Low distribution of synaptic vesicle protein 2A and synaptotagimin-1 in the cerebral cortex and hippocampus of spontaneously epileptic rats exhibiting both tonic convulsion and absence seizure

Neuroscience 221 (2012) 12–20

LOW DISTRIBUTION OF SYNAPTIC VESICLE PROTEIN 2A AND SYNAPTOTAGIMIN-1 IN THE CEREBRAL CORTEX AND HIPPOCAMPUS OF SPONTANEOUSLY EPILEPTIC RATS EXHIBITING BOTH TONIC CONVULSION AND ABSENCE SEIZURE R. HANAYA, a* H. HOSOYAMA, a S. SUGATA, a M. TOKUDOME, a H. HIRANO, a H. TOKIMURA, a K. KURISU, b T. SERIKAWA, c M. SASA d AND K. ARITA a

cortices (47%). Lower synaptotagmin-1 expression (vs Wistar rats) was located in the frontal (31%), piriform (13%) and entorhinal (39%) cortices, and IML of the DG (38%) in SER. Focal low distribution of synaptotagmin-1 accompanying low SV2A expression may contribute to epileptogenesis and seizure propagation in SER. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 734-8551, Japan

b

Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan

Key words: synaptic vesicle protein 2A (SV2A), synaptotagimin-1, GABAergic dysfunction, epileptogenesis, cerebral cortex, BDNF.

c Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan d

Nagisa Clinic, Hirakata 573-1183, Japan

INTRODUCTION

Abstract—The spontaneously epileptic rat (SER) is a double mutant (zi/zi, tm/tm) which begins to exhibit tonic convulsions and absence seizures after 6 weeks of age, and repetitive tonic seizures over time induce sclerosis-like changes in SER hippocampus with high brain-derived neurotrophic factor (BDNF) expression. Levetiracetam, which binds to synaptic vesicle protein 2A (SV2A), inhibited both tonic convulsions and absence seizures in SERs. We studied SER brains histologically and immunohistochemically after verification by electroencephalography (EEG), as SERs exhibit seizure-related alterations in the cerebral cortex and hippocampus. SERs did not show interictal abnormal spikes and slow waves typical of focal epilepsy or symptomatic generalized epilepsy. The difference in neuronal density of the cerebral cortex was insignificant between SER and Wistar rats, and apoptotic neurons did not appear in SERs. BDNF distributions portrayed higher values in the entorhinal and piriform cortices which would relate with hippocampal sclerosis-like changes. Similar synaptophysin expression in the cerebral cortex and hippocampus was found in both animals. Low and diffuse SV2A distribution portrayed in the cerebral cortex and hippocampus of SERs was significantly less than that of all cerebral lobes and inner molecular layer (IML) of the dentate gyrus (DG) of Wistar rats. The extent of low SV2A expression/distribution in SERs was particularly remarkable in the frontal (51% of control) and entorhinal

Synaptic vesicle protein 2A (SV2A) belongs to a family of transporters, and it is expressed throughout the brain at varying levels (Bajjalieh et al., 1994). SV2A binds to synaptotagmin-1 (Syt1), and is thought to regulate synaptic vesicle traffic by interacting with Syt1 (Schivell et al., 2005; Zeng et al., 2009). On the other hand, SV2s are not involved in the uptake, storage, and release of neurotransmitters (Janz et al., 1999). The loss of SV2A leads to a reduction in action potential dependent inhibitory neurotransmission induced by gamma-aminobutyric acid (GABA) (Crowder et al., 1999), and SV2A decreases during epileptogenesis and in cases with chronic epilepsy (van Vliet et al., 2009). SV2A is the binding site for the antiepileptic drug levetiracetam (Lynch et al., 2004), and role of SV2A has attracted notice in expression of epilepsy. Levetiracetam inhibits both tonic convulsion and absence seizures observed in spontaneously epileptic rats (SER) (Ji-qun et al., 2005; Yan et al., 2005). SER is a double mutant (zi/zi, tm/tm) obtained originally by mating a heterozygote tremor rat (tm/+), a mutant found in an inbred colony of Kyoto-Wistar rats (Yamada et al., 1985), with a homozygote zitter rat found in a Sprague–Dawley colony (Rhem et al., 1982). After 6 weeks of age, SERs begin to show spontaneous tonic convulsions with low voltage fast waves, and absence seizures characterized by sudden behavioral changes such as immobility and staring with simultaneous appearance of paroxysms of 5–7 Hz spike-wave complexes in cortical and hippocampal electroencephalography (EEG) (Serikawa and Yamada, 1986; Sasa et al., 1988). The Sasa et al. study showed drugs that are likely to work in humans for absence seizures or tonic seizures, are the same ones that are likely to work for seizures in SERs (Sasa et al., 1988).

*Corresponding author. Address: Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Tel: +81-99-275-5375; fax: +81-99-265-4041. E-mail address: [email protected] (R. Hanaya). Abbreviations: BDNF, brain-derived neurotrophic factor; DG, dentate gyrus; EEG, electroencephalography; FDG-PET, 18F-fluorodeoxyglucose-positron emission tomography; IML, inner molecular layer; LCGU, local cerebral glucose utilization; MF, mossy fibers; OD, optical density; SAB, streptavidin biotin; SERs, spontaneously epileptic rats; SV2A, synaptic vesicle protein 2A; Syt1, synaptotagmin-1; TLE, temporal lobe epilepsy.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.06.058 12

R. Hanaya et al. / Neuroscience 221 (2012) 12–20

Hippocampal CA3 pyramidal neurons in mature SERs display abnormal firings which are attributable to abnormalities of the Ca2+ channels when the mossy fibers (MF) are mimicked with a single electrostimulation (Ishihara et al., 1993; Hanaya et al., 1998, 2010; Yan et al., 2007). Generalized tonic convulsion induces sclerosis-like change with high brain-derived neurotrophic factor (BDNF) expression in SER hippocampus. These suggest SERs have both genetic and focal components of epilepsy, although the SER brain does not have focal lesions (Inui et al., 1990). In the present study, we thoroughly verified EEG, histological findings which indicated the type of seizures in SER. In addition, we evaluated immunohistochemical expression of SV2A, Syt1 and BDNF to elucidate if the cerebral cortex and hippocampus exhibited SER seizurerelated characteristics.

EXPERIMENTAL PROCEDURES Experimental animals We used 15 SERs containing each both sex, weighing 150–220 g (age: 10–14-weeks) and 6 normal age-matched Wistar rats. They were kept in shoebox type cages on laminar flow shelves in a conventional room maintained at 23 ± 2 °C and 55 ± 5%. They were provided with standard rat chow (MF, Oriental Yeast, Tokyo) and tap water ad libitum. The protocols for animal treatment received prior approval from our institutional review board: animals were treated in accordance with the guideline for animal experiments stipulated by the Ministry of Education, Culture, Sports, Science and Technology in Japan, and all efforts were made to minimize animal suffering.

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complexes was less than 1 s, the two events were regarded as 2 separate single-seizures. EEG recording was performed for 120 min/day between 15:00 and 19:00 h.

Staining and cell count Both groups of SER and Wistar rats of 6 each were sacrificed under deep anesthesia with 60 mg/kg sodium pentobarbital. Their brains were then perfused transcardially with heparinized saline, followed by ice-cold fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) before isolation. After isolation, isolated brains were post-fixed in the same fixative at 4 °C. Paraffin-embedded coronal sections (thickness: 4 lm) were serially sliced in an antero-posterior fashion, starting from the anterior level. Sections of interest were selected according to their stereotactic coordinates for each animal. After hematoxylin and eosin (H–E) staining, the cell density of each coronal section was determined via microscopy: i.e. a square box (10  10) with a fixed microscopic grid (1 cm2). Briefly, the grid for counting was placed on a well-defined area of the cerebral structure of interest, and counting was performed at 200-fold magnification for each defined brain structure. Cell-counting was performed twice on each side of three antero-posterior adjacent sections of a specific brain region by the same observer. The mean values of cell counts obtained for all sites of the same brain structure were derived for the respective structures. This procedure was used to minimize potential errors from double counting. Neurons touching the inferior and right edges of the grid were not counted. Counts involved only neurons with a cell body larger than 10 lm; any cells with a smaller cell body were considered as glial cells and were therefore neglected.

Immunohistochemistry EEG recording Under sodium pentobarbital anesthesia (30 mg/kg i.p.; NembutalÒ, Dainippon Sumitomo, Japan), all electrodes were stereotaxically and chronically placed (in mm) according to coordinates of the brain atlas (P: posterior from bregma; L: lateral from midline; D: depth from brain surface) as previously described (Paxinos and Watson, 1996). A monopolar silver-tipped electrode was chronically implanted in the frontal cortex (P, 2.0; L, 3.0), parietal cortex (P, 1.0; L, 5.5), and occipital cortex (P, 6.5; L, 5.0). Enamel-coated stainless-steel electrodes were each placed in the following brain sites: temporal cortex (P, 4.5; L, 7.0; D, 4.5), CA3 of the hippocampus (P, 4.0; L, 4.0; D, 3.5), amygdala (P, 3.5; L, 5.0; D, 7.5), thalamus (P, 2.5; L, 3.5; D, 5.5), substantia nigra (P, 5.5; L, 2.5; D, 7.0), and reticular formation (P, 9.5; L, 0.5; D, 7.8). A total of 9 SERs had 6 or 8 electrodes combined with 2 patterns (bilaterally frontal, parietal, temporal, or occipital cortex; unilateral parietal cortex, CA3, amygdala, thalamus, substantia nigra, and reticular formation). A reference electrode was fixed on the surface of frontal cranium (P: 3.5; L: 0). After a 7-day recovery period, each animal was placed in a sound-proof box (H40  L40  W40 cm) with a small window (H11  W6 cm) to allow behavioral observation. After 30-min acclimatization in the sound-proof box, EEG was recorded for 120 min, using an EEG-monitoring system (BMSI 5000, Nicolet, USA). Changes in EEG during the absence seizure consistently correlated with the respective abnormal changes in behavior, as described previously (Serikawa and Yamada, 1986; Sasa et al., 1988). When 5–7 Hz spike-wave complexes in the cortical and hippocampal EEG tracings lasted for more than 1 s, the animal was considered to have exhibited an absence seizure. When the time interval between two independent 5–7 Hz spike-wave

Tissue sections were deparaffinized with xylene, and antigen retrieval was carried out using the heat-induced epitope retrieval method with a citrate buffer solution of pH 6.0. Endogenous peroxidase blocking was conducted by dipping the slides into a solution containing 10 ml 30% H2O2 and 90 ml 99% methanol for 30 min. Thereafter, slides were serially rinsed and washed three times with phosphate-buffered saline solution (pH 7.5), each time lasting 5 min. The labeled streptavidin biotin (SAB) method, an indirect assay using histofine simplestain (Nichirei Company, Tokyo, Japan) was employed for antibody incubation. Briefly, immunostaining was performed with the following antibodies: goat polyclonal anti-SV2A (1:100 dilution, Santa Cruz Biotechnology, CA, USA), goat polyclonal anti-Syt1 (1:100, Santa Cruz Biotechnology), mouse monoclonal anti-synaptophysin (1:100, Dako-Cytomation, Glostrup, Denmark), and rabbit polyclonal anti-BDNF (1:100, Dako-Cytomation). Primary antibody incubation was performed overnight at 4 °C, followed by 30-min incubation in a secondary antibody (biotinylated secondary antibody, SAB kit; Nichirei Company). Primary and secondary antibodies were incubated for 5–10 min in a mixture containing 0.02% diaminobenzidine (DAB tablet; Wako Pure Chemical Industry, Osaka, Japan) and 0.05% H2O2 in phosphate buffer serum on slides. All slides were mounted with coverslips for storage purposes. Except for exposure to the primary antibody, control sections from two animals for immunohistochemical evaluation were exposed to all antibodies and underwent the above-mentioned procedures, accordingly. To exclude the artifact and uniform the staining, we noted the following points in the operation: (1) staining parallel in each series to be compared, (2) using the same attenuated antibody solution and the other solution for staining in each series.

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R. Hanaya et al. / Neuroscience 221 (2012) 12–20

Detection of apoptosis

a

L Fcx RFcx

Apoptosis was morphologically assessed by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay using the In Situ Apoptosis Detection Kit (catalogue MK500; Takara Bio Inc., Otsu, Japan). The xylene-deparaffinized 4-lm-thick sections were treated with proteinase K for 15 min using phosphate buffer. Endogenous peroxidase blocking was the performed for 5 min. Thereafter, tissue slices were stained according to the manufacturer’s instructions. Briefly, slides were incubated for 5–10 min in a mixture containing 0.02% diaminobenzidine (DAB tablet) and 0.05% H2O2 in phosphate-buffered saline, and treated with methyl green as a counterstain for better cytoplasmic visualization.

L Pcx RPcx L Tcx RTcx L Ocx ROcx Absence seizure

Densitometric analysis of immunoreactivity

b

Unlike in SE-induced epilepsy model, histological change in animals and human material is mild, and raw immunostained slice does not show dramatic result (Li et al., 2002; de Groot et al., 2011). We used optical density (OD) method to histologically evaluate the expression of SV2A, Stg1, synaptophysin, and BDNF. Images captured by a 3-CCD (charge-coupled device) color video camera (DP 70; Olympus, Tokyo, Japan), which was mounted on a light microscope (BH 2; Olympus), were transmitted to both a video monitor and a computer equipped with imaging software (DPC controller, Olympus). The brain sites of interest were selected on the relevant sections with their respective ODs, and measured by the same reader of the study in a

a L Fcx R Fcx L Pcx

1s

200µV

P cx CA3 AMY RT SN RF Absence seizure

1s

200µV

Fig. 2. Electroencephalography (EGG) tracings of absence-like seizures in spontaneously epileptic rats (SERs). (a) 5–7 Hz spikewave complexes recorded from the left (L) and right (R) frontal (F), parietal (P), temporal (T), and occipital (O) cortices (cx). (b) 5–7 Hz spike-wave complexes recorded from unilateral parietal cortex, CA3, amygdala (AMY), reticulothalamus (RT), substantia nigra (SN), and reticular formation (RF) were brain sites investigated. The horizontal and vertical scale bars indicate 1 s and 200 lV, respectively. All spike-wave complexes were synchronized in all electrodes.

R Pcx

SER

Wistar

L Tcx R Tcx

F

F

L Ocx R Ocx Tonic convulsion

b

1s

200µV

P

P cx

P

CA3

Ent

AMY

Ent Pir

RT

Pir

SN

O

RF Tonic convulsion

1s

O

200µV

Fig. 1. Electroencephalography (EEG) recordings of spontaneous tonic convulsion in spontaneously epileptic rats (SERs). (a) Low voltage fast waves recorded respectively from the left (L) and right (R) frontal (F), parietal (P), temporal (T), and occipital (O) cortices (cx). (b) Unilateral recordings of low voltage fast waves from the parietal cortex, CA3, amygdala (AMY), reticulothalamus (RT), substantia nigra (SN), and reticular formation (RF). The horizontal and vertical scale bars indicate 1 s and 200 lV, respectively. The interictal abnormal wave and leading spike before spontaneous tonic seizure were not recorded.

T

T

Fig. 3. H–E staining of the cerebral cortex in the spontaneously epileptic rats (left) and Wistar rat (right). Neuronal counts did not show significant differences between both animals in frontal (F), parietal (P), temporal (T), occipital (O), entorhinal (Ent), and Piriform (Pir) cortices. Quantification of these results can be found in Table 1. The scale bar represents 500 lm.

R. Hanaya et al. / Neuroscience 221 (2012) 12–20 Table 1. Number of cerebral neurons in SER and Wistar rats

Frontal cx Parietal cx Temporal cx Occipital cx Piriform cx Entorhinal cx

SER

Wistar

34.3 ± 2.2 34.6 ± 2.8 30.9 ± 4.1 29.7 ± 3.6 32.4 ± 4.0 31.2 ± 2.8

35.5 ± 7.0 32.4 ± 5.3 34.6 ± 5.4 33.0 ± 5.4 34.9 ± 5.8 32.1 ± 3.8

blind fashion. Images were captured at 40 or 100 for all structures of interest. The color images were transformed to black/ white images and analyzed accordingly (Image J software; NIH, Bethesda, USA). The same observer analyzed each side of three antero-posterior adjacent sections of a specific brain region. Standard transfer curves of the gray level produced by filters of known OD (Kodak, New York, USA) were used to calibrate the system.

Statistical analysis The mean value for each area/structure of each rat was calculated using the values from three serial sections. Statistical comparison of both strains was performed by an analysis of variance for the independent groups followed by the Scheffe’s test.

RESULTS EEG recording from the cerebral cortex Based on 3 observations allocated for EEG recordings of the cerebral cortex for each SER, 6 SERs showed spontaneous tonic convulsions characterized by low voltage fast waves: viz., the mean ± SEM frequencies

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and durations of the spontaneous tonic convulsions without stimulation were 0.44 ± 0.05 times/30 min and 15.11 ± 1.75 s in these SERs, respectively. Video-EEG recorded at least one bout of spontaneous tonic seizure in each SER. None of the electrodes including that of hippocampal CA3 showed interictal abnormal spikes and slow waves typical of focal epilepsy or symptomatic generalized epilepsy (Fig. 1a, b). EEG monitoring did not display obvious leading spikes or other predictive waves typical of focal seizures manifesting low voltage fast waves. SERs were immobile and staring blank during the spike-wave complexes which symptomatically indicated absence seizures. The mean frequencies and the duration of absence seizures were 70.56 ± 3.20 times/ 30 min and 2.65 ± 0.10 s, respectively. Spike-wave complexes of 5–7 Hz synchronously appeared in all electrodes implanted in the cerebral cortex and thalamus (Fig. 2a, b). These values of both seizures were similar to what we had reported previously (Sasa et al., 1988). Cell count and TUNEL assay in cerebral cortices of SER In tissue slices of each cerebral cortex stained by H-E, neuronal densities did not show any significant difference between SERs and Wistar rats. Pathological vacuolation was observed in SERs as shown in the previous reports (Inui et al., 1990). There were no significant differences between the cerebral cortices in both SERs and Wistar rats (Fig. 3, Table 1). Data from the TUNEL assay could not locate any apoptotic neuron in the brain structures/ regions examined in all 6 SERs (Fig. 4).

Fig. 4. TUNEL assay of cerebral cortex in spontaneously epileptic rats (SER) and Wistar rats. TUNEL assay was performed to SER (a), Wistar rats (b), positive control (c), and negative control (d). Small and dense nucleus with DNA fragmentation was stained in brown (illustrated by the arrows). The scale bar represents 200 lm.

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R. Hanaya et al. / Neuroscience 221 (2012) 12–20

(a)

(b)

(c)

(d)

(e)

(f)

higher BDNF expression in the hippocampus and piriform and entorhinal cortex (i.e. regions related with hippocampal sclerosis). BDNF expression in the hippocampus was similar to findings in our previous report (Sugata et al., 2011). SV2A expression in SERs was entirely low. All cerebral cortices and inner molecular layers (IMLs) of the dentate gyrus (DG) in SERs exhibited significantly lower distribution of SV2A than that found in Wistar rats. The SV2A expression in SERs was remarkably lower than values displayed in the frontal cortex and in layers III/IV of entorhinal cortices; registering 50.9% and 46.9% of the values of Wistar rats, respectively (Fig. 3). Moreover, Syt1 expression indicated lower in SERs as well: i.e. 31.2% in the frontal cortex, 29.9% and 13.2% in layer II and III/IV of piriform cortex, and 38.3% and 39.3% in layer II and III/IV of entorhinal cortex (vs values of Wistar rats), respectively. In SER hippocampus, the distribution of Syt1 was significantly (p < 0.05) lower in the IML of DG, registering 38.2% and 39.0% in the supragranular layer and outer part of IMLs of controls, respectively (Fig. 6).

DISCUSSION Abnormal neurotransmission related to epileptic seizures in SER

Fig. 5. Immunoreactivity to synaptotagmin-1 (Syt1) in spontaneously epileptic rats (SER) and Wistar rats. Raw picture image of frontal cortex (Fcx: a) and entorhinal cortex (Ent: b) did not show specific change. Optimized microscopic image suggested lower Syt1 distribution in Fcx (c) and Ent (d) of SER than FCx (e) and Ent (f) of Wistar rats, respectively. The scale bar represents 500 lm. Quantification of these results can be found in Tables 2 and 3.

Immunoreactivity of SV2A, synaptotagmin-1, synaptophysin, and BDNF Optimized picture images of slices revealed the difference of each protein distribution between two animals, however raw picture of immunostained slice did not show optical change as described in Methods (Fig. 5). We obtained the OD value from the raw immunostained slice to compare the results as numerical value. Analyses were performed after correction for background density. The corpus callosum, which has less synaptic terminals, was selected as the background reference, because it was included in slices examined in this study and did not show any variation in synaptophysin expression. The value of BDNF was derived from the difference between the value of region-of-interest and that of white matter of parietal cortex, as previously described (Sugata et al., 2011). All values measured for each structure/region were listed (Tables 2 and 3). Synaptophysin density in the cerebral cortex and hippocampus between SER and Wistar rats did not differ significantly. SERs exhibited significantly

The core of neurotransmitter release is formed by the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes (Rothman, 1994; Rizo and Rosenmund, 2008). SV2 functions in a maturation step and primes vesicles which convert themselves to a Ca2+- and Stg-responsive state (Chang and Su¨dhof, 2009). SV2A could act on upstream of Ca2+-triggering by priming synaptic vesicles, and SNARE is then reduced in SV2A-knockout brains (Xu and Bajjalieh, 2001). Syt1 is mainly expressed in the cytoplasm and cytomembrane of neurons and acts as a synaptic sensor at the synaptic terminal. SV2–Syt interaction regulates exocytosis and endocytosis of the synaptic vesicle traffic (Schivell et al., 2005; Zeng et al., 2009). Therefore, it is likely that low SV2A and Syt1 expressions/distributions in SERs may induce dysfunction of neurotransmitter release. Every electrode in this study recorded low voltage fast waves and 5–7 Hz spike-wave complexes in each tonic convulsion and absence seizures respectively. These findings and the semiology of observed seizures indicated that both seizures of SERs were generalized seizures. Generalized seizures and focal intractable epilepsy exhibited respectively diffuse and focal hypometabolism in interictal state (Herholz and Heiss, 2004; Goffin et al., 2008). A study using 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) has demonstrated diffuse cerebral glucose hypometabolism in epileptic encephalopathy patients, and hypometabolism is a consequence of reduced neuronal activity (Zhai et al., 2010). In addition, LCGU in the interictal state of SERs is decreased in the cerebral cortex and hippocampus (Saji et al., 1993), while cortical and hippocampal GABA concentrations are higher than those in normal Wistar rats (Fukao et al., 1998). GABA agonists produce dosedependent decreases in LCGU of the cerebral cortex

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R. Hanaya et al. / Neuroscience 221 (2012) 12–20 Table 2. Expression of synaptophysin, synaptic vesicle protein 2A (SV2A), and synaptotagmin-1 in SER and Wistar rats Synaptophysin

[Cortex] Frontal cx Parietal cx Temporal cx Occipital cx Piriform cx layer II layer III and IV Entorhinal cx layer II layer III and IV [Hippocampus] CA1 Pyramidal layer Stratum oriens CA3 Pyramidal layer Stratum oriens Hilus S. radiatum Dentate gyrus Granular layer IML: sgl IML: outer OML: inner OML: outer

SV2A

Synaptotagmin-1

SER

Wistar

SER

Wistar

SER

Wistar

2.64 ± 0.35 2.35 ± 0.44 1.84 ± 0.57 2.21 ± 0.46

2.37 ± 0.64 2.23 ± 0.41 2.28 ± 0.56 2.17 ± 0.20

1.15 ± 0.63* 1.32 ± 0.57* 1.13 ± 0.40* 1.35 ± 0.46*

2.26 ± 0.58 2.29 ± 0.62 1.92 ± 0.51 2.18 ± 0.58

0.35 ± 0.10* 1.16 ± 0.39 1.04 ± 0.63 1.08 ± 0.37

1.06 ± 0.36 1.29 ± 0.38 1.06 ± 0.65 1.22 ± 0.47

2.25 ± 0.59 1.89 ± 0.62

2.93 ± 0.82 2.14 ± 0.56

1.60 ± 0.23* 1.10 ± 0.35*

2.12 ± 0.39 1.80 ± 0.39

0.90 ± 0.51# 0.51 ± 0.35#

2.19 ± 0.95 1.58 ± 0.56

2.54 ± 0.47 2.17 ± 0.36

2.91 ± 0.45 2.43 ± 0.19

1.29 ± 0.17* 0.89 ± 0.33

2.11 ± 0.67 1.90 ± 0.43

1.19 ± 0.53# 0.95 ± 0.61*

2.51 ± 1.08 1.85 ± 0.81

0.71 ± 0.55 2.94 ± 0.82

0.42 ± 0.46 3.16 ± 0.26

1.03 ± 0.70 1.77 ± 0.67

1.26 ± 0.56 2.34 ± 0.66

0.58 ± 0.48 0.76 ± 0.19

0.64 ± 0.16 0.77 ± 0.21

1.03 ± 0.35 2.92 ± 0.28 1.97 ± 0.63 1.93 ± 0.35

0.81 ± 0.28 3.32 ± 0.56 2.54 ± 0.37 2.27 ± 0.17

1.18 ± 0.21 1.53 ± 0.19 1.08 ± 0.25 1.24 ± 0.16

1.28 ± 0.40 1.94 ± 0.54 1.64 ± 0.67 1.47 ± 0.34

0.77 ± 0.34 0.76 ± 0.28 0.05 ± 0.42 0.71 ± 0.47

0.71 ± 0.28 0.73 ± 0.26 0.21 ± 0.40 0.61 ± 0.31

0.53 ± 0.37 2.40 ± 0.55 2.24 ± 0.68 1.89 ± 0.56 1.91 ± 0.53

0.45 ± 0.29 2.61 ± 0.51 2.53 ± 0.49 2.22 ± 0.45 2.14 ± 0.46

0.66 ± 0.18 0.98 ± 0.20* 0.97 ± 0.13* 1.24 ± 0.17 1.31 ± 0.17

0.79 ± 0.31 1.64 ± 0.48 1.69 ± 0.56 1.72 ± 0.51 1.73 ± 0.48

0.28 ± 0.33 0.28 ± 0.20* 0.26 ± 0.21* 0.52 ± 0.24 0.62 ± 0.29

0.28 ± 0.17 0.74 ± 0.24 0.67 ± 0.16 0.61 ± 0.12 0.66 ± 0.15

Average ± SD (10 2). IML, inner molecular layer; OML, outer molecular layer; sgl, supragranular layer. * P < 0.05 significantly difference from each control. # P < 0.01 significantly difference from each control.

(Palacios et al., 1981). SV2A expression is reduced in focal cortical dysplasia and cortical tubers of patients with tuberous sclerosis complex, indicating that SV2A may contribute to the epileptogenicity of these malformations in cortical development (Toering et al., 2009). Downregulation of SV2A likely occurs in TLE patients (Feng et al., 2009). In SER cerebral cortices, SV2A expression levels were lower than those found in Wistar rats. Diffusedly low distribution of SV2A might have been due to the feedback inhibition of higher GABA concentration in the SER brain, because GABA receptors and GABAergic neurons are functional in SERs (Fukao et al., 1998). Low expression of SV2A and synaptotagmin-1levels in SER cerebral cortex Low Syt1 distribution is limited to the frontal, entorhinal, and piriform cortices in the SER brain. Additionally, low expression of SV2A with low Stg1 contents in these cortical areas may be involved in epileptogenesis, probably without feedback inhibition in the other cortical regions of SERs. Absence seizures may be triggered by widespread cortical (frontal and parietal) areas and sustained in the subcortical thalamic regions, suggesting that the examined patients could have cortical onset epilepsy with subsequent propagation to the thalamus (Westmijse et al., 2009; Szaflarski et al., 2010).

Ictal FDG-PET performed in a patient of pure absence status shows bilateral thalamic and frontal hypometabolism (Bilo et al., 2010). In an experimental model of absence seizure, an increase in synaptic excitability mediated by N-methyl-d-aspartyl (NMDA) receptors has been located in deep neocortical layers (D’Antuono et al., 2006). Low SV2A with low Syt1 expression levels in the frontal lobe could potentiate absence-like seizures in SERs via attenuated GABAergic dysfunction. Although local findings of partial seizures have not been reported in EEG studies, decreases of SV2A and Syt1 expression levels may be related to tonic seizures, because tonic seizure displays Lennox-Gastaut seizure-like symptoms resembling those of symptomatic or cryptogenic epilepsy (Arzimanoglou et al., 2009). Multiple dysfunction of SNARE-related protein seems to induce symptomatic seizures, and this may help to distinguish whether it be primary generalized or symptomatic generalized. Further investigations are warranted to clarify the seizures relevant to these results. Entorhinal cortex, especially layer III, provided a predominant excitatory drive to the hippocampal CA1 and subicular neurons in chronic epilepsy (Kobayashi et al., 2003). GABAergic dysfunction in layer III and dendritic excitability may increase neuronal firings (Shah et al., 2004). Low SV2A and Syt1 expression/distribution levels occur with subsequent exacerbation of hippocampal

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R. Hanaya et al. / Neuroscience 221 (2012) 12–20

Table 3. BDNF expression in SER and Wistar rats BDNF

[Cortex] Frontal cx Parietal cx Temporal cx Occipital cx Piriform cx layer II layer III and IV Entorhinal cx layer II layer III and IV [Hippocampus] CA1 Pyramidal layer Stratum oriens CA3 Pyramidal layer Stratum oriens Hilus S. radiatum Dentate gyrus Granular layer IML: sgl IML: outer OML: inner OML: outer

SER

Wistar

0.49 ± 0.08 0.50 ± 0.07 0.48 ± 0.09 0.48 ± 0.06

0.49 ± 0.25 0.49 ± 0.05 0.48 ± 0.11 0.49 ± 0.08

1.19 ± 0.22 0.98 ± 0.24*

0.52 ± 0.14 0.46 ± 0.18

1.30 ± 0.21# 1.21 ± 0.32#

0.47 ± 0.19 0.43 ± 0.17

1.23 ± 0.24# 1.15 ± 0.24#

0.40 ± 0.11 0.19 ± 0.09

1.68 ± 0.17# 1.51 ± 0.26# 0.79 ± 0.26* 1.03 ± 0.29#

0.49 ± 0.08 0.28 ± 0.11 0.41 ± 0.13 0.45 ± 0.20

0.89 ± 0.22# 1.37 ± 0.22# 0.83 ± 0.06# 0.41 ± 0.12 0.42 ± 0.17

0.09 ± 0.05 0.74 ± 0.09 0.33 ± 0.14 0.29 ± 0.16 0.27 ± 0.16

Average ± SD (10 2). IML, inner molecular layer; OML, outer molecular layer; sgl, supragranular layer. * P < 0.05 significantly difference from each control. # P < 0.01 significantly difference from each control.

Frontal cx

100

Parietal cx Temporal cx

*

% of control

80 60

#

Occipital cx

* ** **

Piriform cx II

** #

40

#*

# *

#

Piriform cx III&IV

**

20

Entorhinal cx II Entorhinal cx III&IV IML:sgl IML:outer

0

Synaptophysin

SV2A

Synaptotagmin-1

Fig. 6. Expression of synaptophysin, synaptic vesicle (SV2A) and synaptotagmin-1 (Syt1). Values are expressed as percentages (%) of control. Brain sites such as the cortex (cx), inner molecular layer (IML), and supragranular layer (sgl) are indicated. Differences of statistical comparison of both strains where P < 0.05 () and <0.01 (#) were verified by a variance analysis for the independent groups followed by the Scheffe’s test.

sclerosis in SERs. The piriform cortex would have been affected by changes in the other limbic structures, because it is the brain structure most sensitive to brain damage inflicted by continuous and/or frequent stimulations of the amygdala or hippocampus (Lo¨scher and Ebert, 1996). BDNF distributions portrayed higher values in the entorhinal and piriform cortices in our results. In

amygdale-kindled rats, mRNA expression of BDNF is found in neurons of the limbic cortical area, a site which is supposed to harbor projecting glutamatergic cortical neurons (Foster et al., 2004). This therefore supports the difference between SV2A-Syt1 and BDNF expression levels. Taken together, both phenomena enhance hippocampal sclerosis in the SER brain.

R. Hanaya et al. / Neuroscience 221 (2012) 12–20

Low SV2A and synaptotagmin-1 expression levels in the dentate gyrus of SERs Electrophysiological studies in the CA3 of SV2A-knockout mice have revealed that the loss of SV2A leads to a reduction in action potential-dependent GABAergic neurotransmission without affecting action potentialindependent neurotransmission (Crowder et al., 1999). In pentylentetrazole (PTZ)-kindling model animals, SV2A expression increases in the hippocampus hilar region of the DG by activating inhibitory GABAergic neurotransmission (Ohno et al., 2009). Syt1 increases in the hippocampus of phenytoin-resistant kindled rats (Zeng et al., 2009). These events are thought to have resulted from MF sprouting; results resembling the increases of SV2A and SNARE in the kindling models (Matveeva et al., 2007; Ohno et al., 2009). In the SER hippocampus, SV2A and Syt1 distribution/expression levels decreased in IML of DG. This change in the SER hippocampus did not elicit epileptic activity as shown in EEG findings of Figs. 1 and 2, while the number of vulnerable CA3 neurons was decreased, accompanied by mild MF sprouting correlating to a similar phenomenon observed in tonic convulsion (Hanaya et al., 2010). SV2A was insignificantly increased in the granular layer of the SER brain, thus revealing that the GABAergic interneurons may have reacted to MF sprouting. A microdialysis experiment has recently demonstrated that GABA levels are increased by 293% in the hippocampus of temporal lobe epilepsy (TLE) patients with hippocampal sclerosis (Go¨ren et al., 2001). In a similar tendency, SV2A expression/distribution decreases in TLE patients with hippocampal sclerosis as well (van Vliet et al., 2009). The low SV2A expression/distribution would induce feedback derived from high GABA levels in chronic mesial TLE patients. Syt1 expression/distribution in mesial TLE is lower than in controls (Yang et al., 2006). As shown in SER cortices and the IML of SERs (Fig. 3), low Syt1 expression/distribution may be associated with epileptogenesis-related GABAergic dysfunction.

CONCLUSION Diffusedly low SV2A distribution is expressed in irritative SER cerebral cortices that exhibit generalized seizure with IML of hippocampus manifesting sclerotic changes. Decreases of both SV2A and Syt1 expression/distribution levels, which cause dysfunction of synaptic vesicle fusion, may result in impairment of GABAergic dysfunction, thus contributing to the expression and propagation of epileptic seizures in SERs.

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(Accepted 25 June 2012) (Available online 3 July 2012)