Article Figures & Data
Figures
- Figure 1.
Experimental design. Mice were derived from sedentary (F0SED) or exercised (F0RUN) grandfathers. The F0RUN animals were submitted to treadmill exercise of moderate intensity for 6 consecutive weeks, while the F0SED animals remained in their home cage without any access to wheel or treadmill. At the end of these 6 weeks, their sperm was collected and used to produce offspring (F1SED and F1RUN animals, respectively) via IVF methods. Both F1 groups lived until 4.5 months of age when their sperm was collected. The F1 animals remained sedentary throughout the entire period. Then, a new offspring was generated via IVF (F2SED and F2RUN animals). These litters were analyzed from 3.5 months of age when the behavioral tests were conducted. At the end of the behavioral testing, the animals were killed for histological and epigenetic analyses. Both groups (F2SED and F2RUN animals) remained sedentary throughout the entire experiment.
- Figure 2.
Behavioral analysis. a–e, Basal activity. The F2 animals (F2SED, n = 15; F2RUN, n = 8) were subjected to activity measurements in activity cages for 5 min. A scheme of the actimeter test is shown. Both groups showed similar levels of horizontal activity (a) in a novel environment (Day 1, d1) and a known environment (Day 2, d2). Also, both groups showed a significant decrease in activity in the known environment compared with the novel environment. These were also true for the vertical activity (b), total distance traveled (c), and time in movement (d). Both groups spent a similar time in the margins of the cage (e), with no significant differences between them in d1 or d2. The F2SED animals spent significantly less time in the margins on the second day of the test. f–j, NOR test. A scheme of the test is shown. Evaluation of discrimination indexes (DIs, F2SED, n = 15; F2RUN, n = 8) reports a better performance of the F2RUN animals (f). Groups did not have a preference for any object in the training phase (TR), as they had DIs proximal to zero. In the short-term phase (STM), the F2RUN animals showed a trend for a better discrimination than the F2SED animals, but only the mean DI of the F2RUN animals fulfilled the standard criteria to consider that discrimination has occurred (DI > 0.2, red line). In the long-term phase (LTM), the F2RUN animals also showed slightly higher DIs compared with the F2SED animals (not significant). F2RUN showed the trend of a higher DI compared with the training phase, while F2SED did not. Also, in the LTM phase, only the F2RUN animals fulfilled the standard criteria. The time spent exploring objects was also checked (g). The F2RUN animals showed higher levels of exploration in the LTM phase. The correlational analysis between exploration time and DIs in STM and LTM phases was performed either in all F2 animals (h) showing a positive and significant correlation (Spearman's rho; ρ = 0.597; p < 0.001) or by group (i, j). Only the F2SED animals showed a significant positive correlation between time exploring objects and DIs (i; Spearman's rho; ρ = 0.654; p < 0.001), and no correlation was found in the F2RUN animals (j; Pearson's coefficient; r = 0.276; p = 0.3). These results indicate that the higher exploratory behavior of the F2RUN animals does not explain their better performance in the NOR. An OL test was performed to check spatial pattern separation abilities (k–o). A scheme of the test is shown. Evaluation of DIs (F2SED, n = 12; F2RUN, n = 8) reports a better performance of the F2RUN animals (k). Both groups did not prefer any of the columns in the TR phase. In the test phase (TS), the F2RUN animals had significantly higher DIs than the F2SED group and fulfilled the standard criteria for discrimination. In this test, the groups showed similar levels of exploration in both phases (l). The correlational analysis did not show a positive relation between time exploring columns and DIs in this test (m). This was also true for analysis by group (n, o). CFC was applied to study memory and discrimination abilities in an emotional-aversive context (p). A scheme of the test is shown. Both groups (F2SED, n = 14, F2RUN, n = 8) had similar levels of freezing in the training phase (TR), showing similar abilities for contextual classic conditioning. They also exhibited similar levels of freezing when placed again in the conditioning chamber 24 h later (TS). For both groups, the time freezing in TS was significantly higher compared with TR, as expected. In the context change phase (CC), the animals were placed again in the conditioning chamber, but with different walls (plain white vs checkers pattern). In this phase, both groups showed context discrimination, as indicated by a significant reduction in freezing levels compared with the TS phase, although the time freezing in CC was significantly higher compared with TR in both groups, as expected. A CI (q) was calculated to summarize the cognitive performance. We can see how only F2RUNs show a CI above 0.2, illustrating a significantly higher cognitive performance than F2SED. All data are shown as mean ± SEM. For comparisons between independent groups: *p < 0.05, **p < 0.01, ***p < 0.001; *’0.05 > p < 0.099. For comparisons between dependent groups: +p < 0.05, ++p < 0.01, +++p < 0.001, +’0.05 > p < 0.09. Full statistical information can be found in the Extended Data tables (including all statistical information) labeled from Extended Data Tables 2-1 to 2-17.
- Figure 3.
Histological analysis. Immunohistochemistry against a battery of markers was used to characterize the AHN. Total cell numbers were estimated by stereological unbiased protocols (F2SED, n = 13; F2RUN, n = 8). Mean total cell numbers are shown. Neural stem cells/precursors (a, b) were analyzed by the Sox2+/GFAP+ cell number in the subgranular zone of the hippocampal dentate gyrus (a). No significant differences between experimental groups were found (b). Cell proliferation (c,d) in the dentate gyrus granule cell layer was assessed by pH3 immunohistochemistry. pH3+ cells shown in c were counted, but no significant differences between groups were found (d). The immature, differentiating cell subpopulation in the granule cell layer (e,f) showed no significant differences in DCX+/CLR−, DCX+/CLR+, and DCX−/CLR+ cell subpopulations (f, from left to right respectively). Similarly, no differences were found in the subgranular zone area (g).
- Figure 4.
smallRNA analysis. miRNAseq differential expression analysis between the F2 mice, grandsons of either sedentary or exercised grandfathers. a, Clustered heat map of differentially expressed microRNAs between the two groups (p < 0.05). Samples labeled with “R” are from grandsons of exercised grandfathers, and samples labeled with “S” are from grandsons of sedentary grandfathers. All samples have been grouped by unsupervised hierarchical clustering (Pearson's correlation) according to the expression level. Note that the CI of each mouse is depicted above the heat map. b, Volcano plot showing the global expression change between the two groups. microRNAs with a p value <0.05 and positive log2 fold change (upregulated) are represented as blue dots. microRNAs with a p value <0.05 and negative log2 fold change (downregulated) are represented as red dots. The x-axis represents the log2-transformed fold change of the microRNA expression (runner/sedentary). The y-axis is the p value (−log10-transformed). c, Gene Ontology (GO) overrepresentation analysis (ORA) on the differentially expressed miRNA gene targets. Bar plot shows the top 10 enriched GO terms with a p < 0.05. d–e, Significant negative correlation (Pearson's) plots between CI and microRNA expression levels of 144-v1_5p/144-v2_5p* (R −0.68, p < 0.01) and 298_5p (R −0.64, p = 0.025). f, PCA analysis plot showing the separation of both experimental groups.
- Figure 5.
Comparison of F2 smallRNAseq results with F0–F1 RNAseq. a, Illustration of the two different approaches taken to compare F0–F1 with F2. Approach 1 compares putative F0–F1 miRNAs (from the RNAseq GSEA) to F2 miRNA (from miRNAseq). Approach 2 compares F0–F1 enriched genes (from the RNAseq DAVID) with high-probability target genes of the F2 sDE miRNA (from miRNAseq). b, The gene sets from “Molecular Signatures Database collection MIR: microRNA targets” enriched after a GSEA analysis of F0 and F1 RNAseq were used to detect coincidences with the F2 sDE miRNAs. We found the number of genes represented in the Venn diagram for each of the generations. Eleven F2 miRNAs or their target genes in F0 and F1 appeared consistently different across three generations. This diagram shows 27 microRNAs because each sDE miRNA from Figure 4 is represented here just one time for clarity, while Figure 4 shows 35 because all the different sDE miRNA arms are represented (e.g., Fig. 4 represents both 3p and 5p arms of some miRNA when both forms have been found as sDE, while Fig. 5 represents one time that of miRNA). Venn diagram licensed by BioRender.
Tables
- Table 1.
List of F2 sDE miRNAs whose target genes are sDEGs in the RNAseq of F0 or F1 (David analysis)
miRNA Gene target Differentially expressed in Gene description 144_3p Srsf10 F1 Serine/arginine-rich splicing factor 10 144_3p Hnrnpl F1 Heterogeneous nuclear ribonucleoprotein L-like 1981_5p Vipr2 F0 Vasoactive intestinal peptide receptor 2 1981_5p Pnrc2 F1 Proline-rich nuclear receptor coactivator 2 370_3p Hnrnpl F1 Heterogeneous nuclear ribonucleoprotein L-like 544_5p Pou4f1 F0 POU domain, class 4, transcription factor 1 582_5p Irx2 F0 Iroquois homeobox 2 770_3p Zmiz2 F1 Zinc finger, MIZ-type containing 2 770_5p Chrna6 F0 Cholinergic receptor, nicotinic, alpha polypeptide 6 Six microRNAs were found. Three of them appeared with both arms coregulated, thereby 9 different forms are represented in this table.
- Table 2.
Number of miRNAs detected per animal in the miRNAseq and the number of sDE miRNAs
Sample name(s) Unique miRNAs R311_raw 508 R312_raw 525 R313_raw 524 R411_raw 506 R412_raw 518 R413_raw 517 S111_raw 497 S112_raw 518 S113_raw 511 S411_raw 525 S412_raw 537 S413_raw 520 Total miRNAs detected 513 DE miRNAs 35 %DE miRNAs 682
Table 2-1
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-1, DOCX file.
Table 2-2
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-2, DOCX file.
Table 2-3
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-3, DOCX file.
Table 2-4
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-4, DOCX file.
Table 2-5
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-5, DOCX file.
Table 2-6
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-6, DOCX file.
Table 2-7
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-7, DOCX file.
Table 2-8
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-8, DOCX file.
Table 2-9
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-9, DOCX file.
Table 2-10
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-10, DOCX file.
Table 2-11
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-11, DOCX file.
Table 2-12
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-12, DOCX file.
Table 2-13
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-13, DOCX file.
Table 2-14
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-14, DOCX file.
Table 2-15
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-15, DOCX file.
Table 2-16
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-16, DOCX file.
Table 2-17
Detailed statistical tables of all data into the figure 2 are shown, including actimeters, novel object recognition test, object location test, contextual fear conditioning, and cognitive index. Information about the animals removed, statistical analysis performed for each case, and statistical results have been listed. Download Table 2-17, DOCX file.
