Gossamer: Scaling Image Processing and Reconstruction to Whole Brains.

Neuronal reconstruction-a process that transforms image volumes into 3D geometries and skeletons of cells-bottlenecks the study of brain function, connectomics and pathology. Unlike artistic domains with similar challenges (e.g., hair modeling), scientists need exact and complete segmentations to study subtle topological differences. Existing methods are disk-bound, dense-access, coupled, single-threaded, algorithmically unscalable and require manual cropping of small windows and proofreading of skeletons due to low topological accuracy. Designing a data-intensive parallel solution suited to a neurons' shape, topology and far-ranging connectivity is particularly challenging due to I/O and load-balance, yet by abstracting vision tasks such as segmentation and skeletonization into strategically ordered specializations of search, we progressively lower memory by 4 orders of magnitude. This enables 1 mouse brain to be fully processed in-memory on a single server, at 67× the scale with 870× less memory while having 78% higher automated yield than the highest performing alternative methods.

Application of AAV1 for Anterograde Transsynaptic Circuit Mapping and Input-Dependent Neuronal Cataloging.

Viruses that spread transsynaptically provide a powerful means to study interconnected circuits in the brain. Here we describe the use of adeno-associated virus, serotype 1 (AAV1), as a tool to achieve robust, anterograde transsynaptic spread in a variety of unidirectional pathways. A protocol for performing intracranial AAV1 injections in mice is presented, along with additional guidance for planning experiments, sourcing materials, and optimizing the approach to achieve the most successful outcomes. By following the methods presented here, researchers will be able to reveal postsynaptically connected neurons downstream of a given AAV1 injection site and access these input-defined cells for subsequent mapping, recording, and manipulation to characterize their anatomical and functional properties. © 2022 Wiley Periodicals LLC. Basic Protocol: Stereotaxic injection of AAV1 for anterograde transsynaptic spread.

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.

The mouse cortico-basal ganglia-thalamic network.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

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.

Organization of the inputs and outputs of the mouse superior colliculus.

The superior colliculus (SC) receives diverse and robust cortical inputs to drive a range of cognitive and sensorimotor behaviors. However, it remains unclear how descending cortical input arising from higher-order associative areas coordinate with SC sensorimotor networks to influence its outputs. Here, we construct a comprehensive map of all cortico-tectal projections and identify four collicular zones with differential cortical inputs: medial (SC.m), centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). Further, we delineate the distinctive brain-wide input/output organization of each collicular zone, assemble multiple parallel cortico-tecto-thalamic subnetworks, and identify the somatotopic map in the SC that displays distinguishable spatial properties from the somatotopic maps in the neocortex and basal ganglia. Finally, we characterize interactions between those cortico-tecto-thalamic and cortico-basal ganglia-thalamic subnetworks. This study provides a structural basis for understanding how SC is involved in integrating different sensory modalities, translating sensory information to motor command, and coordinating different actions in goal-directed behaviors.

Corticostriatal control of defense behavior in mice induced by auditory looming cues.

Animals exhibit innate defense behaviors in response to approaching threats cued by the dynamics of sensory inputs of various modalities. The underlying neural circuits have been mostly studied in the visual system, but remain unclear for other modalities. Here, by utilizing sounds with increasing (vs. decreasing) loudness to mimic looming (vs. receding) objects, we find that looming sounds elicit stereotypical sequential defensive reactions: freezing followed by flight. Both behaviors require the activity of auditory cortex, in particular the sustained type of responses, but are differentially mediated by corticostriatal projections primarily innervating D2 neurons in the tail of the striatum and corticocollicular projections to the superior colliculus, respectively. The behavioral transition from freezing to flight can be attributed to the differential temporal dynamics of the striatal and collicular neurons in their responses to looming sound stimuli. Our results reveal an essential role of the striatum in the innate defense control.

An open access mouse brain flatmap and upgraded rat and human brain flatmaps based on current reference atlases.

Here we present a flatmap of the mouse central nervous system (CNS) (brain) and substantially enhanced flatmaps of the rat and human brain. Also included are enhanced representations of nervous system white matter tracts, ganglia, and nerves, and an enhanced series of 10 flatmaps showing different stages of rat brain development. The adult mouse and rat brain flatmaps provide layered diagrammatic representation of CNS divisions, according to their arrangement in corresponding reference atlases: Brain Maps 4.0 (BM4, rat) (Swanson, The Journal of Comparative Neurology, 2018, 526, 935-943), and the first version of the Allen Reference Atlas (mouse) (Dong, The Allen reference atlas, (book + CD-ROM): A digital color brain atlas of the C57BL/6J male mouse, 2007). To facilitate comparative analysis, both flatmaps are scaled equally, and the divisional hierarchy of gray matter follows a topographic arrangement used in BM4. Also included with the mouse and rat brain flatmaps are cerebral cortex atlas level contours based on the reference atlases, and direct graphical and tabular comparison of regional parcellation. To encourage use of the brain flatmaps, they were designed and organized, with supporting reference tables, for ease-of-use and to be amenable to computational applications. We demonstrate how they can be adapted to represent novel parcellations resulting from experimental data, and we provide a proof-of-concept for how they could form the basis of a web-based graphical data viewer and analysis platform. The mouse, rat, and human brain flatmap vector graphics files (Adobe Reader/Acrobat viewable and Adobe Illustrator editable) and supporting tables are provided open access; they constitute a broadly applicable neuroscience toolbox resource for researchers seeking to map and perform comparative analysis of brain data.

Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry.

Revealing the organization and function of neural circuits is greatly facilitated by viral tools that spread transsynaptically. Adeno-associated virus (AAV) exhibits anterograde transneuronal transport, however, the synaptic specificity of this spread and its broad application within a diverse set of circuits remains to be explored. Here, using anatomic, functional, and molecular approaches, we provide evidence for the preferential transport of AAV1 to postsynaptically connected neurons and reveal its spread is strongly dependent on synaptic transmitter release. In addition to glutamatergic pathways, AAV1 also spreads through GABAergic synapses to both excitatory and inhibitory cell types. We observed little or no transport, however, through neuromodulatory projections (e.g., serotonergic, cholinergic, and noradrenergic). In addition, we found that AAV1 can be transported through long-distance descending projections from various brain regions to effectively transduce spinal cord neurons. Combined with newly designed intersectional and sparse labeling strategies, AAV1 can be applied within a wide variety of pathways to categorize neurons according to their input sources, morphology, and molecular identities. These properties make AAV1 a promising anterograde transsynaptic tool for establishing a comprehensive cell-atlas of the brain, although its capacity for retrograde transport currently limits its use to unidirectional circuits.SIGNIFICANCE STATEMENT The discovery of anterograde transneuronal spread of AAV1 generates great promise for its application as a unique tool for manipulating input-defined cell populations and mapping their outputs. However, several outstanding questions remain for anterograde transsynaptic approaches in the field: (1) whether AAV1 spreads exclusively or specifically to synaptically connected neurons, and (2) how broad its application could be in various types of neural circuits in the brain. This study provides several lines of evidence in terms of anatomy, functional innervation, and underlying mechanisms, to strongly support that AAV1 anterograde transneuronal spread is highly synapse specific. In addition, several potentially important applications of transsynaptic AAV1 in probing neural circuits are described.

Input-output organization of the mouse claustrum.

Progress in determining the precise organization and function of the claustrum (CLA) has been hindered by the difficulty in reliably targeting these neurons. To overcome this, we used a projection-based targeting strategy to selectively label CLA principal neurons. Combined with adeno-associated virus (AAV) and monosynaptic rabies tracing techniques, we systematically examined the pre-synaptic input and axonal output of this structure. We found that CLA neurons projecting to retrosplenial cortex (RSP) collateralize extensively to innervate a variety of higher-order cortical regions. No subcortical labeling was found, with the exception of sparse terminals in the basolateral amygdala (BLA). This pattern of output was similar to cingulate- and visual cortex-projecting CLA neurons, suggesting a common targeting scheme among these projection-defined populations. Rabies virus tracing directly demonstrated widespread synaptic inputs to RSP-projecting CLA neurons from both cortical and subcortical areas. The strongest inputs arose from classically defined limbic regions, including medial prefrontal cortex, anterior cingulate, BLA, ventral hippocampus, and neuromodulatory systems such as the dorsal raphe and cholinergic basal forebrain. These results suggest that the CLA may integrate information related to the emotional salience of stimuli and may globally modulate cortical state by broadcasting its output uniformly across a variety of higher cognitive centers.

Inhibitory gain modulation of defense behaviors by zona incerta.

Zona incerta (ZI) is a functionally mysterious subthalamic nucleus containing mostly inhibitory neurons. Here, we discover that GABAergic neurons in the rostral sector of ZI (ZIr) directly innervate excitatory but not inhibitory neurons in the dorsolateral and ventrolateral compartments of periaqueductal gray (PAG), which can drive flight and freezing behaviors respectively. Optogenetic activation of ZIr neurons or their projections to PAG reduces both sound-induced innate flight response and conditioned freezing response, while optogenetic suppression of these neurons enhances these defensive behaviors, likely through a mechanism of gain modulation. ZIr activity progressively increases during extinction of conditioned freezing response, and suppressing ZIr activity impairs the expression of fear extinction. Furthermore, ZIr is innervated by the medial prefrontal cortex (mPFC), and silencing mPFC prevents the increase of ZIr activity during extinction and the expression of fear extinction. Together, our results suggest that ZIr is engaged in modulating defense behaviors.

A Non-canonical Reticular-Limbic Central Auditory Pathway via Medial Septum Contributes to Fear Conditioning.

In the mammalian brain, auditory information is known to be processed along a central ascending pathway leading to auditory cortex (AC). Whether there exist any major pathways beyond this canonical auditory neuraxis remains unclear. In awake mice, we found that auditory responses in entorhinal cortex (EC) cannot be explained by a previously proposed relay from AC based on response properties. By combining anatomical tracing and optogenetic/pharmacological manipulations, we discovered that EC received auditory input primarily from the medial septum (MS), rather than AC. A previously uncharacterized auditory pathway was then revealed: it branched from the cochlear nucleus, and via caudal pontine reticular nucleus, pontine central gray, and MS, reached EC. Neurons along this non-canonical auditory pathway responded selectively to high-intensity broadband noise, but not pure tones. Disruption of the pathway resulted in an impairment of specifically noise-cued fear conditioning. This reticular-limbic pathway may thus function in processing aversive acoustic signals.

AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors.

To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits.

The mouse cortico-striatal projectome.

Different cortical areas are organized into distinct intracortical subnetworks. The manner in which descending pathways from the entire cortex interact subcortically as a network remains unclear. We developed an open-access comprehensive mesoscale mouse cortico-striatal projectome: a detailed connectivity projection map from the entire cerebral cortex to the dorsal striatum or caudoputamen (CP) in rodents. On the basis of these projections, we used new computational neuroanatomical tools to identify 29 distinct functional striatal domains. Furthermore, we characterized different cortico-striatal networks and how they reconfigure across the rostral-caudal extent of the CP. The workflow was also applied to select cortico-striatal connections in two different mouse models of disconnection syndromes to demonstrate its utility for characterizing circuitry-specific connectopathies. Together, our results provide the structural basis for studying the functional diversity of the dorsal striatum and disruptions of cortico-basal ganglia networks across a broad range of disorders.

Cross-Modality Sharpening of Visual Cortical Processing through Layer-1-Mediated Inhibition and Disinhibition.

Cross-modality interaction in sensory perception is advantageous for animals' survival. How cortical sensory processing is cross-modally modulated and what are the underlying neural circuits remain poorly understood. In mouse primary visual cortex (V1), we discovered that orientation selectivity of layer (L)2/3, but not L4, excitatory neurons was sharpened in the presence of sound or optogenetic activation of projections from primary auditory cortex (A1) to V1. The effect was manifested by decreased average visual responses yet increased responses at the preferred orientation. It was more pronounced at lower visual contrast and was diminished by suppressing L1 activity. L1 neurons were strongly innervated by A1-V1 axons and excited by sound, while visual responses of L2/L3 vasoactive intestinal peptide (VIP) neurons were suppressed by sound, both preferentially at the cell's preferred orientation. These results suggest that the cross-modality modulation is achieved primarily through L1 neuron- and L2/L3 VIP-cell-mediated inhibitory and disinhibitory circuits.

Thalamocortical Innervation Pattern in Mouse Auditory and Visual Cortex: Laminar and Cell-Type Specificity.

Despite many previous studies, the functional innervation pattern of thalamic axons and their target specificity remains to be investigated thoroughly. Here, in primary auditory cortical slices, we examined thalamic innervation patterns for excitatory and different types of inhibitory neurons across laminae, by optogenetically stimulating axons from the medial geniculate body. We found that excitatory cells and parvalbumin (PV)-expressing inhibitory neurons across layer 2/3 (L2/3) to L6 are directly innervated by thalamic projections, with the strongest innervation occurring in L4. The innervation of PV neurons is stronger than that of excitatory neurons in the same layer, with a relatively constant ratio between their innervation strengths across layers. For somatostatin and vasoactive intestinal peptide inhibitory neurons, essentially only L4 neurons were innervated by thalamic axons and the innervation was much weaker compared with excitatory and PV cells. In addition, more than half of inhibitory neurons in L1 were innervated, relatively strongly, by thalamic axons. Similar innervation patterns were also observed in the primary visual cortex. Thus, thalamic information can be processed independently and differentially by different cortical layers, in addition to the generally thought hierarchical processing starting from L4. This parallel processing is likely shaped by feedforward inhibition from PV neurons in each individual lamina, and may extend the computation power of sensory cortices.

Auditory cortex controls sound-driven innate defense behaviour through corticofugal projections to inferior colliculus.

Defense against environmental threats is essential for animal survival. However, the neural circuits responsible for transforming unconditioned sensory stimuli and generating defensive behaviours remain largely unclear. Here, we show that corticofugal neurons in the auditory cortex (ACx) targeting the inferior colliculus (IC) mediate an innate, sound-induced flight behaviour. Optogenetic activation of these neurons, or their projection terminals in the IC, is sufficient for initiating flight responses, while the inhibition of these projections reduces sound-induced flight responses. Corticocollicular axons monosynaptically innervate neurons in the cortex of the IC (ICx), and optogenetic activation of the projections from the ICx to the dorsal periaqueductal gray is sufficient for provoking flight behaviours. Our results suggest that ACx can both amplify innate acoustic-motor responses and directly drive flight behaviours in the absence of sound input through corticocollicular projections to ICx. Such corticofugal control may be a general feature of innate defense circuits across sensory modalities.

Sensory Cortical Control of a Visually Induced Arrest Behavior via Corticotectal Projections.

Innate defense behaviors (IDBs) evoked by threatening sensory stimuli are essential for animal survival. Although subcortical circuits are implicated in IDBs, it remains largely unclear whether sensory cortex modulates IDBs and what the underlying neural pathways are. Here, we show that optogenetic silencing of corticotectal projections from layer 5 (L5) of the mouse primary visual cortex (V1) to the superior colliculus (SC) significantly reduces an SC-dependent innate behavior (i.e., temporary suspension of locomotion upon a sudden flash of light as short as milliseconds). Surprisingly, optogenetic activation of SC-projecting neurons in V1 or their axon terminals in SC sufficiently elicits the behavior, in contrast to other major L5 corticofugal projections. Thus, via the same corticofugal projection, visual cortex not only modulates the light-induced arrest behavior, but also can directly drive the behavior. Our results suggest that sensory cortex may play a previously unrecognized role in the top-down initiation of sensory-motor behaviors.

Neural networks of the mouse neocortex.

Numerous studies have examined the neuronal inputs and outputs of many areas within the mammalian cerebral cortex, but how these areas are organized into neural networks that communicate across the entire cortex is unclear. Over 600 labeled neuronal pathways acquired from tracer injections placed across the entire mouse neocortex enabled us to generate a cortical connectivity atlas. A total of 240 intracortical connections were manually reconstructed within a common neuroanatomic framework, forming a cortico-cortical connectivity map that facilitates comparison of connections from different cortical targets. Connectivity matrices were generated to provide an overview of all intracortical connections and subnetwork clusterings. The connectivity matrices and cortical map revealed that the entire cortex is organized into four somatic sensorimotor, two medial, and two lateral subnetworks that display unique topologies and can interact through select cortical areas. Together, these data provide a resource that can be used to further investigate cortical networks and their corresponding functions.

Comprehensive connectivity of the mouse main olfactory bulb: analysis and online digital atlas.

We introduce the first open resource for mouse olfactory connectivity data produced as part of the Mouse Connectome Project (MCP) at UCLA. The MCP aims to assemble a whole-brain connectivity atlas for the C57Bl/6J mouse using a double coinjection tracing method. Each coinjection consists of one anterograde and one retrograde tracer, which affords the advantage of simultaneously identifying efferent and afferent pathways and directly identifying reciprocal connectivity of injection sites. The systematic application of double coinjections potentially reveals interaction stations between injections and allows for the study of connectivity at the network level. To facilitate use of the data, raw images are made publicly accessible through our online interactive visualization tool, the iConnectome, where users can view and annotate the high-resolution, multi-fluorescent connectivity data (www.MouseConnectome.org). Systematic double coinjections were made into different regions of the main olfactory bulb (MOB) and data from 18 MOB cases (~72 pathways; 36 efferent/36 afferent) currently are available to view in iConnectome within their corresponding atlas level and their own bright-field cytoarchitectural background. Additional MOB injections and injections of the accessory olfactory bulb (AOB), anterior olfactory nucleus (AON), and other olfactory cortical areas gradually will be made available. Analysis of connections from different regions of the MOB revealed a novel, topographically arranged MOB projection roadmap, demonstrated disparate MOB connectivity with anterior versus posterior piriform cortical area (PIR), and exposed some novel aspects of well-established cortical olfactory projections.