03; ELE group: F(3,102) = 4.42, p = 0.006). There was a significant interaction between training duration and ELE group, supported by prior analyses (p < 0.0001); however, there were no other significant interactions (ELE group x sex, training duration x sex, ELE group x training duration x sex: all with p > 0.05). These results suggest that in both sexes, exercise taking place during a specific juvenile developmental period (P21–27) is sufficient for enhancing long-term memory for an OLM acquisition trial that is usually insufficient for long-term memory in sedentary controls, and juv ELE-induced enabling of long-term memory is present at least 2 weeks after the juvenile exercise period ends.

Synaptic plasticity and hippocampal excitability in CA1 are modulated in male mice after early-life exercise. Prior studies have confirmed that memory for object location requires an intact, functioning dorsal hippocampus46. Therefore, synaptic plasticity was examined in the CA3-CA1 Schafer collateral pathway of the hippocampus in sedentary and ELE mice, to test the hypothesis that ELE also leads to an enhancement in synaptic strength in the same region involved in enabled memory performance after juv and juv-Adol ELE. Long-term potentiation (LTP) studies were performed in acute hippocampal slices from juvenile, adolescent, and juvenile-adolescent ELE mice. Only male mice were used in electrophysiological studies. Mice were sacrificed between P42-P51. LTP was induced with a single train of 5 theta bursts to Schafer collateral inputs and extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from apical dendrites of CA1b. LTP was notably increased in juv-Adol ELE mice but not in the juv ELE or adol ELE groups (Fig. 3a; F(3,42) = 7.20, p = 0.0005). Te enhanced LTP in the juv-Adol ELE group was sustained for 50–60 minutes post-TBS (Fig. 3b; compared to sedentary: p = 0.0006). This finding is consistent with prior studies demonstrating that one week of voluntary physical activity is typically an insufficient duration of exercise for increasing LTP in adult rodents (specifically in dentate gyrus10).

Input/output curves were generated by recording fEPSP slopes in response to increasing stimulus intensities in order to test the hypothesis that ELE may enhance hippocampal synaptic transmission. Te input/output rela- tionship for fEPSP versus stimulus intensity (current) revealed a main efect of ELE group (Fig. 3c; F(3,38) = 5.93, p = 0.002), efect of stimulus intensity (F(9,342) = 444.3, p < 0.0001), and signifcant interaction (F(27,342) = 5.08, p < 0.0001). Post-hoc analysis revealed enhanced fEPSP slopes in the juv ELE and juv-adol ELE groups compared to controls, seen only at the higher stimulus intensities (between 7–10 (x10) uA; p < 0.05). Additionally, aferent fber volley amplitudes (which are considered to represent presynaptic physiological responses within the slice) were also plotted against stimulus intensity, and again demonstrated main efects of ELE group (Fig. 3d; F(3,420) = 34.07, p < 0.0001), main efect of stimulus intensity (F(9,420) = 148.2, p < 0.0001), and signifcant interaction (F(27,420) = 2.02, p = 0.002). To evaluate the relationship between the fEPSP slope and the corresponding pre- synaptic fber volley amplitude, we plotted these values against each other and conducted a linear regression

Figure 3. Early-life exercise enhances hippocampal synaptic plasticity in CA1. (a) Hippocampal slices from mice undergoing 3 weeks of exercise (just-adol ELE) had a significant increase in LTP in response to theta-burst stimulation (TBS) compared to sedentary controls, 1-week exercise in juvenile mice during P21–27 (juv ELE), and 1-week exercise in adolescent mice during P35–41 (Adol ELE). (b) This enhanced potentiation in juv-Adol ELE slices was sustained 50–60 min post-TBS. (c) I/O curve plotting EPSP slope against current generated in the Schafer collaterals showed a significant increase in fEPSP slopes in both juv ELE and juv-Adol ELE compared to adol ELE and sedentary controls. (d) I/O curve plotting EPSP slope against presynaptic fiber volleys also shows a significant effect of early-life exercise on fiber volley amplitude compared to sedentary controls. (e) I/O curve plotting the relationship between fiber volley amplitude and EPSP slope. (f) No significant differences were found between groups following paired-pulse facilitation. ***p ≤0.005.

analysis (Fig. 3e). This analysis revealed no change in the fiber volley amplitude – fEPSP slope relationship in the juv ELE group when compared to control (p > 0.05) but a significant difference was present in juv-Adol ELE (p = 0.002) and adol ELE (p < 0.0001) groups. Paired-pulse facilitation experiments were also performed to interrogate presynaptic function. Tese demonstrated no difference between groups (Fig. 3f; F(3,41) = 1.75, p = 0.171). Overall, these data suggest a significant impact of ELE on enhancing hippocampal long-term potentiation. Although this finding is in contrast to data in adult mice demonstrating lack of enhancement in CA1-LTP afer chronic voluntary wheel running9, it may implicate a specific, 1-week period of juvenile exercise that can lead to lasting changes in hippocampal CA3-CA1 circuit function and plasticity.

Discussion

We have successfully designed a mouse model of early-life voluntary exercise for the evaluation of hippocampus-dependent memory and synaptic plasticity during postnatal development. Our goal was to explore the possibility that physical exercise during early life can lead to enduring benefits in hippocampal function. To that end, our model was designed to target early life periods of hippocampal maturation to uncover temporally-specific cellular and molecular mechanisms uniquely engaged by early-life exercise. This experimental design allowed us to test the hypothesis that juvenile exercise for one week (P21–27) is sufficient to influence hippocampal function in a lasting manner. We discovered that exercise for 4th -6th postnatal weeks (juv-Adol ELE), as well as a shorter exercise experience only during the 4th postnatal week (juv ELE) enable hippocampal-dependent spatial memory formation in male mice. Tis was not true for mice that exercised only during the 6th postnatal week (adol ELE), suggesting that the 4th postnatal week of development was particularly sensitive to the exercise experience concerning enabling long-term memory function in a lasting manner. CA1-LTP was signifcantly increased in mice that exercised for 3-weeks but were not further enhanced in the 1-week juvenile nor 1-week adolescent exercisers when compared to sedentary controls. An interesting finding was that properties refecting hippocampal excitability (I/O curves) were signifcantly modulated in both groups of mice that exercised dur- ing the 4th postnatal week; furthermore, these changes endured for at least 2 weeks in the juv ELE group. This result corresponded with the lasting efect of juv ELE on enhancing memory function in OLM, which was also demonstrated two weeks afer exercise cessation. To our knowledge, this fnding has not been previously tested or described in other existing models of rodent early life or adult exercise.

All mice that exercised during the juvenile period formed long-term memory of a learning event that is typically subthreshold for long-term memory formation45. Most notably, the efect of juv ELE on long-term memory formation persisted in both male and female mice. Our data from the juv-adol ELE group is consistent with prior work demonstrating that in adult male mice, voluntary wheel running for 3-weeks enabled memory in the same hippocampus-dependent OLM paradigm42 . In that study, administration of the histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) produced effects similar to exercise on memory and maintained memory enhancements for a longer duration than exercise. Further, both exercise and NaB treatment were associated with increased expression of BDNF as well as enriched BDNF promoter H4K8 acetylation (a marker of transcriptional activation42,47). Tese results suggest that exercise enables memory formation by modifying chromatin structure, potentially to permit transcriptional activation of genes required for exercise-induced plasticity and long-term memory formation. Previous work has alluded to this concept as a “molecular memory” of exercise17,43,48. In Abel and Rissman18, 1-week of adolescent exercise (P46–52) induced several hippocampal genes related to synaptic plasticity and cell signaling (including Bdnf, Cbln1, Syn1, and Syp), increased global H3 acetylation, and down-regulated several HDAC genes9 . Given the crucial role of histone-modifying mechanisms in memory and synaptic plasticity49,50 and our novel finding that juvenile exercise can lead to persistent effects on memory function, a future direction will be to assess whether there is an underlying persistence of chromatin modica- tions that can increase the efficiency of long-term memory formation afer ELE.

Recent data suggest that adult exercise may persistently enable memory function after a brief exercise re-exposure43. Using the same OLM acquisition paradigm used in this study, a minimum of two weeks of the voluntary exercise was required for memory enhancements to persist. Also, these memory enhancements persisted for about three days afer returning to sedentary activity. 2wk exercised mice given an additional 2 day “reactivating exercise” (typically insufficient for enhancing OLM performance) within a specific temporal window (seven days, but not 14 days, afer return to sedentary activity) which could re-enhance memory performance in OLM43. This study, taken with our current findings, both highlights that timing and duration of exercise can have differing impacts on memory performance, and can reveal novel neurobiological mechanisms underlying the persistence of exercises’ effects on memory42.

OLM requires an intact dorsal hippocampus46. Enabled OLM performance in juv-Adol ELE mice comple- ments our finding of enhanced CA1-LTP in these mice. Studies focusing on the effects of exercise on LTP in hippocampal CA1 are relatively underrepresented in the literature since the funding that LTP was unchanged afer chronic voluntary wheel running in adult rats9,10, but exercise can rescue CA1-LTP impairments in the settings of stress13 or sleep deprivation14. In this study, LTP was enhanced afer theta-burst stimulation in the group of animals that underwent three weeks of exercise (just-adol ELE), but not in those mice that underwent one week of exercise (just ELE and Adol ELE groups, Fig. 3a). This suggests that during juvenile and adolescent periods, LTP enhancements afer ELE may require long-term (greater than 1-week) exercise exposure, which has been suggested previously9. LTP is considered to be the cellular correlate of learning and memory51. Teta-burst stimulation was our chosen LTP induction protocol because this form of LTP is dependent on BDNF52,53, and potentially mimics circuit firing patterns occurring during learning in rodents54 and humans55. This study also presents the novel finding that hippocampal excitability was persistently augmented afer just ELE, as refected in enhanced fEPSP slopes and fiber volley amplitudes with increasing stimulus intensity in both juv ELE and juv-Adol ELE groups. Further experiments characterizing electrophysiological properties of the hippocampal network afer juvenile exercise, particularly when tested well afer the exercise experience has ended, would shed light on how hippocampal synaptic function may be changed in an enduring manner.

ELE has similar effects on OLM performance in females in the juv ELE (but not the juv-Adol ELE) group when compared to sedentary controls. In general, voluntary aerobic physical activity in both male and female rodents during adulthood produces similar benefits in hippocampus-dependent memory tasks56. Although it has been well established that BDNF is one of the key mediators facilitating the effects of exercise on hippocampal func- tion in male mice57, there may be sex-dependent variations in BDNF splice-variants afer exercise influencing memory performance58. Estrogens have been shown to modulate structural plasticity within hippocampus59 and can enhance hippocampus-dependent memory via BDNF60. In the current study, adolescent females were not formally checked for the stage of estrous cycling, but they could have been in varying cycle stages at the time of ELE or memory testing, thereby influencing their memory performance61,62.

In this study, both male and female mice run similar cumulative distances regardless of exercise timing. Adult female mice typically run greater distances than males in voluntary running wheel paradigms63 and estrous cycling impacts running64. The lack of sex differences in distance ran may be due to cohort effects of the phase of estrous cycling. Another interesting finding was that female stationery (sta) mice gained more weight than mice that were sedentary or exercised during the adolescent period (Fig. 1b), which could reflect skeletal muscle adaptations from climbing on the wheel. More studies are needed to characterize the physiological adaptations to ELE in male and female mice and whether they impact long-term outcomes in exercise behaviors (motivation to exercise, or the rewarding effects of exercise) and memory functions.

In summary, it is well known that exercise is a highly potent, positive modulator of cognitive function in adults. Yet exercise parameters for optimal cognitive development in typically developing children, as well as cognitively impaired children, currently do not exist. Furthermore, how early life exercise may contribute to later-life cognitive functions, and the molecular mechanisms responsible for the persistent effects of ELE, is an area of research ripe for further exploration19. Because of the paucity of preclinical and clinical data on the subject, exercise guidelines for children are currently vague65. If findings in this study are relevant to humans, they implicate a lasting effect of ELE on learning and memory functions. A mechanistic understanding of ELE can be leveraged to inform exercise-based interventions for improving and preserving cognitive functions throughout the lives of children.