Digital Reconstruction of the Neuro-Glia-Vascular Architecture

Abstract

Astrocytes connect the vasculature to neurons mediating the supply of nutrients and biochemicals. They are involved in a growing number of physiological and pathophysiological processes that result from biophysical, physiological, and molecular interactions in this neuro-glia-vascular ensemble (NGV). The lack of a detailed cytoarchitecture severely restricts the understanding of how they support brain function. To address this problem, we used data from multiple sources to create a data-driven digital reconstruction of the NGV at micrometer anatomical resolution. We reconstructed 0.2 mm3 of the rat somatosensory cortex with 16 000 morphologically detailed neurons, 2500 protoplasmic astrocytes, and its microvasculature. The consistency of the reconstruction with a wide array of experimental measurements allows novel predictions of the NGV organization, allowing the anatomical reconstruction of overlapping astrocytic microdomains and the quantification of endfeet connecting each astrocyte to the vasculature, as well as the extent to which they cover the latter. Structural analysis showed that astrocytes optimize their positions to provide uniform vascular coverage for trophic support and signaling. However, this optimal organization rapidly declines as their density increases. The NGV digital reconstruction is a resource that will enable a better understanding of the anatomical principles and geometric constraints, which govern how astrocytes support brain function.

Keywords: 3D models; astrocyte; morphology; neuro-glia-vasculature; neuroanatomy; simulation.

Figure 1

Neuro—glia—vascular architecture overview. (A) Astrocytes contact and wrap around synapses and project their perivascular processes to the surface of the vasculature, where they form endfeet. (B) Astrocytes establish anatomically exclusive domains, which minimally overlap with their astrocyte neighbors. (C) In silico digital morphologies are tree structures that connect to a central soma geometry. (D) In silico digital reconstruction of neuronal neocortical microcircuit of the somatosensory cortex. (E) Experimental reconstruction of cerebral microvasculature. (F) Overlap of neuronal mesocircuit and vasculature dataset. (G) Percentage of existing volume occupancy in the circuit space.

Figure 2

Example of the decomposition of astrocytic trees into persistence barcodes. (A) Two small trees of perivascular (blue) and perisynaptic (red) types were selected for demonstration purposes from an experimentally reconstructed astrocyte morphology (soma in black). (B) Each tree is decomposed into a barcode, with each branch being represented as a horizontal line (bar) marking the start and end path length from the soma. The barcode in (B) can also be represented as points in the persistence diagram (C), in which the start and end path lengths of each bar are shown as y and x coordinates, respectively. (D) By applying a kernel density estimator on the persistence diagram, the persistence image is generated, which shows the bar density of the branching structure.

Figure 3

Reconstruction algorithms summary. (A) Cell placement. Astrocytic somata (green) were placed inside a voxelized atlas (grid), sampling from a spatial probability density that combined the cortical density with the distancing between astrocytes (red lines). (B) Astrocytic microdomains were generated from the location and size of astrocytic somas, modeled as a tessellation. (C) NGV connectome. The microdomains determined the accessible space for each astrocyte to establish connections with the vasculature (red) and the neuronal synapses (blue). Connectivity (black dots) between astrocytes (green) was determined after the morphologies have been grown, from the geometrical overlapping. (D) Growing of endfeet areas. Starting from the initial endfeet target points (D1), waves propagated following the geodesics of the vasculature surface, until they either reached an already occupied area or exceeded a maximum growing radius (D2). The converged areas were then truncated so that they match the input area distribution (D3–D4). (E) Morphology synthesis. Experimentally reconstructed astrocytic morphologies were converted into persistence barcodes, which determined the branching probabilities of in silico synthesized morphologies. The local directions of the processes was determined by the local attraction to synaptic terminals. (F) The synthesized morphologies were used to determine the connectivity between astrocytes and their neighbors, via geometric proximity.

Figure 4

NGV data validation—population level. NGV circuit results are represented with orange, whereas literature data with gray, unless stated otherwise. (A) Astrocytic soma density comparison between the NGV circuit and reported values from Appaix et al. (2012). (B) Bar plot comparison between the circuit’s soma radius distribution and literature measurements. (C) The respective histogram of soma radius distribution. (D) Circuit’s histogram of the nearest neighbor distance distribution compared with the input constraint of López-Hidalgo et al. (2016) (gray line). (E) Average astrocyte density in context with measurements from various literature sources. (F) Comparison of volume distribution between tiling (gray) and overlapping (orange) microdomains. (G) Microdomain volume distribution comparison versus literature sources. (H) Pruned endfeet areas histogram and validation of its ECDF versus the target distribution (I) from Calì et al. (2019). (J) The surface area distribution results in a 60% coverage of the total vasculature surface area, leaving 40% uncovered (blue). (K and L) The shortest path length distribution from the soma to the vessel surface was validated against the measurements in Moye et al. (2019). (M) Reproduction of the endfeet volumes and areas relationship as measured in Calì et al. (2019). (N) Comparison of the number of primary and perivascular processes with respect to the literature.

Figure 5

NGV data validation. (A) Entire circuit of synthesized astrocytic morphologies. (B) Example of six morphologies and a closeup (C) of a synthesized astrocyte and its respective endfoot on the vascular surface. (D) Feature comparison between synthesized (orange) and experimental (blue) astrocytes for perisynaptic and perivascular processes. (E) Per layer persistence diagrams overlap between the synthesized and experimental astrocytes. The topological distance of each layer’s persistence diagram compared with the experimental (black continuous line) and its respective standard error (black dashed line). Path distance units are in microns.

Figure 6

Spatial kernel density estimates plots of large vessels (A), capillaries (B), astrocytic somata coordinates (C), and endfeet targets on the surface of the vasculature (D). (E) Homogeneous distribution of endfeet targets in layer I. (F) A 30 μm slice in layer I of endfeet targets (black) and the vasculature surface mesh points (red).

Figure 7

Effect of astrocytic proliferation on the feasibility of perivascular processes in the same bounding space. The red data points correspond to the reference circuit with the biological parameters. (A) Increasing the astrocytic density resulted in an increase in the endfeet numbers. (B) The number of endfeet per astrocyte decreased despite their total numbers. (C) The denser packing resulted in smaller distances to the vessels and domain extents. (D) Classifying the astrocytes into astrocytes with and without endfeet, we measured that as the number of astrocytes increased, astrocytes with no endfeet increased in number, (E) their distance to the closest vessel became smaller, and (F) because of the packing, there is a bias for smaller soma sizes. (G) The increase in astrocytic density within the same volume results in a lower percentage of encapsulated synapses compared with the reference circuit. (H) Relationship between endfeet and percentage of encapsulated synapses per astrocyte, which is a consequence of the shrinking of astrocytic domains (I) as density increases.

Figure 8

NGV data predictions. (A) Neuron (blue), astrocyte (green), and vasculature (red) total process length, surface areas (B), and volume fractions (C) per layer. (D, E) Total endfeet area and volume per astrocyte. (F, G) Number of neurons and synapses connected per astrocyte. (H, I) Number of neighbors and gap junctional connections per astrocyte. (J–L) Total neuron process length, surface area, and volume per astrocytic microdomain across the cortical depth.