Somatostatin release in the hippocampus in the kindling model of epilepsy: a microdialysis study

Somatostatin biosynthesis in the hippocampus is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon is associated with enhanced somatostatin release in vivo. Experiments have been run in awake, freely moving rats, implanted with a bipolar electrode in the right amygdala (for kindling stimulation), and with a recording electrode and a microdialysis probe in the left hippocampus. Basal somatostatin-like immunoreactivity (-LI) release was significantly greater in kindled than naive rats. In naive rats, a 2-min perfusion with 100 mM K(+) did not affect behavior and EEG recordings and nonsignificantly increased somatostatin-LI release; a 10-min K(+) perfusion evoked numerous wet dog shakes, electrical seizures (class 0; latency congruent with 8 min, duration congruent with 8 min), and somatostatin-LI release ( congruent with 350% of basal); and a single kindling after-discharge (4 +/- 3-s duration in the hippocampus) also evoked somatostatin-LI release ( congruent with 200% of basal). In kindled rats, a 2-min 100 mM K(+) perfusion evoked hippocampal discharges in three of seven animals (latency congruent with 2 min, mean duration congruent with 1.5 min) and increased somatostatin-LI release ( congruent with 250% of basal); a 10-min K(+) perfusion evoked behavioral seizures (class 1 to 5, latency congruent with 4 min, mean duration congruent with 12 min) with numerous wet dog shakes and robust somatostatin-LI release ( congruent with 350% of basal); and a kindling stimulation evoked generalized seizures (class 4 or 5, 77 +/- 15-s duration in the hippocampus) with remarkable somatostatin-LI release ( congruent with 300% of basal). These data demonstrate that hippocampal somatostatin release is increased in the kindling model in vivo.     

Amygdala Kindling Modifies Extracellular Opioid Peptide Content in Rat Hippocampus Measured by Microdialysis

Abstract: Opioid peptide release in the hippocampus was shown to be increased immediately following amygdala kindling stimulation in freely moving rats using microdialysis combined with a universal opioid peptide radioimmunoassay (RIA). Extracellular opioid peptide levels were elevated (55% above basal levels) within the first 10 min after electrical stimulation-induced partial seizures in previously nonkindled animals. Fully kindled rats showed lower extracellular opioid peptide levels (40% reduction) during the interictal period [16 ± 2.1 days (mean ± SEM) after the last stage V seizure], in comparison with values obtained from the sham-kindled group under basal conditions. However, opioid peptide release in fully kindled rats increased above 152% of interictal levels within the first 20 min after onset of fully kindled seizures, attaining peak levels equal to that of the partial kindled group and returning to prestimulation conditions 40–60 min following the ictal events. The majority of the immunoreactive material recovered from the hippocampus within the first 20 min following partial and generalized kindled seizures coeluted with dynorphin-A (1–6), dynorphin-A (1–8), and Leu-enkephalin by HPLC/RIA analysis. It is proposed that the enhanced opioid peptide release in hippocampus induced by amygdala kindling stimulation might be associated with either enhanced excitability or seizure suppression as seizure susceptibility fluctuates. The reduced interictal opioid peptide levels may also underlie some interictal behavioral disturbances.     

Involvement of nitric oxide in pentylenetetrazole-induced kindling in rats

We investigated the role of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) in the pentylenetetrazole (PTZ)-induced kindling in rats. Seizures were induced by single administration of PTZ, which was associated with an increase in levels of NO metabolites (NOx) in the hippocampus. Pretreatment with a neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), diminished the PTZ-induced increase in NOx levels without affecting the seizure intensity. Repeated administration of PTZ produced a gradual increase in the seizure intensity, leading to the development of kindling. In the kindled rats, PTZ at a dose of 40 mg/kg increased NOx levels in the hippocampus, whereas it had no effect in control animals. Cotreatment of 7-NI with PTZ blocked the development of kindling and attenuated the PTZ-induced increase in NOx levels. A significant increase in BDNF levels was observed in the hippocampus of the kindled rats, which returned to the control levels following seizures induced by PTZ. 7-NI reduced the hippocampal BDNF levels in control rats and suppressed the increase of BDNF levels in the kindled rats. Our findings suggest that NO plays a role in the development of PTZ-induced kindling and that BDNF may contribute to the NO-dependent plastic changes in neuronal excitability.     

The role of the opiate mechanisms of the hippocampus and substantia nigra in the behavioral and convulsive disorders in picrotoxin-induced kindling]

It was shown in the experiments on rats that the repeated picrotoxin administration resulted in the kindling of generalized seizures. Generalized convulsions were followed by the development of either postictal depression or explosiveness. The injection of mu-opiate agonist met-enkephalin into hippocampus of kindled rats resulted in the increase in the severity of seizure reactions which were induced by picrotoxin and also in the increase in the number of animals with postictal explosiveness. The injection of dynorphin-A-1-13 (kappa-opiate agonist) into substantia nigra reticulata induced the locomotor depression which was like one in postictal period and resulted in the decrease of picrotoxin-induced seizures severity. It was concluded that mu-opiate system of hippocampus took part in the formation of generator of pathologically enhanced excitation in the structure during kindling and the development of seizure syndrome, providing also the postictal explosiveness. Kappa-opiate system of substantia nigra plays an important role in the activation of the antiepileptic system, limitation of seizures and the development of postictal depression.     

Time- and Region-Specific Variations in Somatostatin Release Following Amygdala Kindling in the Rat

Abstract: Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin-like immunoreactivity (somatostatin-LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham-stimulated controls, (a) in the hippocampus, high-K⁺-evoked somatostatin-LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin-LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin-LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.     

Structure of membrane‐associated neuronal SNARE complex: implication in neurotransmitter release

To enable fusion between biological membranes, t‐SNAREs and v‐SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring‐complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t‐/v‐SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE‐ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40–50 nm in diameter membrane‐bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12–17 nm in diameter cup‐shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t‐SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t‐/v‐SNARE complex, 50 nm t‐and v‐SNARE proteoliposomes were mixed, allowing t‐SNARE‐vesicles to interact with v‐SNARE vesicles, followed by detergent solubilization and imaging of the resultant t‐/v‐SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6–7 nm membrane‐directed self‐assembled t‐/v‐SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3–4 nm base diameter, where 40–50 nm in diameter v‐SNARE‐associated synaptic vesicle transiently dock and fuse to release neurotransmitters.     

Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling

The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.     

Crystal structure of the endosomal SNARE complex reveals common structural principles of all SNAREs.

SNARE proteins are crucial for intracellular membrane fusion in all eukaryotes. These proteins assemble into tight complexes that connect membranes and may induce fusion. The crystal structure of the neuronal core complex is represented by an unusually long bundle of four alpha-helices connected by 16 layers of mostly hydrophobic amino acids. Here we report the 1.9 A resolution crystal structure of an endosomal SNARE core complex containing four SNAREs: syntaxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8. Despite limited sequence homology, the helix alignment and the layer structure of the endosomal complex are remarkably similar to those of the neuronal complex. However, subtle variations are evident that characterize different SNARE subfamilies. We conclude that the structure of the SNARE core complex is an evolutionarily conserved hallmark of all SNARE complexes and is intimately associated with the general role of SNAREs in membrane fusion.     

Metformin Plus Caloric Restriction Show Anti-epileptic Effects Mediated by mTOR Pathway Inhibition

Caloric restriction (CR) has anti-epileptic effects in different animal models, at least partially due to inhibition of the mechanistic or mammalian target of rapamycin (mTOR) signaling pathway. Adenosine monophosphate-activated protein kinase (AMPK) inhibits mTOR cascade function if energy levels are low. Since hyper-activation of mTOR participates in epilepsy, its inhibition results in beneficial anti-convulsive effects. A way to attain this is to activate AMPK with metformin. The effects of metformin, alone or combined with CR, on the electrical kindling epilepsy model and the mTOR cascade in the hippocampus and the neocortex were studied. Combined metformin plus CR beneficially affected many kindling aspects, especially those relating to generalized convulsive seizures. Therefore, metformin plus CR could decrease measures of epileptic activity in patients with generalized convulsive seizures. Patients that are obese, overweight or that have metabolic syndrome in addition to having an epileptic disease are an ideal population for clinical trials to test the effectiveness of metformin plus CR.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.     

Wet-dog shaking behavior in rats in hippocampal kindling

The kindling phenomenon, induced by repetitive electrical stimulation of the hippocampus of the rat is associated with the production of a particular behavior: the Wet Dog Shakes (WDS). The evolution of WDS is strongly and negatively correlated with the intensification of motor seizures. Some differences can be seen in the occurrence of WDS according to whether the stimulation is applied in dorsal or in ventral hippocampus. Although the anatomical substrate responsible for this behavior is not clearly defined, the relationship between WDS and kindling let us consider it as an index of the generalization of the epilepsy.     

The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.     

Kindling of the limbic structures with a shortened interstimulus interval

We investigated the possibility to produce hippocampal or amygdala kindling syndrome in rabbits which had been electrically stimulated at a fixed interval between stimuli at 5 min. Animals were prepared with chronically implanted electrodes (neocortex, hippocampus, amygdala, nucleus caudatus). The initial stimuli produced only localized effect, but repeated applications of the stimuli progressively increased the seizure activity resulting in generalized kindled convulsions after 2-4 h period. At the first stage generalized seizures were followed by long lasting refractory period, but at the end of the procedure almost all stimuli evoke major motor seizures and recurrent widely spread electrographic epileptic changes. The most noteworthy findings emerging from this study is the inhibition of postictal seizure inhibition period. This effect was independent of whether stimulated the electrode was positioned in the hippocampus or amygdala, but the hippocampal formation occupied the central position for the once and propagation of the seizure activity in all cases. When established this syndrome persisted without any attenuation for some weeks. It was concluded that this model of rapid development of kindling syndrome is useful for investigation of the nature of epilepsy and postictal seizure inhibition.     

Changes in benzodiazepine receptors in the limbic system following kindling of the olfactory bulb in rats

Repeated electrical stimulations of the olfactory bulb led to the progressive development of a generalized epilepsy (kindling effect). One week after the last stimulation eliciting a stage 5 seizure, diazepam-(3H) binding was studied in olfactory bulb-kindled rats. Numbers of benzodiazepine receptors were increased in kindled olfactory bulb and amygdala. No significant change was observed in hippocampus. This modification could be considered as a response of the inhibitory mechanisms to repeated seizures which is insufficient to counteract the installation of the kindling effect.     

Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain

The vesicular protein synaptobrevin contributes to two mutually exclusive complexes in mature synapses. Synaptobrevin tightly interacts with the plasma membrane proteins syntaxin and SNAP 25 forming the SNARE complex as a prerequisite for exocytotic membrane fusion. Alternatively, synaptobrevin binds to the vesicular protein synaptophysin. It is unclear whether SNARE complex formation is diminished or facilitated when synaptobrevin is bound to synaptophysin. Here we show that the synaptophysin-synaptobrevin complex is increased in adult rat brain after repeated synaptic hyperactivity in the kindling model of epilepsy. Two days after the last kindling-induced stage V seizure the relative amount of synaptophysin-synaptobrevin complex obtained by co-immunoprecipitation from cortical and hippocampal membranes was increased twofold compared to controls. By contrast the relative amounts of various synaptic proteins as well as that of the SNARE complex did not change in membrane preparations from kindled rats compared to controls. The increased amount of synaptophysin-synaptobrevin complex in kindled rats supports the idea that this complex represents a reserve pool for synaptobrevin enabling synaptic vesicles to adjust to an increased demand for synaptic efficiency. We conclude that the synaptophysin-synaptobrevin interaction is involved in activity-dependent plastic changes in adult rat brain.     

Sodium nitroprusside-induced seizure and taurine release from rat hippocampus

Summary. We have recently reported that the nitric oxide (NO) donor, sodium nitroprusside (SNP), induces seizures which are associated with an increase in the basal release of aspartate and glutamate from rat hippocampus (Kaku et al., 1998). In order to determine whether taurine release occurs with SNP-induced seizures, we examined the effects of NO-related compounds, i.e., the NO trapper, diethyldithiocarbamate (DETC), the superoxide radical scavenger, dithiothreitol (DTT), the xanthine oxidase inhibitor, oxypurinol and the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ), on SNP-induced seizures and in vivo taurine release from rat hippocampus using microdialysis. Perfusion with 0.5 mM SNP provoked seizures and significantly increased taurine release, with the increase in release occurring primarily during reperfusion with artificial cerebrospinal fluid lacking SNP. Perfusion with 5 mM DETC significantly abolished the SNP-induced seizures and reduced taurine release during and after perfusion with the drugs. Perfusion with 1 mM DTT significantly reduced both the frequency of the SNP-induced seizures and taurine release during and after perfusion with the drugs. Perfusion with 1 mM oxypurinol or 0.5 mM ODQ did not reduce the frequency of the SNP-induced seizures, but tended to decrease taurine release during and after perfusion with the drugs. These results demonstrate that SNP-induced seizures are triggered by an increase in both NO and peroxynitrite and are related to an increase in taurine release from rat hippocampus. Received January 25, 2000/Accepted January 31, 2000