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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labelling [bulk RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE210173 | GEO | 2024/02/25

REPOSITORIES: GEO

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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labelling [scRNA-seq]

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

Virally Encoded Connectivity Transgenic Overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Project description:Purpose: Establish a high-throughput method to transcriptionally define projection neurons, VECTORseq, that reimagines transgenes expressed by widely used retrogradely infecting viruses as multiplexed RNA barcodes that are detected in single-cell sequencing. Methods: mRNA profiles of adult mouse brains Conclusions: Retrograde viruses express mRNA at levels detectable in single-cell sequencing. Different transgenes can be multiplexed in a single sequencing run. VECTORseq identifies both cortical and subcortical projection neurons. VECTORseq defined new superior colliculus and zona incerta projection populations. Established a high-throughput method to transcriptionally define projection neurons, VECTORseq, that reimagines transgenes expressed by widely used retrogradely infecting viruses as multiplexed RNA barcodes that are detected in single-cell sequencing.

2021-12-02 | GSE189907 | GEO

Prefrontal D2- dopamine receptor deactivates top-down inhibitory control by regulating striatal cholinergic interneurons

Project description:Inhibitory control is a core cognitive function preventing the expression of maladaptive behaviors. This function is overridden in mental illnesses including bipolar disorder, autism spectrum disorders and schizophrenia, causing maladaptive psychomotor agitation. We developed intersectional CRISPR/Cas9 targeting approaches in adult mice to identify receptor/circuit selective mechanisms responsible for inactivation of inhibitory control in two models of amphetamine-induced psychomotor agitation. Inactivation of dopamine D2-receptors in the medial-prefrontal cortex (mPFC) abolished amphetamine-induced: psychomotor agitation, motor sensitization, and cAMP-associated transcriptome changes in the striatum. Further characterization using intersectional projection preparations indicate that these deficits implicate D2-receptors expressed on projection neurons innervating cholinergic interneurons of the dorsomedial striatum (DMS). Trans-synaptic targeting of acetylcholine production in DMS neurons receiving these projections restored psychomotor agitation in mice lacking mPFC D2- receptor expression. These findings identify a circuit by which activation of cortical D2-receptor circumvent an acetylcholine-mediated inhibition of DMS functions by the mPFC to promote psychomotor activation.

2023-06-17 | GSE234994 | GEO

Developmental cell death of cortical projection neurons is controlled by a Bcl11a/Bcl6-dependent pathway I

Project description:Developmental neuron death plays a pivotal role in refining organization and wiring during neocortex formation. Aberrant regulation of this process results in neurodevelopmental disorders including impaired learning and memory. Underlying molecular pathways are incompletely determined. Loss of Bcl11a in cortical projection neurons induces pronounced cell death in upper-layer cortical projection neurons during postnatal corticogenesis. We used this genetic model to explore genetic mechanisms by which developmental neuron death is controlled. Unexpectedly, we found Bcl6, previously shown to be involved in transition of cortical neurons from progenitor to postmitotic differentiation state to provide a major check point regulating neuron survival during late cortical development. We show that Bcl11a is a direct transcriptional regulator of Bcl6. Deletion of Bcl6 exerts death of cortical projection neurons. In turn, reintroduction of Bcl6 into Bcl11a mutants prevents induction of cell death in these neurons. Together, our data identify a novel Bcl11a/Bcl6-dependent molecular pathway in regulation of developmental cell death during corticogenesis.

2022-06-24 | GSE185287 | GEO

Developmental cell death of cortical projection neurons is controlled by a Bcl11a/Bcl6-dependent pathway II

2022-06-24 | GSE185288 | GEO

Transcription profiling of mouse FACS-purified cortical projection neurons using Affymetrix 430 2.0 microarrays

Project description:3 subtypes of cortical projection neurons were purified by fluorescence-activated cell sorting (FACS) at 4 different stages of development from mouse cortex. A detailed description of the data set is described in Arlotta, P et al (2005) and Molyneaux, BJ et al (2009). The hybridization cocktails used here were originally applied to the Affymetrix mouse 430A arrays and submitted as GEO accession number GSE2039. The same hybridization cocktails were then applied to the Affymetrix mouse 430 2.0 arrays, and those data are contained in this series. Experiment Overall Design: Three subtypes of cortical neurons were purified by FACS at multiple stages of mouse brain development. The neuron subtypes are: corticospinal motor neurons (CSMN), callosal projection neurons (CPN), and corticotectal projection neurons (CTPN). The stages of development included embryonic day 18 (E18), postnatal day 3 (P3), postnatal day 6 (P6), and postnatal day 14 (P14). CSMN and CPN were analyzed at all four stages, while CTPN were only analyzed at P14. The replicates included in the data set are all true biological replicates with independent sample collection for each.

2009-10-09 | E-GEOD-17783 | biostudies-arrayexpress

Effect of phox2a knockout on the molecular profiles of hindbrain vestibular neurons in the larval zebrafish (bulk RNA-Seq)

Project description:Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor partner populations shape the subtype fate and connectivity of their pre-motor counterparts remains controversial. Here, we discovered that motor partners are dispensable for proper connectivity across an entire vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in pre-motor projection neurons after constitutive loss of their extraocular motor neuron partners.We observed normal responses and topography consistent with unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their motor partners. Lastly, we show that the transcriptional signatures of projection neuron subtypes develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to canonical sensorimotor circuit assembly, our work speaks to comparable processes in spinal circuits and advances our understanding of general principles of neural development.

2024-01-27 | GSE254345 | GEO

Identification of molecular controls of Corticothalamic projection neurons differentiatiion

Project description:Molecular mechanisms controlling specification and differentiation of distinct neuron subtypes in the cerebral cortex are not well understood. Corticothalamic projection neurons (CThPN) are a diverse set of neurons, critical for function of the neocortex, but little is known about the molecular mechansims controlling their development. We used microarrays to detail CThPN gene expression at 3 developmental time points. We then compared CThPN gene expression, with the gene expression of other neuron subtypes in the cerebral cortex at the same stages (previously described in Arlotta et a., 2005) CThPN were retrogradely label with fluorescent microespheres injected into the thalamus, and were FACS purified at 3 developmental stages for RNA extraction and hybridization on Affymetrix microarrays.

2016-06-28 | E-GEOD-61711 | biostudies-arrayexpress

Analysis of changes in the BLA and vmPFC brain region of control mice and global cerebral ischemia mice after acute stress

Project description:It is known that the eCB 2-AG is synthesized in postsynaptic cells and released into synaptic clefts when needed. As we observed that acute stress prominently increased eCBs in vmPFC-BLA synapses and that inhibited expression of 2-AG synthase gene Dagla in vmPFC-BLA synapses blocked stress-induced PGCID, we plausibly hypothesized that transient GCI increased 2-AG biosynthesis in BLA neurons, leading to stress-induced increase of 2-AG release into vmPFC-BLA synapses. To test this hypothesis, we employed single-cell sequencing to examine whether GCI mice, in comparison with sham mice, showed increased mRNA expression of the Dagla in c-fos positive BLA and vmPFC cells of acute stress-treated mice

2024-01-31 | GSE248200 | GEO

Spatial enhancer activation determines inhibitory neuron identity (ChIP-seq of MEIS transcription factors)

Project description:The mammalian telencephalon contains a tremendous diversity of GABAergic projection neuron and interneuron types, that originate in a germinal zone of the embryonic basal ganglia. How genetic information in this transient structure is transformed into different cell types is not yet fully understood. Using a combination of in vivo lineage tracing, CRISPR perturbation and ChIP-seq in mice, we found that the transcription factor MEIS2 favors the development of projection neurons through genomic binding sites in regulatory enhancers of projection neuron specific genes. MEIS2 requires the presence of the homeodomain transcription factor DLX5 to direct its functional activity towards these sites. In interneurons, the activation of projection neuron specific enhancers by MEIS2 and DLX5 is repressed by the transcription factor LHX6. When MEIS2 carries a mutation associated with intellectual disability in humans, it is less effective at activating enhancers involved in projection neuron development. This suggests that GABAergic differentiation may be impaired in patients carrying this mutation. Our research has uncovered a mechanism by which the selective activation of enhancers plays a crucial role in the establishment of neuronal identity, as well as in potential pathological mechanisms

2024-02-19 | GSE231777 | GEO

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