Department of Cellular Neurophysiology
Department of Cellular Neurophysiology
Vedoucí: Ing. Miroslava Anděrová, CSc.
Tel.: +420 241 062 050
The Department of Cellular Neurophysiology is focused on membrane and morphological characteristics of glial cells after ischemic brain injury and in the progression of neurodegenerative diseases, especially of Alzheimer’s disease. Research is oriented towards astrocytes, both at the level of gene and protein expression, as well as at the level of astrocytic functional properties of ion channels and receptors, which are necessary for maintaining the homeostasis of ions and neurotransmitters in the extracellular environment. Another cell type which is at the centre of interest are NG2 glial cells, also called polydendrocytes, that during development and in the adult nervous tissue function primarily as precursors of oligodendrocytes, but following injury of the central nervous system they proliferate and differentiate into other cell types. The research aims to characterize their membrane properties in post-ischemic tissue and in the progression of Alzheimer’s disease and to clarify the role of Wnt- and Shh-signaling pathways in proliferation/differentiation of NG2 glial cells.
Miroslava Anděrová, PhD.
Petra Suchá, MSc
Markéta Hemerová, MSc
Important results in 2015
1. Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors
Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP+) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP+ glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP+ glial cells and that its expression is increased after ischemia. In situ and in vitro Ca2+ imaging revealed that Ca2+ elevations evoked by the application of NMDA were diminished in GFAP/EGFP+ glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP+ glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.
Focal ischemia increases expression of most of the glutamate receptors (GluRs) in GFAP/EGFP glia. As for the NMDA receptor subunits, immunohistochemical analysis confirmed their presence in GFAP/EGFP glia, and their detection was even increased after ischemic insult. The Ca2+ imaging results indicate diminished NMDA receptor Ca2+ permeability after focal ischemia, which is probably due to the involvement of GluN3A subunit.
Immunohistochemical analysis of the GluN1, GluN2A-D and GluN3A-B subunits of the NMDA receptors in the cortex of adult GFAP/EGFP mice under control conditions (CTRL) and 14 days after MCAo (D14). Coronal brain sections from CTRL (A) and D14 (B) animals stained with triphenyltetrazolium chloride. The white color in B indicates the volume of ischemic tissue at D14. The boxed areas indicate the regions in which the immunohistochemical analysis was performed. The arrowheads in C - P indicate the overlay of GFAP/EGFP+ cells and NMDA subunit staining – see figure insets for detailed images of cells in white rectangles. Note the overlap of the EGFP signal with GluN3A staining in CTRL tissue and GluN1, GluN2B-D and GluN3A staining at D14. The same scale bar applies to all non-inset images.
Important results in 2014
1. Increased expression of hyperpolarization-activated cationic channels in reactive astrocytes following ischemia
Following cerebral ischemia we have identified hyperpolarization-activated (HCN) cationic channels in astrocytes. Until now, these channels were described only in neurons. Since HCN channels are mainly permeable for sodium and potassium ions, their increased expression in reactive astrocytes indicates that they may markedly influence the basic astrocytic functions in central nervous system, and consequently, an extent of nervous tissue damage following ischemia. Astrocytic HCN channels could therefore be an important therapeutic target in post-stroke therapy.
The expression of Hcn genes is strikingly increased in cortical astrocytes from GFAP/EGFP mice following focal cerebral ischemia – single-cell RT-qPCR profiling. (A) Scheme depicting the brain regions, which were used for EGFP+ cells isolations. These brain slices were stained with tetrazolium chloride to visualize the ischemic regions. (B) Percentage of EGFP+ cells in the postischemic mouse cortex (7 and 14 days after focal cerebral ischemia; D7, 2W) expressing Hcn1, 2, 3 and 4. (C)The relative expression of Hcn1–4 genes in EGFP+ cells in the control mouse cortex and in the post-ischemic cortex revealed the strong upregulation of Hcn1–4 expression 2W after FCI. HCN1 staining in the CA1 region of the rat hippocampus in controls and five weeks after global cerebral ischemia. Arrowheads indicate the HCN-positive astrocytes after ischemia (s.p., stratum pyramidale; s.r., stratum radiatum). Scale bars, 50 μm.
HCN1 staining in the CA1 region of the rat hippocampus in controls and five weeks after global cerebral ischemia. Brain slices were stained with anti-HCN1 antibodies and an antibody directed against glial fibrillary acidic protein (GFAP) in controls and fi ve weeks (5W) after global cerebral ischemia (GCI). Arrowheads indicate the HCN-positive astrocytes after ischemia (s.p., stratum pyramidale; s.r., stratum radiatum). Scale bars, 50 μm.
Collaboration: Institute of Biotechnology AS CR, v.v.i.
Honsa P, Pivoňková H, Harantová L, Butenko O, Kriška J, Džamba D, Rusnaková V, Valihrach L, Kubista M. and Anděrová M, (2014): Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia. Glia 62 (12), 2004–2021. IF 5,466
2. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice
We have showed that knockout of α-syntrophin, which is a protein responsible for aquaporin-4 anchoring on the astrocytic membrane, aff ects cell swelling/volume regulation in individual astrocytes in situ when exposed to pathological stimuli. Astrocyte volume quantifi cation revealed that α-syntrophin deletion results in signifi cantly smaller/slower astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K+.
Volume changes in the astrocytic soma and processes during hypotonic stress, increased extracellular K+ concentration and oxygen-glucose deprivation. (A–C) Time-dependent changes in the volume of the astrocytic soma (top) and processes (bottom) in GFAP/EGFP (red) and GFAP/EGFP/α-Syn-/- mice (green) during a 30-minute application of aCSFH-100 (A), a 20-minute application of aCSFK+50 (B) or 20-minute OGD (C), followed by a 60- or 40-minute washout. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn-/- mice.
The contribution of processes and cell soma to total astrocyte swelling is altered in astrocytes lacking α-syntrophin. A scheme depicting differences in swelling of astrocytic processes and cell soma in response to severe hypoosmotic stress, 50 mM K+ and OGD (oxygen-glucose deprivation), highlighting smaller swelling of astrocytic processes in GFAP/EGFP/α-Syn-/- mice compared to those in GFAP/EGFP mice due to altered distribution of aquaporin4 and Kir4.1 channels.
3. Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes
Using patch clamp technique we have demonstrated that in primary cultured rat cortical astrocytes, elevations of [Na+]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity-evoked current mediated by volume-regulated anion channel (VRAC). These results provide the first evidence that intracellular Na+ dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na+ accumulation might be a favorable strategy to counteract the development of brain edema.
High elevation of intracellular sodium (Na+) concentration in cultured rat astrocytes decreases the activity of volume-regulated anion channels (VRAC) measured by patch clamp technique. We speculate that in ischemic conditions intracellular Na+ elevation either through the augmented activity of the Na+-dependent glutamate transporter (GLT-1) or by Na+-permeable channels could modulate the astrocytic volume and glutamate release (Glu-) regulated by VRAC.
Important results in 2013
1. Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles
The mouse Dach1 gene is expressed by neural stem cells during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles of the adult brain. In this study we showed that Dach1-expressing cells participate predominantly in the gliogenesis under physiological conditions, while after ischemia these cells express also neurogenic potential; nevetherless, we never observed their migration into the ischemic region.
Ischemia increased the percentage of DCX+/GFP+ cells in the dorsal part of the lateral ventricles. (A) Graph showing the percentage of DCX+ cells from all GFP+ cells in the dorsal part of the LV. Asterisks indicate significant differences between control and post-ischemic dorsal parts of the LV. (B) An image showing DCX+ and GFP+ cells in the dorsal part of the LV in a control brain. Note the localization of GFP+ cells only in a narrow band in the dorsal part of the LV. (C) Image showing DCX+ and GFP+ cells in the dorsal part of the LV on the ipsilateral side of an ischemic brain. Note the increased number of GFP+ cells in the dorsal part of the LV and the greater incidence of DCX+ cells around the ipsilateral LV. (D) Image showing DCX+ and GFP+ cells in the dorsal part of the LV on the contralateral side of an ischemic brain. Note the significantly increased number of GFP+ cells in the dorsal part of the LV and the greater incidence of DCX+ cells around the contralateral LV. Arrows indicate GFP+/DCX+ cells. Scale bars, 50 μm.
Honsa P, Pivonkova H, Anderova M, Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles, Neuroscience 240 (2013)39–53, IF 3,38
2. Heterogeneity of astrocytes: from development to injury - single cell gene expression
Astrocytes perform control and regulatory functions in the central nervous system and heterogeneity among them is still a matter of debate. To unravel this heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling. We identified three astrocytic subpopulations during postnatal development and additional subpopulations were identified during 14 days after ischemia: resting glia, early reactive glia and permanent reactive glia.
Rusnakova V, Honsa P,Dzamba D, Stahlberg A, Kubista M, Anderova M, Heterogeneity of astrocytes: from development to injury - single cell gene expression, PLoS One 2013 Aug 5;8(8), IF 3,703
3. NMDA Receptors in Glial Cells: Pending Questions
Discoveries of the last 25 years have demonstrated active role of NMDA receptors in glial cells. However, there are many unresolved questions connected with NMDA receptors in glia. The main objective of this work is to shed light on these controversies by summarizing results from all relevant works concerning astrocytes, oligodendrocytes and polydendrocytes in experimental animals, further extended by studies performed on human glia.
The most probable composition of NMDA receptors in astrocytes, oligodendrocytes and polydendrocytes in particular CNS regions under physiological and pathological conditions. Question marks indicate unknown NMDA receptor composition.
Pivoňková, H., Anděrová, M.: (2017) Altered Homeostatic Functions in Reactive Astrocytes and Their Potential as a Therapeutic Target After Brain Ischemic Injury. Current Pharmaceutical Design. IN PRESS
Syková, E., Rychmach, P., Drahorádová, I., Konrádová, Š., Růžičková, K., Voříšek, I., Forostyak, S., Homola, A., Bojar, M.: (2017) Transplantation of Mesenchymal Stromal Cells in Patients With Amyotrophic Lateral Sclerosis: Results of Phase I/IIa Clinical Trial. Cell Transplantation. 26(4): 647-658.
Valný, M., Honsa, P., Kriška, J., Anděrová, M.: (2017) Multipotency and therapeutic potential of NG2 cells. Biochemical Pharmacology. 141(SI): 42-55.
Voříšek, I., Syka, M., Vargová, L.: (2017) Brain Diffusivity and Structural Changes in the R6/2 Mouse Model of Huntington Disease. Journal of Neuroscience Research. 95(7): 1474-1484.
Džamba D., Harantová L., Butenko O., Anděrová, M.: (2015) Glial cells – the key elements of Alzheimer´s disease. Curr. Alzheimer Res., 13(8): 894-911.
Džamba, D., Valihrach, L., Kubista, M., Anděrová, M.: (2016) The correlation between expression profiles measured in single cells and in traditional bulk samples. Scientific Reports, 16(6): 37022.
Forostyak, O., Butenko, O., Anděrová, M., Forostyak, S., Syková, E., Verkhratsky, A., Dayanithi, G.: (2016) Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells. Stem Cell Res., 16(3):622-634.
Honsa, P., Valný, M., Kriška, J., Matušková, H., Harantová, L., Kirdajová, D., Valihrach, L., Androvic, P., Kubista, M., Anděrová, M.: (2016) Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog. Glia, 64(9): 1518-1531.
Janečková, L., Fafilek, B., Krausová, M., Horazná, M., Vojtěchová, M., Alberich-Jorda, M., Sloncová, E., Galusková, K., Sedláček, R., Anděrová, M., Kořínek, V.: (2016) Wnt Signaling Inhibition Deprives Small Intestinal Stem Cells of Clonogenic Capacity. Genesis, 54(3): 101-114.
Kriška, J., Honsa, P., Džamba, D., Butenko, O., Koleničová, D., Janečková, L., Nahacká, Z., Anděra, L., Kozmik, Z., Taketo, M.M., Kořínek, V., Anděrová, M.: (2016) Manipulating Wnt signaling at different subcellular levels affects the fate of neonatal neural stem/progenitor cells. Brain Res., 1651: 73-87.
Lee, C.Y., Dallérac, G., Ezan, P., Anděrová, M., Rouach, N.: (2016) Glucose Tightly Controls Morphological and Functional Properties of Astrocytes. Front. Aging Neurosci., 8(82): 1-12.
Valný,M., Honsa, P., Kirdajová,D., Kamenik,Z., Anděrová, M.: (2016) Tamoxifen in the Mouse Brain: Implications for Fate-Mapping Studies Using the Tamoxifen-Inducible Cre-loxP System. Front. Cell. Neurosci., 10: 243.
Džamba, D., Honsa, P., Valný, M., Kriška, J., Valihrach, L., Novosadová, V., Kubista, M., Anděrová,M.: (2015) Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors. Cell Mol Neurobiol. 35(8): 1187-1202.
Džamba D., Harantová L., Butenko O., Anděrová, M.: (2015) Glial cells – the key elements of Alzheimer´s disease. Current Alzheimer Research IN PRESS
Chvátal, A.: (2015) Discovering the structure of nrve tissue: part 1: from Marcello Malpighi to Christian Berres. J. Hist. Neurosci., 24(3): 268-291.
Chvátal, A.: (2015) Jiří Procháska (1749-1820): Part 2: "De structura nervorum"--studies on a structure of the nervous system. J. Hist. Neurosci., 24(1): 1-25.
Chvátal, A.: (2015) Jan Křtitel Boháč (1724-1768) a jeho disertace o bolesti z roku 1746. (Jan Křtitel Boháč (1724-1768) and dissertation on pain from 1746). Bolest, 18(1): 8-20.
Chvátal A. : (2015) Výzkum struktury nervové tkáně III: od Jana Evangelisty Purkyně (1787–1869) k Ludwigovi Mauthnerovi (1840–1894). Československá fyziologie, 2: 52-72.
Minieri, L., Pivoňková, H., Harantová, L., Anděrová, M., Ferroni S.: (2014) Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes. J. Neurochem. 132(3): 286-300.
Anděrová, M., Benešová, J., Mikesová, M., Džamba, D., Honsa, P., Kriška, J., Butenko, O., Novosadová, V., Valihrach, L., Kubista, M., Dmytrenko, L., Cicanič, M., Vargová, L.: (2014) Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice. PloS One. 9(11): e113444.
Honsa, P., Pivoňková, H., Harantová, L., Butenko, O., Kriška, J., Džamba, D., Rusňáková, V., Valihrach, L., Kubista, M., Anděrová, M.: (2014) Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia. Glia. 62(12): 2004-2021
Chvátal, A.: (2014) Jiří Procháska (1749-1820): Part 1: A Significant Czech Anatomist, Physiologist and Neuroscientist of the Eighteenth Century. J. Hist. Neurosci. 23(4): 367-376
Chvátal, A.: (2014) Discovering the Structure of Nerve Tissue: Part 1: From Marcello Malpighi to Christian Berres. J. Hist. Neurosci.In press.
Mazurová, Y., Anděrová, M., Němečková, I., Bezrouk, A.: (2014) Transgenic Rat Model of Huntington's Disease: A Histopathological Study and Correlations with Neurodegenerative Process in the Brain of HD Patients. Biomed. Res. Int. 2014: 291531.
Dmytrenko, L., Cicanič, M., Anděrová, M., Voříšek, I., Ottersen, O. P., Syková, E., Vargová, L.: (2013) The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions. PLoS One. 8(7): e68044.
Džamba, D., Honsa, P., Anděrová, M.: (2013) NMDA Receptors in Glial Cells: Pending Questions. Curr. Neuropharmacol. 11: 250-262.
Honsa, P., Pivoňková, H., Anděrová, M.: (2013) Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles. Neuroscience. 240: 39-53.
Minieri, L., Pivoňková, H., Caprini, M., Harantová, L., Anděrová, M., Ferroni, S.: (2013) The inhibitor of volume regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes. Br. J. Pharmacol. 168(5): 1240-1254.
Rusnaková, V., Honsa, P., Džamba, D., Stählberg, A., Kubista, M., Anděrová, M.: (2013) Heterogeneity of Astrocytes: From Development to Injury – Single Cell Gene Expression. PLoS One 8(8): e69734.
Stählberg, A., Rusnaková, V., Forootan, A., Anděrová, M., Kubista, M.: (2013) RT-qPCR work-flow for single-cell data analysis.Methods. 59(1): 80-88.
Institute of Molecular Genetics Czech Academy of Science
2nd Faculty of Medicine – Charles University
Institute of Biotechnology Czech Academy of Science