Astroglial Cell-to-Cell Interaction with Autoreactive Immune Cells in Experimental Autoimmune Encephalomyelitis Involves P2X7 Receptor, ß3-Integrin, and Connexin-43.

In multiple sclerosis (MS), glial cells astrocytes interact with the autoreactive immune cells that attack the central nervous system (CNS), which causes and sustains neuroinflammation. However, little is known about the direct interaction between these cells when they are in close proximity in the inflamed CNS. By using an experimental autoimmune encephalomyelitis (EAE) model of MS, we previously found that in the proximity of autoreactive CNS-infiltrated immune cells (CNS-IICs), astrocytes respond with a rapid calcium increase that is mediated by the autocrine P2X7 receptor (P2X7R) activation. We now reveal that the mechanisms regulating this direct interaction of astrocytes and CNS-IICs involve the coupling between P2X7R, connexin-43, and ß3-integrin. We found that P2X7R and astroglial connexin-43 interact and concentrate in the immediate proximity of the CNS-IICs in EAE. P2X7R also interacts with ß3-integrin, and the block of astroglial αvß3-integrin reduces the P2X7R-dependent calcium response of astrocytes upon encountering CNS-IICs. This interaction was dependent on astroglial mitochondrial activity, which regulated the ATP-driven P2X7R activation and facilitated the termination of the astrocytic calcium response evoked by CNS-IICs. By further defining the interactions between the CNS and the immune system, our findings provide a novel perspective toward expanding integrin-targeting therapeutic approaches for MS treatment by controlling the cell-cell interactions between astrocytes and CNS-IICs.

Removal of nitrogen and phosphorus by aboveground biomass of Phragmites australis in Constructed Wetland System under the conditions of temperate continental climate.

In this paper, aboveground biomass and basic nutrients removal, nitrogen (N) and phosphorus (P), was analyzed by the use of reed as the main component of Constructed Wetland System (CWS) "Glozan". In almost ideal conditions of temperate continental climate, with favorable substrate humidity, due to the constant inflow of municipal wastewater, reed populations reach a high density, on average 217 ind/m2. The reed produces significant aboveground biomass, fresh weight (FW) of 144.21 g/plant and dry weight (DW) of 77.04 g/plant, with the largest share being per tree (87.49 g FW/plant, 48.17 g DW/plant), then leaf (49.45 g FW/plant, 24.89 g DW/plant) and the smallest inflorescence (7.27 g FW/plant, 3.99 g DW/plant). The results obtained in this way indicate that the largest amount of nitrogen was removed by leaves, then by stems and, the smallest by inflorescences, 181.07 g/m2, 97.73 g/m2, 23.41 g/m2, respectively. Thus, an average of 302.21 g/m2 of nitrogen was removed by the entire aboveground part of the reed. Also, the largest amount of phosphorus was removed by leaves, then by stems, and the smallest by inflorescences, 5.72 g/m2, 4.82 g/m2 and 2.57 g/m2, respectively, while the entire aboveground part of the reed is on average about 13.11 g/m2.

The contribution of this paper is reflected in the obtained results for population density, reed biomass, and reed efficiency in the process of accumulation and removal of nitrogen and phosphorus as the main factors of accelerated eutrophication of aquatic ecosystems, the recipients of municipal wastewater. These results are significant because there is not enough data concerning this topic in the temperate continental climate of Southeast Europe. In addition to this, Constructed Wetland System is the first system of this kind, developed in our country that has been functioning for past 18 years. Due to this, the results are encouraging the application of CWS for a large number of smaller settlements in Southeast Europe as well as in other similar areas. Also, the results obtained in this paper can be useful to all those who are committed to the environmental approach and are engaged in research related to the use of reed in the process of municipal wastewater treatment.

Can glial cells save neurons in epilepsy?

Epilepsy is a neurological disorder caused by the pathological hyper-synchronization of neuronal discharges. The fundamental research of epilepsy mechanisms and the targets of drug design options for its treatment have focused on neurons. However, approximately 30% of patients suffering from epilepsy show resistance to standard anti-epileptic chemotherapeutic agents while the symptoms of the remaining 70% of patients can be alleviated but not completely removed by the current medications. Thus, new strategies for the treatment of epilepsy are in urgent demand. Over the past decades, with the increase in knowledge on the role of glia in the genesis and development of epilepsy, glial cells are receiving renewed attention. In a normal brain, glial cells maintain neuronal health and in partnership with neurons regulate virtually every aspect of brain function. In epilepsy, however, the supportive roles of glial cells are compromised, and their interaction with neurons is altered, which disrupts brain function. In this review, we will focus on the role of glia-related processes in epileptogenesis and their contribution to abnormal neuronal activity, with the major focus on the dysfunction of astroglial potassium channels, water channels, gap junctions, glutamate transporters, purinergic signaling, synaptogenesis, on the roles of microglial inflammatory cytokines, microglia-astrocyte interactions in epilepsy, and on the oligodendroglial potassium channels and myelin abnormalities in the epileptic brain. These recent findings suggest that glia should be considered as the promising next-generation targets for designing anti-epileptic drugs that may improve epilepsy and drug-resistant epilepsy.

Therapeutic Potential of Astrocyte Purinergic Signalling in Epilepsy and Multiple Sclerosis.

Epilepsy and multiple sclerosis (MS), two of the most common neurological diseases, are characterized by the establishment of inflammatory environment in the central nervous system that drives disease progression and impacts on neurodegeneration. Current therapeutic approaches in the treatments of epilepsy and MS are targeting neuronal activity and immune cell response, respectively. However, the lack of fully efficient responses to the available treatments obviously shows the need to search for novel therapeutic candidates that will not exclusively target neurons or immune cells. Accumulating knowledge on epilepsy and MS in humans and analysis of relevant animal models, reveals that astrocytes are promising therapeutic candidates to target as they participate in the modulation of the neuroinflammatory response in both diseases from the initial stages and may play an important role in their development. Indeed, astrocytes respond to reactive immune cells and contribute to the neuronal hyperactivity in the inflamed brain. Mechanistically, these astrocytic cell to cell interactions are fundamentally mediated by the purinergic signalling and involve metabotropic P2Y1 receptors in case of astrocyte interactions with neurons, while ionotropic P2X7 receptors are mainly involved in astrocyte interactions with autoreactive immune cells. Herein, we review the potential of targeting astrocytic purinergic signalling mediated by P2Y1 and P2X7 receptors to develop novel approaches for treatments of epilepsy and MS at very early stages.

Approach for patch-clamping using an upright microscope with z-axis movable stage.

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.

Dysfunction of oligodendrocyte inwardly rectifying potassium channel in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1G93A rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+ . First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1G93A rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1G93A spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1G93A oligodendrocytes. In addition, the Ba2+ -sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1G93A spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology.

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling.

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca2+ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca2+ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca2+ increase is specifically triggered by the autoreactive CD4+ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca2+ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.

Ferrous iron binding to epinephrine promotes the oxidation of iron and impedes activation of adrenergic receptors.

Upon release in response to stress, epinephrine (Epi) may interact with labile iron pool in human plasma with potentially important (patho)physiological consequences. We have shown that Epi and Fe3+ build stable 1:1 high-spin bidentate complex at physiological pH, and that Epi does not undergo degradation in the presence of iron. However, the interactions of Epi with the more soluble Fe2+, and the impact of iron on biological activity of Epi are still not known. Herein we showed that Epi and Fe2+ build colorless complex which is stable under anaerobic conditions. In the presence of O2, Epi promoted the oxidation of Fe2+ and the formation of Epi-Fe3+ complex. Cyclic voltammetry showed that mid-point potential of Epi-Fe2+ complex is very low (-582 mV vs. standard hydrogen electrode), which explains catalyzed oxidation of Fe2+. Next, we examined the impact of iron binding on biological performance of Epi using patch clamping in cell culture with constitutive expression of adrenergic receptors. Epi alone evoked an increase of outward currents, whereas Epi in the complex with Fe3+ did not. This implies that the binding of Epi to adrenergic receptors and their activation is prevented by the formation of complex with iron. Pro-oxidative activity of Epi-Fe2+ complex may represent a link between chronic stress and cardiovascular problems. On the other hand, labile iron could serve as a modulator of biological activity of ligands. Such interactions may be important in human pathologies that are related to iron overload or deficiency.

Role of astrocyte purinergic signaling in epilepsy.

Epilepsy is characterized by unpredictable recurrent seizures resulting from hypersynchronous discharges from neuron assemblies. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability. Purinergic signaling is central in neuron-glia and glia-glia interactions and dysfunctions in communication pathways involving purinergic receptors have been reported in various CNS pathologies, such as Alzheimer disease, stroke, major depression, schizophrenia, and epilepsy. In the present review we will first discuss the mechanisms by which astrocytes influence neuronal activity. We will then review in more details recent evidence indicating that dysregulation of astrocyte purinergic signaling actively contributes to the appearance of abnormal neuronal activity in epilepsy.

Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte-derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα-driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

An autocrine purinergic signaling controls astrocyte-induced neuronal excitation.

Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. Using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a calcium-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDA receptors. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal.

The effect of amyotrophic lateral sclerosis-linked exogenous SOD1-G93A on electrophysiological properties and intracellular calcium in cultured rat astrocytes.

Over 150 mutations in the SOD1 gene that encodes Cu/Zn superoxide dismutase (SOD1) cause 20-25% of familial ALS, albeit without a known gain-of-function mechanism. ALS is also non-cell-autonomous, the interactions between motor neurons and their glial neighbours being implicated in disease progression. The aim here was to investigate the biophysical effects of the exogenous human mutant SOD1-G93A on rat astrocytes in culture. Primary cortical astrocyte cultures were treated with recombinant human apo- mSOD1-G93A vs. wild-type control (wtSOD1) and recorded by patch-clamp and calcium imaging. Results showed that exogenous mSOD1 as well as wtSOD1 induced a decrease of membrane resistance, the effect being persistent (up to 13 min) only for the mutant form. Similarly, whole-cell inward currents in astrocytes were augmented by both wt and mSOD1, but the effect was twice larger and only progressed continuously for the latter. Both forms of SOD1 also induced a rise in intracellular Ca(2+) activity, the effect being dependent on external Ca(2+) and again only persisted with mSOD1, becoming significantly different from wtSOD1 only at longer times (14 min). In conclusion, this study points to membrane permeability and Ca(2+) signalling as processes affected by SOD1-G93A that presents the humoral factor triggering the role of astrocytes in ALS pathophysiology.

Short- and long-term exposure to alternating magnetic field (50 Hz, 0.5 mT) affects rat pituitary ACTH cells: Stereological study.

The aim of the present study was to determine does extremely low frequency magnetic field (ELF-MF, 50 Hz, 0.5 mT) affect pituitary adrenocorticotroph (ACTH) cells in adult animals. We performed two series of experiments: (1) short-term exposure of 3-month-old rats to ELF-MF for 1 and 7 days, and (2) long-term exposure of rats to ELF-MF from their conception to 3 months of age. Stereological study was performed on immunolabeled pituitary ACTH cells. The total number and volume of ACTH cells, the volume of their nuclei and pituitary volume were measured. ELF-MF exposure for 1 day significantly decreased total number and volume of ACTH cells, the volume of their nuclei, as well as pituitary volume. ELF-MF exposure for 7 days significantly reduced only the volume of ACTH cells. Life-long exposure to ELF-MF induced decrease in the volume of ACTH cells and pituitary volume. We can conclude that the applied ELF-MF has a strong influence on morphometrical parameters of the pituitary ACTH cells and could be considered as a stressogenic factor.

Osmotic swelling activates a novel anionic current with VRAC-like properties in a cytoplasmic droplet membrane from Phycomyces blakesleeanus sporangiophores.

We describe here whole-cell currents of droplets prepared from the apical region of growing Phycomyces blakesleeanus sporangiophores. Whole-cell current recordings revealed the osmotically activated, outwardly rectifying, fast inactivating instantaneous current (ORIC) with biophysical properties closely resembling volume-regulated anionic current (VRAC). ORIC is activated under conditions of osmotically induced swelling and shows strong selectivity for anions over cations. In addition, ORIC shows voltage and time-dependent inactivation at positive potentials and recovery from inactivation at negative potentials. ORIC is blocked by anthracene-9-carboxylic acid, an anion channel blocker, in a voltage-dependent manner. This is the first report of the presence of VRAC-like current in an organism outside the chordate lineage.

Cell death parameters as revealed by whole-cell patch-clamp and interval weighted spectra averaging: changes in membrane properties and current frequency of cultured mouse microglial cells induced by glutaraldehyde.

The physiological and biochemical factors that lead to cell death have not been recognized completely. To our knowledge, there are no data on the bioelectric parameters that characterize early period of cell death, as well as on the appearance of related membrane current frequencies. We studied early parameters of glutaraldehyde (GA)-induced cell death, by examining the membrane properties of mouse microglia using the whole-cell patch-clamp technique. In addition, we investigated the GA-induced changes in the membrane current frequency, to see if characteristic frequencies would appear in dying cell. For data analysis, we applied a new approach, an improved multiple moving window length analysis and interval weighted spectra averaging (IWSA). We chose GA for its ability to induce almost instantaneous cell death. The 0.6% GA did not induce changes in the bioelectric membrane properties of microglia. However, the 3% GA caused significant decrease of membrane capacitance and resistance accompanied by the prominent increase in the membrane currents and nearly ohmic current response of microglial cells. These data indicate that 3% GA caused complete loss of the membrane function consequently inducing instantaneous cell death. The membrane function loss was characterized by appearance of the 1.26-4.62 Hz frequency peak in the IWSA spectra, while no significant increase of amplitudes could be observed for cells treated with 0.6% GA. To our knowledge, this is the first record of a frequency associated with complete loss of the membrane function and thus can be considered as an early indicator of cell death.

Modification of glial response in hibernation: a patch-clamp study on glial cells acutely isolated from hibernating land snail.

Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain rhythmic activity and survival of the animal.

Ouabain modulation of snail Br neuron bursting activity after the exposure to 10 mT static magnetic field revealed by Higuchi fractal dimension.

Aim of this study was to investigate the application of normalized mean of the empirical Higuchi fractal dimension (FD) distributions, as a new approach to analyze the spontaneous bioelectrical activity of garden snail (Helix pomatia) Br neuron. The effect of ouabain on modulation of Br neuron bursting activity before and after the exposure to 10 mT static magnetic field (SMF) was observed by analyzing the following parameters: action potential (AP), interspike interval (ISI) and interbursting interval (IBI) components. Normalized mean of the empirical FD distributions were formed for the following experimental conditions: Control 1, Ouabain 1, Control 2, SMF 2, ASMF 2, Control 3, SMF 3 and Ouabain ASMF 3. Our main results have shown that ouabain without SMF induced increase in participation of AP and a decrease in participation of IBI components compared to the first control condition. However, in the presence of 10 mT SMF, ouabain-induced changes of measured parameters of Br neuron activity were less pronounced compared to the third control condition. We have shown that normalized mean of the empirical FD distributions is a useful method for detecting the changes in AP, ISI, and IBI components of complex bursting activity in altered physiological states.

Changes in the expression and current of the Na+/K+ pump in the snail nervous system after exposure to a static magnetic field.

Compelling evidence supports the use of a moderate static magnetic field (SMF) for therapeutic purposes. In order to provide insight into the mechanisms underlying SMF treatment, it is essential to examine the cellular responses elicited by therapeutically applied SMF, especially in the nervous system. The Na(+)/K(+) pump, by creating and maintaining the gradient of Na(+) and K(+) ions across the plasma membrane, regulates the physiological properties of neurons. In this study, we examined the expression of the Na(+)/K(+) pump in the isolated brain-subesophageal ganglion complex of the garden snail Helix pomatia, along with the immunoreactivity and current of the Na(+)/K(+) pump in isolated snail neurons after 15 min exposure to a moderate (10 mT) SMF. Western blot and immunofluorescence analysis revealed that 10 mT SMF did not significantly change the expression of the Na(+)/K(+) pump α-subunit in the snail brain and the neuronal cell body. However, our immunofluorescence data showed that SMF treatment induced a significant increase in the Na(+)/K(+) pump α-subunit expression in the neuronal plasma membrane area. This change in Na(+)/K(+) pump expression was reflected in pump activity as demonstrated by the pump current measurements. Whole-cell patch-clamp recordings from isolated snail neurons revealed that Na(+)/K(+) pump current density was significantly increased after the 10 mT SMF treatment. The SMF-induced increase was different in the two groups of control snail neurons, as defined by the pump current level. The results obtained could represent a physiologically important response of neurons to 10 mT SMF comparable in strength to therapeutic applications.