Neurogliaform Cells Exhibit Laminar-specific Responses in the Visual Cortex and Modulate Behavioral State-dependent Cortical Activity.

Neurogliaform cells are a distinct type of GABAergic cortical interneurons known for their 'volume transmission' output property. However, their activity and function within cortical circuits remain unclear. Here, we developed two genetic tools to target these neurons and examine their function in the primary visual cortex. We found that the spontaneous activity of neurogliaform cells positively correlated with locomotion. Silencing these neurons increased spontaneous activity during locomotion and impaired visual responses in L2/3 pyramidal neurons. Furthermore, the contrast-dependent visual response of neurogliaform cells varies with their laminar location and is constrained by their morphology and input connectivity. These findings demonstrate the importance of neurogliaform cells in regulating cortical behavioral state-dependent spontaneous activity and indicate that their functional engagement during visual stimuli is influenced by their laminar positioning and connectivity.

Neocortical long-range inhibition promotes cortical synchrony and sleep.

Behavioral states such as sleep and wake are highly correlated with specific patterns of rhythmic activity in the cortex. During low arousal states such as slow wave sleep, the cortex is synchronized and dominated by low frequency rhythms coordinated across multiple regions. Although recent evidence suggests that GABAergic inhibitory neurons are key players in cortical state modulation, the in vivo circuit mechanisms coordinating synchronized activity among local and distant neocortical networks are not well understood. Here, we show that somatostatin and chondrolectin co-expressing cells (Sst-Chodl cells), a sparse and unique class of neocortical inhibitory neurons, are selectively active during low arousal states and are largely silent during periods of high arousal. In contrast to other neocortical inhibitory neurons, we show these neurons have long-range axons that project across neocortical areas. Activation of Sst-Chodl cells is sufficient to promote synchronized cortical states characteristic of low arousal, with increased spike co-firing and low frequency brain rhythms, and to alter behavioral states by promoting sleep. Contrary to the prevailing belief that sleep is exclusively driven by subcortical mechanisms, our findings reveal that these long-range inhibitory neurons not only track changes in behavioral state but are sufficient to induce both sleep-like cortical states and sleep behavior, establishing a crucial circuit component in regulating behavioral states.

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Neurogliaform Cells Exhibit Laminar-specific Responses in the Visual Cortex and Modulate Behavioral State-dependent Cortical Activity.

Neurogliaform cells are a distinct type of GABAergic cortical interneurons known for their "volume transmission" output property. However, their activity and function within cortical circuits remain unclear. Here, we developed two genetic tools to target these neurons and examine their function in the primary visual cortex. We found that the spontaneous activity of neurogliaform cells positively correlated with locomotion. Silencing these neurons increased spontaneous activity during locomotion and impaired visual responses in L2/3 pyramidal neurons. Furthermore, the contrast-dependent visual response of neurogliaform cells varies with their laminar location and is constrained by their morphology and input connectivity. These findings demonstrate the importance of neurogliaform cells in regulating cortical behavioral state-dependent spontaneous activity and indicate that their functional engagement during visual stimuli is influenced by their laminar positioning and connectivity.

Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses.

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

All-optical recreation of naturalistic neural activity with a multifunctional transgenic reporter mouse.

Determining which features of the neural code drive behavior requires the ability to simultaneously read out and write in neural activity patterns with high precision across many neurons. All-optical systems that combine two-photon calcium imaging and targeted photostimulation enable the activation of specific, functionally defined groups of neurons. However, these techniques are unable to test how patterns of activity across a population contribute to computation because of an inability to both read and write cell-specific firing rates. To overcome this challenge, we make two advances: first, we introduce a genetic line of mice for Cre-dependent co-expression of a calcium indicator and a potent soma-targeted microbial opsin. Second, using this line, we develop a method for read-out and write-in of precise population vectors of neural activity by calibrating the photostimulation to each cell. These advances offer a powerful and convenient platform for investigating the neural codes of computation and behavior.

A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex.

Identification of structural connections between neurons is a prerequisite to understanding brain function. Here we developed a pipeline to systematically map brain-wide monosynaptic input connections to genetically defined neuronal populations using an optimized rabies tracing system. We used mouse visual cortex as the exemplar system and revealed quantitative target-specific, layer-specific and cell-class-specific differences in its presynaptic connectomes. The retrograde connectivity indicates the presence of ventral and dorsal visual streams and further reveals topographically organized and continuously varying subnetworks mediated by different higher visual areas. The visual cortex hierarchy can be derived from intracortical feedforward and feedback pathways mediated by upper-layer and lower-layer input neurons. We also identify a new role for layer 6 neurons in mediating reciprocal interhemispheric connections. This study expands our knowledge of the visual system connectomes and demonstrates that the pipeline can be scaled up to dissect connectivity of different cell populations across the mouse brain.

Connecting single-cell transcriptomes to projectomes in mouse visual cortex.

The mammalian brain is composed of diverse neuron types that play different functional roles. Recent single-cell RNA sequencing approaches have led to a whole brain taxonomy of transcriptomically-defined cell types, yet cell type definitions that include multiple cellular properties can offer additional insights into a neuron's role in brain circuits. While the Patch-seq method can investigate how transcriptomic properties relate to the local morphological and electrophysiological properties of cell types, linking transcriptomic identities to long-range projections is a major unresolved challenge. To address this, we collected coordinated Patch-seq and whole brain morphology data sets of excitatory neurons in mouse visual cortex. From the Patch-seq data, we defined 16 integrated morpho-electric-transcriptomic (MET)-types; in parallel, we reconstructed the complete morphologies of 300 neurons. We unified the two data sets with a multi-step classifier, to integrate cell type assignments and interrogate cross-modality relationships. We find that transcriptomic variations within and across MET-types correspond with morphological and electrophysiological phenotypes. In addition, this variation, along with the anatomical location of the cell, can be used to predict the projection targets of individual neurons. We also shed new light on infragranular cell types and circuits, including cell-type-specific, interhemispheric projections. With this approach, we establish a comprehensive, integrated taxonomy of excitatory neuron types in mouse visual cortex and create a system for integrated, high-dimensional cell type classification that can be extended to the whole brain and potentially across species.

Ventral tegmental area glutamate neurons mediate nonassociative consequences of stress.

Exposure to trauma is a risk factor for the development of a number of mood disorders, and may enhance vulnerability to future adverse life events. Recent data demonstrate that ventral tegmental area (VTA) neurons expressing the vesicular glutamate transporter 2 (VGluT2) signal and causally contribute to behaviors that involve aversive or threatening stimuli. However, it is unknown whether VTA VGluT2 neurons regulate transsituational outcomes of stress and whether these neurons are sensitive to stressor controllability. This work adapted an operant mouse paradigm to examine the impact of stressor controllability on VTA VGluT2 neuron function as well as the role of VTA VGluT2 neurons in mediating transsituational stressor outcomes. Uncontrollable (inescapable) stress, but not physically identical controllable (escapable) stress, produced social avoidance and exaggerated fear in male mice. Uncontrollable stress in females led to exploratory avoidance of a novel brightly lit environment. Both controllable and uncontrollable stressors increased VTA VGluT2 neuronal activity, and chemogenetic silencing of VTA VGluT2 neurons prevented the behavioral sequelae of uncontrollable stress in male and female mice. Further, we show that stress activates multiple genetically-distinct subtypes of VTA VGluT2 neurons, especially those that are VGluT2+VGaT+, as well as lateral habenula neurons receiving synaptic input from VTA VGluT2 neurons. Our results provide causal evidence that mice can be used for identifying stressor controllability circuitry and that VTA VGluT2 neurons contribute to transsituational stressor outcomes, such as social avoidance, exaggerated fear, or anxiety-like behavior that are observed within trauma-related disorders.

Intersectional mapping of multi-transmitter neurons and other cell types in the brain.

Recent developments in intersectional strategies have greatly advanced our ability to precisely target brain cell types based on unique co-expression patterns. To accelerate the application of intersectional genetics, we perform a brain-wide characterization of 13 Flp and tTA mouse driver lines and selected seven for further analysis based on expression of vesicular neurotransmitter transporters. Using selective Cre driver lines, we created more than 10 Cre/tTA combinational lines for cell type targeting and circuit analysis. We then used VGLUT-Cre/VGAT-Flp combinational lines to identify and map 30 brain regions containing neurons that co-express vesicular glutamate and gamma-aminobutyric acid (GABA) transporters, followed by tracing their projections with intersectional viral vectors. Focusing on the lateral habenula (LHb) as a target, we identified glutamatergic, GABAergic, or co-glutamatergic/GABAergic innervations from ∼40 brain regions. These data provide an important resource for the future application of intersectional strategies and expand our understanding of the neuronal subtypes in the brain.

Local connectivity and synaptic dynamics in mouse and human neocortex.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Laminar distribution and arbor density of two functional classes of thalamic inputs to primary visual cortex.

Motion/direction-sensitive and location-sensitive neurons are the two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It is under debate whether motion/direction-sensitive inputs preferentially target the superficial layers in V1, as opposed to the location-sensitive inputs, which preferentially target the middle layers. Here, by using calcium imaging to measure the activity of motion/direction-sensitive and location-sensitive axons in V1, we find evidence against these cell-type-specific laminar biases at the population level. Furthermore, using an approach to reconstruct axon arbors with identified in vivo response types, we show that, at the single-axon level, the motion/direction-sensitive axons project more densely to the middle layers than the location-sensitive axons. Overall, our results demonstrate that motion/direction-sensitive thalamic neurons project extensively to the middle layers of V1 at both the population and single-cell levels, providing further insight into the organization of thalamocortical projection in the mouse visual system.

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.

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.

Signature morpho-electric, transcriptomic, and dendritic properties of human layer 5 neocortical pyramidal neurons.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.

Enhancer viruses for combinatorial cell-subclass-specific labeling.

Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

Alternating sources of perisomatic inhibition during behavior.

Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.

Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells.

Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.