Centripetal integration of past events in hippocampal astrocytes regulated by locus coeruleus.

An essential feature of neurons is their ability to centrally integrate information from their dendrites. The activity of astrocytes, in contrast, has been described as mostly uncoordinated across cellular compartments without clear central integration. Here we report conditional integration of calcium signals in astrocytic distal processes at their soma. In the hippocampus of adult mice of both sexes, we found that global astrocytic activity, as recorded with population calcium imaging, reflected past neuronal and behavioral events on a timescale of seconds. Salient past events, indicated by pupil dilations, facilitated the propagation of calcium signals from distal processes to the soma. Centripetal propagation to the soma was reproduced by optogenetic activation of the locus coeruleus, a key regulator of arousal, and reduced by pharmacological inhibition of α1-adrenergic receptors. Together, our results suggest that astrocytes are computational units of the brain that slowly and conditionally integrate calcium signals upon behaviorally relevant events.

Exploratory Rearing Is Governed by Hypothalamic Melanin-Concentrating Hormone Neurons According to Locus Ceruleus.

Information seeking, such as standing on tiptoes to look around in humans, is observed across animals and helps survival. Its rodent analog-unsupported rearing on hind legs-was a classic model in deciphering neural signals of cognition and is of intense renewed interest in preclinical modeling of neuropsychiatric states. Neural signals and circuits controlling this dedicated decision to seek information remain largely unknown. While studying subsecond timing of spontaneous behavioral acts and activity of melanin-concentrating hormone (MCH) neurons (MNs) in behaving male and female mice, we observed large MN activity spikes that aligned to unsupported rears. Complementary causal, loss and gain of function, analyses revealed specific control of rear frequency and duration by MNs and MCHR1 receptors. Activity in a key stress center of the brain-the locus ceruleus noradrenaline cells-rapidly inhibited MNs and required functional MCH receptors for its endogenous modulation of rearing. By defining a neural module that both tracks and controls rearing, these findings may facilitate further insights into biology of information seeking.

Noradrenaline release from the locus coeruleus shapes stress-induced hippocampal gene expression.

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 (LC-NA) system to the acute stress response in mice. We reveal that NA release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via ß-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, and 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 how NA impacts brain function in health and disease.

Harnessing PROTAC technology to combat stress hormone receptor activation.

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GR's genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

Single-Nucleus RNA-Sequencing in Brain Tissue.

Neuroscience research greatly benefits from single-cell sequencing technologies, which can reveal transcriptional alterations on a cellular level. However, preparing single-cell suspensions is technically challenging, requires experience, and has several limitations that can influence the transcriptional readout. Performing sequencing of single nuclei instead of single cells alleviates several of the challenges of sample preparation and highlights acute nuclear transcription. Here, we provide a protocol to prepare a nuclei suspension for single-nucleus RNA-sequencing for cell type-specific transcriptional profiling of brain tissue using the 10x Genomics single-cell gene expression assay. Furthermore, we highlight important aspects to consider during experimental design and data analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of single-nucleus suspension Basic Protocol 2: Preparation and sequencing of single-nucleus libraries for RNA-seq.

Neuromodulation of striatal D1 cells shapes BOLD fluctuations in anatomically connected thalamic and cortical regions.

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 in mice, are shaped by chemogenetically exciting or inhibiting D1 medium spiny neurons (MSNs) of the right dorsomedial caudate putamen (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, changes in local dynamics were strongest in unimodal regions (which process information from a single sensory modality) and weakened along a hierarchical gradient towards transmodal regions. In contrast, a decrease in functional connectivity was observed only for cortico-striatal connections after D1 excitation. Our results show that targeted cellular-level manipulations affect local BOLD dynamics at the macroscale, such as by making BOLD dynamics more predictable over time by increasing its self-correlation structure. This contributes to ongoing attempts to understand the influence of structure-function relationships in shaping inter-regional communication at subcortical and cortical levels.

Sensitive multicolor indicators for monitoring norepinephrine in vivo.

Genetically encoded indicators engineered from G-protein-coupled receptors are important tools that enable high-resolution in vivo neuromodulator imaging. Here, we introduce a family of sensitive multicolor norepinephrine (NE) indicators, which includes nLightG (green) and nLightR (red). These tools report endogenous NE release in vitro, ex vivo and in vivo with improved sensitivity, ligand selectivity and kinetics, as well as a distinct pharmacological profile compared with previous state-of-the-art GRABNE indicators. Using in vivo multisite fiber photometry recordings of nLightG, we could simultaneously monitor optogenetically evoked NE release in the mouse locus coeruleus and hippocampus. Two-photon imaging of nLightG revealed locomotion and reward-related NE transients in the dorsal CA1 area of the hippocampus. Thus, the sensitive NE indicators introduced here represent an important addition to the current repertoire of indicators and provide the means for a thorough investigation of the NE system.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

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.

Deep-learning based identification, tracking, pose estimation, and behavior classification of interacting primates and mice in complex environments.

The quantification of behaviors of interest from video data is commonly used to study brain function, the effects of pharmacological interventions, and genetic alterations. Existing approaches lack the capability to analyze the behavior of groups of animals in complex environments. We present a novel deep learning architecture for classifying individual and social animal behavior, even in complex environments directly from raw video frames, while requiring no intervention after initial human supervision. Our behavioral classifier is embedded in a pipeline (SIPEC) that performs segmentation, identification, pose-estimation, and classification of complex behavior, outperforming the state of the art. SIPEC successfully recognizes multiple behaviors of freely moving individual mice as well as socially interacting non-human primates in 3D, using data only from simple mono-vision cameras in home-cage setups.

Multiomic profiling of the acute stress response in the mouse hippocampus.

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.

Optogenetic activation of striatal D1R and D2R cells differentially engages downstream connected areas beyond the basal ganglia.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor and executive function. Central to BG function are striatal cells expressing D1 (D1R) and D2 (D2R) dopamine receptors. D1R and D2R cells are considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. However, whether they maintain a uniform function across the striatum and what influence they exert outside the BG is unclear. Here, we address these questions by combining optogenetic activation of D1R and D2R cells in the mouse ventrolateral caudoputamen with fMRI. Striatal D1R/D2R stimulation evokes distinct activity within the BG-thalamocortical network and differentially engages cerebellar and prefrontal regions. Computational modeling of effective connectivity confirms that changes in D1R/D2R output drive functional relationships between these regions. Our results suggest a complex functional organization of striatal D1R/D2R cells and hint toward an interconnected fronto-BG-cerebellar network modulated by striatal D1R and D2R cells.

Chronic adolescent stress increases exploratory behavior but does not appear to change the acute stress response in adult male C57BL/6 mice.

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 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 in adulthood and is unlikely to alter the function of the noradrenergic system.

Single paternal dexamethasone challenge programs offspring metabolism and reveals multiple candidates in RNA-mediated inheritance.

Single traumatic events that elicit an exaggerated stress response can lead to the development of neuropsychiatric conditions. Rodent studies suggested 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 to be seen. We find that acute administration of an agonist for the stress-sensitive Glucocorticoid receptor, using the common corticosteroid dexamethasone, affects the RNA payload of mature sperm as soon as 3 hr after exposure. It further impacts early embryonic transcriptional trajectories, as determined by single-embryo sequencing, and metabolism in the offspring. We show persistent regulation of tRNA fragments in sperm and descendant 2-cell embryos, suggesting transmission from sperm to embryo. Lastly, we unravel environmentally induced alterations in sperm circRNAs and their targets in the early embryo, highlighting this class as an additional candidate in RNA-mediated inheritance of disease risk.

Pervasive compartment-specific regulation of gene expression during homeostatic synaptic scaling.

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.

The Acute Stress Response in the Multiomic Era.

Studying the stress response is a major pillar of neuroscience research not only because stress is a daily reality but also because the exquisitely fine-tuned bodily changes triggered by stress are a neuroendocrinological marvel. While the genome-wide changes induced by chronic stress have been extensively studied, we know surprisingly little about the complex molecular cascades triggered by acute stressors, the building blocks of chronic stress. The acute stress (or fight-or-flight) response mobilizes organismal energy resources to meet situational demands. However, successful stress coping also requires the efficient termination of the stress response. Maladaptive coping-particularly in response to severe or repeated stressors-can lead to allostatic (over)load, causing wear and tear on tissues, exhaustion, and disease. We propose that deep molecular profiling of the changes triggered by acute stressors could provide molecular correlates for allostatic load and predict healthy or maladaptive stress responses. We present a theoretical framework to interpret multiomic data in light of energy homeostasis and activity-dependent gene regulation, and we review the signaling cascades and molecular changes rapidly induced by acute stress in different cell types in the brain. In addition, we review and reanalyze recent data from multiomic screens conducted mainly in the rodent hippocampus and amygdala after acute psychophysical stressors. We identify challenges surrounding experimental design and data analysis, and we highlight promising new research directions to better understand the stress response on a multiomic level.

Big behavior: challenges and opportunities in a new era of deep behavior profiling.

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.

A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.

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.

Deep learning-based behavioral analysis reaches human accuracy and is capable of outperforming commercial solutions.

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.

Sperm RNA: Quo vadis?

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.