Why do oligodendrocyte lineage cells express glutamate receptors?

The function of glutamate receptors on oligodendrocytes and their precursor cells is poorly understood, with their only clear action being to damage these cells in pathological conditions. Here we review recent studies of glutamate signalling to oligodendrocyte lineage cells, and explore what its physiological function may be

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

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

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

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

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 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. Keywords: Mural cells; calcium signaling; mouse; neuroscience; pericyte; vasculature

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.ISSN:2399-364

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

Genetic background affects human glial fibrillary acidic protein promoter activity.

The human glial fibrillary acidic protein (hGFAP) promoter has been used to generate numerous transgenic mouse lines, which has facilitated the analysis of astrocyte function in health and disease. Here, we evaluated the expression levels of various hGFAP transgenes at different ages in the two most commonly used inbred mouse strains, FVB/N (FVB) and C57BL/6N (B6N). In general, transgenic mice maintained on the B6N background displayed weaker transgene expression compared with transgenic FVB mice. Higher level of transgene expression in B6N mice could be regained by crossbreeding to FVB wild type mice. However, the endogenous murine GFAP expression was equivalent in both strains. In addition, we found that endogenous GFAP expression was increased in transgenic mice in comparison to wild type mice. The activities of the hGFAP transgenes were not age-dependently regulated. Our data highlight the importance of proper expression analysis when non-homologous recombination transgenesis is used

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.ISSN:1662-510