Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity.

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.

Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²âº entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.

Oligodendrocyte-axon metabolic coupling is mediated by extracellular K+ and maintains axonal health.

The integrity of myelinated axons relies on homeostatic support from oligodendrocytes (OLs). To determine how OLs detect axonal spiking and how rapid axon-OL metabolic coupling is regulated in the white matter, we studied activity-dependent calcium (Ca2+) and metabolite fluxes in the mouse optic nerve. We show that fast axonal spiking triggers Ca2+ signaling and glycolysis in OLs. OLs detect axonal activity through increases in extracellular potassium (K+) concentrations and activation of Kir4.1 channels, thereby regulating metabolite supply to axons. Both pharmacological inhibition and OL-specific inactivation of Kir4.1 reduce the activity-induced axonal lactate surge. Mice lacking oligodendroglial Kir4.1 exhibit lower resting lactate levels and altered glucose metabolism in axons. These early deficits in axonal energy metabolism are associated with late-onset axonopathy. Our findings reveal that OLs detect fast axonal spiking through K+ signaling, making acute metabolic coupling possible and adjusting the axon-OL metabolic unit to promote axonal health.

Astrocytes adopt a progenitor-like migratory strategy for regeneration in adult brain.

Mature astrocytes become activated upon non-specific tissue damage and contribute to glial scar formation. Proliferation and migration of adult reactive astrocytes after injury is considered very limited. However, the regenerative behavior of individual astrocytes following selective astroglial loss, as seen in astrocytopathies, such as neuromyelitis optica spectrum disorder, remains unexplored. Here, we performed longitudinal in vivo imaging of cortical astrocytes after focal astrocyte ablation in mice. We discovered that perilesional astrocytes develop a remarkable plasticity for efficient lesion repopulation. A subset of mature astrocytes transforms into reactive progenitor-like (REPL) astrocytes that not only undergo multiple asymmetric divisions but also remain in a multinucleated interstage. This regenerative response facilitates efficient migration of newly formed daughter cell nuclei towards unoccupied astrocyte territories. Our findings define the cellular principles of astrocyte plasticity upon focal lesion, unravelling the REPL phenotype as a fundamental regenerative strategy of mature astrocytes to restore astrocytic networks in the adult mammalian brain. Promoting this regenerative phenotype bears therapeutic potential for neurological conditions involving glial dysfunction.

Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation.

BACKGROUND: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. METHODS: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. RESULTS: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. CONCLUSION: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.

Direct vascular contact is a hallmark of cerebral astrocytes.

Astrocytes establish extensive networks via gap junctions that allow each astrocyte to connect indirectly to the vasculature. However, the proportion of astrocytes directly associated with blood vessels is unknown. Here, we quantify structural contacts of cortical astrocytes with the vasculature in vivo. We show that all cortical astrocytes are connected to at least one blood vessel. Moreover, astrocytes contact more vessels in deeper cortical layers where vessel density is known to be higher. Further examination of different brain regions reveals that only the hippocampus, which has the lowest vessel density of all investigated brain regions, harbors single astrocytes with no apparent vascular connection. In summary, we show that almost all gray matter astrocytes have direct contact to the vasculature. In addition to the glial network, a direct vascular access may represent a complementary pathway for metabolite uptake and distribution.

Decoupling astrocytes in adult mice impairs synaptic plasticity and spatial learning.

The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition.

Distinct signatures of calcium activity in brain mural cells.

Pericytes have been implicated in various neuropathologies, yet little is known about their function and signaling pathways in health. Here, we characterized calcium dynamics of cortical mural cells in anesthetized or awake Pdgfrb-CreERT2;Rosa26< LSL-GCaMP6s > mice and in acute brain slices. Smooth muscle cells (SMCs) and ensheathing pericytes (EPs), also named as terminal vascular SMCs, revealed similar calcium dynamics in vivo. In contrast, calcium signals in capillary pericytes (CPs) were irregular, higher in frequency, and occurred in cellular microdomains. In the absence of the vessel constricting agent U46619 in acute slices, SMCs and EPs revealed only sparse calcium signals, whereas CPs retained their spontaneous calcium activity. Interestingly, chemogenetic activation of neurons in vivo and acute elevations of extracellular potassium in brain slices strongly decreased calcium activity in CPs. We propose that neuronal activation and an extracellular increase in potassium suppress calcium activity in CPs, likely mediated by Kir2.2 and KATP channels.

Shear-stress sensing by PIEZO1 regulates tendon stiffness in rodents and influences jumping performance in humans.

Athletic performance relies on tendons, which enable movement by transferring forces from muscles to the skeleton. Yet, how load-bearing structures in tendons sense and adapt to physical demands is not understood. Here, by performing calcium (Ca2+) imaging in mechanically loaded tendon explants from rats and in primary tendon cells from rats and humans, we show that tenocytes detect mechanical forces through the mechanosensitive ion channel PIEZO1, which senses shear stresses induced by collagen-fibre sliding. Through tenocyte-targeted loss-of-function and gain-of-function experiments in rodents, we show that reduced PIEZO1 activity decreased tendon stiffness and that elevated PIEZO1 mechanosignalling increased tendon stiffness and strength, seemingly through upregulated collagen cross-linking. We also show that humans carrying the PIEZO1 E756del gain-of-function mutation display a 13.2% average increase in normalized jumping height, presumably due to a higher rate of force generation or to the release of a larger amount of stored elastic energy. Further understanding of the PIEZO1-mediated mechanoregulation of tendon stiffness should aid research on musculoskeletal medicine and on sports performance.

Phosphatidylinositol 4,5-bisphosphate-dependent interaction of myelin basic protein with the plasma membrane in oligodendroglial cells and its rapid perturbation by elevated calcium.

Myelin basic protein (MBP) is an essential structural component of CNS myelin. The electrostatic association of this positively charged protein with myelin-forming membranes is a crucial step in myelination, but the mechanism that regulates myelin membrane targeting is not known. Here, we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) is important for the stable association of MBP with cellular membranes. In oligodendrocytes, overexpression of synaptojanin 1-derived phosphoinositide 5-phosphatase, which selectively hydrolyzes membrane PIP2, causes the detachment of MBP from the plasma membrane. In addition, constitutively active Arf6/Q67L induces the formation of PIP2-enriched endosomal vacuoles, leading to the redistribution of MBP to intracellular vesicles. Fluorescence resonance energy transfer imaging revealed an interaction of the PIP2 sensing probe PH-PLCdelta1 with wild-type MBP, but not with a mutant MBP isoform that fails to associate with the plasma membrane. Moreover, increasing intracellular Ca(2+), followed by phospholipase C-mediated PIP2 hydrolysis, as well as reduction of the membrane charge by ATP depletion, resulted in the dissociation of MBP from the glial plasma membrane. When the corpus callosum of mice was analyzed in acute brain slices by electron microscopy, the reduction of membrane surface charge led to the loss of myelin compaction and rapid vesiculation. Together, these results establish that PIP2 is an essential determinant for stable membrane binding of MBP and provide a novel link between glial phosphoinositol metabolism and MBP function in development and disease.

Arousal-induced cortical activity triggers lactate release from astrocytes.

It has been suggested that, in states of arousal, release of noradrenaline and ß-adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of ß-adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousal-induced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when ß-adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by ß-adrenergic signalling and the mobilization of lactate from glycogen stores.

Differences in glutamate uptake between cortical regions impact neuronal NMDA receptor activation.

Removal of synaptically-released glutamate by astrocytes is necessary to spatially and temporally limit neuronal activation. Recent evidence suggests that astrocytes may have specialized functions in specific circuits, but the extent and significance of such specialization are unclear. By performing direct patch-clamp recordings and two-photon glutamate imaging, we report that in the somatosensory cortex, glutamate uptake by astrocytes is slower during sustained synaptic stimulation when compared to lower stimulation frequencies. Conversely, glutamate uptake capacity is increased in the frontal cortex during higher frequency synaptic stimulation, thereby limiting extracellular buildup of glutamate and NMDA receptor activation in layer 5 pyramidal neurons. This efficient glutamate clearance relies on Na+/K+-ATPase function and both GLT-1 and non-GLT-1 transporters. Thus, by enhancing their glutamate uptake capacity, astrocytes in the frontal cortex may prevent excessive neuronal excitation during intense synaptic activity. These results may explain why diseases associated with network hyperexcitability differentially affect individual brain areas.

Non-Canonical Control of Neuronal Energy Status by the Na+ Pump.

Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors in vitro and in vivo. A short theta burst triggered immediate Na+ entry, followed by a delayed stimulation of the Na+/K+ ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na+ pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na+ pump stimulation. Experiments revealed that the Na+ pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca2+, but by a mechanism commanded by the Na+ pump.

Intravitreal AAV-Delivery of Genetically Encoded Sensors Enabling Simultaneous Two-Photon Imaging and Electrophysiology of Optic Nerve Axons.

Myelination of axons by oligodendrocytes is a key feature of the remarkably fast operating CNS. Oligodendrocytes not only tune axonal conduction speed but are also suggested to maintain long-term axonal integrity by providing metabolic support to the axons they ensheath. However, how myelinating oligodendrocytes impact axonal energy homeostasis remains poorly understood and difficult to investigate. Here, we provide a method of how to study electrically active myelinated axons expressing genetically encoded sensors by combining electrophysiology and two-photon imaging of acutely isolated optic nerves. We show that intravitreal adeno-associated viral (AAV) vector delivery is an efficient tool to achieve functional sensor expression in optic nerve axons, which is demonstrated by measuring axonal ATP dynamics following AAV-mediated sensor expression. This novel approach allows for fast expression of any optical sensor of interest to be studied in optic nerve axons without the need to go through the laborious process of producing new transgenic mouse lines. Viral-mediated biosensor expression in myelinated axons and the subsequent combination of nerve recordings and sensor imaging outlines a powerful method to investigate oligodendroglial support functions and to further interrogate cellular mechanisms governing axonal energy homeostasis under physiological and pathological conditions.

Myelin dynamics: protecting and shaping neuronal functions.

Myelinating glial cells are well-known to insulate axons and to speed up action potential propagation. Through adjustments in the axonal coverage with myelin, myelin sheath thickness and possibly nodal/internode length oligodendrocytes are involved in fine-tuning the brain's computational power throughout life. Be it motor skill learning or social behaviors in higher vertebrates, proper myelination is critical in shaping brain functions. Neurons rely on their myelinating partners not only for setting conduction speed, but also for regulating the ionic environment and fueling their energy demands with metabolites. Also, long-term axonal integrity and neuronal survival are maintained by oligodendrocytes and loss of this well-coordinated axon-glial interplay contributes to neuropsychiatric diseases. Better insight into how myelination and oligodendrocyte functions are constantly fine-tuned in the adult CNS, which includes sensing of neuronal activity and adjusting glial metabolic support, will be critical for understanding higher brain functions and cognitive decline associated with myelin abnormalities in the aging brain.

Monitoring ATP dynamics in electrically active white matter tracts.

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders.

Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons.

Design and performance of an ultra-flexible two-photon microscope for in vivo research.

We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.