Virus&Barcode : The combined application of anatomy and transcriptomics in neural structure analysis
Release time：2024-10-15 17:13:07
Barcode technology in neuroscience research
Barcoding is a technique used in high-throughput sequencing to distinguish between multiple samples by attaching specific "tags" to each sample's sequence. These tags allow for the separation of data from different samples during subsequent data analysis. In other words, a barcode is a short sequence used to differentiate samples within the same "lane" of sequencing. Each sample is assigned a unique barcode, serving as an "identification card" for mixed samples in sequencing experiments.
How to identify and label specific neural circuits within the network of billions of neurons and map connections between different types of neurons? High-throughput sequencing techniques based on barcode sequencing and neuroanatomical techniques offer the potential to map circuits at the cellular resolution and whole-brain scale.
On October 17, 2019, neuroscientist Anthony M. Zador and his team at Cold Spring Harbor Laboratory published a research paper titled "High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing" in the journal Cell. The paper introduced a method called BARseq (barcoded anatomy resolved by sequencing), which combines MAPseq and cell barcode in situ sequencing. This method utilizes high-throughput, multiplexed RNA barcoding to achieve anatomical and transcriptomic connectivity at cellular resolution. It has the potential to uncover the organizational principles of neural circuits, such as the discovery of different, transcriptionally defined subtypes of projection in the mouse auditory cortex. BARseq not only identifies the connectivity of neurons but also determines their gene expression patterns and physiological activities.
Using BARseq, mapping 3,579 neurons projecting to 11 regions of the mouse auditory cortex confirmed the laminar organization of projecting neurons into three top-level categories: intratelencephalic (IT), pyramidal tract-like (PT-like), and corticothalamic (CT). Further analysis revealed a projection type almost entirely confined to transcriptionally defined subtypes of IT neurons.
Figure 1: BARseq for multi-pathway projection studies
Combining barcode technology with rabies virus (RBV) enables the labeling and tracing of specific types of neurons
On April 23, 2021, the team led by Professor Francisco J. Quintana from the Department of Neurology at Harvard University published a research paper titled "Barcoded viral tracing of single-cell interactions in central nervous system inflammation" in the journal Science. The authors designed and developed a cell-cell interaction profiling technology named RABID-seq (rabies barcode interaction detection followed by sequencing), based on rabies virus barcoding. This technique enabled the investigation of cell-cell interactions in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis within the context of central nervous system inflammation.
Rab△G is a powerful tool for studying cell interactions because it can target specific cell types, including astrocytes and other glial cells. To investigate the interactions of astrocytes, the authors designed Rab△G virus to express a mRNA barcode library (Rab△G-mCherry-BC). Fluorescently labeled barcode cells were isolated by flow cytometry, and the transcribed mRNA barcodes in the labeled cells were analyzed using scRNA-seq. The Rab△G-mCherry-BC plasmid library was packaged with envelope protein ENVA, which only infects cells expressing the EnvA receptor and TVA, allowing for genetic targeting of interested cells in vivo. In mice expressing glycoprotein G and TVA under the control of the GFAP promoter, the authors analyzed the cell interaction network of astrocytes. The results showed that RABID-seq can simultaneously identify cell interactions and interaction transcriptomic features of astrocytes at the single-cell level.
Figure 2: Reconstruction of single-cell transcriptomics and connectomics using RABID-seq.
Introducing AAV viruses with molecular barcodes enables the analysis of peripheral vagal neuron networks
On March 16, 2022, a research paper titled "A multidimensional coding architecture of the vagal interoceptive system" was published in the journal Nature by a team led by Dr. Rui Chang, Assistant Professor in the Department of Neurobiology and the Department of Cellular and Molecular Physiology at the Yale School of Medicine, and Dr. Le Zhang, Assistant Professor in the Department of Neurology at Yale School of Medicine.
To decipher how vagal neurons encode signals from different visceral organs, the authors developed a novel single-cell sequencing technology called Projection-seq, which enables unbiased, high-throughput genetic and anatomical analysis of complex neural circuits. Firstly, they used molecular cloning techniques to introduce DNA molecular barcodes called Unique Projection Barcodes (UPBs) into retrogradely tracing adeno-associated viruses (AAVrg). Secondly, the authors simultaneously injected AAV viruses containing different DNA molecular barcodes into various organs of the same mouse, including the lungs, heart, esophagus, stomach, duodenum, colon, and pancreas. One week later, vagal neurons were isolated for single-cell sequencing analysis.
Comprehensive experimental analysis revealed that vagal neurons can utilize different dimensions to encode the three most important feature elements of an interoceptive signal: visceral organs, tissue levels, and stimulus modalities. The multidimensional coding architecture of the mammalian vagal interoceptive system facilitates effective signal communication.
Figure 3: Projection-seq: Unbiased, high-throughput genetic and anatomical analysis of complex neural circuits.
The research on rabies virus(Rbv) barcode technology conducted by the Wickersham lab
On June 8, 2023, a collaborative study led by Xiaoyin Chen from the Allen Institute for Brain Science and Ian Wickersham's research group at the Massachusetts Institute of Technology was published in eLife, titled "Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing". The study validated the feasibility of using rabies virus libraries carrying barcodes for neural pathway resolution in the mouse brain.
This technology applied single-cell RNA sequencing and in situ sequencing to determine the transcriptomic information of cells infected by the rabies virus and distinguish cell types of remote projection cortical cells from multiple cortical regions, identifying cell types with different synaptic connections. RNA barcodes can be combined with two rabies virus-based tracing methods: 1) Using the RV reverse labeling system, rabies viruses lacking glycoprotein G encoding different mRNA libraries (RVΔG) were injected into different brain locations, and single-cell sequencing was used to analyze the barcode information carried by cells infected by upstream virus, distinguishing the different areas projected by the cell; 2) Using the RV reverse transsynaptic system, based on the sequencing analysis of neuronal synaptic connection networks, providing information about neuronal synaptic connections rather than just their axonal projection patterns.
Figure 4: Barcoded rabies multi-pathway retrograde labeling and monosynaptic tracing
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