The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Mnemonic persistent activity in the prefrontal cortex (PFC) constitutes the neural basis of working memory. To understand how neuromodulators contribute to the generation of persistent activity, it is necessary to identify the intrinsic properties of the layer V pyramidal neurons that transfer this information to downstream networks. Here we show that the somatic dynamic and integrative properties of layer V pyramidal neurons in the rat medial PFC depend on whether they project subcortically to the pons [corticopontine (CPn)] or to the contralateral cortex [commissural (COM)]. CPn neurons display low temporal summation and accelerate in firing frequency when depolarized, whereas COM neurons have high temporal summation and display spike frequency accommodation. In response to dynamic stimuli, COM neurons act as low-pass filters, whereas CPn neurons act as bandpass filters, resonating in the theta frequency range (3-6 Hz). The disparate subthreshold properties of COM and CPn neurons can be accounted for by differences in the hyperpolarizationactivated cyclic nucleotide gated cation h-current. Interestingly, neuromodulators hypothesized to enhance mnemonic persistent activity affect COM and CPn neurons distinctly. Adrenergic modulation shifts the dynamic properties of CPn but not COM neurons and increases the excitability of CPn neurons significantly more than COM neurons. In response to cholinergic modulation, CPn neurons were much more likely to display activity-dependent intrinsic persistent firing than COM neurons. Together, these data suggest that the two categories of projection neurons may subserve separate functions in PFC and may be engaged differently during working memory processes.

Neocortical pyramidal neurons with somata in layers 5 and 6 are among the most visually striking and enigmatic neurons in the brain. These deep-layer pyramidal neurons (DLPNs) integrate a plethora of cortical and extracortical synaptic inputs along their impressive dendritic arbors. The pattern of cortical output to both local and long-distance targets is sculpted by the unique physiological properties of specific DLPN subpopulations. Here we revisit two broad DLPN subpopulations: those that send their axons within the telencephalon (intratelencephalic neurons) and those that project to additional target areas outside the telencephalon (extratelencephalic neurons). While neuroscientists across many subdisciplines have characterized the intrinsic and synaptic physiological properties of DLPN subpopulations, our increasing ability to selectively target and manipulate these output neuron subtypes advances our understanding of their distinct functional contributions. This Viewpoints article summarizes our current knowledge about DLPNs and highlights recent work elucidating the functional differences between DLPN subpopulations.