Regional and cell type-specific afferent and efferent projections of the mouse claustrum

Abstract

The claustrum (CLA) is a conspicuous subcortical structure interconnected with cortical and subcortical regions. However, its regional anatomy and cell-type-specific connections in the mouse remain largely undetermined. Here, we accurately delineated the boundary of the mouse CLA and quantitatively investigated its inputs and outputs brain-wide using anterograde and retrograde viral tracing and fully reconstructed single claustral principal neurons. At a population level, the CLA reciprocally connects with all isocortical modules. It also receives inputs from at least 35 subcortical structures but sends projections back to only a few of them. We found that cell types projecting to the CLA are differentiated by cortical areas and layers. We classified single CLA principal neurons into at least 9 cell types that innervate the diverse sets of functionally linked cortical targets. Axons of interneurons within the CLA arborize along almost its entire anteroposterior extent. Together, this detailed wiring diagram of the cell-type-specific connections of the mouse CLA lays a foundation for studying its functions.

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

“…This could indicate that Esr2+ CLA neurons are more likely to innervate RSP than PFC. This would be consistent with the finding that neurons in what some refer to as the CLA "core", which is likely to correspond with the location of these Esr2+ neurons, project strongly to RSP (Marriott et al, 2020;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. For example, while Jackson et al (2018) found that claustrum neurons innervated layer 6 of PFC more strongly than layer 2/3, other studies have found the opposite pattern (Fodoulian et al, 2020;Wang et al, 2017;Wang et al 2022), which may explain differences between their findings of a primarily inhibitory effect, and ours and those of Fodoulian et al (2020) of a primarily excitatory effect.…”

“…Though the Gnb4-IRES2-CreERT2 transgenic line expresses Cre largely specifically to CLA, there are still some non-CLA cells that express Cre, particularly in the deep layers of the overlying insular cortex (Peng et al, 2021;Wang et al, 2017). Therefore, we sought to replicate the above experiments in a different transgenic mouse line, Esr2-IRES2-Cre, which more selectively expresses Cre in CLA than the Gnb4 line, albeit less completely (Figure 4A; Wang et al, 2022). We crossed the Esr2-IRES-Cre line to the channelrhodopsin reporter line, Ai32, and implanted optic fiber cannula as with the Gnb4-Ai32 mice (Figure 4A,B).…”

“…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.…”

“…Whole-brain reconstructions of single CLA neurons have revealed that individual neurons project to an average of 21 cortical regions (Peng et al, 2021;Wang et al, 2022). In addition, CLA neurons are arranged topographically; projections from anterior CLA neurons are biased to innervate anterior cortical areas, while posterior CLA neurons project to posterior cortex (Wang et al, 2022). Slice electrophysiology experiments have demonstrated that CLA neurons can be categorized into several different types based on their intrinsic electrical properties (Chia et al, 2017;Graf et al, 2020); though it is unknown whether these correlate with differences in cortical projection targets.…”