Astrocytes in kindling: relevance to epileptogenesis

EPILEPSY

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RESEARCH ELSEVIER

Epileps.~ Research 26 11'-~i6~ 1¢~3-175

Astrocytes in kindling: relevance to epileptogenesis Moshe Khurgel a., Gwen O. Ivy a.b " Department A,:latomy aml Cell Biology, Faculty ¢{fMedicim', Scarboro,.~h ('amlm~. Unitcr.~it', ~)" T~,ro~Jto, Scarborol~gh, Ont, MiC IA4. b Department t{fPsychology. Scarborough Campus. U~lirersity qfToron;o, Scarbolough. Ore. MIC IA4. Cam~da

Received 15 November 1995: accepted 26 February 1996

Abstract Astrogliosis is a prominent feature of epileptic ~bci, and may play a causal role in the development of seizures and the persistance of seizure disorders. We have studied morphological changes in astrocytes with respect to the evolution of seizures using the kindling model of epilepsy. Kindling-induced seizures result in a prominent hypertrophy of astrocytes that is accompanied by a reorganization of astrocytic cytoskeleton. The change in the morphology of astrocytes appears to be seizure-intensity dependent, occurs early in the kindling process, and persists for weeks following the last seizure. In addition to hypertrophy, we have observed an increase in proliferation of astrocytes in hippocampus, amygdala and pirifonn cortex, but no change in the expression of connexin-43 following kindling. Significantly, induction of a localized astrocyte hyoerlrophy prior to initiation of kindling does not result in seizures and does not facilitate kindling. Altogether these data suggest that 'gliosis' is an adaptive response to seizures. Keywords: Seizures; Gliosis: Vimentin; Gap junctions; bFGF: Proliferatkm

I. Introduction The possibility of a causal relationship between a disfunction of astrocytes and epileptogenesis has been raised occasionally throughout the history of the study of epilepsy. However, astrocytes have been traditionally viewed as passive 'support cells' in the mammalian brain, and as such, not actively involved in neurotransmission. Thus, apart from a few reports

* Corresponding author. Present address: Department of Neuroscience, University of Virginia, Health Sciences Center, P.O. Box 5148, MR4, Charlottesville, VA 22908, USA. Tel.: +1 (804) 924-9111; fax: +1 (804) 982-4380" e-mail: [email protected]

advancing an alternative point of view, the development of hyperactivity and hypersynchronicity characteristic of epileptic seizures have been usually thought to represent abnormalities in neuronal connectivity or intrinsic properties of neurons, rather than possible aberrations in the properties of astrocytes. In the past few years the role of astrocytes in neural activity has been receiving increasing attention. Recent findings suggest that certain characteristics of astrocytes are critical for the maintenance of normal neural activity and that a disruption of proper astrocytic function may result in, among other events, development of seizures. We, and several other research groups, have used the 'dndling model of epilepsy to study the nature

0920-12 ! 1/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI! S0920-1211(96)0005 I-4

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M. Khurgel, G.O. It')'~ Epilepsy Research 26(1996) 163-175

and the significance of some of the changes in astrccytes with respect to epileptogenesis. Kindlinginduced seizures have been shown to result in hypertrophy of astrocytes in conjunction with prominent cytoskeletal changes in these cells. These structural changes take place early in the development of kindling, intensify during the weeks following the last seizure and persist for long periods of time. The 'gliosis' is confined to the stimulated and seizurepropagating brain regions, such as the piriform cortex, amygdala, entorhinal cortex and certain sectors of the hippocampus. Regionally specific patterns of astrocyte hypertrophy can be evoked through kindling of different brain sites. In addition to the hypertrophy, there is a limited but significant proliferation of these cells. Reactive astrogliosis is a prominent morphological feature in seizure foci of human patients and has been observed in many animal models of seizures and of epilepsy. Traditionally, this phenomenon has been regarded as a response of astrocytes to neuronal degeneration, and as such, not directly relevant to the process of epileptogenesis. Using several histological approaches we have been able to demonstrate that certain changes in astrocytes can occur in the absence of neuronal degeneration; probably in response to signals associated with seizure activity. Since hypertrophied astrocytes often appear to constitute the only prominent structural change at the seizuregenerating sites, it is conceivable that these cells play a crucial role in the evolution of a seizure-prone state. In this paper we review selected studies on astrocytes with the focus on morphological changes in these cells, and discuss the possible relevance of these changes to epilepsy. We also present some of the recent findings from our work with the kindling model which may shed new light on the role of astrocytes in epileptogenesis.

2. General properties of astrocytes in a mature brain Astrocytes are possibly the most multifaceted and enigmatic cells in the mammalian brain. The extent of participation of astrocytes in neural activity only recently has began to be appreciated due to the

Fig. i. Spatial relationship between astrocytes and other neural elements in the gray matter. Activated vimentin * astrocytes in the A m y / P i t area of a kindled rat maintaiJi the normal positioning and some of the structural interactions of astrocytes among themselves, with neurons (N), and blood vessels. Vimentin immunohistochemistry, counter-stained with cresyl violet. Scale bar: approx. 15 pm.

rapidly accumulating knowledge about the properties of these cells and the inevitable realization that the comprehension of brain processes will depend on understanding the individual characteristics of and interactions between all cellular elements. Several classes of astrocytes are now recognized, based on cell morphology and localization in the brain. Among those° the 'typical' gray matter astrocytes occupy a seemingly strategic position. Their cell bodies are dispersed more or less evenly throughout most regions, while the multiple processes, which we propose to call 'stellae' (to reflect the star-shaped morphology of these cells and to differentiate these formations from neuronal dendrites and cellular processes of oligodendrocytes and microglia), stretch out to form multiple contacts with other neural elements (Fig. 1) [12,48]. The functional significance of these structural associations is not completely understood: the ensheathing of synapses and neuronal somas is thought to limit the spread of ions and neurotransmitters, the ensheathing of blood capillaries by astrocytic 'end feet' may be important for the maintenance of the blood-brain barrier, while the contacts between astrocytes themselves, which often contain gap junctions, may play a role in spatial redistribution of molecules and, ultimately, communication between these cells. Physiologically, astrocytes are thought to fulfil several crucial functions. Astrocytes have been hy-

M. Ktmrgel. G.O. h y/Epih'l~sy Research 26 f/996~/63. / 75

pothesized for a long time to play a sig~fificant role in ionic homeostasis, in particular the maintenance of proper extracetluar concentrations of K ~ [29,70]. The selective localization of glutamine synthetase [44] and possibly glutamate dehydrogenase [38] in astrocytes designates these cells as key players in the metabolism of glutamate, while the significant presence of monoamine oxidases in astrocytes implicates them in the metabolism of monoamine neurotransmitters. In additiom astrocytes have been proposed to be a significant source of trophic factors, the presence of which may influence neuronal function and survival [39]. Astrocytes have been also hypothesized to participate in the immune responses in the CNS, possibly through interactions with microglia [191. Altogether, the spatial positioning of astrocytes in the central nervous system and the metabolic interactions with other cell types suggest that astrocytes continuously 'sense' and perhaps even regulate the activity of other neural elements.

3. Reactive astrogliosis Undoubtedly, the most conspicuous property of astrocytes is their morphological transformation in response to virtually any type. of neural insult. This aspect of astrocytic function has been studied extensively (see [15,27,40,43,51,53] for recent reviews), but its functional significance is vague in most types of CNS perturbations. The most common reaction of astrocytes to injury in CNS is the striking hypertrophy and, occasionally, proliferation of these cells that is refe:~ed to by several synonymous terms such as 'reactive astrogliosis', 'reactive astrocytosis', 'fibril~ary gliosis' or simply 'gliosis'. The hypertrophy "involves an increase in the size of the soma as well as an increase in the length and width of astrocytic stellae, and is accompanied by a rapid reorganization of the cytoskeleton, with the hallmark accumulation of intermediate filaments (IFs). The principal protein, which constitutes IFs in astrocytes in mature organisms is glial fibrillary acidic protein (GFAP) [16]. Antibodies to GFAP are widely used to assess the 'reactive state' of astrocytes; an increase in the amount of this protein has been shown to coincide

165

with the hlcrease in immuporeactivity (GFAP-IR) [31,69]. While it has not been shown uneqtfivocaHy that this cytoskeletaB change always represents an actual hypertrophy of astrocytes, indirect evidence from many studies suggests that this is the case. Another IF protein, vimentin (Vim). which is present in several cell types of mesenchymal origin, is expressed in astrocytes early in development, but appears to be gradually replaced by GFAP during development [10,58]. In a normal adult brain, subtle amounts of vimentin can be detected immunocytochemically in white matter astrocytes as well as in the stellae of radial astrocytes in selective brain regions, while most gray matter astrocytes are not immunoreactive with antibodies to vimentin ~see [35]). Under certain pathological conditions astrocyles re-acquire immunoreactivity to vimentin [56,64], most likely through an upregulation of vimentin synthesis [47]. A considerable amount of knowledge exists on the composition and distribution of intermediate filaments, but their functional role in astrocytes and other cell types has been elusive. Intermediate filaments are known for their exceptional chemical stability: they seem to withstand harsh extracting procedures resulting in removal of all other cellular components, including other components of the cytoskeleton [4]. This stability suggests a role for IFs in providing structural rigidity to cells. Another distinctive characteristic of IFs is their molecular composition: they are made up of different, albeit highly homologous, cell type-specific proteins [63]. This cell type specificity may hold additional clues to the functional significance of IFs, since different cell t,,,,,~ have distinct structural requirements. GFAP is thought to provide structural stability to astrocytes and their processes [16]. The suppression of GFAP synthesis with an antisense construct has been shown to prevent the formation of stable astrocytic processes in vitro, while a re-introduction of a fully encoding GFAP cDNA into those cells resulted in re-expression of GFAP, formation of a filamentous network, and extention of processes in the presence of neurons [7]. Also, the slow turnover rate of GFAP seems consistent with a structural role (see [16]). On the other hand, very recent studies, utilizing mice with a 'knocked-out' Gfap gene, demonstrated that animals with cells devoid of GFAP de-

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M. Khurgel, G.O. h'y / Epilepsy Research 26 ( 19961 163-175

velop normally [20,54]. While the morphological data are still scant, the abundance and ultrastructural characteristics of astrocytes as well as their response to certain injuries, such as the needle-wound and scrapie infection, appeared normal [20,54]. There was no apparent overcompensatory expression of vimentin in astrocytes in GFAP- mice [54]. Mice lacking vimentin were also recently generated, with no apparent compensatory upregulation of other intermediate filament proteins [9]. Altogether, the continuing lack of knowledge about the function of intermediate filaments in normal cells precludes the understanding of the functional implications of certain cytoskeletal changes, such as the upregulation of GFAP and vimentin syntheses, in reactive astrocytes. At this point, the only certain conclusion to be drawn is that the morphological transformation signals an alteration in the functional state of astrocytes.

4. Astrocytes in epilepsy Studies of tissue fi'om patients with various forms of epilepsy and the experimental work on animals reveal that an apparent gliosis is almost always present in brain regions which exhibit epileptiforrn activity [45,61,72]. A number of studies in which an absence of observable changes in astrocytes has been reported [6,49] may have utilized inadequate methodology, resulting in negative findings. Since necrotic tissue is usually present in most preparations, hypertrophy of astrocytes has been traditionally viewed as the response of these cells to neuronal degeneration. However, based on his studies of the alumina model of epilepsy, Harris [26] proposed that astrocytes hypertrophy in response to hyperactivity of neurons as opposed to acute neuronal degeneration. Also, Meldrum et al. [46] described gliosis as the only pathological change in the hilus of baboons in which seizures were induced with pentylenetetrazol, while Norenberg and Chu [52] observed morphological alterations in astrocytes as the only structural change in the preictal stage in rats treated with aminophylline. The earliest mention which links morphological alterations in astrocytes and epilepsy is attributed to Chaslin (1889, cf. [45]). Later, Penfield [55] sug-

gested an active role for astrocytes in epileptogenesis by proposing that the 'scar' tissue, which surrounds the area of cortical damage and with reactive astrocytes as a prominent component, constitutes the structural basis of post-traumatic epilepsy through 'irritation' of neurons. The concept of physical interference of hypertrophied astrocytes with neuronal activity was also argued by Ward [71], who suggested that gliosis caused a mechanical deformation of dendrites, thereby resulting in enduring depolarizations, which in turn gave rise to high frequency neuronal discharge, possibly evoking seizure activity. Pollen and Trachtenberg [59] advanced an alternative hypothesis that the decisive factor in the development of post-traumatic focal epilepsy was a breakdown of the ability of astrocytes to control extracellular K + levels, whereby a build up in K + woulo result in hyperactivity of neurons. This hypothesis, while provocative, has riot received much experimental support [30,42]. Instead, morphologically modified astrocytes may directly contribute to the process of epileptogenesis through resulting changes in extraceilular space or changes in the metabolic properties of these cells. Indeed, astrocytes at the epileptogenic foci have been shown to present an altered enzymatic profile. Du et al. [13] recently have presented evidence that reactive astrocytes in the stimulation-induced status epilepticus model display elevated immunoreactivity with antibodies to kynurenine aminotrasferase and other enzymes. Similarly, Tiffany-Castiglioni et al. [67] have shown an increase in glutamine synthetase activity following FeCl2/pentylenetetrazol-induced seizures. There may also be alterations in the levels of carbonic anhydrase, which is thought to be vital for the propea" anion and acid-base balance in the brain [23].

5. Astrocytes in kindling The kindling model of epilepsy is particularly well suited to study the involvement of astrocytes in epilepsy because (1) the development of seizures is subject to experimental control, and therefore allows evaluation of changes in astrocytes at different stages of epileptogenesis, (2) this model is widely accepted as a valuable tool for testing epileptogenesis follow-

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ing experimemal interventions, and thereR~re allows evaluation o f strategies designed to directly tes~ the significance o f gliosis in epilepsy. Brotchi and colleagues [61 were the first to use the kindling model to address the involvement of astrocytes in epileptogenesis. Previous work by that group has shown that epileptic loci contain a distinct subtype of reactive astrocytes, which has been referred to as "activated' astrocytes [5]. Activated astrocytes apparently contain elevated levels of certain dehy-

167

drogenases, bu¢ ~d~erwise are indistinguishable flom "reactive + celts, ~he +ader +'+ + reactin~ ~o a mechanical trauma of CNS, when examined with either CajaFs gold-suNima~e method or amiserum to G F A P [14]. (+o~sequen~Iy, activmi~>.+~ of astrocy~es was proposed to be closely rela~ed +to the development of an epileptogenic focus: however, no e n z y m a t i c a l y or morphologicalb' activated asIrocytes were found in the brains of kindled rats [6]. |rl contrast, we observed a striking hypertrophy of astrocytes in the kindling model •

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Fig. 2, Kindling causes changes in the composition of the astrocytic cytoskeleton, hnmunoreactivity to astrocytic cytoskeletal pl'oteiils ill conlralateral P i t / A m y ( A - D ) and in hippocampus (E-F), (A) astrocytes are only lightly immunostained with anti-GFAP in a control animal as compared to GFAP + astrocytes in a kindled animal, (B) (arrows point to examples), (C) only blood vessels are immunoreactive to anti-Vim in a control animal. Note the complete absence of Vim + astrocytes, (D) in a kindled animal, Vim + astrocytes are abundant (example is at'rowed), (E) a few Vim + radial glia-like processes (arrows) +.'anbe seen traversing the ventral leaf of dentate gyrus in a control animal, (F) in a kindled animal, many mote of these processes (long am>w) can be seen throughout the granule cell layer, in addition to the many Vim + astrocytes (short arrow) in the hilus. (E-F) lightly counterstained with cresyl violet to demonstrate the granule cell layer. Scale bar: 50 p,m for ( A - F ) (from Khurgel et al. [34], with permission),

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M. Khurgel, G.O. It'y / Epilepsy Research 26 f 1996) 163-17.5

immunocytochemical results reflect a true hypertrophy of these cells. Morphological changes in astrocytes can be observed in the early stages of epileptogenesis and may be seizure-intensity dependent, with a higher intensity of Vim-lR and a more widespread activation of astrocytes occurring in filly kindled animals, compared to p,-,viz,!iy kindled ones (i.e. those with only stage 1 or 2 seizurcs~ [36]. Howcver, in some cases, changes in GFAP are similar in partially a,,,,! in fidJy kindled anm,als [24,37]. Levels of GFAP are stil! significantly elevated between l and 2 weeks [25,68], and up to a month [34] following the last seizure, but seem to subside by two months following the completion of kindling in most areas [25]. The long-term persistence of Vim-IR has not been addressed yet. Regional variation in activation of astrocytes likely reflects differences in the spread of seizures due to the choice of a particular kindling paradigm. In most experiments, astrocytic reaction occurs bilaterally [24,34,35], but activation confined to the stimulated side only has also been observed [37,65] (see Fig. 3). Astrocytes in Amy and Pir are activated in most preparations: this phenomenon has been observed in amygdala kindled rats in our laboratory [34,35,37,60], in hippocampus kindled rats [24] and, recently, in septum kindled rats [36], whereas activation of astro-

of epilepsy. Kindling-induced seizures produced long lasting structural changes in astrocytes within brain regions that participate in kindling-evoked seizures, most notably hippocampus (Hipp), piriform cortex (Pir) and amygdaloid nuclei (Amy) [34-37,60]. In addition to the increase in GFAP-IR (Fig. 2a and b" Fig. 3), a strong de novo immunoreactivity to vimentin (Vim-!R) was found [35] (Fig. 2c-f). While vimentin might be present under certain conditions in activated microglia [21], in kindled brains the newly expressed vimentin was localized to astrocytes: all Vim + cells were also GFAP + [35]. Also, double-labelling with lectin revealed no Vim + microglia, in agreement with our finding that microglia were not morphologically activated in kindling [37]. The fact that activated astrocytes in an epileptogenic focus re-express vimentin is potentially significant for the pathological assessment of gliosis in epileptic patients. Since traditional methods of evaluating gliosis are usually not satisfactory [45], de novo expression of vimentin in astrocytes may prove helpful in differentiating between reactive and otherwise apparently hypertrophied astrocytes. The increase in GFAP-IR in kindling is accompanied by increases in GFAP mRNA [65] and total GFAP [24,68], as well as an increase in the volume fraction of astrocytic stellae [28], suggesting that the % increace GFAP-IR

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Fig. 3. The effect of kindling on GFAP-immunoreactivity (IR). An increase in GFAP-IR (expressed as percent difference in GFAP-IR [optical density units] between kindled animals and unstimulated controls) is evident in all measured regions, and is significant, ( ' ) p < 9.05, ( * * ) p < 0.01, on the ~timulated side of kindled animals in pirife~ cortex (PIR), ent'arhinai c~rtex (FNT), has,medial amygdaloid nucleus (AMY), hilar region of hinpoeampus (H!L) and molecular layers of dentate gyms (DG) (from Khurgel et al. [36] with permission).

M. Ktmrgel. G.O. h 3' / Epilepsy Re~ear~'h 26 ~1996 ~

cytes in other brain regions may depend on the placement of the stimulating electrode. Not all proteins are upregulated in astrocytes in kindling: Hanson etal. [24] have observed a decrease in the levels of S l 0 0 in Pit and Amy, and no change in the l o r d s of glutamine synthetase 24 h following hippocampal kindling, while Tiffany-Castiglioni et al. [67] have reported a significant decrease in glutamine synthetase activity in the ipsilateral neocortex 4 weeks following amygdala k i n d l i n g . It is important to stress that the changes in astrocytes described above have been observed in the absence o f overt neuronal degeneration [28,37,65]. [n addition, we have been able to demonstrate that activation of astrocytes in kindting occurs in the absence of microglial activation, which otherwise would be indicative of neural trauma [37].

Based on their finding of unchanged levels of glutamine synthetase following kindling, Hanson et

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a!. [24] concluded that a change in the number of as~rocytes is "'-~: '~ . . . . a_i. . .~ . .c .~. .~tudy by ~l J'a' -J -o j u ~ H ~ l q e" ~ "" ,v . ~-nsv,, et at. [ i l l failed ~o uncover significant differences in the number of G F A P " profiles in selective areas of the hippocampus in kindled rats. We used a direct test of cell proliferation by injecting the animMs with bromodeoxyuridine (BrdU), an analog of thymidine [22], 7 days after completion of amygadMa kindling and 2 h prior to eulhanasia. Frozen brain sections were reacted with anti-BrdU (1:206~ Boehringer Mannheim), antiGFAP or anti-Vim [35] or processed for lee,in his,ochemistry [37,66]; several double-labelling combinations were also performed. Some of the resul,s are shown in Fig. 4. Compared to the low levels of cell proliferation in normal brains (Fig. 4a and c), there was a dramatic increase in the number of proliferating cells following kindlh~g, mainly in the P i r / A m y region (Fig. 4b) and in Hipp (Fig 4d). It should be noted that since the animals were 'pulsed' with BrdU on one occasion only, the current evidence of kindling-induced cell proliferation is probably an underestimation of the

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Fig. 4. Kindling induces cell proliferation• Occasional proliferating cells (arrows) are labeled with BrdU in the Amy/Pir region CA) or hippocampal hilus (C) of unstimulatedcontrol animals. Following kindhng, there is all increase in the number of BrdU+ cells in Amy/Pir (B} and hippocampus(D) and other brain regions• Note the large increase in the number of proliferatingcells in the subgranularlayer of the hilus between control rats Cstraight arrows in (C) and kindled rats (D). Many cell pairs Ccurvedarrow in CC))can be seen in the Amy/Pir and hilus of kindled rats, suggesting that BrdU+ cells are indeed proliferatingcells. Open arrow in CA) and CB) outlines the pyramidalcell layer of the piriform cortex (pir). Scale bars: approx. 120 tzm for (A), CB); 60 v,m for (C), (D).

170

M. Khurgel. G.O. h'y / Epih,psy Research 26 (1996) 163-17.5

number of new cells. Some of the BrdU + cells were also GFAP + or Vim +, but none were lectin +, while others could not be identified with the methods used here (Khurgel et al., in preparatiot~). It is conceivable that in addition to cell division in astrocytes, seizure-associated factors induce proliferation of precursor cells which later differentiate into astrocytes. On the other hand, the dramatic increase in the number of BrdU + cells in the subgranular layers of dentate gyrus (Fig. 4d), may be indicative of an increase in proliferation of granule ceils, which continue dividing in low numbers in adult rats [32]. The latter is a plausible explanation, especially since we have previously reported an increase in Vim + radial gila-like processes in DG foiiowing kindling [35] (Fig. 2f). Incorporation of BrdU into DNA may be indicative of an extensive DNA repair in cells presumably damaged by the seizures in addition to (or instead of) DNA replication/cell division. This possibility remains to be explored. However, the presence of adjacent BrdU + 'cell pairs" (curved arrow, Fig. 4c) in normal and kindled animals suggests that BrdU + cells are indeed proliferating cells. Altogether, these findings show that kindling-induced seizures result in increased cell division and present an intriguing possibility that proliferation of astrocytes can be upregulated with elevated neural activity, in the absence of extensive neural damage.

increased coupling between these cells. An augmentation in coupling between astrocytes could contribute to epileptogenesis by participating in hypersynchronization of neuronal firing. This argument is

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7. Expression of connexin-43 Changes in the astrocytic network, partially represented by astrocyte hypertrophy and proliferation, potentially have important implications for epileptogenesis. A related significant change could be an alteration in coupling between astrocytes. Increased numbers of cellular connections, both desmosomes and gap junctions, between astrocytes seem to be present in alumina-induced epileptic foci [26]. Naus et al. [50] have shown an increase in the expression of connexin-43 (Cx43), the main protein in astrocytie gap junctions, in tissue excised from epileptic patients. It has been argued that astrocytes cultured from such tissue exhibit repeated oscillations in response to a pulse of glutamate [8], suggesting an increase in excitability of, and perhaps an

Fig. 5. In situ hybridization with probes to connexin-43 mRNA shows no discernable differences between control (A) and kindled animals (B). In contrast, intense KA-induced seizure activity results in absence of hybridization in brain regions which also display intense gliosis (arrows in (C)). In amygdala, there is a depletion of GFAP + cells in the area marked by asterisk (C), suggestive of astrocytic degeneration, which would explain absence of Cx43 mRNA. However, adjacent areas, with reactive astrocytes surrounding the lesion, also show absence of hybridization. Scale bar: 1.2 mm for (A-C).

M. Khu rgel, G. 0 h 3/EpikTzS3' Re.search 26 ~t 996 ~ 163-175

supported by the data from a recent study in wt:ich astrocyteso isolated from hyperexcitable tissue, have been shown to display a fast f~uorescence recovery after photobleach, and therefi~re thought to be exle~sively coupled [41 ]. We and our colleagues have conducted a pilot study aimed at assessing the expression of connexin43 in kindling. Frozen brain sections from kindled rats were processed for in situ hybridization using Cx43 cRNA probes as described elsewhere [2]. The sections from kindled animals were compared to those from implanted, but unstimulated control and a positive control, in which I h of status epilepricus was induced with systemically-administered kainic acid (KA). The examples of respective patterns of hybridization are presented in Fig. 5. No aifferences in Cx43 mRNA distribution were apparent between the unstimulated control and kindled animals. However, a clear depletion in Cx43 mRNA was observed in areas ot i nfense gliosis ip the KA-treated animal. Specifically, regions where a marked neuronal degeneration is known to take place, such as midline thalamic nuclei, nucleus reuniens, hippocampal areas CAI and CA3, neocortex, and the A m y / P i t area all show an absence of hybridized probe (arrows, Fig. 5c). Since a prolound astrocyte hypertrophy is present in these areas, the lack of hybridization suggests a shut-down of Cx43 synthesis in reactive astrocytes in the KA model. At the same time, the absence of a significant difference in hybridization between control and kindled animals suggests that kindlingactivated astrocytes do not significantly upregu|ate or downregulate their expression of Cx43. It is possible, of course, that the method employed here is not sensitive enough to detect subtle changes in levels of this mRNA. Thus, additional work is required for precise quantification of potential changes in expression of Cx43. It is also conceivable that there are changes in physiological coupling between astrocytes independent of levels of Cx43. This possibility awaits experimental confirmation.

8. Recent advances The evidence is accumulating that astrocytes undergo a variety of changes during or following kindling-mediated epileptogenesis (see Table 1.)

t 71

Table I Summary of cha~lges m aslrocy'les il~ kindbng Observed change

Ref.

Prolifcralion of astrocyles ?

I Khurgel and I',y}

GFAP-IR ~

[I 1,35,37]

Levels of GFAP ~ Levels of GFAP mRNA ~ De novo Vim-|R Hypertrophy of aslrocytes Cx43 mRNA n.c. GS n.c./11 SI {H} 11 Glial muscarinic recept. ~1

[24,25,68] [65.68] {35.37] [28] (Khurgel and ivy) [24,67] [24] [i}

no. No observed change. GS Glulamirm synthetase.

What, then, is the significance of these changes with respect to development of seizures? Is it possible that some of these structural changes reflect a functional transformation of astrocytes, which in turn leads to inadequate control of the extracellular environment, similar to the deficiency in potassium buffering or other metabolic abnormalities proposed for 'glial scar' astrocytes? Recently, we have been attempting to address these questions using novel experimental approaches. We ha,'-, decided to evaluate the significance of altered astrocytk morphology hi epileptogenesis either by activating astrocytes or by selectively removing these cells from the seizure-initiating site prior to commencement of kindling. Seizures have been shown to result in increased levels of various growth factors in the brain regions conveying the seizure activity [3,17,! 8,62]. Among the upregulated proteins is the basic fibroblast growth factor I.bFGF) [ 18,62], which has been shown to be a potent mitogen and stellation factor for astrocytes [57]. It is conceivable that, among its other functions, bFGF serves as a signal for astrocyte hypertrophy following perturbations of CNS. We have injected bFGF (Sigma; 0.25 /xg in 5 /xl artificial cerebrospinal fluid) into Amy of naive rats in an attempt to mimic the seizure- or trauma-mediated induction of gliosis. Injection of bFGF results in increased GFAP-IR and Vim-IR, and apparent hypertrophy of astrocytes throughout the Amy nuclei as early as 48 h post injection (Fig, 6a and b). The

172

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intensity of astroglial reaction is similar to the one observed in kindling. We are currently investigating the effects of this treatment on development of seizures. The results obtained so far show that the animals, treated with bFGF, do not display any overt behavioral seizures. Also, monitoring of animals implanted with recording electrodes following similar injections of bFGF reveals an absence of epileptiform activity (Khurgel et al., in preparation). The properties of bFGF-activated astrocytes with respect to the 'type' of induced gliosis remain to be determined. Nevertheless, these preliminary data argue against the hypothesis that gliosis is in itself epileptogenic. Another promising approach that we are pursuing is the examination of neuronal function and development of seizures in the absence of astrocytes. We have recently characterized in vivo the selective astroglial toxicity of a-aminoadipate [33]. Iajections of (~-aminoadipate into Amy of rats result in selective ablation of astrocytes, without affecting neuronal density [33] (Fig. 6c and d). The consequences of

this treatment on the development of seizures are also currently under investigation.

9. Summary and conclusions The role of astrocytes in epilepsy has been a long standing, albeit not very prominent, line of inquiry in the field of epileptology. The kindling model, which is widely used to explore various aspects of seizure development, has been utilized to discover extensive changes in astrocytes during epileptogenesis. The proliferation, hypertrophy and cytoskeletal changes in astrocytes in kindling are reminiscent of the classical 'gliosis', observed in post-traumatic epilepsy and in long-term epilepsies with various aetiologies. However, there may exist e~sential differences between those phenomena. In particular, there is a potential diversity in metabolic coupling and enzymatic profiles of reactive astrocytes in different perturbations of the CNS, which remain to be explored. Circumstantial evidence links the changes in astrocytes to the development of an epileptic state, but

M. Khurget, G.O. b'y / EpihT~sy Research 26 ( t 990) t63- / 75

direct tests of the contribt|~ion of astrocytes to epfleptogenesis have been lacking until recently. New findings suggest that the presence of reactive astrocytes is not necessarily epileptogenic. The research in progress may yield additional answers with respect to the invoDement of these ceils in seizure disorders.

Acknowledgements The authors would like to thank S.C. Barsoum and A.C. Koo for their assistance with some of the above studies, Drs. D.J. Belliveau and C.C. Naus for performing the Cx43 mRNA assays, and Drs, R.J. Racine and N.W. Milgram for their invaluable contributions to this research. We also thank Drs. N.W. Milgram and O. Steward for discussions and helpful comments on this manuscript. Much of the above work has been supported by NSERC grants to G.O.L and, in part, by the Epilepsy Canada 1995 Canadian National Studentship award to M.K. The presentation of this work at WONOEP III was generously supported in part by Ciba-Geigy Ltd, Canada, the Optimist Club of Ajax, Ontario, and a travel grant from the Department of Anatomy and Cell Biology. University of Toronto to M.K.

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