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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.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The highly distinctive morpho-electric properties of these neurons have mainly been described in rodents, where ET neurons can be labeled by retrograde tracers or transgenic lines. Similar labeling strategies are not possible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically-defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology and morphology are assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were also apparent but were often smaller than differences between cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy, but also highlight marked phenotypic divergence possibly related to functional specialization of human neocortex.