Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Single-cell analysis of chromatin accessibility in the adult mouse brain.

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

An atlas of gene regulatory elements in adult mouse cerebrum.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

Joint profiling of histone modifications and transcriptome in single cells from mouse brain.

Genome-wide profiling of histone modifications can reveal not only the location and activity state of regulatory elements, but also the regulatory mechanisms involved in cell-type-specific gene expression during development and disease pathology. Conventional assays to profile histone modifications in bulk tissues lack single-cell resolution. Here we describe an ultra-high-throughput method, Paired-Tag, for joint profiling of histone modifications and transcriptome in single cells to produce cell-type-resolved maps of chromatin state and transcriptome in complex tissues. We used this method to profile five histone modifications jointly with transcriptome in the adult mouse frontal cortex and hippocampus. Integrative analysis of the resulting maps identified distinct groups of genes subject to divergent epigenetic regulatory mechanisms. Our single-cell multiomics approach enables comprehensive analysis of chromatin state and gene regulation in complex tissues and characterization of gene regulatory programs in the constituent cell types.

General, spinal or regional anaesthesia does not affect strength performance 6 months after ACL reconstruction.

PURPOSE: The recovery of strength is a key element in successfully returning to sports after ACL reconstruction. The type of anaesthesia has been suspected an influential factor in the post-operative recovery of muscle function. METHODS: In this retrospective analysis, n = 442 consecutive patients undergoing primary isolated ACL reconstruction using a hamstring autograft were analysed by pre- and post-operative isokinetic tests in a single orthopaedic centre. These were subdivided into four cohorts: (1) general anaesthesia (n = 47), (2) general anaesthesia with prolonged (48 h) on-demand femoral nerve block (n = 37), (3) spinal anaesthesia (n = 169) and (4) spinal anaesthesia with prolonged (48 h) on-demand femoral nerve block (n = 185). Primary outcome was the change from pre- to post-operative isokinetic strength during knee extension and flexion. RESULTS: Using one-way ANOVA, there was no significant influence of the type of anaesthesia. The main effect of anaesthesia on change in extension forces was not significant, and effect sizes were very small (n.s.). Similarly, the main effect of anaesthesia on change in flexion forces was statistically not significant (n.s.). CONCLUSIONS: The findings of this study support the interpretation that the type of anaesthesia has no significant effect on the ability to recover thigh muscle strength 6 months after isolated hamstring ACL reconstruction. With regard to the recovery of athletic performance and return-to-sports testing criteria, there is no reason to avoid regional anaesthesia. LEVEL OF EVIDENCE: III.

Atmosphere-Snow Exchange Explains Surface Snow Isotope Variability.

The climate signal imprinted in the snow isotopic composition allows to infer past climate variability from ice core stable water isotope records. The concurrent evolution of vapor and surface snow isotopic composition between precipitation events indicates that post-depositional atmosphere-snow humidity exchange influences the snow and hence the ice core isotope signal. To date, however, this is not accounted for in paeleoclimate reconstructions from isotope records. Here we show that vapor-snow exchange explains 36% of the summertime day-to-day δ18O variability of the surface snow between precipitation events, and 53% of the δD variability. Through observations from the Greenland Ice Sheet and accompanying modeling we demonstrate that vapor-snow exchange introduces a warm bias on the summertime snow isotope value relevant for ice core records. In case of long-term variability in atmosphere-snow exchange the relevance for the ice core signal is also variable and thus paleoclimate reconstructions from isotope records should be revisited.

A qualitative exploration of the feasibility and acceptability of Meaning-Centered Psychotherapy for Cancer Caregivers.

OBJECTIVE: Caregivers of patients with cancer are at significant risk for existential distress. Such distress negatively impacts caregivers' quality of life and capacity to serve in their role as healthcare proxies, and ultimately, contributes to poor bereavement outcomes. Our team developed Meaning-Centered Psychotherapy for Cancer Caregivers (MCP-C), the first targeted psychosocial intervention that directly addresses existential distress in caregivers. METHOD: Nine caregivers of patients with glioblastoma multiforme (GBM) enrolled in a pilot randomized controlled trial evaluating the feasibility, acceptability, and effects of MCP-C, and completed in-depth interviews about their experience in the therapy. One focus group with three MCP-C interventionists was also completed. RESULTS: Four key themes emerged from interviews: (1) MCP-C validated caregivers' experience of caregiving; (2) MCP-C helped participants reframe their "caregiving identity" as a facet of their larger self-identity, by placing caregiving in the context of their life's journey; (3) MCP-C enabled caregivers to find ways to assert their agency through caregiving; and (4) the structure and sequence of sessions made MCP-C accessible and feasible. Feedback from interventionists highlighted several potential manual changes and overall ways in which MCP-C can help facilitate caregivers' openness to discussing death and engaging in advanced care planning discussions with the patient. SIGNIFICANCE OF RESULTS: The overarching goal of MCP-C is to allow caregivers to concurrently experience meaning and suffering; the intervention does not seek to deny the reality of challenges endured by caregivers, but instead to foster a connection to meaning and purpose alongside their suffering. Through in-depth interviews with caregivers and a focus group with MCP interventionists, we have refined and improved our MCP-C manual so that it can most effectively assist caregivers in experiencing meaning and purpose, despite inevitable suffering.

Maternal immune activation impairs cognitive flexibility and alters transcription in frontal cortex.

BACKGROUND: Epidemiological studies suggest that the risk of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia is increased by prenatal exposure to viral or bacterial infection during pregnancy. It is still unclear how activation of the maternal immune response interacts with underlying genetic factors to influence observed ASD phenotypes. METHODS: The current study investigated how maternal immune activation (MIA) in mice impacts gene expression in the frontal cortex in adulthood, and how these molecular changes relate to deficits in cognitive flexibility and social behavior, and increases in repetitive behavior that are prevalent in ASD. Poly(I:C) (20 mg/kg) was administered to dams on E12.5 and offspring were tested for social approach behavior, repetitive grooming, and probabilistic reversal learning in adulthood (n = 8 vehicle; n = 9 Poly(I:C)). We employed next-generation high-throughput mRNA sequencing (RNA-seq) to comprehensively investigate the transcriptome profile in frontal cortex of adult offspring of Poly(I:C)-exposed dams. RESULTS: Exposure to poly(I:C) during gestation impaired probabilistic reversal learning and decreased social approach in MIA offspring compared to controls. We found long-term effects of MIA on expression of 24 genes, including genes involved in glutamatergic neurotransmission, mTOR signaling and potassium ion channel activity. Correlations between gene expression and specific behavioral measures provided insight into genes that may be responsible for ASD-like behavioral alterations. CONCLUSIONS: These findings suggest that MIA can lead to impairments in cognitive flexibility in mice similar to those exhibited in ASD individuals, and that these impairments are associated with altered gene expression in frontal cortex.

Disruption of mGluR5 in parvalbumin-positive interneurons induces core features of neurodevelopmental disorders.

Alterations in glutamatergic transmission onto developing GABAergic systems, in particular onto parvalbumin-positive (Pv(+)) fast-spiking interneurons, have been proposed as underlying causes of several neurodevelopmental disorders, including schizophrenia and autism. Excitatory glutamatergic transmission, through ionotropic and metabotropic glutamate receptors, is necessary for the correct postnatal development of the Pv(+) GABAergic network. We generated mutant mice in which the metabotropic glutamate receptor 5 (mGluR5) was specifically ablated from Pv(+) interneurons postnatally, and investigated the consequences of such a manipulation at the cellular, network and systems levels. Deletion of mGluR5 from Pv(+) interneurons resulted in reduced numbers of Pv(+) neurons and decreased inhibitory currents, as well as alterations in event-related potentials and brain oscillatory activity. These cellular and sensory changes translated into domain-specific memory deficits and increased compulsive-like behaviors, abnormal sensorimotor gating and altered responsiveness to stimulant agents. Our findings suggest a fundamental role for mGluR5 in the development of Pv(+) neurons and show that alterations in this system can produce broad-spectrum alterations in brain network activity and behavior that are relevant to neurodevelopmental disorders.

Relationship between strength qualities and short track speed skating performance in young athletes.

This study analyzed the relationships between isometric as well as concentric maximum voluntary contraction (MVC) strength of the leg muscles and the times as well as speeds over different distances in 17 young short track speed skaters. Isometric as well as concentric single-joint MVC strength and multi-joint MVC strength in a stable (without skates) and unstable (with skates) condition were tested. Furthermore, time during maximum skating performances on ice was measured. Results indicate that maximum torques during eversion and dorsal flexion have a significant influence on skating speed. Concentric MVC strength of the knee extensors was higher correlated with times as well as speeds over the different distances than isometric MVC strength. Multi-joint MVC testing revealed that the force loss between measurements without and with skates amounts to 25%, while biceps femoris and soleus showed decreased muscle activity and peroneus longus, tibialis anterior, as well as rectus femoris exhibited increased muscle activity. The results of this study depict evidence that the skating times and speeds are primarily influenced by concentric MVC strength of the leg extensors. To be able to transfer the strength onto ice in an optimal way, it is necessary to stabilize the knee and ankle joints.

Early Adolescent Emergence of Reversal Learning Impairments in Isolation-Reared Rats.

Cognitive impairments appear early in the progression of schizophrenia, often preceding the symptoms of psychosis. Thus, the systems subserving these functions may be more vulnerable to, and mechanistically linked with, the initial pathology. Understanding the trajectory of behavioral and anatomical abnormalities relevant to the schizophrenia prodrome and their sensitivity to interventions in relevant models will be critical to identifying early therapeutic strategies. Isolation rearing of rats is an environmental perturbation that deprives rodents of social contact from weaning through adulthood and produces behavioral and neuronal abnormalities that mirror some pathophysiology associated with schizophrenia, e.g. frontal cortex abnormalities and prepulse inhibition (PPI) of startle deficits. Previously, we showed that PPI deficits in isolation-reared rats emerge in mid-adolescence (4 weeks after weaning; approx. postnatal day 52) but are not present when tested at 2 weeks after weaning (approx. postnatal day 38). Because cognitive deficits are reported during early adolescence, are relevant to the prodrome, and are linked to functional outcome, we examined the putative time course of reversal learning deficits in isolation-reared rats. Separate groups of male Sprague Dawley rats were tested in a two-choice discrimination task at 2 and 8 weeks after weaning, on postnatal day 38 and 80, respectively. The isolation-reared rats displayed impaired reversal learning at both time points. Isolation rearing was also associated with deficits in PPI at 4 and 10 weeks after weaning. The reversal learning deficits in the isolated rats were accompanied by reductions in parvalbumin immunoreactivity, a marker for specific subpopulations of GABAergic neurons, in the hippocampus. Hence, isolation rearing of rats may offer a unique model to examine the ontogeny of behavioral and neurobiological alterations that may be relevant to preclinical models of prodromal psychosis. © 2015 S. Karger AG, Basel.

Effect of plyometric training on neural and mechanical properties of the knee extensor muscles.

This study investigated neuromuscular adaptations of the knee extensors after 8 weeks of plyometric training. 23 subjects were randomly assigned to an intervention group and a control group. We measured isometric maximum voluntary torque (iMVT), rate of torque development (RTD) and impulse (IMP) over different time intervals. The neural drive to muscles was estimated with the interpolated twitch technique and normalized root mean square of the EMG signal. Contractile properties, H reflexes as well as jump height in squat jump (SJ) and countermovement jump (CMJ) were evaluated. Neuromuscular testing was performed at 2 knee angles, i. e., 80° and 45° (0° = full extension). The iMVT at 80° knee flexion was 23.1 N · m (95% CI: 0.1-46.1 N · m, P = 0.049) higher at post-test for the intervention group compared with controls. The same was true for RTD and IMP in the time interval 0-50 ms [308.7 N · m · s(-1) (95% CI: 28.8-588.6 N · m · s(-1), P = 0.033) and 0.32 N · m · s (95% CI: 0.05-0.60 N · m · s, P = 0.026), respectively]. These changes were accompanied by enhanced neural drive to the quadriceps muscle. Jump height in SJ and CMJ was higher at post-test for the intervention group compared with controls. Parameters at 45° knee flexion, contractile properties and evoked potentials did not differ between groups. Although hypertrophic changes were not measured, data suggest that the training regime probably induced mainly neural adaptations that were specifically related to the knee angle. The strength gains at 80° knee flexion likely contributed to the enhanced jump height in SJ and CMJ.

Cell-type-specific effects of age and sex on human cortical neurons.

Altered transcriptional and epigenetic regulation of brain cell types may contribute to cognitive changes with advanced age. Using single-nucleus multi-omic DNA methylation and transcriptome sequencing (snmCT-seq) in frontal cortex from young adult and aged donors, we found widespread age- and sex-related variation in specific neuron types. The proportion of inhibitory SST- and VIP-expressing neurons was reduced in aged donors. Excitatory neurons had more profound age-related changes in their gene expression and DNA methylation than inhibitory cells. Hundreds of genes involved in synaptic activity, including EGR1, were less expressed in aged adults. Genes located in subtelomeric regions increased their expression with age and correlated with reduced telomere length. We further mapped cell-type-specific sex differences in gene expression and X-inactivation escape genes. Multi-omic single-nucleus epigenomes and transcriptomes provide new insight into the effects of age and sex on human neurons.

Cell type-specific enrichment of somatic aneuploidy in the mammalian brain.

Somatic mutations alter the genomes of a subset of an individual's brain cells1-3, impacting gene regulation and contributing to disease processes4,5. Mosaic single nucleotide variants have been characterized with single-cell resolution in the brain2,3, but we have limited information about large-scale structural variation, including whole-chromosome duplication or loss1,6,7. We used a dataset of over 415,000 single-cell DNA methylation and chromatin conformation profiles across the adult mouse brain to identify aneuploid cells comprehensively. Whole-chromosome loss or duplication occurred in <1% of cells, with rates up to 1.8% in non-neuronal cell types, including oligodendrocyte precursors and pericytes. Among all aneuploidies, we observed a strong enrichment of trisomy on chromosome 16, which is syntenic with human chromosome 21 and constitutively trisomic in Down syndrome. Chromosome 16 trisomy occurred in multiple cell types and across brain regions, suggesting that nondisjunction is a recurrent feature of somatic variation in the brain.