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The vasculature is emerging as a key contributor to brain function during neurodevelopment and in mature physiological and pathological states. The brain vasculature itself also exhibits regional heterogeneity, highlighting the need to develop approaches for purifying cells from different microregions. Previous approaches for isolation of endothelial cells and pericytes have predominantly required transgenic mice and large amounts of tissue, and have resulted in impure populations. In addition, the prospective purification of brain pericytes has been complicated by the fact that widely used pericyte markers are also expressed by other cell types in the brain. Here, we describe the detailed procedures for simultaneous isolation of pure populations of endothelial cells and pericytes directly from adult mouse brain microregions using fluorescence-activated cell sorting (FACS) with antibodies against CD31 (endothelial cells) and CD13 (pericytes). This protocol is scalable and takes â^¼5 h, including microdissection of the region of interest, enzymatic tissue dissociation, immunostaining, and FACS. This protocol allows the isolation of brain vascular cells from any mouse strain under diverse conditions; these cells can be used for multiple downstream applications, including in vitro and in vivo experiments, and transcriptomic, proteomic, metabolomic, epigenomic, and single-cell analysis.

Neural stem cells (NSCs) in specialized niches in the adult mammalian brain generate neurons throughout life. NSCs in the adult mouse ventricular-subventricular zone (V-SVZ) exhibit a regional identity and, depending on their location, generate distinct olfactory bulb interneuron subtypes. Here, we show that the hypothalamus, a brain area regulating physiological states, provides long-range regionalized input to the V-SVZ niche and can regulate specific NSC subpopulations. Hypothalamic proopiomelanocortin neurons selectively innervate the anterior ventral V-SVZ and promote the proliferation of Nk×2.1⁺ NSCs and the generation of deep granule neurons. Accordingly, hunger and satiety regulate adult neurogenesis by modulating the activity of this hypothalamic–V-SVZ connection. Our findings reveal that neural circuitry, via mosaic innervation of the V-SVZ, can recruit distinct NSC pools, allowing on-demand neurogenesis in response to physiology and environmental signals.

The barrier between the blood and the ventricular cerebrospinal fluid (CSF) is located at the choroid plexuses. At the interface between two circulating fluids, these richly vascularized veil-like structures display a peculiar morphology explained by their developmental origin, and fulfill several functions essential for CNS homeostasis. They form a neuroprotective barrier preventing the accumulation of noxious compounds into the CSF and brain, and secrete CSF, which participates in the maintenance of a stable CNS internal environment. The CSF circulation plays an important role in volume transmission within the developing and adult brain, and CSF compartments are key to the immune surveillance of the CNS. In these contexts, the choroid plexuses are an important source of biologically active molecules involved in brain development, stem cell proliferation and differentiation, and brain repair. By sensing both physiological changes in brain homeostasis and peripheral or central insults such as inflammation, they also act as sentinels for the CNS. Finally, their role in the control of immune cell traffic between the blood and the CSF confers on the choroid plexuses a function in neuroimmune regulation and implicates them in neuroinflammation. The choroid plexuses, therefore, deserve more attention while investigating the pathophysiology of CNS diseases and related comorbidities.

The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP⁺EGFR⁺CD24⁻), niche astrocytes (GFP⁺EGFR⁻CD24⁻), transit amplifying cells (GFP⁻EGFR⁺CD24⁻), and neuroblasts (GFP⁻EGFR⁻CD24low). One in three EGFR⁺ astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphere-forming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.

Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by labelling the endogenous LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are highly abundant in various cell types in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we also show that LDs are abundant during brain development and can be visualized using live imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2. Here the authors describe a fluorescent lipid droplet reporter mouse, which allows the staining-free detection of these important lipid-storing organelles. Using this model, they show that LDs are highly abundant in the developing and adult brain.

Neurogenesis persists in two germinal regions in the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone in the hippocampal formation. Within these two neurogenic niches, specialized astrocytes are neural stem cells, capable of self-renewing and generating neurons and glia. Cues within the niche, from cell-cell interactions to diffusible factors, are spatially and temporally coordinated to regulate proliferation and neurogenesis, ultimately affecting stem cell fate choices. Here, we review the components of adult neural stem cell niches and how they act to regulate neurogenesis in these regions.

Glia are the most numerous cells in the brain, and their many diverse functions highlight their essential role in the nervous system. Recent studies have revealed an unexpected new role for glia in a wide variety of species, that of stem cells/progenitors in the adult and embryonic brain. Differentiation along the glial lineage may be a default state of development reflected in the progression of stem cells along the neuroepithelial→radial glia→astrocyte lineage.

Neuronal precursors reside in the subventricular zone (SVZ) of adult mammals. This region is composed of a network of chains of migrating neuroblasts ensheathed by astrocytes and juxtaposed by clusters of immature precursors (type C cells). Here we show that after antimitotic treatment with cytosine-β -D-arabinofuranoside, neuroblasts and type C cells are eliminated but some astrocytes remain. Remarkably, the SVZ network rapidly regenerates. Soon after cytosine-β -D-arabinofuranoside treatment astrocytes divide. Two days later, type C cells reappear, followed at 4.5 days by migrating neuroblasts. By 10 days the SVZ network is fully regenerated, and the orientation and organization of chains of migrating neuroblasts resemble that of normal mice. This regeneration reveals an unexpected plasticity in the adult central nervous system and should provide a model system to study the early stages of neurogenesis in the adult brain.

Cells in the brains of adult mammals continue to proliferate in the subventricular zone (SVZ) throughout the lateral wall of the lateral ventricle. Here we show, using whole mount dissections of this wall from adult mice, that the SVZ is organized as an extensive network of chains of neuronal precursors. These chains are immunopositive to the polysialylated form of NCAM, a molecule present at sites of plasticity, and TuJ1, an early neuronal marker. The majority of the chains are oriented along the rostrocaudal axis and many join the rostral migratory stream that terminates in the olfactory bulb. Using focal microinjections of DiI and transplantation of SVZ cells carrying a neuron-specific reporter gene, we demonstrate that cells originating at different rostrocaudal levels of the SVZ migrate rostrally and reach the olfactory bulb where they differentiate into neurons. Our results reveal an extensive network of pathways for the tangential chain migration of neuronal precursors throughout the lateral wall of the lateral ventricle in the adult mammalian brain.

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1–3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.