Bilateral lesions of the central but not anterior or posterior parts of the piriform cortex retard amygdala kindling in rats

The piriform cortex is thought to be involved in temporal lobe seizure propagation, such as that occurring during kindling of the amygdala or hippocampus. A number of observations suggested that the circuits of the piriform cortex might act as a critical pathway for limbic seizure discharges to assess motor systems, but direct evidence for this suggestion is scarce. Furthermore, the piriform cortex is not a homogeneous structure, which complicates studies on its role in limbic epileptogenesis. We have previously reported data indicating that the central part of the piriform cortex might be particularly involved during amygdala kindling. In order to further evaluate the role of different parts of the piriform cortex during kindling development, we bilaterally destroyed either the central, anterior or posterior piriform cortex by microinjections of ibotenate two weeks before onset of amygdala kindling. Lesions of the anterior piriform cortex hardly affected kindling acquisition, except that fewer animals exhibited stage 3 (unilateral forelimb) seizures compared to sham controls. Lesions of the central piriform cortex significantly retarded kindling, which was due to a decreased progression from stage 3 to stage 4/5 seizures, i.e. the lesioned rats needed significantly longer for the acquisition of generalized clonic seizures in the late stages of kindling development. Lesions of the posterior piriform cortex did not significantly affect kindling development.The data demonstrate that different parts of the piriform cortex mediate qualitatively different effects on amygdala kindling. The central piriform cortex seems to be a neural substrate involved in the continuous development of kindling from stage 3 to stages 4/5, indicating that this part of the piriform cortex may have preferred access, either directly or indirectly, to structures capable of supporting generalized kindled seizure expression.     

A long-lasting decrease in the inhibitory effect of GABA on glutamate responses of hippocampal pyramidal neurons induced by kindling epileptogenesis

Experiments were carried out to test whether changes in the sensitivity of hippocampal pyramidal neurons to the neurotransmitters glutamate, GABA and noradrenaline may be associated with the establishment of an epileptogenic focus induced by kindling. The effects of iontophoretically applied neurotransmitters on the firing rate of single units were quantified in the rat hippocampal CA1 area in kindled and control animals. Kindling was induced by electrical tetanic stimulation of the Schaffer collateral/commissural fibers. Firing was evoked by local glutamate iontophoresis while simultaneous GABA or noradrenaline application suppressed this response. A significant reduction of the GABAergic inhibitory action on the firing rate in kindled animals studied around four or around 42 days after the last convulsion was found. In the same neurons, the suppressive effect of noradrenaline was not different from controls. The neurons of kindled animals, investigated around four days after the last seizure, had a reduced sensitivity for glutamate; more glutamate ejection current was needed to evoke firing or to evoke the maximum firing rate. In contrast, the responsiveness for glutamate was significantly increased long-term after the last convulsion. These findings demonstrate that hippocampal Schaffer collateral kindling is associated with a long-lasting reduced effectiveness of the GABA-mediated response on glutamate-evoked firing in CA1.     

Bilateral microinjections of vigabatrin in the central piriform cortex retard amygdala kindling in rats

The piriform cortex (PC) is the largest region of the mammalian olfactory cortex with strong connections to limbic structures, including the amygdala, hippocampus, and entorhinal cortex. Various previous studies in rodents suggest that the PC might be very important in the development and maintenance of limbic kindling, i.e. a widely used model of temporal lobe epilepsy. GABAergic inhibition in the transition zone between the anterior and posterior PC, termed here central PC, seems to be particularly involved in the processes leading to progression of kindled seizures. This prompted us to study whether elevation of GABA levels in this subregion of the PC by bilateral microinjection of vigabatrin is capable of suppressing amygdala kindling. Rats were stimulated once daily until fully kindled (stage 5) seizures had developed. Vigabatrin (10 microg) was injected 24 h before the first stimulation as well as 6 h before the 5th and 10th stimulation, which approximately doubled the number of stimulations required for kindling development compared with controls. This marked retardation of kindling acquisition was predominantly due to a significant inhibition of the progression from stage 1 to stage 2 and stage 3 to stage 4 seizures, demonstrating that microinjection of vigabatrin into the central PC markedly inhibits the progression and secondary generalization of focal seizures emanating from the amygdala.     

Acute changes in the neuronal expression of GABA and glutamate decarboxylase isoforms in the rat piriform cortex following status epilepticus

The piriform cortex (PC) is the largest region of the mammalian olfactory cortex with strong connections to other limbic structures, including the amygdala, hippocampus, and entorhinal cortex. In addition to its functional importance in the classification of olfactory stimuli, the PC has been implicated in the study of memory processing, spread of excitatory information, and the facilitation and propagation of seizures within the limbic system. Previous data from the kindling model of epilepsy indicated that alterations in GABAergic inhibition in the transition zone between the anterior and posterior PC, termed here central PC, are particularly involved in the processes underlying seizure propagation. In the present study we studied alterations in GABAergic neurons in different parts of the PC following seizures induced by kainate or pilocarpine in rats. GABA neurons were labeled either immunohistochemically for GABA or its synthesizing enzyme glutamate decarboxylase (GAD) or by in situ hybridization using antisense probes for GAD65 and GAD67 mRNAs. For comparison with the PC, labeled neurons were examined in the basolateral amygdala, substantia nigra pars reticulata, and the hippocampal formation. In the PC of controls, immunohistochemical labeling for GABA and GAD yielded consistently higher neuronal densities in most cell layers than labeling for GAD65 or GAD67 mRNAs, indicating a low basal activity of these neurons. Eight hours following kainate- or pilocarpine-induced seizures, severe neuronal damage was observed in the PC. Counting of GABA neurons in the PC demonstrated significant decreases in densities of neurons labeled for GABA or GAD proteins. However, a significantly increased density of neurons labeled for GAD65 and GAD67 mRNAs was determined in layer II of the central PC, indicating that a subpopulation of remaining neurons up-regulated the mRNAs for the GAD isoenzymes. One likely explanation for this finding is that remaining GABA neurons in layer II of the central PC maintain high levels of activity to control the increased excitability of the region. In line with previous studies, an up-regulation of GAD67 mRNA, but not GAD65 mRNA, was observed in dentate granule cells following seizures, whereas no indication of such up-regulation was determined for the other brain regions examined. The data substantiate the particular susceptibility of the central PC to seizure-induced plasticity and indicate that this brain region provides an interesting tool to study the regulation of GAD isoenzymes.     

Unilateral up-regulation of glutamate receptors in limbic regions of amygdaloid-kindled rats

Summary Quantitative autoradiography was used to examine central binding sites for L-[³H]glutamate in amygdaloid-kindled rats since receptors for excitatory amino acids have been implicated in epileptiform activity and seizure behaviors. In tissue from rats killed five days after two kindled seizures, the ipsilateral hippocampus, entorhinal, perirhinal and parietal cortices had significantly (35–100%) greater densities of binding sites for L-[³H]glutamate than the opposite, contralateral side or operated, unstimulated controls. These regions receive excitatory inputs from the amygdala via the entorhinal cortex. Dissociation constants were not altered and significant differences were not observed in the binding parameters for L-[³H]glutamate between control and kindled rats or ipsilateral and contralateral sides of the amygdala, corpus striatum, nucleus accumbens or substantia nigra. The proportion and affinity of N-methylD-aspartate (NMDA)-sensitive binding sites for L-[³H]glutamate was unchanged after kindling, as were the relative proportions of kainate- and AMPA- (DL-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) sensitive sites. However, the density of NMDA and non-NMDA receptor subtypes was increased in the ipsilateral hippocampus, entorhinal, perirhinal and parietal cortices of kindled rats. These findings of specific, unilateral glutamate receptor up-regulation may indicate adaptive responses to the enhanced excitation found in kindling, and are consistent with other neuronal changes reported in early kindling.     

Amygdaloid kindled seizures induce weight gain that reflects left hemisphere dominance in rats

The present study sought to determine the effects of kindled seizures generated from the left and right amygdala upon weight gain in rat. Seventy-five female Sprague-Dawley rats were implanted with electrodes in basal amygdala of the left and right hemispheres. A kindling paradigm was employed in which electrical stimulation was applied once per day for 30 days after Stage 5 seizures. An electrode was implanted into the basal amygdala of the control rats but no stimulation was applied. All rats were weighed daily during the course of the experiment and changes in weight during this period were recorded for all rats. The results demonstrated that kindling from either the left or right amygdala induced significant increases in weight gain relative to the control rats. However, kindling from the left basal amygdala induced increases in body weight that were four times greater than control rats and two times greater than the rats kindled from the right side of the basal amygdala. Likewise, serum leptin levels, which were highly correlated with weight gain, also showed significantly greater increases in left amygdaloid kindled rats relative to rats kindled from the right amygdala and control rats. These findings demonstrate that basal amygdaloid kindling induces significant increases in weight gain and that the magnitude of these effects is linked to the dominance of the left hemisphere.     

Effects of pentylenetetrazole-induced kindling on thyrotropin-releasing hormone biosynthesis and receptors in rat brain

It has been postulated that changes in thyrotropin-releasing hormone biosynthesis may be involved in the mechanism of kindling--an animal model of epileptogenesis. To test this hypothesis, a time-course study was carried out to investigate the effects of pentylenetetrazole kindling (40 mg/kg i.p., daily for eight days) on the expression of gene coding for preprothyrotropin-releasing hormone, the thyrotropin-releasing hormone tissue level and thyrotropin-releasing hormone receptor parameters in rat brain. As shown by an in situ hybridization study, a single, convulsant dose of pentylenetetrazole (70 mg/kg i.p.) increased the preprothyrotropin-releasing hormone messenger RNA level in the dentate gyrus of the hippocampal formation and piriform cortex after 3 h and, to a greater extent, after 24 h. Those changes were accompanied with increases in the thyrotropin-releasing hormone level in the striatum, hippocampus, amygdala and piriform cortex. Seven days after single pentylenetetrazole administration, the thyrotropin-releasing hormone level was still significantly elevated in the piriform cortex and striatum. Acute pentylenetetrazole decreased the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum after 3 and 24 h, and increased that density in the piriform cortex and amygdala after 24 h and seven days, respectively. The thyrotropin-releasing hormone receptor affinity (Kd) was decreased in the striatum and increased in the amygdala after only 3 h. Kindled rats showed a moderate increase in the preprothyrotropin-releasing hormone messenger RNA content in the dentate gyrus of the hippocampal formation and piriform cortex after 3 and 24 h; however, a significant decrease in those parameters was found after 14 days. After 3 and 24 h, pentylenetetrazole kindling also elevated the thyrotropin-releasing hormone content in the hippocampus, piriform cortex, and striatum (in the latter structure after 24 h only), whereas in the septum the thyrotropin-releasing hormone level was decreased. After seven days, the thyrotropin-releasing hormone level was still elevated in the hippocampus and piriform cortex of kindled rats, but after 14 days it was significantly lowered in the hippocampus. The kindled rats also showed a significant decrease in the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum (after 24 h, seven and 14 days), and an increase in the piriform cortex (after seven days). The thyrotropin-releasing hormone receptor affinity (Kd) value was increased in the hippocampus after seven and 14 days, and in the piriform cortex after seven days. These results indicate that pentylenetetrazole kindling induces long-lasting alterations in the thyrotropin-releasing hormone biosynthesis and thyrotropin-releasing hormone receptor affinity in discrete regions of rat brain. These region-specific changes, in particular down-regulation of the thyrotropin-releasing hormone biosynthesis in the hippocampus, may be involved in chronic neuronal hyperexcitability associated with kindling.     

Unilateral low-frequency stimulation of central piriform cortex delays seizure development induced by amygdaloid kindling in rats

Low-frequency stimulation of the kindling site interferes with the course of kindling epileptogenesis. The present study examined the effect of unilateral low-frequency stimulation of the central piriform cortex on seizure development induced by amygdaloid kindling in rats. The ipsilateral or contralateral central piriform cortex received low-frequency stimulation (15 min train of 0.1 ms pulses at 1 Hz and 50-150 muA) immediately after termination of once daily kindling stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA) in the right amygdala for 30 days. Low-frequency stimulation of either the ipsilateral or contralateral central piriform cortex significantly suppressed the progression of seizure stages and reduced afterdischarge duration throughout the course of amygdaloid kindling. The marked suppression induced by low-frequency stimulation of the central piriform cortex on either side was predominantly due to the significant retardation of progression from stage 0 to stage 1 and stage 3 to stage 4 seizures. In addition, the suppressive effect of low-frequency stimulation did not disappear when the stimulation was stopped; it could persist for at least 10 days. These findings indicate that brain areas other than the kindling focus, such as the central piriform cortex on both sides, can also be used as reasonable targets for low-frequency stimulation to retard seizure development induced by amygdaloid kindling. Secondly, like the ipsilateral central piriform cortex, the contralateral central piriform cortex may also participate in the progression and secondary generalization of focal seizures. The study suggests that unilateral low-frequency stimulation of the central piriform cortex may have a significant antiepileptogenic effect, and may be helpful for exploring effective and long-lasting therapies for human temporal lobe epilepsy.     

Activation of N-methyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling

1. The cellular and synaptic properties of rat dentate gyrus granule cells (GCs) were examined using intra-/extracellular and Ca2+-sensitive microelectrode recordings following epilepsy induced by kindling of the hippocampal commissures or amygdala. 2. The recordings were made in hippocampal slices prepared from sham-stimulated controls and animals that have received daily stimuli to reach stage IV-V of kindling. The average number of stimulation trials (60 Hz/1 s, 100-150 microA) required to reach full motor seizures (stage V) was 23 +/- 2 for commissural kindling and 14 +/- 1 for amygdala kindling. 3. The resting membrane potential of GCs following kindling (RMP; -72 +/- 3 mV) was not significantly different from the RMP of control GCs (-70 +/- 2 mV). Similarly, action potential height and threshold were unaffected by kindling. However, kindling altered other cellular properties of GCs regardless of the site of stimulation (hippocampal commissures or amygdala), the stage of kindling reached (IV or V), or the time elapsed between the last kindling stimulus and preparation of the hippocampal slices (24 h-6 wk). The input resistance of kindled GCs (55 +/- 4 M omega) was significantly higher than that of controls (40 +/- 3 M omega). In contrast to most control GCs, the slope conductance (GS) of kindled neurons, measured with constant-amplitude current injections at various membrane potentials, generally increased at membrane potentials more negative than rest. Furthermore, other voltage-dependent ionic conductances (see below), that were not normally encountered in control GCs, were present in kindled neurons. 4. The intracellularly recorded monosynaptic excitatory postsynaptic potentials (EPSPs) of kindled GCs, evoked through the stimulation of the lateral perforant pathway, differed significantly from the EPSPs of control GCs. The amplitudes of control EPSPs increased upon hyperpolarizations and decreased following depolarizations of the membrane, as expected for conventional EPSPs without contribution from voltage-dependent conductances. In contrast, the EPSPs of kindled GCs invariably increased in amplitude and duration at membrane potentials 5-20 mV depolarized from rest, indicating the presence of a characteristic voltage-dependent component. Frequently, following the synaptically triggered action potentials, kindled GCs displayed depolarizing afterpotentials. 5. Perfusion of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 30 microM) had no effect on the EPSPs of control GCs, but consistently reduced the amplitude and duration of EPSPs in kindled GCs.(ABSTRACT TRUNCATED AT 400 WORDS)     

THE ROLE OF THE PIRIFORM CORTEX IN KINDLING

In epilepsy research, there is growing interest in the role of the piriform cortex (PC) in the development and maintenance of limbic kindling and other types of limbic epileptogenesis leading to complex partial seizures, i.e. the most common type of seizures in human epilepsy. The PC (“primary olfactory cortex”) is the largest area of the mammalian olfactory cortex and receives direct projections from the olfactory bulb via the lateral olfactory tract (LOT). Beside the obvious involvement in olfactory perception and discrimination, the PC, because of its unique intrinsic associative fiber system and its various connections to and from other limbic nuclei, has been implicated in the study of memory processing, spread of excitatory waves, and in the study of brain disorders such as epilepsy with particular emphasis on the kindling model of temporal lobe epilepsy with complex partial seizures. The interest in the kindling model is based primarily on the following observations. (1) the PC contains the most susceptible neural circuits of all forebrain regions for electrical (or chemical) induction of limbic seizures. (2) During electrical stimulation of other limbic brain regions, broad and large afterdischarges can be observed in the ipsilateral PC, indicating that the PC is activated early during the kindling process. (3) The interictal discharge, which many consider to be the hallmark of epilepsy, originates in the PC, independent of which structure serves as the kindled focus. (4) Autoradiographic studies of cerebral metabolism in rat amygdala kindling show that, during focal seizures, the area which exhibits the most consistent increase in glucose utilization is the ipsilateral paleocortex, particularly the PC. (5) During the commonly short initial afterdischarges induced by stimulation of the amygdala at the early stages of kindling, the PC is the first region that exhibits induction of immediate-early genes, such as c-fos. (6) The PC is the most sensitive brain structure to brain damage by continuous or frequent stimulation of the amygdala or hippocampus. (7) Amygdala kindling leads to a circumscribed loss of GABAergic neurons in the ipsilateral PC, which is likely to explain the increase in excitability of PC pyramidal neurons during kindling. (8) Kindling of the amygdala or hippocampus induces astrogliosis in the PC, indicating neuronal death in this brain region. Furthermore, activation of microglia is seen in the PC after amygdala kindling. (9) Complete bilateral lesions of the PC block the generalization of seizures upon kindling from the hippocampus or olfactory bulb. Incomplete or unilateral lesions are less effective in this regard, but large unilateral lesions of the PC and adjacent endopiriform nucleus markedly increase the threshold for induction of focal seizures from stimulation of the basolateral amygdala (BLA) prior to and after kindling, indicating that the PC critically contributes to regulation of excitability in the amygdala. (10) Potentiation of GABAergic neurotransmission in the PC markedly increases the threshold for induction of kindled seizures via stimulation of the BLA, again indicating a critical role of the PC in regulation of seizure susceptibility of the amygdala. Microinjections of NMDA antagonists or sodium channel blockers into the PC block seizure generalization during kindling development. (11) Neurophysiological studies on the amygdala-PC slice preparation from kindled rats showed that kindling of the amygdala induces long-lasting changes in synaptic efficacy in the ipsilateral PC, including spontaneous discharges and enhanced susceptibility to evoked burst responses. The epileptiform potentials in PC slice preparations from kindled rats seem to originate in neurons at the deep boundary of PC. Spontaneous firing and enhanced excitability of PC neurons in response to kindling from other sites is also seen in vivo, substantiating the fact that kindling induces long-lasting changes in the PC comparable to abnormalities seen in primary foci. Taken together, these observations indicate that the PC might be part of an epileptic network which is pivotal in the genesis of kindling, facilitating and intensifying the spread of seizures from a focus in amygdala or hippocampus to cortical and subcortical regions along pathways that also are utilized in normal movements. Although direct evidence implicating the PC in the pathogenesis of human epilepsy is not yet available, the experimental data reviewed in this paper should initiate clinical studies on the potential role of this brain structure as a pacemaker or secondary focus in TLE and other types of epilepsy. Copyright © 1996 Elsevier Science Ltd.     

Unilateral low-frequency stimulation of central piriform cortex inhibits amygdaloid-kindled seizures in Sprague-Dawley rats

The central piriform cortex (cPC) is considered to be critically involved in the generation and propagation of kindled seizures. Our previous study found that low-frequency stimulation (LFS) of the cPC inhibits the development process of amygdala kindling. In this study, we determined whether unilateral LFS of the cPC had an inhibitory effect on amygdaloid-kindled seizures in Sprague-Dawley rats. When fully-kindled seizures were achieved by daily amygdala electrical stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA), LFS (15 min train of 0.1 ms pulses at 1 Hz and 50-150 microA) was applied to the ipsilateral or contralateral cPC 1 s after cessation of kindling stimulation for 10 days. LFS of the ipsilateral cPC significantly decreased the incidence of generalized seizures and seizure stage, and shortened cumulative afterdischarge duration and cumulative generalized seizure duration. LFS of the contralateral cPC also significantly decreased the expression of seizure stage, but had no appreciable effect on the generalized seizure incidence, cumulative afterdischarge duration and cumulative generalized seizure duration. On the other hand, LFS of the ipsilateral cPC significantly increased the afterdischarge threshold and further increased the differences of current intensity between afterdischarge threshold and generalized seizure threshold. Our data suggest that LFS of the cPC may be an effective method of inhibiting kindled seizures by preventing both afterdischarge generation and propagation. It provide further evidence that brain regions like the cPC, other than the seizure focus, can serve as targets for deep brain stimulation treatment of epilepsy.     

Long-term kindling of the basolateral amygdala impairs copulatory behavior in male rats

This experiment examined the consequences of long-term kindling of the basolateral amygdala on male sexual behavior and the frequency of both spontaneous wet dog shakes (WDS) and those induced by the 5-HT_2A receptor agonist DOI. Results demonstrated that following 60 stimulations of the left basolateral amygdala over a 4-week period, male Long–Evans rats exhibited decrements in every aspect of sexual behavior. Specifically, latencies to mount, intromit and ejaculate were all prolonged following long-term kindling, and ejaculation frequencies were significantly reduced. Furthermore, spontaneous peri-copulatory WDS were increased in kindled rats, suggesting a possible role of the 5-HT_2A receptor. However, countering this suggestion, there were no differences between sham and kindled rats on WDS induced by the 5-HT_2A receptor agonist DOI. These results suggest that kindled rats may exhibit elevated levels of endogenous serotonin during exposure to a female rat, which would attenuate copulatory behavior, while concurrently increasing WDS expression.     

Long-lasting potentiation effects induced in rats by kindling with an inverse agonist of the benzodiazepine receptor

The present work analyzed the changes in evoked field potentials of freely moving rats after kindling induced by a convulsant inverse agonist of the GABA(A)-benzodiazepine receptor complex, methyl beta-carboline-3-carboxylate (beta-CCM). Two doses of beta-CCM (2 mg/kg and 4 mg/kg) were used. In kindled and control animals, a stimulating electrode was implanted in the perforant pathway and a recording electrode in the dentate gyrus. Results showed that, after an acutely injected dose of 20 mg/kg pentylenetetrazol (PTZ), all kindled animals showed a decrease in population spike amplitude after 20 min. After 60 min, only fully kindled rats showed a long-lasting potentiation, also visible up to 24 h later, as compared to controls or nonkindled animals. Changes in glutamate and GABA receptor binding measured in previous experiments may explain this potentiation effect observed in fully kindled rats.     

Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats

Enhancement of GABAergic transmission in the substantia nigra has been shown to attenuate motor manifestations of diverse seizure models, including kindling. Similar anticonvulsant effects were reported after bilateral lesions of the substantia nigra, supporting the view that the nigra efferents constitute a critical gating mechanism in the propagation of seizure activity. However, in the lesion studies reported so far the nigra was not destroyed selectively so that regions destroyed in addition to the nigra could have been involved in the anticonvulsant effects observed. We destroyed the nigra selectively in fully kindled rats by bilateral microinjection of small amounts of the neurotoxin ibotenic acid. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the piriform cortex. In both groups, there was no indication of a reduction in seizure susceptibility, seizure severity or seizure duration after bilateral destruction of the nigra. The data thus indicate that, at least in kindled rats, the substantia nigra might be less important for seizure generation and/or propagation than previously thought.     

Decrease in GABA immunoreactivity and alteration of GABA metabolism after kindling in the rat hippocampus

Summary The kindling model of epilepsy, induced by tetanic stimulation of Schaffer collateral/commissural fibers, was studied in the rat hippocampus. Gamma-aminobutyric acid immunoreactivity was used to quantify the number of GABA-immunoreactive somata per mm² in CA1 region, 28 days after the last generalized seizure. Comparison of the numbers obtained from kindled animals with those from controls, showed a significant decrease (18%) on the ipsilateral stimulated side but none on the contralateral side. In control rats injection of the GABA-transaminase inhibitor, amino oxyacetic acid (AOAA), led to a 46% increase in the number of cell somata immunoreactive for GABA. This probably results from an accumulation of GABA, reflecting GABA synthesis by glutamate decarboxylase (GAD) activity, in somata of interneurons that had initially a GABA content below the immunocytochemical detection threshold. In kindled rats, 31 days after the last seizure, the number of GABA-immunoreactive cells that could be observed after AOAA-treatment was significantly lower (35% ipsilateral and 25% contralateral) when compared to AOAA-treated controls. This suggests that in kindled animals a GAD dependent increase in GABA content did not take place in a subpopulation of interneurons. The observations for kindled rats are interpreted as a long-term decrease in GABA content and as an alteration in GABA turnover in a subpopulation of interneuron somata, the latter possibly due to a decrease in GAD activity. The long-term enhanced seizure sensitivity, characteristic for kindled animals, may be due to a decreased GABAergic inhibitory control of the neuronal circuitry in the CA1 region of the hippocampus.     

Aberrant neuronal physiology in the basal nucleus of the amygdala in a model of chronic limbic epilepsy

Limbic epilepsy is a chronic condition associated with a broad zone of seizure onset and pathology. Studies have focused mainly on the hippocampus, but there are indications that changes occur in other regions of the limbic system. This study used in vitro intracellular recording and histology to examine alterations to the physiology and anatomy of the basal nucleus of the amygdala in a rat model of chronic limbic epilepsy characterized by spontaneously recurring seizures. Epileptic pyramidal neuron responses evoked by stria terminalis stimulation revealed hyperexcitability characterized by multiple action potential bursts and no evident inhibitory potentials. In contrast, no hyperexcitability was observed in amygdalar neurons from kindled (included as a control for seizure activity) or control rats. Blockade of ionotropic glutamate receptors unmasked inhibitory postsynaptic potentials in epileptic pyramidal neurons. Control, kindled and epileptic inhibitory potentials were predominantly biphasic, with fast and slow components, but a few cells exhibited only the fast component (2/12 in controls, 0/3 in kindled, 3/10 in epileptic). Epileptic fast inhibitory potentials had a more rapid onset and shorter duration than control and kindled. Approximately 40% of control neurons exhibited spontaneous inhibitory potentials; no spontaneous inhibitory potentials were observed in neurons from kindled or epileptic rats. A preliminary histological examination revealed no gross alterations in the basal amygdala from epileptic animals.These results extend previous findings from this laboratory that hyperexcitability is found in multiple epileptic limbic regions and may be secondary to multiple alterations in excitatory and inhibitory efficacy. Because there were no differences between control and kindled animals, the changes observed in the epileptic animals are unlikely to be secondary to recurrent seizures.     

Abnormal neuronal excitability in hippocampal slices from kindled rats

To determine if electrophysiological properties of hippocampal pathways are altered in kindled rats, extracellular recordings were made from hippocampal slices of rats kindled in the lateral entorhinal cortex and compared with those from implanted but unstimulated controls. Studies were made either 24 h or 28 days after the last kindled seizure and done in normal (3.5 mM) or elevated (7 mM) K+. The preparation of slices, data accumulation, and data analyses were done blind. One day or 28 days after the last kindled seizure, the proportion of slices with spontaneous epileptiform bursts recorded from the CA2/3 region in elevated K+ was significantly (P less than 0.001) increased in the kindled animals. The frequency of spontaneous burst firing was also increased and reached significance (P less than 0.02) at 28 days following the last kindling stimulus. One day after the last kindling stimulus, paired-pulse (GABAergic) inhibition in the CA1 region was decreased (P less than 0.001). Several measures suggested an increased synaptic inhibition in the dentate gyrus of slices from the kindled groups 1 day after kindling. Paired-pulse inhibition was increased (P less than 0.01), the current required to evoke a near-threshold population spike was increased (P less than 0.05), and the population spike amplitude was reduced for a given field excitatory postsynaptic potential (EPSP) (P less than 0.01). Twenty-eight days after the last kindling stimulus, however, paired-pulse inhibition in the dentate was slightly less in slices from kindled rats (P less than 0.005). In other respects the CA1 and dentate regions did not differ between kindled and control groups within 24 h of the last stage V seizure. Thus the maximum amplitudes of presynaptic fiber volley, population spike, and field-excitatory postsynaptic potential (EPSP) slope, and the number of population spikes evoked by a near-maximally effective afferent stimulus, were unchanged. In the CA1 region the input-output curve of field EPSP versus population spike, and the current intensity required to evoke a near-threshold population spike were also unchanged. In addition, no spontaneous bursts were recorded from CA1 in 3.5 mM K+. We conclude that either synapses or neurons intrinsic to the hippocampus are altered by kindling stimuli applied outside this brain area. The transient increase in inhibition in the dentate gyrus suggests that it may reflect a compensatory reaction to kindled seizures. In contrast, the long-lasting (at least 28 days) increase in burst firing in CA2/3 may represent a mechanism for the initiation or propagation of kindled seizures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of kindling in wheat germ agglutinin-horseradish peroxidase [corrected] labeling in neurons of the interamygdaloid pathway in rats

This paper describes in kindled rats an increment in wheat germ agglutinin-horseradish peroxidase labeling in anterior commissure, bed nuclei of stria terminalis and amygdala. Three groups of animals were analyzed: control, sham-operated and kindled animals with ten convulsive generalized seizures. Results show that kindled animals have an increase in fiber labeling in anterior commissure and in the bed nuclei of stria terminalis, as well as a greater number of labeled neurons in amygdala. This label enhancement is related to the hyperexcitability of neurons produced by epilepsy, and could be associated to the propagation and formation of secondary foci and related plastic changes occurring during kindling.     

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: a microdialysis study

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.     

Mechanisms underlying rule learning-induced enhancement of excitatory and inhibitory synaptic transmission

Training rats to perform rapidly and efficiently in an olfactory discrimination task results in robust enhancement of excitatory and inhibitory synaptic connectivity in the rat piriform cortex, which is maintained for days after training. To explore the mechanisms by which such synaptic enhancement occurs, we recorded spontaneous miniature excitatory and inhibitory synaptic events in identified piriform cortex neurons from odor-trained, pseudo-trained, and naive rats. We show that olfactory discrimination learning induces profound enhancement in the averaged amplitude of AMPA receptor-mediated miniature synaptic events in piriform cortex pyramidal neurons. Such physiological modifications are apparent at least 4 days after learning completion and outlast learning-induced modifications in the number of spines on these neurons. Also, the averaged amplitude of GABA(A) receptor-mediated miniature inhibitory synaptic events was significantly enhanced following odor discrimination training. For both excitatory and inhibitory transmission, an increase in miniature postsynaptic current amplitude was evident in most of the recorded neurons; however, some neurons showed an exceptionally great increase in the amplitude of miniature events. For both excitatory and inhibitory transmission, the frequency of spontaneous synaptic events was not modified after learning. These results suggest that olfactory discrimination learning-induced enhancement of synaptic transmission in cortical neurons is mediated by postsynaptic modulation of AMPA receptor-dependent currents and balanced by long-lasting modulation of postsynaptic GABA(A) receptor-mediated currents.