High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex

Chen, Yushu; Chen, Xiaoyin; Başerdem, Batuhan; Zhan, Huiqing; Li, Yan; Davis, Michael F.; Zador, Anthony M.; Koulakov, Alexei A.; Albeanu, Dinu F.

doi:10.1016/j.cell.2022.09.038

Cited by 61 publications

(57 citation statements)

References 132 publications

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“…Because many barcode molecules can be sequenced in parallel, barcoding-based neuroanatomical approaches can determine the long-range projections of tens of thousands of neurons in single animals. These barcoding-based techniques have been applied to various brain regions (Kebschull et al ., 2016; Han et al, 2018; Chen et al ., 2019; Gergues et al, 2020; Klingler et al, 2021; Mathis et al, 2021; Munoz-Castaneda et al, 2021; Sun et al ., 2021; Chen et al, 2022b) and have validated and extended findings by previous studies using conventional neuroanatomical techniques.…”

Section: Introductionsupporting

confidence: 71%

“…Such space-based rules in connectivity are common in vertebrate brains (e.g. retinotopy, cortical column) and many may exist independent of transcriptomic type-based differences in connectivity (Chen et al ., 2022b); specific spatial patterns of connectivity loss are common in many neurodegenerative diseases. By combining in situ sequencing with barcoded rabies tracing, we expand in situ sequencing-based barcoded neuroanatomical approaches to include anterograde projection mapping (BARseq), multiplexed retrograde tracing, and multiplexed transsynaptic tracing.…”

Section: Discussionmentioning

confidence: 99%

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Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

Zhang

Jin

Yao

et al. 2023

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Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to achieve circuit mapping at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 232 transsynaptically labeled cells using single-cell RNAseq, and 4,130 retrogradely labeled cells and 2,914 transsynaptically labeled cells in situ. We determined the transcriptomic identities of rabies virus-infected cells robustly using both single-cell RNA-seq and in situ sequencing. We then distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus thus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

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Section: Introductionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

Zhang

Jin

Yao

et al. 2023

Preprint

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“…While in wild type mice unbiased machine learning approaches identify at least 5 distinct OR expression patterns, in the triple Nfi cKO mice these patterns become intermixed for all but zone 1 ORs. With recent observations arguing that individual mitral cells, the second order neurons in the olfactory circuit, have patterned projections in the brain [49], non-random OR expression in the MOE may contribute to putative hardwired components of odor perception and valance [50].…”

Section: Discussionmentioning

confidence: 99%

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice

Bashkirova

Klimpert

Pourmorady

et al. 2023

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Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in a probabilistic, yet stereotypic fashion. Here, we show that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription defines spatially constrained OR repertoires, among which one OR allele may be selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization preferentially eliminate from this “privileged” repertoire ORs with more dorsal expression destinations, which are ectopically transcribed in neuronal progenitors throughout the olfactory epithelium. Our experiments identify early transcription as an “epigenetic” contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

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“…MAPseq and BARseq have been repeatedly validated using multiple methods in a wide range of brain areas (Zeisler et al 2023; Y. Chen et al 2022; Hausmann et al 2022; Tsoi et al 2022; Webb et al 2022; Klingler et al 2021; Mathis et al 2021; Muñoz-Castañeda et al 2021; Sun et al 2021; Gergues et al 2020; Huang et al 2020; X. Chen et al 2019; Han et al 2018; Kebschull et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Massive Multiplexing of Spatially Resolved Single Neuron Projections with Axonal BARseq

Yuan

Chen

Zhan

et al. 2023

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Neurons in the cortex are heterogenous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens. Here we present axonal BARseq, a high-throughput approach based on reading out nucleic acid barcodes using in situ RNA sequencing, which enables analysis of even densely labeled neurons. As a proof of principle, we have mapped the long-range projections of >8000 mouse primary auditory cortex neurons from a single brain. We identified major cell types based on projection targets and axonal trajectory. The large sample size enabled us to systematically quantify the projections of intratelencephalic (IT) neurons, and revealed that individual IT neurons project to different layers in an area-dependent fashion. Axonal BARseq is a powerful technique for studying the heterogeneity of single neuronal projections at high throughput within individual brains.

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High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex

Cited by 61 publications

References 132 publications

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice

Massive Multiplexing of Spatially Resolved Single Neuron Projections with Axonal BARseq

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