A tridimensional atlas of the developing human head.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

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.

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.

Diffusion-weighted magnetic resonance spectroscopy enables cell-specific monitoring of astrocyte reactivity in vivo.

Reactive astrocytes exhibit hypertrophic morphology and altered metabolism. Deciphering astrocytic status would be of great importance to understand their role and dysregulation in pathologies, but most analytical methods remain highly invasive or destructive. The diffusion of brain metabolites, as non-invasively measured using diffusion-weighted magnetic resonance spectroscopy (DW-MRS) in vivo, depends on the structure of their micro-environment. Here we perform advanced DW-MRS in a mouse model of reactive astrocytes to determine how cellular compartments confining metabolite diffusion are changing. This reveals myo-inositol as a specific intra-astrocytic marker whose diffusion closely reflects astrocytic morphology, enabling non-invasive detection of astrocyte hypertrophy (subsequently confirmed by confocal microscopy ex vivo). Furthermore, we measure massive variations of lactate diffusion properties, suggesting that intracellular lactate is predominantly astrocytic under control conditions, but predominantly neuronal in case of astrocyte reactivity. This indicates massive remodeling of lactate metabolism, as lactate compartmentation is tightly linked to the astrocyte-to-neuron lactate shuttle mechanism.

Efficient GPU-based Monte-Carlo simulation of diffusion in real astrocytes reconstructed from confocal microscopy.

The primary goal of this work is to develop an efficient Monte-Carlo simulation of diffusion-weighted signal in complex cellular structures, such as astrocytes, directly derived from confocal microscopy. In this study, we first use an octree structure for spatial decomposition of surface meshes. Octree structure and radius-search algorithm help to quickly identify the faces that particles can possibly encounter during the next time step, thus speeding up the Monte-Carlo simulation. Furthermore, we propose to use a three-dimensional binary marker to describe the complex cellular structure and optimize the particle trajectory simulation. Finally, a GPU-based version of these two approaches is implemented for more efficient modeling. It is shown that the GPU-based binary marker approach yields unparalleled performance, opening up new possibilities to better understand intracellular diffusion, validate diffusion models, and create dictionaries of intracellular diffusion signatures.

Can we detect the effect of spines and leaflets on the diffusion of brain intracellular metabolites?

Prior models used to clarify which aspects of tissue microstructure mostly affect intracellular diffusion and corresponding diffusion-weighted magnetic resonance (DW-MR) signal have focused on relatively simple geometrical descriptions of the cellular microenvironment (spheres, randomly oriented cylinders, etc…), neglecting finer morphological details which may have an important role. Some types of neurons present high density of spines; and astrocytes and macroglial cells processes present leaflets, which may all impact the diffusion process. Here, we use Monte-Carlo simulations of many particles diffusing in cylindrical compartments with secondary structures mimicking spines and leaflets of neuronal and glial cell fibers, to investigate to what extent the diffusion-weighted signal of intracellular molecules is sensitive to spines/leaflets density and length. In order to study the specificity of DW-MR signal to these kinds of secondary structures, beading-like geometry is simulated as "control" deviation from smooth cylinder too. Results suggest that: a) the estimated intracellular tortuosity increases as spines/leaflets density or length (beading amplitude) increase; b) the tortuosity limit is reached for diffusion time td>200 ms for metabolites and td>70 ms for water molecules, suggesting that the effects of these finer morphological details are negligible at td longer than these threshold values; c) fiber diameter is overestimated, while intracellular diffusivity is underestimated, when simple geometrical models based on hollow smooth cylinders are used; d) apparent surface-to-volume, S/V, ratio estimated by linear fit of high frequency OG data appears to be an excellent estimation of the actual S/V ratio, even in the presence of secondary structures, and it increases as spines and leaflets density or length increase (while decreasing as beadings amplitude increases). Comparison between numerical simulations and multimodal metabolites DW-MRS experiments in vivo in mouse brain shows that these fine structures may affect the DW-MRS signal and the derived diffusion metrics consistently with their expected density and geometrical features. This work suggests that finer structures of cell morphology have non-negligible effects on intracellular molecules' diffusion that may be measured by using multimodal DW-MRS approaches, stimulating future developments and applications.

Insulin Regulates Astrocytic Glucose Handling Through Cooperation With IGF-I.

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes.

The insulin-like growth factor I receptor regulates glucose transport by astrocytes.

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.