Unsupervised restoration of a complex learned behavior after large-scale neuronal perturbation.

Reliable execution of precise behaviors requires that brain circuits are resilient to variations in neuronal dynamics. Genetic perturbation of the majority of excitatory neurons in HVC, a brain region involved in song production, in adult songbirds with stereotypical songs triggered severe degradation of the song. The song fully recovered within 2 weeks, and substantial improvement occurred even when animals were prevented from singing during the recovery period, indicating that offline mechanisms enable recovery in an unsupervised manner. Song restoration was accompanied by increased excitatory synaptic input to neighboring, unmanipulated neurons in the same brain region. A model inspired by the behavioral and electrophysiological findings suggests that unsupervised single-cell and population-level homeostatic plasticity rules can support the functional restoration after large-scale disruption of networks that implement sequential dynamics. These observations suggest the existence of cellular and systems-level restorative mechanisms that ensure behavioral resilience.

Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

Engineering RNA export for measurement and manipulation of living cells.

A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.

Recovery of a learned behavior despite partial restoration of neuronal dynamics after chronic inactivation of inhibitory neurons.

Maintaining motor skills is crucial for an animal's survival, enabling it to endure diverse perturbations throughout its lifespan, such as trauma, disease, and aging. What mechanisms orchestrate brain circuit reorganization and recovery to preserve the stability of behavior despite the continued presence of a disturbance? To investigate this question, we chronically silenced a fraction of inhibitory neurons in a brain circuit necessary for singing in zebra finches. Song in zebra finches is a complex, learned motor behavior and central to reproduction. This manipulation altered brain activity and severely perturbed song for around two months, after which time it was precisely restored. Electrophysiology recordings revealed abnormal offline dynamics, resulting from chronic inhibition loss, some aspects of which returned to normal as the song recovered. However, even after the song had fully recovered, the levels of neuronal firing in the premotor and motor areas did not return to a control-like state. Single-cell RNA sequencing revealed that chronic silencing of interneurons led to elevated levels of microglia and MHC I, which were also observed in normal juveniles during song learning. These experiments demonstrate that the adult brain can overcome extended periods of abnormal activity, and precisely restore a complex behavior, without recovering normal neuronal dynamics. These findings suggest that the successful functional recovery of a brain circuit after a perturbation can involve more than mere restoration to its initial configuration. Instead, the circuit seems to adapt and reorganize into a new state capable of producing the original behavior despite the persistence of some abnormal neuronal dynamics.

Embryo-scale, single-cell spatial transcriptomics.

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.

Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches.

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

Imaging cell lineage with a synthetic digital recording system.

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.

Macropinocytosis-mediated membrane recycling drives neural crest migration by delivering F-actin to the lamellipodium.

Individual cell migration requires front-to-back polarity manifested by lamellipodial extension. At present, it remains debated whether and how membrane motility mediates this cell morphological change. To gain insights into these processes, we perform live imaging and molecular perturbation of migrating chick neural crest cells in vivo. Our results reveal an endocytic loop formed by circular membrane flow and anterograde movement of lipid vesicles, resulting in cell polarization and locomotion. Rather than clathrin-mediated endocytosis, macropinosomes encapsulate F-actin in the cell body, forming vesicles that translocate via microtubules to deliver actin to the anterior. In addition to previously proposed local conversion of actin monomers to polymers, we demonstrate a surprising role for shuttling of F-actin across cells for lamellipodial expansion. Thus, the membrane and cytoskeleton act in concert in distinct subcellular compartments to drive forward cell migration.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Lineage does not regulate the sensory synaptic input of projection neurons in the mouse olfactory bulb.

Lineage regulates the synaptic connections between neurons in some regions of the invertebrate nervous system. In mammals, recent experiments suggest that cell lineage determines the connectivity of pyramidal neurons in the neocortex, but the functional relevance of this phenomenon and whether it occurs in other neuronal types remains controversial. We investigated whether lineage plays a role in the connectivity of mitral and tufted cells, the projection neurons in the mouse olfactory bulb. We used transgenic mice to sparsely label neuronal progenitors and observed that clonally related neurons receive synaptic input from olfactory sensory neurons expressing different olfactory receptors. These results indicate that lineage does not determine the connectivity between olfactory sensory neurons and olfactory bulb projection neurons.

Persistence of neuronal representations through time and damage in the hippocampus.

How do neurons encode long-term memories? Bilateral imaging of neuronal activity in the mouse hippocampus reveals that, from one day to the next, ~40% of neurons change their responsiveness to cues, but thereafter only 1% of cells change per day. Despite these changes, neuronal responses are resilient to a lack of exposure to a previously completed task or to hippocampus lesions. Unlike individual neurons, the responses of which change after a few days, groups of neurons with inter- and intrahemispheric synchronous activity show stable responses for several weeks. The likelihood that a neuron maintains its responsiveness across days is proportional to the number of neurons with which its activity is synchronous. Information stored in individual neurons is relatively labile, but it can be reliably stored in networks of synchronously active neurons.

Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice.

Autism spectrum disorder (ASD) manifests as alterations in complex human behaviors including social communication and stereotypies. In addition to genetic risks, the gut microbiome differs between typically developing (TD) and ASD individuals, though it remains unclear whether the microbiome contributes to symptoms. We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors. The brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes. Microbiome and metabolome profiles of mice harboring human microbiota predict that specific bacterial taxa and their metabolites modulate ASD behaviors. Indeed, treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain. We propose that the gut microbiota regulates behaviors in mice via production of neuroactive metabolites, suggesting that gut-brain connections contribute to the pathophysiology of ASD.

In Vivo Quantitative Imaging Provides Insights into Trunk Neural Crest Migration.

Neural crest (NC) cells undergo extensive migrations during development. Here, we couple in vivo live imaging at high resolution with custom software tools to reveal dynamic migratory behavior in chick embryos. Trunk NC cells migrate as individuals with both stochastic and biased features as they move dorsoventrally to form peripheral ganglia. Their leading edge displays a prominent fan-shaped lamellipodium that reorients upon cell-cell contact. Computational analysis reveals that when the lamellipodium of one cell touches the body of another, the two cells undergo "contact attraction," often moving together and then separating via a pulling force exerted by lamellipodium. Targeted optical manipulation shows that cell interactions coupled with cell density generate a long-range biased random walk behavior, such that cells move from high to low density. In contrast to chain migration noted at other axial levels, the results show that individual trunk NC cells navigate the complex environment without tight coordination between neighbors.

Hierarchical neural architecture underlying thirst regulation.

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.

Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT).

Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to one another. We describe a technique called TRACT (TRAnsneuronal Control of Transcription) based on ligand-induced intramembrane proteolysis to reveal monosynaptic connections arising from genetically labeled neurons of interest. In this strategy, neurons expressing an artificial ligand ('donor' neurons) bind to and activate a genetically-engineered artificial receptor on their synaptic partners ('receiver' neurons). Upon ligand-receptor binding at synapses the receptor is cleaved in its transmembrane domain and releases a protein fragment that activates transcription in the synaptic partners. Using TRACT in Drosophila we have confirmed the connectivity between olfactory receptor neurons and their postsynaptic targets, and have discovered potential new connections between neurons in the circadian circuit. Our results demonstrate that the TRACT method can be used to investigate the connectivity of neuronal circuits in the brain.

Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.

Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.

Methods to investigate the structure and connectivity of the nervous system.

Understanding the computations that take place in neural circuits requires identifying how neurons in those circuits are connected to one another. In addition, recent research indicates that aberrant neuronal wiring may be the cause of several neurodevelopmental disorders, further emphasizing the importance of identifying the wiring diagrams of brain circuits. To address this issue, several new approaches have been recently developed. In this review, we describe several methods that are currently available to investigate the structure and connectivity of the brain, and discuss their strengths and limitations.

Determination of the connectivity of newborn neurons in mammalian olfactory circuits.

The mammalian olfactory bulb is a forebrain structure just one synapse downstream from the olfactory sensory neurons and performs the complex computations of sensory inputs. The formation of this sensory circuit is shaped through activity-dependent and cell-intrinsic mechanisms. Recent studies have revealed that cell-type specific connectivity and the organization of synapses in dendritic compartments are determined through cell-intrinsic programs already preset in progenitor cells. These progenitor programs give rise to subpopulations within a neuron type that have distinct synaptic organizations. The intrinsically determined formation of distinct synaptic organizations requires factors from contacting cells that match the cell-intrinsic programs. While certain genes control wiring within the newly generated neurons, other regulatory genes provide intercellular signals and are only expressed in neurons that will form contacts with the newly generated cells. Here, the olfactory system has provided a useful model circuit to reveal the factors regulating assembly of the highly structured connectivity in mammals.

Unstable neurons underlie a stable learned behavior.

Motor skills can be maintained for decades, but the biological basis of this memory persistence remains largely unknown. The zebra finch, for example, sings a highly stereotyped song that is stable for years, but it is not known whether the precise neural patterns underlying song are stable or shift from day to day. Here we demonstrate that the population of projection neurons coding for song in the premotor nucleus, HVC, change from day to day. The most dramatic shifts occur over intervals of sleep. In contrast to the transient participation of excitatory neurons, ensemble measurements dominated by inhibition persist unchanged even after damage to downstream motor nerves. These observations offer a principle of motor stability: spatiotemporal patterns of inhibition can maintain a stable scaffold for motor dynamics while the population of principal neurons that directly drive behavior shift from one day to the next.