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Might astrocytes play a role in maintaining the seizure-prone state?
Brain Research 1044 (2005) 190 – 196 www.elsevier.com/locate/brainres
Research report
Might astrocytes play a role in maintaining the seizure-prone state? Mani Vessala,b,c,f,T, Chandrasagar B. Duganid,e,1, Dianand A. Solomonf, W. McIntyre Burnhamc,e, Gwen O. Ivyc,f,g a
Institute of Medical Science, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 b Program in Neuroscience, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 c Bloorview Epilepsy Research Program, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 d Department of Biochemistry, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 e Department of Pharmacology, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 f Centre for the Neurobiology of Stress, University of Toronto at Scarborough, Scarborough, ON, Canada M1C 1A4 g Department of Psychology, University of Toronto at Scarborough, Scarborough, ON, Canada M1C 1A4 Accepted 23 February 2005 Available online 19 April 2005
Abstract The amygdala-kindling model is used to study complex partial epilepsy with secondary generalization. The present study was designed to (A) quantify astrocytic changes in the piriform cortex of amygdala-kindled subjects over time and (B) investigate the role that astrocytes might play in maintaining the seizure-prone state. In Study A, once the experimental subjects reached five stage 5 seizures, stimulation was stopped, and both kindled and control rats were allowed to survive for the interval appropriate to their group (7, 18, 30, or 90 days). Following each interval, the kindled and control animals were given 10 intraperitoneal injections of bromodeoxyuridine (BrdU) and sacrificed 24 h following the last injection. Significantly higher numbers of dividing astrocytes (identified by co-labeling for BrdU and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) were found in the kindled brains. All kindled groups had significantly higher numbers of double-labeled cells on the side contralateral to the stimulation site, except for those in the 90 day survival group. In Study B, rats were implanted with chemotrodes, were kindled as in Study A, and were subsequently infused with either saline or with La-AA (to lesion astrocytes) during a further 25 stimulations (1/day). La-AA infused rats had significantly diminished levels of behavioral seizures, higher after discharge thresholds, lower after discharge durations, and decreased numbers of double-labeled astrocytes in piriform cortex than did saline infused rats. Together, the data indicate that astrocytes may play a role in maintaining the seizure-prone state. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal studies Keywords: Epilepsy; Piriform cortex; Amygdala-kindling; Astrocyte proliferation; GFAP; Vimentin; La-aminoadipate
1. Introduction
T Corresponding author. Department of Comparative Medicine, Stanford University School of Medicine, Stanford Brain Research Institute, 300, Pasteur Drive, Edwards Building, Room R342, Stanford, CA 94305-5342, USA. Fax: +1 650 498 5085. E-mail address: [email protected] (M. Vessal). 1 Present address: The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.058
Epilepsy is the most common primary disorder of the brain occurring in about 1% of the population [4]. Epileptic seizures are divided into two major categories: partial and generalized. Partial seizures are localized to a particular area in the brain. They can be simple, without the loss of consciousness, or complex, with a loss of consciousness. Partial seizures can also be secondarily generalized, a
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condition in which seizures spread from a defined focus to other regions of the brain. Generalized seizures, on the other hand, have no defined focus and involve the entire brain [19]. The amygdala-kindling model is an animal model of complex partial seizures with secondary generalizations [1]. This model of temporal lobe epilepsy involves the electrical stimulation of a defined region of the temporal lobe via chronically implanted electrodes and the severity of the resulting seizures may be quantified by EEG recording or behavioral analysis [20,21]. Astrocytes react to various types of injury and stress with rapid cell growth and division [3]. The most common reaction of astrocytes to injury in the CNS is the striking hypertrophy as observed by an increase in the amount of immunoreactivity of intermediate filament proteins, glial fibrillary acidic protein (GFAP), and vimentin [16,25]. Such astrocytic activation has also been observed in the piriform cortex (PC) of amygdala-kindled brains [13,14]. We have been investigating the response of astrocytes to kindling in an attempt to understand their possible involvement in epileptogenesis [14]. In addition to astrocytic hypertrophy, we have recently reported astrocytic proliferation in the PC of amygdala-kindled rats [24]. In that study, we observed a significantly higher number of dividing astrocytes (identified by co-labeling with antibodies to bromodeoxyuridine (BrdU) and to one of the astrocytic intermediate filament proteins, GFAP, for labeling mature astrocytes or vimentin, for labeling immature astrocytes) in both partially kindled (one stage 1) and fully kindled (five stage 5 seizures) brains. Given the robust proliferation response of the astrocytes to full kindling, we decided to investigate further a role that they might play in maintaining this epileptic state by first (Study A) quantifying their proliferation in the piriform cortex ipsilateral vs. contralateral to the kindling site and second (Study B), by ablating them at the kindling site itself using the astrotoxin La-aminoadipate (La-AA). In Study A, we thus compared astrocyte proliferation in kindled (five stage 5 seizures; [21]) rat brains that subsequently had different survival periods (Sp): 7 days (Sp7), 18 days (Sp18), 30 days (Sp30), or 90 days (Sp90). Regions anatomically linked to the site of kindling on both the ipsilateral- and contralateral PC were examined for the relative distribution of proliferating astrocytes. In Study B, we took rats that had been fully kindled via a chemotrode (which consists of a combined electrode and cannula) and subsequently infused them with either saline or the astroglial specific toxin La-amino adipic acid (La-AA) while giving daily amygdala stimulations as before, over 25 days. During these 25 days, we recorded behavioral seizure levels, measured after-discharge thresholds and after-discharge durations, and examined astrocyte proliferation in piriform cortex in order to determine if lesions of astrocytes at the stimulation site affect the behavioral, physiological, or anatomical correlates of the seizure-prone state.
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2. Materials and methods 2.1. Subjects Adult, Long-Evans male rats (n = 68, postnatal day 60, 225–250 g) were obtained from Charles River Laboratories, Saint-Constaint, QC, Canada and housed as previously described [24]. All research protocols were approved by the Animal Care Committee (Faculty of Medicine) of the University of Toronto, Toronto, Canada. 2.2. Implantation of electrodes and chemotrodes One week after arrival from the breeding farm, subjects were anesthetized for surgery with a 1:1 mixture of ketamine/ xylazine (10/10 mg/kg). Pentobarbital (SomnotolR, 0.1 mL, 65 mg/mL) was used periodically during the surgery to increase the depth of anesthesia. To study the role of astrocytes in maintaining the seizure-prone state (Study A), subjects (n = 48) were then unilaterally implanted with single electrodes in the right basolateral amygdala using the following coordinates: 1.0 mm posterior (bregma), 4.8 mm lateral (from midline), and 8.5 mm ventral (from dura) using standard stereotaxic techniques. The incisor bar was set at +5. Teflon coated, bipolar electrodes constructed from stainless steel wires were used for implantation. Three screws were inserted into the skull to provide anchorage for the dental cement. To study the effects of astrocytes ablation at the kindling site, rats (phosphate buffered saline [PBS] = 8, La-AA = 12) were implanted with chemotrodes, which consisted of a combined electrode and cannula (Plastics One, Roanoke, VA) for infusion with La-AA (Study B). Following surgery, incisions were closed using silk sutures and antibiotic dressing (HibitaneR) was applied. After recovering from anesthesia, subjects were returned to their home cages for a 3-week recovery period. 2.3. Kindling Three weeks after implantation, subjects in Study A were randomly divided into four groups: Sp7, Sp18, Sp30, and Sp90, of 12 rats each. In each group, 6 randomly selected rats served as the kindled subjects and the other 6 served as sham kindled control subjects. Kindling to five stage 5 seizures was performed as described previously [24]. 2.4. La-AA infusion in fully kindled brains After kindling to five stage 5 seizures, subjects in Study B were anesthetized with 4% halothane (and maintained with 1.5% halothane) and given 5 AL of La-AA (20 Ag/mL) through the chemotrode using a 10 AL Hamilton syringe. Controls were infused with PBS. Infusions were given intermittently, at a rate of 0.5 AL/min with 1-min delays, for a total time of 10 min. Following infusion, the cannula cap
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was replaced, the halothane flow was stopped, and air was administered until the subjects were fully awake. The procedure was repeated every 72 h for a total of 10 infusions. Concurrently, the rats were stimulated daily for a total of 25 stimulations, as described in the previous section. 2.5. Measurement of Afterdischarge Threshold (ADT), Afterdischarge Duration (ADD), and motor seizure scores in the La-AA study ADT was determined using the ascending series technique [18]. This technique involves the initial administration of a subthreshold electrical stimulus followed by subsequent, step-wise increases in the electrical stimulus. In this study, ADT determination began with a stimulus of 40 AA (peak–peak). The stimulus intensity was then increased in 20 AA steps up to 320 AA, after which the intensity was increased in 40 AA steps. A 1-min interval was allowed between stimuli. ADT was defined as the minimum stimulus intensity needed to provoke an AD of at least 4 s. After subjects were fully kindled and infusion of La-AA or PBS was started, the rats were given 25 additional stimulations at 1/day with ADTs measured at every fifth stimulation. ADDs were measured on paper EEG recordings (in seconds) and motor seizures were classified using Racine’s five seizure stage model [20,21]. 2.6. BrdU injection, perfusion, and immunohistochemistry All procedures were carried out as described previously [24].
cyanate (TRITC) filters were used to visualize the green and red dyes respectively. Analysis was done by an investigator bblindQ to the treatment conditions. Double-labeled BrdU/ GFAP and BrdU/vimentin cells in the PC were counted using the optical dissector technique with a fractionator sampling scheme described by West et al. [26]. BrdU/GFAP or BrdU/vimentin labeled astrocytes were counted as they came into focus while scanning through the section. The dissector height (h) was set to 10 Am and cells within the first 3 Am were not counted. The total number of dividing astrocytes in the PC was estimated using: X N¼ Q t=h 1=asf 1=ssf P where Q is the total number of counted BrdU/GFAP or BrdU/vimentin labeled astrocytes in each PC, t is the average section thickness, asf is the areal sampling fraction (equal to 1/4), h is the dissector height, and ssf is the section sampling fraction equal to 1/12 (every 12th section was counted). 2.8. Statistical analysis The total number of cells was calculated based on 12 sections analyzed from every 12th section per rat brain. Values are reported as mean F SE. All statistical analyses were done using SigmaStat 2.0 (Jandel Scientific Software). When more than two groups were being compared, two-way analysis of variance (two-way ANOVA) was used. Post hoc comparison was done using Tukey’s test. Differences were deemed significant at the P b 0.05 level.
2.7. Microscopy and quantification 3. Results Brain sections from all experimental and control groups were analyzed using a Zeiss LSM510 confocal-laser scanning microscope with a 20 objective lens. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothio-
Histological examination confirmed that all implants were within the basolateral nucleus of the amygdaloid complex, as defined by Paxinos and Watson [17].
Fig. 1. Average number of (A) BrdU/GFAP and (B) BrdU/vimentin double-labeled astrocytes in the PC of amygdala-kindled brains (filled bars) with different survival periods. Data are represented as (mean F SE). (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). Numbers below each bar represent the percentage of dividing astrocytes as a proportion of (A) Total GFAP+ cells and (B) total vimentin+ cells. n = 6 for each condition.
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3.1. Astrocytic proliferation in fully kindled brains Fig. 1A shows the time course of BrdU/GFAP labeled astrocytes in the PC of amygdala-kindled subjects. There was a significant increase in proliferating astrocytes in the kindled subjects with the highest numbers present in Sp18, and a decline thereafter. The controls however showed no significant change over time. There was a significant effect of treatment ( F = 3470.4, P b 0.001), of time ( F = 147.4, P b 0.001), and between treatment and time ( F = 147.4, P b 0.001). Individual pairwise comparisons indicated that, in each group, the kindled subjects had significantly higher numbers ( P b 0.001) than the corresponding controls. As shown in Fig. 1B, the kindled subjects show a significantly higher number ( P b 0.001) of BrdU/vimentin labeled astrocytes, and, as with the BrdU/GFAP cells, the BrdU/vimentin cells show a decline after Sp18. There was a significant effect of treatment ( F = 1743.2, P b 0.001), of time ( F = 60.8, P b 0.001), and a significant interaction between treatment and time ( F = 57.4, P b 0.001). Fig. 2A shows the average number of BrdU/GFAP labeled astrocytes in the ipsilateral- versus the contralateral PC of amygdala-kindled brains. With the exception of Sp90, the contralateral PC showed a higher number of BrdU/ GFAP labeled astrocytes when compared to the ipsilateral side. There was a significant effect on the contralateral side ( F = 48.9, P b 0.001), of time ( F = 153.4, P b 0.001), and a significant interaction between the sides and time ( F = 7.4, P = 0.005). Individual pairwise comparisons indicated that there were significant differences ( P b 0.001) between contralateral and ipsilateral sides in Sp1, Sp7, Sp18, and Sp30, but not in Sp90. Fig. 2B shows the average number of BrdU/vimentin labeled astrocytes in the ipsilateral- versus the contralateral PC of kindled brains. The contralateral PC showed a higher number of BrdU/vimentin labeled astrocytes when compared to the ipsilateral side in Sp1, Sp7, Sp18, and
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Sp30. There was a significant effect on the contralateral side ( F = 54.1, P b 0.001), of time ( F = 68.5, P b 0.001), and a significant interaction between the sides and time ( F = 15.4, P = 0.005). Individual pairwise comparisons indicated that there were significant differences ( P b 0.001) between contralateral and ipsilateral subjects in Sp7, Sp18, and Sp30. 3.2. Effect of La-AA on the maintenance of seizures: infusion of the drug ipsilateral to the stimulation site Fig. 3C shows the behavioral seizures of both experimental (La-AA infused) and control (PBS-infused) subjects as measured on the five-stage scale developed by Racine [21]. All subjects were fully kindled prior to infusion of the drug or PBS. The La-AA infused group showed a general decrease in seizure score as the number of infusions increased. The La-AA infused subjects also showed a significant decrease ( P b 0.001, paired t test analysis) at every 5th stimulation interval compared to the PBS-infused subjects. Compared to the PBS-infused group, the druginfused group showed a significant effect of treatment ( F = 1062, P b 0.001), of time ( F = 10.8, P b 0.001), and a significant interaction between treatment and time ( F = 18.8, P b 0001). The ADT levels in stimulation numbers 5 to 25 were analyzed using a two-way repeated measure ANOVA. Pairwise comparisons indicated at each stimulation interval, La-AA infused subjects had significantly higher ( P b 0.001) ADTs compared to the corresponding controls (Fig. 3B). There was a significant effect of treatment ( F = 1761.2, P b 0.001), of time ( F = 49.5, P b 0.001), and a significant interaction between treatment and time ( F = 59.2, P b 0.001). Observed ADDs supported ADT measurements; individual pairwise comparisons indicated that La-AA infused subjects at 10, 15, 20, and 25 days had significantly ( P b 0.001) lower ADDs than the corresponding controls (Fig. 3A).
Fig. 2. Average number of (A) BrdU/GFAP and (B) BrdU/vimentin double-labeled astrocytes in the ipsilateral (gray) and contralateral (black) PC of kindled brains with different survival periods. Data are represented as mean F SE. (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). Numbers below each bar represent the percentage of dividing astrocytes as a proportion of (A) total GFAP+ cells and (B) total vimentin+ cells. n = 6 for each condition.
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Fig. 3. Effects of administering La-AA (black) compared to PBS-treated controls (clear) with concurrent kindling in subjects kindled to five stage 5 seizures. ADD (A), ADT (B), seizure stage (C), and average numbers of BrdU/GFAP (D) and BrdU/vimentin (E) double-labeled cells were obtained as described in Materials and Methods. Data are represented as mean F SE. (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). n = 8 for PBS-treated controls, and n = 12 for La-AA treated subjects.
The La-AA infused subjects had a significantly lower number of BrdU/GFAP (Fig. 3D) and BrdU/vimentin (Fig. 3E) double-labeled cells in piriform cortex relative to the corresponding controls.
4. Discussion The results of this study provide the first quantitative evidence of increases in the proliferation of astrocytes for at least 90 days as a result of kindling. Our findings extend the past work of Khurgel et al. [13] who showed astrocytic proliferation in PC of amygdala-kindled brains and demonstrated that application of La-AA to the basolateral nucleus of the amygdala caused a selective ablation of astrocytes. We also show that a toxin applied to astrocytes at the stimulation site in fully kindled rat brains causes decreased behavioral, physiological, and anatomical responses to further stimulation at the kindling site, as compared to these responses in rats that received saline. Previous studies have shown that, after a stab wound to the cortex, levels of GFAP and vimentin decline to normal by 10 to 21 days [11,23], which is consistent with the 3 week post operative delay in our study. In the present study, the observed quantitative differences between the number of dividing astrocytes in the control and kindled subjects are clearly not a consequence of the lesion caused by the
penetrating electrode, as both the control and the experimental subjects had implanted electrodes and there was not a large number of double-labeled astrocytes (BrdU/GFAP or BrdU/vimentin) in the non-stimulated subjects. Furthermore, the number of double-labeled cells in the sham kindled control subjects was not noticeably different from non-operated animals (data not shown). Duffy et al. [7] have shown that astrocytes react differently to bstressQ depending on whether they are in vitro or in vivo because of the K+ and neurotransmitters released by neurons. Since our study was done in vivo, the results indicate true astrocytic responses to the bstressQ of kindling. The increased astrocytic proliferation observed in the kindled subjects is not surprising, since immediately following neural trauma, other astrocytic responses such as hypertrophy, including increased GFAP- and vimentinimmunoreactivity, have been reported [5]. Furthermore, Khurgel et al. [14] reported the presence of hypertrophied astrocytes throughout the amygdala and PC in amygdalakindled rat brains with no apparent neuronal degeneration [15], thus providing evidence against the then widely held belief that neuron death was the main cause of astrocyte hypertrophy, but supporting the idea that astrocytes hypertrophy to protect neurons when they are in various forms of stress. Our results indicate a significant difference between the kindled and the control subjects with higher number of
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dividing astrocytes in the experimental animals. For the BrdU/GFAP labeled cells, the highest number of proliferating astrocytes was observed in Sp18 (Fig. 1A). In the BrdU/ vimentin labeled astrocytes however, the highest number was seen in Sp1, a trend that continued at Sp7 and Sp18 post kindling stimulation (Fig. 1B). These results are supported by the work of Aronica et al. [2], who examined the expression of Id3, a protein involved in the regulation of cell proliferation and differentiation. They observed that 1 day following electrically induced status epilepticus in rats, the hippocampal astrocytes showed a marked increase of Id3 protein expression. These levels persisted for up to 3 weeks and dropped to control levels at 90 days. Their time points approximate ours and although their changes were observed in the hippocampus (the PC was not reported), their findings are similar as they are based on up-regulation of gene expression following electrically induced seizures. In addition to observing a higher number of doublelabeled cells (BrdU/GFAP and BrdU/vimentin) in the PC of amygdala-kindled subjects, we also observed differences in the proliferation number between the ipsilateral- and contralateral PC. The contralateral PC in Sp1, Sp7, Sp18, and Sp30 showed higher numbers of dividing astrocytes (BrdU/GFAP and BrdU/vimentin) as compared to the ipsilateral PC (Figs. 2A, B). It is of interest that the rates of proliferation on the contralateral side were higher than any rate of proliferation observed on the ipsilateral side. Since there is no obvious degeneration in such brains [13], one possibility is that the astrocytes in the contralateral PC might play a different role in maintaining the seizure-prone state. Indeed, our previous study [24] which showed that as early as stage 1 of kindling there were more double-labeled astrocytes (both GFAP/BrdU and vimentin/BrdU) in the ipsilateral PC, indicating that there is likely a time course for the transfer of kindling phenomenon from one brain region to another. We hypothesize that this effect is based on the efficacy of electrical and/or chemical signal transfer among regions anatomically connected to the kindling site and that growth and remodeling may be involved. This time course hypothesis is supported by data from Sp90 brains, in which the ipsilateral PC and contralateral PC show no statistical difference in their number of double-labeled BrdU/GFAP and BrdU/vimentin double-labeled cells, possibly indicating that any new growth or remodeling, which may have been occurring earlier to bensureQ the maintenance of a seizureprone state is now nearing an end. Astrocytes are involved in maintaining the chemical environment around synapses during physiological activity. This is especially true of the hypertrophied astrocytes, which surround experience-dependent synaptic changes. In an electron microscopic study, Jones and Greenough [12] observed increased contact between astrocytic processes and synaptic elements within the visual cortex of rats raised in an enriched environment, compared to controls. The subjects also developed increased arboriza-
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tion of pyramidal dendrites in the visual cortex and had increased numbers of synapses on these cells [12]. Chang et al. [6] have shown that new synapses are formed in early stages of kindling of hippocampal subfield CA1. These results are supported by evidence that kindling of hippocampal subfield CA1 induces an increase in shaft synapse density as well as astrocytic hypertrophy [9]. These results combined strengthen the notion that both synaptogenesis and astrocytic changes may contribute to epileptogenesis and persist into the seizure-prone state. When La-AA was infused into subjects with fully generalized seizures (five stage 5), the convulsions were reduced to focal seizures (stage 1 or 2), ADDs were shortened and the ADTs were increased significantly (Fig. 3). Finally, there were a significantly lower number of proliferating astrocytes in the PC following the La-AA infusions in the amygdala (Fig. 3). The effects of La-AA might be explained by several different mechanisms. First, killing astrocytes causes a collapse of the support system necessary for optimal synaptic transmission in amygdala neurons. Since astrocytes play a number of important roles in synaptic transmission, such as buffering K+ and Ca2+ [22], the absence or perturbation of these cells might impede the transmission of the stimuli from the amygdala to other brain regions, thus impeding the seizure generalization. A possible mechanism that could account for the decreased ADT seen in the La-AA infused subjects is a loss of glutamate release. There is a series of studies suggesting that astrocytes in cell culture and in situ can release glutamate [10]. The Ca2+ dependent glutamate release can, in turn, lower the ADTs at the synapses, and in the amygdala as a whole. Thus, eliminating astrocytes at the kindling focus would increase the focal ADT and weaken the signal propagation from the amygdala to the PC. The cell count analysis revealed a decrease in the total number of proliferating astrocytes in the PC of La-AA treated rats to normal levels. This decrease is probably due to the decreased number of astrocytes in the amygdala, which would functionally lesion the nucleus and deprives the PC of a powerful source of input. Since the PC is likely to play a major role in the generalization of kindled seizures [8], preventing astrocyte proliferation in the PC would keep this structure from becoming hyperexcitable (kindling) and prevent enhanced propagation from the PC to other sites. That the rise in ADTs is not a consequence of neuronal death in the infusion site is strongly supported by the fact that, at a month following the last La-AA infusion, the ADTs were back to control levels. Changes related to neuronal death would be permanent, whereas changes related to astrocyte death are not. It has been shown that 1 week following an La-AA injection, astrocytes have started to migrate back to the injection site. Thirty days following our last La-AA infusion, we noted a significant repopulation of the site by astrocytes, and no noticeable effect on neurons with immunohistochemical examination.
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In this study, we report significant differences in astrocytic proliferation among kindled brains with different survival periods, and also between the ipsilateral PC and contralateral PC in kindled brains (except in Sp90). In response to the stimulations, we have shown that the astrocytes in the ipsilateral- and the contralateral PC become activated as well as proliferate and produce new proteins. Since the formation of new synapses in response to seizures is accompanied by dividing astrocytes and since, without these astrocytes, synaptogenesis might not be as efficient, a role for proliferating astrocytes in the maintenance of the seizure-prone state can be appreciated. Furthermore, with lesioning of the astrocytes at the kindling site in fully kindled rats, there are notable decreases in behavioral, physiological, and anatomical correlates of the kindled state, a strong indication that these cells are functionally involved in maintaining the seizure-prone state.
Acknowledgments This study was supported by NSERC (GOI) and by Bloorview Epilepsy Research Foundation (WMB).
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Research report
Might astrocytes play a role in maintaining the seizure-prone state? Mani Vessala,b,c,f,T, Chandrasagar B. Duganid,e,1, Dianand A. Solomonf, W. McIntyre Burnhamc,e, Gwen O. Ivyc,f,g a
Institute of Medical Science, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 b Program in Neuroscience, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 c Bloorview Epilepsy Research Program, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 d Department of Biochemistry, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 e Department of Pharmacology, Medical Science Building, University of Toronto, Toronto, ON, Canada M5S 1A8 f Centre for the Neurobiology of Stress, University of Toronto at Scarborough, Scarborough, ON, Canada M1C 1A4 g Department of Psychology, University of Toronto at Scarborough, Scarborough, ON, Canada M1C 1A4 Accepted 23 February 2005 Available online 19 April 2005
Abstract The amygdala-kindling model is used to study complex partial epilepsy with secondary generalization. The present study was designed to (A) quantify astrocytic changes in the piriform cortex of amygdala-kindled subjects over time and (B) investigate the role that astrocytes might play in maintaining the seizure-prone state. In Study A, once the experimental subjects reached five stage 5 seizures, stimulation was stopped, and both kindled and control rats were allowed to survive for the interval appropriate to their group (7, 18, 30, or 90 days). Following each interval, the kindled and control animals were given 10 intraperitoneal injections of bromodeoxyuridine (BrdU) and sacrificed 24 h following the last injection. Significantly higher numbers of dividing astrocytes (identified by co-labeling for BrdU and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) were found in the kindled brains. All kindled groups had significantly higher numbers of double-labeled cells on the side contralateral to the stimulation site, except for those in the 90 day survival group. In Study B, rats were implanted with chemotrodes, were kindled as in Study A, and were subsequently infused with either saline or with La-AA (to lesion astrocytes) during a further 25 stimulations (1/day). La-AA infused rats had significantly diminished levels of behavioral seizures, higher after discharge thresholds, lower after discharge durations, and decreased numbers of double-labeled astrocytes in piriform cortex than did saline infused rats. Together, the data indicate that astrocytes may play a role in maintaining the seizure-prone state. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal studies Keywords: Epilepsy; Piriform cortex; Amygdala-kindling; Astrocyte proliferation; GFAP; Vimentin; La-aminoadipate
1. Introduction
T Corresponding author. Department of Comparative Medicine, Stanford University School of Medicine, Stanford Brain Research Institute, 300, Pasteur Drive, Edwards Building, Room R342, Stanford, CA 94305-5342, USA. Fax: +1 650 498 5085. E-mail address: [email protected] (M. Vessal). 1 Present address: The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.058
Epilepsy is the most common primary disorder of the brain occurring in about 1% of the population [4]. Epileptic seizures are divided into two major categories: partial and generalized. Partial seizures are localized to a particular area in the brain. They can be simple, without the loss of consciousness, or complex, with a loss of consciousness. Partial seizures can also be secondarily generalized, a
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condition in which seizures spread from a defined focus to other regions of the brain. Generalized seizures, on the other hand, have no defined focus and involve the entire brain [19]. The amygdala-kindling model is an animal model of complex partial seizures with secondary generalizations [1]. This model of temporal lobe epilepsy involves the electrical stimulation of a defined region of the temporal lobe via chronically implanted electrodes and the severity of the resulting seizures may be quantified by EEG recording or behavioral analysis [20,21]. Astrocytes react to various types of injury and stress with rapid cell growth and division [3]. The most common reaction of astrocytes to injury in the CNS is the striking hypertrophy as observed by an increase in the amount of immunoreactivity of intermediate filament proteins, glial fibrillary acidic protein (GFAP), and vimentin [16,25]. Such astrocytic activation has also been observed in the piriform cortex (PC) of amygdala-kindled brains [13,14]. We have been investigating the response of astrocytes to kindling in an attempt to understand their possible involvement in epileptogenesis [14]. In addition to astrocytic hypertrophy, we have recently reported astrocytic proliferation in the PC of amygdala-kindled rats [24]. In that study, we observed a significantly higher number of dividing astrocytes (identified by co-labeling with antibodies to bromodeoxyuridine (BrdU) and to one of the astrocytic intermediate filament proteins, GFAP, for labeling mature astrocytes or vimentin, for labeling immature astrocytes) in both partially kindled (one stage 1) and fully kindled (five stage 5 seizures) brains. Given the robust proliferation response of the astrocytes to full kindling, we decided to investigate further a role that they might play in maintaining this epileptic state by first (Study A) quantifying their proliferation in the piriform cortex ipsilateral vs. contralateral to the kindling site and second (Study B), by ablating them at the kindling site itself using the astrotoxin La-aminoadipate (La-AA). In Study A, we thus compared astrocyte proliferation in kindled (five stage 5 seizures; [21]) rat brains that subsequently had different survival periods (Sp): 7 days (Sp7), 18 days (Sp18), 30 days (Sp30), or 90 days (Sp90). Regions anatomically linked to the site of kindling on both the ipsilateral- and contralateral PC were examined for the relative distribution of proliferating astrocytes. In Study B, we took rats that had been fully kindled via a chemotrode (which consists of a combined electrode and cannula) and subsequently infused them with either saline or the astroglial specific toxin La-amino adipic acid (La-AA) while giving daily amygdala stimulations as before, over 25 days. During these 25 days, we recorded behavioral seizure levels, measured after-discharge thresholds and after-discharge durations, and examined astrocyte proliferation in piriform cortex in order to determine if lesions of astrocytes at the stimulation site affect the behavioral, physiological, or anatomical correlates of the seizure-prone state.
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2. Materials and methods 2.1. Subjects Adult, Long-Evans male rats (n = 68, postnatal day 60, 225–250 g) were obtained from Charles River Laboratories, Saint-Constaint, QC, Canada and housed as previously described [24]. All research protocols were approved by the Animal Care Committee (Faculty of Medicine) of the University of Toronto, Toronto, Canada. 2.2. Implantation of electrodes and chemotrodes One week after arrival from the breeding farm, subjects were anesthetized for surgery with a 1:1 mixture of ketamine/ xylazine (10/10 mg/kg). Pentobarbital (SomnotolR, 0.1 mL, 65 mg/mL) was used periodically during the surgery to increase the depth of anesthesia. To study the role of astrocytes in maintaining the seizure-prone state (Study A), subjects (n = 48) were then unilaterally implanted with single electrodes in the right basolateral amygdala using the following coordinates: 1.0 mm posterior (bregma), 4.8 mm lateral (from midline), and 8.5 mm ventral (from dura) using standard stereotaxic techniques. The incisor bar was set at +5. Teflon coated, bipolar electrodes constructed from stainless steel wires were used for implantation. Three screws were inserted into the skull to provide anchorage for the dental cement. To study the effects of astrocytes ablation at the kindling site, rats (phosphate buffered saline [PBS] = 8, La-AA = 12) were implanted with chemotrodes, which consisted of a combined electrode and cannula (Plastics One, Roanoke, VA) for infusion with La-AA (Study B). Following surgery, incisions were closed using silk sutures and antibiotic dressing (HibitaneR) was applied. After recovering from anesthesia, subjects were returned to their home cages for a 3-week recovery period. 2.3. Kindling Three weeks after implantation, subjects in Study A were randomly divided into four groups: Sp7, Sp18, Sp30, and Sp90, of 12 rats each. In each group, 6 randomly selected rats served as the kindled subjects and the other 6 served as sham kindled control subjects. Kindling to five stage 5 seizures was performed as described previously [24]. 2.4. La-AA infusion in fully kindled brains After kindling to five stage 5 seizures, subjects in Study B were anesthetized with 4% halothane (and maintained with 1.5% halothane) and given 5 AL of La-AA (20 Ag/mL) through the chemotrode using a 10 AL Hamilton syringe. Controls were infused with PBS. Infusions were given intermittently, at a rate of 0.5 AL/min with 1-min delays, for a total time of 10 min. Following infusion, the cannula cap
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was replaced, the halothane flow was stopped, and air was administered until the subjects were fully awake. The procedure was repeated every 72 h for a total of 10 infusions. Concurrently, the rats were stimulated daily for a total of 25 stimulations, as described in the previous section. 2.5. Measurement of Afterdischarge Threshold (ADT), Afterdischarge Duration (ADD), and motor seizure scores in the La-AA study ADT was determined using the ascending series technique [18]. This technique involves the initial administration of a subthreshold electrical stimulus followed by subsequent, step-wise increases in the electrical stimulus. In this study, ADT determination began with a stimulus of 40 AA (peak–peak). The stimulus intensity was then increased in 20 AA steps up to 320 AA, after which the intensity was increased in 40 AA steps. A 1-min interval was allowed between stimuli. ADT was defined as the minimum stimulus intensity needed to provoke an AD of at least 4 s. After subjects were fully kindled and infusion of La-AA or PBS was started, the rats were given 25 additional stimulations at 1/day with ADTs measured at every fifth stimulation. ADDs were measured on paper EEG recordings (in seconds) and motor seizures were classified using Racine’s five seizure stage model [20,21]. 2.6. BrdU injection, perfusion, and immunohistochemistry All procedures were carried out as described previously [24].
cyanate (TRITC) filters were used to visualize the green and red dyes respectively. Analysis was done by an investigator bblindQ to the treatment conditions. Double-labeled BrdU/ GFAP and BrdU/vimentin cells in the PC were counted using the optical dissector technique with a fractionator sampling scheme described by West et al. [26]. BrdU/GFAP or BrdU/vimentin labeled astrocytes were counted as they came into focus while scanning through the section. The dissector height (h) was set to 10 Am and cells within the first 3 Am were not counted. The total number of dividing astrocytes in the PC was estimated using: X N¼ Q t=h 1=asf 1=ssf P where Q is the total number of counted BrdU/GFAP or BrdU/vimentin labeled astrocytes in each PC, t is the average section thickness, asf is the areal sampling fraction (equal to 1/4), h is the dissector height, and ssf is the section sampling fraction equal to 1/12 (every 12th section was counted). 2.8. Statistical analysis The total number of cells was calculated based on 12 sections analyzed from every 12th section per rat brain. Values are reported as mean F SE. All statistical analyses were done using SigmaStat 2.0 (Jandel Scientific Software). When more than two groups were being compared, two-way analysis of variance (two-way ANOVA) was used. Post hoc comparison was done using Tukey’s test. Differences were deemed significant at the P b 0.05 level.
2.7. Microscopy and quantification 3. Results Brain sections from all experimental and control groups were analyzed using a Zeiss LSM510 confocal-laser scanning microscope with a 20 objective lens. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothio-
Histological examination confirmed that all implants were within the basolateral nucleus of the amygdaloid complex, as defined by Paxinos and Watson [17].
Fig. 1. Average number of (A) BrdU/GFAP and (B) BrdU/vimentin double-labeled astrocytes in the PC of amygdala-kindled brains (filled bars) with different survival periods. Data are represented as (mean F SE). (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). Numbers below each bar represent the percentage of dividing astrocytes as a proportion of (A) Total GFAP+ cells and (B) total vimentin+ cells. n = 6 for each condition.
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3.1. Astrocytic proliferation in fully kindled brains Fig. 1A shows the time course of BrdU/GFAP labeled astrocytes in the PC of amygdala-kindled subjects. There was a significant increase in proliferating astrocytes in the kindled subjects with the highest numbers present in Sp18, and a decline thereafter. The controls however showed no significant change over time. There was a significant effect of treatment ( F = 3470.4, P b 0.001), of time ( F = 147.4, P b 0.001), and between treatment and time ( F = 147.4, P b 0.001). Individual pairwise comparisons indicated that, in each group, the kindled subjects had significantly higher numbers ( P b 0.001) than the corresponding controls. As shown in Fig. 1B, the kindled subjects show a significantly higher number ( P b 0.001) of BrdU/vimentin labeled astrocytes, and, as with the BrdU/GFAP cells, the BrdU/vimentin cells show a decline after Sp18. There was a significant effect of treatment ( F = 1743.2, P b 0.001), of time ( F = 60.8, P b 0.001), and a significant interaction between treatment and time ( F = 57.4, P b 0.001). Fig. 2A shows the average number of BrdU/GFAP labeled astrocytes in the ipsilateral- versus the contralateral PC of amygdala-kindled brains. With the exception of Sp90, the contralateral PC showed a higher number of BrdU/ GFAP labeled astrocytes when compared to the ipsilateral side. There was a significant effect on the contralateral side ( F = 48.9, P b 0.001), of time ( F = 153.4, P b 0.001), and a significant interaction between the sides and time ( F = 7.4, P = 0.005). Individual pairwise comparisons indicated that there were significant differences ( P b 0.001) between contralateral and ipsilateral sides in Sp1, Sp7, Sp18, and Sp30, but not in Sp90. Fig. 2B shows the average number of BrdU/vimentin labeled astrocytes in the ipsilateral- versus the contralateral PC of kindled brains. The contralateral PC showed a higher number of BrdU/vimentin labeled astrocytes when compared to the ipsilateral side in Sp1, Sp7, Sp18, and
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Sp30. There was a significant effect on the contralateral side ( F = 54.1, P b 0.001), of time ( F = 68.5, P b 0.001), and a significant interaction between the sides and time ( F = 15.4, P = 0.005). Individual pairwise comparisons indicated that there were significant differences ( P b 0.001) between contralateral and ipsilateral subjects in Sp7, Sp18, and Sp30. 3.2. Effect of La-AA on the maintenance of seizures: infusion of the drug ipsilateral to the stimulation site Fig. 3C shows the behavioral seizures of both experimental (La-AA infused) and control (PBS-infused) subjects as measured on the five-stage scale developed by Racine [21]. All subjects were fully kindled prior to infusion of the drug or PBS. The La-AA infused group showed a general decrease in seizure score as the number of infusions increased. The La-AA infused subjects also showed a significant decrease ( P b 0.001, paired t test analysis) at every 5th stimulation interval compared to the PBS-infused subjects. Compared to the PBS-infused group, the druginfused group showed a significant effect of treatment ( F = 1062, P b 0.001), of time ( F = 10.8, P b 0.001), and a significant interaction between treatment and time ( F = 18.8, P b 0001). The ADT levels in stimulation numbers 5 to 25 were analyzed using a two-way repeated measure ANOVA. Pairwise comparisons indicated at each stimulation interval, La-AA infused subjects had significantly higher ( P b 0.001) ADTs compared to the corresponding controls (Fig. 3B). There was a significant effect of treatment ( F = 1761.2, P b 0.001), of time ( F = 49.5, P b 0.001), and a significant interaction between treatment and time ( F = 59.2, P b 0.001). Observed ADDs supported ADT measurements; individual pairwise comparisons indicated that La-AA infused subjects at 10, 15, 20, and 25 days had significantly ( P b 0.001) lower ADDs than the corresponding controls (Fig. 3A).
Fig. 2. Average number of (A) BrdU/GFAP and (B) BrdU/vimentin double-labeled astrocytes in the ipsilateral (gray) and contralateral (black) PC of kindled brains with different survival periods. Data are represented as mean F SE. (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). Numbers below each bar represent the percentage of dividing astrocytes as a proportion of (A) total GFAP+ cells and (B) total vimentin+ cells. n = 6 for each condition.
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Fig. 3. Effects of administering La-AA (black) compared to PBS-treated controls (clear) with concurrent kindling in subjects kindled to five stage 5 seizures. ADD (A), ADT (B), seizure stage (C), and average numbers of BrdU/GFAP (D) and BrdU/vimentin (E) double-labeled cells were obtained as described in Materials and Methods. Data are represented as mean F SE. (*) indicates a significant difference from the control group, as indicated by a post hoc Tukey’s test ( P b 0.001). n = 8 for PBS-treated controls, and n = 12 for La-AA treated subjects.
The La-AA infused subjects had a significantly lower number of BrdU/GFAP (Fig. 3D) and BrdU/vimentin (Fig. 3E) double-labeled cells in piriform cortex relative to the corresponding controls.
4. Discussion The results of this study provide the first quantitative evidence of increases in the proliferation of astrocytes for at least 90 days as a result of kindling. Our findings extend the past work of Khurgel et al. [13] who showed astrocytic proliferation in PC of amygdala-kindled brains and demonstrated that application of La-AA to the basolateral nucleus of the amygdala caused a selective ablation of astrocytes. We also show that a toxin applied to astrocytes at the stimulation site in fully kindled rat brains causes decreased behavioral, physiological, and anatomical responses to further stimulation at the kindling site, as compared to these responses in rats that received saline. Previous studies have shown that, after a stab wound to the cortex, levels of GFAP and vimentin decline to normal by 10 to 21 days [11,23], which is consistent with the 3 week post operative delay in our study. In the present study, the observed quantitative differences between the number of dividing astrocytes in the control and kindled subjects are clearly not a consequence of the lesion caused by the
penetrating electrode, as both the control and the experimental subjects had implanted electrodes and there was not a large number of double-labeled astrocytes (BrdU/GFAP or BrdU/vimentin) in the non-stimulated subjects. Furthermore, the number of double-labeled cells in the sham kindled control subjects was not noticeably different from non-operated animals (data not shown). Duffy et al. [7] have shown that astrocytes react differently to bstressQ depending on whether they are in vitro or in vivo because of the K+ and neurotransmitters released by neurons. Since our study was done in vivo, the results indicate true astrocytic responses to the bstressQ of kindling. The increased astrocytic proliferation observed in the kindled subjects is not surprising, since immediately following neural trauma, other astrocytic responses such as hypertrophy, including increased GFAP- and vimentinimmunoreactivity, have been reported [5]. Furthermore, Khurgel et al. [14] reported the presence of hypertrophied astrocytes throughout the amygdala and PC in amygdalakindled rat brains with no apparent neuronal degeneration [15], thus providing evidence against the then widely held belief that neuron death was the main cause of astrocyte hypertrophy, but supporting the idea that astrocytes hypertrophy to protect neurons when they are in various forms of stress. Our results indicate a significant difference between the kindled and the control subjects with higher number of
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dividing astrocytes in the experimental animals. For the BrdU/GFAP labeled cells, the highest number of proliferating astrocytes was observed in Sp18 (Fig. 1A). In the BrdU/ vimentin labeled astrocytes however, the highest number was seen in Sp1, a trend that continued at Sp7 and Sp18 post kindling stimulation (Fig. 1B). These results are supported by the work of Aronica et al. [2], who examined the expression of Id3, a protein involved in the regulation of cell proliferation and differentiation. They observed that 1 day following electrically induced status epilepticus in rats, the hippocampal astrocytes showed a marked increase of Id3 protein expression. These levels persisted for up to 3 weeks and dropped to control levels at 90 days. Their time points approximate ours and although their changes were observed in the hippocampus (the PC was not reported), their findings are similar as they are based on up-regulation of gene expression following electrically induced seizures. In addition to observing a higher number of doublelabeled cells (BrdU/GFAP and BrdU/vimentin) in the PC of amygdala-kindled subjects, we also observed differences in the proliferation number between the ipsilateral- and contralateral PC. The contralateral PC in Sp1, Sp7, Sp18, and Sp30 showed higher numbers of dividing astrocytes (BrdU/GFAP and BrdU/vimentin) as compared to the ipsilateral PC (Figs. 2A, B). It is of interest that the rates of proliferation on the contralateral side were higher than any rate of proliferation observed on the ipsilateral side. Since there is no obvious degeneration in such brains [13], one possibility is that the astrocytes in the contralateral PC might play a different role in maintaining the seizure-prone state. Indeed, our previous study [24] which showed that as early as stage 1 of kindling there were more double-labeled astrocytes (both GFAP/BrdU and vimentin/BrdU) in the ipsilateral PC, indicating that there is likely a time course for the transfer of kindling phenomenon from one brain region to another. We hypothesize that this effect is based on the efficacy of electrical and/or chemical signal transfer among regions anatomically connected to the kindling site and that growth and remodeling may be involved. This time course hypothesis is supported by data from Sp90 brains, in which the ipsilateral PC and contralateral PC show no statistical difference in their number of double-labeled BrdU/GFAP and BrdU/vimentin double-labeled cells, possibly indicating that any new growth or remodeling, which may have been occurring earlier to bensureQ the maintenance of a seizureprone state is now nearing an end. Astrocytes are involved in maintaining the chemical environment around synapses during physiological activity. This is especially true of the hypertrophied astrocytes, which surround experience-dependent synaptic changes. In an electron microscopic study, Jones and Greenough [12] observed increased contact between astrocytic processes and synaptic elements within the visual cortex of rats raised in an enriched environment, compared to controls. The subjects also developed increased arboriza-
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tion of pyramidal dendrites in the visual cortex and had increased numbers of synapses on these cells [12]. Chang et al. [6] have shown that new synapses are formed in early stages of kindling of hippocampal subfield CA1. These results are supported by evidence that kindling of hippocampal subfield CA1 induces an increase in shaft synapse density as well as astrocytic hypertrophy [9]. These results combined strengthen the notion that both synaptogenesis and astrocytic changes may contribute to epileptogenesis and persist into the seizure-prone state. When La-AA was infused into subjects with fully generalized seizures (five stage 5), the convulsions were reduced to focal seizures (stage 1 or 2), ADDs were shortened and the ADTs were increased significantly (Fig. 3). Finally, there were a significantly lower number of proliferating astrocytes in the PC following the La-AA infusions in the amygdala (Fig. 3). The effects of La-AA might be explained by several different mechanisms. First, killing astrocytes causes a collapse of the support system necessary for optimal synaptic transmission in amygdala neurons. Since astrocytes play a number of important roles in synaptic transmission, such as buffering K+ and Ca2+ [22], the absence or perturbation of these cells might impede the transmission of the stimuli from the amygdala to other brain regions, thus impeding the seizure generalization. A possible mechanism that could account for the decreased ADT seen in the La-AA infused subjects is a loss of glutamate release. There is a series of studies suggesting that astrocytes in cell culture and in situ can release glutamate [10]. The Ca2+ dependent glutamate release can, in turn, lower the ADTs at the synapses, and in the amygdala as a whole. Thus, eliminating astrocytes at the kindling focus would increase the focal ADT and weaken the signal propagation from the amygdala to the PC. The cell count analysis revealed a decrease in the total number of proliferating astrocytes in the PC of La-AA treated rats to normal levels. This decrease is probably due to the decreased number of astrocytes in the amygdala, which would functionally lesion the nucleus and deprives the PC of a powerful source of input. Since the PC is likely to play a major role in the generalization of kindled seizures [8], preventing astrocyte proliferation in the PC would keep this structure from becoming hyperexcitable (kindling) and prevent enhanced propagation from the PC to other sites. That the rise in ADTs is not a consequence of neuronal death in the infusion site is strongly supported by the fact that, at a month following the last La-AA infusion, the ADTs were back to control levels. Changes related to neuronal death would be permanent, whereas changes related to astrocyte death are not. It has been shown that 1 week following an La-AA injection, astrocytes have started to migrate back to the injection site. Thirty days following our last La-AA infusion, we noted a significant repopulation of the site by astrocytes, and no noticeable effect on neurons with immunohistochemical examination.
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In this study, we report significant differences in astrocytic proliferation among kindled brains with different survival periods, and also between the ipsilateral PC and contralateral PC in kindled brains (except in Sp90). In response to the stimulations, we have shown that the astrocytes in the ipsilateral- and the contralateral PC become activated as well as proliferate and produce new proteins. Since the formation of new synapses in response to seizures is accompanied by dividing astrocytes and since, without these astrocytes, synaptogenesis might not be as efficient, a role for proliferating astrocytes in the maintenance of the seizure-prone state can be appreciated. Furthermore, with lesioning of the astrocytes at the kindling site in fully kindled rats, there are notable decreases in behavioral, physiological, and anatomical correlates of the kindled state, a strong indication that these cells are functionally involved in maintaining the seizure-prone state.
Acknowledgments This study was supported by NSERC (GOI) and by Bloorview Epilepsy Research Foundation (WMB).
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