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Keywords:

  • bicuculline;
  • carbenoxolone;
  • connexins;
  • epilepsy;
  • gap junction

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 μm increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 μm). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 μm), but not its inactive analog, oleanolic acid (100 μm), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Abbreviations used
ACSF

artificial cerebrospinal fluid

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate

APV

2-amino-5-phosphonovaleric acid

BMI

bicuculline methiodide

CNQX

6-cyano-7-nitroquinoxaline-2,3-dione

CREB

cAMP response element binding protein

Cx

connexin

FRAP

fluorescence recovery after photobleaching

IPSC

inhibitory post-synaptic current

MPP

myelin proteolipid protein

PAD

primary afterdischarge

PPD

pair-pulse depression

Syn

synapsin

Epilepsy is a neurological disorder characterized by periodic and unpredictable generation of synchronous neuronal activity, seizures. Various changes in neuronal and network properties have been reported to accompany or induce seizure activity in human or experimental epilepsy (for review see Glass and Dragunow 1995; Jefferys and Traub 1998; McNamara 1999; Dalby and Mody 2001; McCormick and Contreras 2001; Avoli et al. 2002; Engel 2002) including alteration in the properties of ion channels and synaptic receptors. Among non-synaptic mechanisms in seizures (Jefferys 1995; Dudek et al. 1998), which include ion interactions, electric field effects (ephaptic transmission), and electrotonic coupling through gap junctions (for review see Carlen et al. 2000; Perez Velazquez and Carlen 2000), the latter mechanism has attracted growing interest over the last decade.

Gap junctions are clusters of intercellular channels formed from connexin (Cx) proteins (for review see Evans and Martin 2002). Around 20 highly homologous products of connexin genes have been identified in humans and rodents (Willecke et al. 2002), of which Cxs 26, 30, 32, 36, 43, and 47 are the major isoforms expressed in the brain (Dermietzel et al. 1989; Micevych and Abelson 1991; Nadarajah et al. 1996; Nadarajah et al. 1997; Rash et al. 1997; Condorelli et al. 1998; Dermietzel 1998; Dermietzel and Hofstadter 1998; Nadarajah and Parnavelas 1999; Condorelli et al. 2000; Nagy and Rash 2000; Rash et al. 2000; Nagy et al. 2001; Rash et al. 2001b; Meier et al. 2002). Their expression is cell and region specific and developmentally regulated. Moreover, the gap junction number, composition and functionality are highly regulated (Evans and Martin 2002) and can be subject to rapid remodeling according to physiological requirement or under pathological conditions (Jefferys 1995; Carlen et al. 2000; Perez Velazquez and Carlen 2000; Li et al. 2001; Traub et al. 2001b; Rouach et al. 2002; Steinhauser and Seifert 2002; Traub et al. 2002; Zoidl and Dermietzel 2002) including seizures.

Blockade of gap junctions has been recently shown pharmacologically to reduce seizures in different epilepsy models in vitro (de Curtis et al. 1998; Ross et al. 2000; Kohling et al. 2001; Traub et al. 2001a; Jahromi et al. 2002; LeBeau et al. 2002) and in vivo (Szente et al. 2002). Moreover, treatment that favors gap junction opening has been found to promote seizure-like activity in vitro (Kohling et al. 2001). In addition, through analysis of a Cx36 knockout mouse, in which electrotonic coupling between interneurons is drastically reduced (Deans et al. 2001; Hormuzdi et al. 2001), it has been shown that loss of neuronal specific gap junctions reduces kainate-induced (Hormuzdi et al. 2001) and 4-aminopyridine-induced (Maier et al. 2002) seizures. However, it does not completely abolish all types of neuronal synchronization (Hormuzdi et al. 2001; Traub et al. 2003). These data indicate that gap junctional communication plays a key role in neuronal synchronization during seizure, however, the role of different types of gap junctions (formed from different connexin proteins) and their specific cellular localization, as well as their probable involvement in seizure initiation, need further elucidation.

Using the intact whole hippocampus, freshly isolated from young mice, we have previously shown (Li et al. 2001) that exposure to BMI increases the level of Cx32 transcript and protein and, concurrently, causes persistent epileptiform activity, but probable changes in functional coupling through gap junctions remained unexplored. In the present study, we used long-term organotypic hippocampal slice cultures, a chronic in vitro model of epilepsy with electrophysiological and morphological similarities to both human and in vivo models (Gahwiler et al. 1997; Gutierrez et al. 1999; Kovacs et al. 1999; Bausch and McNamara 2000). This preparation preserves the cytoarchitecture and synaptic connections closely resembling those in the acute slice preparation (Gahwiler et al. 1997; Fischer et al. 2002), and has the advantage of permitting long-term treatments. In the present study, we used cultured hippocampal slices exposed to BMI for 18 h to explore the possible parallelism between epileptogenesis, gap junctional coupling measured by fluorescence recovery after photobleaching (FRAP) and up-regulation of Cx26, 32, 36 and 43. The modulation of Cx32 and Cx43 in rodent brain with induced epileptic discharges, as well as in human epileptic brain tissues, has been previously demonstrated (Naus et al. 1991; Vukelic et al. 1991; Khurgel and Ivy 1996; Sohl et al. 2000; Li et al. 2001; Fonseca et al. 2002; Szente et al. 2002). The involvement of gap junctions formed from Cx36, a neuronal-specific Cx, in seizure generation has been recently shown through analysis of the Cx36 knockout mouse (Hormuzdi et al. 2001; Maier et al. 2002). Although the role of Cx26 in neuronal synchronization is poorly understood, several studies indicate that Cx26 can mediate coupling between neurons in immature brain (Venance et al. 2000; Solomon et al. 2001; Bittman et al. 2002). In a preliminary study, we found relatively high levels of Cx26 mRNA and protein expression in cultured hippocampal slices.

Organotypic hippocampal slice culture preparation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

The method for culturing brain slices has been described previously (Stoppini et al. 1991; Adamchik et al. 2000). Briefly, the brains of 7-day-old male Wistar rats were aseptically removed and immersed in ice-cold dissecting medium (pH 7.15) containing 50% minimal essential medium (MEM) (Gibco BRL, Gaithersburg, MD, USA) with no bicarbonate, 50% calcium-free and magnesium-free balanced salt solution, 20 mm HEPES and 7.5 mm d-glucose. Hippocampi were dissected, and coronal sections (400 μm) were obtained using a tissue chopper, then transferred to sterile, porous membrane units (0.4 μm). Membrane units were placed into 6-well trays, and each well contained one membrane unit and 1 mL of culture medium. The culture medium was composed of 50% MEM with Earl's salts, and l-glutamine, 25% balanced salt solution and 25% horse serum with 6.5 g/L d-glucose, 20 mm HEPES buffer and 2% of streptomycin-penicillin (pH adjusted to 7.2). Cultures were kept in the incubator at 37°C in 5% CO2. The medium was exchanged (50% of volume) two times per week.

Bicuculline exposure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

The advantage in using cultured hippocampal slices over acutely prepared slices is that while both preparations preserve the cytoarchitecture and the synaptic connections, only cultured slices permit the observation of changes following chronic treatments above several hours. After 2 weeks in culture, either 0 (control), 1 μm or 10 μm bicuculline methiodide (BMI; Sigma-Aldrich Canada, Oakville, Ont., Canada), a blocker of GABAA receptors, was added to the culture medium for the duration of 18 h. After BMI washout, the treated slices as well as control slices were taken for experiments.

Electrophysiology

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Cultured hippocampal slices (control slices and those exposed to BMI for 18 h) were transferred to an interface-type chamber (PDMI-2; Harvard Apparatus, South Natick, MA, USA) perfused with oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 25 NaHCO3, 10 d-glucose, pH 7.4 (osmolarity 300 ±5 mOsm) at 34°C. After the cultures had been perfused with BMI-free ACSF for at least 15 min, responses were recorded extracellularly in the stratum pyramidale of the CA1 region with NaCl-filled (150 mm) borosilicate glass pipettes. Extracellular stimulation (100-μs pulse) was delivered by placing a bipolar stimulating electrode (twisted enamel-insulated nichrome wire; 125 μm diameter) in the stratum radiatum to stimulate the Schaffer collaterals. Stimulus strength was adjusted by increasing the amplitude to an intensity that produced a maximal response. To assess GABAA-mediated inhibition, we used a pair-pulse depression (PPD) protocol with an interpulse interval of 50 ms as described in a variety of seizure models (Adamec et al. 1981; Sloviter 1983; Tuff et al. 1983; Stringer 2000). To evoke sustained epileptiform discharges (lasting tens of seconds), a train of stimuli (100 Hz, 2-s duration) was delivered to Schaffer collaterals. We refer to this type of discharge as primary afterdischarge (PAD) which is similar to stimulus train-induced bursting (Stasheff et al. 1985; Pelletier and Carlen 1996; Jahromi et al. 2000; Jahromi et al. 2002).

Whole cell recordings were made in the stratum pyramidale of the CA1 region with glass micropipettes having a resistance of 3–5 MΩ. The pipette solution contained (in mM): 140 K-gluconate, 10 HEPES, and 0.1 EGTA (pH 7.25, osmolarity 290 ± 5 mOsm). To isolate the inhibitory response, the excitatory transmission was blocked by glutamatergic antagonists 6-cyano-7-nitroquinoxaline- 2,3-dione (CNQX, 10 μm) and 2-amino-5-phosphonovaleric acid (APV, 30 μm) (both Tocris, Ellisville, MO, USA). Inhibitory post-synaptic currents (IPSCs) were induced by submaximal stimulation of Schaffer collaterals. Electrical signals were recorded using an Axopatch 200B amplifier (Axon Instruments Inc., Foster City, CA, USA); the low pass Bessel filter was set at 1 and 5 KHz for extracellular and whole cell recording, respectively. Evoked responses were digitized via a 12-bit D/A interface (Digidata 1200; Axon Instruments) and acquired and analyzed using pCLAMP version 6.0.3 software (Axon Instruments). Spontaneous epileptiform discharges were recorded on videotape, digitized with VR-10 (Instrutech Corporation) and analysed using Axotape version 2.02 software (Axon Instruments).

RNase protection assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Total RNA isolation, RNA probe preparation and RNase protection assay were carried out as in our previous study (Li et al. 2001). Briefly, total RNA from cultured hippocampal slices was prepared using SV total RNA isolation system (Promega, Madison, WI, USA). RNA probes were biotinylated during synthesis using riboprobe in vitro transcription systems (Promega) according to the manufacturer's protocol. Cx43, 36, 32 and 26 cDNAs were used to generate both sense probe and antisense probe.

RNase protection assay was performed using a RPA II kit (Ambion, Austin, TX, USA). The protected fragments on the membrane were visualized using a BrightStar BioDetect non-isotopic detection kit (Ambion). The protected fragment bands on the film were scanned and quantified by Quantity One software (Bio-Rad Laboratories Canada Ltd., Mississauga, Ont., Canada).

Western blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Western blotting was performed as described earlier (Li et al. 2001). Briefly, both the nuclear fraction and the cellular membrane fraction were prepared from cultured hippocampal slices by super-centrifuge technique. The sample proteins, along with rainbow molecular weight markers (Amersham Pharmacia Biotech, Piscataway, NJ, USA), were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Bio-Rad), transferred from gel to nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) and incubated with affinity purified rabbit polyclonal antibodies and then with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (Promega).

Affinity purified rabbit polyclonal IgG antibodies against Cx26, 32 and 43 proteins were obtained from Chemicon (Temecula, CA, USA). Antibodies against N-methyl-d-aspartate (NMDA) receptor subunit NR2, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, synapsin (Syn), myelin proteolipid protein (MPP), c-fos protein and cAMP response element binding protein (CREB) were obtained from Calbiochem (Cambridge, MA, USA). Individual antibodies were titrated for optimum results. Quantity One software (Bio-Rad) was used for the quantitative analyses of protein and mRNA bands on the film. The integrated intensity of the band (optical density [OD] value) was determined using both band density and band area. We constructed a standard curve in each experiment by using known protein or RNA concentration of mouse cortex so that the results could be quantified relative to the standard tissue and between groups.

Fluorescence recovery after photobleaching (FRAP)

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

FRAP in slice cultures is a very useful technique for assessing gap junctional coupling in vitro, which is not possible in acutely prepared slices or whole hippocampus. The method used for FRAP was similar to that described previously (Lee et al. 1995; Frantseva et al. 2002). Briefly, slices were incubated with 7 μg/mL 5,6-carboxyfluorescein diacetate in culture medium for 30 min. The slices were then rinsed three times (10 min per wash) to remove excess 5,6-carboxyfluorescein. Carbenoxolone (Sigma-Aldrich) was added to the culture medium of BMI-exposed cultures when needed (final concentration of 100 μm) 15 min prior to imaging. Slices were viewed on a Bio-Rad MRC 600 laser-scanning confocal microscope, and images were acquired and analyzed using version 7.0 COMOS software (Bio-Rad). An image was taken prior to photobleaching to establish a baseline. The area of interest (stratum pyramidale of the CA1 region) was bleached to 50–80% of the original fluorescence. Then, imaging was resumed immediately and images were acquired continuously every 30 s. Recovery of fluorescence in the bleached area is due to an influx of unbleached dye from neighboring cells via gap junctions. The extent of gap junctional coupling between a cell and its neighbors was quantified by measuring the initial rate and percentage of fluorescence recovery. As a negative control, we used carboxyfluorescein-dextran, a fluorescent molecule similar to, but larger than, 5, 6-carboxyfluorescein diacetate, loaded with liposomes into the cultures. The large size of carboxyfluorescein-dextran (approximately 4000 Da) prevents it from passing through gap junctions and its recovery after photobleaching was < 10%. This small degree of recovery is attributed to the partially bleached cells located on the periphery of the bleached area where recovery of fluorescence is dependent upon the diffusion of the dye intracellularly.

Persistent epileptiform activity induced by chronic exposure to BMI

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

All electrophysiological recordings were performed using BMI-free ACSF (perfused for a minimum of 15 min prior to recording) in control cultures or cultures exposed previously to BMI (10 μm) for 18 h.

As shown in Fig. 1a, spontaneous epileptiform discharges occurred with significantly greater frequency in chronically BMI-exposed cultures when compared with controls (Table 1). In control cultures, orthodromic stimulation (maximum intensity) evoked post-synaptic potentials possessing a single population spike, whereas in BMI-exposed slices orthodromic stimulation evoked post-synaptic potentials with multiple population spikes (Fig. 1b), a feature which is consistent with the epileptiform responses recorded extracellularly. The average amplitude of single population spikes was not significantly different in control and BMI-exposed slices (Table 1). As illustrated in Fig. 1c, the duration of PAD, evoked with the tetanic train, was significantly less in control cultures compared with BMI-exposed cultures (Table 1). Altogether these observations indicate that cultured hippocampal slices exhibit increased epileptiform activity after chronic BMI exposure.

image

Figure 1. Persistent epileptiform responses in hippocampal slice cultures exposed chronically to BMI. (a) Continuous record of spontaneous activity in control cultures (left) and cultures exposed for 18 h in BMI (10 μm; right). (b) Left, orthodromic stimulation (maximum intensity) produced post-synaptic potentials (three superimposed records) possessing a single population spike in control cultures, whereas in BMI-exposed cultures (right) orthodromic stimulation evoked post-synaptic potentials with multiple population spikes, a feature consistent with epileptiform responses recorded extracellularly. (c) The duration of PAD, evoked with the tetanic train, was significantly less in control cultures (left) when compared with BMI-incubated cultures (right). (d) Paired-pulse stimulation (interpulse interval of 50 ms) produced a significant depression of the amplitude of the population spike evoked with the second pulse when compared with the population spike evoked with the first pulse (paired-pulse depression; PPD). Representative records (average of three consecutive records) of PPD in control cultures (left) and in BMI-exposed cultures (right). (e) Representative recording of IPSCs induced by sub-threshold stimulation of Schaffer collaterals in control (left) and BMI-exposed (right) cultures. Superimposed current traces from cells held at different holding potentials from −100 to +20 mV are shown. Note that neither IPSC reversal potential nor amplitude is different, suggesting restoration of normal inhibitory transmission in BMI-exposed slices after BMI washout.

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Table 1.  The effect of BMI exposure on electrophysiological characteristics of cultured hippocampal slices
  Control slicesBMI-exposed slices
  • *

    p < 0.05. Values are mean 

  • ±

    SE; n, number of experiments.

Extracellular recordingFrequency of spontaneous epileptiform discharges (Hz)0.04 ± 0.02 n = 100.64 ± 0.11*n = 16
Amplitude of single population spikes (mV)5.5 ± 0.7 n = 134.9 ± 0.5 n = 21
Duration of PAD (s)15.9 ± 3.837.2 ± 6.9*
 n = 13n = 21
Whole cell recordingIPSC reversal potential (mV)− 84.7 ± 5.2− 83.5 ± 1.4
 n = 3n = 7
IPSC amplitude at 0 mV (pA)287 ± 105313 ± 61
 n = 5n = 8
Resting potential (mV)− 53.4 ± 3.0− 46.5 ± 2.5
 n = 5n = 8

In both control and BMI-exposed slices, the paired-pulse paradigm produced a significant reduction of the amplitude of the second post-synaptic potential (0.77 ± 0.05, n = 11 and 0.76 ± 0.04, n = 9, respectively). As the post-synaptic potentials evoked in BMI-exposed cultures typically possessed multiple population spikes, in the determination of PPD we measured only the amplitude of the first-occurring population spike. Nevertheless, note in the example presented in Fig. 1d, that not only the first, but the subsequent population spikes as well were depressed. The magnitude of PPD was not different when control cultures where compared with BMI-exposed cultures, suggesting that GABAA-mediated responses assessed in BMI-free ACSF were not altered persistently after chronic exposure to BMI.

The restoration of GABAA-mediated synaptic transmission in BMI-exposed slices after BMI washout was directly confirmed using whole cell recording from individual CA1 pyramidal neurons. As shown in Fig. 1e, in the presence of glutamatergic synaptic transmission blockers, CNQX and APV, submaximal stimulation induced fast inhibitory post-synaptic currents (IPSCs). These currents are mediated by GABAA receptors, as their reversal potential was in keeping with transmembrane Cl ion distribution (Table 1) and they were blocked by BMI (n = 3 in both control and BMI-exposed slices; data not shown). Neither reversal potential nor amplitude of IPSCs differed significantly (Table 1) in control and BMI-exposed slices. It should be noted that resting membrane potential of CA1 pyramidal neurons was similar in control and BMI-exposed slices.

It is important to note that the above electrophysiological recordings were conducted in BMI-free ACSF, suggesting therefore that the increased epileptiform activity in BMI-exposed slices was due to a persistent network modification attributable to a pro-convulsant mechanism, such as increased neural network synchrony mediated by up-regulation of gap junction channels, rather than a decrease in GABAA-mediated synaptic transmission.

Transcription of Cx43 and Cx32 genes was increased by BMI exposure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

To explore the effect of chronic BMI exposure on Cx gene transcription in slice cultures using the RNase protection assay, we measured the levels of Cx43, Cx36, Cx32 and Cx26 mRNAs. As shown in Fig. 2, Cx mRNAs could be detected with antisense probes but not with the sense probes.

image

Figure 2. Levels of Cx mRNAs in cultured hippocampal slices. Total RNA extracted from the cultures was processed for RNase protection assay. Sense or antisense RNA probe for Cx43, 36, 32 or 26 mRNA was co-precipitated with sample RNA (15 μg) and after hybridization at 56°C overnight, the pellet was digested by RNase T1. The protected fragments were separated with denaturing gel, transferred to nylon membrane, and then visualized using a non-isotopic detection kit. (a) Low concentration exposure of BMI (1 μm) did not change the levels of Cx mRNAs, but high concentration exposure (10 μm) significantly promoted the transcription of Cx43 and Cx32 genes compared with control. (b) Summary of Cx mRNA levels (n = 4 for all groups). Note, these Cx mRNAs could not be detected by using sense RNA probes (*p < 0.05 and **p < 0.01, applying to (a) and (b) respectively).

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Exposure for 18 h to the higher concentration of BMI (10 μm) promoted significantly the transcription of Cx43 and Cx32 genes (Cx43 mRNA, 1.25 ± 0.11 OD/μg; Cx32 mRNA, 1.15 ± 0.12 OD/μg, n = 4 for both) when compared with control cultures (Cx43 mRNA, 0.88 ± 0.08 OD/μg; Cx32 mRNA, 0.66 ± 0.05, n = 4 for both). However, there were no significant differences in the levels of Cx43 and 32 mRNAs produced by exposure to the low concentration of BMI (1 μm). Neither low nor high concentrations of BMI affected the transcription of the Cx36 or the Cx26 genes in cultured hippocampal slices (Fig. 2). These results indicate that BMI exposure selectively affected the transcription of some Cx genes in a concentration-dependent manner.

Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

As an increase in mRNA level does not necessarily imply an increase in the level of the related protein, the level of Cx proteins in cellular membrane is more important from a functional perspective. To assess whether the increased Cx mRNAs was also translated into the related Cx proteins, we measured the levels of Cx proteins in membrane fractions of cultured hippocampal slices using western blotting and immunohistochemistry.

As shown in Fig. 3(a and b), exposing the cultured slices to 1 μm of BMI for 18 h did not significantly change the levels of Cx43, Cx32 and Cx26 proteins in membrane fractions, while 10 μm BMI increased significantly the levels of Cx43 and Cx32 proteins in membrane fractions (Cx43 protein, 0.34 ± 0.04 OD/μg; Cx32 protein, 0.25 ± 0.04OD/μg, n = 6 for both) compared with controls (Cx43 protein, 0.22 ± 0.03 OD/μg; Cx32 protein, 0.12 ± 0.02 OD/μg, n = 6 for both). These results suggest an enhanced translation of Cx43 and Cx32 mRNAs to the related proteins and an enhanced insertion of these proteins into the cellular membranes.

image

Figure 3. Levels of Cx proteins in cultured hippocampal slices. Cellular membrane fraction of the cultures was prepared using ultracentrifugation and processed for western blotting. Rabbit polyclonal antibodies against Cx43, Cx32 and Cx26 proteins were used as the primary antibodies. (a) Levels of Cx26 protein were not different after BMI exposure, whereas exposure to the high concentration of BMI (10 μm) increased significantly the expression of Cx43 and Cx32 proteins in cellular membrane fraction of cultured hippocampal slices compared with control. (b) Summary of Cx protein levels (n = 6 for all groups).

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Up-regulation of c-fos protein in BMI-exposed cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Transcription factors including CREB and c-fos have been implicated in cell growth, differentiation and development. CREB protein is a nuclear phosphoprotein responsible for the transcriptional activation of a number of different genes. Neuronal induction of c-fos protein occurs from electrical stimuli, small biological molecules and pharmacological agents and plays a role in signal transduction (West et al. 2002). Based on the important role of transcription factors in modulating gene expression, we explored the effects of BMI on the levels of CREB and c-fos proteins. We have previously shown that up-regulation of c-fos protein preceded the increase in the level of Cx32 transcript and protein in intact whole hippocampus exposed to BMI (Li et al. 2001).

As illustrated in Fig. 4, data from western blotting showed that exposing the cultured hippocampal slices to either 1 or 10 μm BMI for 18 h did not alter the levels of CREB protein in nuclear fractions, but significantly increased the expression of c-fos (1 μm, 0.53 ± 0.06 OD/μg protein; 10 μm, 0.51 ± 0.06 OD/μg, n = 5 for both) compared with control slices (0.31 ± 0.04 OD/μg, n = 5). There was no significant difference between the effects of two concentrations of BMI.

image

Figure 4. Expression of CREB and c-fos proteins in cultured hippocampal slices. Nuclear fraction of the cultures was prepared using ultracentrifugation and processed for western blotting. Sample protein (100 μg) was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane, which was then incubated with rabbit polyclonal anti-CREB or anti-c-fos antibodies. Specific protein bands were visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG followed by ECL immunoblotting detection reagents. (a) Levels of CREB protein in cultured hippocampal slices were not different after BMI exposure; however, both low (1 μm) and high (10 μm) concentrations of BMI significantly increased the expression of c-fos protein in nuclear fraction of cultured hippocampal slices compared with control (0). (b) Summary of CREB and c-fos protein levels (n = 5 for both).

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Unaltered expression of other neural proteins in BMI-exposed slices

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

To explore the specificity of the effect of BMI on the expression of Cx43 and Cx32, we measured the levels of several neural proteins including NR2, GluR1, Syn and MPP in BMI-exposed hippocampal slice cultures. The NMDA receptor subunit, NR2, and the AMPA receptor subunit, GluR1, mediate a number of neuronal processes involving excitatory synaptic transmission in normal (Dingledine et al. 1999; Kullmann et al. 2000) and pathological conditions, including epilepsy (Porter and Rogawski 1992; Chapman 2000; Kullmann et al. 2000; Meldrum 2000). Syn is a neuron-specific protein that serves as an excellent marker for synaptic terminals and is believed to regulate neurotransmitter release through a phosphorylation–dependent interaction with synaptic vesicles (Hosaka et al. 1999). MPP, a predominant integral membrane protein in the mammalian CNS myelin, is essential for rapid and effective propagation of the action potential along the axon (Regueiro et al. 1996). As illustrated in Fig. 5, western blotting data showed that the expression of these four neural proteins did not differ between the cultured hippocampal slices exposed to 10 μm BMI for 18 h and the control slices, thus favoring the possibility that BMI altered specifically the expression of Cxs.

image

Figure 5. Expression of neural proteins in cultured hippocampal slices. Membrane fraction of the cultures was prepared using ultracentrifugation and processed for western blotting. Rabbit polyclonal antibodies against NR2, GluR1, Syn and MPP proteins were used as the primary antibodies. Levels of these neural proteins were not different when BMI exposed cultures (BMI) were compared with control (Con).

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Increased rate of FRAP produced by chronic BMI exposure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Time-lapse imaging of FRAP was used to assess functional gap junctional coupling in a cultured hippocampal network (Fig. 6a). Following laser photobleaching, the dye (5,6-carboxyfluorescein) can enter the cytoplasm of a bleached cell only via gap junctions from neighboring cells, and result in fluorescence recovery that is directly proportional to the degree of gap junctional coupling. Exposure to 10 μm BMI induced a significant increase in the rate of FRAP compared with control slices. Differences were significant 4 min after photobleaching, and after 8 min BMI-exposed cultures had recovered to 86.7 ± 2.7% (n = 10) of the pre-bleach fluorescence level compared with 70.9 ± 1.7% (n = 15) in control slices (Fig. 6b). The FRAP experiments were performed in the stratum pyramidale of the CA1 region.

image

Figure 6. The rate of fluorescence recovery after photobleaching (FRAP). (a) Representative images acquired at four time points: prior to photobleaching (pre-bleach), photobleaching (0 min), 4 and 8 min after photobleaching in control (left), BMI-exposed (middle) and BMI-exposed cultures with added carbenoxolone (100 μm; right). Scale bar is 50 μm. The images were taken in the stratum pyramidale of the CA1 region. (b) Summary of the time-course of FRAP in control (n = 15), BMI-exposed (n = 10) and BMI/carbenoxolone-exposed (n = 8) cultures. The rate of FRAP was significantly faster in BMI-exposed cultures compared with control beginning after 4 min of photobleaching. Addition of carbenoxolone (15 min prior to imaging) completely blocked FRAP.

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Glycyrrhetinic acid, an aglycone saponin derived from the licorice root, is a potent blocker of gap junctions (Davidson et al. 1986). Carbenoxolone, which is a derivative of glycyrrhetinic acid, has been shown to block gap junctions in human fibroblasts (Davidson and Baumgarten 1988) and in NT2/D1 CNS precursor cells (Bani-Yaghoub et al. 1999). Carbenoxolone is thought to block gap junctions via direct binding, which produces a conformational change in the gap junction protein and subsequent channel closure (Goldberg et al. 1996). To assess whether the increased rate of FRAP was attributable to gap junctional coupling we repeated the above experiments using BMI-exposed slices in the presence of carbenoxolone (100 μm). Dye transfer was inhibited completely within minutes of exposure to carbenoxolone. In the presence of carbenoxolone, BMI-exposed cultures recovered to only 5.6 ± 0.4% (n = 8) of the pre-bleach fluorescence.

Blockade of epileptiform activity by carbenoxolone and octanol

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

We have demonstrated previously the anti-epileptic efficacy of carbenoxolone using repeated tetanic stimulation in acute piriform cortex slices, an in vitro seizure model that is intractable to conventional anti-epileptic drugs (Pelletier and Carlen 1996; Jahromi et al. 2002). Carbenoxolone has also been demonstrated to reduce significantly epileptiform discharges in hippocampus produced by zero Mg2+ (Kohling et al. 2001) or by 4-aminopyridine in an in vitro (Ross et al. 2000) and in vivo (Szente et al. 2002) models. Therefore, to determine whether gap junctional communication played a role in the increased epileptiform activity observed in BMI-exposed slices, we assessed the anti-epileptic efficacy of carbenoxolone (100 μm). As shown in Fig. 7a, spontaneous epileptiform discharges were blocked reversibly by application of carbenoxolone for 20 min, but were again evident after return to normal ACSF for 30 min. Similar results were observed in 6/6 experiments. Longer treatment with carbenoxolone (> 30 min) reduced and finally abolished PAD evoked by tetanic train (Fig. 7b) in 3/3 experiments. Carbenoxolone did not significantly affect the peak amplitude of single population spikes (4.7 ± 0.9 and 4.1 ± 1.2 mV in control and in the presence of carbenoxolone, respectively), although multiple population spikes were apparent (Fig. 7c). These effects of carbenoxolone were partially reversible after over 30 min washout (Fig. 7).

image

Figure 7. Anti-epileptic effect of carbenoxolone in hippocampal slice cultures. (a) Left, continuous record of spontaneous epileptiform discharges from a cultured slice incubated in BMI (10 μm). Middle, after application of carbenoxolone (100 μm) for 20 min, the spontaneous discharges were blocked. Right, spontaneous discharges were again evident after return to control ACSF for 30 min. (b) After 30 min application of carbenoxolone (100 μm) PAD was almost completely abolished (middle) as compared with that in control ACSF (left). The effect of carbenoxolone was partially reversible after return to control ACSF for 30 min (right). (c) Carbenoxolone slightly reduced the amplitude of the first population spike and unmasked multiple population spikes (middle). The effect of carbenoxolone was partially reversible after return to control ACSF for 30 min (right).

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The effects of carbenoxolone have been compared with those of its inactive analog, oleanolic acid (Bani-Yaghoub et al. 1999). In all experiments (n = 3) oleanolic acid (100 μm) did not reduce spontaneous activity (Fig. 8a; there was transient increase in the frequency seen in the figure) or PAD duration (Fig. 8b), even with longer (40–50 min) treatment. Similar to carbenoxolone, oleanolic acid slightly affected the peak amplitude of single population spikes (4.7 ± 0.2 and 4.3 ± 0.1 mV in control and in the presence of oleanolic acid, respectively, Fig. 8c).

image

Figure 8. The absence of anti-epileptic effect of oleanolic acid, inactive analog of carbenoxolone, in hippocampal slice cultures. (a) Left, continuous record of spontaneous epileptiform discharges from a cultured slice incubated in BMI (10 μm). Middle, the frequency of spontaneous discharges was increased after application of oleanolic acid (100 μm) for 30 min, but gradually returned to normal level during further treatment (50 min) (right). (b) After 30 min (middle) and 50 min (right) application of oleanolic acid (100 μm) PAD did not markedly change as compared with that in control ACSF (left). (c) Oleanolic acid did not markedly affect the amplitude of the population spike after 30 min (middle) and 50 min (right) of treatment.

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The effects of another widely used gap junctional blocker, octanol, have been also studied. Octanol (300 μm) did not significantly affect the peak amplitude of single population spikes (5.7 ± 0.9 and 4.7 ± 0.5 mV in control and in the presence of octanol, respectively). At the same time (after 20–30 min application) octanol completely blocked PAD induced by a tetanic train and abolished spontaneous activity in all experiments (n = 4; data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References

Our data show that cultured hippocampal slices exposed to 10 μm BMI for 18 h and washed for at least 15 min developed electrical activity with characteristic epileptiform features. It is noteworthy that this increased activity was observed after BMI washout and was not due to changes in the characteristics of GABAA-mediated inhibition. It has been shown that only much longer (several days) BMI treatment resulted in homeostatic regulation of synaptic receptor expression and function (Galante et al. 2000; Marty et al. 2000). Our data indicate that 18-h exposure to BMI did not significantly alter the expression of the NMDA receptor subunit, NR2 and the AMPA receptor subunit, GluR1, as well as the expression of pre-synaptic vesicle protein, synapsin, and myelin proteolipid protein (Fig. 5). At the same time, chronic exposure to 10 μm BMI selectively promoted the transcription of Cx43 and Cx32 genes (Fig. 2) and the levels of Cx43 and Cx32 proteins (Fig. 3).

The expression of Cx32 and Cx43 in connection with epileptic activity was investigated previously in human epileptic brain tissues and in rats and mice with induced epileptic discharges (Naus et al. 1991; Vukelic et al. 1991; Khurgel and Ivy 1996; Elisevich et al. 1997; Sohl et al. 2000; Aronica et al. 2001; Li et al. 2001; Fonseca et al. 2002; Szente et al. 2002). In a study conducted on human epileptic tissue, Naus et al. (1991) have found that the level of Cx43 mRNA was elevated in samples of temporal lobe neocortex obtained at the time of surgical resection for intractable seizures. Similar changes were also observed for Cx32 mRNA, although the observed differences were less dramatic. Both Cx32 and Cx43 mRNA levels have been found to be significantly elevated in a 4-aminopyridine in vivo model of epilepsy in rats (Szente et al. 2002). In contrast, no significant changes in Cx43 mRNA level were found in hippocampal tissues of kindled rats (Khurgel and Ivy 1996; Sohl et al. 2000) or from patients with a partial seizure disorder (Elisevich et al. 1997). However, it has been recently shown that astrocytosis in mesial temporal lobe epilepsy human hippocampus is accompanied by remarkable up-regulation of Cx43 protein level (Fonseca et al. 2002). The expression of Cx43 and Cx32 proteins was elevated in surgically obtained specimens from patients with chronic medically intractable epilepsy (Aronica et al. 2001). Data on the expression level of Cxs from kainate-treated rats are controversial (Khurgel and Ivy 1996; Sohl et al. 2000). In our previous study in intact whole hippocampus freshly isolated from young mice and exposed to BMI (Li et al. 2001), we observed a significant up-regulation of Cx32 but not Cx43 transcript and protein levels. No change in Cx26 level after chronic BMI exposure was detected in both mouse whole hippocampus (Li et al. 2001) and cultured rat hippocampal slices in the present study. Existing controversies might be explained by the differences in the experimental set-ups, specifically, the time window during which the experimental tissue was taken for analysis. In addition, experimental animal models of epilepsy differed in the various studies. Despite the difference, there is a consensus that gap junctional coupling might be involved in seizure generation and/or propagation.

Data from human studies have directly implicated the role of astrocytes and their gap junctions in the epilepsy (Naus et al. 1991; Aronica et al. 2001; Fonseca et al. 2002). Astrocytes form a syncytium mediated by gap junctions that are composed predominantly of the Cx43 protein (Dermietzel et al. 1991; Dermietzel and Spray 1993; Giaume and Venance 1995; Giaume and McCarthy 1996; Rozental et al. 2000) and allow ions and small molecules to move freely between cells (Dermietzel and Spray 1998). Several studies published in the past decade have indicated that glial cells, particularly astrocytes, can play an important role in modulating integration within the CNS (Araque et al. 2001; Haydon 2001). Alvarez-Maubecin et al. (2000) have shown that heterocellular functional coupling between glia and neurons in the locus ceruleus directly affects the co-ordinated activity of the locus ceruleus, directly demonstrating glial participation in the network properties. Dual intracellular recordings from neuron-glia pairs in the cat neocortex suggest that cortical network oscillations of the slow sleep or paroxysmal type could result from complex glial–neuronal interaction (Amzica and Massimini 2002; Amzica et al. 2002), but the involvement of gap junctional communication was not addressed in this study. In addition, it had been demonstrated in mixed neuronal-glial dissociated cultures that neurons up-regulate gap-junctional communication and Cx43 expression in astrocytes (Rouach et al. 2000). In our study, astrocytes, which proliferate in long-term cultured hippocampal slices, may have contributed to the increase in Cx43 expression in the BMI-exposed slices, but parallel up-regulation of Cx43 in neurons cannot be excluded. Although Cx43 is primarily expressed in astrocytes (Naus et al. 1990; Dermietzel et al. 1991; Giaume et al. 1991; Micevych and Abelson 1991; Belliveau and Naus 1995; Nadarajah et al. 1997; Simburger et al. 1997), it may also be present in neurons (Micevych and Abelson 1991; Nadarajah et al. 1997; Simburger et al. 1997).

While the presence and putative role of heterocellular gap junctional coupling between glia and neurons in specific networks need to be elucidated, paired patch-clamp recordings have revealed the presence of functional electrical synapses between GABAergic interneurons in different CNS structures (for review see Galarreta and Hestrin 2001) and between pyramidal neurons in the hippocampus (Schmitz et al. 2001). Moreover, epileptiform discharges evoked by kainate (Hormuzdi et al. 2001) or by 4-aminopyridine (Maier et al. 2002) in hippocampal slices from Cx36-deficient mice have been found to be markedly attenuated. Interestingly, the deletion of neuronal specific gap junctions does not completely abolish all types of neuronal synchronization (Hormuzdi et al. 2001; Maier et al. 2002; Traub et al. 2003), indicating that, besides Cx36, other connexins play a role in normal and pathological synchronization. In our study using cultured hippocampal slices exposed to BMI, we did not detect changes in mRNA level of Cx26 and Cx36 (Fig. 2) that are primarily expressed in neurons (Condorelli et al. 2000; Rash et al. 2001a; Rash et al. 2001b; Bittman et al. 2002; Condorelli et al. 2002), but the level of another connexin, Cx32, which was also found in neurons (Naus et al. 1990; Micevych and Abelson 1991; Belliveau and Naus 1995; Nadarajah et al. 1996; Alvarez-Maubecin et al. 2000; Venance et al. 2000), was significantly increased in BMI-exposed slices after chronic BMI exposure (Figs 2 and 3). Importantly, strong Cx32 immunolabeling in the neuronal component of the human epileptic tissue has been reported (Aronica et al. 2001). In addition, Cx30, that is normally expressed in glia, was detected in neurons after kainate-induced seizures (Condorelli et al. 2002). This observation indicates that de novo expression of gap junctions can occur in the convulsive brain.

One of the most important findings of the present study is that up-regulation of Cx proteins is accompanied by a significant increase in functional gap junctional coupling, manifested by faster and more complete fluorescence recovery from laser photobleaching (Fig. 6). Similar data has been obtained from tissues surgically excised from medically intractable epilepsy patients (Lee et al. 1995), indicating that the seizure-prone state in epileptic patients may be associated with alterations in gap junctional permeability. We have also shown that carbenoxolone, a gap junctional blocker, effectively decreased functional gap junctional coupling (Fig. 6) as well as the epileptiform discharges (Fig. 7) in cultured hippocampal slices. As shown previously by patch clamp recording (Schmitz et al. 2001; Jahromi et al. 2002), carbenoxolone does not reduce neuronal cell excitability. Moreover, oleanolic acid, an inactive analog of carbenoxolone (Bani-Yaghoub et al. 1999), did not block epileptiform discharges (Fig. 8) in our experiments. Another widely used gap junctional blocker, octanol, completely blocked PADs induced by a tetanic train and abolished spontaneous activity only slightly affecting the peak amplitude of single population spikes (data not shown).

In a good agreement with literature on the appearance of c-fosgene and c-fos protein in the convulsing brain (Herdegen and Leah 1998; Hughes et al. 1999), we have found that chronic BMI-exposure significantly increased the level of the indictable transcription factor, c-fos, protein in cultured hippocampal slices (Fig. 4). It has been previously shown that focal application of BMI in vivo up-regulated the transcription of c-fos gene (Maggio et al. 1993) and the expression of c-fos protein (Gass et al. 1992) in the hippocampus. Using the acutely isolated mouse hippocampus as a model, we also found that short-term exposure (2 h) to bicuculline increased the level of c-fos protein in cell nuclei 2-fold, followed by an up-regulation of Cx32 gene transcription (Li et al. 2001). If the detection of the c-fos protein can serve as a marker of neuronal hyperactivity, one could anticipate that chronic BMI exposure promotes intrinsic neuronal activity in cultured hippocampal slices. The questions of why and how this enhanced neuronal activity per se can induce an up-regulation of gap junction expression resulting in pathological hyperactivity need further studies using different convulsants and cultured hippocampal slices as a reliable model of epileptogenesis. Specific prevention of the transcription of connexin genes and demonstration that this can affect the epileptiform activity could provide new conclusive evidence of the involvement of gap junctions in epileptogenesis. However, this was beyond the scope of the present study.

In conclusion, we found that the chronic exposure to BMI induced simultaneously both up-regulation of gap junctional expression and function, and persistent epileptiform activity in cultured hippocampal slices, and that specific gap junctional blockers could selectively and reversibly suppress the increased spontaneous and evoked epileptiform discharges. These results support the hypothesis that gap junctional communication plays an important role in the development and maintenance of seizures. The present study also provides a reliable model to study molecular and cellular mechanism of epileptogenesis.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Organotypic hippocampal slice culture preparation
  5. Bicuculline exposure
  6. Electrophysiology
  7. RNase protection assay
  8. Western blot analysis
  9. Fluorescence recovery after photobleaching (FRAP)
  10. Statistics
  11. Results
  12. Persistent epileptiform activity induced by chronic exposure to BMI
  13. Transcription of Cx43 and Cx32 genes was increased by BMI exposure
  14. Increased levels of Cx43 and Cx32 proteins in BMI-exposed slices
  15. Up-regulation of c-fos protein in BMI-exposed cultures
  16. Unaltered expression of other neural proteins in BMI-exposed slices
  17. Increased rate of FRAP produced by chronic BMI exposure
  18. Blockade of epileptiform activity by carbenoxolone and octanol
  19. Discussion
  20. Acknowledgements
  21. References