Heterogeneity and terminology
Before discussing details of membrane physiology of astrocytes it is important to note that there are different types of cells with astroglial properties within a given brain region, and that astrocyte properties may vary in different subregions. An approach to demonstrate this heterogeneity is the analysis of glial cells in appropriate transgenic animal models. For example, cells with astroglial properties are labeled in the living brain of mice with human glial fibrillary acidic protein (hGFAP) promoter-driven enhanced green fluorescent protein (EGFP) expression (Tg[hGFAP/EGFP] mice; (Nolte et al., 2001)). In the hippocampus of these mice, a coexistence of two distinct populations of hGFAP/EGFP-positive glial cells has been identified and investigated in detail (Matthias et al., 2003; Wallraff et al., 2004; Jabs et al., 2005). Figure 1 summarizes important properties of these cells. The two cell types were termed GluT and GluR cells for their segregated expression of glutamate transporters (GluT cells) and ionotropic glutamate receptors (GluR cells), respectively. Moreover, GluT cells are characterized by a significant resting K+ conductance, giving rise to very low-input resistances, mostly below 10 MΩ, and almost linear, “passive” whole-cell current patterns (Figs 1A2 and B3). GluT cells are intensively coupled via gap junctions, enwrap blood vessels with their endfeet and bear a sponge-like, heavily branched net of processes. The majority of GluT cells show immunoreactivity for GFAP and S100β but not for NG2. Hence, GluT cells resemble all properties of bona fide protoplasmic astrocytes. Investigation of the role of gap junction coupling for K+ buffering in the hippocampus of adult mice revealed two types of astrocytes differing in morphology and function (Wallraff et al., 2006).
On the other hand, GluR cells lack dye coupling and are characterized by the expression of various voltage- and time-dependent ion channels and input resistances larger than 50 MΩ (Figs 1A1, B1, B2). Their processes often spread in a radial, spider web-like manner and apparently do not touch blood capillaries. GluR cells receive synaptic input from GABAergic interneurons and glutamatergic CA1 pyramidal neurons (Jabs et al., 2005). The majority of GluR cells is immunoreactive for NG2 but not for GFAP. In this article, we refer to both cell types as astrocytes, based on their GFAP promoter activity. However, it is clear that the functional impact of these two cell types with astroglial properties is different, although not yet completely understood. It will be a challenge for future work to define sets of parameters allowing for unequivocal identification and discrimination of glial cell types in the central nervous system (CNS).
Astrocytes express almost the same set of ion channels and receptors as neurons do (Verkhratsky & Steinhäuser, 2000; Seifert & Steinhäuser, 2004; Kettenmann & Steinhäuser, 2005) although the relative strength of expression varies between the two cell types. For example, in astrocytes, K+ channel density exceeds that of Na+ channels by far, preventing generation of glial action potentials. Nevertheless, a glia-specific, or at least preferential expression has been elucidated for some of these channels and carriers. Among them is Kir4.1, a subunit belonging to the family of inwardly rectifying K+ (Kir) channels. In the CNS, this channel is predominantly localized at distant astrocyte processes surrounding synapses or capillaries (Higashi et al., 2001). Recent work suggests a colocalization of Kir4.1 with the water channel aquaporin-4 (AQP4), which in the brain and spinal cord is also expressed by astrocytes but not neurons. Increasing evidence indicates that a coordinated action of both channels is required for the astrocytes to maintain K+ and water homeostasis in the CNS (Nielsen et al., 1997; Verkman, 2005). As illustrated in the following sections, dysfunction of these astroglial transmembrane channels appears to play a key role in epilepsy.
In contrast to the majority of mature neurons, astrocytes are usually coupled through gap junctions to form large intercellular networks. Astrocytic gap junctions are mainly formed by connexins 43 and 30 (Cx43 and Cx30) in a cell type-specific fashion. Through these networks astrocytes can dissipate molecules, such as K+ or glutamate, a process considered important to prevent their detrimental extracellular accumulation (Theis et al., 2005). Recent data suggest that the capacity of K+ clearance is only partially disturbed in the absence of astrocyte gap junctions, presumably because of the existence of “indirect” coupling of elongated astrocytic processes (Wallraff et al., 2006). Connexins also contribute to the propagation of intercellular Ca2+ waves, presumably by enhancing ATP release, rather than by providing an intercellular pathway for signal diffusion (Nedergaard et al., 2003). However, the pathological impact of disturbed astroglial gap junction expression is not well understood yet (Steinhäuser & Seifert, 2002; Seifert et al., 2006).
Another important function of astrocytes is the removal of neurotransmitters released by active neurons. Uptake of glutamate is accomplished by two glia-specific transporters, EAAT1 and EAAT2 (in rodents termed GLAST and GLT-1), the activity of which may shape the kinetics of receptor currents at synapses (Bergles et al., 1999; Danbolt, 2001). Compelling evidence suggests that disturbed glutamate uptake by astrocytes is directly involved in the pathogenesis of epilepsy, as discussed in the following sections.
Astrocytes can also release neuroactive agents, including neurotransmitters. Several studies revealed that such a release is critically dependent on an increase of astroglial [Ca2+]i. Astrocytes express a plethora of neurotransmitter receptors that are coupled through G-proteins (Gq) and phospholipase C to the release of Ca2+ from internal stores (Haydon, 2001). Stimulation of neuronal afferents induces Ca2+ elevations within astrocytes (Dani et al., 1992; Porter & McCarthy, 1996), which can spread to neighboring astrocytes, demonstrating the presence of an astrocyte-to-astrocyte network (Charles et al., 1991; Sul et al., 2004). Thus, although electrically inexcitable, astrocytes contain a chemically based form of excitability that is bidirectionally linked to neuronal activity (Haydon, 2001). Though initially discovered in 1994 (Parpura et al., 1994), the past decade has seen many studies demonstrating that astrocytes release chemical transmitters (“gliotransmitters”), including glutamate, ATP and D-serine (reviewed in Haydon, 2001; Volterra & Meldolesi, 2005). Although the mechanism(s) underlying astroglial transmitter release are open to debate, at least part of the release seems to occur through regulated, Ca2+-dependent exocytosis, a mechanism that in the CNS was previously thought to be exclusive to neurons.
What is the impact of transmitter release from astrocytes? Several reports suggested that gliotransmitters may activate receptors in neurons to modulate the strength of inhibitory and excitatory synaptic transmission (Bezzi et al., 1998; Kang et al., 1998; Parri et al., 2001; Yang et al., 2003; Zhang et al., 2003; Fiacco & McCarthy, 2004; Pascual et al., 2005; reviewed by Volterra and Meldolesi, 2005). Importantly, because the fine terminal processes of single astrocytes reach tens of thousands of synapses simultaneously (Bushong et al., 2002), the release of gliotransmitters may lead to the synchronization of neuronal firing (Angulo et al., 2004; Fellin et al., 2004). The different gliotransmitters that are released from astrocytes have quite distinct functions. Glutamate, the first identified gliotransmitter (Parpura et al., 1994), is able to modulate neuronal excitability (Angulo et al., 2004; Fellin et al., 2004; Tian et al., 2005) through actions on N-methyl-d-aspartate (NMDA) receptors, and can also modulate synaptic transmission (Fiacco & McCarthy, 2004). D-serine is a coagonist of the NMDA receptor. Released D-serine can bind to the glycine-binding site of NMDA receptors and, as a consequence, enhance neuronal NMDA receptor function. In the hypothalamus, the amount of astrocyte-derived D-serine supplied to synapses can regulate forms of synaptic plasticity. In conditions where too little of the coagonist is supplied, long-term synaptic depression can result, whereas when D-serine is released, long-term potentiation is induced (Panatier et al., 2006). Release of ATP from astrocytes can have a several effects. In cultures, release of ATP mediates the propagation of Ca2+ waves through paracrine actions on neighbors, inducing further Ca2+ signals and ATP release (Guthrie et al., 1999). In more intact preparations, because of the presence of a plethora of extracellular ectonucleotidases, newly ATP released is rapidly hydrolyzed to adenosine. As a consequence, ATP released from astrocytes leads to synaptic modulation mediated by adenosine (Pascual et al., 2005; Serrano et al., 2006). In the hippocampus, high-frequency activity of groups of synapses induces Ca2+ signals in neighboring astrocytes, which then release ATP and, after hydrolysis to adenosine, cause presynaptic inhibition of neighboring synapses through A1 receptors (Pascual et al., 2005; Serrano et al., 2006). In this manner, astrocytes may coordinate the strength of synaptic signaling. However, we still await an understanding of the functional consequences of gliotransmission on neural signalling, i.e., on processes such as learning and memory and ultimately behavior. Rapid forms of neuron-glia interactions also seem to be involved in the regulation of local blood flow as demonstrated in cortical brain slices where neuronal stimulation led to glutamate release, activation of metabotropic glutamate receptors (mGluRs) in astrocytes, and regulation of the tone of vessels contacted by processes of the stimulated astrocyte (Zonta et al., 2003; Mulligan & MacVicar, 2004; Takano et al., 2006). Moreover, activity-dependent astrocytic Ca2+ increase led to K+ release through astrocytic BK channels and to vasodilatation of arteriolar smooth muscle cells in the brain (Filosa et al., 2006). Emerging evidence suggests that disturbances of these mechanisms are involved in the pathogenesis of epilepsy, as discussed below.
Several important aspects of neuron-glia interactions are not yet understood. Thus, as outlined above, recent studies corroborated the finding that astrocytes are heterogeneous with respect to antigen profiles and functional properties but it is still unclear which type(s) of astroglial cells are activated and are capable of releasing transmitters, which transmitters can be released by astrocytes, which mechanisms these cells use for the release, and whether the efficiency of neuron-glia signaling changes during development. Intriguingly, a recent report presented evidence that a subtype of cells with astroglial properties even receives direct synaptic input from glutamatergic and GABAergic neurons (Jabs et al., 2005). The physiological impact of this type of interaction remains to be clarified.