The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer’s disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knockins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

The neocortex is disproportionately expanded in human compared to mouse, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers that selectively make connections within the cortex and other telencephalic structures. Single-cell transcriptomic analyses of human and mouse cortex show an increased diversity of glutamatergic neuron types in supragranular cortex in human and pronounced gradients as a function of cortical depth. To probe the functional and anatomical correlates of this transcriptomic diversity, we describe a robust Patch-seq platform using neurosurgically-resected human tissues. We characterize the morphological and physiological properties of five transcriptomically defined human glutamatergic supragranular neuron types. Three of these types have properties that are specialized compared to the more homogeneous properties of transcriptomically defined homologous mouse neuron types. The two remaining supragranular neuron types, located exclusively in deep layer 3, do not have clear mouse homologues in supragranular cortex but are transcriptionally most similar to deep layer mouse intratelencephalic-projecting neuron types. Furthermore, we reveal the transcriptomic types in deep layer 3 that express high levels of non-phosphorylated heavy chain neurofilament protein that label long-range neurons known to be selectively depleted in Alzheimer’s disease. Together, these results demonstrate the power of transcriptomic cell type classification, provide a mechanistic underpinning for increased complexity of cortical function in human cortical evolution, and implicate discrete transcriptomic cell types as selectively vulnerable in disease.

The left temporal lobe is an integral part of the language system and its cortical structure and function associate with general intelligence. However, whether cortical laminar architecture and cellular properties of this brain area relate to verbal intelligence is unknown. Here, we addressed this using histological analysis and cellular recordings of neurosurgically resected temporal cortex in combination with presurgical IQ scores. We find that subjects with higher general and verbal IQ scores have thicker left (but not right) temporal cortex (Brodmann area 21, BA21). The increased thickness is due to the selective increase in layers 2 and 3 thickness, accompanied by lower neuron densities, and larger dendrites and cell body size of pyramidal neurons in these layers. Furthermore, these neurons sustain faster action potential kinetics, which improves information processing. Our results indicate that verbal mental ability associates with selective adaptations of supragranular layers and their cellular micro-architecture and function in left, but not right temporal cortex.

Synaptic transmission constitutes the primary mode of communication between neurons. It is extensively studied in rodent but not human neocortex. We characterized synaptic transmission between pyramidal neurons in layers 2 and 3 using neurosurgically resected human middle temporal gyrus (MTG, Brodmann area 21), which is part of the distributed language circuitry. We find that local connectivity is comparable with mouse layer 2/3 connections in the anatomical homologue (temporal association area), but synaptic connections in human are 3-fold stronger and more reliable (0% vs 25% failure rates, respectively). We developed a theoretical approach to quantify properties of spinous synapses showing that synaptic conductance and voltage change in human dendritic spines are 3–4-folds larger compared with mouse, leading to significant NMDA receptor activation in human unitary connections. This model prediction was validated experimentally by showing that NMDA receptor activation increases the amplitude and prolongs decay of unitary excitatory postsynaptic potentials in human but not in mouse connections. Since NMDA-dependent recurrent excitation facilitates persistent activity (supporting working memory), our data uncovers cortical microcircuit properties in human that may contribute to language processing in MTG.

Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have surprisingly fast input-output properties: rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human pyramidal neurons can explain their fast input-output properties. Human Na+ and K+ currents had depolarized voltage-dependence, slower inactivation and exhibited a faster recovery from inactivation than their mouse counterparts. Computational modeling showed that despite lower Na+ channel densities in human neurons, the biophysical properties of Na+ channels resulted in higher channel availability and contributed to fast AP kinetics stability. Finally, human Na+ channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+ and K+ channels enable fast input-output properties of large human pyramidal neurons.

The expansion of supragranular cortical layers is thought to have enabled evolutionary development of human cognition and language. However, whether increased volume of supragranular cortical layers can actually support greater cognitive and language abilities in humans has not been demonstrated. Here, we find that subjects with higher general and verbal intelligence test (VIQ) scores have selectively expanded layers 2 and 3 only in the left temporal cortex, an area associated with language and IQ-test performance. This expansion is accompanied by lower neuron densities and larger cell-body size. Furthermore, individuals with higher VIQ scores had neurons with larger dendritic trees in left temporal cortex, potentially impacting their function. Indeed, neurons of subjects with higher VIQ scores had faster action potential upstroke kinetics, which improves information processing. These data show that expansion of supragranular layer volume, cortical and cellular micro-architecture and function are associated with improved verbal mental ability in human subjects.

In rodent cortical networks, fast spiking interneurons (FSINs) provide fast inhibition that synchronizes neuronal activity and is critical for cognitive function. Fast synchronization frequencies are evolutionary conserved in the expanded human neocortex, despite larger neuron-to-neuron distances that challenge fast input-output transfer functions of FSINs. Here, we test which mechanistic specializations of large human FSINs explain their fast-signaling properties in human cortex. With morphological reconstructions, multi-patch recordings, and biophysical modeling we find that despite three-fold longer dendritic path lengths, human FSINs maintain fast inhibition between connected pyramidal neurons through several mechanisms: stronger synapse strength of excitatory inputs, larger dendrite diameter with reduced complexity, faster AP initiation, and faster and larger inhibitory output, while Na+ current activation /inactivation properties are similar. These adaptations underlie short input-output delays in fast inhibition of human pyramidal neurons through FSINs, explaining how cortical synchronization frequencies are conserved despite expanded and sparse network topology of human cortex.