Oligodendrocyte death is common in aging and neurodegenerative disease. In these conditions, dying oligodendrocytes must be efficiently removed to allow remyelination and to prevent a feedforward degenerative cascade. Removal of this cellular debris is thought to primarily be carried out by resident microglia. To investigate the cellular dynamics underlying how microglia do this, we use a single-cell cortical demyelination model combined with longitudinal intravital imaging of dual-labeled transgenic mice. Following phagocytosis, single microglia clear the targeted oligodendrocyte and its myelin sheaths in one day via a precise, rapid, and stereotyped sequence. Deletion of the fractalkine receptor, CX3CR1, delays the microglial phagocytosis of the cell soma but has no effect on clearance of myelin sheaths. Unexpectedly, deletion of the phosphatidylserine receptor, MERTK, has no effect on oligodendrocyte or myelin sheath clearance. Thus, separate molecular signals are used to detect, engage, and clear distinct sub-compartments of dying oligodendrocytes to maintain tissue homeostasis.

Myelin degeneration occurs in neurodegenerative diseases and aging. In these conditions, resident oligodendrocyte progenitor cells (OPCs) differentiate into oligodendrocytes that carry out myelin repair. To investigate the cellular dynamics underlying these events, we developed a noninflammatory demyelination model that combines intravital two-photon imaging with a single-cell ablation technique called two-photon apoptotic targeted ablation (2Phatal). Oligodendrocyte 2Phatal in both sexes results in a myelin degeneration cascade that triggers rapid forms of synchronous remyelination on defined axons. This remyelination is driven by oligodendrocytes differentiated from a subset of morphologically distinct, highly branched OPCs. Moreover, remyelination efficiency depends on the initial myelin patterns, as well as the age of the organism. In summary, using 2Phatal, we show a form of rapid synchronous remyelination, mediated by a distinct subset of OPCs, capable of restoring the original myelin patterning in adulthood but not aging.

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-β, yet the methodology for studying efflux remains rudimentary. Here, we develop a method to evaluate glymphatic real-time clearance. Efflux of Direct Blue 53 (DB53, also T-1824 or Evans Blue) injected into the striatum is quantified by imaging the DB53 signal in the vascular compartment, where it is retained due to its high affinity to albumin. The DB53 signal is detectable as early as 15 min after injection and the efflux kinetics are sharply reduced in mice lacking the water channel aquaporin 4 (AQP4). Pharmacokinetic modeling reveal that DB53 efflux is consistent with the existence of two efflux paths, one with fast kinetics (T1/2 = 50 min) and another with slow kinetics (T1/2 = 240 min), in wild-type mice. This in vivo methodology will aid in defining the physiological variables that drive efflux, as well as the impact of brain states or disorders on clearance kinetics.

Cerebral oedema develops after anoxic brain injury. In two models of asphyxial and asystolic cardiac arrest without resuscitation, we found that oedema develops shortly after anoxia secondary to terminal depolarizations and the abnormal entry of CSF. Oedema severity correlated with the availability of CSF with the age-dependent increase in CSF volume worsening the severity of oedema. Oedema was identified primarily in brain regions bordering CSF compartments in mice and humans. The degree of ex vivo tissue swelling was predicted by an osmotic model suggesting that anoxic brain tissue possesses a high intrinsic osmotic potential. This osmotic process was temperature-dependent, proposing an additional mechanism for the beneficial effect of therapeutic hypothermia. These observations show that CSF is a primary source of oedema fluid in anoxic brain. This novel insight offers a mechanistic basis for the future development of alternative strategies to prevent cerebral oedema formation after cardiac arrest.

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-ß and Tau, yet the pathways and mechanisms of clearance remain unclear. Here we develop a new method to evaluate glymphatic clearance in real-time. Direct Blue 53 (DB53), a stable fluorescent compound with low toxicity, is delivered in striatum and imaged in the vascular compartment where it is retained due to its high affinity for albumin. DB53 signal was detectable in femoral veins as early as 20 min post-injection in young mice while aged mice exhibited a sharp reduction in the kinetics of DB53 clearance. Pharmacokinetic modeling revealed that DB53 efflux is consistent with the existence of two efflux paths, with fast (T1/2=32 min) kinetics and slow kinetics (T1/2=170 min), demonstrating that this real-time, in vivo methodology can provide novel insights into the kinetics of brain fluid clearance.

Significance Nerve terminals generally engage in two opposite and essential forms of synaptic plasticity (facilitation or depression) that play critical roles in learning and memory. While the molecular components of both types of terminals are similar with regards to vesicle fusion, much less is known about their molecular control of electrical signaling. Measurements of the electrical impulses (action potentials) underlying these two forms of plasticity have been difficult in small nerve terminals due to their size. In this study we deployed optical physiology measurements to overcome this size barrier. Here, we identify a unique mechanism (Kvβ1 subunit) that enables broadening of the presynaptic action potentials that selectively supports synaptic facilitation, but does not alter any other aspects of nerve terminal function.

ABSTRACTMyelin is critical for neural circuit function and its destruction is widespread in neurodegenerative disease and aging. In these conditions, homeostatic repair mechanisms initiate oligodendrocyte replacement by resident progenitor cells called NG2-glia. To investigate the cellular dynamics of this repair we developed a novel demyelination model by combining intravital myelin imaging with a targeted single-cell ablation technique called 2Phatal. Oligodendrocyte 2Phatal activated a stereotyped degeneration cascade which triggered remyelination by local NG2-glia. Remyelination efficiency was dependent on initial myelin patterning and dynamic imaging revealed rapid repair mechanisms resulting in near-seamless transitions between myelin loss and repair. A subset of morphologically complex NG2-glia executed this remyelination, pointing towards unrecognized functional diversity within this population. Age-related demyelination mirrored the degenerative cascade observed with 2Phatal, while remyelination in aging was defective due to failed oligodendrogenesis. Thus, oligodendrocyte 2Phatal revealed cellular diversity within the oligodendrocyte lineage and uncovered novel forms of rapid remyelination.

Despite the initial promise of immunotherapy for CNS disease, multiple recent clinical trials have failed. This may be due in part to characteristically low penetration of antibodies to cerebrospinal fluid (CSF) and brain parenchyma, resulting in poor target engagement. We here utilized transcranial macroscopic imaging to noninvasively evaluate in vivo delivery pathways of CSF fluorescent tracers. Tracers in CSF proved to be distributed through a brain-wide network of periarterial spaces, previously denoted as the glymphatic system. CSF tracer entry was enhanced approximately 3-fold by increasing plasma osmolality without disruption of the blood-brain barrier. Further, plasma hyperosmolality overrode the inhibition of glymphatic transport that characterizes the awake state and reversed glymphatic suppression in a mouse model of Alzheimer's disease. Plasma hyperosmolality enhanced the delivery of an amyloid-β (Aβ) antibody, obtaining a 5-fold increase in antibody binding to Aβ plaques. Thus, manipulation of glymphatic activity may represent a novel strategy for improving penetration of therapeutic antibodies to the CNS.

Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.