Involvement of Putrescine in the Development of Kindled Seizure in Rats

During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with alpha-difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling.     

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5-4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala-kindled and "yoked" control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of gamma-aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.     

In vitro 1H NMR spectroscopy shows an increase in N-acetylaspartylglutamate and glutamine content in the hippocampus of amygdaloid-kindled rats

We examined energy metabolism and amino acid content in the hippocampus of amygdaloid-kindled rats using (1)H NMR spectroscopy. Three weeks after the last stage 5 seizure, kindled rats were killed by microwave irradiation. The hippocampus was dissected out and subjected to MeOH/CHCl(3) extraction. All (1)H spectra were analyzed to quantify absolute concentrations using a non-linear least squares method, combined with a prior knowledge of chemical shifts. Saturation effects were compensated for by the T1 measurement of each component. Levels of energy metabolism-related compounds, phosphocreatine, creatine, glucose and succinate were the same in both kindled rats and sham controls. Lactate concentration had a tendency to increase, although this was not statistically significant. When compared with sham controls, levels of aspartate, glutamate, glycine and glutamine, as well as GABA and inositol, were increased in the ipsilateral but not the contralateral hippocampus. In contrast, levels of taurine, alanine and threonine were unchanged. Finally, N-acetylaspartylglutamate content was elevated, whereas N-acetyl-l-aspartate content was unaltered in the ipsilateral hippocampus of kindled animals. Our results suggest that amygdala kindling may affects amino acid metabolism, but not energy metabolism.     

Hippocampal Kindling in the Rat Is Associated with Time-Dependent Increases in the Concentration of Glial Fibrillary Acidic Protein

The effect of hippocampal kindling on the regional brain concentration of total glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, was studied in partially kindled rats, and in fully kindled rats after a post-kindling period of 24 h, 1 week, and 2 months. GFAP concentration was measured in arbitrary units by dot-blots. In the hippocampus, dentate gyrus, basolateral amygdala, pyriform cortex, and entorhinal cortex, limbic structures which are known to be involved in the kindling process, there was an increase in GFAP concentration which was maximal in the fully kindled animals studied after 24 h. In most brain areas, GFAP concentration was still elevated 1 week post-kindling, but had declined to control level 2 months post-kindling. A significant increase in GFAP was also found in septum, ventral pallidum/accumbens nucleus, and primary motor cortex of kindled rats with a post-kindling period of 24 h, whereas in several other brain regions GFAP was unchanged. These results suggest that astrocyte activation, indicative of degenerative changes in nearby neurons, is a transient and regional phenomenon in kindling occurring only during the development of the kindled state.     

Estrogens up-regulate type I and type II glucocorticoid receptors in brain regions from ovariectomized rats

The effects of 1-4 days of estradiol (E2) treatment on type I and type II glucocorticoid receptors (GCR) were determined in cytosolic fractions from brain regions of ovariectomized rats. Four days after E2 administration, type I GCR increased in septum, amygdala, hypothalamus and hippocampus, but decreased in the anterior pituitary. Type II GCR increased in septum and hypothalamus only. For both receptor types, changes occurred earlier in septum (1 day) than in the other regions. The E2 increment was due to an increase in Bmax, without changes in Kd. The up-regulation of type II GCR by E2 was also confirmed immunocytochemically in four nuclei of the septal area. In a parallel study, E2 receptors were determined in nuclear and cytosol fractions from the same regions analyzed for GCR. In rats receiving E2, estrogen receptors decreased in cytosol and increased in nuclei from septum, amygdala, hypothalamus and anterior pituitary, but did not change in hippocampus. The results suggest that GCR in certain neuroendocrine regions are regulated by E2, without taking into account whether the areas involved contain high (anterior pituitary), moderate (septum, hypothalamus, amygdala) or low (hippocampus) levels of E2 receptors. Our model may shed light on sex differences in GCR and on E2 regulation of glucocorticoid action in brain and the pituitary.     

Enduring Effects of Early Life Stress on Firing Patterns of Hippocampal and Thalamocortical Neurons in Rats: Implications for Limbic Epilepsy

Early life stress results in an enduring vulnerability to kindling-induced epileptogenesis in rats, but the underlying mechanisms are not well understood. Recent studies indicate the involvement of thalamocortical neuronal circuits in the progression of kindling epileptogenesis. Therefore, we sought to determine in vivo the effects of early life stress and amygdala kindling on the firing pattern of hippocampus as well as thalamic and cortical neurons. Eight week old male Wistar rats, previously exposed to maternal separation (MS) early life stress or early handling (EH), underwent amygdala kindling (or sham kindling). Once fully kindled, in vivo juxtacellular recordings in hippocampal, thalamic and cortical regions were performed under neuroleptic analgesia. In the thalamic reticular nucleus cells both kindling and MS independently lowered firing frequency and enhanced burst firing. Further, burst firing in the thalamic reticular nucleus was significantly increased in kindled MS rats compared to kindled EH rats (p<0.05). In addition, MS enhanced burst firing of hippocampal pyramidal neurons. Following a stimulation-induced seizure, somatosensory cortical neurons exhibited a more pronounced increase in burst firing in MS rats than in EH rats. These data demonstrate changes in firing patterns in thalamocortical and hippocampal regions resulting from both MS and amygdala kindling, which may reflect cellular changes underlying the enhanced vulnerability to kindling in rats that have been exposed to early life stress.     

Failure to kindle seizures after repeated intracerebral administration of arginine vasopressin

In an attempt to kindle seizures with arginine-vasopressin (AVP), we injected AVP into the amygdala or hippocampus of rats. Although behavioral and electrographic alterations were sometimes observed, seizures failed to develop, even in rats that had previously been kindled with electrical stimulation. This and previous failures to kindle seizures by intraventricular injections of AVP call into question the possibility of AVP kindling.     

Amygdala kindled seizure stage is related to altered benzodiazepine binding site density

Benzodiazepine receptor binding was examined in rats at 3 stages of amygdaloid kindling (i.e., initial afterdischarge, Stage 3 and Stage 5) immediately or 24 hr after seizure. 3H-diazepam binding site density (Bmax) was significantly increased 24 hr after Stage 3 and Stage 5 kindled seizures in the hippocampus but not in the amygdala. There were no significant differences in the dissociation constants (KD) between kindled and control rats at any time point examined for either brain region. These results demonstrate that changes in benzodiazepine binding are observed with partial kindled seizures (i.e., Stage 3), indicating that generalized seizures are not prerequisite to increased benzodiazepine receptor site density.     

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.     

Amygdala Kindling Potentiates Seizure-Stimulated Immediate-Early Gene Expression in Rat Cerebral Cortex

Kindling induces long-term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate-early genes (IEGs) c-fos, c-jun, and NGF1-A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c-fos mRNA in cerebral cortex, ECS-stimulated levels of c-fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS-stimulated levels of c-fos mRNA were not significantly increased in stage 2 kindled animals. ECS-stimulated levels of c-jun and NGF1-A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was long-lasting because an acute ECS stimulus significantly elevated levels of c-fos and c-jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS-stimulated levels of c-fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina-specific changes in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of immunoreactive somatostatin and beta-endorphin content in rat brain after amygdaloid kindling

A possible contribution of brain beta-endorphin and somatostatin to the epileptogenicity established by amygdaloid kindling was investigated in rats. Fourteen male rats were chronically implanted with electrodes placed bilaterally into the amygdala. The rats received 1 sec of electrical stimulation to the left amygdala each day. Generalized seizures were observed on average 10 days after initiation of kindling and the electrical stimulation was continued up to twenty-one days. Two months after the completion of the kindling procedure, each kindled and control rat was killed by microwave irradiation and the brains were dissected on ice into thirteen subregions. Each region was homogenized and centrifuged twice in 0.1 N acetic acid. The supernatant extracts were decanted and stored at - 20 degrees C until assay. Immunoreactive beta-endorphin and somatostatin were measured by radioimmunoassays. There were no significant differences in brain beta-endorphin contents between the two groups. In kindled rats, immunoreactive somatostatin was increased significantly in amygdala, sensorimotor, piriform, and entorhinal cortex. The results suggest that changes in somatostatin may be associated with epileptic susceptibility induced by the electrical kindling procedure.     

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.     

Changes in Polyamine Concentrations in Amygdaloid-Kindled Rats

Concentrations of the polyamines putrescine, spermidine, and spermine were investigated in the left and right amygdala and in the remaining cerebrum, in which kindling was induced by repeated application of electrical stimulation of the left amygdala of rats. In kindled rats, the concentrations of spermidine and spermine increased slightly, but elevations did not reach significant levels in any brain regions. The most profound increase was detected in the putrescine concentration in all parts of the cerebrum 1-8 h after the final stimulation. These results suggest that the increases in concentrations of polyamines, particularly of putrescine, are involved in the pathogenesis of amygdaloid kindling.     

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.     

Alterations in Glutamate Transporter Protein Levels in Kindling-Induced Epilepsy

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na⁺-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.     

Cysteamine normalizes cerebral somatostatin level and binding in pentylenetetrazol-kindled rats

Rats were kindled by intraperitoneal injection of pentylenetetrazol (PTZ) (30 mg/Kg) every 48 h. Once kindled, some of the animals received a single injection of cysteamine (200 mg/Kg). Somatostatin-like immunoreactivity (SLI) and 125 I-Tyr11-somatostatin binding were measured in the frontoparietal cortex and hippocampus of the two experimental groups and the control rats. After PTZ kindling the following was observed: 1) SLI content was increased in the two areas; 2) Somatostatin receptor affinity decreased in the frontoparietal cortex and was unaltered in the hippocampus; 3) The number of somatostatin receptors decreased in the hippocampus and was unaltered in the frontoparietal cortex. Cysteamine, an agent which depletes brain somatostatin and suppresses kindled seizures in PTZ-treated rats, reversed the altered SLI levels and binding in these rats.     

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.     

The influence of chronic neurotization on the monoaminergic systems of various brain structures in rats with various typological characteristics

Typological behavioral features of Wistar rats were tested in the open field and in Porsolt test. Rats were assigned to groups with high (HAct), medium (MAct), and low (LAct) behavioral activities. The same rats were assigned to high (HDep), medium (MDep) and low depressive (LDep) groups. The release of norepinephrine, dopamine, serotonin and their metabolites in homogenates obtained from the hypothalamus, hippocampus, frontal cortex and amygdala was assessed by microdialysis and HPLC. In these groups, the monoamine concentrations were different: the level of serotonin was higher in the hypothalamus and norepinephrine and 5-HIAA levels were lower in the hippocampus of MAct - MDep rats as compared to LAct - HDep. Chronic neurotization caused changes in monoamine concentrations in the hypothalamus and amygdala in rats of all groups, whereas in the hippocampus and frontal cortex monoamine changes were observed in HAct - LDep and LAct -HDep rats. The most prominent changes in monoamines levels in neurotized rats with different types of behavior were found in the frontal cortex, amygdala and hippocampus. The results show a correlation between the typological of behavioral characteristics and the reaction to stress of monoaminergic systems of the hypothalamus, hippocampus, frontal cortex and amygdala.     

Decreased benzodiazepine receptor binding in amygdala-kindled rat brains

3H-Flunitrazepam (3H-FLU) binding was measured in multiple brain regions of amygdala-kindled rats two weeks following the sixth Stage 5 convulsion. As compared to 'yoked' controls, the kindled animals displayed significant reductions in 3H-FLU binding in the ipsilateral cortex (20%) and in the hypothalamus (20%). Scatchard plots revealed that these reductions were due to changes in the maximal number of available binding sites (Bmax) rather than to alterations in receptor affinity (KD). No significant changes were found in the contralateral cortex, or in either the contralateral or ipsilateral amygdala, hippocampus or striatum. These data suggest that kindling is associated with long-lasting changes in the benzodiazepine receptor system and possibly with related changes in GABA-mediated neural inhibition.