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Summary: Purpose: Although sound-induced (audiogenic) seizures in the genetically epilepsy-prone rat (GEPR) initially occur independent of the forebrain, repeated audiogenic seizures recruit forebrain seizure circuits in a process referred to as audiogenic kindling. In GEPR-3s, audiogenic kindling results in facial and forelimb (F&F) clonic seizures that are typical of forebrain seizures. However, in GEPR-9s, audiogenic kindling produces posttonic all-limb clonus not usually observed during forebrain seizures. We hypothesized that the more severe brainstem seizures of the GEPR-9 prevent the expression of F&F clonic seizures during audiogenic kindling. Therefore attenuation of audiogenic seizures during audiogenic kindling in GEPR-9s should allow F&F clonic seizures to be expressed. Likewise, intensifying audiogenic seizure severity in GEPR-3s should inhibit audiogenically kindled F&F clonic seizures. We have tested this hypothesis in the present study.
Methods: Lesions of the superior colliculus or treatment with low-dose phenytoin were used to suppress audiogenic seizure severity in GEPR-9s. Depletion of brain serotonin was used to increase the seizure severity in GEPR-3s. All GEPRs were then subjected to audiogenic kindling. Behavioral and electrographic seizures were assessed.
Results: Suppression of audiogenic seizure severity during audiogenic kindling in GEPR-9s increased the incidence forebrain seizure behavior. Kindled GEPR-9s that continued to display full tonic seizures did not exhibit forebrain convulsions, but did show posttonic clonus and forebrain seizure activity in the EEG. GEPR-3s chronically depleted of brain serotonin, along with displaying tonic brainstem seizures, tended to display less severe forebrain seizures during audiogenic kindling.
Conclusions: These findings support the concept that severe brainstem seizures prevent the behavioral expression of forebrain seizures in audiogenically kindled GEPR-9s. It appears that the severe brainstem seizure of the GEPR-9 does not allow the forebrain seizure to manifest its typical behavioral concomitants despite electrographic evidence that spike–wave discharge is occurring in the forebrain.
The sound-induced seizures of the genetically epilepsy-prone rat (GEPR) are initiated and driven by a brainstem network independent of the forebrain (1). Two strains of GEPRs differ in seizure severity in response to intense auditory stimulation. GEPR-3s exhibit seizures characterized by an explosive running episode followed by loss of posture, paraxial extension, and generalized clonus of all limbs. GEPR-9s exhibit a more severe seizure characterized by an explosive run, followed by paraxial flexion and complete tonic extension of all limbs (see ref. 2 for a more complete description of the convulsive behavior). Earlier studies have shown that repetitive (daily) audiogenic-induced seizures result in the addition of seizure behavior that phenotypically mimics forebrain-seizure behavior (3–6). This activation of forebrain seizure circuits and the manifestation of forebrain seizure behavior after repeated audiogenic-induced seizures has been referred to as “audiogenic kindling” (3,6,7).
Interestingly, GEPR-3s and GEPR-9s do not respond identically to audiogenic kindling. Repetition of audiogenic-induced seizures in GEPR-3s leads to a progressive appearance of facial and forelimb (F&F) clonus typical of forebrain evoked seizures (4). Unlike the all-limb clonus characteristic of a brainstem seizure in the GEPR-3, the forebrain clonus occurs in the presence of an intact righting reflex. In contrast, audiogenic kindling does not produce F&F clonus in the GEPR-9 (4). GEPR-9s display clonus of all four limbs, ultimately being expressed beyond the duration of tonic extension, and in the presence of continued loss of righting posture. Although the sustained clonus of all the limbs (with loss of righting reflex) is not characteristic of forebrain seizure behavior, it is accompanied by an increasingly evident EEG representation of cortical spike–wave discharge (4).
The reason for the different behavioral manifestations after seizure repetition in GEPR-3s and GEPR-9s is not known. However, Garcia-Cairasco et al. (8) found that repeated audiogenic-induced seizures caused a decrease in brainstem seizure severity in the GEPR-3 that paralleled the development of forebrain seizure behavior (i.e., F&F clonus with rearing). Noting repetition-induced suppression of brainstem seizure behavior in GEPR-3s, these investigators hypothesized that only after suppression of the brainstem seizure will the forebrain seizure become behaviorally apparent (8). The lack of expression of forebrain behavior in the GEPR-9, subsequent to repeated audiogenic-induced seizures, may be due to insufficient suppression of the more intense brainstem seizure in GEPR-9s.
Given that brainstem seizure networks have dominant control of motor output, when both seizure networks are activated simultaneously, forebrain seizure behavior cannot manifest unless the brainstem seizure is suppressed (9–11). Thus we have hypothesized that GEPR-9s, subjected to repeated audiogenic seizures, fail to display the classic forebrain-driven convulsive behavior because of either the prolonged brainstem discharge associated with tonic hindlimb extension or the more intense postictal depression that follows tonic hindlimb extension. According to this hypothesis, attenuation of the brainstem seizure in a GEPR-9 during audiogenic kindling may result in the emergence of more typical forebrain seizure behavior, similar to that observed in GEPR-3s.
In the present study, we tested this hypothesis by carrying out the audiogenic kindling paradigm in GEPR-9s and GEPR-3s after lesions of the superior colliculus, which are known to attenuate the brainstem seizure severity markedly (12). We also examined this paradigm in GEPR-9s pretreated with phenytoin (PHT) to attenuate brainstem seizures, a pharmacologic technique we previously used to allow expression of limbic motor seizures in GEPR-9s receiving corneal electroshock (9). Last, we produced widespread serotonin depletion in GEPR-3s, which is known to result in more severe brainstem seizures (13), to examine further the relation between brainstem and forebrain seizure severity on audiogenic-induced seizure repetition.
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Previous research has shown that repeated sound-induced brainstem seizures can trigger (or kindle) limbic motor seizures (3–5). The propagation of seizure discharge into the forebrain is evidenced by the gradual development of cortical epileptiform discharge, the appearance of forebrain seizure behaviors, and a marked increase in Fos-like immunoreactivity in the forebrain, none of which is seen in animals experiencing their first audiogenic seizure (3,4,21–23). The amygdala appears to be a critical structure in the recruitment of the forebrain seizure circuitry during repeated audiogenic seizures (24–27).
The severity of the brainstem seizure in response to the audiogenic stimulation appears to influence the expression of the kindled forebrain seizure during the audiogenic kindling paradigm. Rats that display clonic brainstem seizures in response to an audiogenic stimulus, such as the GEPR-3 or Wistar audiogenic rat (WARs), typically show F&F clonus after repeated audiogenic stimulation (3–5). Furthermore, in GEPR-3s and WARs, audiogenic kindling causes a decrease in the severity of brainstem seizure behavior, which parallels the increase in the kindled forebrain seizure severity (8,28,29). Conversely, GEPR-9s expressing tonic seizures in response to audiogenic stimulation display a posttonic clonus (not a typical kindled forebrain seizure behavior) after audiogenic kindling, even in the presence of EEG seizure discharge in the forebrain (4). The present experiments were aimed at examining the relation between the audiogenic-induced brainstem seizure severity and the kindled forebrain seizure behavior. This was accomplished by conducting the audiogenic kindling paradigm in GEPR-9s after two treatments known to reduce brainstem seizure severity and in GEPR-3s after a treatment known to increase brainstem seizure severity.
We hypothesized that tonic brainstem seizures preclude the behavioral expression of forebrain seizures because of either a postictal depression in the brainstem or continuous brainstem seizure activity that prevents expression of forebrain discharge. If this hypothesis is correct, suppression of the brainstem seizure during repeated audiogenic stimulation in GEPR-9s should result in the expression of forebrain seizure behavior that is typically seen in kindled GEPR-3s (i.e., F&F clonus). In the present study, lesions of the SC were found to reduce markedly the severity of brainstem seizure behavior in GEPR-9s and GEPR-3s (12). In support of our hypothesis, SC-lesioned GEPR-9s subjected to audiogenic kindling displayed an increased incidence and rate of development of forebrain seizure behavior. Remarkably, SC-lesioned GEPR-9s displayed an average of 14 F&F clonic seizures, whereas the control animals averaged only one F&F clonic seizure. In contrast to the marked facilitation of forebrain seizures in SC-lesioned GEPR-9s, SC lesions did not alter the rate of appearance or severity of audiogenically kindled forebrain seizures in GEPR-3s. However, as reported by Garcia-Cairasco et al. (8), repeated audiogenic seizures resulted in a decrease in brainstem seizure severity in some GEPR-3s accompanied by an increase in forebrain seizure severity. Ablating the SC before audiogenic kindling provided information about the role of the SC in the spread of seizure discharge from the brainstem to the forebrain. Although the SC is an obligatory structure of the brainstem seizure circuitry of the GEPR (12), it is not required for propagating brainstem seizure discharge into the forebrain because lesioned GEPRs displayed kindled forebrain seizures.
GEPR-9s treated daily (just before audiogenic stimulation) with PHT to reduce brainstem seizure severity developed forebrain seizure behavior that included F&F clonus in response to audiogenic kindling. Furthermore, a high negative correlation (−0.86) occurred between brainstem seizure severity and forebrain seizure severity (as measured on the Racine scale) in the PHT-treated GEPR-9s. In contrast, saline-treated GEPR-9s did not develop F&F clonic seizures but displayed posttonic clonus and the less severe forebrain seizure behaviors, facial clonus, and head bobbing, as described by Naritoku et al. (4). Whereas the saline-treated GEPR-9s did not display F&F clonus after the tonic brainstem seizure, EEG evidence of spike–wave discharge was noted in the cerebral cortex (i.e., seizure discharge in the forebrain) after 9–11 days of audiogenic kindling. Despite the marked difference in forebrain seizure behavior between PHT-treated and saline-treated GEPR-9s, the EEG spike–wave discharge looked similar whether the animal displayed F&F clonus or a posttonic clonus. These findings further support the notion that brainstem seizures inhibit or mask the behavioral expression of kindled forebrain seizures in GEPR-9s.
Further to test the hypothesis that the expression of forebrain seizure behavior is inversely related to the severity of brainstem seizures, we increased the severity of brainstem seizures in GEPR-3s in an attempt to decrease the incidence of forebrain-kindled behavior. From previous studies in our laboratory, it was known that widespread depletion of 5-HT by intracerebral administration of 5,7-DHT increased sound-induced brainstem seizure severity in GEPR-3s (13,30). As predicted, GEPR-3s treated with 5,7-DHT displayed less severe forebrain seizures, including a lower incidence of F&F clonus, during audiogenic kindling. These data also support the hypothesis that tonic brainstem seizures reduce the expression of forebrain seizure behavior. Interpretation of this finding is, however, confounded by the widespread depletion of 5-HT. Although the role of 5-HT in propagation of seizure discharge in to the forebrain is not clear, 5-HT depletion appears to have a minor (and perhaps facilitative) affect on kindling (31–34). Therefore the diminution of forebrain seizure behavior after 5-HT depletion is likely due to brainstem seizure severity regulating the expression of seizure behavior in the GEPR.
In conclusion, the three experiments (SC lesion, PHT, and 5,7-DHT) taken together provide considerable support for the hypothesis that tonic brainstem seizures prevent the expression of kindled forebrain seizure behavior. Given that the spike–wave discharged in the forebrain looked similar whether the GEPR-9 displayed a posttonic clonus or F&F clonus, the lack of expression of the forebrain convulsion after a tonic seizure in kindled GEPR-9s is due to either continuous brainstem seizure activity or the postictal state of the brainstem. Thus the altered state of the brainstem network in GEPR-9s appears to preclude motor expression of forebrain seizure activity (i.e., F&F clonus).