Epilepsy or the occurrence of recurrent seizures is a behavioural syndrome, that is, a component of several neurological conditions. Several brain foci associated with seizure generation are populated by increased numbers of astrocytes. Such foci include hippocampal seizure foci in temporal lobe epilepsy, several types of mass lesions in the brain (low-grade astrocytomas, oligodendrogliomas and arteriovenous malformations) and tuberous sclerosis. Gliotic scar formation is a prominent feature of human epilepsy (Foerster and Penfield, 1930
; Penfield and Humphreys, 1940
). What is the role of astrocytes in epileptogenesis in these neurological conditions?
Epileptogenesis literally means the beginning of epilepsy or spontaneous recurrent seizures. Do astrocytes play a role in the mechanisms necessary for spontaneous seizures to first appear or do astrocytes play a role in seizure maintenance in the chronic stages of epilepsy? There is some evidence to support a primary role of astrocytes in seizure generation, particularly in animal models. In the EL (epileptic) mouse the hippocampus is important for the generation of behavioural seizures. However, there is no neuronal injury or loss in this model but an increase in expression of astrocytes around the age when seizures appear (Drage et al., 2002
), and these astrocytes have reduced glutamate transporters, suggesting a primary role for astrocytes, perhaps through defective glutamate clearance at the seizure focus (Ingram et al., 2001
). More direct evidence is found mice in which there is astrocyte-specific inactivation of the Tsc1 gene (Tsc1 cKO mice). In these mice, there is an increase in proliferating GFAP-immunoreactive astrocytes throughout the brain (Uhlmann et al., 2002
), with the most distinctive histological alterations seen in the hippocampus. What is significant is that these astrocyte-specific cKO mutant mice did not show cortical tubers or defects in neocortical lamination. These mice have electrographically confirmed seizures shortly after astrocyte proliferation has begun (Uhlmann et al., 2002
). Further, the astrocytes in Tsc1 cKO exhibit decreased expression of the glutamate transporters GLT-1 and GLAST and a functional decrease in glutamate transport current in astrocytes in hippocampal slices and astrocyte cultures (Wong et al., 2003
). Such changes in glutamate transporters may lead to the extracellular accumulation of glutamate, which could cause hyperexcitability of neurons and seizures. Cultured Tsc1-deficient astrocytes and hippocampal slices from cKO mice also exhibited reduced Kir currents and decreased expression of specific Kir
channel protein subunits Kir2.1 and Kir6.1. Thus impaired extracellular K+
uptake by astrocytes may also contribute to neuronal hyperexcitability and epileptogenesis in this Tsc1 cKO mouse model (Jansen et al., 2005
Much of our understanding of the role of glia in human epilepsy is obtained from the study of seizure foci surgically removed for the control of medically intractable seizures (de Lanerolle et al., 2010
). The one limitation in studying human tissue that it is taken from patients is that they have had seizures for a considerable period (6–20 years) prior to surgery. Thus it is more difficult to draw conclusions from this patient group on the role of astrocytes in the early (acute) stages of epileptogenesis.
The seizure focus that has received the most study is the hippocampus from patients with medically intractable temporal lobe epilepsy. The examination of such hippocampi indicates that approximately 40–65% of these hippocampi have hippocampal sclerosis. Eighty percent of these sclerotic patients have an excellent surgical outcome. The sclerotic hippocampi have a very high density of astrocytes and these astrocytes have many distinctive properties compared with astrocytes from non-sclerotic hippocampi. Differences in these astrocytes are seen in their cell membrane properties – they show increased expression of the glutamate receptors mGluR2/3 (metabotropic glutamate receptor 2/3); mGluR4, mGluR8 and GluR1 receptors that have an elevated ratio of flip-to-flop mRNA splice variants (Seifert et al., 2002
). The expression of membrane transporter molecules is also altered. Prominent among these are aquaporin 4 molecules, where their polarity of distribution on the astrocytes is altered with reduced expression on the perivascular end feet and unchanged on the membrane facing the neuropil. The GABA (γ-aminobutyric acid) transporter GAT-3 (GABA transporter 3) expression is increased on protoplasmic astrocytes in regions of relative neuronal sparing such as dentate gyrus and hilus. There is some disagreement in the literature as to whether the glutamate transporters EAAT1 (excitatory amino acid transporter 1) and EAAT2 are also reduced. The membrane Na+
channels and α1C subunit of the calcium ion channels are also up-regulated, suggesting that astrocytes in sclerotic hippocampi have a significant change in their membrane current characteristics. The inwardly rectifying potassium ion (Kir
4.1) channels are also shown to be impaired, significantly impeding removal of K+
ions from the extracellular space (Bordey and Sontheimer, 1998
; Hinterkeuser et al., 2000
; Schroder et al., 2000
). Among the astrocyte specific enzymes, glutamine synthetase activity is reduced (Eid et al., 2004
), impeding glutamate clearance and thus leading to increases in extracellular glutamate levels (During and Spencer, 1993
). There is also a reduced capacity for glutamine synthesis and ammonia detoxification. Levels of other astrocyte specific enzymes such as GDH (glutamate dehydrogenase), aspartate aminotransferase and lactate dehydrogenase (Malthankar-Phatak et al., 2006
) are also altered in astrocytes in sclerotic hippocampi.
Gene expression studies in sclerotic hippocampi have also suggested changes in the expression of several genes associated with astrocytes. Among those up-regulated are those involved with immune and inflammatory functions, including several chemokines and cytokines, class II MHC antigen genes and interleukins and complement factors (Aronica et al., 2007
; Lee et al., 2007
). Several molecules associated with the astrocyte/microvascular interface are also altered, in particular increases in EPO-r (EPO receptor), the MDR1 (multidrug resistance gene-1) encoded P-glycoprotein (Tishler et al., 1995
), CD44 and plectin 1 (Lee et al., 2007
), among others (de Lanerolle et al., 2010
Associated with the above molecular anatomical changes in astrocytes in sclerotic hippocampi are also changes in their function. Some astrocytes in primary cultures derived from sclerotic hippocampi and in vitro
hippocampal slices are capable of generating action potential-like responses in response to depolarizing currents (Bordey and Sontheimer, 1998
; O'Connor et al., 1998
). Astrocytes from sclerotic hippocampi respond to glutamate with elevated intracellular Ca2+
release and Ca2+
oscillations and waves (Lee et al., 1995
). Additionally, several lines of evidence suggest that the altered properties of sclerotic astrocytes, particularly down-regulation of glutamine synthetase, also alter glutamine–glutamate cycling in hippocampal seizure foci resulting in increased extracellular glutamate levels before and during seizures ().
Expression of glutamine synthetase immunoreactivity in a non-sclerotic and sclerotic hippocampus
More recent studies suggest the recognition of two functional classes of astrocytes. One type, sometimes referred to as GluR cells, is weakly positive for GFAP, expresses AMPA-type glutamate receptors and properties akin to NG2 cells. These cells are excitable (O'Connor et al., 1998
). A second type of cell, the GluT cell, is more strongly GFAP positive, is more fibrous in appearance and expresses K+
channels, but lack glutamate receptors (Matthias et al., 2003
). Cells similar to GluR and GluT cells have been recognized in the human hippocampus, and though both types are found in normal hippocampi, an almost complete loss GluT cells is reported in sclerotic hippocampi (Hinterkeuser et al., 2000
). It is most likely that it is these cells that have impaired Kir
channels. Further, the GluR cells in sclerotic hippocampus have increased levels of the Flip isoform of GluR1 receptor, suggesting an increased potential for excitability.
What role do astrocytes play in a mature hippocampal seizure focus? As the above review suggests, they may play several roles. (i) Astrocytes may contribute to the high glutamate levels at seizure foci through defective glutamate clearance, and additionally active release of glutamate from GluR (NG2)-like cells due to enhanced intracellular Ca2+
release or by astrocyte swelling due to reduced aquaporin 4 transporters on perivascular end feet (de Lanerolle et al., 2010
). These elevated glutamate levels may activate neurons in surrounding or adjacent undamaged regions such as the subiculum to generate seizure activity. (ii) Defective astrocytes may contribute increased extracellular potassium in the seizure focus. Impaired inwardly rectifying K+
channels and decreased water flux due to reduced AQP transporters in astrocytic end feet may be contributory factors. (iii) The presence of excitable GluR or NG2-like cells with more glutamate-sensitive GluR1 receptors in the sclerotic seizure focus may directly contribute to an excitable focus. (iv) Astrocytes may also modulate the microvasculature, leading to vascular permeability and promoting entry of substances such as albumin or circulating leucocytes into the brain parenchyma with consequent seizure promoting effects (de Lanerolle et al., 2010
). (v) The release of inflammatory and immune factors by astrocytes may also contribute to the development of the seizure focus in ways that are only just beginning to be understood.
Another interesting aspect of epilepsy is the destruction and loss of astrocytic domain organization (). Several groups have shown that in the normal brain cortical and hippocampal astrocytes are organized in non-overlapping spatial domains with limited interdigitation of processes of adjacent cells (Bushong et al., 2002
; Ogata and Kosaka, 2002
; Halassa et al., 2007a
). Through a process termed ‘tiling,’ astrocytic processes grow within exclusive territories during development when neuronal and vascular territories are also being established. In the rodent brain, one astrocytic domain encompasses ~100000 synapses, whereas this number rises to 2000000 synapses in the brain of homo sapiens (Bushong et al., 2002
; Oberheim et al., 2006
). Each domain represents an area of the neuropil that is under control of a single astrocyte, being also an entity of synaptic modulation that is independent of neural networking. All synapses within one territory will be contacted by processes from only one single astrocyte. Reactive astrocytes in three very different murine models of epilepsy (post-traumatic injury, genetic susceptibility and systemic kainate exposure) all were associated with a 10-15-fold increase in overlap of processes of neighbouring astrocytes (Oberheim et al., 2008
). A similar loss of astrocytic domain organization was noted in tissue surgically resected from patients resilient to medical treatment. It is important to note that astroglial domain organization was preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a striking up-regulation in GFAP expression. Thus, while the functional consequences of loss of astrocytic territories have not been established, it appears to be specifically linked to epilepsy. It is tempting to speculate that synapses receiving input from more than one astrocyte may not function optimally.
Organization of reactive astrocytes in a model of post-traumatic epilepsy induced by cortical injection of a ferrous chloride solution
Are these changes in astrocytes in the hippocampal seizure focus secondary mechanisms in seizure development or are they causative? Comparison of these observations with animal studies discussed above, where astrocytes appear to have a more primary role in epilepsy, show that at least some of the astrocytic changes in the human focus may be causative, in particular impairment of Kir channels and decreased expression of astrocytic glutamate transporters.