Epileptic activity triggers rapid ROCK1-dependent astrocyte morphology changes.

Long-term modifications of astrocyte function and morphology are well known to occur in epilepsy. They are implicated in the development and manifestation of the disease, but the relevant mechanisms and their pathophysiological role are not firmly established. For instance, it is unclear how quickly the onset of epileptic activity triggers astrocyte morphology changes and what the relevant molecular signals are. We therefore used two-photon excitation fluorescence microscopy to monitor astrocyte morphology in parallel to the induction of epileptiform activity. We uncovered astrocyte morphology changes within 10-20 min under various experimental conditions in acute hippocampal slices. In vivo, induction of status epilepticus resulted in similarly altered astrocyte morphology within 30 min. Further analysis in vitro revealed a persistent volume reduction of peripheral astrocyte processes triggered by induction of epileptiform activity. In addition, an impaired diffusion within astrocytes and within the astrocyte network was observed, which most likely is a direct consequence of the astrocyte remodeling. These astrocyte morphology changes were prevented by inhibition of the Rho GTPase RhoA and of the Rho-associated kinase (ROCK). Selective deletion of ROCK1 but not ROCK2 from astrocytes also prevented the morphology change after induction of epileptiform activity and reduced epileptiform activity. Together these observations reveal that epileptic activity triggers a rapid ROCK1-dependent astrocyte morphology change, which is mechanistically linked to the strength of epileptiform activity. This suggests that astrocytic ROCK1 signaling is a maladaptive response of astrocytes to the onset of epileptic activity.

Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy.

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.

Overview Article Astrocytes as Initiators of Epilepsy.

Astrocytes play a dual role in the brain. On the one hand, they are active signaling partners of neurons and can for instance control synaptic transmission and its plasticity. On the other hand, they fulfill various homeostatic functions such as clearance of glutamate and K+ released from neurons. The latter is for instance important for limiting neuronal excitability. Therefore, an impairment or failure of glutamate and K+ clearance will lead to increased neuronal excitability, which could trigger or aggravate brain diseases such as epilepsy, in which neuronal hyperexcitability plays a role. Experimental data indicate that astrocytes could have such a causal role in epilepsy, but the role of astrocytes as initiators of epilepsy and the relevant mechanisms are under debate. In this overview, we will discuss the potential mechanisms with focus on K+ clearance, glutamate uptake and homoeostasis and related mechanisms, and the evidence for their causative role in epilepsy.

Cell death of hippocampal CA1 astrocytes during early epileptogenesis.

OBJECTIVE: Growing evidence suggests that dysfunctional astrocytes are crucial players in the development of mesial temporal lobe epilepsy (MTLE). Using a mouse model closely recapitulating key alterations of chronic human MTLE with hippocampal sclerosis, here we asked whether death of astrocytes contributes to the initiation of the disease and investigated potential underlying molecular mechanisms. METHODS: Antibody staining was combined with confocal imaging and semiquantitative real-time polymerase chain reaction analysis to identify markers of different cellular death mechanisms between 4 h and 3 days after epilepsy induction. RESULTS: Four hours after kainate-mediated induction of status epilepticus (SE), we found a significant reduction in the density of astrocytes in the CA1 stratum radiatum (SR) of the ipsilateral hippocampus. This reduction was transient, as within the next 3 days, astrocyte cell numbers recovered to the initial values, which was accompanied by enhanced proliferation. Four hours after SE induction, a small proportion of astrocytes in the ipsilateral CA1 SR expressed autophagy-related genes and proteins, whereas we did not find astrocytes positive for cleaved caspase 3 or terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling, ruling out apoptosis-related astrocytic death. Importantly, at the same early time point post-SE, many astrocytes in the ipsilateral CA1 SR showed strong expression of genes encoding pro-necroptosis factors, including receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Phosphorylation of MLKL (pMLKL), formation of necrosome complexes composed of RIPK3 and pMLKL, and translocation of pMLKL to the nucleus and to the plasma membrane were often observed in astrocytes of the ipsilateral hippocampus 4 h post-SE. SIGNIFICANCE: The present study revealed that astrocytes die shortly after induction of SE. Our expression data and immunohistochemistry suggest that necroptosis and autophagy contribute to astrocytic death. These findings help to better understand how dysfunctional and pathological remodeling of astrocytes contributes to the initiation of temporal lobe epilepsy.

A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Properties of human astrocytes and NG2 glia.

Since animal models are inevitable for medical research, information on species differences in glial cell properties is critical for successful translational research. Here, we review current knowledge about morphological and functional properties of human astrocytes and NG2 glial cells and compare these data with those obtained for the comparable cells in rodents. Morphological analyses of astrocytes in the neocortex of rodents versus humans have demonstrated clear differences. In contrast, the functional properties of astrocytes or NG2 glial cells in these species are surprisingly similar. However, these findings should be interpreted with caution, as so far functional analyses of human cells are only available from neocortex and hippocampus, and it is known from rodent studies that the properties of astrocytes in different brain regions may vary considerably. Moreover, technical challenges render astrocyte electrophysiological measurements in situ unreliable, and human cell properties may be affected by medications. Nevertheless, based on the limited data currently available, there is substantial similarity between human and rodent astrocytes with regard to those functional properties studied to date. The unique morphological characteristics of astrocytes in human neocortex call for further physiological analysis. The basic properties for NG2 glia are even less completely evaluated with regard to the question of species differences but no glaring differences have been reported so far. In conclusion, it remains justifiable to employ mouse or rat models to investigate the etiology of human CNS diseases that might involve astrocytes or NG2 glia.

Constitutive deletion of astrocytic connexins aggravates kainate-induced epilepsy.

The astroglial gap junctional network formed by connexin (Cx) channels plays a central role in regulating neuronal activity and network synchronization. However, its involvement in the development and progression of epilepsy is not yet understood. Loss of interastrocytic gap junction (GJ) coupling has been observed in the sclerotic hippocampus of patients with mesial temporal lobe epilepsy (MTLE) and in mouse models of MTLE, leading to the suggestion that it plays a causative role in the pathogenesis. To further elucidate this clinically relevant question, we investigated consequences of astrocyte disconnection on the time course and severity of kainate-induced MTLE with hippocampal sclerosis (HS) by comparing mice deficient for astrocytic Cx proteins with wild-type mice (WT). Continuous telemetric EEG recordings and video monitoring performed over a period of 4 weeks after epilepsy induction revealed substantially higher seizure and interictal spike activity during the chronic phase in Cx deficient versus WT mice, while the severity of status epilepticus was not different. Immunohistochemical analysis showed that, despite the elevated chronic seizure activity, astrocyte disconnection did not aggravate the severity of HS. Indeed, the extent of CA1 pyramidal cell loss was similar between the experimental groups, while astrogliosis, granule cell dispersion, angiogenesis, and microglia activation were even reduced in Cx deficient as compared to WT mice. Interestingly, seizure-induced neurogenesis in the adult dentate gyrus was also independent of astrocytic Cxs. Together, our data indicate that constitutive loss of GJ coupling between astrocytes promotes neuronal hyperexcitability and attenuates seizure-induced histopathological outcomes.

Lipoprotein receptor loss in forebrain radial glia results in neurological deficits and severe seizures.

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.

Plaque-dependent morphological and electrophysiological heterogeneity of microglia in an Alzheimer's disease mouse model.

Microglia, the central nervous system resident innate immune cells, cluster around Aß plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aß plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aß plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes. In addition, patch-clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K+ currents in plaque-associated but not plaque-distant microglia. Within the subgroup of plaque-associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single-cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in- and outward K+ conductance in plaque-associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.

Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy.

Dysfunctional astrocytes are increasingly recognized as key players in the development and progression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-µm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.

Experimental febrile seizures impair interastrocytic gap junction coupling in juvenile mice.

Prolonged and focal febrile seizures (FSs) have been associated with the development of temporal lobe epilepsy (TLE), although the underlying mechanism and the contribution of predisposing risk factors are still poorly understood. Using a kainate model of TLE, we previously provided strong evidence that interruption of astrocyte gap junction-mediated intercellular communication represents a crucial event in epileptogenesis. To elucidate this aspect further, we induced seizures in immature mice by hyperthermia (HT) to study the consequences of FSs on the hippocampal astrocytic network. Changes in interastrocytic coupling were assessed by tracer diffusion studies in acute slices from mice 5 days after experimental FS induction. The results reveal that HT-induced FSs cause a pronounced reduction of astrocyte gap junctional coupling in the hippocampus by more than 50%. Western blot analysis indicated that reduced connexin43 protein expression and/or changes in the phosphorylation status account for this astrocyte dysfunction. Remarkably, uncoupling occurred in the absence of neuronal death and reactive gliosis. These data provide a mechanistic link between FSs and the subsequent development of TLE and further strengthen the emerging view that astrocytes have a central role in the pathogenesis of this disorder. © 2016 Wiley Periodicals, Inc.

Astrocyte uncoupling as a cause of human temporal lobe epilepsy.

Glial cells are now recognized as active communication partners in the central nervous system, and this new perspective has rekindled the question of their role in pathology. In the present study we analysed functional properties of astrocytes in hippocampal specimens from patients with mesial temporal lobe epilepsy without (n = 44) and with sclerosis (n = 75) combining patch clamp recording, K(+) concentration analysis, electroencephalography/video-monitoring, and fate mapping analysis. We found that the hippocampus of patients with mesial temporal lobe epilepsy with sclerosis is completely devoid of bona fide astrocytes and gap junction coupling, whereas coupled astrocytes were abundantly present in non-sclerotic specimens. To decide whether these glial changes represent cause or effect of mesial temporal lobe epilepsy with sclerosis, we developed a mouse model that reproduced key features of human mesial temporal lobe epilepsy with sclerosis. In this model, uncoupling impaired K(+) buffering and temporally preceded apoptotic neuronal death and the generation of spontaneous seizures. Uncoupling was induced through intraperitoneal injection of lipopolysaccharide, prevented in Toll-like receptor4 knockout mice and reproduced in situ through acute cytokine or lipopolysaccharide incubation. Fate mapping confirmed that in the course of mesial temporal lobe epilepsy with sclerosis, astrocytes acquire an atypical functional phenotype and lose coupling. These data suggest that astrocyte dysfunction might be a prime cause of mesial temporal lobe epilepsy with sclerosis and identify novel targets for anti-epileptogenic therapeutic intervention.

Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells.

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.

Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

Functional redundancy and compensation among members of gap junction protein families?

Gap junctions are intercellular conduits for small molecules made up by protein subunits called connexins. A large number of connexin genes were found in mouse and man, and most cell types express several connexins, lending support to the view that redundancy and compensation among family members exist. This review gives an overview of the current knowledge on redundancy and functional compensation - or lack thereof. It takes into account the different properties of connexin subunits which comprise gap junctional intercellular channels, but also the compatibility of connexins in gap junctions. Most insight has been gained by the investigation of mice deficient for one or more connexins and transgenic mice with functional replacement of one connexin gene by another. Most single deficient mice show phenotypical alterations limited to critical developmental time points or to specific organs and tissues, while mice doubly deficient for connexins expressed in the same cell type usually show more severe phenotypical alterations. Replacement of a connexin by another connexin in some cases gave rise to rescue of phenotypical alterations of connexin deficiencies, which were restricted to specific tissues. In many tissues, connexin substitution did not restore phenotypical alterations of connexin deficiencies, indicating that connexins are specialized in function. In some cases, fatal consequences arose from the replacement. The current consensus gained from such studies is that redundancy and compensation among connexins exists at least to a limited extent. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Role of Impaired Astrocyte Gap Junction Coupling in Epileptogenesis.

The gap-junction-coupled astroglial network plays a central role in the regulation of neuronal activity and synchronisation, but its involvement in the pathogenesis of neuronal diseases is not yet understood. Here, we present the current state of knowledge about the impact of impaired glial coupling in the development and progression of epilepsy and discuss whether astrocytes represent alternative therapeutic targets. We focus mainly on temporal lobe epilepsy (TLE), which is the most common form of epilepsy in adults and is characterised by high therapy resistance. Functional data from TLE patients and corresponding experimental models point to a complete loss of astrocytic coupling, but preservation of the gap junction forming proteins connexin43 and connexin30 in hippocampal sclerosis. Several studies further indicate that astrocyte uncoupling is a causal event in the initiation of TLE, as it occurs very early in epileptogenesis, clearly preceding dysfunctional changes in neurons. However, more research is needed to fully understand the role of gap junction channels in epilepsy and to develop safe and effective therapeutic strategies targeting astrocytes.

TGF-ß Activated Kinase 1 (TAK1) Is Activated in Microglia After Experimental Epilepsy and Contributes to Epileptogenesis.

Increasing evidence suggests that inflammation promotes epileptogenesis. TAK1 is a central enzyme in the upstream pathway of NF-κB and is known to play a central role in promoting neuroinflammation in neurodegenerative diseases. Here, we investigated the cellular role of TAK1 in experimental epilepsy. C57Bl6 and transgenic mice with inducible and microglia-specific deletion of Tak1 (Cx3cr1CreER:Tak1fl/fl) were subjected to the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE). Immunohistochemical staining was performed to quantify different cell populations. The epileptic activity was monitored by continuous telemetric electroencephalogram (EEG) recordings over a period of 4 weeks. The results show that TAK1 was activated predominantly in microglia at an early stage of kainate-induced epileptogenesis. Tak1 deletion in microglia resulted in reduced hippocampal reactive microgliosis and a significant decrease in chronic epileptic activity. Overall, our data suggest that TAK1-dependent microglial activation contributes to the pathogenesis of chronic epilepsy.

Astrocyte dysfunction in temporal lobe epilepsy: K+ channels and gap junction coupling.

Astrocytes are endowed with the machinery to sense and respond to neuronal activity. Recent work has demonstrated that astrocytes play important physiological roles in the CNS, e.g., they synchronize action potential firing, ensure ion homeostasis, transmitter clearance and glucose metabolism, and regulate the vascular tone. Astrocytes are abundantly coupled through gap junctions, which is a prerequisite to redistribute elevated K(+) from sites of excessive neuronal activity to sites of lower extracellular K(+) concentration. Recent studies identified dysfunctional astrocytes as crucial players in epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial inwardly rectifying K(+) (Kir) channels, particularly Kir4.1, which is suspected to entail impaired K(+) buffering. Gap junctions in astrocytes appear to play a dual role: on the one hand they counteract the generation of hyperactivity by facilitating clearance of elevated extracellular K(+) levels while in contrast, they constitute a pathway for energetic substrate delivery to fuel neuronal (hyper)activity. Recent work suggests that astrocyte dysfunction is causative of the generation or spread of seizure activity. Thus, astrocytes should be considered as promising targets for alternative antiepileptic therapies.

Albumin is taken up by hippocampal NG2 cells and astrocytes and decreases gap junction coupling.

PURPOSE: Dysfunction of the blood-brain barrier (BBB) and albumin extravasation have been suggested to play a role in the etiology of human epilepsy. In this context, dysfunction of glial cells attracts increasing attention. Our study was aimed to analyze in the hippocampus (1) which cell types internalize albumin injected into the lateral ventricle in vivo, (2) whether internalization into astrocytes impacts their coupling and expression of connexin 43 (Cx43), and (3) whether expression of Kir4.1, the predominating astrocytic K(+) channel subunit, is altered by albumin. METHODS: The patch-clamp method was combined with single cell tracer filling to investigate electrophysiologic properties and gap junction coupling (GJC). For cell identification, mice with cell type-specific expression of a fluorescent protein (NG2kiEYFP mice) and immunohistochemistry were employed. Semiquantitative real time polymerase chain reaction (RT-PCR) allowed analysis of Kir4.1 and Cx43 transcript levels. KEY FINDINGS: We show that fluorescently labeled albumin is taken up by astrocytes, NG2 cells, and neurons, with NG2 cells standing out in terms of the quantity of uptake. Within 5 days postinjection (dpi), intracellular albumin accumulation was largely reduced suggesting rapid degradation. Electrophysiologic analysis of astrocytes and NG2 cells revealed no changes in their membrane properties at either time point. However, astrocytic GJC was significantly decreased at 1 dpi but returned to control levels within 5 dpi. We found no changes in hippocampal Cx43 transcript expression, suggesting that other mechanisms account for the observed changes in coupling. Kir4.1 mRNA was regulated oppositely in the CA1 stratum radiatum, with a strong increase at 1 dpi followed by a decrease at 5 dpi. SIGNIFICANCE: The present study demonstrates that extravasal albumin in the hippocampus induces rapid changes of astrocyte function, which can be expected to impair ion and transmitter homeostasis and contribute to hyperactivity and epileptogenesis. Therefore, astrocytes may represent alternative targets for antiepileptic therapeutic approaches.