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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 remain 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 μm2area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+transients. 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.

Congenital heart malformations often include mitral valve defects which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal (AVC) undergoes endothelial to mesenchymal transition (EndMT) to give rise to mitral valvular cells. However, the identity, fate decisions of these progenitors as well as the distribution of their derivatives in valve leaflets remain unknown.Here, we use scRNA-seq of genetically labeled mouse AVC endocardial cells and of micro-dissected embryonic and postnatal mitral valves to characterize the developmental road. We uncovered the genetic, cell signaling and metabolic processes underlying specification of the progenitors and how they contribute to subtypes of endothelial and interstitial embryonic and postnatal valvular cells. Using clonal genetic tracing with multicolor reporter, we describe specific modes of growth of endocardial cell-derived clones which build up in a proper manner functional valve leaflets.Our data reveal how both genetic and metabolic specification mechanisms specifically drive the fate of a subset of endocardial cells toward valve progenitors and their distinct clonal contribution to the formation of the valve.

International audience; 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.

In mammals, the connections between motor neurons and muscle fibers profoundly reorganize in the early postnatal period. To better understand this synaptic rewiring we traced out all the connectivity in muscles at successive ages in the mouse using serial section scanning electron microscopy in a muscle at birth and Brainbow-based and XFP-based fluorescent reconstructions in neonatal and older muscles respectively. Our data indicate that axons prune about 85% of their branches in the first two weeks of postnatal life, and that while much of this pruning leaves neuromuscular junctions with only one remaining axon (a ∼8-fold reduction), it also causes a ∼6-fold reduction in the number of muscle fibers that possess more than one neuromuscular junction. Unexpectedly, the simplification of the wiring diagram was not haphazard but rather was constrained by the tendency for neurons to maintain co-innervation the longest with other neurons based on their proximity in an abstract rank order. This synaptic ordering preference was even significant at birth when connectivity was the most overlapping but became more striking as development proceeded and was even obvious in the few adult muscle fibers that retained more than one axon at different neuromuscular junctions. Analysis of properties of muscle fibers sharing axons at developing ages and changes in the physical distance between neuromuscular junctions that were maintained in young versus older muscles suggests that the rank order of motor neurons is based on their relative similarity in activity patterns. This same ranking governs both the close-proximity synaptic competitions within neuromuscular junctions and the long-distance competitions that remove or maintain synapses millimeters apart meaning that all neuromuscular rewiring is based on the same global activity ordering rule. We think it is likely that this ranking is related to the ultimate recruitment order of motor axon activity as first described by (Henneman, 1957). Thus the emerging structure of neuromuscular circuitry is a product of its function: initial nearly all-to-all connectivity gives rise to a well-organized system of axons, allowing for the orderly recruitment of neurons during a smoothly graded behavior.

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., Previous studies on astrocyte development have led to controversial results due to a lack of pertinent tools. Here, authors analyze large numbers of astrocyte clones generated by nearby cortical progenitors using the MAGIC Markers strategy and ChroMS 3D imaging, and show that clonally-related astrocytes organize in a non-stereotyped manner and that cortical astrocyte subtypes are not intrinsically specified.

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.

International audience

Since its first demonstrations, two-photon microscopy has enabled discoveries in many fields of biology since it is a uniquely suited imaging method for live / deep fluorescence imaging of biological tissues with sub-cellular resolution. However, imaging depth remains a crucial limitation for investigating scattering tissues such as the brain. To overcome this limitation and access deeper areas, one recent and very promising approach consists in using three-photon excitation (3PE) and shifting the excitation to the SWIR (Short-Wavelength InfraRed) range. Indeed, scattering decreases with increasing wavelength, following a typical 1/λ3 law. However water absorption should be avoided to prevent tissue from heating, so that it was realized recently that two optimal spectral excitation windows for deep multiphoton imaging lie in the 1300 and 1700 nm regions as shown in fig. 1 [1]. We are presenting here the first dual-color SWIR source optimized for 3PE microscopy. The source is based on an OPCPA (optical parametric chirped pulse amplification) injected by a high-power Yb-fiber system. It operates at 1.25 MHz and emits concomitantly the two optimal 1.3 and 1.7 μm wavelengths with 69 fs, 65 fs pulse durations and with energies of 710 nJ and 3100 nJ respectively. These characteristics enabled us to record dual-color 3PE microscopy images in chick spinal cord, and in zebrafish and mouse brain.

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. We thank Minh-Son Phan, Samuel Tozer, and Z. Josh Huang for scientific discussions and comments on the manuscript. We thank TissueVision for providing technical advice for the implementation and customization of the block-face system. We thank Bastien Binet from Polytechnique Polymedia center for his generous help in the creation of 3D graphics. We thank IdV core imaging, animal experimentation and histology facilities for technical assistance. This work was supported by fellowships from Universite Paris-Saclay (Initiatives Doctorales Interdisciplinaires) to L.A., Ecole des Neurosciences de Paris to K.M., Region Ile-de-France and Fondation ARC pour la Recherche sur le Cancer to S.C. by Agence Nationale de la Recherche under contracts ANR-11-EQPX-0029 (Equipex Morphoscope2), ANR-10-INBS-04 (France BioImaging), and ANR-10-LABX-65 (LabEx LifeSenses), by Fondation pour la Recherche Medicale (grant DBI20141231328), by Target ALS and by the European Research Council (ERC-CoG 649117).

International audience; The cells composing brain tissue, neurons, and glia, form extraordinarily complex networks that support cognitive functions. Understanding the organization and development of these networks requires quantitative data resolved at the single cell level. To this aim, we apply novel large-scale 3D multicolor microscopy methodologies in combination with “Brainbow”, a transgenic approach enabling to label neural cells with diverse combinations of spectrally distinct fluorescent proteins. In this paper, we present a pipeline based on Convolutional Neural Network (CNN) to detect and segment individual astrocytes, the main type of glial cells of the brain, and map the domains occupied by their fine processes. This bioimage analysis approach successfully handles the challenging variety of astrocyte shape, color, size and their overlap with background elements. Our method shows significant improvement compared with classical techniques, opening the way to varied biological inquiries.

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.

International audience; We present a novel ultrafast laser design providing simultaneous excitation in the two water-transparency windows at 1.3 and 1.7 µm, and we demonstrate its potentiality for efficient 3-photon microscopy of nervous tissues.

WOS:000402561100001; International audience; The spectral window lying between 1.6 and 1.7 mu m is interesting for in-depth multiphoton microscopy of intact tissues due to reduced scattering and absorption in this wavelength range. However, wide adoption of this excitation range will rely on the availability of robust and cost-effective high peak power pulsed lasers operating at these wavelengths. In this communication, we report on a monolithically integrated high repetition rate (50 MHz) all-fiber femtosecond laser based on a soliton self-frequency shift providing 9 nJ, 75 fs pulses at 1650 nm. We illustrate its potential for biological microscopy by recording three-photon-excited fluorescence and third-harmonic generation images of mouse nervous tissue and developing Drosophila embryos labeled with a red fluorescent protein.

We propose an innovative OPCPA source for deep-tissue multiphoton microscopy providing μJ pulses at 1700 nm. We demonstrate its performances for in-depth 3-photon imaging of mouse brain tissue and compare them with 2-photon microscopy.

Reconstructing neural circuits and cell lineage raises the challenge of imaging large volumes of tissue with high contrast, specificity and resolution. Our work aims at combining Brainbow labeling and large-volume microscopy to reach this goal.

SummaryWe 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.

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.

SUMMARY 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., Graphical Abstract, In Brief Tenney et al. demonstrate that embryonic facial motor neurons are transcriptionally diverse as they establish somatotopic innervation of the facial muscles, a process that requires the transcription factor ETV1. Facial-motor axon-targeting errors in Etv1 mutants cause coordination of whisking and eyeblink evocative of human blepharospasm.

Summary Background The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. Results When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. Conclusions The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

We report on a monolithically integrated high repetition rate all-fiber format femtosecond laser operating at 1650 nm. We explore its potential for biological microscopy by imaging mouse brain tissue.

Sparse, random labelling of individual cells is a key approach to study brain circuit organisation and development. An array of methods based on genetic engineering now complements older methods such as Golgi staining, facilitating analysis while providing higher information content. Increasingly refined expression strategies based on transcriptional modulators and site-specific recombinases are used to distribute markers or combinations of markers within specific neuronal subsets. Several trends are emerging: first, increasing labelling density with multiplexed markers to allow more cells to be reliably distinguished; second, using labels to report lineage relationships among defined cells in addition to anatomy; third, coupling cell labelling with genetic manipulations that reveal or perturb cell function. These strategies offer new opportunities for characterizing the fine scale architecture of neuronal circuits, and understanding lineage and functional relations among their cellular components in normal or experimental situations.

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.

Infrared emissivity is a necessary parameter for computing models to predict road surface status and pavement temperature irrespective of the weather situation. In this work a new experimental device based on the indirect method was developed for the measurement of surface emissivities. The surface is exposed to modulated isotropic infrared radiation. The intensity reflected by the surface of the sample in a given direction is measured by a detector operating in the spectral range 1–40 µm. This large spectral range allows measurement of the total hemispheric emissivity defined in this case as emissivity. The effect of the temperature modulation frequency, surface composition and surface roughness on the emissivity measurements was investigated. The results show good ability of the device for the determination of emissivities at room temperature.

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, we analysed neurogenesis, neuronal differentiation and motoneuron migration in Hoxa1 , Hoxb1 and Hoxb2 mutant mice. This comparison revealed that neurogenesis and differentiation of specific neuronal subpopulations in r4 was impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory were re-specified towards an r2-like identity. Motoneurons derived from that territory resembled trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory underwent changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially formed ectopic nuclei that were subsequently cleared. Their survival could be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, we have delineated the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1 , Hoxb1 and Hoxb2 .

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. Stem Cells 2014;32:3046–3054

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFRα1 (considered to be the favored coreceptor for GDNF) or GFRα2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2. © 2001 Wiley-Liss, Inc.

Glial cell line-derived neurotrophic factor (GDNF) family ligands promote the survival of developing motor neurons in vivo and in vitro. However, not all neurons survive with any single ligand in culture and GDNF null mutant mice display only a partial motor neuron loss. An interesting possibility is that subpopulations of motor neurons based on their function and/or their myotopic organization require distinct members of GDNF family ligands. Because responsiveness to the different ligands depends on the expression of their cognate ligand-binding receptor we have herein addressed this issue by examining the expression of GDNF-family receptors (gfr) during development and in the adult in cranial motor nuclei subpopulations. We have furthermore examined the in vivo role of GDNF for cranial motor neuron subpopulations. The shared ret receptor was expressed in all somatic, branchial and visceral cranial embryonic motor nuclei examined, showing that they are all competent to respond to GDNF family ligands during development. At early stages of development both the GDNF receptor, gfralpha1, and the neurturin (NTN) receptor, gfralpha2, were expressed in the oculomotor, facial and spinal accessory, and only gfralpha1 in the trochlear, superior salivatory, trigeminal, hypoglossal and weakly in the dorsal motor nucleus of the vagus and the ambiguous nucleus. The abducens nucleus was negative for both gfralpha1 and gfralpha2. The artemin (ART) receptor, gfralpha3, was expressed only in the superior salivatory nucleus. A motor neuron subnuclei-specific expression of gfralpha1 and gfralpha2 was seen in the facial and trigeminal nuclei which corresponded to their dependence on GDNF in null mutant mice. We found that the expression was dynamic in these nuclei, which may reflect developmental changes in their trophic factor dependency. Analysis of GDNF null mutant mice revealed that the dynamic receptor expression is regulated by the ligand in vivo, indicating that the attainment of changes in dependency could be ligand induced. Our results indicate that specific GDNF family ligands support selective muscle-motor neuron circuits during development.

Muscle-derived factors are known to be important for the survival of developing spinal motoneurons, but the molecules involved have not been characterized. Hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in muscle development and motoneuron axon outgrowth. We show that HGF/SF has potent neurotrophic activity (EC50=2 pM) for a subpopulation (40%) of purified embryonic rat motoneurons. Moreover, HGF/SF is an essential component of muscle-derived support for motoneurons, since blocking antibodies to HGF/SF specifically inhibited 65% of the trophic activity of media conditioned by C2/C7 skeletal myotubes, but did not inhibit the trophic activity secreted by Schwann cell lines. High levels of expression of the HGF/SF receptor c-Met in the spinal cord are restricted to subsets of motoneurons, mainly in limb-innervating segments. Consistent with this distribution, cultured motoneurons from limb-innervating brachial and lumbar segments showed a more potent response to HGF/SF than did thoracic motoneurons. By the end of the period of motoneuron cell death, levels of c-Met mRNA in motoneurons were markedly reduced, suggesting that the effects of HGF/SF may be limited to the period of motoneuron cell death. HGF/SF may play an important role during motoneuron development as a muscle-derived survival factor for a subpopulation of limb-innervating motoneurons.

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.

International audience; 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.

INTRODUCTIONVisualizing the precise morphology of closely juxtaposed cells and their interactions can be highly informative, particularly when studying the complex organization of neuronal and glial networks in the nervous system. To this end, one can use optical approaches to image-distinct markers that are differentially distributed among the cells of interest, such as fluorescent proteins of various colors (XFPs). The Brainbow strategies use Cre/lox recombination to stochastically express two to four XFPs in a cellular population from a single promoter. Integration of multiple Brainbow transgene copies results in combinatorial expression of these XFPs, creating a wide range of hues. In the nervous system, the multicolor labeling thus generated can be used to distinguish adjacent neuronal or glial cells and to verify the identity of neuronal processes while tracing circuitry. This article describes the generation of Brainbow transgenes and mice as well as their use to image and digitally reconstruct nerve cells and their interactions in fixed samples. This method also holds potential for studies in other tissues and model organisms as well as live imaging in vivo.

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.

In the spinal cord, motor neurons (MNs) with similar muscle targets and sensory inputs are grouped together into motor pools. To date, relatively little is known about the molecular mechanisms that control the establishment of pool-specific circuitry. Semaphorins, a large family of secreted and cell surface proteins, are important mediators of developmental processes such as axon guidance and cell migration. Here, we used mRNA in situ hybridization to study the expression patterns of semaphorins and their receptors, neuropilins and plexins, in the embryonic mouse spinal cord. Our data show that semaphorins and their receptors are differentially expressed in MNs that lie in distinct locations within the spinal cord. Furthermore, we report a combinatorial expression of class 3 (secreted) semaphorins and their receptors that characterizes distinct motor pools within the brachial and lumbar spinal cord. Finally, we found that a secreted semaphorin, Sema3A, elicits differential collapse responses in topologically distinct subpopulations of spinal MNs. These findings lead us to propose that semaphorins and their receptors might play important roles in the sorting of motor pools and the patterning of their afferent and efferent projections.

The development of a patterned vasculature is essential for normal organogenesis. We found that signaling by semaphorin 3E (Sema3E) and its receptor plexin-D1 controls endothelial cell positioning and the patterning of the developing vasculature in the mouse. Sema3E is highly expressed in developing somites, where it acts as a repulsive cue for plexin-D1–expressing endothelial cells of adjacent intersomitic vessels. Sema3E–plexin-D1 signaling did not require neuropilins, which were previously presumed to be obligate Sema3 coreceptors. Moreover, genetic ablation of Sema3E or plexin-D1 but not neuropilin-mediated Sema3 signaling disrupted vascular patterning. These findings reveal an unexpected semaphorin signaling pathway and define a mechanism for controlling vascular patterning.

In the future, roads will have to tell the drivers what they are, whatever the conditions are. The accent is specially put on on-board infrared vision so as to understand how weather phenomena can change the surface of the road (presence of ice, …). The determination of the emissivity is necessary to reach the road surface temperature. Some research has been undertaken on several conventional materials. Their emissivities were determined by the indirect method considering the conditions to apply Kirchhoff's law were met. An infrared hemispheric and isotropic source was used to create a periodic modulated heat flux to which the considered roads materials were submitted to. Measurements presented in this study have shown differences according to road structure (average particle diameter size) and composition. Emissivity measurements conducted on different salt types used for winter maintenance have shown they can be sorted according to their origin.

The projection of developing axons to their targets is a crucial step in the assembly of neuronal circuits. In the spinal cord, the differentiation of specific motor neuron pools is associated with the expression of ETS class transcription factors, notably PEA3 and ER81. Their initial expression coincides with the arrival of motor axons in the vicinity of muscle targets and depends on limb-derived signals. We show that in Pea3 mutant mice, the axons of specific motor neuron pools fail to branch normally within their target muscles, and the cell bodies of these motor neurons are mispositioned within the spinal cord. Thus, the induction of an intrinsic program of ETS gene expression by peripheral signals is required to coordinate the central position and terminal arborization of specific sets of spinal motor neurons.

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a glycosyl-phosphatidylinositol-linked co-receptor, either GFRα1 or GFRα2. By whole-mountin situhybridization on embryonic mouse spinal cord, we demonstrate that whereasRetis expressed by nearly all motoneurons,Gfra1 andGfra2 exhibit complex and distinct patterns of expression. Most motoneurons purified fromGfra1 null mutant mice had lost their responsiveness to both GDNF and NTN. However, a minority of them (∼25%) retained their ability to respond to both factors, perhaps because they express GFRα2. Surprisingly,Gfra2−/−motoneurons showed normal survival responses to both GDNF and NTN. Thus, GFRα1, but not GFRα2, is absolutely required for the survival response of a majority of motoneurons to both GDNF and NTN. In accordance with the phenotype of the mutant motoneurons observed in culture we found the loss of distinct groups of motoneurons, identified by several markers, in theGfra1−/−spinal cords but no gross defects in theGfra2−/−mutant. During their natural programmed cell death period, motoneurons in theGfra1−/−mutant mice undertook increased apoptosis. Taken together these findings support the existence of subpopulations of motoneuron with different trophic requirements, some of them being dependent on the GDNF family.