Gerald Seifert

Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca2+- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca2+-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Astrocyte dysfunction in neurological disorders: a molecular perspective

Recent work on glial cell physiology has revealed that glial cells, and astrocytes in particular, are much more actively involved in brain information processing than previously thought. This finding has stimulated the view that the active brain should no longer be regarded solely as a network of neuronal contacts, but instead as a circuit of integrated, interactive neurons and glial cells. Consequently, glial cells could also have as yet unexpected roles in the diseased brain. An improved understanding of astrocyte biology and heterogeneity and the involvement of these cells in pathogenesis offers the potential for developing novel strategies to treat neurological disorders.

Viscoelastic properties of individual glial cells and neurons in the CNS

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “γλια” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances

Functional properties of astrocytes were investigated with the patch‐clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco‐resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammons horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin‐sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to ‐130 mV. Comparative recordings were performed in astrocytes from patients with lesion‐associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion‐associated TLE.

Astrocyte dysfunction in epilepsy

Epilepsy comprises a group of disorders characterized by the periodic occurrence of seizures. Currently available anticonvulsant drugs and therapies are insufficient to controlling seizure activity in about one third of epilepsy patients. Thus, there is an urgent need for new therapies that prevent the genesis of the disorder and improve seizure control in individuals already afflicted. The vast majority of epileptic cases are of idiopathic origin, and a deeper understanding of the cellular basis of hyperactivity and synchronization is essential. Neurosurgical specimens from patients with temporal lobe epilepsy typically demonstrate marked reactive gliosis. Since recent studies have implicated astrocytes in important physiological roles in the CNS, such as synchronization of neuronal firing, it is plausible that they may also have a role in seizure generation or seizure spread. In support of this view, several membrane channels, receptors and transporters in the astrocytic membrane have been found to be deeply altered in the epileptic brain, and they are now gradually emerging as new potential targets for antiepileptic therapeutic strategies. This review summarizes current evidence regarding astroglial dysfunction in epilepsy and discusses presumed underlying mechanisms.

Analysis of Astroglial K+ Channel Expression in the Developing Hippocampus Reveals a Predominant Role of the Kir4.1 Subunit

Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express time- and voltage-independent currents, but the underlying ion channels are not well defined. This ignorance is partly attributable to abundant intercellular coupling of these cells through gap junctions, impeding quantitative analyses of intrinsic membrane properties. Moreover, distinct types of cells with astroglial properties coexist in a given brain area, a finding that confused previous analyses. In the present study, we investigated expression of inwardly rectifying (Kir) and two-pore-domain (K2P) K+ channels in astrocytes, which are thought to be instrumental in the regulation of K+ homeostasis. Freshly isolated astrocytes were used to improve space-clamp conditions and allow for quantitative assessment of functional parameters. Patch-clamp recordings were combined with immunocytochemistry, Western blot analysis, and semiquantitative transcript analysis. Comparative measurements were performed in different CA1 subregions of astrocyte-targeted transgenic mice. While confirming weak Ba2+ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba2+ concentrations. Upregulation of Kir4.1 transcripts and protein during the first 10 postnatal days was accompanied by a fourfold increase in astrocyte inward current density. Hippocampal astrocytes from Kir4.1−/− mice lacked Ba2+-sensitive currents. In addition, we report functional expression of K2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K+ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the “passive” current pattern of hippocampal astrocytes.

Functional and Molecular Properties of Human Astrocytes in Acute Hippocampal Slices Obtained from Patients with Temporal Lobe Epilepsy

Summary: Purpose: The specific role of glial cells in epilepsy is still elusive. In this study, functional properties of astrocytes were investigated in acute hippocampal brain slices obtained from surgical specimens of patients with drug‐resistant temporal lobe epilepsy (TLE).

Glial membrane channels and receptors in epilepsy: impact for generation and spread of seizure activity

Epilepsy is a condition in the brain characterized by repetitively occurring seizures. While various changes in neuronal properties have been reported to accompany or induce seizure activity in human or experimental epilepsy, other studies suggested that glial cells might be involved in epileptogenesis. Recent findings demonstrate that in the course of the disease, glial cells not only undergo structural alterations but also display distinct functional properties. Several studies identified reduced inwardly rectifying K(+) currents in astrocytes of epileptic tissue, which probably results in disturbances of the K(+) homeostasis. Other data hinted at an abnormal increase in [Ca(2+)](i) in astrocytes through enhanced activity of glial glutamate receptors. This review summarizes current knowledge of alterations of plasma membrane channels and receptors of macroglial cells in epilepsy and discusses the putative importance of these changes for the generation and spread of seizure activity.

AMPA receptor-mediated modulation of inward rectifier K+ channels in astrocytes of mouse hippocampus.

Astrocytes and neurons are tightly associated and recent data suggest a direct signaling between neuronal and glial cells in vivo. To further analyze these interactions, the patch-clamp technique was combined with single-cell RT-PCR in acute hippocampal brain slices. Subsequent to functional analysis, the cytoplasm of the same cell was harvested to perform transcript analysis and identify subunits that underlie inwardly rectifying K+ currents (I(Kir)) in astrocytes of the CA1 stratum radiatum. Transcripts encoding Kir2.1, Kir2.2, or Kir2.3, were encountered in a majority of cells, while Kir4.1 was less frequent. Further investigation revealed that glial Kir channels are rapidly inhibited upon activation of AMPA-type glutamate receptors, most probably due a receptor-mediated influx of Na+, which plugs the channels from the intracellular side. A transient inhibition of I(Kir) in astrocytes in response to neuronal glutamate release and glial AMPA receptor activation represents a further, so far undetected mechanism to balance neuronal excitability.

Astrocytic function and its alteration in the epileptic brain

Currently available anticonvulsant drugs and complementary therapies are insufficient to control seizures in about a third of epileptic patients. Thus, there is an urgent need for new treatments that prevent the development of epilepsy and control it better in patients already afflicted with the disease. A prerequisite to reach this goal is a deeper understanding of the cellular basis of hyperexcitability and synchronization in the affected tissue. Epilepsy is often accompanied by massive reactive gliosis. Although the significance of this alteration is poorly understood, recent findings suggest that modified astroglial function may have a role in the generation and spread of seizure activity. Here we summarize properties of astrocytes as well as their changes that can be associated with epileptic tissue. The goal is to provide an understanding of the current knowledge of these cells with the long‐term view of providing a foundation for the development of novel hypotheses about the role of glia in epilepsy.