Spatially patterned excitatory neuron subtypes and projections of the claustrum

Abstract

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

Influence of claustrum on cortex varies by area, layer, and cell type

“…In terms of the cell populations, the three previous studies that include CLA stimulation and neural recordings in the cortex used three different methods for expressing channelrhodpsin in CLA (Fodoulian et al, 2020, Vglut2-Cre mouse with AAV-DIO-ChR2-YFP injection in CLA; Jackson et al, 2018, AAVretro-syn-Cre in PFC, AAV5-DIO-ChR2-YFP in CLA; Narikiyo et al, 2020, Cla-Cre mouse with AAV-EF1a-DIO-ChR2-EYFP injection in CLA). One reason for these differences is that the mouse CLA is difficult to isolate, and its exact cellular and anatomical definition remains unsettled, so there is not one agreed upon operational method for labeling CLA neurons (Erwin et al, 2021; Mathur et al, 2009; Wang et al, 2022). It is quite likely that these different molecular targeting methods resulted in different populations of CLA or non-CLA neurons expressing ChR2, which when stimulated could produce different effects in the cortex.…”

Developmental Patterning and Neurogenetic Gradients of Nurr1 Positive Neurons in the Rat Claustrum and Lateral Cortex

“…In several anatomical studies (e.g., Witter et al, 1988 ), the claustrum is relatively broadly defined, encompassing a range of nuclei that are now identified as distinct subregions such as the dorsal claustrum (dCL), ventral claustrum (vCL), and the dorsal endopiriform nucleus (DEn). Much like other cortical regions, the claustrum consists of roughly 90% glutamatergic neurons ( Druga, 2014 ), and typical claustrum markers like Nurr1 (also known as Nr4a2 ) label a large fraction of these neurons ( Erwin et al, 2021 ). Recent studies of the claustrum using circuit-based and transcriptomic approaches ( Wang et al, 2017 ; Erwin et al, 2021 ; Marriott et al, 2021 ; Peng et al, 2021 ) allow a more precise definition of the claustrum and related nuclei.…”

“…Much like other cortical regions, the claustrum consists of roughly 90% glutamatergic neurons ( Druga, 2014 ), and typical claustrum markers like Nurr1 (also known as Nr4a2 ) label a large fraction of these neurons ( Erwin et al, 2021 ). Recent studies of the claustrum using circuit-based and transcriptomic approaches ( Wang et al, 2017 ; Erwin et al, 2021 ; Marriott et al, 2021 ; Peng et al, 2021 ) allow a more precise definition of the claustrum and related nuclei. Intriguingly, while there is an ever expanding list of gene markers mainly expressed in the claustrum ( Zeisel et al, 2015 ; Wang et al, 2017 ; Smith et al, 2019 ; Bruguier et al, 2020 ; Erwin et al, 2021 ; Yao et al, 2021 ), a large fraction of these genes are expressed not only in the claustrum proper, but also consistently in other regions, such as the DEn, the subplate (Sp), and – in rodents – in the deep layers of lateral neocortex ( Arimatsu, 1994 ; Puelles, 2014 ; Bruguier et al, 2020 ; Ratié et al, 2020 ).…”

“…Recent studies of the claustrum using circuit-based and transcriptomic approaches ( Wang et al, 2017 ; Erwin et al, 2021 ; Marriott et al, 2021 ; Peng et al, 2021 ) allow a more precise definition of the claustrum and related nuclei. Intriguingly, while there is an ever expanding list of gene markers mainly expressed in the claustrum ( Zeisel et al, 2015 ; Wang et al, 2017 ; Smith et al, 2019 ; Bruguier et al, 2020 ; Erwin et al, 2021 ; Yao et al, 2021 ), a large fraction of these genes are expressed not only in the claustrum proper, but also consistently in other regions, such as the DEn, the subplate (Sp), and – in rodents – in the deep layers of lateral neocortex ( Arimatsu, 1994 ; Puelles, 2014 ; Bruguier et al, 2020 ; Ratié et al, 2020 ). Knowledge about the developmental patterning of claustrum cell types could help to understand their adult distribution and function.…”

Global Spatial Transcriptome of Macaque Brain at Single-Cell Resolution

“…From snRNA-seq data on tissues that contained claustrum and the adjacent cortex, we identified two distinct groups of excitatory neuron clusters based on the expression of previously identified claustrum marker genes(Erwin et al, 2021) ( GNB4 , NTNG2 and NR4A2 ; Figure S10B, C). Our snRNA-seq analysis revealed no inhibitory claustrum neurons, in line with previous reports (Erwin et al, 2021; Graf et al, 2020; Kim et al, 2016). However, our Stereo-seq data revealed a small population of claustrum neurons expressing GAD1 or GAD2 (Figure S10D), indicating the existence of inhibitory neurons that were not detected by the snRNA-seq method.…”