Whole-cortex in situ sequencing reveals peripheral input-dependent cell type-defined area identity

Abstract

The cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture1–3, connectivity4,5, and neuronal activity6–10, are wired into modular networks4,5,11. However, it remains unclear whether cortical areas and their modular organization can be similarly defined by their transcriptomic signatures. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 107 cell type marker genes in 1.2 million cells over a mouse forebrain hemisphere at cellular resolution.De novoclustering of gene expression in single neurons revealed transcriptomic types that were consistent with previous single-cell RNAseq studies12,13. Within medium-grained cell types that are shared across all cortical areas, gene expression and the distribution of fine-grained cell types vary along the contours of cortical areas. The compositions of transcriptomic types are highly predictive of cortical area identity. We grouped cortical areas into modules so that areas within a module, but not across modules, had similar compositions of transcriptomic types. Strikingly, these modules match cortical subnetworks that are highly interconnected4,5,11, suggesting that cortical areas that are similar in cell types are also wired together. This “wire-by-similarity” rule reflects a novel organizing principle for the connectivity of cortical areas. Our BARseq-based strategy is high-throughput and low-cost, and scaling up this approach to many animals can potentially reveal the brain-wide molecular architecture across individuals, developmental times, and disease models.

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

“…Our in situ sequencing approach is based on BARseq, a high-throughput technique that can determine both long-range projections of neurons and their gene expression by in situ sequencing of both endogenous gene expression and an mRNA barcode encoded in the genome of Sindbis virus (Chen et al ., 2019; Sun et al ., 2021). We have previously shown that BARseq-style in situ sequencing can distinguish transcriptomic types of cortical neurons with high transcriptomic resolution in a uninfected mouse brain (Chen et al ., 2022a). To adapt BARseq to rabies barcodes ( Fig.…”

“…In situ sequencing oligos RT primers, sequencing primers, and padlock probes for both endogenous genes and rabies transcripts are designed as previously described (Sun et al, 2021;Chen et al, 2022a). A list of oligos used in in situ sequencing are provided in Table S3.…”

“…As anatomical borders between brain areas (e.g. cortical areas) usually correspond to distinct changes in cytoarchitecture (Brodmann, 1909; Vogt and Vogt, 1919; Von Bonin, 1947) and transcriptomically defined neuronal types (Chen et al, 2022a), retrograde labeling is particularly valuable for understanding how long-range connectivity is associated with cell types and anatomical boundaries.…”

“…Solving these problems will not only allow barcoded rabies virus-based connectivity mapping, but also provide a foundation for potential future techniques based on a wide range of transsynaptic viruses, such as herpes simplex virus (Ugolini et al, 1989; Xiong et al, 2022; Fischer et al, 2023), pseudorabies virus (Martin and Dolivo, 1983), vesicular stomatitis virus (Beier et al, 2013), and yellow fever virus (Li et al, 2021). Because in situ sequencing (Ke et al, 2013; Chen et al ., 2022a) can read out both endogenous mRNAs and random RNA barcodes (Chen et al ., 2019; Sun et al ., 2021) with high throughput and low cost, and does not rely on tissue dissociation (Chen et al ., 2019; Sun et al ., 2021), it is uniquely suited to overcome the challenges associated with barcoded rabies tracing.…”

Circuit-specific selective vulnerability in the DMN persists in the face of widespread amyloid burden

“…We propose that in AD, the selective vulnerability of the DMN is a consequence of intrinsic vulnerability to injury and is not explained by the anatomical specificity of amyloid accumulation. Within DMN regions, the vulnerability of individual neurons to functional disruption is like to be determined by the gene expression underlying connectivity patterns (Whitesell et al, 2021; Chen et al, 2022). The advent of new technologies in measuring mesoscale connectivity, recording neuronal activity, and resolving spatial transcriptomics will make it possible to further disentangle to cellular and circuit rules disease spread through the DMN.…”