Kindling antagonism: a role for hindbrain norepinephrine in the development of site suppression following concurrent, alternate stimulation

The concurrent, alternate electrical stimulation of the septal nucleus and the entorhinal cortex results in the development of fully generalized seizures at one site (dominant site) and the lack of development of kindled seizures at the other (suppressed or antagonized site). We have labeled this phenomenon kindling antagonism. Previous work from our laboratory has demonstrated that the whole brain depletion of norepinephrine (NE) eliminates the development of kindling antagonism. In the present study animals were treated with the neurotoxin 6-hydroxydopamine (6-OHDA) as neonates. The neonatal administration of 6-OHDA produced robust increases in brainstem and cerebellar NE levels and depletions of forebrain NE levels when assayed at maturity. Striatal dopamine levels were spared by this treatment. Neonatal 6-OHDA did not alter the development of the kindling antagonism phenomenon which is typically observed following concurrent, alternate stimulation of the septal nucleus and entorhinal cortex. Neonatal 6-OHDA treatment significantly facilitated the rate of kindled seizure development at dominant sites but failed to alter thresholds for the elicitation of afterdischarges (AD) or patterns of development of AD durations. Other characteristics of kindling antagonism were similarly unaffected by 6-OHDA treatment. These data suggest that brainstem and/or cerebellar NE are sufficient to mediate the development of kindling antagonism in the relative absence of forebrain NE.     

Potentiation of amygdala kindling in adult or infants rats by injections of 6-hydroxydopamine.

Rats with bilateral amygdala electrodes were pretreated with 6-hydroxydopamine and/or desmethylimipramine (DMI) to deplete norepinephrine (NE) and/or dopamine (DA). Subsequently they were subjected to kindling procedures in which daily low-intensity amygdala stimulation resulted eventually in the development of clonic motor seizures. The NE depletion, but not the DA depletion, resulted in a marked reduction in the number of stimulations required to kindle convulsions (i.e., faster kindling), but increased the latency to onset of convulsions; the convulsion or after-discharge durations were unaffected. Interestingly, the control animals that received DMI alone manifested no obvious permanent neurochemical effect yet exhibited retarded after-discharge evolution. The above data were replicated in two experiments using adult- and infant-injected rats. These data confirm previous reports that the central catecholamines are capable of inhibiting a variety of forms of seizure.     

Potentiation of amygdala kindling in adult or infant rats by injections of 6-hydroxydopamine

Rats with bilateral amygdala electrodes were pretreated with 6-hydroxydopamine and/or desmethylimipramine (DMI) to deplete norepinephrine (NE) and/or dopamine (DA). Subsequently they were subjected to kindling procedures in which daily low-intensity amygdala stimulation resulted eventually in the development of clonic motor seizures. The NE depletion, but not the DA depletion, resulted in a marked reduction in the number of stimulations required to kindle convulsions (i.e., faster kindling), but increased the latency to onset of convulsions; the convulsion or after-discharge durations were unaffected. Interestingly, the control animals that received DMI alone manifested no obvious permanent neurochemical effect yet exhibited retarded after-discharge evolution. The above data were replicated in two experiments using adult- and infant-injected rats. These data confirm previous reports that the central catecholamines are capable of inhibiting a variety of forms of seizure.     

Unilateral peri-substantia nigra catecholaminergic lesion and amygdala kindling

Recent evidence suggests that the substantia nigra (SN) may be involved in the modulation of kindled seizures in adult rats. In this report we investigated the role of the dopaminergic nigrostriatal pathway in mediating the SN effect on seizures by lesioning this pathway with unilateral infusions of 6-hydroxydopamine (6-OHDA) in the vicinity of the right SN with or without desmethylimipramine pretreatment. Our data suggest that the facilitation of amygdala kindling observed following 6-OHDA lesion in the vicinity of the ipsilateral SN is due to norepinephrine depletion of the ipsilateral forebrain. Selective destruction of the nigrostriatal dopaminergic neurons neither facilitates nor inhibits the development of amygdala-kindled convulsions in adult rats.     

Alteration of seizures resulting from pinealectomy in the rat after neonatal treatment with 6-hydroxydopamine

Convulsions from combined pinealectomy and parathyroidectomy were examined in rats treated as neonates with 6-hydroxydopamine (6-OHDA). Injection of 6-OHDA caused a significant reduction in the average latency to onset of clonic-tonic convulsions. Moreover, of the 6-OHDA-treated rats which convulsed, nearly half of them died during a seizure. In sham-pinealectomized rats, neonatal treatment with 6-OHDA reduced norepinephrine (NE) and increased dopamine levels in the forebrain while brainstem levels of NE were increased. Pretreatment with desmethylimipramine reversed the effects of 6-OHDA on convulsions and catecholamine levels. The results indicate that catecholamine lesions with 6-OHDA exacerbate convulsions from pinealectomy.     

Facilitation of amygdala kindling after norepinephrine depletion with 6-hydroxydopamine in rats

It is unclear whether rats pretreated with desmethylimipramine (DMI) and 6-hydroxydopamine (6-OH) kindled at a rate similar to controls because the DMI protected the brain norepinephrine (NE) from 6-OH, or whether it was because DMI exhibited a long-term antiepileptic effect, independent of NE protection, which counteracted the facilitative action on kindling of 6-OH alone. Rats with bipolar amygdala electrodes were pretreated bilaterally with intraventricular 6-OH and/or subcutaneous DMI to deplete brain NE and/or dopamine (DA). All rats were subsequently subjected to daily low-intensity electrical stimulation of the amygdala which resulted in the development of clonic motor seizures (kindling). With injections of 6-OH alone or DMI 1 week before or 2 weeks after 6-OH, extensive depletion of NE occurred, paralleled by very rapid kindling. Importantly, the form and duration of these convulsions was the same as for all other groups. DMI administered 30 min before 6-OH (30-min-6-OH group) substantially protected the NE but not the DA fibers and resulted in kindling similar to the saline- or DMI-injected control rats. Unlike our previous observation, DMI alone did not retard the spread of afterdischarge. Collectively these data confirm the importance of NE in amygdala kindling, and indicate that the normalcy in kindling of the 30-min-6-OH group was related to the protection of their NE by DMI and not to a long-term antiepileptic action of DMI.     

Microdialysates of amines and metabolites from core nucleus accumbens of freely moving rats are altered by dizocilpine

In vivo microdialysis combined with high performance liquid chromatography (HPLC) with electrochemical detection, was used to study the effect of MK-801 (0.1 mg/kg i.p.) on extracellular concentrations of dopamine (DA) 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), norepinephrine (NE) and DOPAC/DA ratio in intact, 6-hydroxydopamine (6-OHDA)-lesioned, DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine hydrochloride)-lesioned and reserpine-treated rats. The results revealed high basal DA (0.735+/-0.05 fmol/microl), DOPAC (195.93+/-20.18 fmol/microl) and NE (0.585+/-0.01 fmol/microl), low 5-HT (0.334+/-0.032 fmol/microl) and high DOPAC/DA ratio (265.11+/-20.73) in intact cACC. 6-OHDA alone (8 microg/2 microl) depleted DA (-66%), DOPAC (-65%), and NE (-62%). On the other hand, in desipramine (DMI)-pretreated rats, 6-OHDA induced a large depletion of DA (-94%), DOPAC (-97%) and reduced DOPAC/DA ratio (-73%), but increased NE to 142% of intact and 369% of 6-OHDA-lesioned rats. DSP4 (50 mg/kg) decreased NE (-97%), DOPAC (-75%) and DOPAC/DA ratio (-69%). Reserpine (5 mg/kg s.c.) significantly decreased DOPAC (-84%), DOPAC/DA ratio (-81%), 5-HT (-69%) and NE (-86%), but nonsignificantly increased DA. In the intact rats, MK-801 did not change DA, but increased DOPAC and DOPAC/DA ratio. In 6-OHDA-lesioned rats, MK-801 increased DA, whereas in 6-OHDA+DMI rats MK-801 additionally increased DOPAC and DOPAC/DA ratio. DSP4 and reserpine reduced the ability of MK-801 to increase DOPAC and DOPAC/DA ratio. MK-801 did not change NE concentration in dialysates collected from intact rats, but increased that from 6-OHDA+DMI-lesioned rats. In DSP4-lesioned and reserpine-treated rats, MK-801 increased NE but to a level lower than that observed in the intact rats. These results suggest that systemic administration of a low dose of MK-801, which induces profound locomotor stimulation without stereotypy, increases DOPAC and DOPAC/DA ratio in the cACC of intact rats, whereas it additionally increases the depleted DA and NE concentrations especially in 6-OHDA-lesioned rats pretreated with DMI.     

Brain norepinephrine reductions in soman-intoxicated rats: association with convulsions and AChE inhibition, time course, and relation to other monoamines.

The organophosphate chemical nerve agent, soman, causes convulsions, neuropathology, and, ultimately, death. A major problem in treating soman intoxication is that peripherally acting pharmacological agents which prevent death do not prevent seizures. Although a primary cause of these symptoms is the excess of acetylcholine which follows acetylcholinesterase (AChE) inhibition, centrally acting muscarinic blockers, such as atropine, alleviate, but do not block, the convulsive actions of soman. Moreover, there is a relatively weak relationship between CNS reductions of AChE and the incidence of convulsions. There is evidence suggesting that soman intoxication stimulates the release of norepinephrine (NE) in the brain. Recent evidence has implicated NE in the induction and/or maintenance of seizures. Thus, in the present study the relations among soman-induced convulsions, AChE inhibition, and brain NE and other monoamine changes were examined. The time course of brain NE recovery was also determined. Rats were injected (im) with a single dose (78 micrograms/kg) of soman. At this dose 68% of the injected rats developed convulsions. Both convulsive and nonconvulsive rats were sacrificed between 1 and 96 h following soman injection and NE levels in the rostral forebrain and olfactory bulb were determined by HPLC with electrochemical detection. In all convulsive rats NE levels declined substantially. Forebrain NE levels were decreased by 50% at 1 h and 70% at 2 h following soman injection. Recovery of NE began at 8 h and was complete by 96 h following soman administration. Although nonconvulsive rats showed other signs of intoxication, NE levels in these rats were unchanged. Dopamine (DA) and serotonin (5-HT) levels were not significantly affected in either convulsive or nonconvulsive rats. However, 5-hydroxyindoleacetic acid, the major metabolite of 5-HT, and homovanillic acid and 3,4-dihydroxyphenylacetic acid, the two major metabolites of DA, were increased significantly in the forebrain of convulsive, but not nonconvulsive rats, indicating an increase in 5-HT and DA turnover. However, in contrast to the abrupt decline in NE, these increases in DA and 5-HT metabolites were slow and progressive. Taken together, the present results and other recent findings suggest that rapid, sustained NE release could play a role in the induction and/or maintenance of soman-induced convulsions, whereas increased release of 5-HT and DA may be a consequence of seizures. Further investigation of the role of NE in soman-induced convulsions may lead to improved treatment of soman intoxication and a better understanding of the role of NE in other forms of seizures, including human epilepsy.     

Secondary generalization of hippocampal kindled seizures in rats: examining the role of the piriform cortex

A primary feature of epilepsy is the potential for focal seizures to recruit distant structures and generalize into convulsions. Key to understanding generalization is to identify critical structures facilitating the transition from focal to generalized seizures. In kindling, development of a primary site leads progressively to secondarily generalized convulsions. In addition, subsequent kindling of a secondary site results in rapid kindling from that site, presumably because of its facilitated access to the primary kindled network. Here, we investigated the role of the piriform cortex in convulsive generalization from a secondary site kindled in the hippocampus after primary site amygdala kindling. In a necessarily complicated design, rats initially experienced forebrain commissurotomy to lateralize the experiment to one hemisphere. Then the amygdala was kindled and, 3 weeks later, it was electrically-triggered into status epilepticus, which destroyed the ipsilateral piriform cortex. This experience occurred several days before secondary site kindling of the dorsal hippocampus. In rats with complete piriform cortex loss, there was no disruption in kindling or convulsive seizure expression from the hippocampus. However, when damage also involved parts of the perirhinal, insular and entorhinal cortices, convulsive expression was blocked. Although other evidence suggests that piriform lesions affect generalization of primary site kindling, the present study shows that they do not alter secondary site kindling in the dorsal hippocampus. The additional involvement of parahippocampal cortical areas in convulsive expression suggests an important functional association between these cortical regions and the hippocampus in seizure propagation and clinical expression.     

Estrogen Alters the Acquisition of Seizures Kindled by Repeated Amygdala Stimulation or Pentylenetetrazol Administration in Ovariectomized Female Rats

Ovariectomized female rats received estradiol (E2) replacement by means of subcutaneous silastic capsules (10% E2 in cholesterol). E2-replaced rats required fewer daily amygdala stimulations to develop fully kindled seizures as compared with ovariectomized rats implanted with cholesterol-only capsules. Other rats were injected with pentylenetetrazol (PTZ), 40 mg/kg i.p., every 48 h. E2-replaced rats showed a progressive increase in convulsive severity from minimal responses after the first injection to tonic convulsions after eight injections. Most rats without E2 replacement failed to progress past clonic convulsive responses after 22 injections. The results support a marked influence of E2 on seizure processes as demonstrated in two different models of seizure acquisition.     

The effect of regional differences in noradrenergic neuron growth patterns on juvenile kindling

Kindling can be altered by a variety of lesions designed to deplete norepinephrine (NE). However, the effect of the regional alteration in NE concentration on seizure susceptibility has not been studied. Two different concentrations of 6-hydroxydopamine (6-OHDA) were administered to one-day-old rat pups. At age 18 days, rat with significant rostral brain NE loss, due to high dose 6-OHDA, had a faster rate of electrical kindling in the entorhinal cortex than controls. In contrast rats receiving low dose 6-OHDA which resulted in comparable forebrain NE depletion but with a dramatic hindbrain noradrenergic overgrowth showed no enhancement of kindling. These results suggest that in the immature rat the proconvulsant effect of forebrain NE depletion can be overridden by an augmentation of hindbrain NE growth patterns.     

Suppression of clinical weakness in experimental autoimmune encephalomyelitis associated with weight changes, and post-decapitation convulsions after intracisternal-ventricular administration of 6-hydroxydopamine

Selective depletion of central nervous system norepinephrine (NE) by the neurotoxin 6-hydroxydopamine (6-OHDA) in rats subsequently inoculated with myelin basic protein (MBP) and complete Freund's adjuvant (CFA) produced experimental autoimmune encephalomyelitis (EAE) without the usual expected degree of weakness. The preservation of strength occurred in spite of continued weight loss. Post-decapitation myoclonic convulsive kick latency and kick number, which are known to depend on spinal cord NE, agreed well with the degree of weakness through the clinical disease course. The only difference between EAE groups was that the stronger 6-OHDA pretreated EAE animals did not have an elevated pons-medulla NE compared to saline intracisternal-ventricular (i.c.v.) pretreated controls. We conclude that 6-OHDA can influence the clinical course of weakness by interfering with central noradrenergic activity independent of other features associated with disease in EAE. This effect of 6-OHDA may be exerted through alteration of the blood-spinal cord barrier function and/or central nervous system blood flow.     

Aggravation of Penicillin-Induced Epilepsy in Rats with Locus Ceruleus Lesions

The rate and pattern of development of seizures induced by penicillin injected intraperitoneally were determined in rats that had been depleted of brain norepinephrine (NE) by bilateral injections of the neurotoxin 6-OH dopamine into the locus ceruleus. Behavioral observations and scalp electrographic recordings were made after injection and the efficacy of NE depletion was determined by high performance liquid chromatography measurement of cortical levels of NE and its metabolites. We found that in comparison to sham-operated control rats, NE-depleted rats had a significantly shorter latency to first observable myoclonic jerk, the first epileptic discharge, the first convulsion with sustained epileptic discharges, and a longer duration of convulsions. We observed a similar electrographic pattern of multifocal spikes with bilateral synchrony in both groups. However, more of the control rats (six of 12) had convulsions as compared to the lesioned rats (four of 12). These findings are consistent with previous evidence that depletion of neocortical NE facilitates the development of epileptiform activity in the CNS; however, a convulsive state was not induced by NE depletion.     

Effects of midbrain raphe lesions or systemic p-chlorophenylalanine on the development of kindled seizures in rats

RACINE, R. AND D. V. COSCINA. Effects of midbrain raphe lesions or systemic p-chlorophenylalanine on the developmentof kindled seizures in rats. BRAIN RES. BULL. 4(1) 1–7, 1979.—Previous research has suggested that brain serotonin (5-hydroxytryptamine or 5-HT) neurons inhibit epileptiform seizure activity. To test further this possibility, experiments were performed to determine if brain 5-HT depletion would enhance the occurrence and/or magnitude of seizures “kindled” from the amygdala or neocortex of rats. Two modes of 5-HT depletion were used: (1) radiofrequency heat lesions of the midbrain dorsal and median raphe nuclei, and (2) systemic injection of the 5-HT synthesis inhibitor, p-chlorophenylalanine (pCPA). Both modes of 5-HT depletion reliably enhanced the strength of motor convulsions kindled from the cortex. Systemic pCPA also reduced the duration of after-discharges (ADs) in cortically-stimulated rats. However, pCPA reduced rather than enhanced convulsions kindled from the amygdala. In contrast to this, raphe lesions appeared to sensitize rats to the effects of amygdaloid kindling, i.e., lesions lowered AD thresholds, AD durations and number of ADs to elicit motor convulsions. Viewed together, these data support the hypothesis that 5-HT neurons can serve to inhibit seizures. However, the lack of robustness across parameters of epileptogenesis as well as discrepant findings related to 5-HT depletion mode additionally suggest that kindled seizures affect other neuronal populations in addition to those under serotonergic influence.     

Evidence for a norepinephrine-dependent brain-stem substrate in the development of kindling antagonism

Kindling antagonism' is a modification of the standard kindling paradigm in which two limbic system structures are stimulated on alternate trials. The consistent outcome of this procedure is that one site ('dominant site') develops typical generalized seizures, while kindled seizure development from the other site ('suppressed site') is arrested at an intermediate stage. Our recent research suggests that kindling antagonism is dependent on hindbrain norepinephrine (NE) systems. The present study explores this relationship further. Neonatal rats were treated with intracerebral injections of 6-hydroxydopamine (6-OHDA). At adulthood, these animals received either (1) a low dose of 6-OHDA (75 micrograms), (2) a high dose of 6-OHDA (200 micrograms), or (3) vehicle. Regional NE concentrations were determined by high pressure liquid chromatography. Neonatal 6-OHDA followed by vehicle resulted in decreases in forebrain and increases in hindbrain NE concentrations. Low dose 6-OHDA at adulthood depleted cerebellar, but not pontine-medullary NE. High dose 6-OHDA resulted in depletions of both cerebellar and pontine-medullary NE. Only high dose 6-OHDA significantly interfered with the development of antagonism. Neonatal 6-OHDA facilitated the rate of dominant site kindling independently of subsequent adult treatment regimens. Results suggest that the influence of NE on kindling antagonism is mediated through a brain-stem substrate and that the influence of NE on kindling rate is mediated through a forebrain substrate.     

Amygdala kindling in juvenile rats following neonatal administration of 6-hydroxydopamine

The role of catecholamines in mediating the acquisition of amygdala-kindled seizures was investigated in juvenile rats administered intracisternal injections of 6-hydroxydopamine (6-OHDA) on postnatal days 1 and 2. Amygdala kindling was initiated on day 28, using stimulations delivered each hour through two consecutive stage V seizures. The 6-OHDA treatment resulted in a 53% increase in the overall rate of kindling in juvenile rats. This acceleration was confined primarily to the early phases of kindling in that the 6-OHDA-treated rats skipped the early kindling stages, and the later stages of kindling were unaffected. These findings support evidence from adult rats that catecholamines play a role in initially limiting the spread of seizure activity during kindled seizure acquisition; however, when the seizures have begun to generalize, the ability of catecholaminergic systems to inhibit seizure spread diminishes.     

The effect of procaine hydrochloride and diazepam, separately or in combination, on cortico-generalized kindled seizures.

The effects of cortically kindled seizure responses of procaine hydrochloride, diazepam and combinations of these two drugs were tested in this study. The cortex was stimulated until seizure responses developed past the focal stage (accompanied primarily by brief tonic convulsions) to the cortico-generalized stage (accompanied, typically, by an early brief tonus followed by a longer clonic seizure that is characteristic of subcortically triggered seizures). Diazepam was found to block the generalized component of the cortico-generalized electrographic and motor seizure leaving the tonus only slightly suppressed. Procaine blocked the tonus leaving the clonic seizure and discharge that is characteristic of the generalized response relatively intact. Combinations of half doses of the two drugs completely blocked all electrographic and motor seizure responses in about half the animals. The remaining animals had a very brief discharge with no convulsive responses.     

The anticonvulsant effects of progesterone and 5alpha-dihydroprogesterone on amygdala-kindled seizures in rats

PURPOSE: Progesterone has been shown to be anticonvulsant in several animal seizure models. The purpose of the present study was to investigate the anticonvulsant actions of progesterone and its primary metabolite 5alpha-dihydroprogesterone in the amygdala kindling model. METHODS: Female Wistar rats were implanted in the right basolateral amygdala with a long-term, bipolar electrode. The subjects were kindled to 30 stage 5 seizures and stability tested. Multiple doses of progesterone and 5alpha-dihydroprogesterone were then tested for anticonvulsant activity against focal electrographic and generalized convulsive kindled seizures. The time course of progesterone's anticonvulsant action also was examined. RESULTS: Progesterone had a median effective dose (ED50) of 103 mg/kg against generalized convulsions at 15 min after injection. Subjects were not sedated at the time of seizure testing, although sedation developed later (40-60 min after injection). In time-course experiments, it was found that 120 mg/kg of progesterone caused complete suppression of the generalized convulsion from 20 to 160 min after injection. Suppression of the focal discharge also was seen in some animals between 20 and 160 min. 5alpha-dihydroprogesterone had an ED50 of 2.9 mg/kg against generalized kindled convulsions and an ED50 of 4.3 mg/kg against focal afterdischarge 15 min after injection. 5alpha-dihydroprogesterone did not produce sedation 15 min after injection, or at any later time interval. CONCLUSIONS: Progesterone is anticonvulsant only at high doses when tested against amygdala kindled seizures. 5alpha-dihydroprogesterone is considerably more potent than progesterone. At low, nonsedative doses, it was effective against both the kindled amygdala focal afterdischarge and the generalized convulsion.     

Role of the Claustrum in Convulsive Evolution of Visual Afferent and Partial Nonconvulsive Seizure in Primates

: Purpose: We tested cross-species validity of the role of the claustrum in the convulsive evolution of the visual afferent and amygdaloid seizure and the specificity of the claustral lesioning effect. Methods: In 7 Senegalese baboons, we examined the effect of unilateral claustral lesioning on generalized convulsive seizures either kindled from the amygdaloid nucleus (AM) and cingulate cortex (CG) or induced by intermittent photic stimulation (IPS) after systemic administration of D,L-allylglycine (AG). Results: A lesioned area common to all animals was the anterior half of the left claustrum. Postoperative restimulation of the kindled left AM or CG evoked only nonconvulsive seizures. When few convulsive seizures emerged in 1 CG-kindled animal, they were mirror image of the kindled seizure and arose from the nonlesioned right hemisphere. Restimulation of the kindled right AM or CG reactivated kindled seizures. An IPS-induced generalized convulsive seizure was transformed into a secondarily generalized seizure arising from the nonlesioned right hemisphere. Conclusions: The primate claustrum regulates the convulsive evolution of partial seizures originating from nonmotor structures such as the AM and CG and also regulates the convulsive development that follows IPS. Our findings suggest that predisposed susceptibility expressed at the claustrum may be involved in the clinical variation with respect to convulsive evolution of nonmotor partial seizures and convulsive susceptibility to IPS in human primates.     

Metrazol thresholds in inbred and non-inbred audiosensitive mice

Mice of the O'Grady strain which were inbred for susceptibility to audiogenic seizures were found to be more sensitive to pentamethylenetetrazol (Metrazol) convulsions than mice of the parent Swiss-Webster strain from which they were originally derived. A dose of 35 mg/kg Metrazol was required to produce a 50% convulsive response (CD50) in O'Grady mice and 50 mg/kg in the control parent strain. No differences in convulsive thresholds to Metrazol were obtained, however, between untreated Swiss-Webster mice and mice of the same strain made audiosuseptible by rubidium chloride intake or magnesium deficiency.