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This protocol describes how to use the Brainbow strategy to label neurons in many different hues. The Brainbow system uses a random Cre/lox recombination to create varied combinations of red, blue, and green fluorescent proteins in each cell. The differences in color allow users to follow multiple cells, regardless of how closely they are positioned. This protocol describes how to use Brainbow imaging in zebrafish and provides examples of how to use color as a guide to trace axonal processes. We use the zebrafish trigeminal sensory ganglion as an example and discuss potential modifications for the general use of this technique., Molecular and Cellular Biology

This protocol describes how to generate Brainbow transgenes and mice, how to induce recombination in Brainbow mice with tamoxifen, and how to fix tissue samples from these animals. It also describes how to acquire multichannel images from Brainbow samples and use those images to digitally trace cellular contours and connections., Molecular and Cellular Biology

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

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, GRAB ATP1.0 , 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 μm 2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca 2+ transients, and intracellular Ca 2+ 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 Ca 2+ -independent manners, thus potentially regulating hundreds of synapses all together., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no competing interest.

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., (© 2020 Wiley Periodicals, Inc.)

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., Competing Interests: Declaration of Interests J.L., T.K., R.B.-M., F.M., S.T., K.L., and M. Le have filed a patent application related to the technology described in this article (number EP18305623)., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)