Similar Datasets
Project description:Deciphering patterns of connectivity between neurons in the mammalian brain is a critical step toward understanding brain function. Conventional imaging based neuroanatomical tracing methods identify area-to-area or sparse neuron-to-neuron connectivity patterns, but with extremely limited throughput. Recently developed barcode-based connectomics methods can efficiently map large numbers of single-neuron projections, but linking these data to single-cell transcriptomics remains a challenge. Here, we established a retro-AAV barcode-based multiplexed tracing method called MERGE-seq (Multiplexed projection neuRons retroGrade barcodE sequencing), which is capable of simultaneously characterizing the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets (AI, DMS, BLA, MD and LH). We found that projection-defined vmPFC neurons are molecularly heterogeneous, which are composed of different neuronal subtypes. We further identified transcriptional signatures of various dedicated and bifurcated vmPFC neurons, and verified Pou3f1 as the marker gene of neurons sending collateral axons to DMS and LH. Finally, we fitted our single-neuron connectome/transcriptome data into a machine learning-based model and revealed groups of genes that were predictive of certain projection pattern. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.
2024-02-25 | GSE210172 | GEO
Project description:Deciphering patterns of connectivity between neurons in the mammalian brain is a critical step toward understanding brain function. Conventional imaging based neuroanatomical tracing methods identify area-to-area or sparse neuron-to-neuron connectivity patterns, but with extremely limited throughput. Recently developed barcode-based connectomics methods can efficiently map large numbers of single-neuron projections, but linking these data to single-cell transcriptomics remains a challenge. Here, we established a retro-AAV barcode-based multiplexed tracing method called MERGE-seq (Multiplexed projection neuRons retroGrade barcodE sequencing), which is capable of simultaneously characterizing the projectome and transcriptome at the single neuron level. We uncovered dedicated and collateral projection patterns of ventromedial prefrontal cortex (vmPFC) neurons to five downstream targets (AI, DMS, BLA, MD and LH). We found that projection-defined vmPFC neurons are molecularly heterogeneous, which are composed of different neuronal subtypes. We further identified transcriptional signatures of various dedicated and bifurcated vmPFC neurons, and verified Pou3f1 as the marker gene of neurons sending collateral axons to DMS and LH. Finally, we fitted our single-neuron connectome/transcriptome data into a machine learning-based model and revealed groups of genes that were predictive of certain projection pattern. In summary, we have developed a new multiplexed technique whose paired connectome and gene expression data can help reveal organizational principles that form neural circuits and process information.
2024-02-25 | GSE210173 | GEO
Project description:Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits.
| S-EPMC6640135 | biostudies-literature
Project description:Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.
| S-EPMC7836778 | biostudies-literature
Project description:Understanding the structure and function of neural circuits underlying speech and language is a vital step toward better treatments for diseases of these systems. Songbirds, among the few animal orders that share with humans the ability to learn vocalizations from a conspecific, have provided many insights into the neural mechanisms of vocal development. However, research into vocal learning circuits has been hindered by a lack of tools for rapid genetic targeting of specific neuron populations to meet the quick pace of developmental learning. Here, we present a viral tool that enables fast and efficient retrograde access to projection neuron populations. In zebra finches, Bengalese finches, canaries, and mice, we demonstrate fast retrograde labeling of cortical or dopaminergic neurons. We further demonstrate the suitability of our construct for detailed morphological analysis, for in vivo imaging of calcium activity, and for multi-color brainbow labeling.
| S-EPMC8236207 | biostudies-literature
Project description:To characterize the molecular diversity of olfactory bulb projection neurons we used viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for piriform cortex-projecting or AON-projecting neurons, and bulk RNA deep sequencing (bulk RNA deep seq) to comprehensively characterize their transcriptomes.
2020-12-05 | GSE162655 | GEO
Project description:Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We present a microfluidic system for the rapid alignment of multifunctional hydrogel microparticles designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that (1) allows for a high particle throughput, (2) ensures proper particle alignment for decoding and target quantification, and (3) can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of approximately 50 cm s(-1) and with throughputs of approximately 40 particles s(-1). Simple physical models and CFD simulations have been used to investigate flow behavior in the device.
| S-EPMC2814334 | biostudies-literature
Project description:Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we use complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes complimenting missing biochemical activity. Although assay effectiveness ranged with respect to source genome (41% recovery), even distant E. coli relatives Bacillus subtilis and Bacteroides thetaiotaomicron showed success. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq's utility is demonstrated, with future applications in ecology, health, and engineering.
| S-SCDT-10_1038-S44320-024-00068-Z | biostudies-other
Project description:Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.
| S-EPMC5338412 | biostudies-literature
Project description:Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion, and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.
| S-EPMC8352594 | biostudies-literature