A stable proportion of Purkinje cell inputs from parallel fibers are silent during cerebellar maturation.

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.

Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses.

Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRABATP1.0 , we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron-glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50-250 µm2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+ transients, and intracellular Ca2+ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca2+ -independent manners, thus potentially regulating hundreds of synapses all together.

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.

The intellectual disability protein Oligophrenin-1 controls astrocyte morphology and migration.

Astrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID.

Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling.

Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.

Multicolor analysis of oligodendrocyte morphology, interactions, and development with Brainbow.

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single-out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre-lox recombination, previously used for visualizing axons and neurons. We used tamoxifen-inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single-cell resolution.

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.

Concise review: understanding clonal dynamics in homeostasis and injury through multicolor lineage tracing.

Lineage tracing is an essential tool to study stem cell fate. Although traditional lineage tracing techniques have considerably advanced our understanding of stem cell behavior, they pose significant limitations for identification and longitudinal tracking of the progeny of individual stem cells, to compare their behaviors. This is of importance given the well-established heterogeneity among stem cells both in terms of potentialities and proliferative capacities. The recent development of multicolor genetic reporters addressable to specific cell populations largely overcomes these issues. These new "rainbow" technologies provide increased resolution in clonal identification and offer the possibility to study the relative distribution, contacts, tiled arrangement, and competitive interactions among cells or groups of cells of the same type.

Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve.

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.

A technicolour approach to the connectome.

A central aim of neuroscience is to map neural circuits, in order to learn how they account for mental activities and behaviours and how alterations in them lead to neurological and psychiatric disorders. However, the methods that are currently available for visualizing circuits have severe limitations that make it extremely difficult to extract precise wiring diagrams from histological images. Here we review recent advances in this area, along with some of the opportunities that these advances present and the obstacles that remain.

Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.

Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.

Chromatically Corrected Multicolor Multiphoton Microscopy.

Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this light-efficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging.

Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development.

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch.

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.

Cortical astrocytes develop in a plastic manner at both clonal and cellular levels.

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.

Etv1 Controls the Establishment of Non-overlapping Motor Innervation of Neighboring Facial Muscles during Development.

The somatotopic motor-neuron projections onto their cognate target muscles are essential for coordinated movement, but how that occurs for facial motor circuits, which have critical roles in respiratory and interactive behaviors, is poorly understood. We report extensive molecular heterogeneity in developing facial motor neurons in the mouse and identify markers of subnuclei and the motor pools innervating specific facial muscles. Facial subnuclei differentiate during migration to the ventral hindbrain, where neurons with progressively later birth dates-and evolutionarily more recent functions-settle in more-lateral positions. One subpopulation marker, ETV1, determines both positional and target muscle identity for neurons of the dorsolateral (DL) subnucleus. In Etv1 mutants, many markers of DL differentiation are lost, and individual motor pools project indifferently to their own and neighboring muscle targets. The resulting aberrant activation patterns are reminiscent of the facial synkinesis observed in humans after facial nerve injury.

Adult Neural Stem Cells and Multiciliated Ependymal Cells Share a Common Lineage Regulated by the Geminin Family Members.

Adult neural stem cells and multiciliated ependymal cells are glial cells essential for neurological functions. Together, they make up the adult neurogenic niche. Using both high-throughput clonal analysis and single-cell resolution of progenitor division patterns and fate, we show that these two components of the neurogenic niche are lineally related: adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. Unexpectedly, we found that the antagonist regulators of DNA replication, GemC1 and Geminin, can tune the proportion of neural stem cells and ependymal cells. Our findings reveal the controlled dynamic of the neurogenic niche ontogeny and identify the Geminin family members as key regulators of the initial pool of adult neural stem cells.

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.

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.