Calcium oscillations in neocortical astrocytes under epileptiform conditions.

Morphological and functional alterations in astrocytic glia are often found in epileptic syndromes, although the exact role of astrocytes in epilepsy is poorly understood. During calcium imaging of epileptiform events in juvenile neocortical slices we previously discovered cells with spontaneous oscillations in their intracellular free calcium concentration ([Ca(2+)](i)). We have now characterized these oscillations using two in vitro models of epilepsy and find that they are produced by astrocytes. Astrocytic oscillations are widespread throughout the imaged territories, are remarkably regular and have long periods, averaging 100 s, which become shorter during development. Astrocytic oscillations are uncorrelated among themselves and with epileptiform events, are blocked by internal release antagonists and are stimulated by caffeine. Astrocytic calcium oscillations could mediate reactive astrogliosis, contribute to the pathogenesis of chronic epileptic syndromes, and be used as a diagnostic test for epileptic tissue.     

Calcium dynamics of cortical astrocytic networks in vivo

Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca²⁺]_i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca²⁺]_i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca²⁺]_i activity in individual cells and a robust coordination of [Ca²⁺]_i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.     

New In Vitro Phenotypic Assay for Epilepsy: Fluorescent Measurement of Synchronized Neuronal Calcium Oscillations

Research in the epilepsy field is moving from a primary focus on controlling seizures to addressing disease pathophysiology. This requires the adoption of resource- and time-consuming animal models of chronic epilepsy which are no longer able to sustain the testing of even moderate numbers of compounds. Therefore, new in vitro functional assays of epilepsy are needed that are able to provide a medium throughput while still preserving sufficient biological context to allow for the identification of compounds with new modes of action. Here we describe a robust and simple fluorescence-based calcium assay to measure epileptiform network activity using rat primary cortical cultures in a 96-well format. The assay measures synchronized intracellular calcium oscillations occurring in the population of primary neurons and is amenable to medium throughput screening. We have adapted this assay format to the low magnesium and the 4-aminopyridine epilepsy models and confirmed the contribution of voltage-gated ion channels and AMPA, NMDA and GABA receptors to epileptiform activity in both models. We have also evaluated its translatability using a panel of antiepileptic drugs with a variety of modes of action. Given its throughput and translatability, the calcium oscillations assay bridges the gap between simplified target-based screenings and compound testing in animal models of epilepsy. This phenotypic assay also has the potential to be used directly as a functional screen to help identify novel antiepileptic compounds with new modes of action, as well as pathways with previously unknown contribution to disease pathophysiology.     

Gain-of-function mutation in Gnao1: A murine model of epileptiform encephalopathy (EIEE17)?

G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gα_o, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1 ^+/G184S) that prevents G_o turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41–70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.     

Epileptic activity during early postnatal life in the AY-9944 model of atypical absence epilepsy

Atypical absence epilepsy (AAE) is an intractable disorder characterized by slow spike-and-wave discharges in electroencephalograms (EEGs) and accompanied by severe cognitive dysfunction and neurodevelopmental or neurological deficits in humans. Administration of the cholesterol biosynthesis inhibitor AY-9944 (AY) during the postnatal developmental period induces AAE in animals; however, the neural mechanism of seizure development remains largely unknown. In this study, we characterized the cellular manifestations of AY-induced AAE in the mouse. Treatment of brain slices with AY increased membrane excitability of hippocampal CA1 neurons. AY treatment also increased input resistance of CA1 neurons during early postnatal days (PND) 5–10. However, these effects were not observed during late PND (14–21) or in adulthood (7–10 weeks). Notably, AY treatment elicited paroxysmal depolarizing shift (PDS)-like epileptiform discharges during the early postnatal period, but not during late PND or in adults. The PDS-like events were not compromised by application of glutamate or GABA receptor antagonists. However, the PDS-like events were abolished by blockage of voltage-gated Na⁺ channels. Hippocampal neurons isolated from an in vivo AY model of AAE showed similar PDS-like epileptiform discharges. Further, AY-treated neurons from T-type Ca²⁺ channel α1G knockout (Ca_v3.1^−/−) mice, which do not exhibit typical absence seizures, showed similar PDS-like epileptiform discharges. These results demonstrate that PDS-like epileptiform discharges during the early postnatal period are dependent upon Na⁺ channels and are involved in the generation of AY-induced AAE, which is distinct from typical absence epilepsy. Our findings may aid our understanding of the pathophysiological mechanisms of clinical AAE in individuals, such as those with Lennox–Gastaut syndrome.     

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.     

Intracellular Free Calcium Dynamics in Stretch-Injured Astrocytes

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca²⁺]_i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca²⁺]_i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca²⁺]_i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca²⁺]_i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca²⁺]_i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca²⁺]_i, is responsible for some of the pathological consequences of traumatic brain injury.     

Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

Dynorphins act as endogenous anticonvulsants via activation of kappa opioid receptor (KOR). However, the mechanism underlying the anticonvulsant role remains elusive. This study aims to investigate whether the potential protection of KOR activation by dynorphin against epilepsy was associated with the regulation of PI3K/Akt/Nrf2/HO-1 pathway. Here, a pilocarpine-induced rat model of epilepsy and Mg²⁺-free-induced epileptiform hippocampal neurons were established. Decreased prodynorphin (PDYN) expression, suppressed PI3K/Akt pathway, and activated Nrf2/HO-1 pathway were observed in rat epileptiform hippocampal tissues and in vitro neurons. Furthermore, dynorphin activation of KOR alleviated in vitro seizure-like neuron injury via activation of PI3K/Akt/Nrf2/HO-1 pathway. Further in vivo investigation revealed that PDYN overexpression by intra-hippocampus injection of PDYN-overexpressing lentiviruses decreased hippocampal neuronal apoptosis and serum levels of inflammatory cytokines and malondialdehyde (MDA) content, and increased serum superoxide dismutase (SOD) level, in pilocarpine-induced epileptic rats. The protection of PDYN in vivo was associated with the activation of PI3K/Akt/Nrf2/HO-1 pathway. In conclusion, dynorphin activation of KOR protects against epilepsy and seizure-induced brain injury, which is associated with activation of the PI3K/Akt/Nrf2/HO-1 pathway.     

Enhancement of asynchronous release from fast-spiking interneuron in human and rat epileptic neocortex

Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca²⁺ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca²⁺ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.     

Altered Kir and gap junction channels in temporal lobe epilepsy

Since astrocytes may sense and respond to neuronal activity these cells are now considered important players in brain signaling. Astrocytes form large gap junction coupled syncytia allowing them to clear the extracellular space from K⁺ and neurotransmitters accumulating during neuronal activity, and redistribute it to sites of lower extracellular concentrations. Increasing evidence suggests a crucial role for dysfunctional astrocytes in the etiology of epilepsy. Notably, alterations in expression, localization and function of astroglial K⁺ channels as well as impaired K⁺ buffering was observed in specimens from patients with pharmacoresistant temporal lobe epilepsy and in chronic epilepsy models. Altered astroglial gap junction coupling has also been reported in epileptic tissue which, however, seems to play a dual role: (i) junctional coupling counteracts hyperactivity by facilitating clearance of elevated extracellular K⁺ and glutamate while (ii) it also provides a pathway for energetic substrates and fuels neuronal activity. Dysfunctional astrocytes should be considered promising targets for new therapeutic strategies.     

Behavioral Deficit and Decreased GABA Receptor Functional Regulation in the Hippocampus of Epileptic Rats: Effect of <Emphasis Type="Italic">Bacopa monnieri</Emphasis>

In the present study, alterations of the General GABA and GABA_A receptors in the hippocampus of pilocarpine-induced temporal lobe epileptic rats and the therapeutic application of Bacopa monnieri and its active component Bacoside-A were investigated. Bacopa monnieri (Linn.) is a herbaceous plant belonging to the family Scrophulariaceae. Hippocampus is the major region of the brain belonging to the limbic system and plays an important role in epileptogenesis, memory and learning. Scatchard analysis of [³H]GABA and [³H]bicuculline in the hippocampus of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA_A receptor sub-units such as GABA_Aά1, GABA_Aά5, GABA_Aδ, and GAD were down regulated (P < 0.001) in the hippocampus of the epileptic rats compared to control. GABA_Aγ subunit was up regulated. Epileptic rats have deficit in the radial arm and Y maze performance. Bacopa monnieri and Bacoside-A treatment reverses all these changes near to control. Our results suggest that decreased GABA receptors in the hippocampus have an important role in epilepsy associated behavioral deficit, Bacopa monnieri and Bacoside-A have clinical significance in the management of epilepsy.     

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.     

Changes in TWIK-related Acid Sensitive K+-1 and -3 Channel Expressions from Neurons to Glia in the Hippocampus of Temporal Lobe Epilepsy Patients and Experimental Animal Model

In the present study, we analyzed expressions of tandem of P domains in a Weak Inwardly rectifying K⁺ channel (TWIK)-related Acid-Sensitive K⁺ (TASK) channel-1 and -3 in the hippocampus of patients with temporal lobe epilepsy (TLE) and in rat model. In the control human subjects, TASK-1, and -3 immunoreactivity was observed in pyramidal neurons and dentate granule cells. In TLE patients, TASK-1 and -3 immunoreactivity was rarely observed in neurons. However, TASK-1 immunoreactivity was observed in astrocytes, and TASK-3 immunoreactivity was detected in both astrocytes and microglia. In the rat hippocampus, TASK-1 immunoreactivity was observed in astrocytes within normal and epileptic hippocampus. The alterations in TASK-3 immunoreactivity in the rat hippocampus were similar to those in the human hippocampus. These findings reveal that TASK-1 and -3 are differentially expressed in the normal and epileptic hippocampus, and suggest that TASK channels may contribute to the properties of the epileptic hippocampus.     

Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus

Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1) receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus.     

In vivo optical mapping of epileptic foci and surround inhibition in ferret cerebral cortex

The population of neurons participating in an epileptiform event varies from moment to moment. Most techniques currently used to localize epileptiform events in vivo have spatial and/or temporal sampling limitations. Here we show in an animal model that optical imaging based on intrinsic signals is an excellent method for in vivo mapping of clinically relevant epileptiform events, such as interictal spikes, ictal onsets, ictal spread and secondary homotopic foci. In addition, a decrease in the optical signal correlates spatially with a decrease in neuronal activity recorded from cortex surrounding an epileptic focus. Optical mapping of epilepsy might be a useful adjunct in the surgical treatment of neocortical epilepsy, which critically depends on the precise localization of intrinsically epileptogenic neurons.     

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.     

Calcium sensitive non-selective cation current promotes seizure-like discharges and spreading depression in a model neuron

As described by others, an extracellular calcium-sensitive non-selective cation channel ([Ca²⁺]_o-sensitive NSCC) of central neurons opens when extracellular calcium level decreases. An other non-selective current is activated by rising intracellular calcium ([Ca²⁺] _i ). The [Ca²⁺]_o-sensitive NSCC is not dependent on voltage and while it is permeable by monovalent cations, it is blocked by divalent cations. We tested the hypothesis that activation of this channel can promote seizures and spreading depression (SD). We used a computer model of a neuron surrounded by interstitial space and enveloped in a glia-endothelial “buffer” system. Na⁺, K⁺, Ca²⁺ and Cl⁻ concentrations, ion fluxes and osmotically driven volume changes were computed. Conventional ion channels and the NSCC were incorporated in the neuron membrane. Activation of NSCC conductance caused the appearance of paroxysmal afterdischarges (ADs) at parameter settings that did not produce AD in the absence of NSCC. The duration of the AD depended on the amplitude of the NSCC. Similarly, NSCC also enabled the generation of SD. We conclude that NSCC can contribute to the generation of epileptiform events and to spreading depression.     

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.