Cell-Type Specific Responses to Associative Learning in the Primary Motor Cortex

The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue-reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca2+ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both cue and reward stimuli, and their response properties undergo cell-type specific modifications after associative learning. PV-INs' responses became more reliable to the cue stimulus, while VIP-INs' responses became more reliable to the reward stimulus. PNs only showed robust response to the novel reward stimulus, and they habituated to it after associative learning. Lastly, SOM-IN responses emerged and became more reliable to both conditioned cue and reward stimuli after conditioning. These observations suggest that cue- and reward-related signals are represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.

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Comparative cellular analysis of motor cortex in human, marmoset and mouse

Nature ◽

10.1038/s41586-021-03465-8 ◽

2021 ◽

Vol 598 (7879) ◽

pp. 111-119 ◽

Cited By ~ 1

Author(s):

Trygve E. Bakken ◽

Nikolas L. Jorstad ◽

Qiwen Hu ◽

Blue B. Lake ◽

Wei Tian ◽

...

Keyword(s):

Motor Cortex ◽

Primary Motor Cortex ◽

Neuronal Cell ◽

Cell Types ◽

Morphological Characterization ◽

Fine Motor ◽

Marker Genes ◽

Cell Type ◽

Regulatory Pathways ◽

Cellular Analysis

AbstractThe primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

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Multiscale Co-Expression in the Brain

10.1101/2020.03.31.018630 ◽

2020 ◽

Author(s):

Benjamin D. Harris ◽

Megan Crow ◽

Stephan Fischer ◽

Jesse Gillis

Keyword(s):

Motor Cortex ◽

Single Cell ◽

Rna Sequencing ◽

Primary Motor Cortex ◽

Cell Types ◽

Cell Type ◽

Single Cell Rna Sequencing ◽

Cell Type Specific ◽

The Brain ◽

Regulatory Landscape

ABSTRACTSingle-cell RNA-sequencing (scRNAseq) data can reveal co-regulatory relationships between genes that may be hidden in bulk RNAseq due to cell type confounding. Using the primary motor cortex data from the Brain Initiative Cell Census Network (BICCN), we study cell type specific co-expression across 500,000 cells. Surprisingly, we find that the same gene-gene relationships that differentiate cell types are evident at finer and broader scales, suggesting a consistent multiscale regulatory landscape.

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

Nature ◽

10.1038/s41586-021-03500-8 ◽

2021 ◽

Vol 598 (7879) ◽

pp. 103-110 ◽

Cited By ~ 6

Author(s):

Zizhen Yao ◽

Hanqing Liu ◽

Fangming Xie ◽

Stephan Fischer ◽

Ricky S. Adkins ◽

...

Keyword(s):

Motor Cortex ◽

Primary Motor Cortex ◽

Neuronal Cell ◽

Pyramidal Cells ◽

Cell Types ◽

Regulatory Elements ◽

Marker Genes ◽

Specific Cell ◽

Cell Type ◽

The Brain

AbstractSingle-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.

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A multimodal cell census and atlas of the mammalian primary motor cortex

10.1101/2020.10.19.343129 ◽

2020 ◽

Cited By ~ 5

Author(s):

◽

Ricky S. Adkins ◽

Andrew I. Aldridge ◽

Shona Allen ◽

Seth A. Ament ◽

...

Keyword(s):

Motor Cortex ◽

Single Cell ◽

Primary Motor Cortex ◽

Spatial Information ◽

Molecular Genetic ◽

Cell Types ◽

Developmental Trajectory ◽

Open Chromatin ◽

Cell Type ◽

Spatially Resolved

ABSTRACTWe report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

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Complexity and graded regulation of neuronal cell-type–specific alternative splicing revealed by single-cell RNA sequencing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013056118 ◽

2021 ◽

Vol 118 (10) ◽

pp. e2013056118

Author(s):

Huijuan Feng ◽

Daniel F. Moakley ◽

Shuonan Chen ◽

Melissa G. McKenzie ◽

Vilas Menon ◽

...

Keyword(s):

Alternative Splicing ◽

Single Cell ◽

Neuronal Cell ◽

Cell Types ◽

Gabaergic Neurons ◽

Cell Type ◽

Differential Splicing ◽

Single Cell Rna Sequencing ◽

Hierarchical Levels ◽

Cell Type Specific

The enormous cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell-type–specific gene-regulatory programs at the molecular level. Although prevalent in the brain, the contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here, we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single-cell RNA-sequencing data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class. These programs consist of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2, and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs contribute to splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which may provide a molecular mechanism to specify neuronal identity and function that are orthogonal to established classifications based on transcriptional regulation.

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Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

10.1101/2021.08.03.454921 ◽

2021 ◽

Author(s):

Sruti Rayaprolu ◽

Sara Bitarafan ◽

Ranjita Betarbet ◽

Sydney N Sunna ◽

Lihong Cheng ◽

...

Keyword(s):

Mouse Brain ◽

Neurological Diseases ◽

Neuronal Cell ◽

Cell Types ◽

Proteomic Profiling ◽

Cell Type ◽

Specific Expression ◽

Cell Type Specific ◽

The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

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Single nucleus analysis of the chromatin landscape in mouse forebrain development

10.1101/159137 ◽

2017 ◽

Cited By ~ 2

Author(s):

Sebastian Preissl ◽

Rongxin Fang ◽

Yuan Zhao ◽

Ramya Raviram ◽

Yanxiao Zhang ◽

...

Keyword(s):

Neuronal Cell ◽

Cell Types ◽

Cellular Heterogeneity ◽

Regulatory Sequences ◽

Specific Cell ◽

Cell Type ◽

Tissue Samples ◽

Forebrain Development ◽

Cell Type Specific ◽

Accessible Chromatin

ABSTRACTGenome-wide analysis of chromatin accessibility in primary tissues has uncovered millions of candidate regulatory sequences in the human and mouse genomes1–4. However, the heterogeneity of biological samples used in previous studies has prevented a precise understanding of the dynamic chromatin landscape in specific cell types. Here, we show that analysis of the transposase-accessible-chromatin in single nuclei isolated from frozen tissue samples can resolve cellular heterogeneity and delineate transcriptional regulatory sequences in the constituent cell types. Our strategy is based on a combinatorial barcoding assisted single cell assay for transposase-accessible chromatin5 and is optimized for nuclei from flash-frozen primary tissue samples (snATAC-seq). We used this method to examine the mouse forebrain at seven development stages and in adults. From snATAC-seq profiles of more than 15,000 high quality nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell-types in foetal and adult forebrains. We further define cell-type specific cis regulatory sequences and infer potential master transcriptional regulators of each cell population. Our results demonstrate the feasibility of a general approach for identifying cell-type-specific cis regulatory sequences in heterogeneous tissue samples, and provide a rich resource for understanding forebrain development in mammals.

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An integrated transcriptomic and epigenomic atlas of mouse primary motor cortex cell types

10.1101/2020.02.29.970558 ◽

2020 ◽

Cited By ~ 15

Author(s):

Zizhen Yao ◽

Hanqing Liu ◽

Fangming Xie ◽

Stephan Fischer ◽

A. Sina Booeshaghi ◽

...

Keyword(s):

Motor Cortex ◽

Single Cell ◽

Primary Motor Cortex ◽

Neuronal Cell ◽

Cell Types ◽

Regulatory Elements ◽

Brain Cell ◽

Molecular Signatures ◽

Sequencing Technologies ◽

Cell Transcriptome

AbstractSingle cell transcriptomics has transformed the characterization of brain cell identity by providing quantitative molecular signatures for large, unbiased samples of brain cell populations. With the proliferation of taxonomies based on individual datasets, a major challenge is to integrate and validate results toward defining biologically meaningful cell types. We used a battery of single-cell transcriptome and epigenome measurements generated by the BRAIN Initiative Cell Census Network (BICCN) to comprehensively assess the molecular signatures of cell types in the mouse primary motor cortex (MOp). We further developed computational and statistical methods to integrate these multimodal data and quantitatively validate the reproducibility of the cell types. The reference atlas, based on more than 600,000 high quality single-cell or -nucleus samples assayed by six molecular modalities, is a comprehensive molecular account of the diverse neuronal and non-neuronal cell types in MOp. Collectively, our study indicates that the mouse primary motor cortex contains over 55 neuronal cell types that are highly replicable across analysis methods, sequencing technologies, and modalities. We find many concordant multimodal markers for each cell type, as well as thousands of genes and gene regulatory elements with discrepant transcriptomic and epigenomic signatures. These data highlight the complex molecular regulation of brain cell types and will directly enable design of reagents to target specific MOp cell types for functional analysis.

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A multimodal cell census and atlas of the mammalian primary motor cortex

Nature ◽

10.1038/s41586-021-03950-0 ◽

2021 ◽

Vol 598 (7879) ◽

pp. 86-102 ◽

Cited By ~ 7

Author(s):

◽

Edward M. Callaway ◽

Hong-Wei Dong ◽

Joseph R. Ecker ◽

Michael J. Hawrylycz ◽

...

Keyword(s):

Motor Cortex ◽

Single Cell ◽

Primary Motor Cortex ◽

Spatial Information ◽

Molecular Genetic ◽

Cortical Cell ◽

Neuronal Cell ◽

Open Chromatin ◽

Cell Type ◽

Spatially Resolved

AbstractHere we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization1–5. First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

Download Full-text