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Changes in the mRNAs encoding voltage-gated sodium channel types II and III in human epileptic hippocampus
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Neuroscience Vol. 106, No. 2, pp. 275^285, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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CHANGES IN THE mRNAs ENCODING VOLTAGE-GATED SODIUM CHANNEL TYPES II AND III IN HUMAN EPILEPTIC HIPPOCAMPUS W. R. J. WHITAKER,a R. L. M. FAULL,b M. DRAGUNOW,b E. W. MEE,c P. C. EMSONa and J. J. CLAREd * a b
Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
Departments of Anatomy and Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand c
d
Department of Neurosurgery, Auckland Hospital, Auckland, New Zealand
Department of Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
AbstractöStudies with animal seizure models have indicated that changes in temporal and spatial expression of voltagegated sodium channels may be important in the pathology of epilepsy. Here, by using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes [Whitaker et al. (2000) J. Comp. Neurol. 422, 123^139], we have compared the cellular expression of all four brain K-subunit sodium channel mRNAs in `normal' and epileptic hippocampi from humans. Neuronal cell loss was observed in all regions of the hippocampus of diseased patients, indicating that sclerosis had occurred. Losses of up to 40% compared to post-mortem controls were observed which were statistically signi¢cant in all regions studied (dentate gyrus, hilus, and CA1^3). To assess mRNA levels of the di¡erent K-subtypes in speci¢c subregions, control and diseased tissue sections were hybridised to subtype-speci¢c probes. To quantify any changes in expression while allowing for cell loss, the sections were processed for liquid emulsion autoradiography and grain counts were performed on populations of individual neurones in di¡erent subregions. No signi¢cant di¡erences were found in the expression of type I and VI mRNAs. In contrast, a signi¢cant down-regulation of type II mRNA was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71 þ 7% of control, P 6 0.01), CA2 (81 þ 8% of control, P 6 0.05) and CA1 (72 þ 6% of control, P 6 0.05) compared with control tissue. Additionally, a signi¢cant up-regulation in type III mRNA in epileptic CA4 pyramidal cells (145 þ 7% of control, P 6 0.05) was observed. It is not clear whether these changes play a causal role in human epilepsy or whether they are secondary to seizures or drug treatment; further studies are necessary to investigate these alternatives. However, it is likely that such changes would a¡ect the intrinsic excitability of hippocampal neurones. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: sodium channels, human brain, mRNA distribution, epilepsy.
K-subunit, of which four subtypes (types I, II, III and VI) are found in brain, associated with one or more auxiliary L-subunits. Analysis of sodium channel K-subunit mRNAs following kainic acid-induced seizures in rat hippocampus revealed a marked increase in type III mRNA expression, a modest increase in type II mRNA signal and no change in type I mRNA expression (Bartolomei et al., 1997). Studies using reverse transcriptase-polymerase chain reaction and in situ hybridisation indicate that seizure activity induces alterations in the developmental splicing of neonatal and adult type II and III isoforms (Gastaldi et al., 1997). Seizures were found to re-activate the neonatal splicing event, causing an increase in the presence of the neonatal type II and III isoforms in localised regions of the adult rat brain. More recently, a biphasic response of L2-subunit transcription has been reported following kainic acid administration (Gastaldi et al., 1998). An initial increase in L2-subunit expression was followed by a decrease over a time course that paralleled the increase seen in K-subunit expression. Other studies, using non-selective sodium channel probes, have indicated increases in sodium channel
Voltage-gated sodium channels play a critical role in the initiation and transmission of action potentials and are therefore likely to be important in the aetiology of epilepsy. This is illustrated by the fact that several established anti-epileptic drugs are known to inhibit sodium channels, e.g. phenytoin, carbemazepine, lamotrigine (Ragsdale and Avoli, 1998; Clare et al., 2000). Additionally, it has recently been found that sodium channel mutations are responsible for certain inherited forms of epilepsy (generalised epilepsy with febrile seizures plus, GEFS+, types I and II; Wallace et al., 1998; Escayg et al., 2000). Studies with animal seizure models have also indicated the involvement of voltage-gated sodium channels in epilepsy. The channels consist of a large pore forming
*Corresponding author. Tel.: +44-1438-763834; fax: +44-1438764488. E-mail address: [email protected] (J. J. Clare). Abbreviations : DRG, dorsal rot ganglion; MCID, micro computer imaging device; UTR, untranslated region. 275
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mRNA and protein in the cortex of genetically seizure susceptible E1 mice brains (Sashihara et al., 1992). Conversely, in the brains of tottering (Tg) mice, which spontaneously undergo spike wave seizures due to mutations of the P/Q-type calcium channel (Fletcher et al., 1996), a decrease in sodium channel proteins in nerve terminals was observed as detected by [3 H]saxitoxin binding (Willow et al., 1986). Interestingly, the remaining channels showed an 80% increase in activity, as measured by radioisotopic sodium in£ux. Whilst studies with animal models have indicated that changes in the temporal and spatial expression of sodium channels may be important in the pathology of epilepsy, only a single human study has been performed to date. Such studies are important, not only in furthering our understanding of the human epileptic condition, but also for the development of more selective anti-epileptic sodium channel inhibitors by identifying subtypes of key relevance to the disease. The previous human study uncovered an altered ratio of types I and II mRNA in hippocampi resected from temporal lobe epilepsy patients compared to control tissue (Lombardo et al., 1996). However, this was performed using the ligase detection assay, which is not a well-established technique, did not include types III and VI and did not provide information at the cellular level. Thus, a major aim of the present study was to compare the expression of all four brain K-subunit mRNAs in the `normal' and epileptic hippocampal formation from humans. By using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes (Whitaker et al., 2000) we have carried out the ¢rst detailed investigation of this type at a cellular level.
EXPERIMENTAL PROCEDURES
Human tissue and sample preparation Human brain tissue was obtained from the New Zealand Neurological Foundation Human Brain Bank at the University of Auckland using ethical consent procedures approved by the University of Auckland Human Subjects Ethics Committee and the Auckland Ethics Committees. Samples used in this study were obtained from eight temporal lobe resection patients and seven post-mortem control cases. Tables 1 and 2 summarise the available clinico-pathological data for these samples. Tissue was obtained as snap frozen blocks stored at 380³C which were sectioned for in situ hybridisation on a freezing cryostat (Bright Instruments, Cambs, UK). Blocks were removed from storage at 380³C and left in the cryostat chamber for 1 h at 320³C to equilibrate prior to sectioning. Sections (15 Wm) were dissected from the block and mounted onto electrostatically charged microscope slides (Superfrost plus, Gurr, BDH-Merck, Poole, UK) and then dried using a hair-dryer before storage at 370³C until use. Two-dimensional cell counting Cell counts were performed using a Viglen PC running micro computer imaging device (MCID) software (Imaging Research, ON, Canada) linked to a Leica DMRBE microscope via a high resolution charged couple device (CCD) digital camera (MTI CCD 72) following the manufacturer's protocol. Populations of cells were counted in a de¢ned area of 100 Wm by 50 Wm for dentate granule cells and 250 Wm by 250 Wm for CA pyra-
Table 1. Summary of clinico-pathological information of patients having undergone surgery for intractable idiopathic epilepsy, whose tissue was used in in situ hybridisation studies Case no.
Age (years)
Gender (M/F)
Drug therapy
E19
20
F
E45 E40 E23 E41
29 32 34 43
M M F F
E47 E50
25 42
M F
E51
25
F
carbemazepine, vigabatrin carbemazepine carbemazepine carbemazepine carbemazepine, vigabatrin carbemazepine carbemazepine, benzodiazipine, ACE inhibitor phenytoin
midal cells. As the software was calibrated in both the horizontal and vertical planes, neurone densities were calculated by dividing cell counts with the total scan area. Cell counts were obtained from ¢ve to eight cases. All values were expressed as mean þ S.E.M., and statistical comparisons were made using the unpaired Student's t-test (two-tailed). In all instances P 6 0.05 was considered statistically signi¢cant. Radioactive in situ hybridisation histochemistry The oligonucleotide probes and in situ hybridisation procedures used in this study were all as described previously (Whitaker et al., 2000). Two speci¢c antisense oligonucleotide probes (36-mers) were used for each K-subunit subtype, one located within the coding region and the other in the non-conserved 3P untranslated region (UTR). The coding region probes were chosen from regions of very low homology to avoid crosshybridisation; the 3P UTR probes shared no homology. The coding region probes were shown to be highly speci¢c by northern blot analysis using cell lines stably expressing each of the cloned subtypes (Whitaker et al., 2000). Control in situ hybridisations were carried out to ensure that, for all subtypes, each of the two oligonucleotides gave qualitatively similar results when used individually. Following synthesis the oligonucleotides were puri¢ed by reverse-phase, high performance liquid chromatography and 3P end-labelled with 25 WCi of [K-35 S]dATP (NEN, Hounslow, Middlesex, UK) using terminal deoxynucleotidyl transferase (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Cryostat sections (15 Wm) of the fresh frozen tissue were ¢xed in 4% neutral phosphate-bu¡ered paraformaldehyde (pH 7.4) and then hybridised with 5 fmol/ml of labelled probe as previously described (Whitaker et al., 2000). To de¢ne non-speci¢c hybridisation, human cerebellum sections were incubated with a hybridisation mixture containing 100-fold excess (500 fmol/ml) of unlabelled probe. With each probe speci¢c mRNA hybridisation was abolished in these experiments. Liquid emulsion radiography Sections were emulsion-dipped and counter-stained with Methylene Blue to obtain cellular resolution. Brie£y, slides were emulsion-dipped at 37³C in Ilford K5 emulsion and exposed for 4 weeks at 4³C. After exposure sections were processed in Ilford phenisol developer for 5 min, washed in 2% chrome alum, 2% sodium metabisulphite for 5 min, ¢xed in 30% sodium thiosulphate for 10 min and rinsed with distilled H2 O. Dried sections were counter-stained with Methylene Blue, dehydrated in a graded ethanol series, cleared with xylene, coverslipped with DePex medium (Gurr, BDH-Merck, Poole, UK) and visualised using light microscopy.
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Grain count analysis Grain counts were performed using the MCID system (see Two-dimensional cell counting). The software was `distance'calibrated by measuring a set distance of 100 Wm in both the horizontal and vertical planes, and all subsequent grain counts were expressed as grains/Wm2 /cell. A segmentation range was applied to di¡erentiate grain targets from background detritus, the software was adjusted to disregard grains of an area of less than 0.25 Wm2 and to estimate the number of grains present in clumps based upon this minimum grain area. These criteria were kept constant for each section studied. Following calibration `redirected counting' was performed which involved capturing the same image twice and instructing the software to count only silver grains overlying counter-stained cell bodies. For analysis of the stratum granulosum of the dentate gyrus a box of a ¢xed area of 370 Wm2 was used due to the di¤culty of counting individual cells that are so densely packed. For each section, grain counts were obtained from 50 cells in hilus, CA3, CA2 and CA1 (50 ¢xed areas for dentate gyrus) for each region studied from between ¢ve and eight cases. All values were expressed as mean þ S.E.M., and statistical comparisons were made using the unpaired Student's t-test (two-tailed).
RESULTS
Neuronal cell loss in resected epilepsy tissue Brain tissue for this study was obtained from eight temporal lobe resection patients and seven post-mortem control cases as indicated in Tables 1 and 2. To assess neuronal cell loss in the epilepsy cases, two-dimensional cell counts were compared in the normal and diseased tissue sections. Cell loss was observed in all regions of the hippocampal formation of the diseased patients, indicating that hippocampal sclerosis had occurred. These cell losses were statistically signi¢cant in all regions studied (P 9 0.05 unpaired Student's t-test, two-tailed) (Fig. 1). In the dentate granule cells, cell loss to 73 þ 14% control was observed. In the hilus, CA3 and CA1 cell counts in the epileptic tissue represented 60% ( þ 14%, 20% and 16% respectively) of control counts. In the CA2 sub¢eld cell counts performed upon epileptic resections were found to be 74 þ 14% of control tissue. In situ hybridisation of sodium channel K-subunits in human temporal lobe resections In situ hybridisation experiments were performed on both post-mortem and epileptic resection tissue using individual probe cocktails to types I, II, III and VI sodium channels as described previously (Whitaker et al., 2000). In post-mortem hippocampal tissue, gross dis-
Fig. 1. Mean cell loss seen in hippocampal neurones in resected hippocampal tissue (open bars; n = 5^7) compared with control tissue (grey bars; n = 7). Two-dimensional cell counts were performed on frozen sections (15 Wm thickness). Data are represented as mean þ S.E.M. Statistical analysis used unpaired Student's t-test (two-tailed), where *P 6 0.05. Abbreviations : DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus.
tribution of K-subunit mRNAs was identical to that seen in the previous study (Whitaker et al., 2000). Thus, types II and VI showed robust expression in the dentate gyrus and CA1^3 regions whereas types I and III showed moderate expression in the dentate gyrus and weak expression in the CA1^3 regions (Fig. 2). At the macroscopic level, autoradiographs obtained with the epileptic tissue from di¡erent patients showed consistent hybridisation patterns with a lower overall signal intensity compared to controls, probably due to the observed cell loss in the hippocampal subregions. Thus, to quantitate mRNA levels of the di¡erent subtypes expressed in speci¢c subregions, the sections were processed for liquid emulsion autoradiography and grain counts were performed on neuronal populations in both normal and epileptic tissues. Representative photomicrographs of silver grain deposition in dentate granule cells and hippocampal pyramidal cells from normal and epileptic human brains are shown in Fig. 3A^D. Grain counts were statistically analysed using the unpaired Student's t-test (two-tailed) and the results are summarised in Fig. 4. No signi¢cant di¡erences were found in the expression of types I and VI sodium channel mRNAs in epileptic tissue when compared with controls (P 9 0.05) (Fig. 4A, D). In contrast, for type II mRNA, a signi¢cant reduction of signal was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71 þ 7% of control, P 6 0.01), CA2 (81 þ 8% of control, P 6 0.05) and CA1 (72 þ 6% of control, P 6 0.05) as compared with control tissue (Fig. 3B). Additionally a signi¢cant increase in type III mRNA in
Table 2. Summary of clinico-pathological information of post-mortem tissue used in in situ hybridisation studies Case no.
Age (years)
Gender (M/F)
Post-mortem interval (h)
Cause of death
H98 H100 H76 H116 H108 H117 H111
60 55 67 18 58 64 46
M M M F M F M
18 23 6 10 16 17 10
myocardial infarction carbon monoxide poisoning ruptured aorta asphyxia coronary atherosclerosis coronary atherosclerosis coronary atherosclerosis
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Fig. 2. Macroscopic images showing the distribution of types I (A), II (B), III (C) and VI (D) mRNA in representative 15 Wm sections of post-mortem `control' (case H108) and `epileptic' (case E51) hippocampal formation. Abbreviations: DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus. Scale bars = 5 mm.
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Fig. 3. Cellular distribution of sodium channel types I (A), II (B), III (C) and VI (D) in representative tissue sections of post-mortem `control' (case H108) and `epileptic' (case E51) dentate gyrus (DG), hilar/CA4 region (H) and CA3, sub¢elds of the hippocampus (CA2, CA1 subregions are not shown but were similar to CA3 in each case). Note the observable cell loss seen in the epileptic tissue when compared with control. Scale bars = 100 Wm.
epileptic CA4 pyramidal cells (145 þ 7% of control, P 6 0.05) was observed (Fig. 4C).
DISCUSSION
The aim of this study was to investigate possible cellular changes in sodium channel K-subunit mRNA expression in the human epileptic hippocampus. Previous studies have indicated that a relative increase in the type I/II transcript ratio occurred in human epileptic hippocampi (Lombardo et al., 1996). The data presented here have revealed a signi¢cant down-regulation of the type II channel in pyramidal cells of CA3, CA2 and CA1 with no signi¢cant changes observed in the expression of the
type I channel. Down-regulation of the type II transcript may account for the previously observed increase in the type I/II ratio. The present report also provides the ¢rst investigation into the cellular distribution of the type III and VI channels in human epilepsy. Type III mRNA was found to be expressed at signi¢cantly higher levels in CA4 hilar cells in the epileptic hippocampus when compared with control. No signi¢cant changes in the expression of type VI mRNA were observed. Alternative interpretations of these results can be proposed. It is possible that the observed changes in sodium channel mRNA expression contribute to the pathophysiology of the epileptic condition. Recent studies using the whole-cell voltage clamp technique indicated there were changes in the sodium currents of kindled rat CA1 neu-
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Fig. 3 (B).
rones, when compared with matched controls, which might account for the hyperexcitability of these cells (Vreugdenhil et al., 1998). A small but signi¢cant positive shift in the voltage-dependence of steady-state inactivation (3 mV) coupled with an increased current amplitude (20%) was observed. It was proposed that such changes could enhance the excitability of these neurones, and contribute to the burst ¢ring seen in CA1 neurones following kindling. These changes could re£ect the down-regulation of the type II channel observed in CA1 in the present study. Increased burst ¢ring also occurs in the CA1 neurones from human temporal lobe epilepsy patients with hippocampal sclerosis (Knowles et al., 1992). Comparison of the properties of the cloned human brain K-subunits when expressed in mammalian cells shows that the type II inactivates at more depolarised potentials than the other subtypes (Clare et al., 2000). Based on this, a reduction in type II level might
be expected to cause a negative shift in inactivation of conglomerate sodium currents in CA1 neurones, rather than the positive shift reported. However, the e¡ects of type II down-regulation might not be detected in these experiments since it is present in axons rather than cell bodies (Westenbroek et al., 1989; Gong et al., 1999; Clare et al., 2000) where these recordings were made. The selective increase of type III mRNA observed in the hilus of resected hippocampi may also have consequences for overall cellular hyperexcitability. The rat type III sodium channel exhibits bi-modal gating when expressed in Xenopus oocytes, producing both fast and slow kinetics (Moorman et al., 1990). In a mammalian cell host, the human type III K-subunit gave currents with a prominent persistent component (Chen et al., 2000). Thus, it is possible that type III could contribute to the persistent sodium currents observed in hippocampal neurones (Hotson et al., 1979; French and Gage,
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Fig. 3 (C).
1985). Consistent with this, the rat type III (Westenbroek et al., 1992) and human type III (Whitaker et al., in press) sodium channel proteins are expressed in the soma of neurones, where persistent currents are predominantly generated (Llinas and Sugimori, 1980). If type III channels also contribute to persistent currents in vivo, it is conceivable that the observed up-regulation in epilepsy could lead to neuronal excitability by lowering the threshold for ¢ring of action potentials in hilar neurones. An alternative interpretation is that the observed alterations in expression of sodium channel mRNAs are not causative, but are secondary changes arising from the epileptic condition. For example, such changes may occur as a consequence of the neuronal loss and ensuing morphological rearrangements, which are a hallmark of the epileptic condition. In the dentate gyrus, excitatory mossy cells, the most numerous cells of the hilus, are
highly sensitive to seizure-induced cell death, when compared with GABAergic interneurones (Sloviter, 1987; Babb et al., 1989). It has been hypothesised that this selective loss of excitatory interneurones may remove excitatory input to inhibitory basket cells, leading them to remain `dormant' with subsequent disinhibition of dentate granule cells (Sloviter, 1991, 1994). This would lead to excessive ¢ring of granule cells, progressive cell death and the emergence of an epileptic condition. An alternative hypothesis postulates that hyperexcitability is caused by sprouting of mossy ¢bres to form synapses with dentate granule cells replacing the synapses lost due to the death of mossy cells. This mossy ¢bre sprouting may lead to excitatory granule cells innervating themselves resulting in a recurrent excitatory circuit (Nadler et al., 1980; Tauck and Nadler, 1985). Such excitatory innervation may cause the simultaneous sprouting of inhibitory interneurone ¢bres. The observed
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Fig. 3 (D).
increase of type III mRNA in polymorphic cells of the hilus in this study could serve to boost the post-synaptic responses of GABAergic interneurones in response to hyperexcitability of dentate granule cells. Interestingly, studies of dorsal root ganglion (DRG) neurones following axotomy have also indicated an increase in type III transcripts, with the DRG neurones reverting to an embryonic mode of sodium channel expression (Iwahashi et al., 1994; Waxman et al., 1994). This suggests the type III channel plays a role in regeneration and plasticity following neuronal injury. Other compensatory events could explain the changes in type II and type III mRNAs that we have observed in resected human hippocampi. A number of reports have suggested that a regulatory feedback may exist between the electrical activity of a neurone and sodium channel
density. Experiments using in vivo and in vitro administration of drugs and toxins that block electrical activity have revealed an up-regulation of the expression of rat cardiac (Taouis et al., 1991; Du¡ et al., 1992), skeletal muscle (Sherman and Catterall, 1984; Brodie et al., 1989; O¡ord and Catterall, 1989) and mouse brain (Sashihara et al., 1994) sodium channels. Conversely, down-regulation of sodium channels has been found to occur when channel activators, which increase electrical activity, are used (O¡ord and Catterall, 1989; Dargent and Couraud, 1990; Du¡ et al., 1992). This is consistent with the increased human type III mRNA level observed in the epileptic hilus, since all the brain samples were from patients undergoing treatment with sodium channel blockers (see Table 1). However, the down-regulation of human type II mRNA in the CA1, CA2 and CA3
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Fig. 4. Expression levels of sodium channel type I (A), II (B), III (C) and VI (D) mRNAs in resected hippocampal tissue (open bars; n = 5^8) as compared with post-mortem control hippocampal samples (grey bars; n = 7). Data are represented as mean þ S.E.M. Statistical analysis used unpaired Student's t-test (two-tailed), where *P 6 0.05 and **P 6 0.01. Abbreviations: DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus.
pyramidal cells cannot be so explained. Other studies of sodium channel expression in tottering mice (Tg), which show prolonged hyperexcitability and spontaneous epilepsy, have also revealed a down-regulation of sodium channels in nerve terminals (Willow et al., 1986). Unfortunately, it is not possible to control for drug e¡ects with human epilepsy tissue since resections are only carried out following drug therapy. Adjacent tissues that are also removed with the hippocampus, e.g. temporal cortex, do not necessarily provide reliable controls since changes in gene expression can occur within these regions also (Grigorenko et al., 1997). There is evidence that the high levels of neuronal activity occurring during seizures are associated with changes in gene expression. Examples include seizure-induced upregulation of a number of `immediate early genes' (Morgan and Curran, 1991; Kiessling and Gass, 1993; Labiner et al., 1993) and neurotrophic factors (Gall and Isackson, 1989; Enfors et al., 1991; Gall, 1993; Gall et al., 1994). Altered sodium channel levels seen in resected human tissue may be directly related to the increased expression of such genes. Indeed, it has been shown that type II mRNA and proteins are up-regulated in response to nerve growth factor in PC-12 cells (Mandel et al., 1988; D'Arcangelo et al., 1993) and cultured DRG neurones (Zur et al., 1995). Additionally, changes in sodium channel expression could be in response to the altered levels of other ion channels. For example, a decrease in the expression of voltage dependent potassium channels in rat brain during pentylenetetrazol-
induced seizures has been observed (Tsaur et al., 1992). This may cause increased neuronal excitability, in turn bringing about changes in sodium channel expression. In considering these alternative interpretations certain factors must be taken into account. A number of uncontrollable factors are inherent with studies using human tissue, which may a¡ect interpretation of the data. Due to a lack of surgically removed `normal' tissue, post-mortem samples must be used as controls making it di¤cult to control for factors such as age and post-mortem interval. The mean age of post-mortem tissue used in this study was 53 years compared with 31 years for postsurgical tissue. To date no e¡ect of age on the expression of voltage-gated sodium channels in human brain has been observed (Whitaker et al., 2000). Although temporal changes in expression of both mRNA and protein have been reported for rat, the most striking changes occurred at the late embryonic and early post-natal stages (Beckh et al., 1989; Scheinman et al., 1989; Felts et al., 1997; Gong et al., 1999). The relative stability of mRNA in post-mortem tissue compared to postsurgical tissue may also have an in£uence. However, it has previously been reported that a post-mortem interval of up to 36 h did not a¡ect RNA stability (Barton et al., 1993) and in our study the longest interval was only 23 h. Pre-mortem events that a¡ect the agonal state of tissue could also be important in the stability of RNA in postmortem tissue. Tissue pH is reported to be an indicator of agonal state and mRNA preservation (Kingsbury et al., 1995), a lower pH indicating a greater extent of ter-
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minal hypoxia. Tissue pH for post-mortem tissue used in this study was between 6 and 7 (data not shown).
CONCLUSION
This study reports the cellular expression of sodium channel K-subunit mRNAs in resected human epilepsy tissue compared with control tissue. Grain count analysis of the epilepsy tissue revealed a signi¢cant reduction of type II sodium channel mRNAs in CA3^CA1 pyramidal cells and an increase in type III mRNA in the hilus when compared with control tissue. Whether these changes play a causal role in human epilepsy or whether they
occur secondary to seizures and/or drug treatment is not known. However, it is likely that such changes would a¡ect the intrinsic excitability of hippocampal neurones. More research into the functions that these channels play in vivo is necessary to further characterise their roles in human epilepsy.
AcknowledgementsöThis work was supported by a BBSRC/ GlaxoWellcome studentship awarded to W.R.J.W. and grants from the New Zealand Health Research Council and the New Zealand Neurological Foundation to R.L.M.F. We would like to thank Mr Ian King for help with the emulsion dipping and preparation of sections for grain counting.
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A threshold sodium current in pyramidal cells in rat hippocampus. Neurosci. Lett. 56, 289^293. Gall, C.M., 1993. Seizure-induced changes in neurotrophin expression: Implications for epilepsy. Exp. Neurol. 124, 150^166. Gall, C.M., Isackson, P.J., 1989. Limbic seizures increase neuronal production of messenger RNA for nerve growth factor. Science 245, 758^761. Gall, C.M., Berschauer, R., Isackson, P.J., 1994. Seizures increase basic ¢broblast growth factor mRNA in adult rat forebrain neurons and glia. Mol. Brain Res. 21, 190^205. Gastaldi, M., Bartolomei, F., Massacrier, A., Planells, R., Robaglia-Schlupp, A., Cau, P., 1997. Increase in mRNAs encoding neonatal II and III sodium channel K-isoforms during kainate-induced seizures in adult rat hippocampus. Mol. Brain Res. 44, 179^190. Gastaldi, M., Robaglia-Schlupp, A., Massacrier, A., Planells, R., Cau, P., 1998. mRNA coding for voltage-gated sodium channel L2 subunit in rat central nervous system : Cellular distribution and changes following kainate-induced seizures. Neurosci. Lett. 249, 53^56. Gong, B., Rhodes, K.J., Bekele-Arcuri, Z., Trimmer, J.S., 1999. Type I and Type II Na channel K-subunit polypeptides exhibit distinct spatial and temporal patterning, and association with auxiliary subunits in rat brain. J. Comp. Neurol. 412, 342^352. Grigorenko, E., Glazier, S., Bell, W., Tytell, M., Nosel, E., Pons, T., Deadwyler, S.A., 1997. Changes in glutamate receptor subunit composition in hippocampus and cortex in patients with refractory epilepsy. J. Neurol. Sci. 153, 35^45. Hotson, J.R., Prince, D.A., Schwartzkroin, P.A., 1979. Anomalous inward recti¢cation in hippocampal neurons. J. Neurophysiol. 42, 889^895. Iwahashi, Y., Furuyama, T., Inagaki, S., Morita, Y., Takagi, H., 1994. Distinct regulation of sodium channel types I, II and III following nerve transection. Mol. Brain Res. 22, 341^345. Kiessling, M., Gass, P., 1993. Immediate early gene expression in experimental epilepsy. Brain Pathol. 3, 381^393. Kingsbury, A.E., Foster, O.J.F., Nisbet, A.P., Cairns, N., Bray, L., Eve, D.J., Lees, A.J., Marsden, C.D., 1995. Tissue pH as an indicator of mRNA preservation in human post-mortem brain. Mol. Brain Res. 28, 311^318. Knowles, W.D., Awad, I.A., Nayel, M.H., 1992. Di¡erences of in vitro electrophysiology of hippocampal neurons from epileptic patients with mesiotemporal sclerosis versus structural lesions. Epilepsia 33, 601^609. Labiner, D.M., Butler, L.S., Cau, Z., Hosford, D.A., Shin, C., McNamara, J.O., 1993. Induction of c-fos mRNA by kindled seizures: Complex relationship with neuronal burst ¢ring. J. Neurosci. 13, 744^751. Llinas, R., Sugimori, M., 1980. Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. J. 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Lombardo, A.J., Kuzniecky, R., Powers, R.E., Brown, G.B., 1996. Altered brain sodium channel transcript levels in human epilepsy. Mol. Brain Res. 35, 84^90. Mandel, G., Cooperman, S.S., Maue, R.A., Goodman, R.H., Brehm, P., 1988. Selective induction of brain type II Na channels by nerve growth factor. Proc. Natl. Acad. Sci. USA 85, 924^928. Moorman, J.R., Kirsch, G.E., VanDongen, A.M.J., Joho, R.H., Brown, A.M., 1990. Fast and slow gating of sodium channels encoded by a single mRNA. Neuron 4, 243^252. Morgan, J.I., Curran, T., 1991. Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci. 14, 421^451. Nadler, J.V., Perry, B.W., Cotman, C.W., 1980. Selective reinnervation of hippocampal area CA1 and the fascia dentata after destruction of CA3CA4 a¡erents with kainic acid. Brain Res. 182, 1^9. O¡ord, J., Catterall, W.A., 1989. Electrical activity, cAMP, and cytosolic calcium regulate mRNA encoding sodium channel K subunits in rat muscle cells. Neuron 2, 1447^1452. Ragsdale, D.S., Avoli, M., 1998. Sodium channels as molecular targets for antiepileptic drugs. Brain Res. Rev. 26, 16^28. Sashihara, S., Yanagihara, N., Kobayashi, H., Izumi, F., Tsuji, S., Murai, Y., Mita, T., 1992. Overproduction of voltage-dependent Na channels in the developing brain of genetically seizure-susceptible El mice. Neuroscience 48, 285^291. Sashihara, S., Yanagihara, N., Izumi, F., Murai, Y., Mita, T., 1994. Di¡erential up-regulation of voltage-dependent Na channels induced by phenytoin in brains of genetically seizure-susceptible (E1) and control (ddY) mice. Neuroscience 62, 803^811. Scheinman, R.I., Auld, V.J., Goldin, A.L., Davidson, N., Dunn, R.J., Catterall, W.A., 1989. Developmental regulation of sodium channel expression in the rat forebrain. J. Biol. Chem. 264, 10660^10666. Sherman, S.J., Catterall, W.A., 1984. Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells. Proc. Natl. Acad. Sci. USA 81, 262^266. Sloviter, R.S., 1987. Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy. Science 235, 73^76. Sloviter, R.S., 1991. Feedforward and feedback inhibition of hippocampal principal cell activity evoked by perforant path stimulation: GABAmediated mechanisms that regulate excitability in vivo. Hippocampus 1, 31^40. Sloviter, R.S., 1994. The functional organization of the hippocampal dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy. Ann. Neurol. 35, 640^654. Taouis, M., Sheldon, R.S., Du¡, H.J., 1991. Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug. J. Clin. Invest. 88, 375^378. Tauck, D.L., Nadler, J.V., 1985. Evidence of functional mossy sprouting in hippocampal formation of kainic acid-treated rats. J. Neurosci. 5, 1016^1022. Tsaur, M.-L., Sheng, M., Lowenstein, D.H., Jan, Y.N., Jan, L.Y., 1992. Di¡erential expression of K channel mRNAs in the rat brain and downregulation in the hippocampus following seizures. Neuron 8, 1055^1067. Vreugdenhil, M., Faas, G.C., Wadman, W.J., 1998. Sodium currents in isolated rat CA1 neurons after kindling epileptogenesis. Neuroscience 86, 99^107. Wallace, R.H., Wang, D.W., Singh, R., Sche¡er, I.E., George, A.L., Phillips, H.A., Saar, K., Reis, A., Johnson, E.W., Sutherland, G.R., Berkovic, S.F., Mulley, J.C., 1998. Febrile seizures and generalized epilepsy associated with a mutation in the Na -channel beta1 subunit gene SCN1B. Nat. Genet. 19, 337^366. Waxman, S.G., Kocsis, J.D., Black, J.A., 1994. Type III channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy. J. Neurophysiol. 72, 466^470. Westenbroek, R.E., Merrick, D.K., Catterall, W.A., 1989. Di¡erential subcellular localization of the RI and RII Na channel subtypes in central neurons. Neuron 3, 695^704. Westenbroek, R.E., Noebels, J.L., Catterall, W.A., 1992. Elevated expression of type II Na channels in hypomyelinated axons of Shiverer mouse brain. J. Neurosci. 12, 2259^2267. Whitaker, W.R.J., Faull, R.L.M., Waldvogel, H., Plumpton, C.J., Emson, P.C., Clare, J.J., Comparative distribution of the voltage-gated sodium channel proteins in human brain. Mol. Brain Res. 88, 37^53. Whitaker, W.R.J., Clare, J.J., Powell, A.J., Chen, Y.H., Faull, R.L.M., Emson, P.C., 2000. Distribution of voltage-gated sodium channel K and L subunit mRNAs in human cerebellum, cortex and hippocampal formation. J. Comp. Neurol. 422, 123^139. Willow, M., Taylor, S.M., Catterall, W.A., Finnell, R.H., 1986. Down regulation of sodium channels in nerve terminals of spontaneously epileptic mice. Cell. Mol. Neurobiol. 6, 213^220. Zur, K.B., Oh, Y., Waxman, S.G., Black, J.A., 1995. Di¡erential up-regulation of sodium channel K- and L1 subunit mRNAs in cultured embryonic DRG neurons following exposure to NGF. Mol. Brain Res. 30, 97^103. (Accepted 2 May 2001)
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www.elsevier.com/locate/neuroscience
CHANGES IN THE mRNAs ENCODING VOLTAGE-GATED SODIUM CHANNEL TYPES II AND III IN HUMAN EPILEPTIC HIPPOCAMPUS W. R. J. WHITAKER,a R. L. M. FAULL,b M. DRAGUNOW,b E. W. MEE,c P. C. EMSONa and J. J. CLAREd * a b
Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
Departments of Anatomy and Pharmacology, University of Auckland, Private Bag 92019, Auckland, New Zealand c
d
Department of Neurosurgery, Auckland Hospital, Auckland, New Zealand
Department of Molecular Pharmacology, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
AbstractöStudies with animal seizure models have indicated that changes in temporal and spatial expression of voltagegated sodium channels may be important in the pathology of epilepsy. Here, by using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes [Whitaker et al. (2000) J. Comp. Neurol. 422, 123^139], we have compared the cellular expression of all four brain K-subunit sodium channel mRNAs in `normal' and epileptic hippocampi from humans. Neuronal cell loss was observed in all regions of the hippocampus of diseased patients, indicating that sclerosis had occurred. Losses of up to 40% compared to post-mortem controls were observed which were statistically signi¢cant in all regions studied (dentate gyrus, hilus, and CA1^3). To assess mRNA levels of the di¡erent K-subtypes in speci¢c subregions, control and diseased tissue sections were hybridised to subtype-speci¢c probes. To quantify any changes in expression while allowing for cell loss, the sections were processed for liquid emulsion autoradiography and grain counts were performed on populations of individual neurones in di¡erent subregions. No signi¢cant di¡erences were found in the expression of type I and VI mRNAs. In contrast, a signi¢cant down-regulation of type II mRNA was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71 þ 7% of control, P 6 0.01), CA2 (81 þ 8% of control, P 6 0.05) and CA1 (72 þ 6% of control, P 6 0.05) compared with control tissue. Additionally, a signi¢cant up-regulation in type III mRNA in epileptic CA4 pyramidal cells (145 þ 7% of control, P 6 0.05) was observed. It is not clear whether these changes play a causal role in human epilepsy or whether they are secondary to seizures or drug treatment; further studies are necessary to investigate these alternatives. However, it is likely that such changes would a¡ect the intrinsic excitability of hippocampal neurones. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: sodium channels, human brain, mRNA distribution, epilepsy.
K-subunit, of which four subtypes (types I, II, III and VI) are found in brain, associated with one or more auxiliary L-subunits. Analysis of sodium channel K-subunit mRNAs following kainic acid-induced seizures in rat hippocampus revealed a marked increase in type III mRNA expression, a modest increase in type II mRNA signal and no change in type I mRNA expression (Bartolomei et al., 1997). Studies using reverse transcriptase-polymerase chain reaction and in situ hybridisation indicate that seizure activity induces alterations in the developmental splicing of neonatal and adult type II and III isoforms (Gastaldi et al., 1997). Seizures were found to re-activate the neonatal splicing event, causing an increase in the presence of the neonatal type II and III isoforms in localised regions of the adult rat brain. More recently, a biphasic response of L2-subunit transcription has been reported following kainic acid administration (Gastaldi et al., 1998). An initial increase in L2-subunit expression was followed by a decrease over a time course that paralleled the increase seen in K-subunit expression. Other studies, using non-selective sodium channel probes, have indicated increases in sodium channel
Voltage-gated sodium channels play a critical role in the initiation and transmission of action potentials and are therefore likely to be important in the aetiology of epilepsy. This is illustrated by the fact that several established anti-epileptic drugs are known to inhibit sodium channels, e.g. phenytoin, carbemazepine, lamotrigine (Ragsdale and Avoli, 1998; Clare et al., 2000). Additionally, it has recently been found that sodium channel mutations are responsible for certain inherited forms of epilepsy (generalised epilepsy with febrile seizures plus, GEFS+, types I and II; Wallace et al., 1998; Escayg et al., 2000). Studies with animal seizure models have also indicated the involvement of voltage-gated sodium channels in epilepsy. The channels consist of a large pore forming
*Corresponding author. Tel.: +44-1438-763834; fax: +44-1438764488. E-mail address: [email protected] (J. J. Clare). Abbreviations : DRG, dorsal rot ganglion; MCID, micro computer imaging device; UTR, untranslated region. 275
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mRNA and protein in the cortex of genetically seizure susceptible E1 mice brains (Sashihara et al., 1992). Conversely, in the brains of tottering (Tg) mice, which spontaneously undergo spike wave seizures due to mutations of the P/Q-type calcium channel (Fletcher et al., 1996), a decrease in sodium channel proteins in nerve terminals was observed as detected by [3 H]saxitoxin binding (Willow et al., 1986). Interestingly, the remaining channels showed an 80% increase in activity, as measured by radioisotopic sodium in£ux. Whilst studies with animal models have indicated that changes in the temporal and spatial expression of sodium channels may be important in the pathology of epilepsy, only a single human study has been performed to date. Such studies are important, not only in furthering our understanding of the human epileptic condition, but also for the development of more selective anti-epileptic sodium channel inhibitors by identifying subtypes of key relevance to the disease. The previous human study uncovered an altered ratio of types I and II mRNA in hippocampi resected from temporal lobe epilepsy patients compared to control tissue (Lombardo et al., 1996). However, this was performed using the ligase detection assay, which is not a well-established technique, did not include types III and VI and did not provide information at the cellular level. Thus, a major aim of the present study was to compare the expression of all four brain K-subunit mRNAs in the `normal' and epileptic hippocampal formation from humans. By using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes (Whitaker et al., 2000) we have carried out the ¢rst detailed investigation of this type at a cellular level.
EXPERIMENTAL PROCEDURES
Human tissue and sample preparation Human brain tissue was obtained from the New Zealand Neurological Foundation Human Brain Bank at the University of Auckland using ethical consent procedures approved by the University of Auckland Human Subjects Ethics Committee and the Auckland Ethics Committees. Samples used in this study were obtained from eight temporal lobe resection patients and seven post-mortem control cases. Tables 1 and 2 summarise the available clinico-pathological data for these samples. Tissue was obtained as snap frozen blocks stored at 380³C which were sectioned for in situ hybridisation on a freezing cryostat (Bright Instruments, Cambs, UK). Blocks were removed from storage at 380³C and left in the cryostat chamber for 1 h at 320³C to equilibrate prior to sectioning. Sections (15 Wm) were dissected from the block and mounted onto electrostatically charged microscope slides (Superfrost plus, Gurr, BDH-Merck, Poole, UK) and then dried using a hair-dryer before storage at 370³C until use. Two-dimensional cell counting Cell counts were performed using a Viglen PC running micro computer imaging device (MCID) software (Imaging Research, ON, Canada) linked to a Leica DMRBE microscope via a high resolution charged couple device (CCD) digital camera (MTI CCD 72) following the manufacturer's protocol. Populations of cells were counted in a de¢ned area of 100 Wm by 50 Wm for dentate granule cells and 250 Wm by 250 Wm for CA pyra-
Table 1. Summary of clinico-pathological information of patients having undergone surgery for intractable idiopathic epilepsy, whose tissue was used in in situ hybridisation studies Case no.
Age (years)
Gender (M/F)
Drug therapy
E19
20
F
E45 E40 E23 E41
29 32 34 43
M M F F
E47 E50
25 42
M F
E51
25
F
carbemazepine, vigabatrin carbemazepine carbemazepine carbemazepine carbemazepine, vigabatrin carbemazepine carbemazepine, benzodiazipine, ACE inhibitor phenytoin
midal cells. As the software was calibrated in both the horizontal and vertical planes, neurone densities were calculated by dividing cell counts with the total scan area. Cell counts were obtained from ¢ve to eight cases. All values were expressed as mean þ S.E.M., and statistical comparisons were made using the unpaired Student's t-test (two-tailed). In all instances P 6 0.05 was considered statistically signi¢cant. Radioactive in situ hybridisation histochemistry The oligonucleotide probes and in situ hybridisation procedures used in this study were all as described previously (Whitaker et al., 2000). Two speci¢c antisense oligonucleotide probes (36-mers) were used for each K-subunit subtype, one located within the coding region and the other in the non-conserved 3P untranslated region (UTR). The coding region probes were chosen from regions of very low homology to avoid crosshybridisation; the 3P UTR probes shared no homology. The coding region probes were shown to be highly speci¢c by northern blot analysis using cell lines stably expressing each of the cloned subtypes (Whitaker et al., 2000). Control in situ hybridisations were carried out to ensure that, for all subtypes, each of the two oligonucleotides gave qualitatively similar results when used individually. Following synthesis the oligonucleotides were puri¢ed by reverse-phase, high performance liquid chromatography and 3P end-labelled with 25 WCi of [K-35 S]dATP (NEN, Hounslow, Middlesex, UK) using terminal deoxynucleotidyl transferase (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). Cryostat sections (15 Wm) of the fresh frozen tissue were ¢xed in 4% neutral phosphate-bu¡ered paraformaldehyde (pH 7.4) and then hybridised with 5 fmol/ml of labelled probe as previously described (Whitaker et al., 2000). To de¢ne non-speci¢c hybridisation, human cerebellum sections were incubated with a hybridisation mixture containing 100-fold excess (500 fmol/ml) of unlabelled probe. With each probe speci¢c mRNA hybridisation was abolished in these experiments. Liquid emulsion radiography Sections were emulsion-dipped and counter-stained with Methylene Blue to obtain cellular resolution. Brie£y, slides were emulsion-dipped at 37³C in Ilford K5 emulsion and exposed for 4 weeks at 4³C. After exposure sections were processed in Ilford phenisol developer for 5 min, washed in 2% chrome alum, 2% sodium metabisulphite for 5 min, ¢xed in 30% sodium thiosulphate for 10 min and rinsed with distilled H2 O. Dried sections were counter-stained with Methylene Blue, dehydrated in a graded ethanol series, cleared with xylene, coverslipped with DePex medium (Gurr, BDH-Merck, Poole, UK) and visualised using light microscopy.
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Grain count analysis Grain counts were performed using the MCID system (see Two-dimensional cell counting). The software was `distance'calibrated by measuring a set distance of 100 Wm in both the horizontal and vertical planes, and all subsequent grain counts were expressed as grains/Wm2 /cell. A segmentation range was applied to di¡erentiate grain targets from background detritus, the software was adjusted to disregard grains of an area of less than 0.25 Wm2 and to estimate the number of grains present in clumps based upon this minimum grain area. These criteria were kept constant for each section studied. Following calibration `redirected counting' was performed which involved capturing the same image twice and instructing the software to count only silver grains overlying counter-stained cell bodies. For analysis of the stratum granulosum of the dentate gyrus a box of a ¢xed area of 370 Wm2 was used due to the di¤culty of counting individual cells that are so densely packed. For each section, grain counts were obtained from 50 cells in hilus, CA3, CA2 and CA1 (50 ¢xed areas for dentate gyrus) for each region studied from between ¢ve and eight cases. All values were expressed as mean þ S.E.M., and statistical comparisons were made using the unpaired Student's t-test (two-tailed).
RESULTS
Neuronal cell loss in resected epilepsy tissue Brain tissue for this study was obtained from eight temporal lobe resection patients and seven post-mortem control cases as indicated in Tables 1 and 2. To assess neuronal cell loss in the epilepsy cases, two-dimensional cell counts were compared in the normal and diseased tissue sections. Cell loss was observed in all regions of the hippocampal formation of the diseased patients, indicating that hippocampal sclerosis had occurred. These cell losses were statistically signi¢cant in all regions studied (P 9 0.05 unpaired Student's t-test, two-tailed) (Fig. 1). In the dentate granule cells, cell loss to 73 þ 14% control was observed. In the hilus, CA3 and CA1 cell counts in the epileptic tissue represented 60% ( þ 14%, 20% and 16% respectively) of control counts. In the CA2 sub¢eld cell counts performed upon epileptic resections were found to be 74 þ 14% of control tissue. In situ hybridisation of sodium channel K-subunits in human temporal lobe resections In situ hybridisation experiments were performed on both post-mortem and epileptic resection tissue using individual probe cocktails to types I, II, III and VI sodium channels as described previously (Whitaker et al., 2000). In post-mortem hippocampal tissue, gross dis-
Fig. 1. Mean cell loss seen in hippocampal neurones in resected hippocampal tissue (open bars; n = 5^7) compared with control tissue (grey bars; n = 7). Two-dimensional cell counts were performed on frozen sections (15 Wm thickness). Data are represented as mean þ S.E.M. Statistical analysis used unpaired Student's t-test (two-tailed), where *P 6 0.05. Abbreviations : DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus.
tribution of K-subunit mRNAs was identical to that seen in the previous study (Whitaker et al., 2000). Thus, types II and VI showed robust expression in the dentate gyrus and CA1^3 regions whereas types I and III showed moderate expression in the dentate gyrus and weak expression in the CA1^3 regions (Fig. 2). At the macroscopic level, autoradiographs obtained with the epileptic tissue from di¡erent patients showed consistent hybridisation patterns with a lower overall signal intensity compared to controls, probably due to the observed cell loss in the hippocampal subregions. Thus, to quantitate mRNA levels of the di¡erent subtypes expressed in speci¢c subregions, the sections were processed for liquid emulsion autoradiography and grain counts were performed on neuronal populations in both normal and epileptic tissues. Representative photomicrographs of silver grain deposition in dentate granule cells and hippocampal pyramidal cells from normal and epileptic human brains are shown in Fig. 3A^D. Grain counts were statistically analysed using the unpaired Student's t-test (two-tailed) and the results are summarised in Fig. 4. No signi¢cant di¡erences were found in the expression of types I and VI sodium channel mRNAs in epileptic tissue when compared with controls (P 9 0.05) (Fig. 4A, D). In contrast, for type II mRNA, a signi¢cant reduction of signal was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71 þ 7% of control, P 6 0.01), CA2 (81 þ 8% of control, P 6 0.05) and CA1 (72 þ 6% of control, P 6 0.05) as compared with control tissue (Fig. 3B). Additionally a signi¢cant increase in type III mRNA in
Table 2. Summary of clinico-pathological information of post-mortem tissue used in in situ hybridisation studies Case no.
Age (years)
Gender (M/F)
Post-mortem interval (h)
Cause of death
H98 H100 H76 H116 H108 H117 H111
60 55 67 18 58 64 46
M M M F M F M
18 23 6 10 16 17 10
myocardial infarction carbon monoxide poisoning ruptured aorta asphyxia coronary atherosclerosis coronary atherosclerosis coronary atherosclerosis
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Fig. 2. Macroscopic images showing the distribution of types I (A), II (B), III (C) and VI (D) mRNA in representative 15 Wm sections of post-mortem `control' (case H108) and `epileptic' (case E51) hippocampal formation. Abbreviations: DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus. Scale bars = 5 mm.
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Fig. 3. Cellular distribution of sodium channel types I (A), II (B), III (C) and VI (D) in representative tissue sections of post-mortem `control' (case H108) and `epileptic' (case E51) dentate gyrus (DG), hilar/CA4 region (H) and CA3, sub¢elds of the hippocampus (CA2, CA1 subregions are not shown but were similar to CA3 in each case). Note the observable cell loss seen in the epileptic tissue when compared with control. Scale bars = 100 Wm.
epileptic CA4 pyramidal cells (145 þ 7% of control, P 6 0.05) was observed (Fig. 4C).
DISCUSSION
The aim of this study was to investigate possible cellular changes in sodium channel K-subunit mRNA expression in the human epileptic hippocampus. Previous studies have indicated that a relative increase in the type I/II transcript ratio occurred in human epileptic hippocampi (Lombardo et al., 1996). The data presented here have revealed a signi¢cant down-regulation of the type II channel in pyramidal cells of CA3, CA2 and CA1 with no signi¢cant changes observed in the expression of the
type I channel. Down-regulation of the type II transcript may account for the previously observed increase in the type I/II ratio. The present report also provides the ¢rst investigation into the cellular distribution of the type III and VI channels in human epilepsy. Type III mRNA was found to be expressed at signi¢cantly higher levels in CA4 hilar cells in the epileptic hippocampus when compared with control. No signi¢cant changes in the expression of type VI mRNA were observed. Alternative interpretations of these results can be proposed. It is possible that the observed changes in sodium channel mRNA expression contribute to the pathophysiology of the epileptic condition. Recent studies using the whole-cell voltage clamp technique indicated there were changes in the sodium currents of kindled rat CA1 neu-
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Fig. 3 (B).
rones, when compared with matched controls, which might account for the hyperexcitability of these cells (Vreugdenhil et al., 1998). A small but signi¢cant positive shift in the voltage-dependence of steady-state inactivation (3 mV) coupled with an increased current amplitude (20%) was observed. It was proposed that such changes could enhance the excitability of these neurones, and contribute to the burst ¢ring seen in CA1 neurones following kindling. These changes could re£ect the down-regulation of the type II channel observed in CA1 in the present study. Increased burst ¢ring also occurs in the CA1 neurones from human temporal lobe epilepsy patients with hippocampal sclerosis (Knowles et al., 1992). Comparison of the properties of the cloned human brain K-subunits when expressed in mammalian cells shows that the type II inactivates at more depolarised potentials than the other subtypes (Clare et al., 2000). Based on this, a reduction in type II level might
be expected to cause a negative shift in inactivation of conglomerate sodium currents in CA1 neurones, rather than the positive shift reported. However, the e¡ects of type II down-regulation might not be detected in these experiments since it is present in axons rather than cell bodies (Westenbroek et al., 1989; Gong et al., 1999; Clare et al., 2000) where these recordings were made. The selective increase of type III mRNA observed in the hilus of resected hippocampi may also have consequences for overall cellular hyperexcitability. The rat type III sodium channel exhibits bi-modal gating when expressed in Xenopus oocytes, producing both fast and slow kinetics (Moorman et al., 1990). In a mammalian cell host, the human type III K-subunit gave currents with a prominent persistent component (Chen et al., 2000). Thus, it is possible that type III could contribute to the persistent sodium currents observed in hippocampal neurones (Hotson et al., 1979; French and Gage,
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Fig. 3 (C).
1985). Consistent with this, the rat type III (Westenbroek et al., 1992) and human type III (Whitaker et al., in press) sodium channel proteins are expressed in the soma of neurones, where persistent currents are predominantly generated (Llinas and Sugimori, 1980). If type III channels also contribute to persistent currents in vivo, it is conceivable that the observed up-regulation in epilepsy could lead to neuronal excitability by lowering the threshold for ¢ring of action potentials in hilar neurones. An alternative interpretation is that the observed alterations in expression of sodium channel mRNAs are not causative, but are secondary changes arising from the epileptic condition. For example, such changes may occur as a consequence of the neuronal loss and ensuing morphological rearrangements, which are a hallmark of the epileptic condition. In the dentate gyrus, excitatory mossy cells, the most numerous cells of the hilus, are
highly sensitive to seizure-induced cell death, when compared with GABAergic interneurones (Sloviter, 1987; Babb et al., 1989). It has been hypothesised that this selective loss of excitatory interneurones may remove excitatory input to inhibitory basket cells, leading them to remain `dormant' with subsequent disinhibition of dentate granule cells (Sloviter, 1991, 1994). This would lead to excessive ¢ring of granule cells, progressive cell death and the emergence of an epileptic condition. An alternative hypothesis postulates that hyperexcitability is caused by sprouting of mossy ¢bres to form synapses with dentate granule cells replacing the synapses lost due to the death of mossy cells. This mossy ¢bre sprouting may lead to excitatory granule cells innervating themselves resulting in a recurrent excitatory circuit (Nadler et al., 1980; Tauck and Nadler, 1985). Such excitatory innervation may cause the simultaneous sprouting of inhibitory interneurone ¢bres. The observed
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Fig. 3 (D).
increase of type III mRNA in polymorphic cells of the hilus in this study could serve to boost the post-synaptic responses of GABAergic interneurones in response to hyperexcitability of dentate granule cells. Interestingly, studies of dorsal root ganglion (DRG) neurones following axotomy have also indicated an increase in type III transcripts, with the DRG neurones reverting to an embryonic mode of sodium channel expression (Iwahashi et al., 1994; Waxman et al., 1994). This suggests the type III channel plays a role in regeneration and plasticity following neuronal injury. Other compensatory events could explain the changes in type II and type III mRNAs that we have observed in resected human hippocampi. A number of reports have suggested that a regulatory feedback may exist between the electrical activity of a neurone and sodium channel
density. Experiments using in vivo and in vitro administration of drugs and toxins that block electrical activity have revealed an up-regulation of the expression of rat cardiac (Taouis et al., 1991; Du¡ et al., 1992), skeletal muscle (Sherman and Catterall, 1984; Brodie et al., 1989; O¡ord and Catterall, 1989) and mouse brain (Sashihara et al., 1994) sodium channels. Conversely, down-regulation of sodium channels has been found to occur when channel activators, which increase electrical activity, are used (O¡ord and Catterall, 1989; Dargent and Couraud, 1990; Du¡ et al., 1992). This is consistent with the increased human type III mRNA level observed in the epileptic hilus, since all the brain samples were from patients undergoing treatment with sodium channel blockers (see Table 1). However, the down-regulation of human type II mRNA in the CA1, CA2 and CA3
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Fig. 4. Expression levels of sodium channel type I (A), II (B), III (C) and VI (D) mRNAs in resected hippocampal tissue (open bars; n = 5^8) as compared with post-mortem control hippocampal samples (grey bars; n = 7). Data are represented as mean þ S.E.M. Statistical analysis used unpaired Student's t-test (two-tailed), where *P 6 0.05 and **P 6 0.01. Abbreviations: DG, stratum granulosum of dentate gyrus; H, hilar/CA4 region of dentate gyrus; CA3, CA2, CA1, sub¢elds of the hippocampus.
pyramidal cells cannot be so explained. Other studies of sodium channel expression in tottering mice (Tg), which show prolonged hyperexcitability and spontaneous epilepsy, have also revealed a down-regulation of sodium channels in nerve terminals (Willow et al., 1986). Unfortunately, it is not possible to control for drug e¡ects with human epilepsy tissue since resections are only carried out following drug therapy. Adjacent tissues that are also removed with the hippocampus, e.g. temporal cortex, do not necessarily provide reliable controls since changes in gene expression can occur within these regions also (Grigorenko et al., 1997). There is evidence that the high levels of neuronal activity occurring during seizures are associated with changes in gene expression. Examples include seizure-induced upregulation of a number of `immediate early genes' (Morgan and Curran, 1991; Kiessling and Gass, 1993; Labiner et al., 1993) and neurotrophic factors (Gall and Isackson, 1989; Enfors et al., 1991; Gall, 1993; Gall et al., 1994). Altered sodium channel levels seen in resected human tissue may be directly related to the increased expression of such genes. Indeed, it has been shown that type II mRNA and proteins are up-regulated in response to nerve growth factor in PC-12 cells (Mandel et al., 1988; D'Arcangelo et al., 1993) and cultured DRG neurones (Zur et al., 1995). Additionally, changes in sodium channel expression could be in response to the altered levels of other ion channels. For example, a decrease in the expression of voltage dependent potassium channels in rat brain during pentylenetetrazol-
induced seizures has been observed (Tsaur et al., 1992). This may cause increased neuronal excitability, in turn bringing about changes in sodium channel expression. In considering these alternative interpretations certain factors must be taken into account. A number of uncontrollable factors are inherent with studies using human tissue, which may a¡ect interpretation of the data. Due to a lack of surgically removed `normal' tissue, post-mortem samples must be used as controls making it di¤cult to control for factors such as age and post-mortem interval. The mean age of post-mortem tissue used in this study was 53 years compared with 31 years for postsurgical tissue. To date no e¡ect of age on the expression of voltage-gated sodium channels in human brain has been observed (Whitaker et al., 2000). Although temporal changes in expression of both mRNA and protein have been reported for rat, the most striking changes occurred at the late embryonic and early post-natal stages (Beckh et al., 1989; Scheinman et al., 1989; Felts et al., 1997; Gong et al., 1999). The relative stability of mRNA in post-mortem tissue compared to postsurgical tissue may also have an in£uence. However, it has previously been reported that a post-mortem interval of up to 36 h did not a¡ect RNA stability (Barton et al., 1993) and in our study the longest interval was only 23 h. Pre-mortem events that a¡ect the agonal state of tissue could also be important in the stability of RNA in postmortem tissue. Tissue pH is reported to be an indicator of agonal state and mRNA preservation (Kingsbury et al., 1995), a lower pH indicating a greater extent of ter-
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minal hypoxia. Tissue pH for post-mortem tissue used in this study was between 6 and 7 (data not shown).
CONCLUSION
This study reports the cellular expression of sodium channel K-subunit mRNAs in resected human epilepsy tissue compared with control tissue. Grain count analysis of the epilepsy tissue revealed a signi¢cant reduction of type II sodium channel mRNAs in CA3^CA1 pyramidal cells and an increase in type III mRNA in the hilus when compared with control tissue. Whether these changes play a causal role in human epilepsy or whether they
occur secondary to seizures and/or drug treatment is not known. However, it is likely that such changes would a¡ect the intrinsic excitability of hippocampal neurones. More research into the functions that these channels play in vivo is necessary to further characterise their roles in human epilepsy.
AcknowledgementsöThis work was supported by a BBSRC/ GlaxoWellcome studentship awarded to W.R.J.W. and grants from the New Zealand Health Research Council and the New Zealand Neurological Foundation to R.L.M.F. We would like to thank Mr Ian King for help with the emulsion dipping and preparation of sections for grain counting.
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