Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity

Astrocytes produce a complex repertoire of Ca²⁺ events that coordinate their major functions. The principle of Ca²⁺ events integration in astrocytes, however, is unknown. Here we analyze whole Ca²⁺ events, which were defined as spatiotemporally interconnected transient Ca²⁺ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca²⁺ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca²⁺ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (α) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in α indicated an increase in proportion of large Ca²⁺ events. Notably, mGluRs activation did not increase the frequency of whole Ca²⁺ events. This result suggests that neuronal activity does not trigger new Ca²⁺ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca²⁺ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow.     

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca²⁺ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca²⁺ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.     

Modeling study of the effects of membrane surface charge on calcium microdomains and neurotransmitter release

Synchronous neurotransmitter release is mediated by the opening of voltage-gated Ca²⁺ channels and the build-up of submembrane Ca²⁺ microdomains. Previous models of Ca²⁺ microdomains have neglected possible electrostatic interactions between Ca²⁺ ions and negative surface charges on the inner leaflet of the plasma membrane. To address the effects of these interactions, we built a computational model of ion electrodiffusion described by the Nernst-Planck and Poisson equations. We found that inclusion of a negative surface charge significantly alters the spatial characteristics of Ca²⁺ microdomains. Specifically, close to the membrane, Ca²⁺ ions accumulate, as expected from the strong electrostatic attraction exerted on positively charged Ca²⁺ ions. Farther away from the membrane, increasing the surface charge density results in a reduction of the Ca²⁺ concentration because of the preferential spread of Ca²⁺ ions along lateral directions. The model also predicts that the negative surface charge will decrease the spatial gradient of the Ca²⁺ microdomain in the lateral direction, resulting in increased overlap of microdomains originating from different Ca²⁺ channels. Finally, we found that surface charge increases the probability of vesicle release if the Ca²⁺ sensor is located within the electrical double layer, whereas this probability is decreased if the Ca²⁺ sensor lies at greater distances from the membrane. Our data suggest that membrane surface charges exert a significant influence on the profile of Ca²⁺ microdomains, and should be taken into account in models of neurotransmitter release.     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Spatiotemporal pattern of calcium activity in astrocytic network

Ca²⁺ influx through an astrocyte plasma membrane is mediated by ionotropic receptors and Ca²⁺ channels according the electrochemical gradient. These conductances allow astrocytes to sense the levels of neuronal activity and environmental changes. Na⁺/Ca²⁺ exchanger (NCX) removes elevated Ca²⁺ from the cell but can reverse and bring Ca²⁺ in. Ca²⁺ entry through the plasma membrane produces local Ca²⁺ elevations that can be further amplified by Ca²⁺ induced activation of inositol-3-phosphate (IP₃) receptors and subsequent Ca²⁺ release from intracellular Ca²⁺ stores. These Ca²⁺ stores are located in astrocytic processes called branchlets, while perisynaptic astrocytic processes are formed by organelle-free leaflets. Such morphological structure suggests separate synaptic and extrasynaptic mechanisms of Ca²⁺ signaling in astrocytes. Astrocytic leaflets sense synaptic activity, astrocytic branchlets integrate signals arriving from the leaflets and from extrasynaptic inputs. The surface-to-volume ratio (SVR) of the branchlets sets the threshold for generation of spreading Ca²⁺ events. Therefore, morphological remodeling of the processes is an important regulator of astrocytic Ca²⁺ activity. Ca²⁺ events can propagate beyond single astrocytes and form complex spatiotemporal patterns of Ca²⁺ activity in the astrocytic network. Ca²⁺ events spread intercellularly through gap-junctions and via extracellular ATP diffusion. Spatially and temporarily organized Ca²⁺ events in astrocytic network influence variable numbers of synapses and neuronal compartments, gate excitation flow and synaptic plasticity in the neuronal network through the release of gliotransmitters. Thus, multiple patterns of Ca²⁺ activity in the astrocytic network (guiding templates) determine multiple states of the neuronal network. This phenomenon may be linked to learning, memory and information processing in the brain.     

Failure of calcium microdomain generation and pathological consequences

Normal physiological regulation depends on Ca²⁺ microdomains, because there is a need to spatially separate Ca²⁺ regulation of different cellular processes. It is only possible to generate local Ca²⁺ signals transiently; so, there is an important functional link between Ca²⁺ spiking and microdomains. The pancreatic acinar cell provides a useful cell biological model, because of its clear structural and functional polarization. Although local Ca²⁺ spiking in the apical (granular) microdomain regulates fluid and enzyme secretion, prolonged global elevations of the cytosolic Ca²⁺ concentration are associated with the human disease acute pancreatitis, in which proteases in the granular region become inappropriately activated and digest the pancreas and its surroundings. A major cause of pancreatitis is alcohol abuse and it has now been established that fatty acid ethyl esters and fatty acids, non-oxidative alcohol metabolites, are principally responsible for causing the acinar cell damage. The fatty acid ethyl esters release Ca²⁺ from the endoplasmic reticulum and the fatty acids inhibit markedly mitochondrial ATP generation, which prevents the acinar cell from disposing of the excess Ca²⁺ in the cytosol. Because of the abolition of ATP-dependent Ca²⁺ pump activity, all intracellular Ca²⁺ concentration gradients disappear and the most important part of the normal regulatory machinery is thereby destroyed. The end stage is necrosis.     

Infrared Excitation Induces Heating and Calcium Microdomain Hyperactivity in Cortical Astrocytes

Unraveling how neural networks process and represent sensory information and how these cellular signals instruct behavioral output is a main goal in neuroscience. Two-photon activation of optogenetic actuators and calcium (Ca²⁺) imaging with genetically encoded indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques locally expose the brain to high near-infrared light doses, raising the concern of light-induced adverse effects on the biology under study. Combining 2P imaging of Ca²⁺ transients in GCaMP6f-expressing cortical astrocytes and unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca²⁺ transients in the fine astroglial processes that depended on the average rather than peak laser power. Illumination conditions routinely being used in biological 2P microscopy (920-nm excitation, ∼100-fs, and ∼10 mW average power) increased the frequency of microdomain Ca²⁺ events but left their amplitude, area, and duration largely unchanged. Ca²⁺ transients in the otherwise silent soma were secondary to this peripheral hyperactivity that occurred without overt morphological damage. Continuous-wave (nonpulsed) 920-nm illumination at the same average power was as damaging as femtosecond pulses, unraveling the dominance of a heating-mediated damage mechanism. In an astrocyte-specific inositol 3-phosphate receptor type-2 knockout mouse, near-infrared light-induced Ca²⁺ microdomains persisted in the small processes, underpinning their resemblance to physiological inositol 3-phosphate receptor type-2-independent Ca²⁺ signals, whereas somatic hyperactivity was abolished. We conclude that, contrary to what has generally been believed in the field, shorter pulses and lower average power can help to alleviate damage and allow for longer recording windows at 920 nm.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.     

Is the intrasomal phase of fast axonal transport driven by oscillations of intracellular calcium?

An hypothesis is presented suggesting that the delivery of vesicle-packaged protein from the neuronal soma to the axonal transport system is physiologically coupled to spontaneous fluctuations of intracellular calcium (Ca_i). Evidence is reviewed that oscillations of Ca_i, commonly detected as agonist-or voltage-triggered waves and spikes propagating through the cytosol, also occur as spontaneous events. Endogenously-generated oscillations are examined since intrasomal transport persists in the absence of extracellular signals or nerve impulse activity. Vesicle budding from the endoplasmic reticulum (ER) may be a key step at which anterograde transport is regulated by events related to the release and reuptake of ER stores of Ca²⁺.Special-issue dedicated to Dr. Sidney Ochs.     

Astrocytes mediate in vivo cholinergic-induced synaptic plasticity

Long-term potentiation (LTP) of synaptic transmission represents the cellular basis of learning and memory. Astrocytes have been shown to regulate synaptic transmission and plasticity. However, their involvement in specific physiological processes that induce LTP in vivo remains unknown. Here we show that in vivo cholinergic activity evoked by sensory stimulation or electrical stimulation of the septal nucleus increases Ca²⁺ in hippocampal astrocytes and induces LTP of CA3-CA1 synapses, which requires cholinergic muscarinic (mAChR) and metabotropic glutamate receptor (mGluR) activation. Stimulation of cholinergic pathways in hippocampal slices evokes astrocyte Ca²⁺ elevations, postsynaptic depolarizations of CA1 pyramidal neurons, and LTP of transmitter release at single CA3-CA1 synapses. Like in vivo, these effects are mediated by mAChRs, and this cholinergic-induced LTP (c-LTP) also involves mGluR activation. Astrocyte Ca²⁺ elevations and LTP are absent in IP₃R2 knock-out mice. Downregulating astrocyte Ca²⁺ signal by loading astrocytes with BAPTA or GDPβS also prevents LTP, which is restored by simultaneous astrocyte Ca²⁺ uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²⁺ elevations, which stimulate astrocyte glutamate release that activates mGluRs. The cholinergic-induced LTP results from the temporal coincidence of the postsynaptic activity and the astrocyte Ca²⁺ signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²⁺ signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information.     

Stimulation of GABA-Induced Ca2+ Influx Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Neurons

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca²⁺ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca²⁺ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca²⁺ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca²⁺ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca²⁺ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca²⁺ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.     

Calcium‐induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca²⁺)‐induced Ca²⁺ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1‐43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1‐43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1‐43 produced a frequency‐dependent number of fluorescent spots. While a 1‐Hz train produced 19.5 ± 5.0 spots/soma, a 10‐Hz train produced 146.7 ± 20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca²⁺ release from intracellular stores without electrical stimulation and external Ca²⁺, produced 168 ± 21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca²⁺‐ ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 μM) to block Ca²⁺‐induced Ca²⁺ release, FM1‐43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca²⁺‐induced Ca²⁺ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Ouabain enhances exocytosis through the regulation of calcium handling by the endoplasmic reticulum of chromaffin cells

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na⁺/K⁺-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K⁺ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca²⁺ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) triggered by K⁺ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K⁺ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K⁺-elicited [Ca²⁺]_c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca²⁺ load and facilitate the Ca²⁺-induced-Ca²⁺ release (CICR) component of the [Ca²⁺]_c signal generated during K⁺ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.     

Glutamate Mediated Astrocytic Filtering of Neuronal Activity

Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca²⁺ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca²⁺ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca²⁺ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP₃ and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca²⁺ activity as a nonlinear integrator of glutamate-dependent neuronal activity.     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue

Brain function relies in large part on Ca²⁺-dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca²⁺ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca²⁺ −sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spike-evoked presynaptic Ca²⁺ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca²⁺ dynamics and transmitter release in an intact brain at the level of individual synapses.     

Bidirectional Scaling of Astrocytic Metabotropic Glutamate Receptor Signaling following Long-Term Changes in Neuronal Firing Rates

Very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of changes in neuronal activity. Here we provide evidence for bidirectional scaling of astrocytic group I metabotropic glutamate receptor signaling in acute mouse hippocampal slices following long-term changes in neuronal firing rates. Plasticity of astrocytic mGluRs was measured by recording spontaneous and evoked Ca²⁺ elevations in both astrocytic somata and processes. An exogenous astrocytic Gq G protein-coupled receptor was resistant to scaling, suggesting that the alterations in astrocyte Ca²⁺ signaling result from changes in activity of the surface mGluRs rather than a change in intracellular G protein signaling molecules. These findings suggest that astrocytes actively detect shifts in neuronal firing rates and adjust their receptor signaling accordingly. This type of long-term plasticity in astrocytes resembles neuronal homeostatic plasticity and might be important to ensure an optimal or expected level of input from neurons.