Structural covariation between cerebellum and neocortex intrinsic structural covariation links cerebellum subregions to the cerebral cortex.

The human cerebellum is increasingly recognized to be involved in nonmotor and higher-order cognitive functions. Yet, its ties with the entire cerebral cortex have not been holistically studied in a whole brain exploration with a unified analytical framework. Here, we characterized dissociable cortical-cerebellar structural covariation patterns based on regional gray matter volume (GMV) across the brain in n = 38,527 UK Biobank participants. Our results invigorate previous observations in that important shares of cortical-cerebellar structural covariation are described as 1) a dissociation between the higher-level cognitive system and lower-level sensorimotor system and 2) an anticorrelation between the visual-attention system and advanced associative networks within the cerebellum. We also discovered a novel pattern of ipsilateral, rather than contralateral, cerebral-cerebellar associations. Furthermore, phenome-wide association assays revealed key phenotypes, including cognitive phenotypes, lifestyle, physical properties, and blood assays, associated with each decomposed covariation pattern, helping to understand their real-world implications. This systems neuroscience view paves the way for future studies to explore the implications of these structural covariations, potentially illuminating new pathways in our understanding of neurological and cognitive disorders.NEW & NOTEWORTHY Cerebellum's association with the entire cerebral cortex has not been holistically studied in a unified way. Here, we conjointly characterize the population-level cortical-cerebellar structural covariation patterns leveraging ∼40,000 UK Biobank participants whole brain structural scans and ∼1,000 phenotypes. We revitalize the previous hypothesis of an anticorrelation between the visual-attention system and advanced associative networks within the cerebellum. We also discovered a novel ipsilateral cerebral-cerebellar associations. Phenome-wide association (PheWAS) revealed real-world implications of the structural covariation patterns.

Where Do Core Thalamocortical Axons Terminate in Mammalian Neocortex When There Is No Cytoarchitecturally Distinct Layer 4?

Although the mammalian cerebral cortex is most often described as a hexalaminar structure, there are cortical areas (primary motor cortex) and species (elephants, cetaceans, and hippopotami), where a cytoarchitecturally indistinct, or absent, layer 4 is noted. Thalamocortical projections from the core, or first order, thalamic system terminate primarily in layers 4/inner 3. We explored the termination sites of core thalamocortical projections in cortical areas and in species where there is no cytoarchitecturally distinct layer 4 using the immunolocalization of vesicular glutamate transporter 2, a known marker of core thalamocortical axon terminals, in 31 mammal species spanning the eutherian radiation. Several variations from the canonical cortical column outline of layer 4 and core thalamocortical inputs were noted. In shrews/microchiropterans, layer 4 was present, but many core thalamocortical projections terminated in layer 1 in addition to layers 4 and inner 3. In primate primary visual cortex, the sublaminated layer 4 was associated with a specialized core thalamocortical projection pattern. In primate primary motor cortex, no cytoarchitecturally distinct layer 4 was evident and the core thalamocortical projections terminated throughout layer 3. In the African elephant, cetaceans, and river hippopotamus, no cytoarchitecturally distinct layer 4 was observed and core thalamocortical projections terminated primarily in inner layer 3 and less densely in outer layer 3. These findings are contextualized in terms of cortical processing, perception, and the evolutionary trajectory leading to an indistinct or absent cortical layer 4.

MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size.

Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small non-coding RNAs known to play a role in human brain development and neurogenesis. Here, we performed small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, measured 907 expressed miRNAs, discovering 111 of which were novel, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and brain size phenotypes, where the miRNA expression increasing allele was associated with decreased brain size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells decreased expression of predicted targets and increased cell proliferation, indicating miR-4707-3p modulates progenitor gene regulation and cell fate decisions. Integrating miRNA-eQTLs with existing GWAS yielded evidence of a miRNA that may influence human brain size and function via modulation of neocortical brain development.

Patterned cPCDH expression regulates the fine organization of the neocortex.

The neocortex consists of a vast number of diverse neurons that form distinct layers and intricate circuits at the single-cell resolution to support complex brain functions1. Diverse cell-surface molecules are thought to be key for defining neuronal identity, and they mediate interneuronal interactions for structural and functional organization2-6. However, the precise mechanisms that control the fine neuronal organization of the neocortex remain largely unclear. Here, by integrating in-depth single-cell RNA-sequencing analysis, progenitor lineage labelling and mosaic functional analysis, we report that the diverse yet patterned expression of clustered protocadherins (cPCDHs)-the largest subgroup of the cadherin superfamily of cell-adhesion molecules7-regulates the precise spatial arrangement and synaptic connectivity of excitatory neurons in the mouse neocortex. The expression of cPcdh genes in individual neocortical excitatory neurons is diverse yet exhibits distinct composition patterns linked to their developmental origin and spatial positioning. A reduction in functional cPCDH expression causes a lateral clustering of clonally related excitatory neurons originating from the same neural progenitor and a significant increase in synaptic connectivity. By contrast, overexpression of a single cPCDH isoform leads to a lateral dispersion of clonally related excitatory neurons and a considerable decrease in synaptic connectivity. These results suggest that patterned cPCDH expression biases fine spatial and functional organization of individual neocortical excitatory neurons in the mammalian brain.

Phenotypic Alterations in Cortical Organization and Connectivity across Different Time Scales.

In the following review, we describe the types of phenotypic changes to the neocortex that occur over the longer time scale of evolution, and over the shorter time scale of an individual lifetime. To understand how phenotypic variability emerges in the neocortex, it is important to consider the cortex as part of an integrated system of the brain, the body, the environment in which the brain and body develops and evolves, and the affordances available within a particular environmental context; changes in any part of this brain/body/environment network impact the neocortex. We provide data from comparative studies on a wide variety of mammals that demonstrate that body morphology, the sensory epithelium, and the use of a particular morphological structure have a profound impact on neocortical organization and connections. We then discuss the genetic and epigenetic factors that contribute to the development of the neocortex, as well as the role of spontaneous and sensory driven activity in constructing a nervous system. Although the evolution of the neocortex cannot be studied directly, studies in which developmental processes are experimentally manipulated provide important insights into how phenotypic transformations could occur over the course of evolution and demonstrate that relatively small alterations to the body and/or the environment in which an individual develops can manifest as large changes to the neocortex. Finally, we discuss how these phenotypic alterations to the neocortex impact an important target of selection - behavior.

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.

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.

Topographic gradients of intrinsic dynamics across neocortex.

The intrinsic dynamics of neuronal populations are shaped by both microscale attributes and macroscale connectome architecture. Here we comprehensively characterize the rich temporal patterns of neural activity throughout the human brain. Applying massive temporal feature extraction to regional haemodynamic activity, we systematically estimate over 6000 statistical properties of individual brain regions' time-series across the neocortex. We identify two robust spatial gradients of intrinsic dynamics, one spanning a ventromedial-dorsolateral axis and dominated by measures of signal autocorrelation, and the other spanning a unimodal-transmodal axis and dominated by measures of dynamic range. These gradients reflect spatial patterns of gene expression, intracortical myelin and cortical thickness, as well as structural and functional network embedding. Importantly, these gradients are correlated with patterns of meta-analytic functional activation, differentiating cognitive versus affective processing and sensory versus higher-order cognitive processing. Altogether, these findings demonstrate a link between microscale and macroscale architecture, intrinsic dynamics, and cognition.

The human cerebellum has almost 80% of the surface area of the neocortex.

The surface of the human cerebellar cortex is much more tightly folded than the cerebral cortex. It was computationally reconstructed for the first time to the level of all individual folia from multicontrast high-resolution postmortem MRI scans. Its total shrinkage-corrected surface area (1,590 cm2) was larger than expected or previously reported, equal to 78% of the total surface area of the human neocortex. The unfolded and flattened surface comprised a narrow strip 10 cm wide but almost 1 m long. By applying the same methods to the neocortex and cerebellum of the macaque monkey, we found that its cerebellum was relatively much smaller, approximately 33% of the total surface area of its neocortex. This suggests a prominent role for the cerebellum in the evolution of distinctively human behaviors and cognition.

Human-specific ARHGAP11B increases size and folding of primate neocortex in the fetal marmoset.

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

Individual-Level Identification of Gene Expression Associated with Volume Differences among Neocortical Areas.

The human cerebral cortex is the source of many complex behaviors and is a vulnerable target of various neuropsychiatric disorders, but transcriptional profiles linked to cerebral cortical volume (CCV) differences across brain areas remain unknown. Here, we screened CCV-related genes using an across-sample spatial correlation analysis in 6 postmortem brains and then individually validated these correlations in 1091 subjects with different ages and ethnicities. We identified 62 genes whose transcriptional profiles were repeatedly associated with CCV in more than 90% of individuals. CCV-related genes were specifically expressed in neurons and in developmental periods from middle childhood to young adulthood, were enriched in ion channels and developmental processes, and showed significant overlap with genes linked to brain functional activity and mental disorders. The identified genes represent the conserved transcriptional architecture of the human cerebral cortex, suggesting a link between conserved gene transcription and neocortical structural properties.

Sequential pattern of sublayer formation in the paleocortex and neocortex.

The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.

Comparative neocortical neuromorphology in felids: African lion, African leopard, and cheetah.

The present study examines cortical neuronal morphology in the African lion (Panthera leo leo), African leopard (Panthera pardus pardus), and cheetah (Acinonyx jubatus jubatus). Tissue samples were removed from prefrontal, primary motor, and primary visual cortices and investigated with a Golgi stain and computer-assisted morphometry to provide somatodendritic measures of 652 neurons. Although neurons in the African lion were insufficiently impregnated for accurate quantitative dendritic measurements, descriptions of neuronal morphologies were still possible. Qualitatively, the range of spiny and aspiny neurons across the three species was similar to those observed in other felids, with typical pyramidal neurons being the most prominent neuronal type. Quantitatively, somatodendritic measures of typical pyramidal neurons in the cheetah were generally larger than in the African leopard, despite similar brain sizes. A MARsplines analysis of dendritic measures correctly differentiated 87.4% of complete typical pyramidal neurons between the African leopard and cheetah. In addition, unbiased stereology was used to compare the soma size of typical pyramidal neurons (n = 2,238) across all three cortical regions and gigantopyramidal neurons (n = 1,189) in primary motor and primary visual cortices. Both morphological and stereological analyses indicated that primary motor gigantopyramidal neurons were exceptionally large across all three felids compared to other carnivores, possibly due to specializations related to the felid musculoskeletal systems. The large size of these neurons in the cheetah which, unlike lions and leopards, does not belong to the Panthera genus, suggests that exceptionally enlarged primary motor gigantopyramidal neurons evolved independently in these felid species.

The origin and evolution of neocortex: From early mammals to modern humans.

Human neocortex evolved in a series of ancestors with less neocortex and fewer cortical areas. Thus, early mammals had little neocortex and roughly 20 cortical areas, while early primates had much more cortex and around 50 cortical areas. Humans have the largest of primate brains that is 80% neocortex with about 200 areas. Other changes include more and more complex cortical networks, such as those for language, and modular and cellular specializations within areas. These and other changes allow the impressive mental abilities of humans.

Allometry, evolution and development of neocortex size in mammals.

Variation in neocortex size is one of the defining features of mammalian brain evolution. The paramount assumption has been that neocortex size indicates a monotonic allometric relationship with brain size. This assumption holds the concomitant neurodevelopmental assumption that the ontogenetic trajectory of neocortex size is so stable across species that it restrains changes in the direction of evolution. Here we test this fundamental assumption. Whereas previous research has focused exclusively on changes in mean size among groups (i.e., intercept), we additionally investigate changes in covariation (i.e., slope) and strength of allometric integration (i.e., residual variation). We further increase data resolution by investigating 350 species representing 11 mammalian orders. Results identify nine shifts in covariation between neocortex and brainstem in different mammalian groups, indicate that these shifts occur independently of shifts in size, and demonstrate that the strength of allometric integration across different neocortical regions in primates is inversely related to the neurodevelopmental gradient such that later developing regions underwent more evolutionary change. Although our results confirm that variation in brain organization is structured along a neurodevelopmental gradient, our results suggest two additional principles of size reorganization in brain evolution: (1) repatterning of growth allocation among brain regions may occur independently of size and (2) later developing regions indicate faster evolution, not necessarily directional evolution toward larger size. We conclude that the evolution of neocortex size in mammals is far more variable than previously assumed, in turn suggesting a higher degree of evolutionary flexibility in neurodevelopmental patterning than commonly suggested.

Distinct Patterns of Hippocampal and Neocortical Evolution in Primates.

Because of the central role of the hippocampus in representing spatial and temporal details of experience, comparative studies of its volume and structure are relevant to understanding the evolution of representational memory across species. The hippocampal formation, however, is organized into separate anatomical subregions with distinct functions, and little is known about the evolutionary diversification of these subregions. We investigate relative volumetric changes in hippocampal subregions across a large sample of primate species. We then compare the evolution of the hippocampal formation to the neocortex. Results across hippocampal subregions indicate that, compared to strepsirrhines, the anthropoid lineage displays a decrease in relative CA3, fascia dentata, subiculum, and rhinal cortex volume in tandem with an increase in relative neocortical volume. These findings indicate that hippocampal function in anthropoids might be substantially augmented by the executive decision-making functions of the neocortex. Humans are found to have a unique cerebral organization combining increased relative CA3, subiculum, and rhinal cortex with increased relative neocortical volumes, suggesting that these regions may play a role in behaviors that are uniquely specialized in humans.

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling approach for optically cleared human cortex samples, which can be applied to large (up to 5 mm thick) formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dye protocols in combination with new refractive index matching solutions allows deep volume imaging. This greatly reduces time and cost of imaging cytoarchitecture in thick samples and enables classification of cytoarchitectonic layers over the full cortical depth. We demonstrate application of MASH to large archival samples of human visual areas, characterizing cortical architecture in 3D from the scale of cortical areas to that of single cells. In combination with scalable light sheet imaging and data analysis, MASH could open the door to investigation of large human cortical systems at cellular resolution and in the context of their complex 3-dimensional geometry.

Evolution of neocortical folding: A phylogenetic comparative analysis of MRI from 34 primate species.

We conducted a comparative analysis of primate cerebral size and neocortical folding using magnetic resonance imaging data from 65 individuals belonging to 34 different species. We measured several neocortical folding parameters and studied their evolution using phylogenetic comparative methods. Our results suggest that the most likely model for neuroanatomical evolution is one where differences appear randomly (the Brownian Motion model), however, alternative models cannot be completely ruled out. We present estimations of the ancestral primate phenotypes as well as estimations of the rates of phenotypic change. Based on the Brownian Motion model, the common ancestor of primates may have had a folded cerebrum similar to that of a small lemur such as the aye-aye. Finally, we observed a non-linear relationship between fold wavelength and fold depth with cerebral volume. In particular, gyrencephalic primate neocortices across different groups exhibited a strikingly stable fold wavelength of about 12 mm (±20%), despite a 20-fold variation in cerebral volume. We discuss our results in the context of current theories of neocortical folding.