Early cortical GABAergic interneurons determine the projection patterns of L4 excitatory neurons.

During cerebral cortex development, excitatory pyramidal neurons (PNs) establish specific projection patterns while receiving inputs from GABAergic inhibitory interneurons (INs). Whether these inhibitory inputs can shape PNs' projection patterns is, however, unknown. While layer 4 (L4) PNs of the primary somatosensory (S1) cortex are all born as long-range callosal projection neurons (CPNs), most of them acquire local connectivity upon activity-dependent elimination of their interhemispheric axons during postnatal development. Here, we demonstrate that precise developmental regulation of inhibition is key for the retraction of S1L4 PNs' callosal projections. Ablation of somatostatin INs leads to premature inhibition from parvalbumin INs onto S1L4 PNs and prevents them from acquiring their barrel-restricted local connectivity pattern. As a result, adult S1L4 PNs retain interhemispheric projections responding to tactile stimuli, and the mice lose whisker-based texture discrimination. Overall, we show that temporally ordered IN activity during development is key to shaping local ipsilateral S1L4 PNs' projection pattern, which is required for fine somatosensory processing.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Regulation of early cerebellar development.

The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.

A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum.

SignificanceOutside the neurogenic niches, the adult brain lacks multipotent progenitor cells. In this study, we performed a series of in vivo screens and reveal that a single factor can induce resident brain astrocytes to become induced neural progenitor cells (iNPCs), which then generate neurons, astrocytes, and oligodendrocytes. Such a conclusion is supported by single-cell RNA sequencing and multiple lineage-tracing experiments. Our discovery of iNPCs is fundamentally important for regenerative medicine since neural injuries or degeneration often lead to loss/dysfunction of all three neural lineages. Our findings also provide insights into cell plasticity in the adult mammalian brain, which has largely lost the regenerative capacity.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

Individual human cortical progenitors can produce excitatory and inhibitory neurons.

The cerebral cortex is a cellularly complex structure comprising a rich diversity of neuronal and glial cell types. Cortical neurons can be broadly categorized into two classes-excitatory neurons that use the neurotransmitter glutamate, and inhibitory interneurons that use γ-aminobutyric acid (GABA). Previous developmental studies in rodents have led to a prevailing model in which excitatory neurons are born from progenitors located in the cortex, whereas cortical interneurons are born from a separate population of progenitors located outside the developing cortex in the ganglionic eminences1-5. However, the developmental potential of human cortical progenitors has not been thoroughly explored. Here we show that, in addition to excitatory neurons and glia, human cortical progenitors are also capable of producing GABAergic neurons with the transcriptional characteristics and morphologies of cortical interneurons. By developing a cellular barcoding tool called 'single-cell-RNA-sequencing-compatible tracer for identifying clonal relationships' (STICR), we were able to carry out clonal lineage tracing of 1,912 primary human cortical progenitors from six specimens, and to capture both the transcriptional identities and the clonal relationships of their progeny. A subpopulation of cortically born GABAergic neurons was transcriptionally similar to cortical interneurons born from the caudal ganglionic eminence, and these cells were frequently related to excitatory neurons and glia. Our results show that individual human cortical progenitors can generate both excitatory neurons and cortical interneurons, providing a new framework for understanding the origins of neuronal diversity in the human cortex.

Single-cell delineation of lineage and genetic identity in the mouse brain.

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of postmitotic neurons and glia1,2. The link between developmental lineage and cell-type diversity remains an open question. Here we used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence across all major cell classes postnatally, and found diverse types of GABAergic neuron that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis upon cell-cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor cell level.

Dentate gyrus and CA3 GABAergic interneurons bidirectionally modulate signatures of internal and external drive to CA1.

Specific classes of GABAergic neurons play specific roles in regulating information processing in the brain. In the hippocampus, two major classes, parvalbumin-expressing (PV+) and somatostatin-expressing (SST+), differentially regulate endogenous firing patterns and target subcellular compartments of principal cells. How these classes regulate the flow of information throughout the hippocampus is poorly understood. We hypothesize that PV+ and SST+ interneurons in the dentate gyrus (DG) and CA3 differentially modulate CA3 patterns of output, thereby altering the influence of CA3 on CA1. We find that while suppressing either interneuron class increases DG and CA3 output, the effects on CA1 were very different. Suppressing PV+ interneurons increases local field potential signatures of coupling from CA3 to CA1 and decreases signatures of coupling from entorhinal cortex to CA1; suppressing SST+ interneurons has the opposite effect. Thus, DG and CA3 PV+ and SST+ interneurons bidirectionally modulate the flow of information through the hippocampal circuit.

Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks.

Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons.

Spatially resolved cell atlas of the mouse primary motor cortex by MERFISH.

A mammalian brain is composed of numerous cell types organized in an intricate manner to form functional neural circuits. Single-cell RNA sequencing allows systematic identification of cell types based on their gene expression profiles and has revealed many distinct cell populations in the brain1,2. Single-cell epigenomic profiling3,4 further provides information on gene-regulatory signatures of different cell types. Understanding how different cell types contribute to brain function, however, requires knowledge of their spatial organization and connectivity, which is not preserved in sequencing-based methods that involve cell dissociation. Here we used a single-cell transcriptome-imaging method, multiplexed error-robust fluorescence in situ hybridization (MERFISH)5, to generate a molecularly defined and spatially resolved cell atlas of the mouse primary motor cortex. We profiled approximately 300,000 cells in the mouse primary motor cortex and its adjacent areas, identified 95 neuronal and non-neuronal cell clusters, and revealed a complex spatial map in which not only excitatory but also most inhibitory neuronal clusters adopted laminar organizations. Intratelencephalic neurons formed a largely continuous gradient along the cortical depth axis, in which the gene expression of individual cells correlated with their cortical depths. Furthermore, we integrated MERFISH with retrograde labelling to probe projection targets of neurons of the mouse primary motor cortex and found that their cortical projections formed a complex network in which individual neuronal clusters project to multiple target regions and individual target regions receive inputs from multiple neuronal clusters.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

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.

Isoform cell-type specificity in the mouse primary motor cortex.

Full-length SMART-seq1 single-cell RNA sequencing can be used to measure gene expression at isoform resolution, making possible the identification of specific isoform markers for different cell types. Used in conjunction with spatial RNA capture and gene-tagging methods, this enables the inference of spatially resolved isoform expression for different cell types. Here, in a comprehensive analysis of 6,160 mouse primary motor cortex cells assayed with SMART-seq, 280,327 cells assayed with MERFISH2 and 94,162 cells assayed with 10x Genomics sequencing3, we find examples of isoform specificity in cell types-including isoform shifts between cell types that are masked in gene-level analysis-as well as examples of transcriptional regulation. Additionally, we show that isoform specificity helps to refine cell types, and that a multi-platform analysis of single-cell transcriptomic data leveraging multiple measurements provides a comprehensive atlas of transcription in the mouse primary motor cortex that improves on the possibilities offered by any single technology.

Cellular anatomy of the mouse primary motor cortex.

An essential step toward understanding brain function is to establish a structural framework with cellular resolution on which multi-scale datasets spanning molecules, cells, circuits and systems can be integrated and interpreted1. Here, as part of the collaborative Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based anatomical description of one exemplar brain structure, the mouse primary motor cortex, upper limb area (MOp-ul). Using genetic and viral labelling, barcoded anatomy resolved by sequencing, single-neuron reconstruction, whole-brain imaging and cloud-based neuroinformatics tools, we delineated the MOp-ul in 3D and refined its sublaminar organization. We defined around two dozen projection neuron types in the MOp-ul and derived an input-output wiring diagram, which will facilitate future analyses of motor control circuitry across molecular, cellular and system levels. This work provides a roadmap towards a comprehensive cellular-resolution description of mammalian brain architecture.

Tonic GABAA Receptor-Mediated Currents of Human Cortical GABAergic Interneurons Vary Amongst Cell Types.

Persistent anion conductances through GABAA receptors (GABAARs) are important modulators of neuronal excitability. However, it is currently unknown how the amplitudes of these currents vary among different cell types in the human neocortex, particularly among diverse GABAergic interneurons. We have recorded 101 interneurons in and near layer 1 from cortical tissue surgically resected from both male and female patients, visualized 84 of them and measured tonic GABAAR currents in 48 cells with an intracellular [Cl-] of 65 mm and in the presence of 5 µm GABA. We compare these tonic currents among five groups of interneurons divided by firing properties and four types of interneuron defined by axonal distributions; rosehip, neurogliaform, stalked-bouton, layer 2-3 innervating and a pool of other cells. Interestingly, the rosehip cell, a type of interneuron only described thus far in human tissue, and layer 2-3 innervating cells exhibit larger tonic currents than other layer 1 interneurons, such as neurogliaform and stalked-bouton cells; the latter two groups showing no difference. The positive allosteric modulators of GABAARs allopregnanolone and DS2 also induced larger current shifts in the rosehip and layer 2-3 innervating cells, consistent with higher expression of the δ subunit of the GABAAR in these neurons. We have also examined how patient parameters, such as age, seizures, type of cancer and anticonvulsant treatment may alter tonic inhibitory currents in human neurons. The cell type-specific differences in tonic inhibitory currents could potentially be used to selectively modulate cortical circuitry.SIGNIFICANCE STATEMENT Tonic currents through GABAA receptors (GABAARs) are a potential therapeutic target for a number of neurologic and psychiatric conditions. Here, we show that these currents in human cerebral cortical GABAergic neurons display cell type-specific differences in their amplitudes which implies differential modulation of their excitability. Additionally, we examine whether the amplitudes of the tonic currents measured in our study show any differences between patient populations, finding some evidence that age, seizures, type of cancer, and anticonvulsant treatment may alter tonic inhibition in human tissue. These results advance our understanding of how pathology affects neuronal excitability and could potentially be used to selectively modulate cortical circuitry.

Homeostatic control of nuclear-encoded mitochondrial gene expression by the histone variant H2A.Z is essential for neuronal survival.

Histone variants are crucial regulators of chromatin structure and gene transcription, yet their functions within the brain remain largely unexplored. Here, we show that the H2A histone variant H2A.Z is essential for neuronal survival. Mice lacking H2A.Z in GABAergic neurons or Purkinje cells (PCs) present with a progressive cerebellar ataxia accompanied by widespread degeneration of PCs. Ablation of H2A.Z in other neuronal subtypes also triggers cell death. H2A.Z binds to the promoters of key nuclear-encoded mitochondrial genes to regulate their expression and promote organelle function. Bolstering mitochondrial activity genetically or by organelle transplant enhances the survival of H2A.Z-ablated neurons. Changes in bioenergetic status alter H2A.Z occupancy at the promoters of nuclear-encoded mitochondrial genes, an adaptive response essential for cell survival. Our results highlight that H2A.Z fulfills a key, conserved role in neuronal survival by acting as a transcriptional rheostat to regulate the expression of genes critical to mitochondrial function.

Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Direct Differentiation of Functional Neurons from Human Pluripotent Stem Cells (hPSCs).

Somatic cell nuclear transfer and in vitro induction of pluripotency in somatic cells by defined factors provided unambiguous evidence that the epigenetic state of terminally differentiated somatic cells is not static and can be reversed to a more primitive one. Inspired by these results, stem cell biologists have identified approaches to directly convert fibroblasts into induced neuronal (iN) cells, indicating that direct lineage conversions are possible between distantly related cell types. More recently, we took advantages of pro-neurogenic capacity of iN factors and developed methods to rapidly derive functionally mature neurons directly from human pluripotent stem cells (hPSCs) through a brief induction of defined transcription factors. In this chapter, we describe the detailed methods used to attain the direct conversion from hPSCs to glutamatergic and GABAergic iN cells.

FoxG1 regulates the formation of cortical GABAergic circuit during an early postnatal critical period resulting in autism spectrum disorder-like phenotypes.

Abnormalities in GABAergic inhibitory circuits have been implicated in the aetiology of autism spectrum disorder (ASD). ASD is caused by genetic and environmental factors. Several genes have been associated with syndromic forms of ASD, including FOXG1. However, when and how dysregulation of FOXG1 can result in defects in inhibitory circuit development and ASD-like social impairments is unclear. Here, we show that increased or decreased FoxG1 expression in both excitatory and inhibitory neurons results in ASD-related circuit and social behavior deficits in our mouse models. We observe that the second postnatal week is the critical period when regulation of FoxG1 expression is required to prevent subsequent ASD-like social impairments. Transplantation of GABAergic precursor cells prior to this critical period and reduction in GABAergic tone via Gad2 mutation ameliorates and exacerbates circuit functionality and social behavioral defects, respectively. Our results provide mechanistic insight into the developmental timing of inhibitory circuit formation underlying ASD-like phenotypes in mouse models.

A functional model of adult dentate gyrus neurogenesis.

In adult dentate gyrus neurogenesis, the link between maturation of newborn neurons and their function, such as behavioral pattern separation, has remained puzzling. By analyzing a theoretical model, we show that the switch from excitation to inhibition of the GABAergic input onto maturing newborn cells is crucial for their proper functional integration. When the GABAergic input is excitatory, cooperativity drives the growth of synapses such that newborn cells become sensitive to stimuli similar to those that activate mature cells. When GABAergic input switches to inhibitory, competition pushes the configuration of synapses onto newborn cells toward stimuli that are different from previously stored ones. This enables the maturing newborn cells to code for concepts that are novel, yet similar to familiar ones. Our theory of newborn cell maturation explains both how adult-born dentate granule cells integrate into the preexisting network and why they promote separation of similar but not distinct patterns.

A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.