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Keywords:

  • Status epilepticus;
  • Temporal lobe epilepsy;
  • Keppra;
  • Pharmacoresistance;
  • Tolerance.

Summary

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Purpose: We previously showed that gene expression of synaptic vesicle protein 2A (SV2A), the binding site for the antiepileptic drug levetiracetam, is reduced during epileptogenesis in the rat. Since absence of SV2A has been associated with increased epileptogenicity, changes in expression of SV2A could have consequences for the progression of epilepsy. Therefore we investigated hippocampal SV2A protein expression of temporal lobe epilepsy (TLE) patients and in rats during epileptogenesis and in the chronic epileptic phase.

Methods: SV2A immunocytochemistry and Western blot analysis were performed on the hippocampus of autopsy controls, patients that died from status epilepticus (SE), and pharmacoresistant TLE patients. In addition, in epileptic rats, SV2A expression was determined after SE during the acute, latent, and chronic epileptic phase.

Results: In control tissue, presynaptic SV2A was expressed in all hippocampal subfields, with strongest expression in mossy fiber terminals. SV2A positive puncta were distributed in a patchy pattern over the somata and dendrites of neurons. SV2A decreased throughout the hippocampus of TLE patients with hippocampal sclerosis (HS), compared to autopsy control, SE, and non-HS tissue. In most rats, SV2A was already decreased in the latent period especially in the inner molecular layer and stratum lucidum. Similarly as in humans, SV2A was also decreased throughout the hippocampus of chronic epileptic rats, specifically in rats with a progressive form of epilepsy.

Discussion: These data support previous findings that reduced expression of SV2A could contribute to the increased epileptogenicity. Whether this affects the effectiveness of levetiracetam needs to be further investigated.

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein present in synaptic vesicles of neurons and endocrine cells (Buckley & Kelly, 1985; Noyer et al., 1995). In mammals, three different SV2 genes are identified: SV2A, SV2B, and SV2C. The most abundant and widely expressed isoform is SV2A (Bajjalieh et al., 1993, 1994; Janz & Sudhof, 1999). This protein was identified as the binding site for the antiepileptic drug levetiracetam (LEV), both in animals as in humans (Lynch et al., 2004; Gillard et al., 2006). Recent data show that the novel LEV analogs, brivaracetam and seletracetam, also bind to SV2A (Bennett et al., 2007; von Rosenstiel, 2007). The positive correlation between binding of LEV analogs in rat brain and antiseizure potency (Noyer et al., 1995; Gillard et al., 2003) indicated that SV2A could be a novel target for antiepileptic drug therapy. Although the molecular action of SV2A is not known, it has been suggested to regulate synaptic vesicle exocytosis and to play a role in the homeostasis of synaptic vesicle constituents, such as ATP or calcium (Crowder et al., 1999; Janz et al., 1999). Knockout studies showed that SV2A gene disruption leads to a reduction in the action potential-dependent GABAergic neurotransmission in the CA3 subfield of the hippocampus (Crowder et al., 1999). In SV2A/SV2B knockouts, a sustained increase in Ca2+-dependent synaptic transmission was found in cultured hippocampal neurons (Janz et al., 1999). This led to the hypothesis that SV2 regulates presynaptic calcium and that loss of SV2 can lead to accumulation of calcium, causing an abnormal increase in neurotransmitter release that destabilizes neuronal circuits and induces epilepsy (Janz et al., 1999). SV2A knockout mice have spontaneous seizures and die within 3 weeks after birth (Crowder et al., 1999; Janz et al., 1999). Interestingly, we showed in our previous microarray study that gene expression of SV2A is reduced shortly after status epilepticus (SE) during epileptogenesis in the rat (Gorter et al., 2006). This suggests that changes in SV2A could have consequences for the progression of epilepsy. Therefore we investigated SV2A protein expression in rats during epileptogenesis and in the chronic epileptic phase and compared this with SV2A expression in human epileptic brain.

Materials and Methods

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Human material

Brain material was obtained from the files of the department of neuropathology of the Academic Medical Center (University of Amsterdam). Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. All tissue was obtained and used in a manner compliant with the Declaration of Helsinki. Two neuropathologists reviewed all cases independently. Patients underwent resection of the hippocampus (n = 16) for medically intractable epilepsy. In 11 patients, a pathological diagnosis of hippocampal sclerosis (HS) was made. In the other five patients, a focal lesion was observed (Table 1), but this did not involve the hippocampus (non-HS cases). This group was used as a comparison group to HS cases. In addition, autopsy material was used from two epilepsy patients that died during an acute SE. These patients had history of epilepsy and died before pharmacological treatment was started to stop the SE. Pathological examination excluded encephalitis or meningitis in these patients. Control hippocampal tissue was obtained at autopsy from six patients without history of seizures or other neurological diseases. All autopsies were performed within 12 h after death. Table 1 summarizes the clinical features of all patients.

Table 1.   Summary of the clinical and neuropathological data of the patients with epilepsy
Patient/ageClinical/pathologicalSeizure frequency/Age atDurationSeizure Follow-upEngel
(year)/sexdiagnosismonthsonset (year)epilepsy (year)typeAEDs(yr)class
  1. TLE, temporal lobe epilepsy; HS, hippocampal sclerosis; W, Wyler grading system; FCD, focal cortical dysplasia; AVM, arteriovenous malformation; MD, microdysgenesis; ODG, oligodendroglioma; DNT, dysembryoplastic neuroepithelial tumor; SW, Sturge-Weber syndrome; HMEG, hemimegalencephaly; ND, pathology not defined at autopsy; *, autopsy patients; CPS, complex partial seizures; SGS, secondary generalized seizures; SE, status epilepticus; AEDs, antiepileptic drugs; PHT, phenytoin; PB, phenobarbital; CBZ, carbamazepine; VPA, valproate; LEV, levetiracetam; OXC, oxcarbazepine.

  2. The hippocampi of patients 8–11 were used for Western blot analysis; all other material was used for immunocytochemistry.

1/17/MTLE/HS (W3)<511 6CPSPHT/CBZ/PB6I A
2/21/MTLE/HS (W3)>20 219CPS/SGSPHT/CBZ/PB1I A
3/19/FTLE/HS (W3)10 613CPSCBZ/PB/VPA1I A
4/33/FTLE/HS (W4)10–20 825CPSCBZ/PB/VPA2I A
5/27/MTLE/HS (W3)10–201017CPSCBZ/PB/VPA3I A
6/48/MTLE/HS (W3)5–101731CPS/SGSPHT/CBZ/PB1I A
7/9/MTLE/FCD/HS (W3)20–30 3 6CPSCBZ/LEV4I A
8/24/FTLE/HS (W4)5–101311CPSCBZ/PB/VPA4I A
9/28/MTLE/HS (W3)>20 622CPSPHT/CBZ/PB3I A
10/31/MTLE/HS (W3)5–10 818CPSCBZ/PB/VPA4I A
11/19/FTLE/HS (W3)10–20 514CPS/SGSCBZ/PB/VPA4I A
12/51/FTLE/non-HS/AVM<52229CPSPHT/OXC3IA
13/13/FTLE/non-HS/MD10–20 7 6CPSCBZ/VPA4IA
14/23/MTLE/non-HS/ODG10–2022 1CPSCBZ/LEV3IIA
15/35/MTLE/non-HS/DNT20–301520CPSCBZ/PB/VPA3IA
16/1/FTLE/non-HS/SW>20<1<1CPS/SGSCBZ/VPA2IIA
17/18/M*TLE/HMEG30<118CPS/SGS/SECBZ/PB
18/23/M*Epilepsy/ND10–20 914CPS/SGS/SECBZ/LEV

Experimental animals

To evaluate whether changes in SV2A protein expression occurred during epileptogenesis, the SE rat model for temporal lobe epilepsy (TLE) was used (Gorter et al., 2001). Adult male Sprague Dawley rats (Harlan CPB laboratories, Zeist, The Netherlands) weighing 400–550 g were housed individually in a controlled environment (21° ± 1°C, humidity 60%, lights on 8:00 a.m. to 8:00 p.m., food and water available ad libitum). All procedures were approved by the University Animal Welfare committee.

Electrode implantation

Rats were anesthetized with ketamine (57 mg/kg; Alfasan, Woerden, The Netherlands) and xylazine (9 mg/kg; Bayer AG, Leverkusen, Germany) and placed in a stereotactic frame. In order to record hippocampal electroencephalography (EEG), a pair of insulated stainless steel electrodes (70 μm wire diameter, tips were 0.8 mm apart) were implanted into the left dentate gyrus under electrophysiological control as previously described (Gorter et al., 2001). A pair of stimulation electrodes was implanted in the angular bundle.

SE induction

Two weeks after electrode implantation, each rat was transferred to a recording cage (40 × 40 × 80 cm) and connected to a recording and stimulation system (NeuroData Digital Stimulator, Cygnus Technology, Delaware Water Gap, NJ, U.S.A.) with a shielded multistrand cable and electrical swivel (Air Precision, Le Plessis Robinson, France). A week after habituation to the new condition, rats underwent tetanic stimulation (50 Hz) of the hippocampus in the form of a succession of trains of pulses every 13 s. Each train had a duration of 10 s and consisted of biphasic pulses (pulse duration 0.5 ms, maximal intensity 500 μA). Stimulation was stopped when the rats (n = 24) displayed sustained forelimb clonus and salivation for minutes, which usually occurred within 1 h. However, stimulation never lasted longer than 90 min. Behavior was continuously monitored during electrical stimulation and several hours thereafter. Immediately after termination of the stimulation, periodic epileptiform discharges (PEDs) occurred at a frequency of 1–2 Hz and were accompanied by behavioral generalized seizures and EEG seizures (SE). The total PEDs duration was considered as the total SE duration. On average, the SE lasted 10.5 ± 0.6 h. Electrode-implanted control rats (n = 9) were handled and recorded identically, but did not receive electrical stimulation.

EEG monitoring

Hippocampal EEG signals were amplified (10×) via a field effect transistor (FET) that connected the headset of the rat to a differential amplifier (20×; CyberAmp, Axon Instruments, Burlingame, CA, U.S.A.), filtered (1–60 Hz), and digitized by a computer. A seizure detection program (Harmonie, Stellate Systems, Montreal, Canada) sampled the incoming signal at a frequency of 200 Hz per channel. All EEG recordings were visually screened, and seizures were confirmed by trained human observers. All rats that were killed in the acute (1 day after SE) or latent phase (1 week after SE) were monitored continuously. Chronic epileptic rats were monitored from the SE onwards, until the first spontaneous seizure appeared (on average 11 ± 3 days after stimulation). Hereafter, some rats were disconnected from the setup. All rats were connected again 6–8 months later, and continuous EEG recordings (24 h/day) were started to confirm the occurrence of spontaneous seizures.

Brain dissection

Rats were disconnected from the EEG recording setup and deeply anesthetized with pentobarbital (Nembutal, intraperitoneally, 60 mg/kg). For immunocytochemistry and Timm stain, the animals were perfused through the ascending aorta with 300 ml 0.37% Na2S solution, followed by 300 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Hereafter, the brains were dissected, incubated for 72 h in 0.3 M EDTA, pH 6.7 (Merck, Amsterdam, The Netherlands), and paraffin-embedded.

For Western blot analysis, animals were decapitated, and the hippocampus was dissected and immediately frozen on dry ice. All material was stored at −80°C until use.

SV2A immunocytochemistry in human and rat brain

Brain tissue of humans [control, n = 6; HS, n = 7 (cases 1–7); non-HS, n = 5; SE, n = 2] and rats [control, n = 5; acute (1 day after SE), n = 5; latent (1 week after SE), n = 6; chronic (6–8 months after SE) nonprogressive epilepsy, n = 5; progressive epilepsy, n = 5] was paraffin-embedded, sectioned at 6 μm, and mounted on organosilane-coated slides (Sigma, St. Louis, MO, U.S.A.). Sections were deparaffinated in xylene, rinsed in ethanol (100%, 95%, 70%), and incubated for 20 min in 0.3% hydrogen peroxide diluted in methanol. For the rat, two sagittal sections were used (2.90–3.18 mm lateral to bregma) (Paxinos & Watson, 1998). Slides were then incubated for 10 min at 120°C in 0.01 M Tris-EDTA buffer, washed with 0.1 M phosphate-buffered saline (PBS), pH 7.4, and incubated for 30 min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK). This was followed by overnight incubation in anti-SV2A (rabbit anti-SV2A, 1:100; Synaptic Systems, Goettingen, Germany; or mouse anti-SV2A, 15E11, 1:50; Abcam, Cambridge, UK) at 4°C. Hereafter, sections were washed in PBS and stained with a polymer-based peroxidase immunocytochemistry detection kit (PowerVision Peroxidase System; ImmunoVision, Brisbane, CA, U.S.A.). After washing, sections were stained with 50 mg 3,3-diaminobenzidin tetrahydrochloride (DAB; Sigma-Aldrich, Zwijndrecht, The Netherlands) and 5 μl 30% hydrogen peroxide in a 10-ml solution Tris-HCl. Sections were counterstained with hematoxylin, dehydrated in alcohol and xylene, and coverslipped. Sections incubated without anti-SV2A or with preimmune serum were essentially blank. The intensity of SV2A immunoreactivity (IR) was estimated semiquantitatively. The intensity was classified as (+/−), absent or very weak; (+), weak; +, moderate; or ++, strong.

Fluorescent immunocytochemistry

A subset of sections were deparaffinated in xylene, rinsed in ethanol (100%, 95%, 70%), and washed (2 × 10 min) in 0.1 M PBS. Slides were then incubated for 10 min at 121°C in 0.01 M Tris-EDTA buffer, washed with 0.1 M PBS, pH 7.4, and incubated for 30 min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK). Hereafter, sections were incubated with mouse anti-SV2A (15E11, 1:50; Abcam, Cambridge, U.K.) and rabbit anti-synaptophysin (A0010, 1:200; DAKO, Glostrup, Denmark). Twenty-four hours later, the sections were washed in PBS (three times for 10 min each) and incubated for 2 h in anti-mouse Alexa Fluor 546 (1:200; Molecular Probes, Invitrogen, Breda, The Netherlands) and anti-rabbit Alexa Fluor 488 (1:200; Molecular Probes, Invitrogen, Breda, The Netherlands). Following three additional washes in PBS, sections were coverslipped with mounting medium for fluorescence, (Vectashield; Vector Laboratories, Burlingame, CA, U.S.A.). Images were acquired  using a confocal laser-scanning microscope and Adobe Photoshop.

Timm’s histochemistry

The mossy fibers were visualized using a Timm's stain (Danscher, 1981; Sloviter, 1982). A subset of rat slides were incubated for 1 h in the dark in a 200 ml solution of 30% Gum arabic containing 154 mM hydrochinone, 120 mM citric acid monohydrate, 80 mM citric acid tri-sodium salt dehydrate, and 5 mM silver nitrate (all from Merck, Amsterdam, The Netherlands). To terminate development, slides were washed for 10 min in running tap water in the dark. Hereafter, slides were immersed for 10 min in 200 mM thiosulphate pentahydrate (Acros Organics, Geel, Belgium). After washing in aquadest, slides were dehydrated in alcohol and xylene and coverslipped.

Western blot analysis

Freshly frozen human [control, n = 7; MTS (cases 8–11), n = 4] and rat hippocampi [control, n = 4; chronic (6–8 months after SE with progressive epilepsy), n = 5] were homogenized in lysis buffer containing, per 20 ml: 200 μl 1 M Tris, pH 8.0, 1 ml 3 M NaCl, 2 ml 10% Nonidet P-40 (NP-40), 4 ml 50% glycerol, 800 μl Na-orthovanadate (10 mg/ml), 200 μl 0.5 M EDTA, pH 8.0, 400 μl protease inhibitors, 200 μl 0.5 M NaF, 11.2 ml water. Fifty micrograms total protein per lane, as determined using the bicinchoninic acid method (Smith et al., 1985), were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide) and transferred to nitrocellulose by electroblotting (Transblot SD; Bio-Rad Laboratories, Hercules, CA, U.S.A.). Blots were incubated with primary antibodies (rabbit anti-SV2A, 1:1000; Synaptic Systems, Goettingen, Germany; or mouse anti-SV2A, 15E11, 1:500; Abcam, Cambridge, U.K.; and mouse anti-β-actin, clone AC-15, 1:50000; Sigma, St. Louis, MO, U.S.A.) and the secondary antibody, anti-mouse (1:2500; Dako, Glostrup, Denmark) or anti-rabbit (1:2500; Invitrogen, Breda, The Netherlands) labeled with horseradish peroxidase. IR was visualized with lumi-light plus Western blot analysis substrate (Roche Diagnostics, Mannheim, Germany), and the blots were digitized using a Luminescent Image Analyzer, LAS-3000 (Fuji Film, Tokyo, Japan). The optical density of each sample was measured using Scion Image analysis software (Release beta 3b; Scion Corporation, Frederick, MD, U.S.A.). For each sample, the optical density of the SV2A was calculated relative to the optical density of β-actin.

Results

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

SV2A IR in human brain

In autopsy controls (n = 6) SV2A IR was present throughout all subfields of the hippocampus (Figs. 1A, 2A, 2C, 3E, and Table 3). The neuropil was moderately stained, while the somata of the granule and pyramidal cells were devoid of staining. The strongest IR was observed in stratum lucidum of CA3 (mossy fibers) and in the hilar region of the dentate gyrus. In this region, strong staining was present around the somata of neurons, as well as along their dendrites (Fig. 2G) in a patchy distribution (Fig. 5A). SV2A was colocalized in these puncta with the presynaptic marker synaptophysin (Fig. 5A–5C).

image

Figure 1.   SV2A immunocytochemistry in the human (A, autopsy control) and rat (B, control) hippocampus. High magnification pictures of the dentate gyrus, CA3, and CA1 (black rectangles) are shown in the following figures.

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image

Figure 2.   SV2A immunocytochemistry in the human hippocampus. SV2A immunoreactivity (IR) was present throughout all subfields of the dentate gyrus (A), CA3 (C), and CA1 (E) in autopsy control tissue. The neuropil was moderately stained, while the somata of the granule and pyramidal cells were devoid of staining. The strongest IR was observed in stratum lucidum of CA3 (mossy fiber terminals) and in the hilar region of the dentate gyrus (arrows in panels A and C). In this region, strong staining was present around the somata of interneurons, as well as along their dendrites (G). In resected hippocampi of patients with HS, SV2A IR was decreased in the neuropil throughout the dentate gyrus (B), CA3 (D), and CA1 (F), except in the inner molecular layer of the dentate gyrus, where moderate IR was observed. Interneurons in the hilus (arrows in panel B), if any remained, had moderate IR around the somata (arrowhead in panel H) and dendrites (arrows in panel H). ml, molecular layer; gcl, granule cell layer; sl, stratum lucidum; pcl, pyramidal cell layer; so, stratum oriens. Scale bar: AF, 130 μm; G and H, 30 μm.

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Table 2.   SV2A expression in the human brain
 ControlSEnon-HSHS
 n = 6n = 2n = 5n = 7
  1. The intensity of immunoreactivity was classified as (+), weak; +, moderate; or ++, strong.

  2. SE, status epilepticus; HS, hippocampal sclerosis; DG, dentate gyrus; Oml, outer molecular layer; Mml, middle molecular layer; Iml, inner molecular layer; Gcl, granule cell layer; So, stratum oriens; Pcl, pyramidal cell layer; Sl, stratum lucidum; Sr, stratum radiatium.

DG
 Oml+++(+)
 Mml+++(+)
 Iml++++
 Gcl(+)(+)(+)(+)
 Hilus++++++(+)
CA3
 So+++(+)
 Pcl(+)(+)(+)(+)
 Sl++++++(+)
 Sr+++(+)
CA1
 So+++(+)
 Pcl(+)(+)(+)(+)
 Sr+++(+)
image

Figure 5.   Subcellular localization of SV2A in human brain. SV2A staining (red) was present in the human hippocampus (autopsy control tissue, panels AC) around the somata of hilar neurons (arrowheads in panel C), as well as along their dendrites (arrow in panel C) in a patchy distribution. SV2A was colocalized with the presynaptic marker synaptophysin (green, panels B and C). In the hilus of resected hippocampi from TLE patients with HS, many hilar cells were lost. Remaining cells were surrounded by SV2A IR particles around the soma and dendrite (D). SV2A was also colocalized with synaptophysin (arrowheads in F), but synaptophysin-positive/SV2A-negative particles were also evident around the somata of these hilar neurons (arrows in F). Scale bar, 5 μm.

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Non-HS cases (n = 5) and autopsy material of patients that had died during SE (n = 2) were similar to autopsy controls (Table 2).

In resected hippocampi of patients with HS (n = 7), a large loss of IR was found in the neuropil throughout the hippocampus (Fig. 2B, 2D, 2F, and Table 2), except in the inner molecular layer of the dentate gyrus where moderate IR was observed. Interneurons in the hilus, if any remained, had moderate IR around the somata and dendrites (Fig. 2H) in a patchy distribution (Fig. 5D).

The staining pattern of the monoclonal SV2A antibody was similar to the polyclonal antibody, except for a few scattered SV2A positive astrocytes in HS-hippocampi that stained with the polyclonal antibody and were not observed when the monoclonal antibody was used.

SV2A IR in rat brain

Control rats

In control rats, SV2A IR was present throughout all subfields of the hippocampus, except in the somata of granule and pyramidal cells (Fig. 1B and Table 3). All synaptic layers were moderately stained, while strong IR was observed in stratum lucidum of CA3 (mossy fiber endings) (Fig. 4A) and in the hilar region of the dentate gyrus (Fig. 3A), similarly as in the human hippocampus. Strong SV2A IR puncta were present around the somata of neurons in the hilus, as well as along their dendrites (Fig. 3A). Pyramidal cell dendrites in stratum radiatum of CA1 were surrounded by moderate SV2A staining (Fig. 3B).

Table 3.   SV2A expression in the rat brain
    ChronicChronic
 ControlAcuteLatentnonprogressiveprogressive
 n = 6n = 5n = 6n = 5n = 5
  1. The intensity of immunoreactivity was classified as (+/−), absent or very weak; (+), weak; +, moderate; or ++, strong.

  2. SE, status epilepticus; HS, hippocampal sclerosis; DG, dentate gyrus; Oml, outer molecular layer; Mml, middle molecular layer; Iml, inner molecular layer; Gcl, granule cell layer; So, stratum oriens; Pcl, pyramidal cell layer; Sl, stratum lucidum; Sr, stratum radiatium.

DG
 Oml++++(+)
 Mml++++(+)
 Iml+(+)(+/−)++
 Gcl(+)(+)(+)(+)(+)
 Hilus++(+)(+/−)++(+)
CA3
 So++(+)+(+)
 Pcl(+)(+)(+)(+)(+)
 Sl++++(+)++(+)
 Sr++(+)+(+)
CA1
 So++(+)+(+)
 Pcl(+)(+)(+)(+)(+)
 Sr++(+)+(+)
image

Figure 4.   SV2A immunocytochemistry and Timm's staining in the rat hippocampus (CA3). In control rats, SV2A IR was present throughout all subfields of CA3 (A), except in the somata of pyramidal cells. Strong IR was observed in stratum lucidum (arrows in panel A), which represents the zinc containing mossy fibers (E). In the acute phase (B), the mossy fibers are moderately SV2A IR, whereas in the latent (C) and chronic phase (D), SV2A IR was weak. However, the mossy fibers are still present, as indicated by the Timm stain (F). sr, stratum radiatium; sl, stratum lucidum; pcl, pyramidal cell layer; so, stratum oriens. Scale bar, 100 μm.

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image

Figure 3.   SV2A immunocytochemistry in the rat hippocampus (dentate gyrus and CA1). SV2A IR was present throughout all subfields of the dentate gyrus (A) and CA1 (B) in control rats, except in the somata of granule and pyramidal cells. Around the somata and dendrites of neurons in the hilar region, strong IR was present (arrows in panel A). Pyramidal cell dendrites in stratum radiatum of CA1 (arrows in panel B) were surrounded by moderate SV2A staining. In the acute phase (one day after SE), SV2A staining decreased to some extent in the molecular layer and the hilus of the dentate gyrus (C). However, many neurons were still present in the hilus that were surrounded by moderately stained SV2A fibers. This was also the case for the pyramidal cell dendrites in CA1 (D). In the latent phase (one week after SE), SV2A IR decreased, particularly in the inner molecular layer and hilus (E). The arrowhead in panel E indicates the border between the inner molecular layer and middle molecular layer. Only a few cells surrounded by strong IR were found in the hilus. In CA1, SV2A IR decreased throughout all layers (F). In chronic epileptic rats (6–8 months after SE), SV2A IR decreased in all hippocampal layers (G and H), although the inner molecular layer was moderately stained (G). In the hilus, only a few neurons were present surrounded by weak SV2A IR (arrows in panel G and in inset). In addition, some neurons were not surrounded by SV2A IR fibers (arrowhead in inset). mml, middle molecular layer; iml, inner molecular layer; gcl, granule cell layer; so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum. Scale bar, 100 μm.

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Acute phase

In the acute seizure phase (one day after SE), SV2A staining decreased in the inner molecular layer and the hilus of the dentate gyrus (Fig. 3C). However, the neurons that were still present in the hilus were surrounded by moderately stained SV2A fibers. This was also the case for the pyramidal cell dendrites in CA3 and CA1.

Latent phase

In the latent phase (1 week after SE), SV2A IR was decreased throughout all CA layers (Figs. 3F and 4C). SV2A IR was also decreased in the dentate gyrus, compared to controls, and was absent in four out of six rats in the inner molecular layer (Fig. 2E and Table 1). Only a few hilar cells were surrounded with strong IR. The lack of presynaptic marker synaptophysin in the inner molecular layer confirmed that the hilar axonal projections had degenerated (data not shown). In contrast, the mossy fiber terminals in stratum lucidum contained less SV2A, while synaptophysin was still present (data not shown). A lower percentage of hilar cells (approximately 30%) was lost in two rats, which coincided with a moderate decrease of SV2A in the inner molecular layer.

Chronic epileptic phase

In chronic epileptic rats that had a progressive form of epilepsy [6–8 months after SE, >3 seizures/day (Gorter et al., 2001; van Vliet et al., 2004), on average 9 ± 2 seizures/day], SV2A IR was decreased compared to controls in almost all hippocampal layers (Table 1 and Figs. 3G, 3H, and 4D). The inner molecular layer was moderately stained (Fig. 3G). Combined SV2A and synaptophysin stainings showed that the inner molecular layer was filled with synaptophysin IR fibers, representing the reorganized mossy fibers. The mossy fiber terminals contained less SV2A (Fig. 4D) compared to control rats (Fig. 4A). A Timm staining confirmed that these mossy fiber terminals were present, but hardly contained SV2A (Fig. 4F). In the hilus, only a few neurons were present that were surrounded by weak IR. SV2A IR puncta were present around the somata of these neurons and colocalized with synaptophysin. In addition, many synaptophysin positive puncta were present that had no SV2A IR. We also investigated chronic epileptic rats with a nonprogressive form of epilepsy [i.e., rats that experienced SE, but have a few seizures/week that do not increase over time (Gorter et al., 2001; Van Vliet et al., 2004), on average 0.6 ± 0.1 seizures/day]. These rats showed very similar protein expression patterns as control rats (Table 1).

Western blot analysis

Western blot analysis was used to quantify the expression of SV2A in the human and rat hippocampus. The monoclonal SV2A antibody, as well as the polyclonal antibody, stained a protein of approximately 90 kDa (Fig. 6A), in accordance with the molecular weight of SV2A (Lynch et al., 2004). The optical density of SV2A/β-actin was significantly decreased (approximately 32%; Student's t-test, p < 0.05) in the hippocampus of TLE patients (Fig. 6B) in comparison with autopsy controls (1.92 ± 0.07 versus 1.32 ± 0.08). In chronic epileptic rats that had a progressive form of epilepsy (Fig. 6C), the optical density was significantly decreased (approximately 28%; Student's t-test, p < 0.05) compared to control rats (1.34 ± 0.08 versus 0.97 ± 0.7).

image

Figure 6.   Western blot analyses of human and rat hippocampi. Western blot analysis of the human hippocampus using the monoclonal SV2A antibody identified the 90 kDa protein SV2A (A) and showed that less SV2A is present in TLE tissue compared to autopsy control tissue. Optical densitometry indicated that the SV2A/β-actin ratio (mean ±sem) was significantly decreased by approximately 32% (Student's t-test, p < 0.05) in the hippocampus of TLE patients (B) in comparison to autopsy controls (1.92 ± 0.07 versus 1.32 ± 0.08). In the hippocampus of chronic epileptic rats (C), the optical density decreased significantly by approximately 38% (Student's t-test, p < 0.05) compared to control rats (1.34 ± 0.08 versus 0.97 ± 0.7). TLE, temporal lobe epilepsy, O.D., optical density. *, indicates significant difference compared to control values (Student's t-test, p < 0.05).

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Discussion

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study, we have characterized SV2A protein expression in the hippocampus of humans and rats. SV2A was decreased in the hippocampus of TLE patients with HS. Similarly, SV2A was decreased throughout the hippocampus of epileptic rats, particularly in the mossy fiber terminals in the latent and chronic epileptic phase. In chronic epileptic rats, SV2A was specifically decreased in rats with frequent seizures and a progressive form of epilepsy.

SV2A expression has been investigated in various brain structures of control rats (Bajjalieh et al., 1994; Janz & Sudhof, 1999). Nevertheless, no detailed description has been provided on SV2A protein expression and its subcellular localization in the hippocampus. Moreover, there are no detailed studies on SV2A expression in human brain. The present study shows that SV2A is abundantly present in presynaptic terminals throughout the human and rat hippocampus and that striking similarities are observed between SV2A expression in rats and humans. In control tissue, mossy fiber terminals contain the strongest IR. This is most obvious around hilar neurons and in stratum lucidum in the CA3 to which the mossy fibers project. We demonstrated that SV2A positive puncta are distributed in a patchy pattern around the somata and dendrites of these neurons. SV2A expression decreased during epileptogenesis in the rat and was still decreased in the chronic epileptic phase in all hippocampal subfields, including stratum lucidum (mossy fibers), both in rats that had a progressive form of epilepsy and in humans with TLE.

The acute loss of SV2A can partly be explained by neuronal degeneration that occured after SE. In rats, cell death mainly occurs within the latent period (approximately 1 week after SE) (Gorter et al., 2003), which parallels the decrease of SV2A. This is particularly evident in the inner molecular layer to which the hilar cells project. However, the loss of SV2A cannot be explained by degeneration only, since we show that synaptophysin and Timm positive terminals were still present in the hippocampus during the chronic epileptic phase, but expressed less SV2A protein compared to control expression.

Since the molecular action of SV2A is unknown, we can only speculate on the function of SV2A and the consequences of its decrease in epileptic tissue. It has been suggested that SV2A acts as a modulator of vesicles (Crowder et al., 1999; Janz et al., 1999; Xu & Bajjalieh, 2001) and enhances the release probability at quiescent synapses (Custer et al., 2006). The fact that SV2A gene disruption leads to a reduction in inhibitory neurotransmission (Crowder et al., 1999) and that SV2A/SV2B knockouts have increased excitatory neurotransmission compared to SV2B knockouts (Janz et al., 1999) implicates that SV2 plays an important role in the stability of the neuronal networks. This is illustrated by the fact that SV2A knockout mice develop epilepsy (Crowder et al., 1999; Janz et al., 1999). It has been hypothesized that SV2A functions as cytoplasmatic Ca2+ regulator in the nerve terminal during repetitive stimulation, possibly as Ca2+ transporter (Janz et al., 1999). Build-up of residual calcium during repetitive stimulation has been suggested to lead to abnormal oscillations and epilepsy (Janz et al., 1999).

In rats, SV2A expression already had decreased in the latent period and preceded the occurrence of spontaneous seizures. This indicates that decreased expression of SV2A at this stage of the epileptogenesis will not immediately lead to seizures. Interestingly, in two rats that were sacrificed in the latent period, a lower percentage of hilar cells was lost (approximately 30%), which coincided with a more moderate decrease of SV2A in the inner molecular layer. Since the SE duration was comparable between all animals that were sacrificed in the latent phase, the decrease of SV2A apparently does not only occur as a direct result of sustained epileptic activity during SE. Thus, sustained seizure activity can reduce SV2A expression but will not always lead to a complete loss of SV2A IR in the inner molecular layer. Since granule cells and their mossy fibers mostly survive after SE in rats, we can assume that the decrease of SV2A expression in the (CA3) region represents a decreased expression per cell (mossy fiber terminal). SV2A expression in rats with a nonprogressive form of epilepsy was very similar to control expression. This is partially related to the decreased cell death in these animals. Of course we cannot exclude that frequent seizure activity also contributes to a decrease in SV2A expression and that the decrease is just a consequence of seizure activity. However, with the knowledge that the absence of SV2A in knockout mice is also associated with the development of seizures, we speculate that the observed decrease of mossy fiber SV2A expression is not only a consequence of seizure activity but could also play a role in the progression of epilepsy. In this respect, sprouting of mossy fibers may be an important factor. In epileptic rats, sprouting of these fibers into the inner molecular layer can already be detected in the latent period and continues in the chronic epileptic phase (Gorter et al., 2001, 2002b). Since the mossy fiber terminals lose most of their SV2A, repetitive firing of granule cells may lead to an accumulation of calcium as has been described to occur in SV2 knockout (Janz et al., 1999). Interestingly, increased calcium currents were observed in granule cells of epileptic rats (Gorter et al., 2002a), together with reduced calcium extrusion protein expression in the axon terminals (Ketelaars et al., 2004). Since the mossy fibers not only terminate in stratum lucidum but also gradually and increasingly sprout into the inner molecular layer where they can contact other granule cells, this can contribute to altered hippocampal excitability (Sutula, 2002). Moreover, we previously showed that the expression of neuronal glutamate transporters is specifically and permanently decreased in the inner molecular layer in rats with extensive mossy fiber sprouting and seizure progression (Gorter et al., 2002c), which could further contribute to the instability of these networks. Similarly, we find permanently decreased SV2A expression in rats with frequent seizures but not in rats that do not display seizure progression (but nevertheless still an occasional seizure).

Since SV2A is the binding site for LEV, decrease of SV2A may change the efficacy of LEV. On basis of our data, a diminished effect of LEV is expected in epileptic subjects. Although clinical and animal studies show that LEV has initially strong seizure suppressive effects (Loscher & Honack, 2000; Klitgaard, 2001; Ben-Menachem, 2003; Ben-Menachem et al., 2003; French et al., 2005), a reduced effect of LEV has been observed during chronic treatment in epileptic rats (Glien et al., 2002; van Vliet et al., 2008). Incidental evidence shows that tolerance to LEV can occur in humans (Meencke & Meinke-Jäggi, 2004; Friedman & French, 2006; Loscher & Schmidt, 2006). Whether this is associated with further changes of SV2A expression during treatment needs to be investigated.

In conclusion, this study shows that SV2A protein expression was decreased throughout the hippocampus during epileptogenesis and chronic epileptic phase, particularly in the mossy fiber terminals. Further studies are needed to elucidate the effects of decreased SV2A expression on neuronal excitability and LEV efficacy.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Dr. W.G.M. Spliet and Prof. Dr. D. Troost [neuropathologists; Department of (Neuro)Pathology of University Medical Center Utrecht and University of Amsterdam] and Dr. J.C. Baayen (Department of Neurosurgery, VU University Medical Center, Amsterdam) for the collaboration in the collection of human material. This work was supported by the Epilepsy Institute in The Netherlands—SEIN (EAvV).

Conflict of interest: We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors have no conflicts of interest to report.

References

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References