Genetic liability for internalizing versus externalizing behavior manifests in the developing and adult hippocampus: Insight from a meta-analysis of transcriptional profiling studies in a selectively-bred rat model

Abstract

2For over 16 years, we have selectively-bred rats to react differently to a novel, anxiety-3 inducing environment, exhibiting either high or low exploratory activity. These "bred High 4 Responder" (bHR) and "bred Low Responder" (bLR) rats serve as a general model for extreme 5 manifestations of behavioral inhibition and temperament, showing large differences in a variety 6 of internalizing and externalizing behaviors relevant to both mood and substance abuse 7 disorders. The current study elucidated persistent differences in gene expression related to 8 bHR/bLR phenotype across development (P7, P14, P21) and adulthood within the 9 hippocampus, a brain structure critical for emotional regulation. To do this, we meta-analyzed 10 eight transcriptional profiling datasets (microarray and RNA-Seq) spanning 43 generations of 11 selective breeding (n=2-6 rats per group per dataset; total n per meta-analysis: adult: n=46, P7: 12 n=22, P14: n=49, P21: n=21). By cross-referencing these results with a concurrent exome 13 sequencing study performed on our colony, we pinpointed several genes that are strongly 14 implicated in bHR/bLR behavioral phenotype, including two genes previously associated with 15 energy metabolism and mood: Thyrotropin releasing hormone receptor (Trhr) and the 16 mitochondrial protein Uncoupling protein 2 (Ucp2). Our meta-analysis also highlighted robust 17 bHR/bLR functional differences in the hippocampus, including a network essential for 18 neurodevelopmental programming, cell proliferation, and differentiation, which centered on the 19 hub genes Bone morphogenetic protein 4 (Bmp4) and the canonical Marker of proliferation 20 (Mki67). Another functional theme was microglial activation and phagocytosis, including 21 differential expression of pro-inflammatory Complement C1q A chain (C1qa) and the anti- 22inflammatory Milk fat globule-EGF factor 8 (Mfge8), situated within a chromosomal loci 23 implicated by our concurrent genetic study. Given the newly-discovered role of microglia in 24 synaptic pruning in relationship to neuronal activity during both development and adulthood, we 25 propose that these functional pathways have the capability to not only direct bHR and bLR rats 26 along a different developmental trajectory, but to set the stage for a widely-different reactivity to 27 the environment. 28 29 30 35 36 Both mood disorders and substance abuse disorders affect approximately 8-10% of 37 adults in the United States each year (1, 2). Due to high comorbidity, these disorders are often 38 classified as either internalizing and externalizing. Internalizing disorders are associated with 39 neuroticism, anxiety, and depression, whereas externalizing disorders are associated with 40 greater risk-taking and novelty-seeking, as seen in mania, substance abuse, and impulse-41 control disorders (3). This pattern of comorbidity is thought to represent a spectrum of latent 42 liability, which arises from a complex interplay of genetic risk and environmental factors, such as 43 stress and childhood adversity...

Differences in microglia morphological profiles reflect divergent emotional temperaments: Insights from a selective breeding model

“…Compared to bHRs, hippocampus samples from bLRs had higher expression levels of multiple genes within the classical complement cascade (Figure 2, Table S3). These data confirm previously described bLR elevations in the expression of the initiator of the complement cascade, C1qA ( p < 0.0001) and C1qC ( p = 0.0003) (Birt et al, 2020), and extend this pattern to include the downstream effector C3 ( p = 0.0372), as well as the complement receptor molecule cd11b ( p = 0.0021). These data implicate microglia as a source of variation that may underlie divergent behavioral traits or temperament.…”

“…Our first goal was to confirm and extend findings from the meta-analysis of hippocampal gene expression in the bHR/bLR bred lines relating to microglia signaling (Birt et al, 2020). qPCR was used to measure expression levels of multiple genes within the classical complement cascade, including cascade components C1q ( C1qA : NM_001008515; C1qC : NM_001008524) and C3 (NM_016994), and the receptor molecule cd11b (NM_012711).…”

“…One of the top microglia genes identified by this analysis, C1q, plays a critical role in microglia signaling as the initiator of the classical complement cascade (Stevens et al, 2007b; Stephan et al, 2012). The meta-analysis also showed that a genetic variant near C1qa (on chromosome 5) segregates bHR and bLR lines, providing evidence for genetic determinants of the differential expression profiles (Birt et al, 2020).…”

“…Over the fifteen years of research on bHR and bLR lines, multiple molecular pathways have been identified underlying these unique phenotypes (Ballaz et al, 2007, 2008; Perez et al, 2009; Turner et al, 2011, 2017). A recent meta-analysis compared gene expression profiles collected from 8 transcriptional datasets, spanning 43 generations of selective breeding, and found a broad range of hippocampal genes associated with breeding (Birt et al, 2020). Collectively, these studies confirm a role for the hippocampus in the behavioral phenotypes, as well as highlight novel candidate pathways to explore.…”

“…In multiple laboratory assays, bLRs display increased anxiety- and depressive-like behaviors and reduced social exploration and proactive coping, consistent within rodent models of internalizing disorders (Turner et al, 2017). Much research has focused on the neurobiology underlying these temperaments, and a recent meta-analysis of multiple transcriptional studies has identified broadly divergent patterns of gene expression within hippocampus, highlighting a range of cellular processes (Birt et al, 2020). Interestingly, microglia-related pathways emerged as one of the main functional candidates from this analysis, as the bLR phenotype is associated with elevated expression of several key genes related to microglial activation and signaling (Birt et al, 2020).…”