INTRODUCTION: Astrocytic Transient receptor potential vanilloid 4 (TRPV4) channels, together with Aquaporin 4 (AQP4), are suspected to be the key players in cellular volume regulation, and therefore may affect the development and severity of cerebral edema during ischemia. In this study, we examined astrocytic swelling/volume recovery in mice with TRPV4 and/or AQP4 deletion in response to in vitro ischemic conditions, to determine how the deletion of these channels can affect the development of cerebral edema. METHODS: We used three models of ischemia-related pathological conditions: hypoosmotic stress, hyperkalemia, and oxygenglucose deprivation (OGD), and observed their effect on astrocyte volume changes in acute brain slices of Aqp4-/-, Trpv4-/- and double knockouts. In addition, we employed single-cell RT-qPCR to assess the effect of TRPV4 and AQP4 deletion on the expression of other ion channels and transporters involved in the homeostatic functioning of astrocytes. RESULTS: Quantification of astrocyte volume changes during OGD revealed that the deletion of AQP4 reduces astrocyte swelling, while simultaneous deletion of both AQP4 and TRPV4 leads to a disruption of astrocyte volume recovery during the subsequent washout. Of note, astrocyte exposure to hypoosmotic stress or hyperkalemia revealed no differences in astrocyte swelling in the absence of AQP4, TRPV4, or both channels. Moreover, under ischemia-mimicking conditions, we identified two distinct subpopulations of astrocytes with low and high volumetric responses (LRA and HRA), and their analyses revealed that mainly HRA are affected by the deletion of AQP4, TRPV4, or both channels. Furthermore, gene expression analysis revealed reduced expression of the ion transporters KCC1 and ClC2 as well as the receptors GABAB and NMDA in Trpv4-/- mice. The deletion of AQP4 instead caused reduced expression of the serine/cysteine peptidase inhibitor Serpina3n. DISCUSSION: Thus, we showed that in AQP4 or TRPV4 knockouts, not only the specific function of these channels is affected, but also the expression of other proteins, which may modulate the ischemic cascade and thus influence the final impact of ischemia.

• Keywords
• aquaporin 4, astrocytes, brain edema, ischemia, transient receptor potential vanilloid 4,
• Publication type
• Journal Article MeSH

NG2 glia display wide proliferation and differentiation potential under physiological and pathological conditions. Here, we examined these two features following different types of brain disorders such as focal cerebral ischemia (FCI), cortical stab wound (SW), and demyelination (DEMY) in 3-month-old mice, in which NG2 glia are labeled by tdTomato under the Cspg4 promoter. To compare NG2 glia expression profiles following different CNS injuries, we employed single-cell RT-qPCR and self-organizing Kohonen map analysis of tdTomato-positive cells isolated from the uninjured cortex/corpus callosum and those after specific injury. Such approach enabled us to distinguish two main cell populations (NG2 glia, oligodendrocytes), each of them comprising four distinct subpopulations. The gene expression profiling revealed that a subpopulation of NG2 glia expressing GFAP, a marker of reactive astrocytes, is only present transiently after FCI. However, following less severe injuries, namely the SW and DEMY, subpopulations mirroring different stages of oligodendrocyte maturation markedly prevail. Such injury-dependent incidence of distinct subpopulations was also confirmed by immunohistochemistry. To characterize this unique subpopulation of transient astrocyte-like NG2 glia, we used single-cell RNA-sequencing analysis and to disclose their basic membrane properties, the patch-clamp technique was employed. Overall, we have proved that astrocyte-like NG2 glia are a specific subpopulation of NG2 glia emerging transiently only following FCI. These cells, located in the postischemic glial scar, are active in the cell cycle and display a current pattern similar to that identified in cortical astrocytes. Astrocyte-like NG2 glia may represent important players in glial scar formation and repair processes, following ischemia.

• Keywords
• NG2 glia, astrocytes, demyelination, ischemia, oligodendrocytes, stab wound,
• MeSH
• Astrocytes * metabolism   MeSH
• Gliosis pathology   MeSH
• Brain Ischemia * metabolism   MeSH
• Mice MeSH
• Neuroglia metabolism   MeSH
• Oligodendroglia pathology   MeSH
• Animals MeSH
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• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Aquaporin-4 (AQP4) is the primary cellular water channel in the brain and is abundantly expressed by astrocytes along the blood-brain barrier and brain-cerebrospinal fluid interfaces. Water transport via AQP4 contributes to the activity-dependent volume changes of the extracellular space (ECS), which affect extracellular solute concentrations and neuronal excitability. AQP4 is anchored by α-syntrophin (α-syn), the deletion of which leads to reduced AQP4 levels in perivascular and subpial membranes. We used the real-time iontophoretic method and/or diffusion-weighted magnetic resonance imaging to clarify the impact of α-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling in vitro and in vivo. In mice lacking α-syn, we found higher resting values of the apparent diffusion coefficient of water (ADCW) and the extracellular volume fraction (α). No significant differences in tortuosity (λ) or non-specific uptake (k'), were found between α-syn-negative (α-syn -/-) and α-syn-positive (α-syn +/+) mice. The deletion of α-syn resulted in a significantly smaller relative decrease in α observed during elevated K(+) (10 mM) and severe hypotonic stress (-100 mOsmol/l), but not during mild hypotonic stress (-50 mOsmol/l). After the induction of terminal ischemia/anoxia, the final values of ADCW as well as of the ECS volume fraction α indicate milder cell swelling in α-syn -/- in comparison with α-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in λ was faster in α-syn -/- mice, but the final values did not differ between α-syn -/- and α-syn +/+ mice. This study reveals that water transport through AQP4 channels enhances and accelerates astrocyte swelling. The substantially altered ECS diffusion parameters will likely affect the movement of neuroactive substances and/or trophic factors, which in turn may modulate the extent of tissue damage and/or drug distribution.

• MeSH
• Aquaporin 4 metabolism   MeSH
• Astrocytes metabolism   MeSH
• Gene Deletion * MeSH
• Diffusion MeSH
• Potassium metabolism   MeSH
• Extracellular Space metabolism   MeSH
• Genotype MeSH
• Gene Knockout Techniques MeSH
• Ischemia genetics   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Osmotic Pressure MeSH
• Calcium-Binding Proteins genetics   metabolism   MeSH
• Somatosensory Cortex metabolism   MeSH
• Heart Arrest genetics   metabolism   MeSH
• Muscle Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aquaporin 4 MeSH
• Potassium MeSH
• Membrane Proteins MeSH
• Calcium-Binding Proteins MeSH
• Muscle Proteins MeSH
• syntrophin alpha1 MeSH Browser

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.

• MeSH
• Astrocytes physiology   MeSH
• DNA Primers MeSH
• Hippocampus pathology   physiopathology   MeSH
• Immunohistochemistry MeSH
• TRPV Cation Channels physiology   MeSH
• Rats MeSH
• Patch-Clamp Techniques MeSH
• Hypoxia-Ischemia, Brain pathology   physiopathology   MeSH
• Polymerase Chain Reaction MeSH
• Rats, Wistar MeSH
• Base Sequence MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• TRPV Cation Channels MeSH
• Trpv4 protein, rat MeSH Browser

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na(+)-currents and K (A)- and K (DR) currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.

• MeSH
• Bicuculline metabolism   MeSH
• Biomarkers metabolism   MeSH
• Potassium Channel Blockers metabolism   MeSH
• Cell Differentiation physiology   MeSH
• Embryo, Mammalian * anatomy & histology   physiology   MeSH
• GABA Antagonists metabolism   MeSH
• gamma-Aminobutyric Acid metabolism   MeSH
• Stem Cells cytology   physiology   MeSH
• Rats MeSH
• Patch-Clamp Techniques MeSH
• Mice MeSH
• Neurons cytology   physiology   MeSH
• Promoter Regions, Genetic MeSH
• Recombinant Fusion Proteins * genetics   metabolism   MeSH
• Telencephalon * cytology   pathology   physiology   MeSH
• Stem Cell Transplantation * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bicuculline MeSH
• Biomarkers MeSH
• Potassium Channel Blockers MeSH
• GABA Antagonists MeSH
• gamma-Aminobutyric Acid MeSH
• Recombinant Fusion Proteins * MeSH

Pathological states in the central nervous system lead to dramatic changes in the activity of neuroactive substances in the extracellular space, to changes in ionic homeostasis and often to cell swelling. To quantify changes in cell morphology over a certain period of time, we employed a new technique, three-dimensional confocal morphometry. In our experiments, performed on enhanced green fluorescent protein/glial fibrillary acidic protein astrocytes in brain slices in situ and thus preserving the extracellular microenvironment, confocal morphometry revealed that the application of hypotonic solution evoked two types of volume change. In one population of astrocytes, hypotonic stress evoked small cell volume changes followed by a regulatory volume decrease, while in the second population volume changes were significantly larger without subsequent volume regulation. Three-dimensional cell reconstruction revealed that even though the total astrocyte volume increased during hypotonic stress, the morphological changes in various cell compartments and processes were more complex than have been previously shown, including swelling, shrinking and structural rearrangement. Our data show that astrocytes in brain slices in situ during hypotonic stress display complex behaviour. One population of astrocytes is highly capable of cell volume regulation, while the second population is characterized by prominent cell swelling, accompanied by plastic changes in morphology. It is possible to speculate that these two astrocyte populations play different roles during physiological and pathological states.

• MeSH
• Astrocytes pathology   ultrastructure   MeSH
• Animals, Genetically Modified MeSH
• Glial Fibrillary Acidic Protein analysis   MeSH
• Microscopy, Confocal methods   MeSH
• Humans MeSH
• Models, Animal MeSH
• Brain pathology   ultrastructure   MeSH
• Mice MeSH
• Brain Diseases pathology   MeSH
• Green Fluorescent Proteins MeSH
• Imaging, Three-Dimensional * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Glial Fibrillary Acidic Protein MeSH
• Green Fluorescent Proteins MeSH