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Exposure to an acute stressor triggers a complex cascade of neurochemical events in the brain. However, deciphering their individual impact on stress-induced molecular changes remains a major challenge. Here we combine RNA-sequencing with selective pharmacological, chemogenetic and optogenetic manipulations to isolate the contribution of the locus coeruleus - noradrenaline (LN-NA) system to the acute stress response. We reveal that NA-release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via β-adrenergic receptors. For a smaller subset of these genes, we show that NA release triggered by LC stimulation is sufficient to mimic the stress-induced transcriptional response. We observe these effects in both sexes, independent of the pattern and frequency of LC activation. Using a retrograde optogenetic approach, we demonstrate that hippocampus-projecting LC neurons directly regulate hippocampal gene expression. Overall, a highly selective set of astrocyte-enriched genes emerges as key targets of LC-NA activation, most prominently several subunits of protein phosphatase 1 (Ppp1r3c,Ppp1r3d,Ppp1r3g) and type II iodothyronine deiodinase (Dio2). These results highlight the importance of astrocytic energy metabolism and thyroid hormone signaling in LC mediated hippocampal function, and offer new molecular targets for understanding LC function in health and disease.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature, and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1, and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role ofApold1(Apolipoprotein L domain containing 1) in angiogenesisin vivoandin vitro. Single-cell analyses reveal that - across tissues - the expression ofApold1is restricted to the vasculature, and thatApold1expression in endothelial cells (ECs) is highly sensitive to environmental factors. UsingApold1-/-mice, we find thatApold1is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation,Apold1-/-micedisplay dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels ofApold1,and thatApold1deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically,Apold1is activated in ECs upon growth factor stimulation as well as in hypoxia, andApold1intrinsically controls EC proliferation but not migration. Our data demonstrate thatApold1is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Noradrenaline (NA) release from the brainstem nucleus locus coeruleus (LC) changes activity and connectivity in neuronal networks across the brain, thus modulating multiple behavioural states. NA release is mediated by both tonic and burst-like neuronal LC activity. However, it remains unknown whether the functional changes in downstream projection areas depend on these firing patterns. Using optogenetics, pupillometry, photometry, and functional MRI in mice, we show that tonic and burst LC firing patterns elicit brain responses that are specific to the activation frequency and temporal pattern. Tonic activation of the LC evokes nonlinear responses in prefrontal, limbic, and cerebellar regions, in line with the proposed inverted-U relationship between LC activity and behaviour. We further demonstrate that LC activity enhances network integration and acts as a facilitator of brain state transitions, hence increasing brain flexibility. Together, these findings reveal how the LC-NA system achieves a nuanced regulation of global circuit operations.

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1-2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only similar to 300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.

Understanding how the brain’s macroscale dynamics are shaped by underlying microscale mechanisms is a key problem in neuroscience. In animal models, we can now investigate this relationship in unprecedented detail by directly manipulating cellular-level properties while measuring the whole-brain response using resting-state fMRI. Here we focused on understanding how blood-oxygen-level-dependent (BOLD) dynamics, measured within a structurally well-defined striato-thalamo-cortical circuit, are shaped by chemogenetically exciting or inhibiting D1 medium spiny neurons (MSNs) of the right dorsomedial striatum (CPdm). We characterize changes in both the BOLD dynamics of individual cortical and subcortical brain areas, and patterns of inter-regional coupling (functional connectivity) between pairs of areas. Using a classification approach based on a large and diverse set of time-series properties, we found that CPdm neuromodulation alters BOLD dynamics within thalamic subregions that project back to dorsomedial striatum. In the cortex, the strongest changes in local dynamics were observed in unimodal regions, i.e., regions that process information from a single sensory modality, while changes in the local dynamics weakened along a putative cortical hierarchical gradient towards transmodal regions. In contrast, a decrease in functional connectivity was observed only for cortico-striatal connections after D1 excitation. Our results provide a comprehensive understanding of how targeted cellular-level manipulations affect local BOLD dynamics at the macroscale, including the role of a circuit’s structural characteristics and hierarchical cortical level in shaping those dynamics. These findings contribute to ongoing attempts to understand the influence of structure–function relationships in shaping inter-regional communication at subcortical and cortical levels.

Chronic stress exposure in adolescence can lead to a lasting change in stress responsiveness later in life and is associated with increased mental health issues in adulthood. Here we investigate whether the Chronic Social Instability (CSI) paradigm influences the behavioral and molecular responses to novel acute stressors in mice, and whether it alters physiological responses influenced by the noradrenergic system. Using large cohorts of mice, we show that CSI mice display a persistent increase in exploratory behaviors in the open field test alongside small but widespread transcriptional changes in the ventral hippocampus. However, both the transcriptomic and behavioral responses to novel acute stressors are indistinguishable between groups. In addition, the pupillo-metric response to a tail shock, known to be mediated by the noradrenergic system, remains unaltered in CSI mice. Ultra-high performance liquid chromatography analysis of monoaminergic neurotransmitter levels in the ventral hippocampus also shows no differences between control or CSI mice at baseline or in response to acute stress. We conclude that CSI exposure during adolescence leads to persistent changes in exploratory behavior and gene expression in the hippocampus, but it does not alter the response to acute stress in adulthood and is unlikely to alter the function of the noradrenergic system., Neurobiology of Stress, 15, ISSN:2352-2895

International audience; Various different classes of RNAs contained in sperm cells have emerged as causal vectors for the transmission of acquired information from father to offspring. This has invigorated research and raised many new questions concerning the heritability of disease risk and the ability to adapt to novel environments. Here, we will focus on recent advances in the field of epigenetic germline inheritance in mammals, with a particular focus on the following three questions: (1) What is the current evidence for an involvement of sperm RNAs in the transmission of acquired information from father to offspring? (2) How can acquired changes in the sperm-RNA payload be induced in the male germline? (3) How can acquired changes be transferred from sperm to oocyte? We propose a novel mechanism for transfer of sperm RNA to the oocyte in a DNA/RNA-hybrid, possibly interacting with DNA-bound proteins, and suggest experiments that should advance our understanding of epigenetic germline inheritance.

Synaptic scaling is a form of homeostatic plasticity which allows neurons to adjust their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell‐wide mechanisms is controversial. Here we perform a comprehensive multi‐omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. We uncover both highly compartment‐specific and correlating changes in the neuronal transcriptome and proteome. Whereas downregulation of crucial regulators of neuronal excitability occurs primarily in the somatic compartment, structural components of excitatory postsynapses are mostly downregulated in processes. Local inhibition of protein synthesis in processes during scaling is confirmed for candidate synaptic proteins. Motif analysis further suggests an important role for trans‐acting post‐transcriptional regulators, including RNA‐binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that, during synaptic scaling, compartmentalized gene expression changes might co‐exist with neuron‐wide mechanisms to allow synaptic computation and homeostasis., Downscaling of excitatory synapses in response to high activity involves compartment‐specific changes in the neuron's transcriptome and proteome. This study supports the view that local gene regulation is important in both Hebbian and homeostatic forms of synaptic plasticity.

Chronic stress exposure in adolescence can lead to a lasting change in stress responsiveness later in life and is associated with increased mental health issues in adulthood. Here we investigate whether the Chronic Social Instability (CSI) paradigm in mice influences the behavioural and molecular responses to novel acute stressors, and whether it alters physiological responses influenced by the noradrenergic system. Using large cohorts of mice, we show that CSI mice display a persistent increase in exploratory behaviors in the open field test alongside small but widespread transcriptional changes in the ventral hippocampus. However, both the transcriptomic and behavioural responses to novel acute stressors are indistinguishable between groups. In addition, the pupillometric response to a tail shock, known to be mediated by the noradrenergic system, remains unaltered in CSI mice. Ultra-high performance liquid chromatography analysis of monoaminergic neurotransmitter levels in the ventral hippocampus also shows no differences between control or CSI mice at baseline or in response to acute stress. We conclude that CSI exposure during adolescence leads to persistent changes in exploratory behavior and gene expression in the hippocampus, but it does not alter the response to acute stress challenges in adulthood and is unlikely to alter the function of the noradrenergic system.

The acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, the underlying molecular cascades are unclear. Here, we use a brief swim exposure to trigger an acute stress response in mice, which transiently increases anxiety, without leading to lasting maladaptive changes. Using multiomic profiling, such as proteomics, phospho-proteomics, bulk mRNA-, single-nuclei mRNA-, small RNA-, and TRAP-sequencing, we characterize the acute stress-induced molecular events in the mouse hippocampus over time. Our results show the complexity and specificity of the response to acute stress, highlighting both the widespread changes in protein phosphorylation and gene transcription, and tightly regulated protein translation. The observed molecular events resolve efficiently within four hours after initiation of stress. We include an interactive app to explore the data, providing a molecular resource that can help us understand how acute stress impacts brain function in response to stress.

SummaryThe acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, little is known about the nature and dynamics of the underlying molecular cascades. We used a brief forced swim exposure to trigger a strong stress response in mice, which transiently increases anxiety, but does not lead to lasting maladaptive behavioral changes. Using multiomic profiling we characterize the stress-induced molecular events in the hippocampus over time, across molecular scales, and down to the level of cell types and single cells. Our results unveil the complexity and specificity of the healthy stress response, with widespread changes in protein phosphorylation and gene transcription, but tightly regulated protein translation. All observed molecular events resolve efficiently within 4 hours after initiation of stress. The data are accessible through an interactive web portal, creating an extensive molecular resource that will help understand how stress impacts brain function in health and disease.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor control, motor learning and executive function. Central to BG function are striatal medium spiny neurons (MSNs) expressing D1 and D2 dopamine receptors. D1 and D2 MSNs are typically considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. While their opposite role is well documented for certain sensorimotor loops of the BG-thalamocortical network, it is unclear whether MSNs maintain a uniform functional role across the striatum and which influence they exert on brain areas outside the BG. Here, we addressed these questions by combining optogenetic activation of D1 and D2 MSNs in the mouse ventrolateral caudoputamen (vl CPu) with whole-brain functional MRI (fMRI) recordings. Neuronal excitation of either cell population in the vl CPu evoked distinct activity patterns in key regions of the BG-thalamocortical network including the pallidum, thalamus and motor cortex. Importantly, we report that striatal D1 and D2 MSN stimulation differentially engaged cerebellar and prefrontal regions. We characterised these long-range interactions by computational modelling of effective connectivity and confirmed that changes in D1 / D2 output drive functional relationships between regions within and beyond the BG. These results suggest a more complex functional organization of MSNs across the striatum than previously anticipated and provide evidence for the existence of an interconnected fronto - BG - cerebellar network modulated by striatal D1 and D2 MSNs.Graphical Abstract

Summary Single traumatic events that elicit an exaggerated stress response can lead to the development of neuropsychiatric conditions. Studies in mice suggests germline RNA as a mediator of effects of chronic environmental exposures to the progeny. The effects of an acute paternal stress exposure on the germline and their potential consequences on offspring remain unknown. We find that acute administration of an agonist for the stress-sensitive Glucocorticoid receptor, using the common corticosteroid Dexamethasone, affects the RNA payload of post-meiotic mature sperm as soon as 3 hours post exposure, challenging the prevalent view that epididymal transit is required to alter sperm RNA. It further impacts early embryonic transcriptional trajectories, as determined by single embryo sequencing, and metabolism in the offspring. Importantly, we show persistent regulation of tRNA fragments in sperm and the descendant 2-cell- embryos, suggesting actual transmission from sperm to embryo. Lastly, we unravel environmentally induced alterations in the previously underconsidered class of sperm circRNAs, and their targets in the early embryo, highlighting this class as a novel candidate in RNA-mediated inheritance.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor control, motor learning and executive function. Central to BG function are striatal medium spiny neurons (MSNs) expressing D1 and D2 dopamine receptors. D1 and D2 MSNs are typically considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. While their opposite role is well documented for certain sensorimotor loops of the BG-thalamocortical network, it is unclear whether MSNs maintain a uniform functional role across the striatum and which influence they exert on brain areas outside the BG. Here, we addressed these questions by combining optogenetic activation of D1 and D2 MSNs in the mouse ventrolateral caudoputamen (vl CPu) with whole-brain functional MRI (fMRI) recordings. Neuronal excitation of either cell population in the vl CPu evoked distinct activity patterns in key regions of the BG-thalamocortical network including the pallidum, thalamus and motor cortex. Importantly, we report that striatal D1 and D2 MSN stimulation differentially engaged cerebellar and prefrontal regions. We characterised these long-range interactions by computational modelling of effective connectivity and confirmed that changes in D1 / D2 output drive functional relationships between regions within and beyond the BG. These results suggest a more complex functional organization of MSNs across the striatum than previously anticipated and provide evidence for the existence of an interconnected fronto - BG - cerebellar network modulated by striatal D1 and D2 MSNs.

The assessment of rodent behavior forms a cornerstone of preclinical assessment in neuroscience research. Nonetheless, the true and almost limitless potential of behavioral analysis has been inaccessible to scientists until very recently. Now, in the age of machine vision and deep learning, it is possible to extract and quantify almost infinite numbers of behavioral variables, to break behaviors down into subcategories and even into small behavioral units, syllables or motifs. However, the rapidly growing field of behavioral neuroethology is experiencing birthing pains. The community has not yet consolidated its methods, and new algorithms transfer poorly between labs. Benchmarking experiments as well as the large, well-annotated behavior datasets required are missing. Meanwhile, big data problems have started arising and we currently lack platforms for sharing large datasets—akin to sequencing repositories in genomics. Additionally, the average behavioral research lab does not have access to the latest tools to extract and analyze behavior, as their implementation requires advanced computational skills. Even so, the field is brimming with excitement and boundless opportunity. This review aims to highlight the potential of recent developments in the field of behavioral analysis, whilst trying to guide a consensus on practical issues concerning data collection and data sharing., Neuropsychopharmacology, 46 (1), ISSN:0893-133X, ISSN:1740-634X

The impact of stress on gene expression in different cell types of the brain remains poorly characterized. Three pioneering studies have recently used translating ribosome affinity purification followed by RNA sequencing (TRAP-seq) to assess the response to stress in CA3 pyramidal neurons of the hippocampus. The results suggest that acute stress alters the translation of thousands of genes in CA3 pyramidal neurons, and that this response is strongly modulated by factors such as sex, genotype and a history of early life stress. However, our reanalysis of these datasets leads to different conclusions. We confirm that acute stress induces robust translational changes in a small set of genes. However, we found no evidence that either early life stress or sex have an effect on gene translation induced by acute stress. Our findings highlight the need for additional studies with adequate sample sizes and proper methods of analysis to assess the impact of stress across cell types in the brain.

Synaptic scaling is a form of homeostatic plasticity which allows neurons to adjust their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell-wide mechanisms is controversial. Here we performed a comprehensive multi-omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. Thereby, we uncovered both highly compartment-specific and correlated changes in the neuronal transcriptome and proteome. Whereas downregulation of crucial regulators of neuronal excitability occurred primarily in the somatic compartment, structural components of excitatory postsynapses were mostly downregulated in processes. Motif analysis further suggests an important role for trans-acting post-transcriptional regulators, including RNA-binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that compartmentalized gene expression changes are widespread in synaptic scaling and might co-exist with neuron-wide mechanisms to allow synaptic computation and homeostasis.

BACKGROUND Endothelial cells regulate the formation of blood clots thus, genes selectively expressed in these cells could primarily determine thrombus formation. Apold1(apolipoprotein L domain containing 1) is a gene expressed by endothelial cells; whether Apold1 directly contributes to arterial thrombosis has not yet been investigated. Here, we assessed the effect of Apold1 deletion on arterial thrombus formation using an in vivo model of carotid thrombosis induced by photochemical injury. MATERIAL AND METHODS Apold1 knockout (Apold1-/- ) mice and wild type (WT) littermates underwent carotid thrombosis induced by photochemical injury and time to occlusion was recorded. Tissue factor (TF) activity as well as activation of mitogen-activated protein kinases (MAPKs) and phosphatidyl-inositol-3 kinase (PI3K)/Akt pathways were analyzed by colorimetric assay and western blotting in both Apold1-/- and WT mice. Finally, platelet reactivity was assessed using light transmission aggregometry. RESULTS After photochemical injury, Apold1$^{-/-}$ mice exhibited shorter time to occlusion as compared to WT mice. Moreover, TF activity was increased in carotid arteries of Apold1$^{-/-}$ when compared to WT mice. Underlying mechanistic markers such as TF mRNA and MAPKs activation were unaffected in Apold1$^{-/-}$ mice. In contrast, phosphorylation of Akt was reduced in Apold1$^{-/-}$ as compared to WT mice. Additionally, Apold1$^{-/-}$ mice displayed increased platelet reactivity to stimulation with collagen compared to WT animals. CONCLUSIONS Deficiency of Apold1 results in a prothrombotic phenotype, accompanied by increased vascular TF activity,decreased PI3K/Akt activation and increased platelet reactivity. These findings suggest Apold1 as an interesting new therapeutic target in the context of arterial thrombosis.

5-hydroxymethylation (5-hmC) is an epigenetic modification on DNA that results from the conversion of 5-methylcytosine by Ten-Eleven Translocation (TET) proteins. 5-hmC is widely present in the brain and is subjected to dynamic regulation during development and upon neuronal activity. It was recently shown to be involved in memory processes but currently, little is known about how it is controlled in the brain during memory formation. Here, we show that Tet3 is selectively up-regulated by activity in hippocampal neurons in vitro, and after formation of fear memory in the hippocampus. This is accompanied by a decrease in miR-29b expression that, through complementary sequences, regulates the level of Tet3 by preferential binding to its 3'UTR. We newly reveal that SAM68, a nuclear RNA-binding protein known to regulate splicing, acts upstream of miR-29 by modulating its biogenesis. Together, these findings identify novel players in the adult brain necessary for the regulation of 5-hmC during memory formation., Scientific Reports, 8 (1), ISSN:2045-2322

To study brain function, preclinical research relies heavily on animal monitoring and the subsequent analyses of behavior. Commercial platforms have enabled semi high-throughput behavioral analyses by providing accurate tracking of animals, yet they often struggle with the analysis of ethologically relevant behaviors and lack the flexibility to adapt to variable testing environments. In the last couple of years, substantial advances in deep learning and machine vision have given researchers the ability to take behavioral analysis entirely into their own hands. Here, we directly compare the performance of commercially available platforms (Ethovision XT14, Noldus; TSE Multi Conditioning System, TSE Systems) to cross-verified human annotation. To this end, we provide a set of videos - carefully annotated by several human raters - of three widely used behavioral tests (open field, elevated plus maze, forced swim test). Using these data, we show that by combining deep learning-based motion tracking (DeepLabCut) with simple post-analysis, we can track animals in a range of classic behavioral tests at similar or even greater accuracy than commercial behavioral solutions. In addition, we integrate the tracking data from DeepLabCut with post analysis supervised machine learning approaches. This combination allows us to score ethologically relevant behaviors with similar accuracy to humans, the current gold standard, thus outperforming commercial solutions. Moreover, the resulting machine learning approach eliminates variation both within and between human annotators. In summary, our approach helps to improve the quality and accuracy of behavioral data, outperforming commercial systems at a fraction of the cost.

To study brain function, preclinical research heavily relies on animal monitoring and the subsequent analyses of behavior. Commercial platforms have enabled semi high-throughput behavioral analyses by automating animal tracking, yet they poorly recognize ethologically relevant behaviors and lack the flexibility to be employed in variable testing environments. Critical advances based on deep-learning and machine vision over the last couple of years now enable markerless tracking of individual body parts of freely moving rodents with high precision. Here, we compare the performance of commercially available platforms (EthoVision XT14, Noldus; TSE Multi-Conditioning System, TSE Systems) to cross-verified human annotation. We provide a set of videos—carefully annotated by several human raters—of three widely used behavioral tests (open field test, elevated plus maze, forced swim test). Using these data, we then deployed the pose estimation software DeepLabCut to extract skeletal mouse representations. Using simple post-analyses, we were able to track animals based on their skeletal representation in a range of classic behavioral tests at similar or greater accuracy than commercial behavioral tracking systems. We then developed supervised machine learning classifiers that integrate the skeletal representation with the manual annotations. This new combined approach allows us to score ethologically relevant behaviors with similar accuracy to humans, the current gold standard, while outperforming commercial solutions. Finally, we show that the resulting machine learning approach eliminates variation both within and between human annotators. In summary, our approach helps to improve the quality and accuracy of behavioral data, while outperforming commercial systems at a fraction of the cost., Neuropsychopharmacology, 45, ISSN:0893-133X, ISSN:1740-634X

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1-2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain, regulates many fundamental brain functions, and is implicated in several neuropsychiatric diseases. Although selective manipulation of the LC is not possible in humans, studies have suggested that strong LC activation might shift network connectivity to favor salience processing. To test this hypothesis, we use a mouse model to study the impact of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term “chemo-connectomics”. LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. We reveal a direct correlation between functional connectivity changes and transcript levels of alpha-1, alpha-2, and beta-1 adrenoceptors across the brain, and a positive correlation between NE turnover and functional connectivity within select brain regions. These results represent the first brain-wide functional connectivity mapping in response to LC activation, and demonstrate a causal link between receptor expression, brain states and functionally connected large-scale networks at rest. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection.

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 β-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.

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.

Several studies have reported that exposure to acute psychophysiological stressors can lead to an increase in blood–brain barrier permeability, but these findings remain controversial and disputed. We thoroughly examined this issue by assessing the effect of several well-established paradigms of acute stress and chronic stress on blood–brain barrier permeability in several brain areas of adult mice. Using cerebral extraction ratio for the small molecule tracer sodium fluorescein (NaF, 376 Da) as a sensitive measure of blood–brain barrier permeability, we find that neither acute swim nor restraint stress lead to increased cerebral extraction ratio. Daily 6-h restraint stress for 21 days, a model for the severe detrimental impact of chronic stress on brain function, also does not alter cerebral extraction ratio. In contrast, we find that cold forced swim and cold restraint stress both lead to a transient, pronounced decrease of cerebral extraction ratio in hippocampus and cortex, suggesting that body temperature can be an important confounding factor in studies of blood–brain barrier permeability. To additionally assess if stress could change blood–brain barrier permeability for macromolecules, we measured cerebral extraction ratio for fluorescein isothiocyanate-dextran (70 kDa). We find that neither acute restraint nor cold swim stress affected blood–brain barrier permeability for macromolecules, thus corroborating our findings that various stressors do not increase blood–brain barrier permeability.

Stressful experiences are linked to anxiety disorders in humans. Similar effects are observed in rodent models, where anxiety is often measured in classic conflict tests such as the open-field test. Spontaneous rearing behavior, in which rodents stand on their hind legs to explore, can also be observed in this test yet is often ignored. We define two forms of rearing, supported rearing (in which the animal rears against the walls of the arena) and unsupported rearing (in which the animal rears without contacting the walls of the arena). Using an automated open-field test, we show that both rearing behaviors appear to be strongly context dependent and show clear sex differences, with females rearing less than males. We show that unsupported rearing is sensitive to acute stress, and is reduced under more averse testing conditions. Repeated testing and handling procedures lead to changes in several parameters over varying test sessions, yet unsupported rearing appears to be rather stable within a given animal. Rearing behaviors could therefore provide an additional measure of anxiety in rodents relevant for behavioral studies, as they appear to be highly sensitive to context and may be used in repeated testing designs.

Background Acutely stressful experiences can trigger neuropsychiatric disorders and impair cognitive processes by altering hippocampal function. Although the intrinsic organization of the hippocampus is highly conserved throughout its long dorsal-ventral axis, the dorsal (anterior) hippocampus mediates spatial navigation and memory formation, whereas the ventral (posterior) hippocampus is involved in emotion regulation. To understand the molecular consequences of stress, detailed genome-wide screens are necessary and need to distinguish between dorsal and ventral hippocampal regions. While transcriptomic screens have become a mainstay in basic and clinical research, proteomic methods are rapidly evolving and hold even greater promise to reveal biologically and clinically relevant biomarkers. Methods Here, we provide the first combined transcriptomic (RNA sequencing) and proteomic (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) profiling of dorsal and ventral hippocampus in mice. We used three different acute stressors (novelty, swim, and restraint) to assess the impact of stress on both regions. Results We demonstrated that both hippocampal regions display radically distinct molecular responses and that the ventral hippocampus is particularly sensitive to the effects of stress. Separately analyzing these structures greatly increased the sensitivity to detect stress-induced changes. For example, protein interaction cluster analyses revealed a stress-responsive epigenetic network around histone demethylase Kdm6b restricted to the ventral hippocampus, and acute stress reduced methylation of its enzymatic target H3K27me3. Selective Kdm6b knockdown in the ventral hippocampus led to behavioral hyperactivity/hyperresponsiveness. Conclusions These findings underscore the importance of considering dorsal and ventral hippocampus separately when conducting high-throughput molecular analyses, which has important implications for fundamental research as well as clinical studies.

Psychiatric diseases are multifaceted disorders with complex etiology, recognized to have strong heritable components. Despite intense research efforts, genetic loci that substantially account for disease heritability have not yet been identified. Over the last several years, epigenetic processes have emerged as important factors for many brain diseases, and the discovery of epigenetic processes in germ cells has raised the possibility that they may contribute to disease heritability and disease risk. This review examines epigenetic mechanisms in complex diseases and summarizes the most illustrative examples of transgenerational epigenetic inheritance in mammals and their relevance for brain function. Environmental factors that can affect molecular processes and behavior in exposed individuals and their offspring, and their potential epigenetic underpinnings, are described. Possible routes and mechanisms of transgenerational transmission are proposed, and the major questions and challenges raised by this emerging field of research are considered.

Epigenetic marks in an organism can be altered by environmental factors throughout life. Although changes in the epigenetic code can be positive, some are associated with severe diseases, in particular, cancer and neuropsychiatric disorders. Recent evidence has indicated that certain epigenetic marks can be inherited, and reshape developmental and cellular features over generations. This review examines the challenging possibility that epigenetic changes induced by environmental factors can contribute to some of the inheritance of disease and disease risk. This concept has immense implications for the understanding of biological functions and disease etiology, and provides potential novel strategies for diagnosis and treatment. Examples of epigenetic inheritance relevant to human disease, such as the detrimental effects of traumatic stress or drug/toxic exposure on brain functions, are reviewed. Different possible routes of transmission of epigenetic information involving the germline or germline-independent transfer are discussed, and different mechanisms for the maintenance and transmission of epigenetic information like chromatin remodeling and small noncoding RNAs are considered. Future research directions and remaining major challenges in this field are also outlined. Finally, the adaptive value of epigenetic inheritance, and the cost and benefit of allowing acquired epigenetic marks to persist across generations is critically evaluated.

The goal of the present study was to explore the effects of long-term hormone deprivation on the ability of subsequent estrogen treatment to affect attention performance on the 5-choice serial reaction time task (5-CSRTT). In an initial experiment to assess estradiol effects in young adults, 2-month-old rats were trained on the 5-CSRTT, then ovariectomized and immediately implanted with capsules containing cholesterol (n=10) or estradiol (n=10). Then rats were tested on the 5-CSRTT under baseline task parameters, under increased task difficulty (behavior challenge condition), and finally in muscarinic and nicotinic drug challenge conditions. In a second experiment, 10-month-old rats were trained on the 5-CSRTT and at 12 or 17 months of age rats were ovariectomized and treated with estradiol or cholesterol, so that one group received continuous cholesterol control treatment, two groups received estradiol treatment immediately following ovariectomy (either at 12 or 17 months), and one group received delayed estradiol treatment initiated 5 months following ovariectomies. At 17 months of age, rats were tested on the 5-CSRTT. Baseline performance was comparable between estradiol- and cholesterol-treated rats of both age groups. However, young estradiol-treated rats outperformed controls when behavior was challenged by shortening the intertrial interval (Short ITI). In the same Short ITI condition, middle-aged rats receiving immediate estradiol treatment beginning at the age of 17 months, but not 12 months, outperformed controls as well as animals receiving delayed estradiol treatment. No differences between groups were found in the cholinergic drug challenge conditions. These data indicate that chronic estradiol treatment for approximately 1 month but not 6 months is able to enhance attention performance, and that prolonged ovarian hormone deprivation attenuates these beneficial effects of subsequent estradiol treatment. These findings have implications for informing clinical research about the importance of timing and duration of hormone treatment.

We determined whether transient exposure to estradiol during middle age in ovariectomized rats would exert lasting effects on cognition and the brain beyond the period of exposure. Two experiments were conducted. Rats 10-11 months of age were ovariectomized and received vehicle control treatment throughout the experiment, continuous estradiol treatment throughout the experiment, or 40 d of transient exposure to estradiol that ended 3 d before behavioral training. In the first experiment, rats were trained on a radial-maze working memory task and killed 2 months after the termination of transient exposure to estradiol. The hippocampus was immunostained for choline acetyltransferase and estrogen receptors alpha (ER alpha) and beta (ER beta) by Western blotting. In a second experiment to determine the durability of treatment effects, rats were behaviorally tested every other month until brains were collected for Western blotting 8 months after the termination of transient exposure to estradiol. Maze testing included delay trials and scopolamine trials, in which dose-effect curves for the muscarinic receptor antagonist were determined. Transient exposure to estradiol enhanced working memory and attenuated amnestic effects of scopolamine as effectively as continuous estradiol exposure. Enhancements persisted for up to 7 months. Transient exposure to estradiol increased hippocampal levels of ER alpha and choline acetyltransferase 2 months and ER alpha 8 months after termination of the exposure. Neither estradiol treatment altered estrogen receptor beta levels. Results demonstrate that short-term treatment with estradiol during middle age enhances working memory well beyond the duration of treatment and suggest ER alpha as a potential mechanism for this effect.

Prenatal exposure to infectious or inflammatory insults is increasingly recognized to contribute to the etiology of psychiatric disorders with neurodevelopmental components, including schizophrenia, autism and bipolar disorder. It remains unknown, however, if such immune-mediated brain anomalies can be transmitted to subsequent generations. Using an established mouse model of prenatal immune activation by the viral mimetic poly(I:C), we show that reduced sociability and increased cued fear expression are similarly present in the first- and second-generation offspring of immune-challenged ancestors. We further demonstrate that sensorimotor gating impairments are confined to the direct descendants of infected mothers, whereas increased behavioral despair emerges as a novel phenotype in the second generation. These transgenerational effects are mediated via the paternal lineage and are stable until the third generation, demonstrating transgenerational non-genetic inheritance of pathological traits following in-utero immune activation. Next-generation sequencing further demonstrated unique and overlapping genome-wide transcriptional changes in first- and second-generation offspring of immune-challenged ancestors. These transcriptional effects mirror the transgenerational effects on behavior, showing that prenatal immune activation leads to a transgenerational transmission (presence of similar phenotypes across generations) and modification (presence of distinct phenotypes across generations) of pathological traits. Together, our study demonstrates for, we believe, the first time that prenatal immune activation can negatively affect brain and behavioral functions in multiple generations. These findings thus highlight a novel pathological aspect of this early-life adversity in shaping disease risk across generations.

Behavioural traits in mammals are influenced by environmental factors, which can interact with the genome and modulate its activity by complex molecular interplay. Environmental experiences can modify social, emotional and cognitive behaviours during an individual's lifetime, and result in acquired behavioural traits that can be transmitted to subsequent generations. This Review discusses the concept of, and experimental support for, non-genetic transgenerational inheritance of acquired traits involving the germ line in mammals. Possible mechanisms of induction and maintenance during development and adulthood are considered along with an interpretation of recent findings showing the involvement of epigenetic modifications and non-coding RNAs in male germ cells.

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.

Life experiences can induce epigenetic changes in mammalian germ cells, which can influence the developmental trajectory of the offspring and impact health and disease across generations. While this concept of epigenetic germline inheritance has long been met with skepticism, evidence in support of this route of information transfer is now overwhelming, and some key mechanisms underlying germline transmission of acquired information are emerging. This review focuses specifically on sperm RNAs as causal vectors of inheritance. We examine how they might become altered in the germline, and how different classes of sperm RNAs might interact with other epimodifications in germ cells or in the zygote. We integrate the latest findings with earlier pioneering work in this field, point out major questions and challenges, and suggest how new experiments could address them.

Traumatic experiences in childhood can alter behavioural responses and increase the risk for psychopathologies across life, not only in the exposed individuals but also in their progeny. In some conditions, such experiences can however be beneficial and facilitate the appraisal of adverse environments later in life. Here we expose newborn mice to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) for 2 weeks and assess the impact on behaviour in the offspring when adult. We show that MSUS in male mice favours goal-directed behaviours and behavioural flexibility in the adult offspring. This effect is accompanied by epigenetic changes involving histone post-translational modifications at the mineralocorticoid receptor (MR) gene and decreased MR expression in the hippocampus. Mimicking these changes pharmacologically in vivo reproduces the behavioural phenotype. These findings highlight the beneficial impact that early adverse experiences can have in adulthood, and the implication of epigenetic modes of gene regulation.