Morphoelectric and transcriptomic divergence of the layer 1 interneuron repertoire in human versus mouse neocortex.

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 neurons with extensive L1 branching, but whether L1 interneurons are similarly diverse is underexplored. Using Patch-seq recordings from human neurosurgical tissue, we identified four transcriptomic subclasses with mouse L1 homologs, along with distinct subtypes and types unmatched in mouse L1. Subclass and subtype comparisons showed stronger transcriptomic differences in human L1 and were correlated with strong morphoelectric variability along dimensions distinct from mouse L1 variability. Accompanied by greater layer thickness and other cytoarchitecture changes, these findings suggest that L1 has diverged in evolution, reflecting the demands of regulating the expanded human neocortical circuit.

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex.

Identification of structural connections between neurons is a prerequisite to understanding brain function. Here we developed a pipeline to systematically map brain-wide monosynaptic input connections to genetically defined neuronal populations using an optimized rabies tracing system. We used mouse visual cortex as the exemplar system and revealed quantitative target-specific, layer-specific and cell-class-specific differences in its presynaptic connectomes. The retrograde connectivity indicates the presence of ventral and dorsal visual streams and further reveals topographically organized and continuously varying subnetworks mediated by different higher visual areas. The visual cortex hierarchy can be derived from intracortical feedforward and feedback pathways mediated by upper-layer and lower-layer input neurons. We also identify a new role for layer 6 neurons in mediating reciprocal interhemispheric connections. This study expands our knowledge of the visual system connectomes and demonstrates that the pipeline can be scaled up to dissect connectivity of different cell populations across the mouse brain.

Connecting single-cell transcriptomes to projectomes in mouse visual cortex.

The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neuron's role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morpho-electric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.

Local connectivity and synaptic dynamics in mouse and human neocortex.

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.

Human neocortical expansion involves glutamatergic neuron diversification.

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.

Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells.

Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.

Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor ß Signaling.

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the gene encoding fibrillin-1 (FBN1); however, the mechanisms through which fibrillin-1 deficiency causes MFS-associated aortopathy are uncertain. Recently, attention was focused on the hypothesis that MFS-associated aortopathy is caused by increased transforming growth factor-ß (TGF-ß) signaling in aortic medial smooth muscle cells (SMC). However, there are many reasons to doubt that TGF-ß signaling drives MFS-associated aortopathy. We used a mouse model to test whether SMC TGF-ß signaling is perturbed by a fibrillin-1 variant that causes MFS and whether blockade of SMC TGF-ß signaling prevents MFS-associated aortopathy. METHODS AND RESULTS: MFS mice (Fbn1C1039G/+ genotype) were genetically modified to allow postnatal SMC-specific deletion of the type II TGF-ß receptor (TBRII; essential for physiologic TGF-ß signaling). In young MFS mice with and without superimposed deletion of SMC-TBRII, we measured aortic dimensions, histopathology, activation of aortic SMC TGF-ß signaling pathways, and changes in aortic SMC gene expression. Young Fbn1C1039G/+ mice had ascending aortic dilation and significant disruption of aortic medial architecture. Both aortic dilation and disrupted medial architecture were exacerbated by superimposed deletion of TBRII. TGF-ß signaling was unaltered in aortic SMC of young MFS mice; however, SMC-specific deletion of TBRII in Fbn1C1039G/+ mice significantly decreased activation of SMC TGF-ß signaling pathways. CONCLUSIONS: In young Fbn1C1039G/+ mice, aortopathy develops in the absence of detectable alterations in SMC TGF-ß signaling. Loss of physiologic SMC TGF-ß signaling exacerbates MFS-associated aortopathy. Our data support a protective role for SMC TGF-ß signaling during early development of MFS-associated aortopathy.

Postnatal Deletion of the Type II Transforming Growth Factor-ß Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice.

OBJECTIVE: Prenatal deletion of the type II transforming growth factor-ß (TGF-ß) receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-ß signaling, and blockade of TGF-ß signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. APPROACH AND RESULTS: We generated mice with either of 2 tamoxifen-inducible SMC-specific Cre (SMC-CreER(T2)) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreER(T2) alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy, including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-ß signaling and reproduced some of the gene expression abnormalities detected in vivo. CONCLUSIONS: SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-ß signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-ß signaling in humans could cause aortic disease rather than prevent it.