Fast imaging of millimeter-scale areas with beam deflection transmission electron microscopy

Zheng, Zhihao; Own, Christopher S.; Wanner, Adrian; Koene, Randal A.; Hammerschmith, Eric W.; Silversmith, William; Kemnitz, Nico; Tank, David W; Seung, H. Sebastian

doi:10.1101/2022.11.23.517701

“…1B). Although other techniques, such as electron microscopy (Bae et al, 2021;Androvic et al, 2022;Zheng et al, 2022;Schneider-Mizell et al, 2023) and multi-patch experiments (Campagnola et al, 2022), can also read out synaptic connectivity, these approaches are difficult to scale up to many neurons across large areas of the brain and/or difficult to relate connectivity to the gene expression of neurons. Thus, achieving barcoded transsynaptic tracing using rabies virus could potentially transform the scale at which synaptic connectivity of transcriptomic types of neurons can be interrogated at cellular resolution.…”

Section: Multiplexed Retrograde Labeling and Monosynaptic Tracing Usi...mentioning

confidence: 99%

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

Zhang

¹

,

Jin

²

,

Yao

³

et al. 2024

eLife

1

0

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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 295 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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“…1B ). Although other techniques, such as electron microscopy (Bae et al, 2021; Androvic et al, 2022; Zheng et al, 2022; Schneider-Mizell et al, 2023) and multi-patch experiments (Campagnola et al, 2022), can also read out synaptic connectivity, these approaches are difficult to scale up to many neurons across large areas of the brain and/or difficult to relate connectivity to the gene expression of neurons. Thus, achieving barcoded transsynaptic tracing using rabies virus could potentially transform the scale at which synaptic connectivity of transcriptomic types of neurons can be interrogated at cellular resolution.…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

¹

,

Jin

²

,

Yao

³

et al. 2023

Preprint

0

View full text Add to dashboard Cite

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.

show abstract

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

Zhang

¹

,

Jin

²

,

Yao

³

et al. 2023

Preprint

View full text Add to dashboard Cite

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 295 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.

show abstract

Fast imaging of millimeter-scale areas with beam deflection transmission electron microscopy

Cited by 4 publications

References 63 publications

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

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

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

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