GSBS Dissertations and Theses

ORCID ID

0000-0002-3533-6538

Publication Date

2020-08-26

Document Type

Doctoral Dissertation

Academic Program

Neuroscience

Department

Neurobiology; Schafer Lab

First Thesis Advisor

Dorothy Schafer

Keywords

Microglia, Blood Vessel, Development, Fractalkine Receptor

Abstract

Microglia, the resident macrophages of the central nervous system (CNS), are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about how microglia interact with the CNS vasculature, particularly under healthy steady-state conditions. Here, I provide the first extensive characterization of juxtavascular microglia in the healthy, postnatal brain and identify a molecular mechanism regulating the timing of these interactions during development. Using the mouse cerebral cortex, I show that microglia are intimately associated with the vasculature in the CNS, directly contacting the basal lamina in vascular sites that are devoid of astrocyte endfeet. I demonstrate a high percentage of microglia are associated with the vasculature during the first week of postnatal development, which is concomitant with a peak in microglial colonization of the cortex and recruitment to synapses. I find that as microglia colonize the cortex, juxtavascular microglia are highly motile along vessels and become largely stationary as the brain matures. 2-photon live imaging in adult mice reveals that these vascular-associated microglia in the mature brain are stable and stationary for several weeks. Further, a decrease in microglia motility along the vasculature is tightly correlated with the expansion of astrocyte endfeet along the vasculature. Finally, I provide evidence that the timing of these microglia-vascular interactions during development is regulated by the microglial fractalkine receptor (CX3CR1). Together, these data support a model by which microglia use the vasculature as a scaffold to migrate and colonize the developing brain and the timing of these associations is modulated by CX3CR1. This migration along the vasculature becomes restricted as astrocyte vascular endfoot territory expands and, upon maturation, vascular-associated microglia become largely stationary.

DOI

10.13028/rgfc-7v07

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Movie1.mp4 (5298 kB)
Movie 1: 3D rendering of juxtavascular microglia in the early postnatal frontal cortex. 3D reconstruction and surface rendering of juxtavascular microglia (green, EGFP) associated with blood vessels (magenta, anti-PECAM) in the P5 frontal cortex. Yellow denotes association area between microglia and blood vessels.

Movie2.mp4 (6533 kB)
Movie 2: 3D rendering of juxtavascular microglia in the P28 frontal cortex. 3D reconstruction and surface rendering of juxtavascular microglia (green, EGFP) associated with blood vessels (magenta, anti-PECAM) in the P28 frontal cortex. Yellow denotes association area between microglia and blood vessels.

Movie3.mp4 (9607 kB)
Movie 3: Juxtavascular microglial migration in the early postnatal somatosensory cortex. Representative live imaging of juxtavascular microglia (green, EGFP) migrating on vessels (magenta; dextran) in the P7 somatosensory. Cx3cr1EGFP/+ mice received a retro-orbital injection of Texas red labeled dextran to label the vasculature 10 minutes prior to euthanasia. Coronal somatosensory cortices were imaged every 5 minutes over 6 hours immediately following slice preparation.

Movie4.mp4 (11425 kB)
Movie 4: Juxtavascular microglial migration in the early postnatal somatosensory cortex. A second representative live imaging of juxtavascular microglia (green, EGFP) migrating on vessels (magenta; dextran) in the P7 somatosensory. Cx3cr1EGFP/+ mice received a retro-orbital injection of Texas red labeled dextran to label the vasculature 10 minutes prior to euthanasia. Coronal somatosensory cortices were imaged every 5 minutes over 6 hours immediately following slice preparation.

Movie5.mp4 (9906 kB)
Movie 5: Juxtavascular microglial migration in the early postnatal somatosensory cortex. A third representative live imaging of juxtavascular microglia (green, EGFP) migrating on vessels (magenta; dextran) in the P7 somatosensory. Cx3cr1EGFP/+ mice received a retro-orbital injection of Texas red labeled dextran to label the vasculature 10 minutes prior to euthanasia. Coronal somatosensory cortices were imaged every 5 minutes over 6 hours immediately following slice preparation.

Movie6.mp4 (2024 kB)
Movie 6: Juxtavascular microglial migration in the adult somatosensory cortex. Representative live imaging of juxtavascular microglia (green, EGFP) stationary on vessels (magenta; dextran) in the P≥120 somatosensory cortex. Cx3cr1EGFP/+ mice received a retro-orbital injection of Texas red labeled dextran to label the vasculature 10 minutes prior to euthanasia. Coronal somatosensory cortices were imaged every 5 minutes over 6 hours immediately following slice preparation.

Movie7.mp4 (255 kB)
Movie 7: 2-photon in vivo live imaging of juxtavascular microglia in the adult cortex. Representative 2-photon in vivo live imaging of juxtavascular microglia (green, EGFP) stationary on blood vessels (magenta, dextran) over 2 hours in vivo in the adult cortex. Cx3cr1EGFP/+ mice received a retro-orbital injection of Texas Red-labeled dextran to visualize the vasculature 10 min prior to each imaging session. EGFP+ juxtavascular microglia were then imaged every 5 minutes for 2 hours.

Movie8.mp4 (5712 kB)
Movie 8: Juxtavascular microglia associate with the cortical vasculature in areas lacking full astrocytic endfoot coverage in the P5 frontal cortex. 3D reconstruction and surface rendering of juxtavascular microglia (green, EGFP) associated with blood vessels (magenta, anti-PDGFRβ) in areas void of astrocytic endfoot labeling (gray, anti-AQP4) in the frontal cortex at P5.

Movie9.mp4 (7992 kB)
Movie 9: Juxtavascular microglia associate with the cortical vasculature in areas lacking full astrocytic endfoot coverage in the P7 frontal cortex. 3D reconstruction and surface rendering of juxtavascular microglia (green, EGFP) associated with blood vessels (magenta, anti-PDGFRβ) in areas void of astrocytic endfoot labeling (gray, anti-AQP4) in the frontal cortex at P7.

Movie10.mp4 (7303 kB)
Movie 10: Juxtavascular microglia associate with the cortical vasculature in areas lacking full astrocytic endfoot coverage in the P28 frontal cortex. 3D reconstruction and surface rendering of juxtavascular microglia (green, EGFP) associated with blood vessels (magenta, anti-PDGFRβ) in areas void of astrocytic endfoot labeling (gray, anti-AQP4) in the frontal cortex at P28.

Movie11.mp4 (8345 kB)
Movie 11: Serial EM 3D reconstruction of juxtavascular microglia in the early postnatal cortex. 3D reconstruction of serial electron microscopy (EM) of juxtavascular microglia (green) contacting a blood vessel in an area void of astrocyte endfeet (blue) in the P5 frontal cortex. Red and tan pseudocoloring denotes a pericyte and vessel lumen, respectively.

Movie12.mp4 (5881 kB)
Movie 12: Serial EM 3D reconstruction of juxtavascular microglia in the P56 cortex. 3D reconstruction of serial electron microscopy (EM) of juxtavascular microglia (green) contacting a blood vessel lacking full astrocyte endfoot (blue) coverage in the P56 frontal cortex. Red and tan pseudocoloring denotes a pericyte and vessel lumen, respectively.

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