153 results on '"MacVicar BA"'
Search Results
2. Astrocyte Regulation of Cerebral Blood Flow in Health and Disease.
- Author
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Mishra A, Gordon GR, MacVicar BA, and Newman EA
- Subjects
- Neurons, Prostaglandins metabolism, Astrocytes metabolism, Cerebrovascular Circulation physiology
- Abstract
Astrocytes play an important role in controlling microvascular diameter and regulating local cerebral blood flow (CBF) in several physiological and pathological scenarios. Neurotransmitters released from active neurons evoke Ca
2+ increases in astrocytes, leading to the release of vasoactive metabolites of arachidonic acid (AA) from astrocyte endfeet. Synthesis of prostaglandin E2 (PGE2 ) and epoxyeicosatrienoic acids (EETs) dilate blood vessels while 20-hydroxyeicosatetraenoic acid (20-HETE) constricts vessels. The release of K+ from astrocyte endfeet also contributes to vasodilation or constriction in a concentration-dependent manner. Whether astrocytes exert a vasodilation or vasoconstriction depends on the local microenvironment, including the metabolic status, the concentration of Ca2+ reached in the endfoot, and the resting vascular tone. Astrocytes also contribute to the generation of steady-state vascular tone. Tonic release of both 20-HETE and ATP from astrocytes constricts vascular smooth muscle cells, generating vessel tone, whereas tone-dependent elevations in endfoot Ca2+ produce tonic prostaglandin dilators to limit the degree of constriction. Under pathological conditions, including Alzheimer's disease, epilepsy, stroke, and diabetes, disruption of normal astrocyte physiology can compromise the regulation of blood flow, with negative consequences for neurological function., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)- Published
- 2024
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3. Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1.
- Author
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Cain SM, Alles SRA, Gopaul R, Bernier LP, Yung AC, Bauman A, Yang Y, Baker GB, Kozlowski P, MacVicar BA, and Snutch TP
- Subjects
- Mice, Animals, Pregabalin pharmacology, Pregabalin therapeutic use, Mice, Transgenic, Hippocampus, Migraine with Aura drug therapy, Migraine with Aura genetics, Migraine Disorders drug therapy, Migraine Disorders genetics
- Abstract
Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Ca
v 2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs., (© 2023. The Author(s).)- Published
- 2023
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4. Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.
- Author
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Weilinger NL, Yang K, Choi HB, Groten CJ, Wendt S, Murugan M, Wicki-Stordeur LE, Bernier LP, Velayudhan PS, Zheng J, LeDue JM, Rungta RL, Tyson JR, Snutch TP, Wu LJ, and MacVicar BA
- Subjects
- Reactive Oxygen Species metabolism, Cell Death, Adenosine Triphosphate metabolism, Microglia metabolism, Connexins metabolism
- Abstract
Neuronal swelling during cytotoxic edema is triggered by Na
+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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5. hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons.
- Author
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Lee C, Frew J, Weilinger NL, Wendt S, Cai W, Sorrentino S, Wu X, MacVicar BA, Willerth SM, and Nygaard HB
- Subjects
- Humans, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Astrocytes metabolism, Progranulins genetics, Neurons metabolism, Mutation, Frontotemporal Dementia pathology, Induced Pluripotent Stem Cells metabolism, Pick Disease of the Brain metabolism
- Abstract
Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X
-/- knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X-/- astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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6. KCC2 drives chloride microdomain formation in dendritic blebbing.
- Author
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Weilinger NL, Wicki-Stordeur LE, Groten CJ, LeDue JM, Kahle KT, and MacVicar BA
- Subjects
- N-Methylaspartate, Bromides, Neurons metabolism, Chlorides metabolism, Symporters
- Abstract
Intracellular chloride ion concentration ([Cl
- ]i ) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl- ]i dynamics during N-methyl-d-aspartate (NMDA) excitotoxicity to determine how Cl- changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl- dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl- ]i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl- ]i subdomains at dendritic blebs in a process that requires Ca2+ influx and the subsequent opening of small-conductance Ca2+ -activated K+ (SK) channels. We propose sustained and localized K+ efflux increased extracellular K+ concentrations ([K+ ]o ) sufficiently at discrete regions to reverse K+ -Cl- cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl- ]i microdomains in blebbing with relevance for multiple neurological disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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7. Simultaneous imaging of redox states in dystrophic neurites and microglia at Aβ plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress.
- Author
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Wendt S, Johnson S, Weilinger NL, Groten C, Sorrentino S, Frew J, Yang L, Choi HB, Nygaard HB, and MacVicar BA
- Subjects
- Amyloid beta-Peptides metabolism, Amyloid beta-Protein Precursor metabolism, Animals, Disease Models, Animal, Lysosomes metabolism, Mice, Mice, Transgenic, Neurites, Oxidation-Reduction, Oxidative Stress, Ubiquitins metabolism, Ubiquitins pharmacology, Alzheimer Disease diagnostic imaging, Alzheimer Disease genetics, Alzheimer Disease metabolism, Plaque, Amyloid metabolism, Plaque, Amyloid pathology
- Abstract
The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer's Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aβ plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aβ plaques but instead create protective barriers around Aβ plaques possibly reducing the spread of Aβ. Intracranial injection of Aβ was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia. In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aβ plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2022
- Full Text
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8. Mitochondrial Ca 2+ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing.
- Author
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Groten CJ and MacVicar BA
- Subjects
- Action Potentials, Calcium metabolism, Mitochondria metabolism, NAD metabolism, Pyramidal Cells metabolism, Calcium Channels metabolism, Mitochondrial Membrane Transport Proteins metabolism
- Abstract
Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca
2+ uptake by the mitochondrial Ca2+ uniporter (MCU) has long been implicated in the control of energy metabolism and intracellular Ca2+ signalling, but its importance to neuronal function in the brain remains unclear. Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca2+ , cytosolic Ca2+ , and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca2+ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca2+ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD+ to NADH. Moreover, Ca2+ buffering by mitochondria attenuated cytosolic Ca2+ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity., (© 2022. The Author(s).)- Published
- 2022
- Full Text
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9. Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.
- Author
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Zuo M, Fettig NM, Bernier LP, Pössnecker E, Spring S, Pu A, Ma XI, Lee DS, Ward LA, Sharma A, Kuhle J, Sled JG, Pröbstel AK, MacVicar BA, Osborne LC, Gommerman JL, and Ramaglia V
- Subjects
- Animals, Gray Matter pathology, Humans, Inflammation, Mice, Neutrophils pathology, Encephalomyelitis, Autoimmune, Experimental, Multiple Sclerosis
- Abstract
People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.
- Published
- 2022
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10. Gut microbes shape microglia and cognitive function during malnutrition.
- Author
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Bauer KC, York EM, Cirstea MS, Radisavljevic N, Petersen C, Huus KE, Brown EM, Bozorgmehr T, Berdún R, Bernier LP, Lee AHY, Woodward SE, Krekhno Z, Han J, Hancock REW, Ayala V, MacVicar BA, and Finlay BB
- Subjects
- Animals, Cognition, Mice, Mice, Inbred C57BL, Microglia, Gastrointestinal Microbiome physiology, Malnutrition complications
- Abstract
Fecal-oral contamination promotes malnutrition pathology. Lasting consequences of early life malnutrition include cognitive impairment, but the underlying pathology and influence of gut microbes remain largely unknown. Here, we utilize an established murine model combining malnutrition and iterative exposure to fecal commensals (MAL-BG). The MAL-BG model was analyzed in comparison to malnourished (MAL mice) and healthy (CON mice) controls. Malnourished mice display poor spatial memory and learning plasticity, as well as altered microglia, non-neuronal CNS cells that regulate neuroimmune responses and brain plasticity. Chronic fecal-oral exposures shaped microglial morphology and transcriptional profile, promoting phagocytic features in MAL-BG mice. Unexpectedly, these changes occurred independently from significant cytokine-induced inflammation or blood-brain barrier (BBB) disruption, key gut-brain pathways. Metabolomic profiling of the MAL-BG cortex revealed altered polyunsaturated fatty acid (PUFA) profiles and systemic lipoxidative stress. In contrast, supplementation with an ω3 PUFA/antioxidant-associated diet (PAO) mitigated cognitive deficits within the MAL-BG model. These findings provide valued insight into the malnourished gut microbiota-brain axis, highlighting PUFA metabolism as a potential therapeutic target., (© 2022 The Authors. GLIA published by Wiley Periodicals LLC.)
- Published
- 2022
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11. Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of Sudden Unexpected Death in Epilepsy.
- Author
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Cain SM, Bernier LP, Zhang Y, Yung AC, Kass J, Bohnet B, Yang Y, Gopaul R, Kozlowski P, MacVicar BA, and Snutch TP
- Abstract
Cardiorespiratory arrest and death in mouse models of sudden unexpected death in epilepsy occur when spreading depolarization is triggered by cortical seizures and then propagates to the brainstem. However, the critical brain regions and the specific changes required to allow spreading depolarization to propagate to the brainstem under the relatively rare circumstances leading to a fatal seizure are unknown. We previously found that following cortical seizure-inducing electrical stimulation, spreading depolarization could occur in both the superior and inferior colliculi in Cacna1a
S218L mice, but was never observed in wild-type animals or following non-seizure-inducing stimuli in Cacna1aS218L mice. Here, we show that optogenetic stimulation of the superior/inferior colliculi in Cacna1aS218L mice induces severe seizures, and resulting spreading depolarization in the superior/inferior colliculi that propagates to the brainstem and correlates with the respiratory arrest followed by cardiac arrest. Further, we show that neurons of the superior colliculus in Cacna1aS218L mice exhibit hyperexcitable properties that we propose underlie a distinct susceptibility to spreading depolarization. Our data suggest that the susceptibility of the superior colliculus to elicit fatal spreading depolarization is a result of either genetic or seizure-related alterations within the superior colliculus that may involve changes to structure, connectivity and/or excitability., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)- Published
- 2022
- Full Text
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12. The Oral and Fecal Microbiota in a Canadian Cohort of Alzheimer's Disease.
- Author
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Cirstea MS, Kliger D, MacLellan AD, Yu AC, Langlois J, Fan M, Boroomand S, Kharazyan F, Hsiung RGY, MacVicar BA, Chertkow H, Whitehead V, Brett Finlay B, and Appel-Cresswell S
- Subjects
- Canada epidemiology, Cross-Sectional Studies, Humans, RNA, Ribosomal, 16S genetics, Alzheimer Disease microbiology, Microbiota
- Abstract
Background: Despite decades of research, our understanding of Alzheimer's disease (AD) etiology remains incomplete. In recent years, appreciation has grown for potential roles for the microbiota in shaping neurological health., Objective: This study aimed to examine associations between the microbiota and AD in a human cross-sectional cohort., Methods: Forty-five AD patients and 54 matched controls were recruited in Vancouver, Canada. Fecal and oral samples underwent 16S microbiota sequencing. A wide array of demographic and clinical data were collected. Differences between participant groups were assessed, and associations between microbes and clinical variables were examined within the AD population., Results: The gut microbiota of AD patients displayed lower diversity relative to controls, although taxonomic differences were sparse. In contrast, the AD oral microbiota displayed higher diversity, with several taxonomic differences relative to controls, including a lower abundance of the families Streptococcaceae and Actinomycetaceae, and a higher abundance of Weeksellaceae, among others. The periodontitis-associated oral microbe Porphyromonas gingivalis was 5 times more prevalent among patients. No significant associations between gut or oral microbes and cognition were detected, but several correlations existed between microbes and mood disorders and BMI among patients, including a strong positive correlation between Alphaproteobacteria and depression score., Conclusion: The gut microbiota of AD patients was not overtly different from controls, although it displayed lower diversity, an overall marker of microbiota health. The oral microbiota did display marked differences. Cognition was not associated with a microbial signature, but other relevant AD factors including mood and BMI did demonstrate an association.
- Published
- 2022
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13. Reactive astrocyte nomenclature, definitions, and future directions.
- Author
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Escartin C, Galea E, Lakatos A, O'Callaghan JP, Petzold GC, Serrano-Pozo A, Steinhäuser C, Volterra A, Carmignoto G, Agarwal A, Allen NJ, Araque A, Barbeito L, Barzilai A, Bergles DE, Bonvento G, Butt AM, Chen WT, Cohen-Salmon M, Cunningham C, Deneen B, De Strooper B, Díaz-Castro B, Farina C, Freeman M, Gallo V, Goldman JE, Goldman SA, Götz M, Gutiérrez A, Haydon PG, Heiland DH, Hol EM, Holt MG, Iino M, Kastanenka KV, Kettenmann H, Khakh BS, Koizumi S, Lee CJ, Liddelow SA, MacVicar BA, Magistretti P, Messing A, Mishra A, Molofsky AV, Murai KK, Norris CM, Okada S, Oliet SHR, Oliveira JF, Panatier A, Parpura V, Pekna M, Pekny M, Pellerin L, Perea G, Pérez-Nievas BG, Pfrieger FW, Poskanzer KE, Quintana FJ, Ransohoff RM, Riquelme-Perez M, Robel S, Rose CR, Rothstein JD, Rouach N, Rowitch DH, Semyanov A, Sirko S, Sontheimer H, Swanson RA, Vitorica J, Wanner IB, Wood LB, Wu J, Zheng B, Zimmer ER, Zorec R, Sofroniew MV, and Verkhratsky A
- Subjects
- Animals, Brain Diseases pathology, Brain Injuries pathology, Humans, Spinal Cord Injuries pathology, Aging pathology, Astrocytes pathology, Brain pathology, Spinal Cord pathology
- Abstract
Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.
- Published
- 2021
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14. Neuroinflammatory inhibition of synaptic long-term potentiation requires immunometabolic reprogramming of microglia.
- Author
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York EM, Zhang J, Choi HB, and MacVicar BA
- Subjects
- Cytokines metabolism, Hippocampus metabolism, Lipopolysaccharides pharmacology, Long-Term Potentiation, Microglia metabolism
- Abstract
Immunometabolism refers to the rearrangement of metabolic pathways in response to immune stimulation, and the ability of these metabolic pathways themselves to control immune functions. Many aspects of immunometabolism have been revealed through studies of peripheral immune cells. However, immunometabolic reprogramming of microglia, the resident immune cell of the central nervous system, and the consequential outcome on neuronal activity have remained difficult to unravel. Microglia are highly sensitive to subtle changes in their environment, limiting the techniques available to study their metabolic and inflammatory profiles. Here, using fluorescence lifetime imaging of endogenous NAD(P)H, we measure the metabolic activity of individual microglia within acute hippocampal slices. We observed an LPS-induced increase in aerobic glycolysis, which was blocked by the addition of 5 mM 2-deoxyglucose (2DG). This LPS-induced glycolysis in microglia was necessary for the stabilization of hypoxia inducible factor-1α (HIF-1α) and production of the proinflammatory cytokine, interleukin-1β (IL-1β). Upon release, IL-1β acted via the neuronal interleukin-1 receptor to inhibit the formation of synaptic long-term potentiation (LTP) following high frequency stimulation. Remarkably, the addition of 2DG to blunt the microglial glycolytic increase also inhibited HIF-1α accumulation and IL-1β production, and therefore rescued LTP in LPS-stimulated slices. Overall, these data reveal the importance of metabolic reprogramming in regulating microglial immune functions, with appreciable outcomes on cytokine release and neuronal activity., (© 2020 Wiley Periodicals LLC.)
- Published
- 2021
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15. Immunometabolism in the Brain: How Metabolism Shapes Microglial Function.
- Author
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Bernier LP, York EM, and MacVicar BA
- Subjects
- Brain, Energy Metabolism, Humans, Inflammation, Microglia
- Abstract
Immune cells react to their environment by flexibly reprogramming intracellular metabolic pathways that subsequently alter immune function, in a process called immunometabolism. However, in the CNS, the impact of metabolic reprogramming on microglia, neuroinflammation, and subsequently on brain function is poorly understood. As brain-resident macrophages, microglia are the CNS immune effectors and share similarities with peripheral immune cells. New tools for studying immunometabolism now allow the analysis of bioenergetic regulation with cellular resolution and, as a result, have uncovered previously unappreciated roles for microglial immunometabolism in shaping neuroinflammation. This review highlights evidence that microglia metabolism adapts to changes in brain energy homeostasis and that metabolic reprogramming regulates microglial polarization, thereby impacting pathological inflammatory responses in the brain., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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16. Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.
- Author
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Radhakrishnan S, Norley J, Wendt S, LeRoy N, Hall H, Norcross S, Doan S, Snaider J, MacVicar BA, Weake VM, Huang L, and Tantama M
- Subjects
- Animals, Cytosol metabolism, Fluorescence Resonance Energy Transfer, Oxidation-Reduction, Rats, Reactive Oxygen Species metabolism, Biosensing Techniques, Neurons
- Abstract
Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.
- Published
- 2020
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17. PANX1 in inflammation heats up: New mechanistic insights with implications for injury and infection.
- Author
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Sanchez Arias JC, Wicki-Stordeur LE, Candlish RC, van der Slagt E, Paci I, Rao PPN, MacVicar BA, and Swayne LA
- Subjects
- Human Umbilical Vein Endothelial Cells metabolism, Humans, Inflammasomes metabolism, Models, Biological, Signal Transduction, Tumor Necrosis Factor-alpha metabolism, Connexins metabolism, Infections metabolism, Infections pathology, Inflammation metabolism, Inflammation pathology, Nerve Tissue Proteins metabolism
- Abstract
A new study by Yang and colleagues has revealed that TNF-alpha regulates PANX1 levels through an NF-kB-dependent mechanism in human endothelial cells. PANX1 modulates Ca
2+ influx contributing to IL-1beta production independent of purinergic signaling. These novel findings expand our understanding of TNF-alpha-mediated upregulation of IL-1beta with implications for responses to tissue injury and infection., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2020
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18. Microglial metabolic flexibility supports immune surveillance of the brain parenchyma.
- Author
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Bernier LP, York EM, Kamyabi A, Choi HB, Weilinger NL, and MacVicar BA
- Subjects
- Animals, Brain pathology, CX3C Chemokine Receptor 1 genetics, Cell Movement, Fatty Acids metabolism, Glucose deficiency, Glucose metabolism, Glutamine metabolism, Immunologic Surveillance, Mice, Mice, Transgenic, Microglia cytology, Microglia immunology, NAD metabolism, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases metabolism, Brain immunology, Energy Metabolism physiology, Microglia metabolism
- Abstract
Microglia are highly motile cells that continuously monitor the brain environment and respond to damage-associated cues. While glucose is the main energy substrate used by neurons in the brain, the nutrients metabolized by microglia to support surveillance of the parenchyma remain unexplored. Here, we use fluorescence lifetime imaging of intracellular NAD(P)H and time-lapse two-photon imaging of microglial dynamics in vivo and in situ, to show unique aspects of the microglial metabolic signature in the brain. Microglia are metabolically flexible and can rapidly adapt to consume glutamine as an alternative metabolic fuel in the absence of glucose. During insulin-induced hypoglycemia in vivo or in aglycemia in acute brain slices, glutaminolysis supports the maintenance of microglial process motility and damage-sensing functions. This metabolic shift sustains mitochondrial metabolism and requires mTOR-dependent signaling. This remarkable plasticity allows microglia to maintain their critical surveillance and phagocytic roles, even after brain neuroenergetic homeostasis is compromised.
- Published
- 2020
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19. Agrin plays a major role in the coalescence of the aquaporin-4 clusters induced by gamma-1-containing laminin.
- Author
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Noël G, Tham DKL, MacVicar BA, and Moukhles H
- Subjects
- Agrin analysis, Animals, Aquaporin 4 analysis, Astrocytes chemistry, Cells, Cultured, Laminin analysis, Mice, Rats, Rats, Sprague-Dawley, Agrin metabolism, Aquaporin 4 metabolism, Astrocytes metabolism, Laminin metabolism
- Abstract
The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with β-dystroglycan (β-DG). Conversely, agrin sliencing profoundly disrupts β-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and β-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2020
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20. Green fluorescent protein emission obscures metabolic fluorescent lifetime imaging of NAD(P)H.
- Author
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York EM, Weilinger NL, LeDue JM, and MacVicar BA
- Abstract
Autofluorescence of endogenous molecules can provide valuable insights in both basic research and clinical applications. One such technique is fluorescence lifetime imaging (FLIM) of NAD(P)H, which serves as a correlate of glycolysis and electron transport chain rates in metabolically active tissue. A powerful advantage of NAD(P)H-FLIM is the ability to measure cell-specific metabolism within heterogeneous tissues. Cell-type specific identification is most commonly achieved with directed green fluorescent protein (GFP) expression. However, we demonstrate that NAD(P)H-FLIM should not be analyzed in GFP-expressing cells, as GFP molecules themselves emit photons in the blue spectrum with short fluorescence lifetimes when imaged using two-photon excitation at 750 nm. This is substantially different from the reported GFP emission wavelength and lifetime after two-photon excitation at 910 nm. These blue GFP photons are indistinguishable from free NAD(P)H by both emission spectra and fluorescence lifetime. Therefore, NAD(P)H-FLIM in GFP-expressing cells will lead to incorrect interpretations of metabolic rates, and thus, these techniques should not be combined., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)
- Published
- 2019
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21. Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia.
- Author
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Bernier LP, Bohlen CJ, York EM, Choi HB, Kamyabi A, Dissing-Olesen L, Hefendehl JK, Collins HY, Stevens B, Barres BA, and MacVicar BA
- Subjects
- Actins metabolism, Adenosine Triphosphate metabolism, Animals, Brain drug effects, Brain metabolism, Cyclic Nucleotide Phosphodiesterases, Type 3 metabolism, Female, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Microglia drug effects, Microtubules metabolism, Potassium Channels, Tandem Pore Domain genetics, Potassium Channels, Tandem Pore Domain metabolism, Pseudopodia drug effects, Rats, Rats, Sprague-Dawley, Signal Transduction, Brain diagnostic imaging, Cyclic AMP metabolism, Microglia metabolism, Pseudopodia metabolism
- Abstract
Microglia, the brain's immune cells, maintain homeostasis and sense pathological changes by continuously surveying the parenchyma with highly motile large processes. Here, we demonstrate that microglia also use thin actin-dependent filopodia that allow fast nanoscale sensing within discrete regions. Filopodia are distinct from large processes by their size, speed, and regulation mechanism. Increasing cyclic AMP (cAMP) by activating norepinephrine G
s -coupled receptors, applying nitric oxide, or inhibiting phosphodiesterases rapidly increases filopodia but collapses large processes. Alternatively, Gi -coupled P2Y12 receptor activation collapses filopodia but triggers large processes extension with bulbous tips. Similar control of cytoskeletal dynamics and microglial morphology by cAMP is observed in ramified primary microglia, suggesting that filopodia are intrinsically generated sensing structures. Therefore, nanoscale surveillance of brain parenchyma by microglia requires localized cAMP increases that drive filopodia formation. Shifting intracellular cAMP levels controls the polarity of microglial responses to changes in brain homeostasis and alters the scale of immunosurveillance., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2019
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22. 3DMorph Automatic Analysis of Microglial Morphology in Three Dimensions from Ex Vivo and In Vivo Imaging.
- Author
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York EM, LeDue JM, Bernier LP, and MacVicar BA
- Subjects
- 6-Cyano-7-nitroquinoxaline-2,3-dione pharmacology, Adenosine Triphosphate pharmacology, Animals, Brain diagnostic imaging, Brain drug effects, CX3C Chemokine Receptor 1 genetics, CX3C Chemokine Receptor 1 metabolism, Cell Size, Excitatory Amino Acid Antagonists pharmacology, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Imaging, Three-Dimensional, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microglia drug effects, Neurons cytology, Neurons drug effects, Rats, Reproducibility of Results, Sodium Channel Blockers pharmacology, Software, Tetrodotoxin pharmacology, Brain cytology, Electronic Data Processing methods, Microglia cytology
- Abstract
Microglia are dynamic immune cells of the central nervous system, and their morphology is commonly used as a readout of cellular function. However, current morphological analysis techniques rely on either tracing of cells or two-dimensional projection analysis, which are time-consuming, subject to bias, and may ignore important three-dimensional (3D) information. Therefore, we have created 3DMorph, a MATLAB-based script that analyzes microglial morphology from 3D data. The program initially requires input of threshold levels, cell size expectations, and preferred methods of skeletonization. This makes 3DMorph easily scalable and adaptable to different imaging parameters or cell types. After these settings are defined, the program is completely automatic and can batch process files without user input. Output data includes cell volume, territorial volume, branch length, number of endpoints and branch points, and average distance between cells. We show that 3DMorph is accurate compared to manual tracing, with significantly decreased user input time. Importantly, 3DMorph is capable of processing in vivo microglial morphology, as well as other 3D branching cell types, from mouse cranial windows or acute hippocampal slices. Therefore, we present a novel, user-friendly, scalable, and semiautomatic method of analyzing cell morphology in 3 dimensions. This method should improve the accuracy of cell measurements, remove user bias between conditions, increase reproducibility between experimenters and labs, and reduce user input time. We provide this open source code on GitHub so that it is free and accessible to all investigators.
- Published
- 2018
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23. Rap2 and TNIK control Plexin-dependent tiled synaptic innervation in C. elegans .
- Author
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Chen X, Shibata AC, Hendi A, Kurashina M, Fortes E, Weilinger NL, MacVicar BA, Murakoshi H, and Mizumoto K
- Subjects
- Animals, Caenorhabditis elegans chemistry, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Guanosine Diphosphate chemistry, Guanosine Triphosphate chemistry, Mutation, Nerve Tissue Proteins genetics, Neurogenesis genetics, Neurons chemistry, Protein Serine-Threonine Kinases genetics, Receptors, Cell Surface genetics, Signal Transduction genetics, Synapses chemistry, Synapses genetics, Synapses pathology, rap GTP-Binding Proteins genetics, Caenorhabditis elegans Proteins chemistry, Nerve Tissue Proteins chemistry, Protein Serine-Threonine Kinases chemistry, Receptors, Cell Surface chemistry, rap GTP-Binding Proteins chemistry
- Abstract
During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase ( rap-2 ) and its effector, TNIK ( mig-15 ), act genetically downstream of Plexin ( plx-1 ) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans . Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo., Competing Interests: XC, AS, AH, MK, EF, NW, BM, HM, KM No competing interests declared, (© 2018, Chen et al.)
- Published
- 2018
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24. Microglial modulation of neuronal activity in the healthy brain.
- Author
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York EM, Bernier LP, and MacVicar BA
- Subjects
- Animals, Cell Communication physiology, Humans, Brain physiology, Microglia physiology, Neurons physiology
- Abstract
Investigations on the role of microglia in the brain have traditionally been focused on their contributions to disease states. However, recent observations have now convincingly shown that microglia in the healthy brain are not passive bystanders, but instead play a critical role in both central nervous system development and homeostasis of synaptic circuits in the adult. Here, we review the various mechanisms by which microglia impact neuronal communication in the healthy adult brain, both by sensing nearby synaptic responses and by actively modulating neuronal function. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 593-603, 2018., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2018
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25. Ca V 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.
- Author
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Cain SM, Tyson JR, Choi HB, Ko R, Lin PJC, LeDue JM, Powell KL, Bernier LP, Rungta RL, Yang Y, Cullis PR, O'Brien TJ, MacVicar BA, and Snutch TP
- Subjects
- Animals, Electroencephalography methods, Epilepsy, Absence genetics, Female, Male, Rats, Rats, Transgenic, Seizures genetics, Action Potentials physiology, Calcium Channels, T-Type physiology, Epilepsy, Absence physiopathology, Neurons physiology, Seizures physiopathology, Thalamus physiopathology
- Abstract
Objective: Genetic alterations have been identified in the CACNA1H gene, encoding the Ca
V 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN., Methods: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings., Results: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures., Significance: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)- Published
- 2018
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26. Astrocytes Provide Metabolic Support for Neuronal Synaptic Function in Response to Extracellular K .
- Author
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MacVicar BA and Choi HB
- Subjects
- Action Potentials drug effects, Action Potentials physiology, Animals, Astrocytes drug effects, Cells, Cultured, Extracellular Fluid drug effects, Glucose pharmacology, Glycogen metabolism, Humans, Neurons drug effects, Organ Culture Techniques, Synapses drug effects, Astrocytes metabolism, Extracellular Fluid metabolism, Neurons physiology, Potassium metabolism, Synapses physiology
- Abstract
It is an honour to have this opportunity write an article in recognition of the immense contributions of Bruce Ransom to the field of glial research. For me (BAM) personally there are many highlights both as a colleague and a friend that come to mind when I reflect on the many years that I have known Bruce. My own entry into the glial field was inspired by the early work by Ransom and his lab showing the sensitivity of astrocytes to neuronal activity. During my PhD and postdoctoral research I read these early papers and was inspired to ask the question when I first set up my independent lab in 1983: what if astrocytes also express some of the multitude of ion channels or transmitter receptors that were beginning to be described in neurons? Could they modify neuronal excitability during seizures or behaviour? As it turned out this was not only true but glial-neuronal interactions continues to be a growing and exciting field that I am still working in. I first met Bruce at the 1984 Society for Neuroscience meeting in Anaheim at my poster describing voltage gated calcium channels in astrocytes in cell culture. That was the start of a great friendship and years of discussions and collaborations. This review describes recent work from my lab led by Hyun Beom Choi that followed and was inspired by the groundbreaking studies by Bruce on electrophysiological and pH recordings from astrocytes and on glycogen mobilization in astrocytes to protect white matter axons.
- Published
- 2017
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27. Pannexin1 knockout and blockade reduces ischemic stroke injury in female, but not in male mice.
- Author
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Freitas-Andrade M, Bechberger JF, MacVicar BA, Viau V, and Naus CC
- Subjects
- Adjuvants, Pharmaceutic pharmacology, Animals, Connexins antagonists & inhibitors, Connexins metabolism, Disease Models, Animal, Female, Humans, Infarction, Middle Cerebral Artery etiology, Infarction, Middle Cerebral Artery metabolism, Male, Mice, Inbred C57BL, Mice, Knockout, Nerve Tissue Proteins antagonists & inhibitors, Nerve Tissue Proteins metabolism, Probenecid pharmacology, Sex Factors, Connexins genetics, Infarction, Middle Cerebral Artery genetics, Ischemia complications, Nerve Tissue Proteins genetics, Stroke complications
- Abstract
The membrane channel Pannexin 1 (Panx1) mediates apoptotic and inflammatory signaling cascades in injured neurons, responses previously shown to be sexually dimorphic under ischemic conditions. We tested the hypothesis that Panx1 plays an underlying role in mediating sex differences in stroke outcome responses. Middle-aged, 8-9 month old male and female wild type and Panx1 KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocyte and microglia activation were assessed 4 days later. The sexually dimorphic nature of Panx1 deletion was also explored by testing the effect of probenecid a known Panx1 blocker to alter stroke volume. Panx1 KO females displayed significantly smaller infarct volumes (~ 50 % reduction) compared to their wild-type counterparts, whereas no such KO effect occurred in males. This sex-specific effect of Panx1 KO was recapitulated by significant reductions in peri-infarct inflammation and astrocyte reactivity, as well as smaller infarct volumes in probenecid treated females, but not males. Finally, females showed overall, higher Panx1 protein levels than males under ischemic conditions. These findings unmask a deleterious role for Panx1 in response to permanent MCA occlusion, that is unique to females, and provide several new frameworks for understanding sex differences in stroke outcome.
- Published
- 2017
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28. The cost of communication in the brain.
- Author
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MacVicar BA, Wicki-Stordeur L, and Bernier LP
- Subjects
- Adenosine Triphosphate, Axons, Brain, Neuroglia, White Matter
- Abstract
Imaging ATP in axons reveals that they rely on glucose from the blood and lactate produced by glial cells as sources of energy.
- Published
- 2017
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29. A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain.
- Author
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Howarth C, Sutherland B, Choi HB, Martin C, Lind BL, Khennouf L, LeDue JM, Pakan JM, Ko RW, Ellis-Davies G, Lauritzen M, Sibson NR, Buchan AM, and MacVicar BA
- Subjects
- Adrenergic alpha-2 Receptor Agonists pharmacology, Adrenergic alpha-Agonists pharmacology, Animals, Animals, Newborn, Carbon Dioxide metabolism, Carbon Dioxide pharmacology, Cerebrovascular Circulation drug effects, Clonidine pharmacology, Cycloleucine analogs & derivatives, Cycloleucine pharmacology, Cyclooxygenase 1 metabolism, Dinoprostone metabolism, Female, Glial Fibrillary Acidic Protein metabolism, Glutathione metabolism, In Vitro Techniques, Male, Membrane Proteins metabolism, Mice, Mice, Inbred C57BL, Neuroprotective Agents pharmacology, Norepinephrine pharmacology, Rats, Rats, Wistar, Vibrissae innervation, Astrocytes metabolism, Hippocampus pathology, Hypercapnia pathology
- Abstract
Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO
2 , arterial O2 , and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E2 (PgE2 ) plays a key role for cerebrovascular CO2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2 ) evokes an increase in astrocyte [Ca2+ ]i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2 is decreased and vasodilation triggered by increased astrocyte [Ca2+ ]i in vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO2 Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage. SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO2 , which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE2 We demonstrate that hypercapnia (increased CO2 ) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO2 -evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage., (Copyright © 2017 Howarth, Sutherland et al.)- Published
- 2017
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30. In vivo imaging reveals that pregabalin inhibits cortical spreading depression and propagation to subcortical brain structures.
- Author
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Cain SM, Bohnet B, LeDue J, Yung AC, Garcia E, Tyson JR, Alles SR, Han H, van den Maagdenberg AM, Kozlowski P, MacVicar BA, and Snutch TP
- Subjects
- Animals, Brain diagnostic imaging, Brain drug effects, Brain metabolism, Brain pathology, Calcium Channels, N-Type metabolism, Cerebellar Ataxia diagnostic imaging, Cerebellar Ataxia metabolism, Cerebellar Ataxia pathology, Diffusion Magnetic Resonance Imaging, Disease Models, Animal, Gene Expression, Humans, Mice, Mice, Transgenic, Migraine Disorders diagnostic imaging, Migraine Disorders metabolism, Migraine Disorders pathology, Migraine with Aura diagnostic imaging, Migraine with Aura metabolism, Migraine with Aura pathology, Mutation, Neurons drug effects, Neurons metabolism, Neurons pathology, Synaptic Transmission, Analgesics pharmacology, Calcium Channel Blockers pharmacology, Calcium Channels, N-Type genetics, Cerebellar Ataxia drug therapy, Cortical Spreading Depression drug effects, Migraine Disorders drug therapy, Migraine with Aura drug therapy, Pregabalin pharmacology
- Abstract
Migraine is characterized by severe headaches that can be preceded by an aura likely caused by cortical spreading depression (SD). The antiepileptic pregabalin (Lyrica) shows clinical promise for migraine therapy, although its efficacy and mechanism of action are unclear. As detected by diffusion-weighted MRI (DW-MRI) in wild-type (WT) mice, the acute systemic administration of pregabalin increased the threshold for SD initiation in vivo. In familial hemiplegic migraine type 1 mutant mice expressing human mutations (R192Q and S218L) in the Ca
V 2.1 (P/Q-type) calcium channel subunit, pregabalin slowed the speed of SD propagation in vivo. Acute systemic administration of pregabalin in vivo also selectively prevented the migration of SD into subcortical striatal and hippocampal regions in the R192Q strain that exhibits a milder phenotype and gain of CaV 2.1 channel function. At the cellular level, pregabalin inhibited glutamatergic synaptic transmission differentially in WT, R192Q, and S218L mice. The study describes a DW-MRI analysis method for tracking the progression of SD and provides support and a mechanism of action for pregabalin as a possible effective therapy in the treatment of migraine.- Published
- 2017
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31. Mitochondrial Calcium Sparkles Light Up Astrocytes.
- Author
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MacVicar BA and Ko RW
- Subjects
- Mitochondria, Mitochondrial Membrane Transport Proteins, Astrocytes, Calcium
- Abstract
Discrete calcium signals in the fine processes of astrocytes are a recent discovery and a new mystery. In a recent issue of Neuron, Agarwal et al. (2017) report that calcium efflux from mitochondria during brief openings of the mitochondrial permeability transition pore (mPTP) contribute to calcium microdomains., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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32. Ca 2+ transients in astrocyte fine processes occur via Ca 2+ influx in the adult mouse hippocampus.
- Author
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Rungta RL, Bernier LP, Dissing-Olesen L, Groten CJ, LeDue JM, Ko R, Drissler S, and MacVicar BA
- Subjects
- Action Potentials drug effects, Animals, Calcium Channels, L-Type genetics, Calcium Channels, L-Type metabolism, Calcium Signaling drug effects, Carbenoxolone pharmacology, Chromones pharmacology, Excitatory Amino Acid Antagonists pharmacology, Excitatory Amino Acid Transporter 1 genetics, Excitatory Amino Acid Transporter 1 metabolism, Female, In Vitro Techniques, Inositol 1,4,5-Trisphosphate Receptors genetics, Inositol 1,4,5-Trisphosphate Receptors metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Male, Membrane Microdomains drug effects, Membrane Microdomains metabolism, Mice, Mice, Inbred C57BL, Mice, Transgenic, Pyridines pharmacology, TRPA1 Cation Channel genetics, TRPA1 Cation Channel metabolism, Astrocytes metabolism, Calcium metabolism, Hippocampus cytology
- Abstract
Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca
2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103., (© 2016 Wiley Periodicals, Inc.)- Published
- 2016
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33. Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aβ plaques by iGluSnFR two-photon imaging.
- Author
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Hefendehl JK, LeDue J, Ko RW, Mahler J, Murphy TH, and MacVicar BA
- Subjects
- Animals, Ceftriaxone, Glutamic Acid, Mice, Mice, Transgenic, Amino Acid Transport System X-AG metabolism, Amyloid beta-Peptides metabolism, Amyloid beta-Protein Precursor metabolism, Diagnostic Imaging methods, Plaque, Amyloid metabolism
- Abstract
Amyloid-β (Aβ) plaques, a hallmark of Alzheimer's disease (AD), are surrounded by regions of neuronal and glial hyperactivity. We use in vivo two-photon and wide-field imaging of the glutamate sensor iGluSnFR to determine whether pathological changes in glutamate dynamics in the immediate vicinity of Aβ deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment. In regions close to Aβ plaques chronic states of high spontaneous glutamate fluctuations are observed and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas. GLT-1 expression is reduced around Aβ plaques and upregulation of GLT-1 expression and activity by ceftriaxone partially restores glutamate dynamics to values in control regions. We conclude that the toxic microenvironment surrounding Aβ plaques results, at least partially, from enhanced glutamate levels and that pharmacologically increasing GLT-1 expression and activity may be a new target for early therapeutic intervention., Competing Interests: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Published
- 2016
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34. Bidirectional Control of Blood Flow by Astrocytes: A Role for Tissue Oxygen and Other Metabolic Factors.
- Author
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Gordon GR, Howarth C, and MacVicar BA
- Subjects
- Animals, Energy Metabolism, Humans, Vasoconstriction, Astrocytes metabolism, Brain metabolism, Cerebrovascular Circulation physiology, Oxygen metabolism
- Abstract
Altering cerebral blood flow through the control of cerebral vessel diameter is critical so that the delivery of molecules important for proper brain functioning is matched to the activity level of neurons. Although the close relationship of brain glia known as astrocytes with cerebral blood vessels has long been recognized, it is only recently that these cells have been demonstrated to translate information on the activity level and energy demands of neurons to the vasculature. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq-protein coupled receptors elevate intracellular calcium via IP3 signaling. A close examination of astrocyte endfeet calcium signals has been shown to cause either vasoconstriction or vasodilation. Common to both vasomotor responses is the generation of arachidonic acid in astrocytes by calcium sensitive phospholipase A2. Vasoconstriction ensues from the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid, while vasodilation ensues from the production of epoxyeicosatrienoic acids or prostaglandins. Factors that determine whether constrictor or dilatory pathways predominate include brain oxygen, lactate, adenosine as well as nitric oxide. Changing the oxygen level itself leads to many downstream changes that facilitate the switch from vasoconstriction at high oxygen to vasodilation at low oxygen. These findings highlight the importance of astrocytes as sensors of neural activity and metabolism to coordinate the delivery of essential nutrients via the blood to the working cells.
- Published
- 2016
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35. Driving the Early Auditory Network the Old-Fashioned Way.
- Author
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MacVicar BA
- Subjects
- Animals, Ear, Inner growth & development, Hair Cells, Auditory cytology
- Abstract
Spontaneous neuronal activity during the development of the auditory sensory system is important in establishing mature connectivity. Wang et al. show that glia-like cells drive spontaneous spiking in neighboring cochlear inner hair cells via a process that involves osmotic cell shrinkage and the secretion of potassium ions., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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36. Microglia: Dynamic Mediators of Synapse Development and Plasticity.
- Author
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Wu Y, Dissing-Olesen L, MacVicar BA, and Stevens B
- Subjects
- Aging genetics, Aging immunology, Aging metabolism, Animals, Brain physiology, Central Nervous System immunology, Central Nervous System metabolism, Humans, Inflammation genetics, Inflammation immunology, Inflammation metabolism, Neuroimmunomodulation, Neuronal Plasticity, Signal Transduction, Synapses physiology, Microglia physiology
- Abstract
Neuronal communication underlies all brain activity and the genesis of complex behavior. Emerging research has revealed an unexpected role for immune molecules in the development and plasticity of neuronal synapses. Moreover microglia, the resident immune cells of the brain, express and secrete immune-related signaling molecules that alter synaptic transmission and plasticity in the absence of inflammation. When inflammation does occur, microglia modify synaptic connections and synaptic plasticity required for learning and memory. Here we review recent findings demonstrating how the dynamic interactions between neurons and microglia shape the circuitry of the nervous system in the healthy brain and how altered neuron-microglia signaling could contribute to disease., (Copyright © 2015. Published by Elsevier Ltd.)
- Published
- 2015
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37. The cellular mechanisms of neuronal swelling underlying cytotoxic edema.
- Author
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Rungta RL, Choi HB, Tyson JR, Malik A, Dissing-Olesen L, Lin PJC, Cain SM, Cullis PR, Snutch TP, and MacVicar BA
- Subjects
- Animals, Brain Edema metabolism, Cell Death, Cells, Cultured, Chloride-Bicarbonate Antiporters chemistry, Humans, In Vitro Techniques, Membrane Transport Proteins chemistry, Membrane Transport Proteins metabolism, Mice, Neurons pathology, Rats, Sodium metabolism, Sulfate Transporters, Brain Edema pathology, Chloride-Bicarbonate Antiporters metabolism, Neurons metabolism
- Abstract
Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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38. Fixation and Immunolabeling of Brain Slices: SNAPSHOT Method.
- Author
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Dissing-Olesen L and MacVicar BA
- Subjects
- Animals, Brain, Clinical Protocols, Immunohistochemistry methods, Microscopy, Fluorescence, Multiphoton methods, Tissue Fixation methods
- Abstract
Acute brain slices are widely used in neuroscience because this preparation enables pharmacological interventions in a timely manner, similar to what is currently done in cultured cell studies, while preserving the natural cytoarchitecture. However, compared with cells in culture and thin cryostat sections, acute brain slices are not commonly used for immunolabeling because of poor fixation and antibody penetration. Thus, we have established a novel protocol to overcome these issues. We named this protocol SNAPSHOT (StaiNing of dynAmic ProcesseS in HOt-fixed Tissue) because it describes a simple approach for preserving the morphology of fine dynamic cellular processes at the exact time of fixation and for improving the penetration of antibodies. We have previously shown that SNAPSHOT preserves the ultrastructure of the tissue and allows for a uniform immunolabeling throughout a 300 μm thick slice. SNAPSHOT has recently proven to be beneficial in addressing several unique biological questions., (Copyright © 2015 John Wiley & Sons, Inc.)
- Published
- 2015
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39. Astrocyte regulation of blood flow in the brain.
- Author
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MacVicar BA and Newman EA
- Subjects
- Brain blood supply, Brain cytology, Brain physiology, Capillaries physiology, Humans, Astrocytes physiology, Cerebrovascular Circulation physiology
- Abstract
Neuronal activity results in increased blood flow in the brain, a response named functional hyperemia. Astrocytes play an important role in mediating this response. Neurotransmitters released from active neurons evoke Ca(2+) increases in astrocytes, leading to the release of vasoactive metabolites of arachidonic acid from astrocyte endfeet onto blood vessels. Synthesis of prostaglandin E2 (PGE2) and epoxyeicosatrienoic acids (EETs) dilate blood vessels, whereas 20-hydroxyeicosatetraenoic acid (20-HETE) constricts vessels. The release of K(+) from astrocyte endfeet may also contribute to vasodilation. Oxygen modulates astrocyte regulation of blood flow. Under normoxic conditions, astrocytic Ca(2+) signaling results in vasodilation, whereas under hyperoxic conditions, vasoconstriction is favored. Astrocytes also contribute to the generation of vascular tone. Tonic release of both 20-HETE and ATP from astrocytes constricts vascular smooth muscle cells, generating vessel tone. Under pathological conditions, including Alzheimer's disease and diabetic retinopathy, disruption of normal astrocyte physiology can compromise the regulation of blood flow., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2015
- Full Text
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40. Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth.
- Author
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Dissing-Olesen L, LeDue JM, Rungta RL, Hefendehl JK, Choi HB, and MacVicar BA
- Subjects
- Animals, Brain drug effects, Calcium-Binding Proteins metabolism, Cell Growth Processes drug effects, Cell Growth Processes genetics, Excitatory Amino Acid Agents pharmacology, Female, In Vitro Techniques, Magnesium pharmacology, Male, Membrane Potentials drug effects, Membrane Potentials genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microfilament Proteins metabolism, Microglia cytology, N-Methylaspartate pharmacology, Neurons drug effects, Patch-Clamp Techniques, Receptors, Purinergic P2Y12 metabolism, Time Factors, Adenosine Triphosphate metabolism, Brain cytology, Microglia metabolism, Neurons metabolism, Receptors, N-Methyl-D-Aspartate metabolism
- Abstract
Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP., (Copyright © 2014 the authors 0270-6474/14/3410511-17$15.00/0.)
- Published
- 2014
- Full Text
- View/download PDF
41. Cognitive flexibility and long-term depression (LTD) are impaired following β-catenin stabilization in vivo.
- Author
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Mills F, Bartlett TE, Dissing-Olesen L, Wisniewska MB, Kuznicki J, Macvicar BA, Wang YT, and Bamji SX
- Subjects
- Animals, Cadherins metabolism, Endocytosis genetics, Endocytosis physiology, Female, Hippocampus metabolism, Hippocampus ultrastructure, Immunoblotting, Long-Term Synaptic Depression drug effects, Long-Term Synaptic Depression genetics, Male, Maze Learning physiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Microscopy, Immunoelectron, N-Methylaspartate pharmacology, Neurons metabolism, Neurons physiology, Protein Binding, Receptors, AMPA metabolism, Synapses metabolism, Synapses physiology, Synaptosomes metabolism, beta Catenin genetics, Cognition physiology, Hippocampus physiopathology, Long-Term Synaptic Depression physiology, beta Catenin metabolism
- Abstract
The cadherin/β-catenin adhesion complex is a key mediator of the bidirectional changes in synapse strength which are believed to underlie complex learning and memory. In the present study, we demonstrate that stabilization of β-catenin in the hippocampus of adult mice results in significant impairments in cognitive flexibility and spatial reversal learning, including impaired extinction during the reversal phase of the Morris water maze and deficits in a delayed nonmatch to place T-maze task. In accordance with these deficits, β-catenin stabilization was found to abolish long-term depression by stabilizing cadherin at the synaptic membrane and impairing AMPA receptor endocytosis, while leaving basal synaptic transmission and long-term potentiation unaffected. These results demonstrate that the β-catenin/cadherin adhesion complex plays an important role in learning and memory and that aberrant increases in synaptic adhesion can have deleterious effects on cognitive function.
- Published
- 2014
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- View/download PDF
42. Microglial CR3 activation triggers long-term synaptic depression in the hippocampus via NADPH oxidase.
- Author
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Zhang J, Malik A, Choi HB, Ko RW, Dissing-Olesen L, and MacVicar BA
- Subjects
- Acetophenones pharmacology, Animals, Animals, Newborn, CD11b Antigen metabolism, Enzyme Inhibitors pharmacology, Excitatory Amino Acid Antagonists pharmacology, Hypoxia physiopathology, In Vitro Techniques, L-Lactate Dehydrogenase metabolism, Lipid Peroxidation genetics, Long-Term Synaptic Depression drug effects, Macrophage-1 Antigen genetics, Mice, Mice, Knockout, Oxygen pharmacology, Rats, Rats, Sprague-Dawley, Hippocampus physiology, Long-Term Synaptic Depression physiology, Macrophage-1 Antigen metabolism, Microglia metabolism, NADPH Oxidases metabolism
- Abstract
Complement receptor 3 (CR3) activation in microglia is involved in neuroinflammation-related brain disorders and pruning of neuronal synapses. Hypoxia, often observed together with neuroinflammation in brain trauma, stroke, and neurodegenerative diseases, is thought to exacerbate inflammatory responses and synergistically enhance brain damage. Here we show that when hypoxia and an inflammatory stimulus (lipopolysaccharide [LPS]) are combined, they act synergistically to trigger long-term synaptic depression (LTD) that requires microglial CR3, activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), and GluA2-mediated A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Microglial CR3-triggered LTD is independent of N-methyl-D-aspartate receptors (NMDARs), metabotropic glutamate receptors (mGluRs), or patterned synaptic activity. This type of LTD may contribute to memory impairments and synaptic disruptions in neuroinflammation-related brain disorders., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
43. Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain.
- Author
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Rungta RL, Choi HB, Lin PJ, Ko RW, Ashby D, Nair J, Manoharan M, Cullis PR, and Macvicar BA
- Abstract
Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.Molecular Therapy-Nucleic Acids (2013) 2, e136; doi:10.1038/mtna.2013.65; published online 3 December 2013.
- Published
- 2013
- Full Text
- View/download PDF
44. Regenerative glutamate release by presynaptic NMDA receptors contributes to spreading depression.
- Author
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Zhou N, Rungta RL, Malik A, Han H, Wu DC, and MacVicar BA
- Subjects
- Animals, Biosensing Techniques, Brain physiopathology, Calcium metabolism, Electrodes, Electrophysiology, Female, In Vitro Techniques, Male, Potassium administration & dosage, Potassium metabolism, Rats, Rats, Sprague-Dawley, Sodium-Calcium Exchanger metabolism, Synaptic Transmission physiology, Brain metabolism, Cortical Spreading Depression physiology, Glutamic Acid metabolism, Receptors, N-Methyl-D-Aspartate metabolism, Receptors, Presynaptic metabolism
- Abstract
Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K(+) concentrations ([K(+)]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K(+)]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na(+)/Ca(2+) exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na(+) influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na(+)/Ca(2+) exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K(+)]o-triggered SD.
- Published
- 2013
- Full Text
- View/download PDF
45. Progranulin promotes activation of microglia/macrophage after pilocarpine-induced status epilepticus.
- Author
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Zhu S, Tai C, Petkau TL, Zhang S, Liao C, Dong Z, Wen W, Chang Q, Tian Wang Y, MacVicar BA, Leavitt BR, Jia W, and Cynader MS
- Subjects
- Animals, Cell Death physiology, Dentate Gyrus metabolism, Dentate Gyrus pathology, Disease Models, Animal, Granulins, Male, Mice, Mice, Knockout, Nerve Degeneration metabolism, Nerve Degeneration pathology, Pilocarpine pharmacology, Progranulins, Rats, Rats, Sprague-Dawley, Status Epilepticus chemically induced, Hippocampus metabolism, Intercellular Signaling Peptides and Proteins metabolism, Macrophages metabolism, Microglia metabolism, Status Epilepticus metabolism
- Abstract
Progranulin (PGRN) haploinsufficiency accounts for up to 10% of frontotemporal lobe dementia. PGRN has also been implicated in neuroinflammation in acute and chronic neurological disorders. Here we report that both protein and mRNA levels of cortical and hippocampal PGRN are significantly enhanced following pilocarpine-induced status epilepticus. We also identify intense PGRN immunoreactivity that colocalizes with CD11b in seizure-induced animals, suggesting that PGRN elevation occurs primarily in activated microglia and macrophages. To test the role of PGRN in activation of microglia/macrophages, we apply recombinant PGRN protein directly into the hippocampal formation, and observe no change in the number of CD11b(+) microglia/macrophages in the dentate gyrus. However, with pilocarpine-induced status epilepticus, PGRN application significantly increases the number of CD11b(+) microglia/macrophages in the dentate gyrus, without affecting the extent of hilar cell death. In addition, the number of CD11b(+) microglia/macrophages induced by status epilepticus is not significantly different between PGRN knockout mice and wildtype. Our findings suggest that status epilepticus induces PGRN expression, and that PGRN potentiates but is not required for seizure-induced microglia/macrophage activation., (Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
46. Increased 20-HETE synthesis explains reduced cerebral blood flow but not impaired neurovascular coupling after cortical spreading depression in rat cerebral cortex.
- Author
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Fordsmann JC, Ko RW, Choi HB, Thomsen K, Witgen BM, Mathiesen C, Lønstrup M, Piilgaard H, MacVicar BA, and Lauritzen M
- Subjects
- Animals, Male, Organ Culture Techniques, Rats, Rats, Sprague-Dawley, Rats, Wistar, Blood Flow Velocity physiology, Cerebral Cortex blood supply, Cerebral Cortex metabolism, Cerebrovascular Circulation physiology, Cortical Spreading Depression physiology, Hydroxyeicosatetraenoic Acids biosynthesis
- Abstract
Cortical spreading depression (CSD) is associated with release of arachidonic acid, impaired neurovascular coupling, and reduced cerebral blood flow (CBF), caused by cortical vasoconstriction. We tested the hypothesis that the released arachidonic acid is metabolized by the cytochrome P450 enzyme to produce the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and that this mechanism explains cortical vasoconstriction and vascular dysfunction after CSD. CSD was induced in the frontal cortex of rats and the cortical electrical activity and local field potentials recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension (tpO(2)) using polarographic microelectrodes. 20-HETE synthesis was measured in parallel experiments in cortical brain slices exposed to CSD. We used the specific inhibitor HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) to block 20-HETE synthesis. CSD increased 20-HETE synthesis in brain slices for 120 min, and the time course of the increase in 20-HETE paralleled the reduction in CBF after CSD in vivo. HET0016 blocked the CSD-induced increase in 20-HETE synthesis and ameliorated the persistent reduction in CBF, but not the impaired neurovascular coupling after CSD. These findings suggest that CSD-induced increments in 20-HETE cause the reduction in CBF after CSD and that the attenuation of stimulation-induced CBF responses after CSD has a different mechanism. We suggest that blockade of 20-HETE synthesis may be clinically relevant to ameliorate reduced CBF in patients with migraine and acute brain cortex injuries.
- Published
- 2013
- Full Text
- View/download PDF
47. Microglia in neuronal circuits.
- Author
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Wu LJ, Stevens B, Duan S, and MacVicar BA
- Subjects
- Animals, Brain physiology, Brain Diseases pathology, Health, Humans, Microglia pathology, Nerve Net cytology, Nerve Net pathology, Microglia physiology, Nerve Net physiology
- Published
- 2013
- Full Text
- View/download PDF
48. Prevention of LPS-induced microglia activation, cytokine production and sickness behavior with TLR4 receptor interfering peptides.
- Author
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Hines DJ, Choi HB, Hines RM, Phillips AG, and MacVicar BA
- Subjects
- Amino Acid Sequence, Animals, Brain drug effects, Brain immunology, Brain pathology, Humans, Immunity, Innate drug effects, Mice, Microglia immunology, Microglia pathology, Molecular Sequence Data, Peptides chemistry, Signal Transduction drug effects, Cytokines immunology, Illness Behavior drug effects, Lipopolysaccharides immunology, Microglia drug effects, Peptides pharmacology, Toll-Like Receptor 4 antagonists & inhibitors, Toll-Like Receptor 4 immunology
- Abstract
The innate immune receptor Toll-like 4 (TLR4) is the receptor activated by lipopolysaccharide (LPS), and TLR4-LPS interaction is well known to induce an innate immune response, triggering sickness behavior. Within the brain, TLR4 is highly expressed in brain microglia, and excessive inflammation resulting from activation of this pathway in the brain has been implicated in depressive disorders and neurodegenerative pathologies. We hypothesized that blocking LPS-induced activation of TLR4 would prevent downstream immune signaling in the brain and suppress the induction of sickness behavior. We used interfering peptides to block TLR4 activation and confirmed their efficacy in preventing second messenger activation and cytokine production normally induced by LPS treatment. Further, these peptides blocked morphological changes in microglia that are typically induced by LPS. We also demonstrated that intraperitoneal (i.p.) injection of Tat-TLR4 interfering peptides prevented LPS-induced sickness behavior, as assessed in home cage behavior and with the intracranial self-stimulation paradigm. These newly synthesised peptides inhibit TLR4 signaling thereby preventing changes in behavior and motivation caused by inflammatory stimuli. These peptides highlight the roll of TLR4 and microglia morphology changes in sickness behavior, and thus may be of therapeutic value in limiting the deleterious impact of excessive inflammation in specific CNS pathologies.
- Published
- 2013
- Full Text
- View/download PDF
49. Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.
- Author
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Choi HB, Gordon GR, Zhou N, Tai C, Rungta RL, Martinez J, Milner TA, Ryu JK, McLarnon JG, Tresguerres M, Levin LR, Buck J, and MacVicar BA
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid pharmacology, Animals, Animals, Newborn, Astrocytes ultrastructure, Coumaric Acids pharmacology, Cyclic AMP metabolism, Dose-Response Relationship, Drug, Excitatory Postsynaptic Potentials drug effects, Glial Fibrillary Acidic Protein metabolism, Glucose deficiency, Glycogen metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, In Vitro Techniques, Lactic Acid metabolism, Microscopy, Immunoelectron, Microtubule-Associated Proteins metabolism, Models, Biological, Monocarboxylic Acid Transporters antagonists & inhibitors, Monocarboxylic Acid Transporters metabolism, Neurons ultrastructure, Patch-Clamp Techniques, Phosphodiesterase Inhibitors pharmacology, Potassium metabolism, Rats, Rats, Sprague-Dawley, Sodium Channel Blockers pharmacology, Tetrodotoxin pharmacology, Adenylyl Cyclases metabolism, Astrocytes drug effects, Astrocytes enzymology, Bicarbonates pharmacology, Hippocampus cytology, Neurons drug effects, Neurons enzymology
- Abstract
Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
50. Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons.
- Author
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Tai C, Hines DJ, Choi HB, and MacVicar BA
- Subjects
- Androstadienes pharmacology, Animals, Boron Compounds pharmacology, Calcium Signaling, Calmodulin antagonists & inhibitors, Dendrites metabolism, Epilepsy physiopathology, Epilepsy therapy, Humans, Membrane Potentials drug effects, Microscopy, Fluorescence, Patch-Clamp Techniques, Phosphoinositide-3 Kinase Inhibitors, Photons, Rats, Rats, Sprague-Dawley, Sulfonamides pharmacology, TRPC Cation Channels antagonists & inhibitors, Wortmannin, CA1 Region, Hippocampal metabolism, Cell Membrane metabolism, Membrane Potentials physiology, Pyramidal Cells metabolism, Receptors, Muscarinic metabolism, TRPC Cation Channels metabolism
- Abstract
In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2-APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I(tail)) and the associated dendritic Ca(2+) influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I(tail). In addition, pretreatment with the calmodulin (CaM) inhibitor W-7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I(tail)) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3-kinase (PI(3)K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I(tail) and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy., (Copyright © 2010 Wiley-Liss, Inc.)
- Published
- 2011
- Full Text
- View/download PDF
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