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Keywords:

  • epilepsy;
  • neurotransmission;
  • NSF;
  • SNAREs;
  • syntaxin;
  • VAMP

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

Kindling is a model of complex partial epilepsy wherein periodic application of an initially subconvulsive stimulus leads to first limbic and then generalized tonic-clonic seizures. Several laboratories have reported that augmented neurotransmitter release of l-glutamate is associated with the chronically kindled state. Neurotransmitter release requires membrane proteins called SNAREs, which form transmembrane complexes that participate in vesicle docking and are required for membrane fusion. We show here that kindling by entorhinal stimulation is associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus. This increase of 7S SNARE complexes appears to begin early in the kindling process, achieves a peak with full kindling, and remains at this level for at least a month following cessation of further kindling stimuli. The increase is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. It is not seen in animals that receive electroconvulsive seizures, suggesting it is related to the kindling process itself. The duration and focality of increased 7S SNARE complexes with entorhinal kindling suggest that this is an altered molecular process associated with epileptogenesis.

Abbreviations used
ECS

electroconvulsive shock

NSF

N-ethylmaleimide sensitive factor

NT

neurotransmitter

PKC

protein kinase C

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SNARE

soluble NSF attachment protein receptor

VAMP

vesicle associated membrane protein

Calcium-dependent neurotransmitter (NT) release from synaptic vesicles is a specialized exocytotic process that is the basis of interneuronal communication. The fusion of the vesicle and plasma membranes is initiated by the formation of a stable ternary 7S soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) complex composed of the vesicular protein synaptobrevin (v-SNARE) and the plasma membrane proteins syntaxin and SNAP 25 (t-SNAREs). The three proteins arrange as a four-helical, coiled-coil that spans the two fusing membranes (trans configuration). After fusion, the 7S complex, now in a cis configuration, is disassembled by NSF, which acts as a ‘protein helicase’ to unwind the spent 7S complexes (reviewed in Whiteheart et al. 2001; Rizo and Südhof 2002). Because SNAREs can accumulate as cis-complexes on either the vesicular or plasma membrane, they must be disassembled to be recycled and therefore maintain further neurotransmission.

Entorhinal kindling, a model of epileptogenesis (Sato et al. 1990; Sutula 1990), is associated with a permanently enhanced release of l-glutamate in the ipsilateral hippocampus (Jarvie et al. 1990). Kindling is a process of progressive and permanent intensification of epileptiform after-discharges culminating in a generalized seizure, in response to repeated subconvulsive stimulation. Development of entorhinal kindling in the rat is characterized by electrographic and behavioral stages (Racine 1972). The behavior in stages 1–2 mimics human complex partial seizures; behavior in later stages 3–5 is consistent with evolution to secondarily generalized motor seizures. Increased efficiency of presynaptic excitatory NT release, which could sustain the high-frequency network discharges associated with epilepsy, may involve changes in the regulation of elements of the presynaptic secretory machinery. The present study was carried out to determine if changes in the levels of 7S SNARE complex formation correlate with enhanced neurotransmitter release in the chronic epileptic state.

Kindling

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

Fourteen-week-old male Sprague–Dawley rats had stimulating electrodes surgically implanted in the right entorhinal cortex as previously described (Slevin and Ferrara 1985). The test group received electrical stimulation resulting in kindled seizures; the control groups received sham operations but no electrical stimulation, two electroconvulsive shocks (ECS), or were left untreated (naive). Animals were killed during the kindling process and up to 30 days after completion of kindling, defined as a stage-5 seizure occurring on two successive days. Full kindling occurred in 20 ± 6 (mean ± SD) daily stimulations. All animal procedures were approved by the Lexington VA IACUC.

Analysis of SNARE complexes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

Synaptosomes were prepared from individual hippocampi as previously described (Dunkley et al. 1988). Extracts were prepared by incubating equal quantities of synaptosomal protein in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer for 30 min at 37°C. Under these conditions, monomeric SNAREs are denatured but the thermally stable 7S complexes fail to disassemble. After electrophoresis, these complexes were probed by sequential western blotting using antibodies against the t-SNARE, syntaxin 1 (HCP-1; Inoue et al. 1992) and the v-SNARE (VAMP-2) (C1 69.1; a generous gift from Dr R. Jahn, Max Planck Institute, Göttingen, Germany). Parallel samples were boiled for 10 min to denature 7S complexes. Bands detected by both antibodies and sensitive to boiling were considered authentic 7S complexes. Because the VAMP-2 and syntaxin 1 antibodies detect the same high molecular weight SNARE complexes, only immunostaining with HPC-1 was used for further analysis. Quantification of 7S complexes was performed using fluorescently labeled secondary antibodies and a Molecular Dynamics Storm Phosphorimager with ImagQuant software (Sunnyvale, CA, USA) following the method of Xu and Bajjalieh (2001). To normalize for protein loading, the fluorescent intensity of all five SNARE complex bands was standardized to the intensity of the syntaxin 1 monomer bands. Data are presented as the ipsilateral/contralateral ratio of normalized SNARE complex intensity in the hippocampus.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

Several high molecular weight SNARE complexes were detected in synaptosomes using the anti-syntaxin 1 antibody, HPC-1 (Fig. 1). To determine which of these bands represents authentic 7S complexes, two criteria were chosen. First, the band must be detected by both a v- and a t-SNARE antibody. Second, the band must be stable in SDS–PAGE buffer at 37°C but not at 100°C (Hayashi et al. 1994). For the first criterion, parallel samples were solubilized at 37°C; complexes were separated by SDS–PAGE, and subjected to sequential western blotting with antibodies to syntaxin 1 and VAMP-2. As shown in Fig. 1, both antibodies detect a similar pattern of bands (see brackets) that are larger than the monomeric SNARE. This suggests that these bands represent stable complexes that contain both v- and t-SNAREs. The presence of SNAP-25 in the complexes is inferred as VAMP-2 and syntaxin 1 do not interact strongly in its absence (Hayashi et al. 1994). For the second criterion, samples were boiled prior to western blotting analysis. Bands resistant to boiling were not considered authentic 7S SNARE complexes and were not analyzed further. The five bands that fit the criteria for 7S complexes (bands indicated in Fig. 1) may represent oligomers, as reported by Tokumaru et al. (2001). From this analysis, the total intensity of all five SNARE complex bands is approximately 4–5% that of the SNARE monomer. This is less than reported by Xu and Bajjalieh (2001), but may reflect the increased resolution of the western blots used for our analyses.

image

Figure 1. 7S SNARE complexes in rat hippocampi. Synaptosomal protein (50 µg) was treated with 2% SDS (SDS–PAGE sample buffer) for 30 min at 37°C or for 10 min at 100°C (boiled). Samples were fractionated by SDS–PAGE on a standard 10% acrylamide gel and then probed by western blotting with either anti-syntaxin 1 antibody (right) or anti-VAMP-2 antibody (left). Bands in the range indicated were detected by both antibodies and disappeared upon boiling, and were considered authentic 7S complexes.

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Predictably, the ratio of right vs. left hippocampal 7S complex levels is near unity in naive animals (bar 1, Fig. 2) and does not change with electrode implantation (bar 2, Fig. 2). However, animals kindled to stage 5 (bar 3, Fig. 2) show an asymmetric accumulation of 7S complexes in the hippocampus ipsilateral to the stimulus site. This is not a result of the effect of an acute generalized seizure as ECS animals do not show the same asymmetric accumulation (bar 4, Fig. 2). Recently, Hinz et al. (2001) demonstrated an increase in synaptophysin–synaptobrevin complexes without a change in 7S complex in rat cortex post amygdala kindling. However, their measures were determined within 2 days after six daily stage-5 seizures without a seizure control; therefore one cannot determine whether the differences reported were a response to seizures or the actual kindling process.

image

Figure 2. 7S complex differences in acutely kindled and control hippocampi. All SNARE complex bands (see bracketed bands in Fig. 1), detected with the HPC-1 antibody to syntaxin 1, were quantified by enhanced chemifluorescence and normalized to monomeric syntaxin 1 intensity. The bars represent the ipsilateral/contralateral ratio of the normalized band intensities in kindled, naive and surgical control rats. Kindled animals at Stage 5 were compared with electroconvulsive shock (ECS) animals that were given two electroshock seizures 24 h apart. ECS animals were killed 24 h after their last seizure. For each group, at least five animals were analyzed. The ratio in kindled animals was greater than in the three control groups; p < 0.01 by anova and post-hoc t-tests.

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The increased ipsilateral 7S complex accumulation appears to represent a change in kindled animals that is permanent. As seen in Fig. 3, the effect persists for at least 1 month following the cessation of stimulation of fully kindled animals. Asymmetric 7S complex accumulation begins early in the kindling process, gradually reaching a peak by stage 5, where it reaches a plateau (Fig. 3). The levels of syntaxin 1 protein did not significantly change during the time course of epileptogenesis as measured by immunoblotting (data not shown). Consistently, no significant changes in syntaxin 1, VAMP-2 or SNAP-25 mRNA expression were seen in hippocampi from kindled animals vs. control (Whiteheart and Slevin, in preparation).

image

Figure 3. 7S SNARE complex accumulation during epileptogenesis. All 7S complex bands (see bands indicated in Fig. 1) were quantified by enhanced chemifluorescence and normalized to the intensity of the monomeric syntaxin 1 in the same lane. The points ± SEM represent the average ipsilateral/contralateral ratio (7SR/L) of the normalized band intensities in animals at the five different behaviorally characterized stages of kindling (see text). To demonstrate that the accumulation of 7S complexes was a stable phenotype, animals kindled to stage 5 were given no additional stimuli for one month prior to analysis (> 30 days). At least five animals were analyzed at each stage of kindling.

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Increased ipsilateral accumulation correlates best with kindling stage and secondarily with number of stimulations, but not with the initially established kindling after-discharge threshold. The after-discharge threshold was similar among animals (mean ± SD, 1040 ± 329 µA). The gradual accumulation suggests this phenomenon is not a threshold effect. It is focal biochemical process even at behavioral stages that clearly indicate bihemispheric electrophysiologic involvement.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

These studies demonstrate that 7S complexes accumulate in the ipsilateral hippocampus of chronically kindled rodents. 7S complexes could accumulate in either the cis or trans configuration. Increased neuronal synaptic activity, i.e. NT release, would cause more cis complexes. Alternatively, heightened levels of primed synaptic vesicles in a prefusion state (‘readily releasable’ pool) would cause an increase in trans complexes, thereby increasing the NT release potential. Both scenarios are consistent with increased SNARE complexes and the increased l-glutamate release reported for several kindling models (Jarvie et al. 1990; Yamagata et al. 1995; Ueda et al. 2000) and observed in human hippocampal epileptic foci of subjects during interictal periods (During and Spencer 1993).

Several molecular reasons could account for an accumulation of 7S complexes, either via heightened formation or via decreased disassembly. Increased activity of SNARE activators, e.g. Munc18, could promote formation thereby increasing trans complexes (discussed in Rizo and Südhof 2002). As yet no data directly implicate this mechanism in epileptogenesis. Presynaptic calcium levels could also promote 7S complex formation by affecting the activity of various regulatory molecules, e.g. synaptotagmins (reviewed in Chapman 2002), DOC2 (reviewed in Duncan et al. 2000), and calmodulin (Chen et al. 1999). Changes in intrasynaptosomal calcium are seen in kindling-induced epilepsy and may reflect faulty control of Ca2+ channels (Heinemann and Hamon 1986).

It is more difficult to understand how a decrease in SNARE complex disassembly by NSF could produce the increase in NT release associated with kindling-induced epilepsy (Jarvie et al. 1990). Acute loss of NSF activity (e.g. Drosophila comatosets mutants) causes a delayed decrease in neurotransmission and requires synaptic stimulation for full inhibition (Littleton et al. 1998; Tolar and Pallanck 1998). In these in vivo studies, monomeric SNAREs become limiting and thus NT release ceases. However, based on studies by Lonart and Südhof (2000), inhibition of NSF in isolated synaptosomes with N-ethylmaleimide causes an increase in the ‘readily releasable’ pool of synaptic vesicles. These ex vivo studies only focus on initial secretion events and would not show the effects of a prolonged failure to disassemble cis complexes. The two apparently paradoxical results suggest that disregulation of NSF could in fact have a positive effect on acute short-term NT release and yet a negative effect on the long-term efficacy of continued release.

A resolution to this paradox lies in the fact that NSF should be able to disassemble both cis and trans SNARE complexes. Disassembly of cis complexes is essential for continued NT release. Ablation of NSF activity might not acutely inhibit the ‘readily releasable’ pool of synaptic vesicles, therefore NT release would continue until that pool is depleted. This occurs in the Drosophila comatosets mutants (Littleton et al. 1998; Tolar and Pallanck 1998). A transient decrease in NSF activity (or lower levels of active enzyme) would slow disassembly of cis complexes, but as long as monomeric SNAREs were not limiting there would be no adverse effect on NT release. Disassembly of trans complexes, however, would also be lessened, thus stabilizing the 7S SNARE complexes associated with the ‘readily releasable’ pool of vesicles. Such an effect would increase the probability of NT release.

A decrease in NSF protein levels correlates with kainic acid-induced epilepsy (Guan et al. 2001; Yu et al. 2002). Guan et al. (2001) have shown reduced ERG1/NSF mRNA in dorsal hippocampus of rats three weeks following a single episode of kainic acid-induced status epilepticus. Alternatively, there could be a disregulation of NSF activity through its phosphorylation. We have shown that NSF, when phosphorylated by PKC at Ser237, is inactivated and does not bind to SNARE complexes (Matveeva et al. 2001). This phosphorylation event is normally induced by calcium influx upon depolarization. One may postulate that chronic alteration of the calcium-dependent phosphorylation of NSF could cause a decrease, but not the ablation of NSF activity.

In summary, the data presented suggest that at least one molecular defect associated with kindled epilepsy is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. That accumulation of 7S complexes persists in the absence of acute seizure activity implies that it is associated not with convulsions but with epileptogenesis and the development of the permanent epileptic state.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References

We acknowledge the technical assistance of Ms Ramona Alcala. We thank Dr Thomas Vanaman for his comments during the development of our experiments. This work is supported by the Department of Veterans Affairs Research Service (JTS) and by grants from the American Heart Association Ohio Valley Affiliate (01450841B) and National Institutes of Health (HL56652) (SWW).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Kindling
  5. Analysis of SNARE complexes
  6. Results
  7. Discussion
  8. Acknowledgements
  9. References
  • Chapman E. R. (2002) Synaptotagmin: a Ca2+ sensor that triggers exocytosis? Nat. Rev. Mol. Cell Biol. 3, 111.
  • Chen Y. A., Duvvuri V., Schulman H. and Scheller R. H. (1999) Calmodulin and protein kinase C increase Ca2+-stimulated secretion by modulating membrane-attached exocytic machinery. J. Biol. Chem. 274, 2646926476.
  • Duncan R. R., Shipston M. J. and Chow R. H. (2000) Double C2 protein. A review. Biochimie 82, 421426.
  • Dunkley P. R., Heath J. W., Harrison S. M., Jarvie P. E., Glenfield P. J. and Rostas J. A. (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res. 441, 5971.
  • During M. J. and Spencer D. D. (1993) Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet 341, 16071610.
  • Guan Z., Lu L., Zheng Z., Liu J. YuF., Lu S., Xin Y., Liu X., Hong J. and Zhang W. (2001) A spontaneous recurrent seizure-related Rattus NSF gene identified by linker capture subtraction. Brain Res. Mol. Brain Res. 87, 117123.
  • Hayashi T., McMahon H., Yamasaki S., Binz T., Hata Y., Südhof T. C. and Niemann H. (1994) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 50515161.
  • Heinemann U. and Hamon B. (1986) Calcium and epileptogenesis. Exp. Brain Res. 65, 110.
  • Hinz B., Becher A., Mitter D., Schulze K., Heinemann U., Draguhn A. and Ahnert-Hilger G. (2001) Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain. Eur. J. Cell Biol. 80, 615619.
  • Inoue A., Obata K. and Akagawa K. (1992) Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-1. J. Biol. Chem. 267, 1061310619.
  • Jarvie P. A., Logan T. C., Geula C. and Slevin J. T. (1990) Entorhinal kindling permanently enhances Ca2+-dependent 1-glutamate release in regio inferior of rat hippocampus. Brain Res. 508, 188193.
  • Littleton J. T., Chapman E. R., Kreber R., Garment M. B., Carlson S. D. and Ganetzky B. (1998) Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly. Neuron 21, 401413.
  • Lonart G. and Südhof T. C. (2000) Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles. J. Biol. Chem. 275, 2770327707.
  • Matveeva E. A., Whiteheart S. W., Vanaman T. C. and Slevin J. T. (2001) Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes. J. Biol. Chem. 276, 1217412181.
  • Racine R. J. (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281294.
  • Rizo J. and Südhof T. C. (2002) SNAREs and Munc18 in synaptic vesicle fusion. Nat. Rev. Neurosci. 3, 641653.
  • Sato M., Racine R. J. and McIntyre D. C. (1990) Kindling: basic mechanisms and clinical validity. Electroencephalogr. Clin. Neurophysiol. 76, 459472.
  • Slevin J. T. and Ferrara L. P. (1985) Lack of effect of entorhinal kindling on L-[3H]glutamic acid presynaptic uptake and postsynaptic binding in hippocampus. Exp. Neurol. 89, 4858.
  • Sutula T. P. (1990) Experimental models of temporal lobe epilepsy: new insights from the study of kindling and synaptic reorganization. Epilepsia 31, S45S54.
  • Tokumaru H., Umayahara K., Pellegrini L. L., Ishizuka T., Saisu H., Betz H., Augustine G. J. and Abe T. (2001) SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis. Cell 104, 421432.
  • Tolar L. A. and Pallanck L. (1998) NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking. J. Neurosci. 18, 1025010256.
  • Ueda Y., Doi T., Tokumaru J., Mitsuyama Y. and Willmore L. J. (2000) Kindling phenomena induced by the repeated short-term high potassium stimuli in the ventral hippocampus of rats: on-line monitoring of extracellular glutamate overflow. Exp. Brain Res. 135, 199203.
  • Whiteheart S. W., Schraw T. and Matveeva E. A. (2001) N-ethylmaleimide sensitive factor (NSF) structure and function. Int. Rev. Cytol. 207, 71112.
  • Xu T. and Bajjalieh S. M. (2001) SV2 modulates the size of the readily releasable pool of secretory vesicles. Nat. Cell Biol. 3, 691698.
  • Yamagata Y., Obata K., Greengard P. and Czernik A. J. (1995) Increase in synapsin I phosphorylation implicates a presynaptic component in septal kindling. Neuroscience 64, 14.
  • Yu F., Guan Z., Zhuo M., Sun L., Zou W., Zheng Z. and Liu X. (2002) Further identification of NSF as an epilepsy related gene. Mol. Brain Res. 99, 141144.