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Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Mosaic organoids where pathological and healthy cells are grown together, reveal the rescue of phenotypes in pathological cells due to communication with healthy cells without harming them, as demonstrated by single-cell RNA-sequencing data.

Despite the importance of the brain's neocortex, we still do not completely understand the diversity and functional connections of its cell types. Jiang et al. recorded, labeled, and classified over 1200 interneurons and more than 400 pyramidal neurons in the mature mouse visual cortex. Fifteen major classes of interneurons fell into three types: some connect to all neurons, some connect to other interneurons, and some form synapses with pyramidal neurons. Science , this issue p. 10.1126/science.aac9462 The connections between more than 10,000 pairs of individually classified neurons in the visual cortex of adult mice are mapped. Since the work of Ramón y Cajal in the late 19th and early 20th centuries, neuroscientists have speculated that a complete understanding of neuronal cell types and their connections is key to explaining complex brain functions. However, a complete census of the constituent cell types and their wiring diagram in mature neocortex remains elusive. By combining octuple whole-cell recordings with an optimized avidin-biotin-peroxidase staining technique, we carried out a morphological and electrophysiological census of neuronal types in layers 1, 2/3, and 5 of mature neocortex and mapped the connectivity between more than 11,000 pairs of identified neurons. We categorized 15 types of interneurons, and each exhibited a characteristic pattern of connectivity with other interneuron types and pyramidal cells. The essential connectivity structure of the neocortical microcircuit could be captured by only a few connectivity motifs.

Optogenetics allows manipulations of genetically and spatially defined neuronal populations with excellent temporal control. However, neurons are coupled with other neurons over multiple length scales, and the effects of localized manipulations thus spread beyond the targeted neurons. We benchmarked several optogenetic methods to inactivate small regions of neocortex. Optogenetic excitation of GABAergic neurons produced more effective inactivation than light-gated ion pumps. Transgenic mice expressing the light-dependent chloride channel GtACR1 produced the most potent inactivation. Generally, inactivation spread substantially beyond the photostimulation light, caused by strong coupling between cortical neurons. Over some range of light intensity, optogenetic excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks ('paradoxical effect'). The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, limiting temporal resolution. Our data offer guidance for the design of in vivo optogenetics experiments.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses 1 , 2 ; however, the molecular mechanisms that govern astrocyte–synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte–neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function. A cell-surface fragment complementation strategy is used to identify the proteome at the junction of astrocytes and synapses in vivo, and shows that NRCAM expressed in astrocytes has a key role in regulating inhibitory synapse function.

Reinforcement learning models postulate that neurons that release dopamine encode information about action and action outcome, and provide a teaching signal to striatal spiny projection neurons in the form of dopamine release 1 . Dopamine is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of spiny projection neuron 2 . However, the real-time relationship between dopamine and PKA in spiny projection neurons remains untested in behaving animals. Here we monitor the activity of dopamine-releasing neurons, extracellular levels of dopamine and net PKA activity in spiny projection neurons in the nucleus accumbens of mice during learning. We find positive and negative modulation of dopamine that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in the PKA activity of spiny projection neurons. Modulations of PKA in spiny projection neurons that express type-1 and type-2 dopamine receptors are dichotomous, such that these neurons are selectively sensitive to increases and decreases, respectively, in dopamine that occur at different phases of learning. Thus, PKA-dependent pathways in each class of spiny projection neuron are asynchronously engaged by positive or negative dopamine signals during learning. The net PKA activities in each class of spiny projection neuron in the nucleus accumbens of the mouse are dichotomously modulated by asynchronous positive and negative dopamine signals during different phases of learning.

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 brain 1 , 2 . Single-cell epigenomic profiling 3 , 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. As part of the BICCN consortium, the authors used a single-cell transcriptomic imaging method to produce a highly defined atlas of cell types across the mouse primary motor cortex.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals 1 . 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. An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties 1 , 2 . Most existing neural taxonomies are based on either transcriptomic 3 , 4 or morpho-electric 5 , 6 criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells 7 . Here we used Patch-seq 8 to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of more than 1,300 neurons in adult mouse primary motor cortex, providing a morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (those expressing Vip , Pvalb , Sst and so on) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neuronal types in the neocortex do not always form discrete entities. Instead, neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families. Single-cell transcriptomic, morphological and electrophysiological characteristics are combined to classify more than 1,300 neurons from mouse motor cortex.

The adult brain contains dozens of different types of interneurons that control and refine neuronal circuits. Mi et al. used single-cell transcriptomics to investigate when these subtypes emerge during interneuron development in the mouse. Transcriptomes of embryonic interneurons showed similarities to adult classes of differentiated interneurons, thus dividing the immature embryonic interneurons themselves into classes. Nearly a dozen classes of embryonic neurons could be identified soon after their last mitosis by transcriptomic similarity with known classes of adult cortical interneurons. Thus, the fate of embryonic interneurons can be read in their transcriptomes well before the neurons migrate and reach their final sites of differentiation and circuit integration. Science , this issue p. 81 Single-cell transcriptomics reveals embryonic correlates of adult interneuron classes. GABAergic interneurons (GABA, γ-aminobutyric acid) regulate neural-circuit activity in the mammalian cerebral cortex. These cortical interneurons are structurally and functionally diverse. Here, we use single-cell transcriptomics to study the origins of this diversity in the mouse. We identify distinct types of progenitor cells and newborn neurons in the ganglionic eminences, the embryonic proliferative regions that give rise to cortical interneurons. These embryonic precursors show temporally and spatially restricted transcriptional patterns that lead to different classes of interneurons in the adult cerebral cortex. Our findings suggest that shortly after the interneurons become postmitotic, their diversity is already patent in their diverse transcriptional programs, which subsequently guide further differentiation in the developing cortex.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates 1 , 2 . Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development 3 . Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells 4 . We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning 5 in the hypothalamus. In particular, loss of SLIT–ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life. Single-cell RNA sequencing reveals molecular determinants of the developmental programs that orchestrate the intermingling of neuronal subtypes in the hypothalamus.