scholarly journals Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats

2020 ◽  
Author(s):  
M.C Chongtham ◽  
H Todorov ◽  
J.E. Wettschereck ◽  
S. Gerber ◽  
J. Winter
Keyword(s):  
Rapid Evolution ◽  
Type System ◽  
Downstream Processing ◽  
Brain Regions ◽  
Mammalian Brain ◽  
Cell Type ◽  
Specific Stimulus ◽  
Isolated Nuclei ◽  
Nuclei Isolation ◽  
Cell Type Specific

AbstractThe mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells is still a biochemically complex process, with every step affecting downstream results. Therefore, it is necessary to use protocols that fit the experimental design to yield nuclei of high quality and quantity. However, the current protocols are not suitable for nuclei isolation of small volumes of micro-dissected brain regions from individual mouse brains.Additionally, the caveats associated with centrifugation steps of nuclei extraction and the effects of different buffers have not been thoroughly investigated. Therefore, in this study, we describe an iodixanol based density gradient ultracentrifugation protocol suitable for micro-dissected brain regions from individual mice using ArccreERT2 (TG/WT).R26CAG-Sun1-sfGFP-Myc (M/WT or M/M). This mouse model shows sfGFP expression (sfGFP+) in the nuclear membrane of specific stimulus activated cells, thereby providing a good basis for the study - nuclei isolation and separation of cell-type-specific nuclei. The study also introduces new tools for rapid visualization and assessment of quality and quantity of nascent extracted nuclei. These tools were then used to examine critical morphological features of nuclei derived from different centrifugation methods and the use of different buffers to uncover underlying effects. Finally, to obtain cell-type-specific nuclei (sfGFP+ nuclei) from the isolated nuclei pool of high viscosity, an optimized protocol for fluorescence activated nuclei sorting (FANS) was established to speed up sorting. Additionally, we present a 1% PFA protocol for fixation of isolated nuclei for long term microscopic visualization.

scholarly journals Cortical structural differences in major depressive disorder correlate with cell type-specific transcriptional signatures

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiao Li ◽  
Jakob Seidlitz ◽  
John Suckling ◽  
Feiyang Fan ◽  
Gong-Jun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Major Depressive Disorder ◽  
Depressive Disorder ◽  
Structural Changes ◽  
Brain Regions ◽  
Cell Type ◽  
Major Depressive ◽  
Structural Differences ◽  
Neuroimaging Data ◽  
Cell Type Specific

AbstractMajor depressive disorder (MDD) has been shown to be associated with structural abnormalities in a variety of spatially diverse brain regions. However, the correlation between brain structural changes in MDD and gene expression is unclear. Here, we examine the link between brain-wide gene expression and morphometric changes in individuals with MDD, using neuroimaging data from two independent cohorts and a publicly available transcriptomic dataset. Morphometric similarity network (MSN) analysis shows replicable cortical structural differences in individuals with MDD compared to control subjects. Using human brain gene expression data, we observe that the expression of MDD-associated genes spatially correlates with MSN differences. Analysis of cell type-specific signature genes suggests that microglia and neuronal specific transcriptional changes account for most of the observed correlation with MDD-specific MSN differences. Collectively, our findings link molecular and structural changes relevant for MDD.

scholarly journals Using multiple measurements of tissue to estimate subject- and cell-type-specific gene expression

2019 ◽  
Vol 36 (3) ◽  
pp. 782-788 ◽  
Author(s):  
Jiebiao Wang ◽  
Bernie Devlin ◽  
Kathryn Roeder
Keyword(s):  
Gene Expression ◽  
Empirical Bayes ◽  
Brain Regions ◽  
Tissue Level ◽  
Supplementary Information ◽  
Specific Gene ◽  
Expression Data ◽  
Cell Type ◽  
Multiple Measurements ◽  
Cell Type Specific

Abstract Motivation Patterns of gene expression, quantified at the level of tissue or cells, can inform on etiology of disease. There are now rich resources for tissue-level (bulk) gene expression data, which have been collected from thousands of subjects, and resources involving single-cell RNA-sequencing (scRNA-seq) data are expanding rapidly. The latter yields cell type information, although the data can be noisy and typically are derived from a small number of subjects. Results Complementing these approaches, we develop a method to estimate subject- and cell-type-specific (CTS) gene expression from tissue using an empirical Bayes method that borrows information across multiple measurements of the same tissue per subject (e.g. multiple regions of the brain). Analyzing expression data from multiple brain regions from the Genotype-Tissue Expression project (GTEx) reveals CTS expression, which then permits downstream analyses, such as identification of CTS expression Quantitative Trait Loci (eQTL). Availability and implementation We implement this method as an R package MIND, hosted on https://github.com/randel/MIND. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Highly efficient and super-bright neurocircuit tracing using vector mixing-based virus cocktail

2019 ◽  
Author(s):  
Pei Sun ◽  
Sen Jin ◽  
Sijue Tao ◽  
Junjun Wang ◽  
Anan Li ◽  
...  
Keyword(s):  
Brain Function ◽  
Individual Cell ◽  
Brain Regions ◽  
Cell Type ◽  
Current Standard ◽  
Functional Studies ◽  
Highly Efficient ◽  
Order Of Magnitude ◽  
Cell Type Specific ◽  
Dependent Vector

ABSTRACTMapping the detailed cell-type-specific input networks and neuronal projectomes are essential to understand brain function in normal and pathological states. However, several properties of current tracing systems, including labeling sensitivity, trans-synaptic efficiencies, reproducibility among different individuals and different Cre-driver animals, still remained unsatisfactory. Here, we developed MAP-ENVIVIDERS, a recombinase system-dependent vector mixing-based strategy for highly efficient neurocircuit tracing. MAP-ENVIVIDERS enhanced tracing efficiency of input networks across the whole brain, with over 10-fold improvement in diverse previously poor-labeled input brain regions and particularly, up to 70-fold enhancement in brainstem compared with the current standard rabies-virus-mediated systems. MAP-ENVIVIDERS was over 10-fold more sensitive for cell-type-specific labeling than previous strategies, enabling us to capture individual cell-type-specific neurons with extremely complex axonal branches and presynaptic axonal boutons, both about one order of magnitude than previously reported and considered. MAP-ENVIVIDERS provides powerful tools for deconstructing novel input/output circuitry towards functional studies and disorders-related mechanisms.

scholarly journals Disease-, region- and cell type specific diversity of α-synuclein carboxy terminal truncations in synucleinopathies

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Ethan W. Hass ◽  
Zachary A. Sorrentino ◽  
Yuxing Xia ◽  
Grace M. Lloyd ◽  
John Q. Trojanowski ◽  
...  
Keyword(s):  
Lewy Body Dementia ◽  
Brain Regions ◽  
Motor Nucleus ◽  
Cell Type ◽  
Post Translational Modification ◽  
Cytoplasmic Inclusions ◽  
Specific Distribution ◽  
Cell Type Specific ◽  
Disease Region ◽  
Carboxy Terminal

AbstractSynucleinopathies, including Parkinson’s disease (PD), Lewy body dementia (LBD), Alzheimer’s disease with amygdala restricted Lewy bodies (AD/ALB), and multiple system atrophy (MSA) comprise a spectrum of neurodegenerative disorders characterized by the presence of distinct pathological α-synuclein (αSyn) inclusions. Experimental and pathological studies support the notion that αSyn aggregates contribute to cellular demise and dysfunction with disease progression associated with a prion-like spread of αSyn aggregates via conformational templating. The initiating event(s) and factors that contribute to diverse forms of synucleinopathies remain poorly understood. A major post-translational modification of αSyn associated with pathological inclusions is a diverse array of specific truncations within the carboxy terminal region. While these modifications have been shown experimentally to induce and promote αSyn aggregation, little is known about their disease-, region- and cell type specific distribution. To this end, we generated a series of monoclonal antibodies specific to neo-epitopes in αSyn truncated after residues 103, 115, 119, 122, 125, and 129. Immunocytochemical investigations using these new tools revealed striking differences in the αSyn truncation pattern between different synucleinopathies, brain regions and specific cellular populations. In LBD, neuronal inclusions in the substantia nigra and amygdala were positive for αSyn cleaved after residues 103, 119, 122, and 125, but not 115. In contrast, in the same patients' brain αSyn cleaved at residue 115, as well as 103, 119 and 122 were abundant in the dorsal motor nucleus of the vagus. In patients with AD/ALB, these modifications were only weakly or not detected in amygdala αSyn inclusions. αSyn truncated at residues 103, 115, 119, and 125 was readily present in MSA glial cytoplasmic inclusions, but 122 cleaved αSyn was only weakly or not present. Conversely, MSA neuronal pathology in the pontine nuclei was strongly reactive to the αSyn x-122 neo-epitope but did not display any reactivity for αSyn 103 cleavage. These studies demonstrate significant disease-, region- and cell type specific differences in carboxy terminal αSyn processing associated with pathological inclusions that likely contributes to their distinct strain-like prion properties and promotes the diversity displayed in the degrees of these insidious diseases.

scholarly journals Cell type-specific spatial and functional coupling between mammalian brain Kv2.1 K+channels and ryanodine receptors

2014 ◽  
Vol 522 (15) ◽  
pp. 3555-3574 ◽  
Author(s):  
Danielle Mandikian ◽  
Elke Bocksteins ◽  
Laxmi Kumar Parajuli ◽  
Hannah I. Bishop ◽  
Oscar Cerda ◽  
...  
Keyword(s):  
Ryanodine Receptors ◽  
Mammalian Brain ◽  
Functional Coupling ◽  
Cell Type ◽  
K Channels ◽  
Cell Type Specific

scholarly journals Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators

2015 ◽  
Vol 112 (11) ◽  
pp. 3445-3450 ◽  
Author(s):  
Qinghong Yan ◽  
Sebastien M. Weyn-Vanhentenryck ◽  
Jie Wu ◽  
Steven A. Sloan ◽  
Ye Zhang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Mammalian Brain ◽  
Cell Type ◽  
Evolutionary Selection ◽  
Chromatin Regulators ◽  
Brain Transcriptome ◽  
Cell Type Specific ◽  
Specific Regulation

Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23–42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.

scholarly journals Acoustically Targeted Chemogenetics for Noninvasive Control of Neural Circuits

2018 ◽  
Author(s):  
Jerzy O. Szablowski ◽  
Brian Lue ◽  
Audrey Lee-Gosselin ◽  
Dina Malounda ◽  
Mikhail G. Shapiro
Keyword(s):  
Neural Circuits ◽  
Brain Regions ◽  
Cell Type ◽  
Spatial Targeting ◽  
Temporal Specificity ◽  
Cell Type Specific ◽  
Orally Bioavailable ◽  
G Protein Coupled ◽  
The Brain ◽  
Spatial Cell

ABSTRACTNeurological and psychiatric diseases often involve the dysfunction of specific neural circuits in particular regions of the brain. Existing treatments, including drugs and implantable brain stimulators, aim to modulate the activity of these circuits, but are typically not cell type-specific, lack spatial targeting or require invasive procedures. Here, we introduce an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, called acoustically targeted chemogenetics, or ATAC, uses transient ultrasonic opening of the blood brain barrier to transduce neurons at specific locations in the brain with virally-encoded engineered G-protein-coupled receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. We demonstrate this concept in mice by using ATAC to noninvasively modify and subsequently activate or inhibit excitatory neurons within the hippocampus, showing that this enables pharmacological control of memory formation. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds, thereby overcoming some of the key limitations of conventional brain therapies.

scholarly journals Addiction-associated genetic variants implicate brain cell type- and region-specific cis-regulatory elements in addiction neurobiology

2020 ◽  
Author(s):  
Chaitanya Srinivasan ◽  
BaDoi N. Phan ◽  
Alyssa J. Lawler ◽  
Easwaran Ramamurthy ◽  
Michael Kleyman ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Cell Types ◽  
Regulatory Elements ◽  
Brain Regions ◽  
Open Chromatin ◽  
Genome Wide Association Studies ◽  
Cell Type ◽  
Coding Regions ◽  
Cell Type Specific ◽  
The Impact

ABSTRACTRecent large genome-wide association studies (GWAS) have identified multiple confident risk loci linked to addiction-associated behavioral traits. Genetic variants linked to addiction-associated traits lie largely in non-coding regions of the genome, likely disrupting cis-regulatory element (CRE) function. CREs tend to be highly cell type-specific and may contribute to the functional development of the neural circuits underlying addiction. Yet, a systematic approach for predicting the impact of risk variants on the CREs of specific cell populations is lacking. To dissect the cell types and brain regions underlying addiction-associated traits, we applied LD score regression to compare GWAS to genomic regions collected from human and mouse assays for open chromatin, which is associated with CRE activity. We found enrichment of addiction-associated variants in putative regulatory elements marked by open chromatin in neuronal (NeuN+) nuclei collected from multiple prefrontal cortical areas and striatal regions known to play major roles in reward and addiction. To further dissect the cell type-specific basis of addiction-associated traits, we also identified enrichments in human orthologs of open chromatin regions of mouse neuron subtypes: cortical excitatory, PV, D1, and D2. Lastly, we developed machine learning models from mouse cell type-specific regions of open chromatin to further dissect human NeuN+ open chromatin regions into cortical excitatory or striatal D1 and D2 neurons and predict the functional impact of addiction-associated genetic variants. Our results suggest that different neuron subtypes within the reward system play distinct roles in the variety of traits that contribute to addiction.Significance StatementOur study on cell types and brain regions contributing to heritability of addiction-associated traits suggests that the conserved non-coding regions within cortical excitatory and striatal medium spiny neurons contribute to genetic predisposition for nicotine, alcohol, and cannabis use behaviors. This computational framework can flexibly integrate epigenomic data across species to screen for putative causal variants in a cell type- and tissue-specific manner across numerous complex traits.

scholarly journals Single-cell deconvolution of 3,000 post-mortem brain samples for eQTL and GWAS dissection in mental disorders

2021 ◽  
Author(s):  
Yongjin Park ◽  
Liang He ◽  
Jose Davila-Velderrain ◽  
Lei Hou ◽  
Shahin Mohammadi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Genetic Variants ◽  
Cell Types ◽  
Brain Regions ◽  
Post Mortem ◽  
Rna Seq ◽  
Cell Type ◽  
Post Mortem Brain ◽  
Cell Type Specific

AbstractThousands of genetic variants acting in multiple cell types underlie complex disorders, yet most gene expression studies profile only bulk tissues, making it hard to resolve where genetic and non-genetic contributors act. This is particularly important for psychiatric and neurodegenerative disorders that impact multiple brain cell types with highly-distinct gene expression patterns and proportions. To address this challenge, we develop a new framework, SPLITR, that integrates single-nucleus and bulk RNA-seq data, enabling phenotype-aware deconvolution and correcting for systematic discrepancies between bulk and single-cell data. We deconvolved 3,387 post-mortem brain samples across 1,127 individuals and in multiple brain regions. We find that cell proportion varies across brain regions, individuals, disease status, and genotype, including genetic variants in TMEM106B that impact inhibitory neuron fraction and 4,757 cell-type-specific eQTLs. Our results demonstrate the power of jointly analyzing bulk and single-cell RNA-seq to provide insights into cell-type-specific mechanisms for complex brain disorders.

Mapping whole-brain seizure network recruitment with optogenetic kindling

2022 ◽  
Author(s):  
James Edward Niemeyer
Keyword(s):  
Functional Imaging ◽  
Epileptic Seizures ◽  
Brain Regions ◽  
Brain Dynamics ◽  
Cell Type ◽  
Whole Brain ◽  
Common View ◽  
Cell Type Specific

Epilepsy is often labelled a network disorder, though a common view of seizures holds that they initiate in a singular onset zone before expanding contiguously outward. A recent report by Choy et al. (2021) leverages new tools to study whole-brain dynamics during epileptic seizures originating in the hippocampus. Cell-type-specific kindling and functional imaging revealed how various brain regions were recruited to seizures and uncovered a novel form of migrating seizure core.

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