Cortical glutamatergic projection neuron types contribute to distinct functional subnetworks.

The cellular basis of cerebral cortex functional architecture remains not well understood. A major challenge is to monitor and decipher neural network dynamics across broad cortical areas yet with projection-neuron-type resolution in real time during behavior. Combining genetic targeting and wide-field imaging, we monitored activity dynamics of subcortical-projecting (PTFezf2) and intratelencephalic-projecting (ITPlxnD1) types across dorsal cortex of mice during different brain states and behaviors. ITPlxnD1 and PTFezf2 neurons showed distinct activation patterns during wakeful resting, during spontaneous movements and upon sensory stimulation. Distinct ITPlxnD1 and PTFezf2 subnetworks were dynamically tuned to different sensorimotor components of a naturalistic feeding behavior, and optogenetic inhibition of ITsPlxnD1 and PTsFezf2 in subnetwork nodes disrupted distinct components of this behavior. Lastly, ITPlxnD1 and PTFezf2 projection patterns are consistent with their subnetwork activation patterns. Our results show that, in addition to the concept of columnar organization, dynamic areal and projection-neuron-type specific subnetworks are a key feature of cortical functional architecture linking microcircuit components with global brain networks.

nAdder: A scale-space approach for the 3D analysis of neuronal traces.

Tridimensional microscopy and algorithms for automated segmentation and tracing are revolutionizing neuroscience through the generation of growing libraries of neuron reconstructions. Innovative computational methods are needed to analyze these neuronal traces. In particular, means to characterize the geometric properties of traced neurites along their trajectory have been lacking. Here, we propose a local tridimensional (3D) scale metric derived from differential geometry, measuring for each point of a curve the characteristic length where it is fully 3D as opposed to being embedded in a 2D plane or 1D line. The larger this metric is and the more complex the local 3D loops and turns of the curve are. Available through the GeNePy3D open-source Python quantitative geometry library (https://genepy3d.gitlab.io), this approach termed nAdder offers new means of describing and comparing axonal and dendritic arbors. We validate this metric on simulated and real traces. By reanalysing a published zebrafish larva whole brain dataset, we show its ability to characterize different population of commissural axons, distinguish afferent connections to a target region and differentiate portions of axons and dendrites according to their behavior, shedding new light on the stereotypical nature of neurites' local geometry.

Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.

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.

Semantic segmentation of microscopic neuroanatomical data by combining topological priors with encoder-decoder deep networks.

Understanding of neuronal circuitry at cellular resolution within the brain has relied on neuron tracing methods which involve careful observation and interpretation by experienced neuroscientists. With recent developments in imaging and digitization, this approach is no longer feasible with the large scale (terabyte to petabyte range) images. Machine learning based techniques, using deep networks, provide an efficient alternative to the problem. However, these methods rely on very large volumes of annotated images for training and have error rates that are too high for scientific data analysis, and thus requires a significant volume of human-in-the-loop proofreading. Here we introduce a hybrid architecture combining prior structure in the form of topological data analysis methods, based on discrete Morse theory, with the best-in-class deep-net architectures for the neuronal connectivity analysis. We show significant performance gains using our hybrid architecture on detection of topological structure (e.g. connectivity of neuronal processes and local intensity maxima on axons corresponding to synaptic swellings) with precision/recall close to 90% compared with human observers. We have adapted our architecture to a high performance pipeline capable of semantic segmentation of light microscopic whole-brain image data into a hierarchy of neuronal compartments. We expect that the hybrid architecture incorporating discrete Morse techniques into deep nets will generalize to other data domains.

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation.

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing.

Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.

Publisher Correction: Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.

Affiliation 4 incorrectly read 'University of the Basque Country (Ikerbasque), University of the Basque Country and Donostia International Physics Center, San Sebastian 20018, Spain.'Also, the affiliations of Ignacio Arganda-Carreras with 'IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain' and 'Donostia International Physics Center (DIPC), San Sebastian, 20018, Spain' were inadvertently omitted.Additionally, the third sentence of the first paragraph of the Results section entitled 'Multicontrast organ-scale imaging with ChroMS microscopy' incorrectly read 'For example, one can choose lambda1 = 850 and lambda2 = 110 nm for optimal two-photon excitation of blue and red chromophores.'. The correct version reads 'lambda2 = 1100 nm' instead of 'lambda2 = 110 nm'. These errors have now been corrected in the PDF and HTML versions of the Article.

Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.

Radial Glial Lineage Progression and Differential Intermediate Progenitor Amplification Underlie Striatal Compartments and Circuit Organization.

The circuitry of the striatum is characterized by two organizational plans: the division into striosome and matrix compartments, thought to mediate evaluation and action, and the direct and indirect pathways, thought to promote or suppress behavior. The developmental origins of these organizations and their developmental relationships are unknown, leaving a conceptual gap in understanding the cortico-basal ganglia system. Through genetic fate mapping, we demonstrate that striosome-matrix compartmentalization arises from a lineage program embedded in lateral ganglionic eminence radial glial progenitors mediating neurogenesis through two distinct types of intermediate progenitors (IPs). The early phase of this program produces striosomal spiny projection neurons (SPNs) through fate-restricted apical IPs (aIPSs) with limited capacity; the late phase produces matrix SPNs through fate-restricted basal IPs (bIPMs) with expanded capacity. Notably, direct and indirect pathway SPNs arise within both aIPS and bIPM pools, suggesting that striosome-matrix architecture is the fundamental organizational plan of basal ganglia circuitry.

Multiplex cell and lineage tracking with combinatorial labels.

We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.

AT2-receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression.

It is widely accepted that the angiotensin AT2-receptor (AT2R) has neuroprotective features. In the present study we tested pharmacological AT2R-stimulation as a therapeutic approach in a model of spinal cord compression injury (SCI) in mice using the novel non-peptide AT2R-agonist, Compound 21 (C21). Complementary experiments in primary neurons and organotypic cultures served to identify underlying mechanisms. Functional recovery and plasticity of corticospinal tract (CST) fibers following SCI were monitored after application of C21 (0.3mg/kg/dayi.p.) or vehicle for 4 weeks. Organotypic co-culture of GFP-positive entorhinal cortices with hippocampal target tissue served to evaluate the impact of C21 on reinnervation. Neuronal differentiation, apoptosis and expression of neurotrophins were investigated in primary murine astrocytes and neuronal cells. C21 significantly improved functional recovery after SCI compared to controls, and this significantly correlated with the increased number of CST fibers caudal to the lesion site. In vitro, C21 significantly promoted reinnervation in organotypic brain slice co-cultures (+50%) and neurite outgrowth of primary neurons (+25%). C21-induced neurite outgrowth was absent in neurons derived from AT2R-KO mice. In primary neurons, treatment with C21 further induced RNA expression of anti-apoptotic Bcl-2 (+75.7%), brain-derived neurotrophic factor (BDNF) (+53.7%), the neurotrophin receptors TrkA (+57.4%) and TrkB (+67.9%) and a marker for neurite growth, GAP43 (+103%), but not TrkC. Our data suggest that selective AT2R-stimulation improves functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms. Thus, AT2R-stimulation may be considered for the development of a novel therapeutic approach for the treatment of spinal cord injury.

Multicolor two-photon tissue imaging by wavelength mixing.

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

Class 3 semaphorins influence oligodendrocyte precursor recruitment and remyelination in adult central nervous system.

Oligodendrocyte precursor cells, which persist in the adult central nervous system, are the main source of central nervous system remyelinating cells. In multiple sclerosis, some demyelinated plaques exhibit an oligodendroglial depopulation, raising the hypothesis of impaired oligodendrocyte precursor cell recruitment. Developmental studies identified semaphorins 3A and 3F as repulsive and attractive guidance cues for oligodendrocyte precursor cells, respectively. We previously reported their increased expression in experimental demyelination and in multiple sclerosis. Here, we show that adult oligodendrocyte precursor cells, like their embryonic counterparts, express class 3 semaphorin receptors, neuropilins and plexins and that neuropilin expression increases after demyelination. Using gain and loss of function experiments in an adult murine demyelination model, we demonstrate that semaphorin 3A impairs oligodendrocyte precursor cell recruitment to the demyelinated area. In contrast, semaphorin 3F overexpression accelerates not only oligodendrocyte precursor cell recruitment, but also remyelination rate. These data open new avenues to understand remyelination failure and promote repair in multiple sclerosis.