Prenatal DEP/MS exposure induces social deficits in male offspring only
We characterized social behavior in offspring following prenatal exposure to either combined DEP/MS (diesel exhaust particles and maternal stress) or control (CON) conditions. No group differences were observed in maternal weight gain during pregnancy, litter size, sex ratio, or offspring body weight (Fig. S1). For complete litters, animal numbers, and statistics throughout the manuscript, see figure legends and Supplementary Tables 1 & 2, respectively).
The adolescent period – ~ postnatal day (P) 25–45 in mice – is one during which social interactions with peers are of heightened importance. Thus, we conducted behavioral testing during this period (Fig. 1a). In the sociability assay, we found that CON males showed a strong preference for a novel sex-, age-, and treatment- matched social stimulus as compared to an object, whereas DEP/MS males did not (Fig. 1b-d). This effect was male-specific as no such difference was observed in females. In the social novelty preference test, CON males showed a strong preference for a novel social stimulus over a cage mate, while DEP/MS males did not (Fig. 1e-g). To ascertain whether social deficits were driven primarily by one prenatal exposure or the other, we tested sociability in offspring following DEP or MS alone. Neither treatment on its own induced social deficits, indicating that synergism between the two is required (Fig. 1h-j). Finally, we observed no treatment effects on marble-burying (Fig. 1k&l) or anxiety-like behavior (Fig. 1m&n), suggesting that the effects of DEP/MS are specific to the social domain, at least during adolescence.
DEP/MS induces a hyper-ramified phenotype in male but not female microglia
Developmental insults have been shown to have a particularly potent impact on microglia, the resident immune cells of the brain. Microglia play a critical role in the organization of neural circuits via synaptic pruning17, trogocytosis19, and other neuronal contracts19. Gestational DEP increases microglial-neuronal interactions in cortex, and DEP/MS impairs microglial pruning of thalamocortical synapses in male offspring31,32. Therefore, we tested how DEP/MS impacts microglia in the NAc. Microglial morphology is often taken as an early indicator of alterations in microglial function. Using a MATLAB-based, semi-automatic program to quantify microglial ramification (3DMorph52), we found that NAc-microglia are hyper-ramified following DEP/MS in males but not in females (Fig. 2a). No difference was observed in microglial density between CON and DEP/MS males or females, assessed using IHC for Iba1 (quantified by cell density; Fig. 2b). To further define changes in microglial morphology in males, we used Imaris 3D image reconstruction software which revealed increased total volume of NAc-microglia following DEP/MS (Fig. 2c). Sholl analysis revealed significantly more branch endpoints and Sholl intersections in DEP/MS male microglia as compared to CON (Fig. 2d-f). In females, no treatment effects were observed on microglial volume, branch endpoints, or Sholl intersections (Fig. S2). These findings demonstrate, using multiple approaches, that male microglia are larger and hyper-ramified in the NAc following DEP/MS.
To gain a deeper understanding of how DEP/MS affects microglial function, we performed RNA sequencing of NAc-microglia. Notably, we found more differentially expressed genes (DEGs) in male microglia following DEP/MS (49; Fig. 2g) than in female microglia (8; Fig. 2h). In males, more genes were significantly up- (34) than down- (15) regulated following DEP/MS. In keeping with the hyper-ramification observed, gene set enrichment analysis (GSEA) revealed enrichment of biological and cellular pathways involved in cell motility, extracellular matrix interactions, and cell remodeling/projection assembly in male microglia following DEP/MS (Fig. 2i, Fig. S2). For example, several of the most highly and differentially transcribed genes encode proteins critical for cell motility/migration (Ahnak, Dnah9, Odad2, Togaram2, Cfap91, Cfap99), extracellular matrix interactions and cell adhesion (CD44, Antxr2, Fbln7, Lgals3), and intracellular remodeling (Lmna, Ezr, Syne3, Spag6l; Fig. 2j). Interestingly, GSEA analysis revealed a distinct set of biological pathways that were enriched in female microglia following DEP/MS, and no such changes in non-microglial cells (CD11b- population; Fig. S2).
DEP/MS alters dopamine circuitry in males but not females
Based on our microglial findings, we hypothesized that NAc-microglia might be interacting more with neuronal populations in the NAc, potentially to alter social behavior. Indeed, microglia eliminate dopamine D1 receptors (D1Rs) in the NAc (with a specific peak during adolescence [at P30]), and this D1R elimination is critical to the developmental trajectory of social play18. Dopamine, endogenous opioids, and oxytocin all mediate social motivation by acting within the NAc18,53,54. Thus, we performed tissue punching and qPCR for mRNA for dopamine receptors (Drd1 and Drd2), opioid receptors (Oprk1, Oprm1) and oxytocin receptor (Oxtr). We found that both Drd1 and Drd2 mRNA were decreased in the NAc of adolescent male offspring following DEP/MS exposure (Fig. 3a), but not in females (Fig. 3b). mRNA was not decreased for Oprm1 or Oxtr although, interestingly, Oprk1 mRNA was lower in males and higher in females (Fig. 3a&b). No changes in these receptors were observed in the amygdala (Fig. S3) – which is also a critical regulator of social behavior via connections with the NAc55.
We next asked whether decreases in D1R might be due to increased microglial phagocytosis of D1R at P30. However, we observed no differences in microglial engulfment of D1R using IMARIS 3D volumetric reconstructions of D1R and microglia (Iba1; Fig. S3). Alternatively, reduced dopaminergic input from the VTA might account for the lower expression of NAc-D1/D2Rs following DEP/MS in males. Therefore, we used immunohistochemistry to quantify tyrosine hydroxylase (Th) fiber density within the NAc as a measure of dopaminergic input. We observed a significant interaction effect whereby Th mean grey value (a measure of density) was decreased in males but increased in females following DEP/MS (Fig. 3c&d). These data show that decreased dopamine receptor expression in DEP/MS males coincides with a decrease, albeit small, in dopamine input from the VTA into the NAc.
Chemogenetic activation of the dopamine system rescues male social deficits following DEP/MS
Given the reduced Th-fiber density in the NAc in male offspring following DEP/MS, we tested whether chemogenetic activation of the dopamine system would rescue social deficits in male offspring following DEP/MS. A Dat-Ires-Cre mouse line was used to generate offspring expressing Cre under the control of the dopamine transporter 1 (DAT) promotor (Fig. 3e). Cre + offspring were exposed to either CON or DEP/MS prenatally, and subsequently underwent stereotaxic viral transfection (P24-25) and social behavior testing (during adolescence; Fig. 3f-g). We found that CON males transfected with the control virus and treated with CNO showed a significant preference for the social stimulus, as we have previously observed (Fig. 3h). As predicted, DEP/MS males transduced with the control virus and treated with CNO displayed no social preference (Fig. 3h). In contrast, DEP/MS males transduced with the excitatory DREADD receptor showed a reinstatement of their preference for a social stimulus following CNO administration (Fig. 3h). Both CON males transduced with a control virus and DEP/MS males transduced with the excitatory DREADD virus spent significantly more time investigating the social stimulus as compared to an object, while DEP/MS males transduced with the control virus spent equal amounts of time investigating the animal and the object (Fig. 3i). Together, these findings suggest that activating VTA-dopamine neurons is sufficient to restore sociability following prenatal DEP/MS in males.
DEP/MS shifts the composition of the gut microbiome and epithelium
Our results so far demonstrate male-specific changes in social behavior, microglial hyper-ramification, and decreased dopaminergic tone within the NAc following DEP/MS. Importantly, changes in all these endpoints are observed following gut microbiome manipulation. In multiple ASD mouse models, supplementation with L. reuteri rescues social behavior deficits by modulating activity of the dopamine system27,28. Microglia are also exquisitely sensitive to gut microbiota25,26. Microglial hyper-ramification, very similar to that observed in DEP/MS males, is reported in germ-free mice25.
Based on these findings, we asked whether DEP/MS impacts the gut microbiome in offspring using bacterial 16S sequencing of cecal contents in offspring during early adulthood (P45). Measures of alpha diversity quantify community richness (how many bacterial taxa are present) or evenness (how evenly abundant the taxa are that form the community) within the gut microbiome of an individual animal. Community evenness was significantly increased in DEP/MS males as compared to CON (Pielou’s evenness; Fig. 4a). Principal Coordinate Analysis (PCoA) of quantitative beta diversity indices revealed distinct clustering of microbiome profiles in CON and DEP/MS males (Fig. 4b). Significant differences were also observed in the phylogenetic relatedness and abundance of microbial communities between CON and DEP/MS males (Permutational multivariate analysis of variance [PERMANOVA]). Differences were not evident at the level of individual taxa. Notably, no changes in either alpha or beta diversity were found in female offspring (Fig. 4c&d).
Microbes within the gut interface directly with the intestinal epithelium and are important determinants of epithelial structure and immunity. The tight-junction proteins Occludin (Ocln) and Zonula occludins-1 (Zo1) stabilize the gap junctions between epithelial cells. Gastrointestinal dysfunction and evidence for disruption of the gut epithelial barrier – including changes in Ocln and Zo1 expression – are reported in patients with ASD56,57. We observed a sex-specific effect of DEP/MS (decreased in males but increased in females) on Ocln and Zo1 mRNA in the ileum (Fig. 4e-g) and duodenum (Fig. S4), but not the colon (Fig. S4). Constipation and diarrhea are predominant components of GI dysfunction in ASD56. Oprm1 mRNA – a critical regulator of gut motility - was reduced following DEP/MS (Fig. 4h). Interestingly, we observed no changes in the proinflammatory genes Tlr4, Tnfα, or Il-1β in either the ileum (Fig. 4i-k) or colon (Fig. S4), suggesting that these effects are not due to current inflammation, per se. The structure of the intestinal epithelium itself is also sensitive to microbial composition58,59,60. Villi length (Fig. 4l-m) and mucosal thickness (Fig. 4n), but not crypt length (Fig. 4o), were increased following DEP/MS exposure. Together, these findings demonstrate pervasive, male-biased changes in the microbiome and intestinal epithelium following DEP/MS – suggestive of decreased gut barrier function specifically in male offspring.
DEP/MS shifts microglial gene expression towards a germ-free phenotype
The DEP/MS-induced hyper-ramification in microglia that we observe following DEP/MS is like that observed in germ-free mice25. This led us to ask whether microglial gene expression also changes in similar ways following DEP/MS and other microbial disruptions. We used stratified Rank-Rank Hypergeometric Overlap (RRHO) analysis61 to compare gene expression changes between two datasets: gene differentials in male microglia following DEP/MS vs. CON (see Fig. 2) to gene differentials in microglia from germ-free vs. conventionally housed male mice in a published dataset26. We observed significant concordance between genes that are differentially transcribed following DEP/MS and those that are altered in germ-free microglia (Fig. 5a). We also compared DEP/MS microglial gene differentials to gene differentials following acute immune activation (2h after lipopolysaccharide: LPS at P6035, Fig. 5b) and across typical development35 (Fig. 5c). Interestingly, we found the opposite pattern (significant discordance) in both comparisons. These findings are in line with the idea that germ-free microglia are immune-incompetent and immature and suggests that a similar phenotype is induced by DEP/MS exposure in male microglia.
Cross-fostering at birth prevents DEP/MS-induced social deficits in male offspring
Given the established link between gut microbiota and social behavior62 we tested whether shifting the gut microbiome towards a CON-typical composition would prevent social deficits in DEP/MS male offspring. Cross-fostering on the day of birth shifts the composition of the offspring gut microbiome towards that of the foster mother63–65. DEP/MS exposed pups were fostered to either a different DEP/MS dam on the day of birth (D → fD) or to a CON dam (D→ fC). Similarly, CON exposed pups were fostered to a different CON dam (C → fC) or to a DEP/MS dam (C→ fD). Offspring were then tested on social behavior assays during adolescence prior to sacrifice and sample collection for microbiome and gut analyses (Fig. 5d). We also assessed maternal behavior to rule out that changes we observed were due to differences in maternal care. We found no differences between DEP/MS and CON dams in time spent on the nest nursing (Fig. 5e) or in licking and grooming behaviors (Fig. 5f; Fig. S5).
To verify that cross-fostering of DEP/MS pups to a CON dam shifted the gut microbiome towards a CON-typical phenotype, we used 16S sequencing of cecal contents at P45. Indeed, alpha diversity differed significantly with foster condition (Pielou’s Evenness, Fig. 5g). D→ fC males had significantly higher evenness than D → fD males (Fig. 5g) that did not differ from C→ fC males. Furthermore, PERMANOVA analysis revealed divergent microbial community structure between D → fD, D→ fC, and C→ fC males in all four beta diversity indices (Fig. 5h, Bray-Curtis, Jaccard dissimilarity, unweighted and weighted UniFrac). The microbiome of D → fD males differed significantly from that of C→ fC, and D→ fC males. Linear discrimination analysis effect size (LEfSe) identified several genera of bacteria that differed between D → fD and D→ fC male offspring (Fig. 5i-j). Among these, Helicobacter, Bacteroides and Parabacteroides were more abundant in D → fD male offspring, while Lachnospiraceae, and Oscillospira were more abundant in D→ fC male offspring. Helicobacter pylori (H. pylori) has been implicated in gut inflammation and acute gastritis66,67. Both Parabacteroides and Bacteroides are differentially abundant in the gut microbiome of human patients with ASD68,69. Neither tight junction protein mRNA nor villi length/mucosal thickness differed between D → fD and D→ fC males in the ileum at P45 (Fig. S6). We also conducted metabolomic analysis of short chain fatty acids (SCFA) to determine whether bacterial metabolites were influenced by our cross-fostering manipulation. We found that 6 SCFAs including acetate and butyrate were increased in D → fD males as compared to C→ fC, and that this increase was abolished in D→ fC males (Fig. 5k).
Newborn offspring acquire microbes from their mother during rearing in the home cage. We hypothesized that DEP/MS-induced changes in the gut microbiome were due to differential transmission of maternal microbes. To our surprise, we found no differences between DEP/MS or CON dams at any timepoint (Fig. S7). Furthermore, there were no differences in the vaginal or milk microbiomes (Fig. S7). These findings point to the intriguing possibility that non-microbial constituents of maternal milk may carry the signal that leads to microbiome restoration in DEP/MS pups fostered to a CON dam, an exciting avenue for future studies.
Next, we tested whether cross-fostering to a CON dam could rescue sociability in DEP/MS-exposed male offspring. As expected, D → fD offspring displayed no preference for a social stimulus (Fig. 5l). However, D→fC males showed significantly higher sociability (Fig. 5m) and spent more time in social investigation as compared to D → fD offspring (Fig. 5l). In the social novelty preference test, D→fC males spent significantly more time in total social investigation as compared to D → fD males, but there was no significant effect on social novelty preference, per se (Fig. S8). This finding may suggest that shifting the gut microbiome increases social motivation across assays, rather than choice of social partner. We also compared the sociability of CON-exposed males fostered to DEP/MS dams (C→ fD) to that of CON-exposed males fostered to CON dams (C→ fC). We found no difference in sociability between these groups, suggesting that cross fostering is insufficient to induce a DEP/MS behavioral phenotype on its own (Fig. S8). In sum, these findings demonstrate that intervening at the level of the microbiome can ameliorate social deficits in male offspring following DEP/MS.
Cross-fostering at birth prevents DEP/MS-induced microglial hyper-ramification, but does not affect the dopamine system, in male offspring
Finally, we asked whether cross-fostering to a CON dam prevented the microglial and dopaminergic phenotypes we observed in DEP/MS-exposed male offspring. Using Imaris 3D image reconstruction, we observed significant main effects of treatment on microglial volume (Fig. 6a), branch endpoints (Fig. 6b) and Sholl intersections (Fig. 6c). D→ fC males had significantly smaller and less ramified microglia as compared to D → fD males (Fig. 6d), indicating that microbial intervention at birth prevents microglial, as well as social, alterations. We also conducted RNA sequencing of NAc-microglia isolated from D → fD and D→ fC males. Gene expression differed dramatically between these two conditions, with many microglial genes both up- and down-regulated following cross-fostering to a CON dam at birth (Fig. 6e). Interestingly, these genes were not the same gene sets that differed between CON and DEP/MS males in our previous assessment. Rather, genes such as Ccrl2, Cxcl9, Mpo, and Cstdc2 were the most up or down regulated (Fig. 6f). These changes suggest, not surprisingly, that a distinct transcriptional profile is associated with returning microglia to a CON morphological phenotype following DEP/MS (D→ fC ) compared to those that differed between CON and DEP/MS independent of cross-fostering. Thus, cross-fostering, on its own, likely shifted the microglial transcriptome. We also assessed D1 and D2 receptor mRNA within the NAc, as well as Th cell number within the VTA, to determine whether cross-fostering to a CON dam prevented the changes we observed in the dopamine system. Interestingly, we found no difference between D → fD and D→ fC males in D1R mRNA (Fig. 6g), D2R mRNA (Fig. 6h), or Th cell number (Fig. 6i&j).