Cohesin prevents cross-domain gene coactivation.

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

The cell-type-specific spatial organization of the anterior thalamic nuclei of the mouse brain.

Understanding the cell-type composition and spatial organization of brain regions is crucial for interpreting brain computation and function. In the thalamus, the anterior thalamic nuclei (ATN) are involved in a wide variety of functions, yet the cell-type composition of the ATN remains unmapped at a single-cell and spatial resolution. Combining single-cell RNA sequencing, spatial transcriptomics, and multiplexed fluorescent in situ hybridization, we identify three discrete excitatory cell-type clusters that correspond to the known nuclei of the ATN and uncover marker genes, molecular pathways, and putative functions of these cell types. We further illustrate graded spatial variation along the dorsomedial-ventrolateral axis for all individual nuclei of the ATN and additionally demonstrate that the anteroventral nucleus exhibits spatially covarying protein products and long-range inputs. Collectively, our study reveals discrete and continuous cell-type organizational principles of the ATN, which will help to guide and interpret experiments on ATN computation and function.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Sharp cell-type-identity changes differentiate the retrosplenial cortex from the neocortex.

The laminae of the neocortex are fundamental processing layers of the mammalian brain. Notably, such laminae are believed to be relatively stereotyped across short spatial scales such that shared laminae between nearby brain regions exhibit similar constituent cells. Here, we consider a potential exception to this rule by studying the retrosplenial cortex (RSC), a brain region known for sharp cytoarchitectonic differences across its granular-dysgranular border. Using a variety of transcriptomics techniques, we identify, spatially map, and interpret the excitatory cell-type landscape of the mouse RSC. In doing so, we uncover that RSC gene expression and cell types change sharply at the granular-dysgranular border. Additionally, supposedly homologous laminae between the RSC and the neocortex are effectively wholly distinct in their cell-type composition. In collection, the RSC exhibits a variety of intrinsic cell-type specializations and embodies an organizational principle wherein cell-type identities can vary sharply within and between brain regions.

Multimodal mapping of cell types and projections in the central nucleus of the amygdala.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Neuronal cell types, projections, and spatial organization of the central amygdala.

The central amygdala (CEA) has been richly studied for interpreting function and behavior according to specific cell types and circuits. Such work has typically defined molecular cell types by classical inhibitory marker genes; consequently, whether marker-gene-defined cell types exhaustively cover the CEA and co-vary with connectivity remains unresolved. Here, we combined single-cell RNA sequencing, multiplexed fluorescent in situ hybridization, immunohistochemistry, and long-range projection mapping to derive a "bottom-up" understanding of CEA cell types. In doing so, we identify two major cell types, encompassing one-third of all CEA neurons, that have gone unresolved in previous studies. In spatially mapping these novel types, we identify a non-canonical CEA subdomain associated with Nr2f2 expression and uncover an Isl1-expressing medial cell type that accounts for many long-range CEA projections. Our results reveal new CEA organizational principles across cell types and spatial scales and provide a framework for future work examining cell-type-specific behavior and function.

EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson's disease.

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

Spatially patterned excitatory neuron subtypes and projections of the claustrum.

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

A novel family of secreted insect proteins linked to plant gall development.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.

The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive "LAMPshade" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

Behavioral state coding by molecularly defined paraventricular hypothalamic cell type ensembles.

Brains encode behaviors using neurons amenable to systematic classification by gene expression. The contribution of molecular identity to neural coding is not understood because of the challenges involved with measuring neural dynamics and molecular information from the same cells. We developed CaRMA (calcium and RNA multiplexed activity) imaging based on recording in vivo single-neuron calcium dynamics followed by gene expression analysis. We simultaneously monitored activity in hundreds of neurons in mouse paraventricular hypothalamus (PVH). Combinations of cell-type marker genes had predictive power for neuronal responses across 11 behavioral states. The PVH uses combinatorial assemblies of molecularly defined neuron populations for grouped-ensemble coding of survival behaviors. The neuropeptide receptor neuropeptide Y receptor type 1 (Npy1r) amalgamated multiple cell types with similar responses. Our results show that molecularly defined neurons are important processing units for brain function.

Extensive and spatially variable within-cell-type heterogeneity across the basolateral amygdala.

The basolateral amygdala complex (BLA), extensively connected with both local amygdalar nuclei as well as long-range circuits, is involved in a diverse array of functional roles. Understanding the mechanisms of such functional diversity will be greatly informed by understanding the cell-type-specific landscape of the BLA. Here, beginning with single-cell RNA sequencing, we identified both discrete and graded continuous gene-expression differences within the mouse BLA. Via in situ hybridization, we next mapped this discrete transcriptomic heterogeneity onto a sharp spatial border between the basal and lateral amygdala nuclei, and identified continuous spatial gene-expression gradients within each of these regions. These discrete and continuous spatial transformations of transcriptomic cell-type identity were recapitulated by local morphology as well as long-range connectivity. Thus, BLA excitatory neurons are a highly heterogenous collection of neurons that spatially covary in molecular, cellular, and circuit properties. This heterogeneity likely drives pronounced spatial variation in BLA computation and function.

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics.

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso and Rubin, 2016). Here, we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

Publisher Correction: A repeated molecular architecture across thalamic pathways.

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

A repeated molecular architecture across thalamic pathways.

The thalamus is the central communication hub of the forebrain and provides the cerebral cortex with inputs from sensory organs, subcortical systems and the cortex itself. Multiple thalamic regions send convergent information to each cortical region, but the organizational logic of thalamic projections has remained elusive. Through comprehensive transcriptional analyses of retrogradely labeled thalamic neurons in adult mice, we identify three major profiles of thalamic pathways. These profiles exist along a continuum that is repeated across all major projection systems, such as those for vision, motor control and cognition. The largest component of gene expression variation in the mouse thalamus is topographically organized, with features conserved in humans. Transcriptional differences between these thalamic neuronal identities are tied to cellular features that are critical for function, such as axonal morphology and membrane properties. Molecular profiling therefore reveals covariation in the properties of thalamic pathways serving all major input modalities and output targets, thus establishing a molecular framework for understanding the thalamus.

Mapping the transcriptional diversity of genetically and anatomically defined cell populations in the mouse brain.

Understanding the principles governing neuronal diversity is a fundamental goal for neuroscience. Here, we provide an anatomical and transcriptomic database of nearly 200 genetically identified cell populations. By separately analyzing the robustness and pattern of expression differences across these cell populations, we identify two gene classes contributing distinctly to neuronal diversity. Short homeobox transcription factors distinguish neuronal populations combinatorially, and exhibit extremely low transcriptional noise, enabling highly robust expression differences. Long neuronal effector genes, such as channels and cell adhesion molecules, contribute disproportionately to neuronal diversity, based on their patterns rather than robustness of expression differences. By linking transcriptional identity to genetic strains and anatomical atlases, we provide an extensive resource for further investigation of mouse neuronal cell types.

The subiculum is a patchwork of discrete subregions.

In the hippocampus, the classical pyramidal cell type of the subiculum acts as a primary output, conveying hippocampal signals to a diverse suite of downstream regions. Accumulating evidence suggests that the subiculum pyramidal cell population may actually be comprised of discrete subclasses. Here, we investigated the extent and organizational principles governing pyramidal cell heterogeneity throughout the mouse subiculum. Using single-cell RNA-seq, we find that the subiculum pyramidal cell population can be deconstructed into eight separable subclasses. These subclasses were mapped onto abutting spatial domains, ultimately producing a complex laminar and columnar organization with heterogeneity across classical dorsal-ventral, proximal-distal, and superficial-deep axes. We further show that these transcriptomically defined subclasses correspond to differential protein products and can be associated with specific projection targets. This work deconstructs the complex landscape of subiculum pyramidal cells into spatially segregated subclasses that may be observed, controlled, and interpreted in future experiments.