Article Figures & Data
Figures
- Figure 1.
Maternal care behaviors are different between EE and SD Sprague Dawley rat dams. A, Timeline of experimental procedures. B, Representative photographs of EE housing and litters. C, Representative photographs of SD housing and litters. D, Average number of pups born (male, female, and total pups) per SD and EE housing group (n = 8). E, Total time (seconds) SD and EE housed dams spent exploring P7 alien pups from different housing conditions (n = 9). Percent of time that dams spent on the nest across P1–P4 and P10 in the (F) light, (G) dark, and (H) light + dark periods combined. Total time (seconds) that dams spent on the nest collapsed across P1–P4 and P10 in the (I) light, (J) dark, and (K) light + dark periods combined. Stacked bars depict the frequency of pup directed nursing behaviors (arched back nursing, blanked/passive nursing, total nursing) collapsed across P1–P4 and P10 in the (L) light, (M) dark, and (N) light + dark periods. Stacked bars depict the frequency of other types of pup directed behaviors (licking/grooming, pup retrievals, nest building behaviors) collapsed across P1–P4 and P10 in the (O) light, (P) dark, and (Q) light + dark periods. Stacked bars depict the frequency of maternal self-directed behaviors (self-grooming, eating/drinking, tail/arm chases) collapsed across P1–P4 and P10 in the (R) light, (S) dark, and (T) light + dark periods (n = 8). Data are expressed as mean ± SEM; SD: open circles versus EE: closed circles. *p < 0.05, **p < 0.01, ***p < 0.001, SD versus EE; aap < 0.01, main effect of housing; bp < 0.05, main effect of postnatal day. See Extended Data Figure 1-1.
- Figure 2.
Nutritional profile of milk and microbiome community distribution in EE and SD Sprague Dawley rat dams. A, Photograph depictions of maternal milk collection. Maternal milk concentrations (n = 8) of (B) % creamatocrit, (C) fat (g/l), (D) energy value (kcal/l), (E) protein (mg/ml), (F) lactose (ng/μl), (G) corticosterone (ng/ml), (H) IgA (mg/ml), and (I) triglycerides (mg/dl). Microbiome sequencing (n = 6) is demonstrated with (J) α diversity along the Shannon index and (K) β diversity using principle coordinate analysis (PCoA). This plot was created using the matrix of pair-wise distance between samples determined by Bray–Curtis dissimilarity using unique amplicon sequencing variants. Each dot represents an individual microbial profile. Samples that are closer together are more similar, while samples that are dissimilar are plotted further away from one another. L, Microbial composition of taxonomy in maternal milk at the family level for SD and EE-housed dams. M, Microbiome biomarkers plot. Taxa identified as significantly more abundant in the milk of the housing group where a bar appears; SD mothers (blue bars) and EE mothers (red bars). Significance was determined by LEfSe analysis, which identified taxa with distributions that were statistically significant (p < 0.05) and where the effect size (LDA score) was >2. Data are expressed as mean ± SEM; SD: open circles versus EE: closed circles. *p < 0.05; **p < 0.01, SD versus EE; bbbp < 0.001, main effect of postnatal day. See Extended Data Figure 2-1.
- Figure 3.
Transcriptomic analyses of P10 milk samples from rat mothers living in EE or standard housing (SD). A, Volcano plot depicting the distribution of 756 genes based on log2 FC and -log10 p values. Each gray dot is a gene, and 110 dots highlighted in blue represent genes that displayed the highest magnitude of significance (padj < 0.05, FC > 1.3). Heatmaps of differentially expressed genes related specifically to milk (B) triglyceride concentration, (C), nutrient transport, (D), oxytocin signaling, (E) GR signaling, (F) GR binding, and (G) epigenetics. Gene expression is represented with the log2 transformation of counts recorded with a z score based on the average across experimental groups. Data are expressed as *p < 0.05 or **adjusted p < 0.05, with FC > 1.3. GR, glucocorticoid receptor. See Extended Data Figures 3-1 and 3-2.
- Figure 4.
Juvenile offspring physiology and behavior following housing in EE or SD laboratory conditions. Data for (left side) male and (right side) female Sprague Dawley rats for (A, B) P21 body weights (grams). C, D, Total distance traveled (centimeters), and (E, F) percent of time spent in the center of an open field. G, H, Percent of time spent in social interaction, and (I, J) latency (seconds) to approach a novel rat in a social preference test (n = 7–8). Data are expressed as mean ± SEM; aap < 0.01, aaap < 0.001, main effect of prenatal experience (SD vs EE); bp < 0.05, bbp < 0.01, main effect of postnatal experience (SD vs EE). See Extended Data Figures 4-1 and 4-2.
- Figure 5.
Summary of findings and proposed mechanisms.
Extended Data
Extended Data Figure 1-1
Maternal factors in rats housed in standard laboratory housing (SD) or EE environments. Plasma concentrations of prolactin (ng/ml; A), P10 body weights (B), and total number of eating bouts (C) from P1 to P4 and P10 during the light and dark phases for dams housed in EE or SD housing. Data are expressed as mean ± SEM; *p < 0.05, main effect of housing (SD vs EE), n = 7–8. SD (open circles) or EE (closed circles). Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Cladogram of milk biomarkers associated with standard laboratory housing (SD) or EE environments (determined by LEfSe). Diameter of the nodes indicates relative abundance of taxa for SD (green) and EE (red) samples. Placement indicates the classification of taxa, where nodes decrease in rank the closer to the center of the diagram. Download Figure 2-1, TIF file.
Extended Data Figure 3-1
Taxonomy heatmap demonstrating the microbial composition of milk samples from standard laboratory housed (SD) or EE dams at the species level with the top 50 most abundant species identified. The colored bar at the top indicates housing condition (blue = SD, red = EE). Each row represents the abundance for each taxon, with the taxonomy ID shown on the right. Each column represents the abundance for each sample. Download Figure 3-1, TIF file.
Extended Data Figure 3-2
Genes identified with DESeq analysis. List of genes that significantly differed based on a padj < 0.05 and FC > 1.3 as indicated in Figure 3A. Download Figure 3-2, DOC file.
Extended Data Figure 4-1
Heatmaps of differentially expressed genes related to glutamate/GABA and prolactin signaling in standard laboratory housed (SD) or EE dams. Gene expression is represented with the log2 transformation of counts recorded with a z-score based on the average across experimental groups. Data are expressed as *p < 0.05 and FC > 1.3, or as ***FC > 1.3 only. Download Figure 4-1, TIF file.
Extended Data Figure 4-2
Pearson correlations between maternal milk quality measures and the behavior of male and female offspring postnatally housed in standard laboratory (SD) or EE environments. Pearson correlations between distance traveled (mm2) and % creamatocrit for (A) postnatally housed SD males and (B) all females regardless of housing. C, Milk energy content (kcal/l) and total distance traveled by all females. Milk fat concentration (g/l; D), % creamatocrit (E), and milk energy content (kcal/l; F) correlations for latency to visit the novel rat by postnatally housed SD females. Data are expressed as p < 0.05. Download Figure 4-2, TIF file.
Extended Data Figure 5-1
Hypothalamic CB1 and densitometric ratios for offspring and their dams housed in standard laboratory (SD) or EE environments. Males (A), females (B), and mothers (C) housed in SD (open circles) or EE (closed circles) housing conditions. Data are expressed as mean ± SEM, n = 7–8; *p < 0.05, main effect of housing (SD vs EE). Download Figure 5-1, TIF file.
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