Astroglial Volume Research Articles

Astroglial Activation Is Exacerbated in a Down Syndrome Mouse Model

Down syndrome (DS), also known as trisomy 21, is one of the most common chromosomal disorders associated with intellectual disability. Mouse models are valuable for mechanistic and therapeutic intervention studies. The purpose of this study was to investigate astroglial anomalies in Dp16, a widely used DS mouse model. Brain sections were prepared from one-month-old Dp16 mice and their littermates, immunostained with an anti-GFAP or anti-S100B antibody, and imaged to reconstruct astroglial morphology in three dimensions. No significant difference in the number of astrocytes was found in either the hippocampal CA1 region or cortex between Dp16 and WT mice. However, the average astroglial volume in Dp16 was significantly (P < 0.05) greater than that in WT, suggesting the astroglial activation. Reanalysis of the single-nucleus RNA sequencing data indicated that the genes differentially expressed between WT and Dp16 astrocytes were associated with synapse organization and neuronal projection. In contrast, in vitro cultured neonatal astrocytes did not exhibit significant morphological changes. The expression of Gfap in in vitro cultured Dp16 astrocytes was not increased as it was in in vivo hippocampal tissue. However, after treatment with lipopolysaccharides, the inflammatory response gene IFNβ increased significantly more in Dp16 astrocytes than in WT astrocytes. Overall, our results showed that the increase in astrogliogenesis in DS was not apparent in the early life of Dp16 mice, while astrocyte activation, which may be partly caused by increased responses to inflammatory stimulation, was significant. The inflammatory response of astrocytes might be a potential therapeutic target for DS intellectual disability.

Existence of FGFR1-5-HT1AR heteroreceptor complexes in hippocampal astrocytes. Putative link to 5-HT and FGF2 modulation of hippocampal gamma oscillations

The majority of the fibroblast growth factor receptor 1-serotonin 1 A receptor (FGFR1-5-HT1AR) heterocomplexes in the hippocampus appeared to be located mainly in the neuronal networks and a relevant target for antidepressant drugs. Through a neurochemical and electrophysiological analysis it was therefore tested in the current study if astrocytic FGFR1-5-HT1AR heterocomplexes also exist in hippocampus. They may modulate the structure and function of astroglia in the hippocampus leading to possible changes in the gamma oscillations. Localization of hippocampal FGFR1-5-HT1AR heterocomplexes in astrocytes was found using in situ proximity ligation assay combined with immunohistochemistry using glial fibrillary acidic protein (GFAP) immunoreactivity as a marker for astroglia. Acute i.c.v. treatment with 8-OH-DPAT alone or together with basic fibroblast growth factor (FGF2) significantly increased FGFR1-5-HT1AR heterocomplexes in the GFAP positive cells, especially in the polymorphic layer of the dentate gyrus (PoDG) but also in the CA3 area upon combined treatment. No other hippocampal regions were studied. Also, structural plasticity changes were observed in the astrocytes, especially in the PoDG region, upon these pharmacological treatments. They may also be of relevance for enhancing the astroglial volume transmission with increased modulation of the neuronal networks in the regions studied. The effects of combined FGF2 and 5-HT agonist treatments on gamma oscillations point to a significant antagonistic interaction in astroglial FGFR1-5-HT1AR heterocomplexes that may contribute to counteraction of the 5-HT1AR-mediated decrease of gamma oscillations.This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes.

Consolidated evidence indicates that astroglial cells are critical in the homeostatic regulation of cellular volume by means of ion channels and aquaporin-4. Volume-regulated anion channel (VRAC) is the chloride channel that is activated upon cell swelling and critically contributes to cell volume regulation in astrocytes. The molecular identity of VRAC has been recently defined, revealing that it belongs to the leucine-rich repeat-containing 8 (LRRC8) protein family. However, there is a lack of evidence demonstrating that LRRC8A underpins VRAC currents in astrocyte. Nonetheless, direct evidence of the role of LRRC8A in astrocytic regulatory volume decrease remains to be proved. Here, we aim to bridge this gap in knowledge by combining RNA interference specific for LRRC8A with patch-clamp analyses and a water-permeability assay. We demonstrated that LRRC8A molecular expression is essential for swelling-activated chloride current via VRAC in primary-cultured cortical astrocytes. The knockdown of LRRC8A with a specific short interference RNA abolished the recovery of the cell volume after swelling induced by hypotonic challenge. In addition, immunoblotting, immunofluorescence, confocal imaging, and immunogold electron microscopy demonstrated that LRRC8A is expressed in the plasma membrane of primary cortical astrocytes and in situ in astrocytes at the perivascular interface with endothelial cells. Collectively, our results suggest that LRRC8A is an essential subunit of VRAC and a key factor for astroglial volume homeostasis.-Formaggio, F., Saracino, E., Mola, M. G., Rao, S. B., Amiry-Moghaddam, M., Muccini, M., Zamboni, R., Nicchia, G. P., Caprini, M., Benfenati, V. LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes.

The role of glutamate in neuronal ion homeostasis: A case study of spreading depolarization.

Simultaneous changes in ion concentrations, glutamate, and cell volume together with exchange of matter between cell network and vasculature are ubiquitous in numerous brain pathologies. A complete understanding of pathological conditions as well as normal brain function, therefore, hinges on elucidating the molecular and cellular pathways involved in these mostly interdependent variations. In this paper, we develop the first computational framework that combines the Hodgkin–Huxley type spiking dynamics, dynamic ion concentrations and glutamate homeostasis, neuronal and astroglial volume changes, and ion exchange with vasculature into a comprehensive model to elucidate the role of glutamate uptake in the dynamics of spreading depolarization (SD)—the electrophysiological event underlying numerous pathologies including migraine, ischemic stroke, aneurysmal subarachnoid hemorrhage, intracerebral hematoma, and trauma. We are particularly interested in investigating the role of glutamate in the duration and termination of SD caused by K+ perfusion and oxygen-glucose deprivation. Our results demonstrate that glutamate signaling plays a key role in the dynamics of SD, and that impaired glutamate uptake leads to recovery failure of neurons from SD. We confirm predictions from our model experimentally by showing that inhibiting astrocytic glutamate uptake using TFB-TBOA nearly quadruples the duration of SD in layers 2-3 of visual cortical slices from juvenile rats. The model equations are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles or a combination of these principles and known physiological facts. Accordingly, we claim that our approach can be used as a future guide to investigate the role of glutamate, ion concentrations, and dynamics cell volume in other brain pathologies and normal brain function.

Anions Govern Cell Volume: A Case Study of Relative Astrocytic and Neuronal Swelling in Spreading Depolarization.

Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little about the dynamics of cell and tissue swelling, and the differential changes in the volumes of neurons and glia during pathological states such as spreading depolarizations (SD) under ischemic and non–ischemic conditions, and epileptic seizures. By combining the Hodgkin–Huxley type spiking dynamics, dynamic ion concentrations, and simultaneous neuronal and astroglial volume changes into a comprehensive model, we elucidate why glial cells swell more than neurons in SD and the special case of anoxic depolarization (AD), and explore the relative contributions of the two cell types to tissue swelling. Our results demonstrate that anion channels, particularly Cl−, are intrinsically connected to cell swelling and blocking these currents prevents changes in cell volume. The model is based on a simple and physiologically realistic description. We introduce model extensions that are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles, or by a phenomenological combination of these principles and known physiological facts. This work provides insights into numerous studies related to neuronal and glial volume changes in SD that otherwise seem contradictory, and is broadly applicable to swelling in other cell types and conditions.

Cell Volume in Brain Pathologies: Anions-Controlled Neural and Glial Swelling in Spreading Depolarization and Increased Neuronal Susceptibility to Ischemic Injury due to Large Extracellular Space

Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little about the dynamics of cell and tissue swelling, and the differential changes in the volumes of neurons and glia during pathological states such as spreading depolarizations (SD) and epileptic seizures. By combining the Hodgkin-Huxley type spiking dynamics, dynamic ion concentrations, and simultaneous neuronal and astroglial volume changes into a comprehensive model, we elucidate why glial cells swell more than neurons in SD and the special case of ischemia-induced anoxic depolarization (AD). We explore the relative contributions of the two cell types to tissue swelling and our results demonstrate that anion channels, particularly Cl-, are intrinsically connected to cell swelling. Blocking these currents prevents changes in cell volume. Our model is simple, physiologically realistic, and derived from the first physical principles of electroneutrality, osmosis and conservation of particles. We provide new insights into numerous studies related to neuronal and glial volume changes in SD that otherwise seem contradictory. The theory is broadly applicable to swelling in other cell types and conditions.In a complimentary research we found that the brain region-specific extracellular volume fraction has a strong effect on the recoverability of neurons from AD. Glial-vascular K+ clearance and Na+ /K+ -exchange pumps are key to the cell's recovery, and the large extracellular space in the upper brain regions leads to impaired Na+/K+-exchange pumps so that they function at reduced capacity. Hence they are unable to bring the cell out of AD after oxygen and glucose is restored, leading to permanent cell damage.

TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia.

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4(-/-) and Aqp4(-/-) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)]i elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)]i elevations but only modestly attenuated the amplitude of Ca(2+) signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4-AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. Significance statement: We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.

Real‐time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two‐photon microscopy

The brain swells over the several minutes that follow stroke onset or acute hypo-osmotic stress because cells take up water. Measuring the volume responses of single neurons and glia has necessarily been confined to isolated or cultured cells. Two-photon laser scanning microscopy enables real-time visualization of cells functioning deep within living neocortex in vivo or in brain slices under physiologically relevant osmotic and ischemic stress. Astrocytes and their processes expressing green fluorescent protein in murine cortical slices swelled in response to 20 min of overhydration (-40 mOsm) and shrank during dehydration (+40 or +80 mOsm) at 32-34 degrees C. Minute-by-minute monitoring revealed no detectable volume regulation during these osmotic challenges, particularly during the first 5 min. Astrocytes also rapidly swelled in response to elevated [K+](o) for 3 min or oxygen/glucose deprivation (OGD) for 10 min. Post-OGD, astroglial volume recovered quickly when slices were re-supplied with oxygen and glucose, while neurons remained swollen with beaded dendrites. In vivo, rapid astroglial swelling was confirmed within 6 min following intraperitoneal water injection or during the 6-12 min following cardiac arrest. While the astrocytic processes were clearly swollen, the extent of the astroglial arbor remained unchanged. Thus, in contrast to osmo-resistant pyramidal neurons (Andrew et al., 2007) that lack known aquaporins, astrocytes passively respond to acute osmotic stress, reflecting functional aquaporins in their plasma membrane. Unlike neurons, astrocytes better recover from brief ischemic insult in cortical slices, probably because their aquaporins facilitate water efflux.

Regulation of Astroglial Volume by Ketamine in Glutamate Induced Cellular Volume Changes

Regulation of Astroglial Volume by Ketamine in Glutamate Induced Cellular Volume Changes

Receptor-mediated volume regulation in astrocytes in primary culture

Changes in astroglial volume were studied after incubating primary cultures from the cerebral cortex of newborn rats for 5 hr in taurine, glutamate or γ-aminobutyric acid (GABA), alone or together with monoamine receptor agonists. Control cell volume was 2.2 μl mg protein. In the presence of taurine or glutamate there was a small increase in cell volume, further augmented when the cells were incubated in isoproterenol plus taurine or phenylephrine, or isoproterenol plus glutamate. After incubation in 5-hydroxytryptamine (5-HT) plus taurine, on the other hand, the cell volume was not different from the control value. In the presence of GABA, alone or together with adrenoceptor agonists (α 1, α 2 or β) or 5-HT, there were no significant changes in cell volume. The regulation of astroglial volume is complicated and affected by ion fluxes, free amino acids and metabolic events including changes in pH. The results are discussed in relation to late data on receptor regulation of active uptake of amino acids. Astroglial volume might be influenced by amino acid transport, partly under control of monoamine receptors.