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Changes in hippocampal connexin 36 mRNA and protein levels during epileptogenesis in the kindling model of epilepsy
Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 510–515
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Changes in hippocampal connexin 36 mRNA and protein levels during epileptogenesis in the kindling model of epilepsy Siamak Beheshti a,b, Mohammad Sayyah b,⁎, Majid Golkar c, Hoori Sepehri a, Jalal Babaie c, Behrouz Vaziri d a
Department of Animal Biology, School of Biology, University College of Science, Tehran University, Tehran, Iran Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran d Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 5 December 2009 Received in revised form 8 February 2010 Accepted 8 February 2010 Available online 12 February 2010 Keywords: Amygdala kindling Connexin 36 Epileptogenesis Hippocampus
a b s t r a c t Objective: Identification of key molecular changes occurring during epileptogenesis provides better understanding of epilepsy and helps to develop strategies to modify those changes and thus, block the epileptogenic process. Gap junctional communication is thought to be involved in epileptogenesis. This communication can be affected by changes in expression of gap junctional protein subunits called connexins (Cxs). One of the main brain regions involved in epileptogenesis is the hippocampus in which there is a network of gap junctional communication between different cell types. Method: Cx36 and Cx43 expressions at both mRNA and protein level were measured in rat hippocampus during epileptogenesis in the kindling model of epilepsy. Results: Cx36 expression at both mRNA and protein level was upregulated during acquisition of focal seizures but returned to basal level after acquisition of secondarily-generalized seizures. No change in Cx43 gene and protein expression was found during kindling epileptogenesis. Conclusion: These results further point out the significance of Cx36 as a target to modify epileptogenic process and to develop antiepileptogenic treatments. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is the most common neurologic disorder after stroke (Porter and Meldrum, 2001). Currently available antiepileptic drugs suppress seizures without altering the underlying course of epilepsy (Herman, 2006). Epileptogenesis is the process by which parts of a normal brain are converted to a hyperexcitable, epileptic brain (Dichter, 2006). Identification of key molecular changes provides better understanding of epileptogenesis and points to targets that can be used to modify the epileptogenic process and develop antiepileptogenic treatments. Seizure is a result of abnormal excessive synchronized electrical activity of a large group of neurons (Porter and Meldrum, 2001). Spreading of the abnormal activity of a small group of cells to adjacent cells involves large cell groups and results in generalization of the Abbreviations: ACSF, Artificial Cerebrospinal Fluid; AD, After-discharge; AMV, Avian Myeloblastoma Virus; bp, base pair; cDNA, Complementary Deoxyribonucleic acid; CNS, Central Nervous System; Cx, Connexin; ECL, Enhanced Chemiluminescence; i.c.v, intracerebroventricular; IgG, Immunoglobulin G; kDa, Kilodalton; mRNA, messenger Ribonucleic Acid; O.D, Optical Density; PVDF, Polyvinylidene Fluoride; RT-PCR, Reverse Transcription Polymerase Chain Reaction; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; TBST, Tris Buffered Saline Tween. ⁎ Corresponding author. Tel./fax: + 98 21 66968854. E-mail address: [email protected] (M. Sayyah). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.02.006
seizures (Fisher et al., 2005). Besides the synaptic contacts between the cells, gap junctional coupling is supposed to be involved in the generation or spreading of the seizures (Perez-Velazquez and Carlen, 2000; Carlen et al., 2000; Kohling et al., 2001; Traub et al., 2001, 2002; Nemani and Binder, 2005). Gap junctions are specialized cell–cell contacts between eukaryotic cells, composed of aggregates of transmembrane channels, which directly connect the cytoplasm of adjacent cells, allowing intercellular movement of ions, metabolites and second messengers (Condorelli et al., 2003; Sohl et al., 2005). Each channel consists of two hemichannels (termed connexons), each of which is composed of six subunit proteins called connexin (Cx) (Sohl et al., 2005). It has been shown that seizures can be blocked by gap junction blockers, and gap junction openers exacerbate seizure activity (Perez-Velazquez et al., 1994; Dudek et al., 1998; Carlen et al., 2000; Gadja et al., 2003, 2005; Nemani and Binder, 2005; Samoilova et al., 2008; He et al., 2009). A number of studies have also established that epilepsy is associated with changes in Cxs expression and intercellular coupling (Rouach et al., 2002). Apart from the results, in most of the epilepsy studies performed in human and animals regarding Cxs expression, the changes have been evaluated in the brain that is already epileptic (Naus et al., 1991; Khurgel and Ivy, 1996; Elisevich et al., 1997b; Sohl et al., 2000; Li et al., 2001; Aronica et al., 2001; Fonseca et al., 2002; Szente et al., 2002; Condorelli et al., 2003; Samoilova et al., 2003; Gadja et al., 2003, 2006; Collignon et al.,
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and there are few studies performed on Cxs expression during epileptogenesis (Elisevich et al., 1997a). The kindling model of epilepsy represents a model of the seizures, which are analogous to human complex partial (focal) seizures with secondarily generalization (McNamara et al., 1980). Kindling is also a well established animal model of epileptogenesis in which periodic tetanic electrical stimulation of a particular brain region leads to a permanent state of hyperexcitability (Goddard and McIntyre, 1969). In the kindling model, with repeated administration of an initially subconvulsive electrical stimulus, seizures generally evolve through five stages: (1) facial clonus (2) head nodding (3) forelimb clonus (4) rearing and (5) rearing and falling accompanied by generalized clonic seizures (Racine, 1972). The behavioral seizure stages 1 and 2 correspond to the activation of ipsilateral limbic structures including— but not necessarily limited to—the hippocampus, and are considered as focal seizures, whereas stages 3–5 represent secondarily-generalized motor seizures and correspond to the propagation of discharge to the contralateral limbic structures and then to the outside of the limbic system (Hewapathirane and Burnham, 2005). Kindling permits the investigator to evaluate any change that occurs at the cellular and molecular levels at any stage of the transformation of the normal brain to a hyperexcitable one. In the brain, Cxs are expressed in a cell type-specific manner (Rash et al., 2001). Among Cxs, Cx36 and Cx43 are well represented throughout the CNS. Cx36 is mainly expressed in neurons, especially interneurons (Condorelli et al., 1998, 2003) while Cx43 is predominantly expressed in astrocytes (Theis et al., 2003). Hippocampus is one of the main brain regions involved in kindling and epileptogenesis (Racine et al., 1989) and there is a network of gap junctional communication between different cell types within the hippocampus (Sohl et al., 2000). There is no report regarding the possible alterations in hippocampal Cx36 and Cx43 expression during epileptogenic process. In the present study, the extent of expression of Cx36 and Cx43 at mRNA and protein level was investigated in the rat hippocampus during epileptogenesis in the amygdala-kindling model of epilepsy. 2. Methods 2.1. Materials Ketamine and xylazine were from Rotex Medica (Germany) and Chanelle (Ireland), respectively. RNX-PLUS Reagent, Agarose, Acrylamide and Bis-Acrylamide were purchased from CinnaGen (Iran). Hot Start Taq DNA polymerase, 2× RNA Loading Dye, Page Ruler™ Prestained Protein Ladder and Protein Loading Buffer Pack were purchased from Fermentas (Lithuania). Protease Inhibitor Cocktail, RNase free DNase I, and 1st strand cDNA synthesis kit were from Roche (Germany). Enhanced Chemiluminescence (ECL) Advance Western Blotting Detection Kit was from Amersham (UK). Monoclonal anti-connexin 36 was from Zymed (USA). Monoclonal anticonnexin 43 was from Upstate (USA). Monoclonal anti-α-tubulin and anti-mouse IgG peroxidase conjugates were from Sigma-Aldrich (USA). Other chemicals were from Applichem (Germany) and Sigma-Aldrich (USA). 2.2. Animals Adult male Wistar rats (300–350 g, Pasteur Institute of Iran) were used throughout the study. Animals were housed in groups of 2 and had free access to food (standard laboratory rodents chow) and drinking water. The animal house temperature was maintained at 23± 1 °C with an alternating 12-hr light/dark cycle (light on from 6 a.m.). All animal experiments were carried out in accordance with the European Communities Council Directive of November 1986 (86/609/EEC) in such a way to minimize the number of animals and their suffering. All the injections were done intraperitoneally (i.p.).
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2.3. Kindling procedure and tissue preparation The rats were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg). Then they were stereotaxically implanted with bipolar stimulating and monopolar recording stainless-steel Teflon-coated electrodes (A.M. Systems, USA, twisted into a tripolar configuration), in the basolateral amygdala (coordinates: A,−2.5 mm from bregma; L, 4.8 mm from bregma; V, 7.5 mm from dura) of the right hemisphere (Paxinos and Watson, 2005). The electrodes were fixed on the skull with dental acrylic. After 7 days of recovery period, after-discharge (AD) threshold was determined in amygdala by a 2-sec, 100 Hz monophasic square-wave stimulus of 1 ms per wave. The stimulation was initially delivered at 50 µA and then at 5-min intervals, increasing stimulus intensity in increments of 50 µA was delivered until at least 5 s of AD was recorded (Sayyah et al., 2007). Then two different groups of animals with known AD threshold (each group consisting of 5–7 rats) were stimulated once daily at AD threshold until they showed behavioral seizure stage 2 in two consecutive days (as partially-kindled rats) or behavioral seizure stage 5 in three consecutive days (as fully kindled rats) according to Racine classification (Racine, 1972). Two other groups of electrode-implanted, non-stimulated rats were used and considered as sham-operated control groups (sham 1 and sham 2 groups corresponds to the partially- and fully kindled rats, respectively). In previous similar studies, the time courses of 3 h or two weeks were used to study the changes in Cxs expression (Elisevich et al., 1997a; Sohl et al., 2000). However, these time courses seem too early or late. Therefore, we chose a 24-hr time point, which was also the interval of our stimulation protocol, to evaluate the changes in Cxs expression at mRNA and protein level. Twenty four hours after showing last behavioral seizure stage of 2 or 5, all the animals and their corresponding controls were decapitated under deep ether anesthesia and their brain were removed immediately. The brains were incubated in chilled artificial cerebrospinal fluid (ACSF) with pH 7.3 consisted of the following composition (in mM): 124 NaCl, 4.4 KCl, 2 CaCl2, 2 MgCl2, 1.2 KH2PO4, 25 NaHCO3 and 10 Glucose. The hippocampi of the right hemisphere of the brains (the side that the electrodes were implanted in the amygdala) were removed and frozen immediately in liquid nitrogen and stored at −80 °C. The rest of the brains were placed in 10% formalin for at least 24 h at room temperature and they were then processed, cut into 10 μm thick slices and stained by the method described before (Sayyah et al., 2007). The stained slices were qualitatively analyzed for electrode position using a stereoscopic microscope (Olympus, Japan). The data of the animals, in which their electrode was in false placement, were not included in the results. 2.4. Gene expression study The frozen hippocampi were removed from −80 °C and pulverized completely. About 200 µl of chilled phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, and 1.4 mM KH2PO4) was added to the pulverized tissues and vortexed for 30 s, then spinned and aliquoted in two separate microtubes equally. One of the so prepared samples was used for gene expression study and the second part for immunoblotting. An appropriate volume of a protease inhibitor cocktail according to the manufacturer′s proposal was added to samples, which were allocated for immunoblotting. Total cellular RNA was isolated from the hippocampus by a modification of the guanidine isothiocyanate phenol-chloroform method (Ausubel et al., 2002) using RNX-PLUS reagent then treated with 10 U RNase free DNase I (Roche, Germany) to avoid any DNA contamination. The integrity of the RNA samples was determined using denaturing agarose gel electrophoresis. The concentrations of the RNAs were determined spectrophotometrically (Nanodrop, USA). The mean 260/280 ratios were 1.94 ± 0.0, while those of 260/230 were 1.98 ± 0.1.
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A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) based strategy was employed, as it was shown to be a quite sensitive method for quantification of connexins expression (Szente et al., 2002; Gadja et al., 2003, 2006). The reverse transcription reaction was performed with 1st strand cDNA synthesis kit (Roche, Germany) using oligo-dT primer, AMV reverse transcriptase and 1 µg total RNA as template, according to the manufacturer′s instructions. The polymerase chain reaction parameters were adjusted to obtain a condition with linear relation between the initial amount of cDNA template and PCR product and with linear relation between the number of PCR cycles and PCR products. PCR amplification was performed on an appropriate dilution of the RT product by using a Hot Start Taq DNA Polymerase and 35 cycles of 95 °C for 50 s, 54 °C for 1 min and 72 °C for 30 s for Cx36, 35 cycles of 95 °C for 50 s, 48 °C for 1 min and 72 °C for 30 s for Cx43 and 26 cycles of 95 °C for 50 s, 60 °C for 1 min and 72 °C for 30 s for β-actin. The final cycles were followed by a 7 min extension step at 72 °C and a soak cycle at 4 °C. The following primers were used: Cx36 forward: 5′- GCA GAG AGA ACG CCG GTA CT -3′ Cx36 reverse: 5′- CTT GGA CCT TGC TGC TGT GC -3′ Cx43 forward: 5′- GAT GAG GAA GGA AGA GAA G -3′ Cx43 reverse: 5′- CGC TAG CTT GCT TGT TGT AA -3′ β-actin forward: 5′- GCA AGA GAG GTA TCC TGA CC -3′ β-actin reverse: 5′- CCC TCG TAG ATG GGC ACA GT -3′
Cx36 and Cx43 proteins were expressed as ratios (Cx36/α-tubulin×100, Cx43/α-tubulin×100). 2.6. Statistical analysis In order to increase the reliability of the measurements, RT-PCR reactions and immunoblottings were performed on total RNAs and total proteins of 5–7 animals, independently and in duplicate. Results are presented as the mean ± S.E.M. The data were analyzed by unpaired Student′s t-test. Correlation of Cxs mRNA expression with Cxs protein expression was analyzed by Spearman's correlation test. Statistical significance was considered at p b 0.05. 3. Results A sample of recorded AD from amygdala is shown in Fig. 1. The mean AD threshold obtained for the animals was 200 ± 20 µA. The number of stimulations to stages 2 and 5 was 8.3 ± 0.2 and 13.6 ± 0.8, respectively. Mean of AD duration in stages 2 and 5 were 24.2 ± 3.5 and 63.6 ± 9.2 s, respectively. Cx36 and Cx43 mRNA and protein levels were quantitated in the same hippocampi tissues. Histological evaluation of the brains confirmed that in at least five rats out of seven in each group, the electrode was in a correct position, the data of which were used for statistical analysis. 3.1. Gene expression analysis
The sequences of the oligonucleotides were adopted from previous studies. Cx36 and Cx43 primers were chosen to lack homology with any published connexin sequences (Szente et al., 2002; Gadja et al., 2003). PCR products of each Cx and β-actin of each sample were mixed in the same well and run on 1.5% agarose gel in order to maintain the same conditions for test and reference genes and to be able to make more precise comparison. Bands were quantified by densitometry using Labworks analyzing software (Ultra Violet Products, U.K). The relative levels of Cx36 and Cx43 mRNAs were expressed as ratios (Cx36/ β-actin × 100, Cx43/β-actin × 100).
Partially-kindled rats showed a mean density value of 610.5 ± 6.3% (P = 0.0043) and 80.1 ± 10.3% (P = 0.29) for Cx36 and Cx43 mRNAs compared to 100.0 ± 40.3% and 100.0 ± 15.8% of their sham controls, respectively. Fully kindled rats exhibited a mean density value of 136.5 ± 20.6% (P = 0.28) and 92.9 ± 4.7% (P = 0.24) for Cx36 and Cx43 mRNAs compared to 100.0 ± 14.3% and 100.0 ± 3.4% of their sham controls, respectively. In the partially-kindled animals, the mRNA expression levels of hippocampal Cx36 increased significantly relative to the control (Fig. 2).
2.5. Immunoblotting
3.2. Immunoblot analysis
The second part of the homogenized hippocampi tissues was removed from −80 °C and centrifuged at 12,000 g, 4 °C for 10 min. The supernatant was collected and total protein concentration was determined using Bio-Rad DC protein assay reagents. Samples were dissolved in protein loading buffer and denatured for 5 min at 95 °C prior to loading. Equal amounts of protein from each animal (5 µg per lane for α-tubulin, 10 µg per lane for Cx36 and 25 µg per lane for Cx43) were resolved by denaturing SDS-Polyacrylamide gel electrophoresis (SDS-PAGE), 12% acrylamide and transferred to a PVDF membrane (Roche, Germany) by electroblotting (Mini trans blot electrophoretic transfer cell, Bio-Rad). The membrane was blocked in TBST buffer (100 mM Tris base, 150 mM NaCl, and 0.2% Tween 20) containing 2% ECL Advance blocking agent at room temperature for 60 min, rinsed briefly with TBST buffer and then incubated for 60 min with the following primary antibodies: mouse monoclonal anti-connexin 36 diluted 1:2000, mouse monoclonal anticonnexin 43 diluted 1:100,000 and mouse monoclonal anti-α-tubulin diluted 1:200,000. The antibodies were diluted in blocking buffer. After washing with TBST buffer 4 times (1× for 15 min and 3× for 5 min), the membrane was incubated with peroxidase conjugated goat anti-mouse IgG (diluted 1:50,000, 1:400,000 and 1:2,000,000 for Cx36, Cx43 and α-tubulin, respectively) for 1 h, then washed with TBST buffer 4 times (1× for 15 min and 3× for 5 min) and reacted with ECL Advance western blotting detection reagents, for 4 min. An X-ray film (Retina, USA) was used for 30 s to 10 min and then developed to visualize the antibody binding. Bands were quantified by densitometry using Labworks analyzing software (Ultra Violet Products, U.K). The relative levels of
Partially-kindled rats exhibited a mean density value of 209.3±10.2% (P =0.01) and 130.0±19.0% (P =0.17) for Cx36 and Cx43 protein compared to 100.0 ±29.4% and 100.0± 25.2% of their sham controls, respectively. Fully kindled rats showed a mean density value of 115.9± 15.1% (P= 0.51) and 121.8 ±22.5% (P= 0.59) for Cx36 and Cx43 protein compared to 100.0 ±14.8% and 100.0± 29.0% of their sham controls, respectively. In the partially-kindled animals, the protein expression levels of hippocampal Cx36 increased significantly relative to the control (Fig. 3). 3.3. Correlation of Cx36 mRNA with Cx36 protein In partially-kindled rats, Cx36 expression at mRNA level correlated positively with Cx36 expression at protein level (P = 0.001, r = 0.725). 4. Discussion The present study indicates that hippocampal Cx36 expression at both mRNA and protein level is upregulated during acquisition of focal seizures and declines to almost the basal level after acquisition of secondarily-generalized seizures. Furthermore, there was a significant positive correlation between Cx36 mRNA expression and Cx36 protein expression during early stages of kindling epileptogenesis. However, hippocampal Cx43 mRNA and protein expression did not change during either focal or secondarily-generalized seizures acquisition.
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Fig. 1. Recorded after-discharges from the amygdala of an animal with partial (A) or secondarily-generalized (B) seizures.
Expression levels of Cx36 have previously been investigated in human epileptic tissue and different experimental animal models of epilepsy. Aronica et al. (2001), have shown an upregulation in Cx36 mRNA in the human epileptic tissue, while Collignon et al. (2006), indicated preservation of Cx36 in human hippocampal subregions suffering from mesial temporal lobe epilepsy. Upregulation of Cx36 mRNA has been detected in the somatosensory cortex of both immature (Gadja et al., 2006) and adult rats (Gadja et al., 2003) 1 h following repeated seizures induced by 4-Aminopyridine, while no change was seen in Cx36 protein after repeated tetanization of
hippocampal slices (Jahromi et al., 2002). On the other hand, down regulation of hippocampal Cx36 mRNA and protein was reported in fully kindled rats 2–3 weeks following the last kindled seizures (Sohl et al., 2000). Also a brief evoked after-discharge produced a significant reduction in Cx36 protein expression in the dorsal hippocampus (McCracken and Roberts, 2006). Moreover, following i.c.v injection of kainic acid, a progressive down regulation in Cx36 mRNA was detected in the hippocampal CA3–CA4 regions but not in the hippocampal CA1 subfield and in the cerebral cortex (Condorelli et al., 2003). The discrepancies regarding Cx36 expression in different studies have been
Fig. 2. Expression of Cx36 (A) and Cx43 (B) genes in the hippocampus of the rats with partial and secondarily-generalized seizures. Connexin mRNA levels were normalized to that of β-actin mRNA. Data are expressed as means ± S.E.M (n = 5). Each polymerase chain reaction was performed in duplicate to increase the reliability of the measurements. Panels C and D show representative RT-PCR products of Cx36 (270 bp) and Cx43 (620 bp) in the partial and full kindled animals. **:p b 0.01.
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Fig. 3. Expression of Cx36 (A) and Cx43 (B) proteins in the hippocampus of the rats with partial and secondarily-generalized seizures. Cx36 and Cx43 protein levels were normalized to that of α-tubulin protein. Data are expressed as means ± S.E.M (n = 6). Each immunoblotting was performed in duplicate to increase the reliability of the measurements. Panels C and D show representative immunoblots of Cx36 (36 kDa) and Cx43 (43 kDa) in the partial and full kindled animals, respectively. *:p b 0.05.
attributed to variations in preparations used, methods of seizures induction, duration of seizure activity, time point examined after seizure activity and different brain regions and subregions used (McCracken and Roberts, 2006). Although these studies indicate a relationship between Cx36 gap junctional coupling and seizure activity, they do not provide information about possible alterations in Cx36 expression during the course of epileptogenesis. The aforementioned epileptic patients had suffered from epilepsy for several years (Aronica et al., 2001; Collignon et al., 2006). This is somewhat the case in experimental animal models of epilepsy, where the expression levels of connexins were studied only after establishment of seizures or epileptic conditions (Sohl et al., 2000; Gadja et al., 2003; Condorelli et al., 2003). Therefore, it is not clear whether the changes in connexins expression had occurred in response to seizures and epileptic conditions, or had been generated during development of epilepsy. Our study is the first study that has investigated patterns of expression of hippocampal Cx36 and Cx43 during epileptogenesis in a chronic in vivo model. Kindling causes long lasting effects on hippocampal physiology and anatomy, ranging from the subcellular level, such as altered hippocampal gene expression, to complicated structural alterations, like mossy fiber sprouting, neurogenesis and cellular apoptosis (Hewapathirane and Burnham, 2005). According to our results, gene and protein expressions of Cx36 are upregulated in hippocampus during kindling development but the
changes are limited to the early stages of kindling. This finding suggests that Cx36 over expression plays a significant role in induction of partial seizures. However, after establishment of focal seizures, Cx36 expression returns to basal level and therefore, ipsilateral Cx36 over expression does not seem to be involved in stage of seizure spreading to the contralateral limbic structures and consequent generalization of the seizures. Cx36 expressing neurons are present in all the regions of the hippocampal formation, including the entorhinal cortex (Condorelli et al., 2000). Cx36 is expressed in the GABAergic interneurons located in the various layers of CA1, CA3 and dentate gyrus, but the principal pyramidal cells express Cx36 only in CA3 region (Condorelli et al., 2000). However, our results do not address whether the Cx36 upregulation happens in all the hippocampal regions or it is confined to a specific subfield. Nonetheless, it can be proposed that the increase in Cx36 containing gap junctions between CA3 pyramidal neurons or GABAergic interneurons has elevated their capability to synchronize input stimuli and facilitate focal seizures induction. Further experiments such as in situ hybridization are necessary to provide concise data regarding the level of expression in different hippocampal regions during kindling. In this study, significant positive correlation of Cx36 expression at mRNA level with Cx36 expression at protein level was found during
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the early stages of kindling epileptogenesis. This indicates that Cx36 expression was regulated mainly at the transcriptional level. Finally, we found that the overall expression of hippocampal Cx43 at the mRNA and protein level did not change during kindling development. Similar to our results, Elisevich et al. (1997a), reported no change in Cx43 mRNA expression in amygdala of the rats during development of amygdala kindling. Moreover, it has been found that hippocampal Cx43 expression at both mRNA and protein level is unchanged or slightly decreased in fully kindled rats 2–6 weeks after the last kindled seizures occurrence (Sohl et al., 2000). Therefore, it seems that the hippocampal Cx43 has no significant role in kindling epileptogenesis. 5. Conclusion Our study is the first report on the potential role of Cx36 in epileptogenesis. Over expression of Cx36 during initial stages of kindling epileptogenesis further points out the significance of Cx36 as a target to modify epileptogenic process and to develop antiepileptogenic treatments. To further elucidate the role of Cx36 in epileptogenesis, Cx36 specific blockers can be administered intra-hippocampally and their effect on kindled seizures acquisition be evaluated. Moreover, studies on epileptogenesis in Cx36 knockout animals as well as using specific gene expression inhibitors such as antisense oligodeoxynucleotides are suggested to extend the results of this study. Acknowledgment Financial support by grant no. 301 from Pasteur Institute of Iran is acknowledged. References Aronica E, Gorter JA, Jansen GH, Leenstra S, Yankaya B, Troost D. Expression of connexin 43 and connexin 32 gap junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex. Acta Neuropathol 2001;101:449–59. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al. Short protocols in molecular biology. Wiley; 2002. Carlen PL, Skinner F, Zhang L, Naus C, Kushnir M, Perez Velazquez JL. The role of gap junctions in seizures. Brain Res Rev 2000;32:235–41. Collignon F, Wetsen NM, Cohen-Gadol AA, Cascino GD, Parisi J, Meyer FB, et al. Altered expression of connexin subtypes in mesial temporal lobe epilepsy in humans. J Neurosurg 2006;105:77–87. Condorelli DF, Parenti R, Spinella F, Trovato Salino A, Belluardo N, Cardile V. Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur J NeuroSci 1998;10:1202–8. Condorelli DF, Belluardo N, Trovato-salinaro A, Mudo G. Expression of Cx36 in the mammalian neurons. Brain Res Rev 2000;32:72–85. Condorelli DF, Trovato-salinaro A, Mudo G, Mirone MB, Belluardo N. Cellular expression of connexins in the rat brain: neuronal localization, effects of kainite-induced seizures and expression in apoptotic neuronal cells. Eur J NeuroSci 2003;18:1807–27. Dichter MA. Models of epileptogenesis in adult animals available for antiepileptogenesis drug screening. Epilepsy Res 2006;68:31–5. Dudek FE, Yasumura T, Rash JE. Non-synaptic mechanisms in seizures and epileptogenesis. Cell Biol Int 1998;22:793–805. Elisevich K, Rempel SA, Smith B, Allar N. Connexin 43 mRNA expression in two experimental models of epilepsy. Mol Chem Neuropathol 1997a;32:75–88. Elisevich K, Rempel SA, Smith BJ, Edvardsen K. Hippocampal connexin 43 expression in human complex partial seizure disorder. Exp Neurol 1997b;145:154–64. Fisher R, van Emde Boas W, Blume W, Elger C, Genton P, Lee P, et al. Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 2005;46:470–2. Fonseca CG, Green CR, Nicholson LFB. Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Res 2002;929:105–16. Gadja Z, Gyengesi E, Hermesz E, Said Ali K, Szente M. Involvement of gap junctions in the manifestation and control of the duration of seizures in rats in vivo. Epilepsia 2003;44:1596–600.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Changes in hippocampal connexin 36 mRNA and protein levels during epileptogenesis in the kindling model of epilepsy Siamak Beheshti a,b, Mohammad Sayyah b,⁎, Majid Golkar c, Hoori Sepehri a, Jalal Babaie c, Behrouz Vaziri d a
Department of Animal Biology, School of Biology, University College of Science, Tehran University, Tehran, Iran Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran d Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 5 December 2009 Received in revised form 8 February 2010 Accepted 8 February 2010 Available online 12 February 2010 Keywords: Amygdala kindling Connexin 36 Epileptogenesis Hippocampus
a b s t r a c t Objective: Identification of key molecular changes occurring during epileptogenesis provides better understanding of epilepsy and helps to develop strategies to modify those changes and thus, block the epileptogenic process. Gap junctional communication is thought to be involved in epileptogenesis. This communication can be affected by changes in expression of gap junctional protein subunits called connexins (Cxs). One of the main brain regions involved in epileptogenesis is the hippocampus in which there is a network of gap junctional communication between different cell types. Method: Cx36 and Cx43 expressions at both mRNA and protein level were measured in rat hippocampus during epileptogenesis in the kindling model of epilepsy. Results: Cx36 expression at both mRNA and protein level was upregulated during acquisition of focal seizures but returned to basal level after acquisition of secondarily-generalized seizures. No change in Cx43 gene and protein expression was found during kindling epileptogenesis. Conclusion: These results further point out the significance of Cx36 as a target to modify epileptogenic process and to develop antiepileptogenic treatments. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is the most common neurologic disorder after stroke (Porter and Meldrum, 2001). Currently available antiepileptic drugs suppress seizures without altering the underlying course of epilepsy (Herman, 2006). Epileptogenesis is the process by which parts of a normal brain are converted to a hyperexcitable, epileptic brain (Dichter, 2006). Identification of key molecular changes provides better understanding of epileptogenesis and points to targets that can be used to modify the epileptogenic process and develop antiepileptogenic treatments. Seizure is a result of abnormal excessive synchronized electrical activity of a large group of neurons (Porter and Meldrum, 2001). Spreading of the abnormal activity of a small group of cells to adjacent cells involves large cell groups and results in generalization of the Abbreviations: ACSF, Artificial Cerebrospinal Fluid; AD, After-discharge; AMV, Avian Myeloblastoma Virus; bp, base pair; cDNA, Complementary Deoxyribonucleic acid; CNS, Central Nervous System; Cx, Connexin; ECL, Enhanced Chemiluminescence; i.c.v, intracerebroventricular; IgG, Immunoglobulin G; kDa, Kilodalton; mRNA, messenger Ribonucleic Acid; O.D, Optical Density; PVDF, Polyvinylidene Fluoride; RT-PCR, Reverse Transcription Polymerase Chain Reaction; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; TBST, Tris Buffered Saline Tween. ⁎ Corresponding author. Tel./fax: + 98 21 66968854. E-mail address: [email protected] (M. Sayyah). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.02.006
seizures (Fisher et al., 2005). Besides the synaptic contacts between the cells, gap junctional coupling is supposed to be involved in the generation or spreading of the seizures (Perez-Velazquez and Carlen, 2000; Carlen et al., 2000; Kohling et al., 2001; Traub et al., 2001, 2002; Nemani and Binder, 2005). Gap junctions are specialized cell–cell contacts between eukaryotic cells, composed of aggregates of transmembrane channels, which directly connect the cytoplasm of adjacent cells, allowing intercellular movement of ions, metabolites and second messengers (Condorelli et al., 2003; Sohl et al., 2005). Each channel consists of two hemichannels (termed connexons), each of which is composed of six subunit proteins called connexin (Cx) (Sohl et al., 2005). It has been shown that seizures can be blocked by gap junction blockers, and gap junction openers exacerbate seizure activity (Perez-Velazquez et al., 1994; Dudek et al., 1998; Carlen et al., 2000; Gadja et al., 2003, 2005; Nemani and Binder, 2005; Samoilova et al., 2008; He et al., 2009). A number of studies have also established that epilepsy is associated with changes in Cxs expression and intercellular coupling (Rouach et al., 2002). Apart from the results, in most of the epilepsy studies performed in human and animals regarding Cxs expression, the changes have been evaluated in the brain that is already epileptic (Naus et al., 1991; Khurgel and Ivy, 1996; Elisevich et al., 1997b; Sohl et al., 2000; Li et al., 2001; Aronica et al., 2001; Fonseca et al., 2002; Szente et al., 2002; Condorelli et al., 2003; Samoilova et al., 2003; Gadja et al., 2003, 2006; Collignon et al.,
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and there are few studies performed on Cxs expression during epileptogenesis (Elisevich et al., 1997a). The kindling model of epilepsy represents a model of the seizures, which are analogous to human complex partial (focal) seizures with secondarily generalization (McNamara et al., 1980). Kindling is also a well established animal model of epileptogenesis in which periodic tetanic electrical stimulation of a particular brain region leads to a permanent state of hyperexcitability (Goddard and McIntyre, 1969). In the kindling model, with repeated administration of an initially subconvulsive electrical stimulus, seizures generally evolve through five stages: (1) facial clonus (2) head nodding (3) forelimb clonus (4) rearing and (5) rearing and falling accompanied by generalized clonic seizures (Racine, 1972). The behavioral seizure stages 1 and 2 correspond to the activation of ipsilateral limbic structures including— but not necessarily limited to—the hippocampus, and are considered as focal seizures, whereas stages 3–5 represent secondarily-generalized motor seizures and correspond to the propagation of discharge to the contralateral limbic structures and then to the outside of the limbic system (Hewapathirane and Burnham, 2005). Kindling permits the investigator to evaluate any change that occurs at the cellular and molecular levels at any stage of the transformation of the normal brain to a hyperexcitable one. In the brain, Cxs are expressed in a cell type-specific manner (Rash et al., 2001). Among Cxs, Cx36 and Cx43 are well represented throughout the CNS. Cx36 is mainly expressed in neurons, especially interneurons (Condorelli et al., 1998, 2003) while Cx43 is predominantly expressed in astrocytes (Theis et al., 2003). Hippocampus is one of the main brain regions involved in kindling and epileptogenesis (Racine et al., 1989) and there is a network of gap junctional communication between different cell types within the hippocampus (Sohl et al., 2000). There is no report regarding the possible alterations in hippocampal Cx36 and Cx43 expression during epileptogenic process. In the present study, the extent of expression of Cx36 and Cx43 at mRNA and protein level was investigated in the rat hippocampus during epileptogenesis in the amygdala-kindling model of epilepsy. 2. Methods 2.1. Materials Ketamine and xylazine were from Rotex Medica (Germany) and Chanelle (Ireland), respectively. RNX-PLUS Reagent, Agarose, Acrylamide and Bis-Acrylamide were purchased from CinnaGen (Iran). Hot Start Taq DNA polymerase, 2× RNA Loading Dye, Page Ruler™ Prestained Protein Ladder and Protein Loading Buffer Pack were purchased from Fermentas (Lithuania). Protease Inhibitor Cocktail, RNase free DNase I, and 1st strand cDNA synthesis kit were from Roche (Germany). Enhanced Chemiluminescence (ECL) Advance Western Blotting Detection Kit was from Amersham (UK). Monoclonal anti-connexin 36 was from Zymed (USA). Monoclonal anticonnexin 43 was from Upstate (USA). Monoclonal anti-α-tubulin and anti-mouse IgG peroxidase conjugates were from Sigma-Aldrich (USA). Other chemicals were from Applichem (Germany) and Sigma-Aldrich (USA). 2.2. Animals Adult male Wistar rats (300–350 g, Pasteur Institute of Iran) were used throughout the study. Animals were housed in groups of 2 and had free access to food (standard laboratory rodents chow) and drinking water. The animal house temperature was maintained at 23± 1 °C with an alternating 12-hr light/dark cycle (light on from 6 a.m.). All animal experiments were carried out in accordance with the European Communities Council Directive of November 1986 (86/609/EEC) in such a way to minimize the number of animals and their suffering. All the injections were done intraperitoneally (i.p.).
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2.3. Kindling procedure and tissue preparation The rats were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg). Then they were stereotaxically implanted with bipolar stimulating and monopolar recording stainless-steel Teflon-coated electrodes (A.M. Systems, USA, twisted into a tripolar configuration), in the basolateral amygdala (coordinates: A,−2.5 mm from bregma; L, 4.8 mm from bregma; V, 7.5 mm from dura) of the right hemisphere (Paxinos and Watson, 2005). The electrodes were fixed on the skull with dental acrylic. After 7 days of recovery period, after-discharge (AD) threshold was determined in amygdala by a 2-sec, 100 Hz monophasic square-wave stimulus of 1 ms per wave. The stimulation was initially delivered at 50 µA and then at 5-min intervals, increasing stimulus intensity in increments of 50 µA was delivered until at least 5 s of AD was recorded (Sayyah et al., 2007). Then two different groups of animals with known AD threshold (each group consisting of 5–7 rats) were stimulated once daily at AD threshold until they showed behavioral seizure stage 2 in two consecutive days (as partially-kindled rats) or behavioral seizure stage 5 in three consecutive days (as fully kindled rats) according to Racine classification (Racine, 1972). Two other groups of electrode-implanted, non-stimulated rats were used and considered as sham-operated control groups (sham 1 and sham 2 groups corresponds to the partially- and fully kindled rats, respectively). In previous similar studies, the time courses of 3 h or two weeks were used to study the changes in Cxs expression (Elisevich et al., 1997a; Sohl et al., 2000). However, these time courses seem too early or late. Therefore, we chose a 24-hr time point, which was also the interval of our stimulation protocol, to evaluate the changes in Cxs expression at mRNA and protein level. Twenty four hours after showing last behavioral seizure stage of 2 or 5, all the animals and their corresponding controls were decapitated under deep ether anesthesia and their brain were removed immediately. The brains were incubated in chilled artificial cerebrospinal fluid (ACSF) with pH 7.3 consisted of the following composition (in mM): 124 NaCl, 4.4 KCl, 2 CaCl2, 2 MgCl2, 1.2 KH2PO4, 25 NaHCO3 and 10 Glucose. The hippocampi of the right hemisphere of the brains (the side that the electrodes were implanted in the amygdala) were removed and frozen immediately in liquid nitrogen and stored at −80 °C. The rest of the brains were placed in 10% formalin for at least 24 h at room temperature and they were then processed, cut into 10 μm thick slices and stained by the method described before (Sayyah et al., 2007). The stained slices were qualitatively analyzed for electrode position using a stereoscopic microscope (Olympus, Japan). The data of the animals, in which their electrode was in false placement, were not included in the results. 2.4. Gene expression study The frozen hippocampi were removed from −80 °C and pulverized completely. About 200 µl of chilled phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, and 1.4 mM KH2PO4) was added to the pulverized tissues and vortexed for 30 s, then spinned and aliquoted in two separate microtubes equally. One of the so prepared samples was used for gene expression study and the second part for immunoblotting. An appropriate volume of a protease inhibitor cocktail according to the manufacturer′s proposal was added to samples, which were allocated for immunoblotting. Total cellular RNA was isolated from the hippocampus by a modification of the guanidine isothiocyanate phenol-chloroform method (Ausubel et al., 2002) using RNX-PLUS reagent then treated with 10 U RNase free DNase I (Roche, Germany) to avoid any DNA contamination. The integrity of the RNA samples was determined using denaturing agarose gel electrophoresis. The concentrations of the RNAs were determined spectrophotometrically (Nanodrop, USA). The mean 260/280 ratios were 1.94 ± 0.0, while those of 260/230 were 1.98 ± 0.1.
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A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) based strategy was employed, as it was shown to be a quite sensitive method for quantification of connexins expression (Szente et al., 2002; Gadja et al., 2003, 2006). The reverse transcription reaction was performed with 1st strand cDNA synthesis kit (Roche, Germany) using oligo-dT primer, AMV reverse transcriptase and 1 µg total RNA as template, according to the manufacturer′s instructions. The polymerase chain reaction parameters were adjusted to obtain a condition with linear relation between the initial amount of cDNA template and PCR product and with linear relation between the number of PCR cycles and PCR products. PCR amplification was performed on an appropriate dilution of the RT product by using a Hot Start Taq DNA Polymerase and 35 cycles of 95 °C for 50 s, 54 °C for 1 min and 72 °C for 30 s for Cx36, 35 cycles of 95 °C for 50 s, 48 °C for 1 min and 72 °C for 30 s for Cx43 and 26 cycles of 95 °C for 50 s, 60 °C for 1 min and 72 °C for 30 s for β-actin. The final cycles were followed by a 7 min extension step at 72 °C and a soak cycle at 4 °C. The following primers were used: Cx36 forward: 5′- GCA GAG AGA ACG CCG GTA CT -3′ Cx36 reverse: 5′- CTT GGA CCT TGC TGC TGT GC -3′ Cx43 forward: 5′- GAT GAG GAA GGA AGA GAA G -3′ Cx43 reverse: 5′- CGC TAG CTT GCT TGT TGT AA -3′ β-actin forward: 5′- GCA AGA GAG GTA TCC TGA CC -3′ β-actin reverse: 5′- CCC TCG TAG ATG GGC ACA GT -3′
Cx36 and Cx43 proteins were expressed as ratios (Cx36/α-tubulin×100, Cx43/α-tubulin×100). 2.6. Statistical analysis In order to increase the reliability of the measurements, RT-PCR reactions and immunoblottings were performed on total RNAs and total proteins of 5–7 animals, independently and in duplicate. Results are presented as the mean ± S.E.M. The data were analyzed by unpaired Student′s t-test. Correlation of Cxs mRNA expression with Cxs protein expression was analyzed by Spearman's correlation test. Statistical significance was considered at p b 0.05. 3. Results A sample of recorded AD from amygdala is shown in Fig. 1. The mean AD threshold obtained for the animals was 200 ± 20 µA. The number of stimulations to stages 2 and 5 was 8.3 ± 0.2 and 13.6 ± 0.8, respectively. Mean of AD duration in stages 2 and 5 were 24.2 ± 3.5 and 63.6 ± 9.2 s, respectively. Cx36 and Cx43 mRNA and protein levels were quantitated in the same hippocampi tissues. Histological evaluation of the brains confirmed that in at least five rats out of seven in each group, the electrode was in a correct position, the data of which were used for statistical analysis. 3.1. Gene expression analysis
The sequences of the oligonucleotides were adopted from previous studies. Cx36 and Cx43 primers were chosen to lack homology with any published connexin sequences (Szente et al., 2002; Gadja et al., 2003). PCR products of each Cx and β-actin of each sample were mixed in the same well and run on 1.5% agarose gel in order to maintain the same conditions for test and reference genes and to be able to make more precise comparison. Bands were quantified by densitometry using Labworks analyzing software (Ultra Violet Products, U.K). The relative levels of Cx36 and Cx43 mRNAs were expressed as ratios (Cx36/ β-actin × 100, Cx43/β-actin × 100).
Partially-kindled rats showed a mean density value of 610.5 ± 6.3% (P = 0.0043) and 80.1 ± 10.3% (P = 0.29) for Cx36 and Cx43 mRNAs compared to 100.0 ± 40.3% and 100.0 ± 15.8% of their sham controls, respectively. Fully kindled rats exhibited a mean density value of 136.5 ± 20.6% (P = 0.28) and 92.9 ± 4.7% (P = 0.24) for Cx36 and Cx43 mRNAs compared to 100.0 ± 14.3% and 100.0 ± 3.4% of their sham controls, respectively. In the partially-kindled animals, the mRNA expression levels of hippocampal Cx36 increased significantly relative to the control (Fig. 2).
2.5. Immunoblotting
3.2. Immunoblot analysis
The second part of the homogenized hippocampi tissues was removed from −80 °C and centrifuged at 12,000 g, 4 °C for 10 min. The supernatant was collected and total protein concentration was determined using Bio-Rad DC protein assay reagents. Samples were dissolved in protein loading buffer and denatured for 5 min at 95 °C prior to loading. Equal amounts of protein from each animal (5 µg per lane for α-tubulin, 10 µg per lane for Cx36 and 25 µg per lane for Cx43) were resolved by denaturing SDS-Polyacrylamide gel electrophoresis (SDS-PAGE), 12% acrylamide and transferred to a PVDF membrane (Roche, Germany) by electroblotting (Mini trans blot electrophoretic transfer cell, Bio-Rad). The membrane was blocked in TBST buffer (100 mM Tris base, 150 mM NaCl, and 0.2% Tween 20) containing 2% ECL Advance blocking agent at room temperature for 60 min, rinsed briefly with TBST buffer and then incubated for 60 min with the following primary antibodies: mouse monoclonal anti-connexin 36 diluted 1:2000, mouse monoclonal anticonnexin 43 diluted 1:100,000 and mouse monoclonal anti-α-tubulin diluted 1:200,000. The antibodies were diluted in blocking buffer. After washing with TBST buffer 4 times (1× for 15 min and 3× for 5 min), the membrane was incubated with peroxidase conjugated goat anti-mouse IgG (diluted 1:50,000, 1:400,000 and 1:2,000,000 for Cx36, Cx43 and α-tubulin, respectively) for 1 h, then washed with TBST buffer 4 times (1× for 15 min and 3× for 5 min) and reacted with ECL Advance western blotting detection reagents, for 4 min. An X-ray film (Retina, USA) was used for 30 s to 10 min and then developed to visualize the antibody binding. Bands were quantified by densitometry using Labworks analyzing software (Ultra Violet Products, U.K). The relative levels of
Partially-kindled rats exhibited a mean density value of 209.3±10.2% (P =0.01) and 130.0±19.0% (P =0.17) for Cx36 and Cx43 protein compared to 100.0 ±29.4% and 100.0± 25.2% of their sham controls, respectively. Fully kindled rats showed a mean density value of 115.9± 15.1% (P= 0.51) and 121.8 ±22.5% (P= 0.59) for Cx36 and Cx43 protein compared to 100.0 ±14.8% and 100.0± 29.0% of their sham controls, respectively. In the partially-kindled animals, the protein expression levels of hippocampal Cx36 increased significantly relative to the control (Fig. 3). 3.3. Correlation of Cx36 mRNA with Cx36 protein In partially-kindled rats, Cx36 expression at mRNA level correlated positively with Cx36 expression at protein level (P = 0.001, r = 0.725). 4. Discussion The present study indicates that hippocampal Cx36 expression at both mRNA and protein level is upregulated during acquisition of focal seizures and declines to almost the basal level after acquisition of secondarily-generalized seizures. Furthermore, there was a significant positive correlation between Cx36 mRNA expression and Cx36 protein expression during early stages of kindling epileptogenesis. However, hippocampal Cx43 mRNA and protein expression did not change during either focal or secondarily-generalized seizures acquisition.
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Fig. 1. Recorded after-discharges from the amygdala of an animal with partial (A) or secondarily-generalized (B) seizures.
Expression levels of Cx36 have previously been investigated in human epileptic tissue and different experimental animal models of epilepsy. Aronica et al. (2001), have shown an upregulation in Cx36 mRNA in the human epileptic tissue, while Collignon et al. (2006), indicated preservation of Cx36 in human hippocampal subregions suffering from mesial temporal lobe epilepsy. Upregulation of Cx36 mRNA has been detected in the somatosensory cortex of both immature (Gadja et al., 2006) and adult rats (Gadja et al., 2003) 1 h following repeated seizures induced by 4-Aminopyridine, while no change was seen in Cx36 protein after repeated tetanization of
hippocampal slices (Jahromi et al., 2002). On the other hand, down regulation of hippocampal Cx36 mRNA and protein was reported in fully kindled rats 2–3 weeks following the last kindled seizures (Sohl et al., 2000). Also a brief evoked after-discharge produced a significant reduction in Cx36 protein expression in the dorsal hippocampus (McCracken and Roberts, 2006). Moreover, following i.c.v injection of kainic acid, a progressive down regulation in Cx36 mRNA was detected in the hippocampal CA3–CA4 regions but not in the hippocampal CA1 subfield and in the cerebral cortex (Condorelli et al., 2003). The discrepancies regarding Cx36 expression in different studies have been
Fig. 2. Expression of Cx36 (A) and Cx43 (B) genes in the hippocampus of the rats with partial and secondarily-generalized seizures. Connexin mRNA levels were normalized to that of β-actin mRNA. Data are expressed as means ± S.E.M (n = 5). Each polymerase chain reaction was performed in duplicate to increase the reliability of the measurements. Panels C and D show representative RT-PCR products of Cx36 (270 bp) and Cx43 (620 bp) in the partial and full kindled animals. **:p b 0.01.
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Fig. 3. Expression of Cx36 (A) and Cx43 (B) proteins in the hippocampus of the rats with partial and secondarily-generalized seizures. Cx36 and Cx43 protein levels were normalized to that of α-tubulin protein. Data are expressed as means ± S.E.M (n = 6). Each immunoblotting was performed in duplicate to increase the reliability of the measurements. Panels C and D show representative immunoblots of Cx36 (36 kDa) and Cx43 (43 kDa) in the partial and full kindled animals, respectively. *:p b 0.05.
attributed to variations in preparations used, methods of seizures induction, duration of seizure activity, time point examined after seizure activity and different brain regions and subregions used (McCracken and Roberts, 2006). Although these studies indicate a relationship between Cx36 gap junctional coupling and seizure activity, they do not provide information about possible alterations in Cx36 expression during the course of epileptogenesis. The aforementioned epileptic patients had suffered from epilepsy for several years (Aronica et al., 2001; Collignon et al., 2006). This is somewhat the case in experimental animal models of epilepsy, where the expression levels of connexins were studied only after establishment of seizures or epileptic conditions (Sohl et al., 2000; Gadja et al., 2003; Condorelli et al., 2003). Therefore, it is not clear whether the changes in connexins expression had occurred in response to seizures and epileptic conditions, or had been generated during development of epilepsy. Our study is the first study that has investigated patterns of expression of hippocampal Cx36 and Cx43 during epileptogenesis in a chronic in vivo model. Kindling causes long lasting effects on hippocampal physiology and anatomy, ranging from the subcellular level, such as altered hippocampal gene expression, to complicated structural alterations, like mossy fiber sprouting, neurogenesis and cellular apoptosis (Hewapathirane and Burnham, 2005). According to our results, gene and protein expressions of Cx36 are upregulated in hippocampus during kindling development but the
changes are limited to the early stages of kindling. This finding suggests that Cx36 over expression plays a significant role in induction of partial seizures. However, after establishment of focal seizures, Cx36 expression returns to basal level and therefore, ipsilateral Cx36 over expression does not seem to be involved in stage of seizure spreading to the contralateral limbic structures and consequent generalization of the seizures. Cx36 expressing neurons are present in all the regions of the hippocampal formation, including the entorhinal cortex (Condorelli et al., 2000). Cx36 is expressed in the GABAergic interneurons located in the various layers of CA1, CA3 and dentate gyrus, but the principal pyramidal cells express Cx36 only in CA3 region (Condorelli et al., 2000). However, our results do not address whether the Cx36 upregulation happens in all the hippocampal regions or it is confined to a specific subfield. Nonetheless, it can be proposed that the increase in Cx36 containing gap junctions between CA3 pyramidal neurons or GABAergic interneurons has elevated their capability to synchronize input stimuli and facilitate focal seizures induction. Further experiments such as in situ hybridization are necessary to provide concise data regarding the level of expression in different hippocampal regions during kindling. In this study, significant positive correlation of Cx36 expression at mRNA level with Cx36 expression at protein level was found during
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the early stages of kindling epileptogenesis. This indicates that Cx36 expression was regulated mainly at the transcriptional level. Finally, we found that the overall expression of hippocampal Cx43 at the mRNA and protein level did not change during kindling development. Similar to our results, Elisevich et al. (1997a), reported no change in Cx43 mRNA expression in amygdala of the rats during development of amygdala kindling. Moreover, it has been found that hippocampal Cx43 expression at both mRNA and protein level is unchanged or slightly decreased in fully kindled rats 2–6 weeks after the last kindled seizures occurrence (Sohl et al., 2000). Therefore, it seems that the hippocampal Cx43 has no significant role in kindling epileptogenesis. 5. Conclusion Our study is the first report on the potential role of Cx36 in epileptogenesis. Over expression of Cx36 during initial stages of kindling epileptogenesis further points out the significance of Cx36 as a target to modify epileptogenic process and to develop antiepileptogenic treatments. To further elucidate the role of Cx36 in epileptogenesis, Cx36 specific blockers can be administered intra-hippocampally and their effect on kindled seizures acquisition be evaluated. Moreover, studies on epileptogenesis in Cx36 knockout animals as well as using specific gene expression inhibitors such as antisense oligodeoxynucleotides are suggested to extend the results of this study. Acknowledgment Financial support by grant no. 301 from Pasteur Institute of Iran is acknowledged. References Aronica E, Gorter JA, Jansen GH, Leenstra S, Yankaya B, Troost D. Expression of connexin 43 and connexin 32 gap junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex. Acta Neuropathol 2001;101:449–59. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al. Short protocols in molecular biology. Wiley; 2002. Carlen PL, Skinner F, Zhang L, Naus C, Kushnir M, Perez Velazquez JL. The role of gap junctions in seizures. Brain Res Rev 2000;32:235–41. Collignon F, Wetsen NM, Cohen-Gadol AA, Cascino GD, Parisi J, Meyer FB, et al. Altered expression of connexin subtypes in mesial temporal lobe epilepsy in humans. J Neurosurg 2006;105:77–87. Condorelli DF, Parenti R, Spinella F, Trovato Salino A, Belluardo N, Cardile V. Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur J NeuroSci 1998;10:1202–8. Condorelli DF, Belluardo N, Trovato-salinaro A, Mudo G. Expression of Cx36 in the mammalian neurons. Brain Res Rev 2000;32:72–85. Condorelli DF, Trovato-salinaro A, Mudo G, Mirone MB, Belluardo N. Cellular expression of connexins in the rat brain: neuronal localization, effects of kainite-induced seizures and expression in apoptotic neuronal cells. Eur J NeuroSci 2003;18:1807–27. Dichter MA. Models of epileptogenesis in adult animals available for antiepileptogenesis drug screening. Epilepsy Res 2006;68:31–5. Dudek FE, Yasumura T, Rash JE. Non-synaptic mechanisms in seizures and epileptogenesis. Cell Biol Int 1998;22:793–805. Elisevich K, Rempel SA, Smith B, Allar N. Connexin 43 mRNA expression in two experimental models of epilepsy. Mol Chem Neuropathol 1997a;32:75–88. Elisevich K, Rempel SA, Smith BJ, Edvardsen K. Hippocampal connexin 43 expression in human complex partial seizure disorder. Exp Neurol 1997b;145:154–64. Fisher R, van Emde Boas W, Blume W, Elger C, Genton P, Lee P, et al. Epileptic seizures and epilepsy: definitions proposed by the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 2005;46:470–2. Fonseca CG, Green CR, Nicholson LFB. Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Res 2002;929:105–16. Gadja Z, Gyengesi E, Hermesz E, Said Ali K, Szente M. Involvement of gap junctions in the manifestation and control of the duration of seizures in rats in vivo. Epilepsia 2003;44:1596–600.
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