Simulation of gap junction formation reveals critical role of Cys disulfide redox state in connexin hemichannel docking.

Video Abstract.

High-Frequency Imaging Reveals Synchronised Delta- and Theta-Band Ca2+ Oscillations in the Astrocytic Soma In Vivo.

One of the major breakthroughs of neurobiology was the identification of distinct ranges of oscillatory activity in the neuronal network that were found to be responsible for specific biological functions, both physiological and pathological in nature. Astrocytes, physically coupled by gap junctions and possessing the ability to simultaneously modulate the functions of a large number of surrounding synapses, are perfectly positioned to introduce synchronised oscillatory activity into the neural network. However, astrocytic somatic calcium signalling has not been investigated to date in the frequency ranges of common neuronal oscillations, since astrocytes are generally considered to be slow responders in terms of Ca2+ signalling. Using high-frequency two-photon imaging, we reveal fast Ca2+ oscillations in the soma of astrocytes in the delta (0.5-4 Hz) and theta (4-8 Hz) frequency bands in vivo in the rat cortex under ketamine-xylazine anaesthesia, which is known to induce permanent slow-wave sleep. The high-frequency astrocytic Ca2+ signals were not observed under fentanyl anaesthesia, excluding the possibility that the signals were introduced by motion artefacts. We also demonstrate that these fast astrocytic Ca2+ signals, previously considered to be exclusive to neurons, are present in a large number of astrocytes and are phase synchronised at the astrocytic network level. We foresee that the disclosure of these high-frequency astrocytic signals may help with understanding the appearance of synchronised oscillatory signals and may open up new avenues of treatment for neurological conditions characterised by altered neuronal oscillations.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Putrescine Intensifies Glu/GABA Exchange Mechanism and Promotes Early Termination of Seizures.

Endogenous anticonvulsant mechanisms represent a reliable and currently underdeveloped strategy against recurrent seizures and may recall novel original therapeutics. Here, we investigated whether the intensification of the astroglial Glu-GABA exchange mechanism by application of the GABA precursor putrescine (PUT) may be effective against convulsive and non-convulsive seizures. We explored the potential of PUT to inhibit spontaneous spike-and-wave discharges (SWDs) in WAG/Rij rats, a genetic model of absence epilepsy. Significant shortening of SWDs in response to intraperitoneally applied PUT has been observed, which could be antagonized by blocking GAT-2/3-mediated astrocytic GABA release with the specific inhibitor SNAP-5114. Direct application of exogenous GABA also reduced SWD duration, suggesting that PUT-triggered astroglial GABA release through GAT-2/3 may be a critical step in limiting seizure duration. PUT application also dose-dependently shortened seizure-like events (SLEs) in the low-[Mg2+] in vitro model of temporal lobe epilepsy. SNAP-5114 reversed the antiepileptic effect of PUT in the in vitro model as well, further confirming that PUT reduces seizure duration by triggering glial GABA release. In accordance, we observed that PUT specifically reduces the frequency of excitatory synaptic potentials, suggesting that it specifically acts at excitatory synapses. We also identified that PUT specifically eliminated the tonic depolarization-induced desynchronization of SLEs. Since PUT is an important source of glial GABA and we previously showed significant GABA release, it is suggested that the astroglial Glu-GABA exchange mechanism plays a key role in limiting ictal discharges, potentially opening up novel pathways to control seizure propagation and generalization.

Copper signalling: causes and consequences.

Copper-containing enzymes perform fundamental functions by activating dioxygen (O2) and therefore allowing chemical energy-transfer for aerobic metabolism. The copper-dependence of O2 transport, metabolism and production of signalling molecules are supported by molecular systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metabolism may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders associated with aberrant copper metabolism.

Correction to: Copper signalling: causes and consequences.

Following publication of the original article [1], the authors reported an error in Table 3. The correct version of Table 3 is shown below:The publishers apologise for this error. The original article [1] has been corrected.

Astrocyte sodium signaling and the regulation of neurotransmission.

The transmembrane Na(+) concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non-excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na(+) -dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long-lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na(+) ([Na(+) ]i ) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio-temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na(+) ]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na(+) ]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na(+) -coupled co-transporter, can strongly influence, or even reverse, other Na(+) -dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655-1666.

Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes.

Supply of major metabolites such as γ-aminobutyric acid (GABA), ß-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core.

The hippocampal CA3 region can generate two distinct types of sharp wave-ripple complexes, in vitro.

Hippocampal sharp wave-ripples (SPW-Rs) occur during slow wave sleep and behavioral immobility and are thought to play an important role in memory formation. We investigated the cellular and network properties of SPW-Rs with simultaneous laminar multielectrode and intracellular recordings in a rat hippocampal slice model, using physiological bathing medium. Spontaneous SPW-Rs were generated in the dentate gyrus (DG), CA3, and CA1 regions. These events were characterized by a local field potential gradient (LFPg) transient, increased fast oscillatory activity and increased multiple unit activity (MUA). Two types of SPW-Rs were distinguished in the CA3 region based on their different LFPg and current source density (CSD) pattern. Type 1 (T1) displayed negative LFPg transient in the pyramidal cell layer, and the associated CSD sink was confined to the proximal dendrites. Type 2 (T2) SPW-Rs were characterized by positive LFPg transient in the cell layer, and showed CSD sinks involving both the apical and basal dendrites. In both types, consistent with the somatic CSD source, only a small subset of CA3 pyramidal cells fired, most pyramidal cells were hyperpolarized, while most interneurons increased firing rate before the LFPg peak. Different neuronal populations, with different proportions of pyramidal cells and distinct subsets of interneurons were activated during T1 and T2 SPW-Rs. Activation of specific inhibitory cell subsets-with the possible leading role of perisomatic interneurons-seems to be crucial to synchronize distinct ensembles of CA3 pyramidal cells finally resulting in the expression of different SPW-R activities. This suggests that the hippocampus can generate dynamic changes in its activity stemming from the same excitatory and inhibitory circuits, and so, might provide the cellular and network basis for an input-specific and activity-dependent information transmission.

Bilayer Charge Reversal and Modification of Lipid Organization by Dendrimers as Observed by Sum-Frequency Vibrational Spectroscopy.

Polyamidoamine (PAMAM) dendrimers are hyperbranched, nanosized polymers with promising biomedical applications as nanocarriers in targeted drug delivery and gene therapy. For the development of safe dendrimer-based biomedical applications it is necessary to gain an understanding of the detailed mechanism of the interactions of both cationic and anionic dendrimers with cell membranes. To characterize dendrimer-membrane interactions we applied solid-supported lipid bilayers as biomembrane models and utilized infrared-visible sum-frequency vibrational spectroscopy to independently probe the interactions of cationic G5-NH2 and anionic G4.5-COONa dendrimers with the two leaflets of the lipid bilayers. Interaction with both dendrimers led to changes in the interfacial water structure and charge density as evidenced by the changes in the OH band intensities in the sum-frequency spectra of the bilayers. Interaction with the G5-NH2 dendrimer also led to a unique inversion of the sign of the OH-stretch amplitudes, in addition to a decrease in their absolute values. We suggest that the positively charged amino groups on the G5-NH2 dendrimer surface bind to the negatively charged bilayer, while uncompensated positive charges not involved in the binding cause a reversal of the electric field and thus an opposite orientation of the interfacial water molecules. More subtle but nonetheless significant changes were seen in the relative magnitudes of the CH amplitudes. The methyl antisymmetric to symmetric stretch amplitude ratios are altered, implying changes in the tilt angles of the phospholipid alkyl chains. The conformational order of the phospholipid alkyl chains of both leaflets is also influenced by the G5-NH2 dendrimer while G4.5-COONa has no effect on the alkyl chain conformation.

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer.

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer-membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na(+)], but not in low-[Ca(2+)] media suggesting the formation of specific Na(+) permeable channels. SFVS measurements on silica supported DPPG-DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na(+) ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na(+) channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10µg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics.

FluoAnalysis: An Open-Source MATLAB Toolbox for Analysis of Calcium Imaging Measurements of Oscillatory Astrocytic and Neuronal Networks.

Calcium imaging, especially two-photon imaging, has become essential in neuroscience for studying neuronal and astrocytic activity under in vivo and in vitro conditions. Current advances in the development of calcium sensors as well as imaging hardware enable high-frequency measurements of calcium signals in hundreds of cells simultaneously. The analysis of these large datasets requires special tools and usually a certain level of programming experience. Despite advancements in calcium imaging analysis software development, significant gaps remain, particularly for data acquired at a high sampling rate that would allow for the spectral analysis of calcium signals. The FluoAnalysis MATLAB toolbox addresses these gaps by offering a comprehensive solution for analyzing simultaneously measured calcium imaging and electrophysiological data. It features both GUI-based and command-line approaches, emphasizing frequency domain analysis to reveal network-level oscillatory signals linked to single-cell activity. In addition, the toolbox puts special emphasis on differentiating between astrocytes and neurons, revealing the interactions between the network activity of the two major cell types of the brain. It facilitates a streamlined workflow for data loading, ROI identification, cell classification, fluorescence intensity calculation, spectral analysis, and report generation, supporting both manual and automated high-throughput analysis. This versatile platform enables the comprehensive analysis of large imaging datasets. In conclusion, the FluoAnalysis MATLAB toolbox provides a robust and versatile platform for the integrated analysis of calcium imaging and electrophysiological data, supporting diverse neuroscience research applications.

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue.

BACKGROUND: The potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH(2)) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH(2) action in brain tissue on neuronal and astroglial cells. RESULTS: In order to describe early mechanisms of G5-NH(2) dendrimer action in brain tissue we assessed G5-NH(2) trafficking, free intracellular Ca(2+) and mitochondrial membrane potential (Ψ(MITO)) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH(2), we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH(2) evoked robust intracellular Ca(2+) enhancements and Ψ(MITO) depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca(2+) enhancements clearly preceded Ψ(MITO) depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient Ψ(MITO) depolarization, respectively. Transient as opposed to lasting Ψ(MITO) changes to short-term G5-NH(2) application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH(2) on astroglial Ψ(MITO) was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH(2) evoked neuronal activation. CONCLUSION: These findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity.

Astrocytes convert network excitation to tonic inhibition of neurons.

BACKGROUND: Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters. RESULTS: Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na+ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg2+] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions. CONCLUSIONS: The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate.

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 µM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

Dual Role for Astroglial Copper-Assisted Polyamine Metabolism during Intense Network Activity.

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 µM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.

Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers.

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to -45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Spontaneous Ca2+ Fluctuations Arise in Thin Astrocytic Processes With Real 3D Geometry.

Fluctuations of cytosolic Ca2+ concentration in astrocytes are regarded as a critical non-neuronal signal to regulate neuronal functions. Although such fluctuations can be evoked by neuronal activity, rhythmic astrocytic Ca2+ oscillations may also spontaneously arise. Experimental studies hint that these spontaneous astrocytic Ca2+ oscillations may lie behind different kinds of emerging neuronal synchronized activities, like epileptogenic bursts or slow-wave rhythms. Despite the potential importance of spontaneous Ca2+ oscillations in astrocytes, the mechanism by which they develop is poorly understood. Using simple 3D synapse models and kinetic data of astrocytic Glu transporters (EAATs) and the Na+/Ca2+ exchanger (NCX), we have previously shown that NCX activity alone can generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet microdomain. Here, we extend that model by incorporating experimentally determined real 3D geometries of 208 excitatory synapses reconstructed from publicly available ultra-resolution electron microscopy datasets. Our simulations predict that the surface/volume ratio (SVR) of peri-synaptic astrocytic processes prominently dictates whether NCX-mediated spontaneous Ca2+ oscillations emerge. We also show that increased levels of intracellular astrocytic Na+ concentration facilitate the appearance of Ca2+ fluctuations. These results further support the principal role of the dynamical reshaping of astrocyte processes in the generation of intrinsic Ca2+ oscillations and their spreading over larger astrocytic compartments.

Critical Role of Astrocytic Polyamine and GABA Metabolism in Epileptogenesis.

Accumulating evidence indicate that astrocytes are essential players of the excitatory and inhibitory signaling during normal and epileptiform activity via uptake and release of gliotransmitters, ions, and other substances. Polyamines can be regarded as gliotransmitters since they are almost exclusively stored in astrocytes and can be released by various mechanisms. The polyamine putrescine (PUT) is utilized to synthesize GABA, which can also be released from astrocytes and provide tonic inhibition on neurons. The polyamine spermine (SPM), synthesized form PUT through spermidine (SPD), is known to unblock astrocytic Cx43 gap junction channels and therefore facilitate astrocytic synchronization. In addition, SPM released from astrocytes may also modulate neuronal NMDA, AMPA, and kainate receptors. As a consequence, astrocytic polyamines possess the capability to significantly modulate epileptiform activity. In this study, we investigated different steps in polyamine metabolism and coupled GABA release to assess their potential to control seizure generation and maintenance in two different epilepsy models: the low-[Mg2+] model of temporal lobe epilepsy in vitro and in the WAG/Rij rat model of absence epilepsy in vivo. We show that SPM is a gliotransmitter that is released from astrocytes and significantly contributes to network excitation. Importantly, we found that inhibition of SPD synthesis completely prevented seizure generation in WAG/Rij rats. We hypothesize that this antiepileptic effect is attributed to the subsequent enhancement of PUT to GABA conversion in astrocytes, leading to GABA release through GAT-2/3 transporters. This interpretation is supported by the observation that antiepileptic potential of the Food and Drug Administration (FDA)-approved drug levetiracetam can be diminished by specifically blocking astrocytic GAT-2/3 with SNAP-5114, suggesting that levetiracetam exerts its effect by increasing surface expression of GAT-2/3. Our findings conclusively suggest that the major pathway through which astrocytic polyamines contribute to epileptiform activity is the production of GABA. Modulation of astrocytic polyamine levels, therefore, may serve for a more effective antiepileptic drug development in the future.

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain.

The synergy between synaptic Glu release and astrocytic Glu-Na+ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na+/Ca2+ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na+ and Ca2+ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na+ symport differently affect intracellular [Na+] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca2+ oscillations.