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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., Combined patch clamp recording, biocytin staining and single-cell RNA-sequencing of human neurocortical neurons shows an expansion of glutamatergic neuron types relative to mouse that characterizes the greater complexity of the human neocortex.

Human cortical interneurons have been challenging to study due to high diversity and lack of mature brain tissue platforms and genetic targeting tools. We employed rapid GABAergic neuron viral labeling plus unbiased Patch-seq sampling in brain slices to define the signature morpho-electric properties of GABAergic neurons in the human neocortex. Viral targeting greatly facilitated sampling of the SST subclass, including primate specialized double bouquet cells which mapped to two SST transcriptomic types. Multimodal analysis uncovered an SST neuron type with properties inconsistent with original subclass assignment; we instead propose reclassification into PVALB subclass. Our findings provide novel insights about functional properties of human cortical GABAergic neuron subclasses and types and highlight the essential role of multimodal annotation for refinement of emerging transcriptomic cell type taxonomies.One Sentence SummaryViral genetic labeling of GABAergic neurons in humanex vivobrain slices paired with Patch-seq recording yields an in-depth functional annotation of human cortical interneuron subclasses and types and highlights the essential role of multimodal functional annotation for refinement of emerging transcriptomic cell type taxonomies.

Neocortical layer 1 (L1) is a site of convergence between pyramidal neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neuron types with extensive branching in L1, but whether L1 interneurons are similarly diverse is underexplored. Using patch-seq recordings from human neurosurgically resected tissues, we identified four transcriptomically defined subclasses, unique subtypes within those subclasses and additional types with no mouse L1 homologue. Compared with mouse, human subclasses were more strongly distinct from each other across all modalities. Accompanied by higher neuron density and more variable cell sizes compared with mouse, these findings suggest L1 is an evolutionary hotspot, reflecting the increasing demands of regulating the expanding human neocortical circuit.One Sentence SummaryUsing transcriptomics and morpho-electric analyses, we describe innovations in human neocortical layer 1 interneurons.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the average connectivity of several interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells, and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type-specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together, these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.

To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

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 activity and connectivity of inhibitory cells has a profound impact on the operation of neuronal networks. While the average connectivity of many inhibitory cell types has been characterized, we still lack an understanding of how individual interneurons distribute their synapses onto their targets and how heterogeneous the inhibition is onto different individual excitatory neurons. Here, we use large-scale volumetric electron microscopy (EM) and functional imaging to address this question for chandelier cells in layer 2/3 of mouse visual cortex. Using dense morphological reconstructions from EM, we mapped the complete chandelier input onto 153 pyramidal neurons. We find that the number of input synapses is highly variable across the population, but the variability is correlated with structural features of the target neuron: soma depth, soma size, and the number of perisomatic synapses received. Functionally, we found that chandelier cell activity in vivo was highly correlated and tracks pupil diameter, a proxy for arousal state. We propose that chandelier cells provide a global signal whose strength is individually adjusted for each target neuron. This approach, combining comprehensive structural analysis with functional recordings of identified cell types, will be a powerful tool to uncover the wiring rules across the diversity of cortical cell types.

Sensory contribution to perception and action depends on both sensory receptors and the organization of pathways (or projections) reaching the central nervous system. Unlike the semicircular canals that are divided into three discrete sensitivity directions, the utricle has a relatively complicated anatomical structure, including sensitivity directions over essentially $$360^{\circ }$$ of a curved, two-dimensional disk. The utricle is not flat, and we do not assume it to be. Directional sensitivity of individual utricular afferents decreases in a cosine-like fashion from peak excitation for movement in one direction to a null or near null response for a movement in an orthogonal direction. Directional sensitivity varies slowly between neighboring cells except within the striolar region that separates the medial from the lateral zone, where the directional selectivity abruptly reverses along the reversal line. Utricular primary afferent pathways reach the vestibular nuclei and cerebellum and, in many cases, converge on target cells with semicircular canal primary afferents and afference from other sources. Mathematically, some canal pathways are known to be characterized by symmetry groups related to physical space. These groups structure rotational information and movement. They divide the target neural center into distinct populations according to the innervation patterns they receive. Like canal pathways, utricular pathways combine symmetries from the utricle with those from target neural centers. This study presents a generic set of transformations drawn from the known structure of the utricle and therefore likely to be found in utricular pathways, but not exhaustive of utricular pathway symmetries. This generic set of transformations forms a 32-element group that is a semi-direct product of two simple abelian groups. Subgroups of the group include order-four elements corresponding to discrete rotations. Evaluation of subgroups allows us to functionally identify the spatial implications of otolith and canal symmetries regarding action and perception. Our results are discussed in relation to observed utricular pathways, including those convergent with canal pathways. Oculomotor and other sensorimotor systems are organized according to canal planes. However, the utricle is evolutionarily prior to the canals and may provide a more fundamental spatial framework for canal pathways as well as for movement. The fullest purely otolithic pathway is likely that which reaches the lumbar spine via Deiters’ cells in the lateral vestibular nucleus. It will be of great interest to see whether symmetries predicted from the utricle are identified within this pathway.