Using mice from different breeding sites fails to improve replicability of results from single-laboratory studies.

Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here we report a multi-laboratory study aimed at investigating whether heterogenization of study populations by using animals from different breeding sites increases the replicability of results from single-laboratory studies. We used male C57BL/6J mice from six different breeding sites to test a standardized against a heterogenized (HET) study design in six independent replicate test laboratories. For the standardized design, each laboratory ordered mice from a single breeding site (each laboratory from a different one), while for the HET design, each laboratory ordered proportionate numbers of mice from the five remaining breeding sites. To test our hypothesis, we assessed 14 outcome variables, including body weight, behavioral measures obtained from a single session on an elevated plus maze, and clinical blood parameters. Both breeding site and test laboratory affected variation in outcome variables, but the effect of test laboratory was more pronounced for most outcome variables. Moreover, heterogenization of study populations by breeding site (HET) did not reduce variation in outcome variables between test laboratories, which was most likely due to the fact that breeding site had only little effect on variation in outcome variables, thereby limiting the scope for HET to reduce between-lab variation. We conclude that heterogenization of study populations by breeding site has limited capacity for improving the replicability of results from single-laboratory animal studies.

EXPLORE: a novel deep learning-based analysis method for exploration behaviour in object recognition tests.

Object recognition tests are widely used in neuroscience to assess memory function in rodents. Despite the experimental simplicity of the task, the interpretation of behavioural features that are counted as object exploration can be complicated. Thus, object exploration is often analysed by manual scoring, which is time-consuming and variable across researchers. Current software using tracking points often lacks precision in capturing complex ethological behaviour. Switching or losing tracking points can bias outcome measures. To overcome these limitations we developed "EXPLORE", a simple, ready-to use and open source pipeline. EXPLORE consists of a convolutional neural network trained in a supervised manner, that extracts features from images and classifies behaviour of rodents near a presented object. EXPLORE achieves human-level accuracy in identifying and scoring exploration behaviour and outperforms commercial software with higher precision, higher versatility and lower time investment, in particular in complex situations. By labeling the respective training data set, users decide by themselves, which types of animal interactions on objects are in- or excluded, ensuring a precise analysis of exploration behaviour. A set of graphical user interfaces (GUIs) provides a beginning-to-end analysis of object recognition tests, accelerating a fast and reproducible data analysis without the need of expertise in programming or deep learning.

Paternal transmission of behavioural and metabolic traits induced by postnatal stress to the 5th generation in mice.

Life experiences and environmental conditions in childhood can change the physiology and behaviour of exposed individuals and, in some cases, of their offspring. In rodent models, stress/trauma, poor diet, and endocrine disruptors in a parent have been shown to cause phenotypes in the direct progeny, suggesting intergenerational inheritance. A few models also examined transmission to further offspring and suggested transgenerational inheritance, but such multigenerational inheritance is not well characterized. Our previous work on a mouse model of early postnatal stress showed that behaviour and metabolism are altered in the offspring of exposed males up to the 4th generation in the patriline and up to the 2nd generation in the matriline. The present study examined if symptoms can be transmitted beyond the 4th generation in the patriline. Analyses of the 5th and 6th generations of mice revealed that altered risk-taking and glucose regulation caused by postnatal stress are still manifested in the 5th generation but are attenuated in the 6th generation. Some of the symptoms are expressed in both males and females, but some are sex-dependent and sometimes opposite. These results indicate that postnatal trauma can affect behaviour and metabolism over many generations, suggesting epigenetic mechanisms of transmission.

Long-term effects of early postnatal stress on Sertoli cells.

Sertoli cells are somatic cells in testis essential for spermatogenesis, that support the development, maturation, and differentiation of germ cells. Sertoli cells are metabolically highly active and physiologically regulated by external signals, particularly factors in the blood stream. In disease conditions, circulating pathological signals may affect Sertoli cells and consequentially, alter germ cells and fertility. While the effects of stress on reproductive cells have been well studied, how Sertoli cells respond to stress remains poorly characterized. We used a mouse model of early postnatal stress to assess the effects of stress on Sertoli cells. We developed an improved strategy based on intracellular stainings and obtained enriched preparations of Sertoli cells from exposed males. We show that adult Sertoli cells have impaired electron transport chain (ETC) pathways and that several components of ETC complexes particularly complex I, III, and IV are persistently affected. We identify serum as potential mediator of the effects of stress on Sertoli cells by showing that it can recapitulate ETC alterations in primary cells. These results highlight Sertoli cells as cellular targets of stress in early life that can keep a trace of exposure until adulthood.

Early life stress affects the miRNA cargo of epididymal extracellular vesicles in mouse†.

Sperm RNA can be modified by environmental factors and has been implicated in communicating signals about changes in a father's environment to the offspring. The small RNA composition of sperm could be changed during its final stage of maturation in the epididymis by extracellular vesicles (EVs) released by epididymal cells. We studied the effect of exposure to stress in early postnatal life on the transcriptome of epididymal EVs using a mouse model of transgenerational transmission. We found that the small RNA signature of epididymal EVs, particularly miRNAs, is altered in adult males exposed to postnatal stress. In some cases, these miRNA changes correlate with differences in the expression of their target genes in sperm and zygotes generated from that sperm. These results suggest that stressful experiences in early life can have persistent biological effects on the male reproductive tract that may in part be responsible for the transmission of the effects of exposure to the offspring.

Involvement of circulating factors in the transmission of paternal experiences through the germline.

Environmental factors can change phenotypes in exposed individuals and offspring and involve the germline, likely via biological signals in the periphery that communicate with germ cells. Here, using a mouse model of paternal exposure to traumatic stress, we identify circulating factors involving peroxisome proliferator-activated receptor (PPAR) pathways in the effects of exposure to the germline. We show that exposure alters metabolic functions and pathways, particularly lipid-derived metabolites, in exposed fathers and their offspring. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed males into controls recapitulates metabolic phenotypes in the offspring. We identify lipid-activated nuclear receptors PPARs as potential mediators of the effects from father to offspring. Pharmacological PPAR activation in vivo reproduces metabolic dysfunctions in the offspring and grand-offspring of injected males and affects the sperm transcriptome in fathers and sons. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.

Transgenerational inheritance of behavioral and metabolic effects of paternal exposure to traumatic stress in early postnatal life: evidence in the 4th generation.

In the past decades, evidence supporting the transmission of acquired traits across generations has reshaped the field of genetics and the understanding of disease susceptibility. In humans, pioneer studies showed that exposure to famine, endocrine disruptors or trauma can affect descendants, and has led to a paradigm shift in thinking about heredity. Studies in humans have however been limited by the low number of successive generations, the different conditions that can be examined, and the lack of mechanistic insight they can provide. Animal models have been instrumental to circumvent these limitations and allowed studies on the mechanisms of inheritance of environmentally induced traits across generations in controlled and reproducible settings. However, most models available today are only intergenerational and do not demonstrate transmission beyond the direct offspring of exposed individuals. Here, we report transgenerational transmission of behavioral and metabolic phenotypes up to the 4th generation in a mouse model of paternal postnatal trauma (MSUS). Based on large animal numbers (up to 124 per group) from several independent breedings conducted 10 years apart by different experimenters, we show that depressive-like behaviors are transmitted to the offspring until the third generation, and risk-taking and glucose dysregulation until the fourth generation via males. The symptoms are consistent and reproducible, and persist with similar severity across generations. These results provide strong evidence that adverse conditions in early postnatal life can have transgenerational effects, and highlight the validity of MSUS as a solid model of transgenerational epigenetic inheritance.

Potential of Environmental Enrichment to Prevent Transgenerational Effects of Paternal Trauma.

Adverse experiences in early life are risk factors for the development of behavioral and physiological symptoms that can lead to psychiatric and cognitive disorders later in life. Some of these symptoms can be transmitted to the offspring, in some cases by non-genomic mechanisms involving germ cells. Using a mouse model of unpredictable maternal separation and maternal stress, we show that postnatal trauma alters coping behaviors in adverse conditions in exposed males when adult and in their adult male progeny. The behavioral changes are accompanied by increased glucocorticoid receptor (GR) expression and decreased DNA methylation of the GR promoter in the hippocampus. DNA methylation is also decreased in sperm cells of exposed males when adult. Transgenerational transmission of behavioral symptoms is prevented by paternal environmental enrichment, an effect associated with the reversal of alterations in GR gene expression and DNA methylation in the hippocampus of the male offspring. These findings highlight the influence of both negative and positive environmental factors on behavior across generations and the plasticity of the epigenome across life.

Rapid stress-induced transcriptomic changes in the brain depend on beta-adrenergic signaling.

Acute exposure to stressful experiences can rapidly increase anxiety and cause neuropsychiatric disorders. The effects of stress result in part from the release of neurotransmitters and hormones, which regulate gene expression in different brain regions. The fast neuroendocrine response to stress is largely mediated by norepinephrine (NE) and corticotropin releasing hormone (CRH), followed by a slower and more sustained release of corticosterone. While corticosterone is an important regulator of gene expression, it is not clear which stress-signals contribute to the rapid regulation of gene expression observed immediately after stress exposure. Here, we demonstrate in mice that 45 min after an acute swim stress challenge, large changes in gene expression occur across the transcriptome in the hippocampus, a region sensitive to the effects of stress. We identify multiple candidate genes that are rapidly and transiently altered in both males and females. Using a pharmacological approach, we show that most of these rapidly induced genes are regulated by NE through ß-adrenergic receptor signaling. We find that CRH and corticosterone can also contribute to rapid changes in gene expression, although these effects appear to be restricted to fewer genes. These results newly reveal a widespread impact of NE on the transcriptome and identify novel genes associated with stress and adrenergic signaling.

Hippocampal gene expression induced by cold swim stress depends on sex and handling.

Stress-related disorders such as PTSD and depression are more prevalent in women than men. One reason for such discordance may be that brain regions involved in stress responses are more sensitive to stress in females. Here, we compared the effects of acute stress on gene transcription in the hippocampus of female and male mice, and also examined the involvement of two key stress-related hormones, corticosterone and corticotropin releasing hormone (Crh). Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), we measured gene expression of Fos, Per1 and Sgk1 45 min after exposure to brief cold swim stress. Stress induced a stronger increase in Fos and Per1 expression in females than males. The handling control procedure increased Fos in both sexes, but occluded the effects of stress in males. Further, handling increased Per1 only in males. Sgk1 was insensitive to handling, and increased in response to stress similarly in males and females. The transcriptional changes observed after swim stress were not mimicked by corticosterone injections, and the stress-induced increase in Fos, Per1 and Sgk1 could neither be prevented by pharmacologically blocking glucocorticoid receptor (GR) nor by blocking Crh receptor 1 (Crhr1) before stress exposure. Finally, we demonstrate that the effects are stressor-specific, as the expression of target genes could not be increased by brief restraint stress in either sex. In summary, we find strong effects of acute swim stress on hippocampal gene expression, complex interactions between handling and sex, and a remarkably unique response pattern for each gene. Overall, females respond to a cold swim challenge with stronger hippocampal gene transcription than males, independent of two classic mediators of the stress response, corticosterone and Crh. These findings may have important implications for understanding the higher vulnerability of women to certain stress-related neuropsychiatric diseases.