Christian Steinhäuser

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.

Ion channels in glial cells.

Functional and molecular analysis of glial voltage- and ligand-gated ion channels underwent tremendous boost over the last 15 years. The traditional image of the glial cell as a passive, structural element of the nervous system was transformed into the concept of a plastic cell, capable of expressing a large variety of ion channels and neurotransmitter receptors. These molecules might enable glial cells to sense neuronal activity and to integrate it within glial networks, e.g., by means of spreading calcium waves. In this review we shall give a comprehensive summary of the main functional properties of ion channels and ionotropic receptors expressed by macroglial cells, i.e., by astrocytes, oligodendrocytes and Schwann cells. In particular we will discuss in detail glial sodium, potassium and anion channels, as well as glutamate, GABA and ATP activated ionotropic receptors. A majority of available data was obtained from primary cell culture, these results have been compared with corresponding studies that used acute tissue slices or freshly isolated cells. In view of these data, an active glial participation in information processing seems increasingly likely and a physiological role for some of the glial channels and receptors is gradually emerging.

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus.

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30−/− mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

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.

News on glutamate receptors in glial cells.

Glutamate (Glu) receptors convey most of the excitatory synaptic transmission in the mammalian CNS. Distinct Glu-receptor genes and different subtypes of glutamate-activated channels are expressed ubiquitously throughout the developing and mature brain in the two major macroglial cell types, astrocytes and oligodendrocytes. These glial receptors are found in acutely isolated cells and in brain slices, and are therefore functional in vivo. Glutamate receptors in glial cells are activated during neuronal activity, and their activation modulates gene expression in astrocytes and oligodendrocytes. The proliferation and differentiation of glial precursor cells are also regulated by activation of Glu receptors, suggesting that the excitatory transmitter might be one of the environmental signals that regulate glial-cell development.

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.

Functional changes in astroglial cells in epilepsy

Epilepsy comprises a group of disorders characterized by the periodic occurrence of seizures, and pathologic specimens from patients with temporal lobe epilepsy demonstrate marked reactive gliosis. Since recent studies have implicated glial cells in novel physiological roles in the CNS, such as modulation of synaptic transmission, it is plausible that glial cells may have a functional role in the hyperexcitability characteristic of epilepsy. Indeed, alterations in distinct astrocyte membrane channels, receptors and transporters have all been associated with the epileptic state. This review integrates the current evidence regarding astroglial dysfunction in epilepsy and the potential underlying mechanisms of hyperexcitability. Functional understanding of the cellular and molecular alterations of astroglia‐dependent hyperexcitability will help to clarify the physiological role of astrocytes in neural function as well as lead to the identification of novel therapeutic targets.

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.

Distinct Types of Astroglial Cells in the Hippocampus Differ in Gap Junction Coupling

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA‐type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter‐controlled EGFP expression and patch‐clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing β‐Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP‐EGFP‐positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes.