Summary The precise regulation of cerebral blood flow is critical for normal brain function and its disruption underlies many neuropathologies. The extent to which smooth muscle-covered arterioles or pericyte-covered capillaries control vasomotion during neurovascular coupling remains controversial. We found that capillary pericytes in mice and humans do not express smooth muscle actin and are morphologically and functionally distinct from adjacent precapillary smooth muscle cells (SMCs). Using optical imaging we investigated blood flow regulation at various sites on the vascular tree in living mice. Optogenetic, whisker stimulation or cortical spreading depolarization caused microvascular diameter or flow changes in SMC but not pericyte-covered microvessels. During early stages of brain ischemia, transient SMC but not pericyte constrictions were a major cause of hypoperfusion leading to thrombosis and distal microvascular occlusions. Thus, capillary pericytes are not contractile and regulation of cerebral blood flow in physiological and pathological conditions is mediated by arteriolar smooth muscle cells.

SUMMARYNG2-expressing glia (NG2 cells, polydendrocytes) appear in the embryonic brain, expand perinatally, and persist widely throughout the gray and white matter of the mature central nervous system. We have previously reported that NG2 cells generate oligodendrocytes in both gray and white matter and a subset of protoplasmic astrocytes in the gray matter of the ventral forebrain and spinal cord. To investigate the temporal changes in NG2 cell fate, we generated NG2creER TM BAC transgenic mice, in which tamoxifen-inducible Cre is expressed in NG2 cells. Cre induction at embryonic day 16.5, postnatal day (P) 2, P30 and P60 in mice that were double transgenic for NG2creER TM BAC and the Cre reporter revealed that NG2 cells in the postnatal brain generate only NG2 cells or oligodendrocytes, whereas NG2 cells in the embryonic brain generate protoplasmic astrocytes in the gray matter of the ventral forebrain in addition to oligodendrocytes and NG2 cells. Analysis of cell clusters from single NG2 cells revealed that more than 80% of the NG2 cells in the P2 brain give rise to clusters consisting exclusively of oligodendrocytes, whereas the majority of the NG2 cells in the P60 brain generate clusters that contain only NG2 cells or a mixture of oligodendrocytes and NG2 cells. Furthermore, live cell imaging of single NG2 cells from early postnatal brain slices revealed that NG2 cells initially divide symmetrically to produce two daughter NG2 cells and that differentiation into oligodendrocytes occurred after 2-3 days.

Axonal myelin increases neural processing speed and efficiency. In adulthood, it is not known if patterns of myelin distribution are fixed or if myelinating oligodendrocytes are continually generated and maintain the capacity for structural remodeling. Using high-resolution intravital label-free and fluorescence optical imaging in the mouse cortex, we demonstrate lifelong oligodendrocyte generation occurring in parallel with structural plasticity of individual myelin internodes. Continuous internode formation occurred on both partially myelinated and unmyelinated axons and the total myelin coverage along individual axons progressed up to two years of age. After peak myelination, gradual oligodendrocyte death and myelin degeneration in aging were associated with pronounced internode loss and myelin debris accumulation within microglia. Thus, cortical myelin remodeling is protracted throughout life, potentially playing critical roles in neuronal network homeostasis. The gradual loss of internodes and myelin degeneration in aging could contribute significantly to brain pathogenesis.

Cell death is prevalent throughout life; however, the coordinated interactions and roles of phagocytes during corpse removal in the live brain are poorly understood. We developed photochemical and viral methodologies to induce death in single cells and combined this with intravital optical imaging. This approach allowed us to track multicellular phagocytic interactions with precise spatiotemporal resolution. Astrocytes and microglia engaged with dying neurons in an orchestrated and synchronized fashion. Each glial cell played specialized roles: Astrocyte processes rapidly polarized and engulfed numerous small dendritic apoptotic bodies, while microglia migrated and engulfed the soma and apical dendrites. The relative involvement and phagocytic specialization of each glial cell was plastic and controlled by the receptor tyrosine kinase Mertk. In aging, there was a marked delay in apoptotic cell removal. Thus, a precisely orchestrated response and cross-talk between glial cells during corpse removal may be critical for maintaining brain homeostasis.

Oligodendrocytes in the mammalian brain are continuously generated from NG2 cells throughout postnatal life. However it has remained unclear when the decision of NG2 cells to self-renew or differentiate into oligodendrocytes occurs after cell division. Using a combination of in vivo and ex vivo imaging and fate analysis of proliferated NG2 cells in fixed tissue, we demonstrate that in the postnatal developing mouse brain, the majority of divided NG2 cells differentiate into oligodendrocytes during a critical age-specific temporal window of 3–8 days. Importantly, within this time period, myelin and oligodendrocyte damage accelerated oligodendrocyte differentiation from divided cells, while whisker removal decreased the survival of divided cells in the deprived somatosensory cortex. These findings indicate that during the critical temporal window of plasticity, the fate of divided NG2 cells is sensitive to modulation by external signals.

We report a new technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we performed chronic brain imaging up to 400 μm deep, capturing for the first time de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.

Glial cells that express the NG2 proteoglycan and the ␣ receptor for PDGF (NG2 cells, polydendrocytes) make up the fifth major cell population that serves as oligodendrocyte progenitor cells in the postnatal CNS. Although recent studies have suggested differences in their proliferation and oligodendrocyte differentiation in gray and white matter, the mechanism underlying the observed differences has been unclear. Using organotypic slice cultures from the forebrain and cerebellum of early postnatal NG2creBAC:ZEG mice, we have compared basal and growth factor-induced proliferation of NG2 cells in gray and white matter. NG2 cells in white matter exhibited greater proliferative response to PDGF AA than those in gray matter. Heterotopic slice transplant and explant cultures suggested intrinsic mechanisms for the differential proliferative response of gray and white matter cells. Additionally, younger white matter NG2 cells showed a more robust proliferative response to PDGF. Basal and PDGF-induced proliferation of gray and white matter NG2 cells was largely dependent on Wnt/␤-catenin and phosphatidylinositol 3-kinase acting through the mammalian target of rapamycin pathway and not through ERK. These data uncover a previously unrecognized divergence between gray and white matter NG2 cells in the developing brain in their proliferative response to PDGF.

NG2 cells represent a unique glial cell population that is distributed widely throughout the developing and adult CNS and is distinct from astrocytes, mature oligodendrocytes and microglia. The ability of NG2 cells to differentiate into myelinating oligodendrocytes has been documented in vivo and in vitro. We reported recently that NG2 cells in the forebrain differentiate into myelinating oligodendrocytes but into a subpopulation of protoplasmic astrocytes (Zhu et al., 2008). However, the in vivo fate of NG2 cells in the spinal cord and cerebellum has remained unknown. To investigate the fate of NG2 cells in caudal central nervous system (CNS) regions in vivo, we examined the phenotype of cells that express EGFP in mice that are double transgenic for NG2CreBAC and the Cre reporter Z/EG. The fate of NG2 cells can be studied in these mice by permanent expression of EGFP in cells that have undergone Cre-mediated recombination in NG2 cells. We find that NG2 cells give rise to oligodendrocytes in both gray and white matter of the spinal cord and cerebellum, and to protoplasmic astrocytes in the gray matter of the spinal cord. However, NG2 cells do not give rise to astrocytes in the white matter of the spinal cord and cerebellum. These observations indicate that NG2 cells serve as precursor cells for oligodendrocytes and a subpopulation of protoplasmic astrocytes throughout the rostrocaudal axis of the CNS.