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.     

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.     

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.     

Calcineurin Mediates Synaptic Scaling Via Synaptic Trafficking of Ca2+-Permeable AMPA Receptors

Homeostatic synaptic plasticity is a negative-feedback mechanism for compensating excessive excitation or inhibition of neuronal activity. When neuronal activity is chronically suppressed, neurons increase synaptic strength across all affected synapses via synaptic scaling. One mechanism for this change is alteration of synaptic AMPA receptor (AMPAR) accumulation. Although decreased intracellular Ca²⁺ levels caused by chronic inhibition of neuronal activity are believed to be an important trigger of synaptic scaling, the mechanism of Ca²⁺-mediated AMPAR-dependent synaptic scaling is not yet understood. Here, we use dissociated mouse cortical neurons and employ Ca²⁺ imaging, electrophysiological, cell biological, and biochemical approaches to describe a novel mechanism in which homeostasis of Ca²⁺ signaling modulates activity deprivation-induced synaptic scaling by three steps: (1) suppression of neuronal activity decreases somatic Ca²⁺ signals; (2) reduced activity of calcineurin, a Ca²⁺-dependent serine/threonine phosphatase, increases synaptic expression of Ca²⁺-permeable AMPARs (CPARs) by stabilizing GluA1 phosphorylation; and (3) Ca²⁺ influx via CPARs restores CREB phosphorylation as a homeostatic response by Ca²⁺-induced Ca²⁺ release from the ER. Therefore, we suggest that synaptic scaling not only maintains neuronal stability by increasing postsynaptic strength but also maintains nuclear Ca²⁺ signaling by synaptic expression of CPARs and ER Ca²⁺ propagation.     

Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store

Mitochondrial Ca²⁺ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics. Recently developed GCaMP genetically-encoded Ca²⁺ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca²⁺ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca²⁺ dynamics in culture, revealing Ca²⁺ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca²⁺ indicators, our results show that mitochondrial Ca²⁺ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca²⁺ dynamics on cytosolic Ca²⁺ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca²⁺ increase using mito-GCaMP5G and a synthetic organic Ca²⁺ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca²⁺ signaling pathway, our results demonstrated that the mitochondrial Ca²⁺ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.     

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.     

α1-Adrenergic receptors mediate coordinated Ca signaling of cortical astrocytes in awake,behaving mice

Astrocyte Ca²⁺ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca²⁺ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca²⁺ signals by activation of astrocytic α₁-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca²⁺ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α₁-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca²⁺ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca²⁺ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca²⁺ signaling being triggered by the α₁-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity.     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson''s disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.     

Multiscale Model of Dynamic Neuromodulation Integrating Neuropeptide-Induced Signaling Pathway Activity with Membrane Electrophysiology

We developed a multiscale model to bridge neuropeptide receptor-activated signaling pathway activity with membrane electrophysiology. Typically, the neuromodulation of biochemical signaling and biophysics have been investigated separately in modeling studies. We studied the effects of Angiotensin II (AngII) on neuronal excitability changes mediated by signaling dynamics and downstream phosphorylation of ion channels. Experiments have shown that AngII binding to the AngII receptor type-1 elicits baseline-dependent regulation of cytosolic Ca²⁺ signaling. Our model simulations revealed a baseline Ca²⁺-dependent response to AngII receptor type-1 activation by AngII. Consistent with experimental observations, AngII evoked a rise in Ca²⁺ when starting at a low baseline Ca²⁺ level, and a decrease in Ca²⁺ when starting at a higher baseline. Our analysis predicted that the kinetics of Ca²⁺ transport into the endoplasmic reticulum play a critical role in shaping the Ca²⁺ response. The Ca²⁺ baseline also influenced the AngII-induced excitability changes such that lower Ca²⁺ levels were associated with a larger firing rate increase. We examined the relative contributions of signaling kinases protein kinase C and Ca²⁺/Calmodulin-dependent protein kinase II to AngII-mediated excitability changes by simulating activity blockade individually and in combination. We found that protein kinase C selectively controlled firing rate adaptation whereas Ca²⁺/Calmodulin-dependent protein kinase II induced a delayed effect on the firing rate increase. We tested whether signaling kinetics were necessary for the dynamic effects of AngII on excitability by simulating three scenarios of AngII-mediated K_DR channel phosphorylation: (1), an increased steady state; (2), a step-change increase; and (3), dynamic modulation. Our results revealed that the kinetics emerging from neuromodulatory activation of the signaling network were required to account for the dynamical changes in excitability. In summary, our integrated multiscale model provides, to our knowledge, a new approach for quantitative investigation of neuromodulatory effects on signaling and electrophysiology.     

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca²⁺]_i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca²⁺]_i oscillations followed by larger and sustained [Ca²⁺]_i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca²⁺]_i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca²⁺]_i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca²⁺]_i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia.     

Quantifying the Uncertainty of Spontaneous Ca Oscillations in Astrocytes: Particulars of Alzheimer's Disease

The quantification of spontaneous calcium (Ca²⁺) oscillations (SCOs) in astrocytes presents a challenge because of the large irregularities in the amplitudes, durations, and initiation times of the underlying events. In this article, we use a stochastic context to account for such SCO variability, which is based on previous models for cellular Ca²⁺ signaling. First, we found that passive Ca²⁺ influx from the extracellular space determine the basal concentration of this ion in the cytosol. Second, we demonstrated the feasibility of estimating both the inositol 1,4,5-trisphosphate (IP₃) production levels and the average number of IP₃ receptor channels in the somatic clusters from epifluorescent Ca²⁺ imaging through the combination of a filtering strategy and a maximum-likelihood criterion. We estimated these two biophysical parameters using data from wild-type adult mice and age-matched transgenic mice overexpressing the 695-amino-acid isoform of human Alzheimer β-amyloid precursor protein. We found that, together with an increase in the passive Ca²⁺ influx, a significant reduction in the sensitivity of G protein-coupled receptors might lie beneath the abnormalities in the astrocytic Ca²⁺ signaling, as was observed in rodent models of Alzheimer's disease. This study provides new, to our knowledge, indices for a quantitative analysis of SCOs in normal and pathological astrocytes.     

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca²⁺-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain''s computational power and behavioural output. These more active functions are endowed by the Ca²⁺-based excitability displayed by astrocytes. An increase in cytosolic Ca²⁺ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca²⁺ dynamics, Ca²⁺-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca²⁺ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca²⁺ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy.     

A Cellular Mechanism for Graded Persistent Activity in a Model Neuron and Its Implications in Working Memory

Working memory represents the ability of the brain to hold information for relatively short periods of time. Working memory is believed mediated by persistent neuronal firing. A broadly accepted hypothesis is that persistent activity is generated by reverberating synaptic input. However, single cortical neurons capable of showing persistent firing were recently reported. In this modeling study, we propose a cellular mechanism to generate persistent firing of multiple firing rates in single neurons. In the proposed mechanism, bistable concentrations of inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ is achieved by IP₃ formation and IP₃-induced Ca²⁺ release from stores in multiple subcellular domains. A postsynaptic firing rate-dependent switching of these bistable elements can demonstrate graded persistent firing of the rat entorhinal neurons. Such a firing rate-dependent switching may be extended to a variety of intracellular Ca²⁺ signaling cascades.     

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca²⁺ (Ca_V) and voltage-gated K⁺ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K⁺ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca²⁺ influx through Ca_V channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca²⁺ influx. At physiological levels of Ca²⁺, this inhibition is specific to Ca_V2 (not Ca_V1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of Ca_V channels, we found that inhibition of Ca_V2 channels limits the Ca²⁺ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K⁺ channels and Ca_V channels to fine-tune Ca²⁺ influx through Ca_V2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.     

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.     

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca²⁺ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca²⁺ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca²⁺ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.