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Keywords:

  • Brainstem seizure;
  • Forebrain seizure;
  • Kindling;
  • Phenytoin

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animals

Male and female (250–450 g) GEPRs of severe (GEPR-9) and moderate strains (GEPR-3) were obtained from the colonies at the University of Illinois College of Medicine at Peoria. All rats were screened for audiogenic seizure susceptibility with 3 audiogenic seizure tests ≥48 h apart. Only rats with consistent severity scores were included in the studies. No differences in seizure response were observed between male and female GEPRs. Animals were housed two per cage on a 12-h light–dark cycle with food and water ad libitum.

Audiogenic seizure testing

To elicit audiogenic seizures, rats were placed in a Plexiglas cylindrical chamber (40 cm in diameter and 50 cm in height) with two doorbells mounted inside the lid. The bell delivered an ∼100-dB sound. Rats received daily audiogenic stimulation for between 19 and 24 days. Rats were subjected to audiogenic seizure testing at approximately the same time each day, and the brainstem seizure behavior was scored by using the method of Jobe et al. (14). Audiogenic response scores (ARSs) ranged from 0 to 9 with 0, no response; 1, one wild run; 2, two wild runs followed by clonus of fore- and hindlimbs; 3, one wild run followed by clonus of fore- and hindlimbs (bouncing clonus); 4, two wild runs followed by tonic forelimb extension and hindlimb flexion; 5, one wild run followed by tonic forelimb extension and hindlimb flexion; 6, two wild runs followed by tonic forelimb and partial hindlimb extension; 7, one wild run followed by tonic forelimb and partial hindlimb extension; 8, two wild runs followed by tonic forelimb and full hindlimb extension; and 9, one wild run followed by complete fore- and hindlimb extension. Forebrain seizure severity was scored according to the method of Racine (15). To keep the audiogenic stimulus uniform between treated and control animals, the daily audiogenic stimulation consisted of 30 s of ∼100-db sound exposure regardless of the occurrence of brainstem convulsion.

Lesions of the superior colliculus

Rats were placed in a Kopf stereotaxic apparatus with the incisor bar at +5.0. Electrolytic lesions were produced bilaterally in the superior colliculus (SC) by passing 4 mA of anodal current for 10 s through an epoxylite-coated electrode. In the sham-operated rats, the electrode was lowered into the SC without passing current. The coordinates for the GEPR-3 were as follows: 6.1 mm caudal to bregma, ±1.5 mm lateral to midline, and 3.3 mm ventral to the dura mater. In the GEPR-9, the coordinates were as follows: 6.5 mm caudal to bregma, ±1.9 mm lateral to midline, and 3.3 mm ventral to the dura mater. GEPR-3s and GEPR-9s are known to have different stereotaxic coordinates because of a slight difference in the shapes of their heads (12). Lesion location was assessed histologically in 40-μm frozen sections stained with thionine. Damage was assessed by using a Nikon Profile Projector, as described previously (12). The histologic criterion for inclusion of a lesioned animal in the analysis was 85% destruction of the SC. This was assessed as described previously (12).

Phenytoin treatment

Selectively to suppress brainstem seizure discharge, GEPR-9s were treated daily with a low dose of PHT (15–25 mg/kg, i.p.) 1 h before each of a series of 24 daily audiogenic seizure tests. PHT preferentially suppresses the tonic brainstem components of seizures, which would allow the forebrain seizure discharge to acquire greater control of spinal motor neurons (see ref. 16). Moreover, evidence suggests that PHT selectively suppresses brainstem seizures in GEPR-9s (9). Although the half-life of PHT in the rat is ∼0.5–1.5 h, it was shown almost to double after five daily systemic treatments (17,18). Because of possible accumulation of PHT in the treated animals, the dose was adjusted daily (based on the response to previous dose) in an attempt to reduce (ARS, 1), but not to abolish, brainstem seizure discharge. To examine the effect of PHT on electrographic EEG seizure discharge, GEPR-9s were implanted with EEG electrodes and subjected to daily audiogenic kindling (described later).

Implantation of electrodes and EEG recording

GEPR-9s were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a Kopf stereotaxic apparatus. Stainless steel screws (0–80 × 1/8 inch) were inserted bilaterally into the skull over the parietal lobe region. A third screw was placed in the skull over the ethmoid sinus to serve as the reference electrode. Braided copper wire was wound around the screws and held in place with dental acrylic. Rats were allowed ≥1 week to recover from surgery. Before seizure testing, rats were placed in a harness that restrained and suspended the animal but allowed for free movement of the limbs. Recording cables were connected to the implanted recording electrodes, and EEG activity was recorded before, during, and after audiogenic stimulation. After the observed development of spike–wave discharge in the EEG (after approximately the ninth bell test), animals were given PHT preceding the daily audiogenic stimulation, as described earlier. EEG recordings were obtained in the restrained (suspended in the harness) rats by using a Grass 8 channel polygraph (Grass Instrument Co., Quincy, MA, U.S.A.; model 6). EEG patterns were evaluated visually from polygraph records.

Depletion of brain serotonin

Widespread depletion of brain serotonin (5-HT) in GEPR-3s was accomplished by intracerebroventricular (ICV) administration of 5,7-dihydroxytryptamine (5,7-DHT). To protect noradrenergic neurons from the neurotoxic effects of 5,7-DHT, rats were pretreated with protriptyline (20 mg/kg, i.p.) 2 h before surgery. Rats were anesthetized with chloral hydrate (350 mg/kg, i.p.) and injected with procaine penicillin (100,000 IU, i.m.) and atropine (0.542 mg/kg, s.c.) to prevent postsurgical infection and minimize respiratory congestion, respectively. Animals were placed in a stereotaxic head holder with the head flat (incisor bar, 3.3) and given either 5,7-DHT (150 μg/30 μl of saline-ascorbate) or 30 μl of saline-ascorbate (vehicle) into the right lateral ventricle. The following coordinates were used: 1.0 mm caudal to bregma, 1.5 mm lateral to midline, and 4 mm ventral to the skull surface. After surgery, rats were allowed to recover for 2 weeks before undergoing 24 consecutive daily audiogenic seizure tests.

Measurement of monoamines

After the last seizure test, the rats were killed by decapitation, the brains quickly removed and dissected into the cortex, midbrain, and pons/medulla on an ice-cold glass plate. The tissue samples were frozen on dry ice and kept at −80°C until assayed. The concentration of brain 5-HT and norepinephrine (NE) were measured in vehicle- and 5,7-DHT–treated animals as previously described (19,20). In brief, samples were homogenized in 0.3N perchloric acid (PCA) and centrifuged. One milliliter of supernatant was injected directly into an high-performance liquid chromatography (HPLC) system to measure 5-HT brain concentration. An additional 2-ml aliquot of supernatant was used for the measurement of brain NE. NE was extracted from the supernatant with aluminum oxide at pH 8.6 and eluted with 0.2N PCA. The monoamines were separated by HPLC (by using a Beckman Ultrasphere C-18 column) and measured by electrochemical detection by using an Ag/AgCl reference electrode against a detector potential of 650 mV.

Statistical analysis

Audiogenic seizure severity and forebrain seizure severity of treated and control animals were compared by using a Mann–Whitney U test. The incidence of forebrain seizures between groups was compared by using a χ2 analysis. The alpha level was set at p < 0.05 to assess significant differences. Pearson product–correlation coefficient was determined correlating brainstem seizure severity and forebrain seizure severity for the PHT-treated GEPR-9s.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The effect of lesions of the SC on audiogenic kindling

In light of the inhibitory effect of the SC lesion on audiogenic seizure severity (12), the effect of the SC lesion on repeated daily audiogenic seizure tests was examined. To accomplish this, GEPR-9s and GEPR-3s with lesions of the SC were subjected to 24 and 19, respectively, daily audiogenic seizure tests. SC lesions produced a marked reduction in audiogenic seizure severity in GEPR-9s that was consistent across all 24 seizure tests (p < 0.05 for all 24 seizure tests, using a Mann–Whitney U nonparametric test; Fig. 1A). After approximately five audiogenic seizure tests, lesioned GEPR-9s began to display forebrain seizure behavior after the brainstem seizure that was scored according to the method of Racine (15). Interestingly, the SC-lesioned GEPR-9s tended to display more severe forebrain seizures than did the nonlesioned control GEPR-9s, and this difference in severity reached significance during 11 of the 24 seizure tests (p < 0.05 with a Mann–Whitney U nonparametric test; Fig. 1B). The lesioned GEPR-9s displayed an average of 14 F&F clonic seizures (3 on the Racine scale) during the 24 audiogenic seizure tests, whereas the control animals averaged only one F&F clonic seizure across the 24 tests (p < 0.05 with a Student's t test; Table 1).

image

Figure 1. Effect of superior colliculus (SC) lesion on brainstem and forebrain seizure severity in genetically epilepsy-prone rat (GEPR)-9s during repeated audiogenic seizure testing. A: Brainstem seizure severity. Lesion of the SC reduced audiogenic seizure severity throughout the 24 seizure tests (n = 12; control; seven, lesion). *p < 0.05 with a Mann–Whitney U nonparametric test. B: Forebrain seizure severity. Lesioned GEPR-9s displayed more severe forebrain seizures. *p < 0.05 compared with controls with a Mann–Whitney U nonparametric test. The mean seizure severity scores ± SEM are presented in the graph.

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Table 1. The effect of the SC lesion on the number of facial and forelimb clonic seizures during audiogenic kindling of GEPR-3s and GPR-9s
TreatmentNumber of facial and forelimb clonic seizures
  1. The number of seizures is presented as mean ± SEM.

  2. GEPR, genetically epilepsy-prone rat; SC, superior colliculus.

  3. ap < 0.05 with a Student's t test. GEPR-9s received 24 audiogenic seizure tests; GEPR-3s received 19 seizure tests.

GEPR-9
 Control (n = 12)1.0 ± 0.5
 Lesion (n = 7)14.0 ± 3.1a
GEPR-3
 Control (n = 13)5.1 ± 1.0
 Lesion (n = 9)4.3 ± 0.6

In agreement with our previous findings (12), the SC lesion reduced audiogenic seizure severity in GEPR-3s consistently across the audiogenic seizure tests (Fig. 2A). However, after 18–19 seizures, brainstem seizure behavior disappeared completely in GEPR-3s (ARS, 0) in response to audiogenic stimulation. At this point, the forebrain seizure behavior appeared to be dependent on antecedent brainstem seizure activity, because in the absence of a brainstem seizure (i.e., no running), no evidence was found of a forebrain seizure. Thus audiogenic kindling of the GEPR-3 was stopped after 19 seizure tests. Lesions of the SC did not affect the severity of brainstem-evoked forebrain seizures (F&F clonus) in GEPR-3s (Fig. 2B). After approximately four audiogenic seizure tests, GEPR-3s began to display forebrain seizure behavior. Furthermore, after ∼11 audiogenic seizures, both SC lesioned and control GEPR-3s displayed F&F clonic seizures. As can be seen in Table 1, no difference was noted between lesioned and control GEPR-3s in the number of F&F clonic seizures displayed across the 19 audiogenic seizure tests.

image

Figure 2. Effect of superior colliculus (SC) lesion on brainstem and forebrain seizure severity in genetically epilepsy-prone rat (GEPR)-3s during repeated audiogenic seizure testing. A: Brainstem seizure severity. Lesion of the SC reduced audiogenic seizure severity throughout the majority of the 19 seizure tests (n = 13, control; nine, lesion). *p < 0.05 with a Mann–Whitney U nonparametric test. B: Forebrain seizure severity. SC lesions did not affect the severity of forebrain seizure behavior evoked by audiogenic kindling. The mean seizure severity scores ± SEM are presented in the graph.

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Effect of brainstem seizure suppression on audiogenic kindling in GEPR-9s

To test the hypothesis that the severe brainstem seizure of the GEPR-9 precludes the expression of forebrain seizures after audiogenic kindling, we injected GEPR-9s daily with low-dose PHT to suppress brainstem seizures to the wild-run component (ARS, 1). Although male and female GEPRs were used in these studies, no differences were seen in the response to PHT or in the seizure response between the sexes. Suppressing the intensity of the brainstem seizure to the wild run (without suppressing all brainstem seizure activity) posed some difficulty. To be included in the data, animals must have had their brainstem seizures successfully attenuated to an ARS of 1 (wild running) for ≥50% of the seizure tests. Animals that fit this criterion displayed more severe forebrain seizure behavior on seizure tests 8, 11, 13, 19, 20, 23, and 24 (p < 0.05 with a Mann–Whitney U nonparametric test; Fig. 3A). As predicted by our hypothesis, audiogenic seizure severity and forebrain seizure severity were negatively correlated (r=−0.86). Only when the audiogenic seizure severity was reduced with PHT to an ARS of 1 did the animals display F&F clonus.

image

Figure 3. The effect of low-dose phenytoin (PHT) on brainstem and forebrain seizure severity of genetically epilepsy-prone rat (GEPR)-9s exposed to daily audiogenic seizures. GEPR-9s were given a dose of PHT 1 h before audiogenic seizure testing in an attempt to reduce brainstem seizure behavior to an audiogenic response score (ARS) of 1 consisting of only the wild-running. A: Brainstem seizure severity. Only GEPR-9s with an ARS of 1 for ≥50% of the audiogenic seizure tests were included in the data (seven of 18 fit this criterion). B: Forebrain seizure severity. PHT-treated GEPR-9s displayed more severe forebrain seizure behavior. *p < 0.05 with Mann–Whitney U nonparametric test. The mean seizure severity scores ± SEM are presented in the graph (n = 11 saline, seven PHT).

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Electrographic evidence of forebrain seizure activity (i.e., cortical spike–wave discharge) was not present after the initial audiogenic seizure test but began to be present in GEPR-9s after nine to 11 seizure tests (Fig. 4A and B). By the eighteenth seizure test, spike–wave discharge was present in rats regardless of the brainstem or forebrain seizure behavior (compare Fig. 4C and D). As can be seen in Fig. 4C, during and after the all-limb tonic extension, spike–wave discharge was present. However, this animal displayed only facial clonus (1 on the Racine scale). On the contrary, the same animal displayed F&F clonus when the brainstem seizure was reduced to the running component with treatment with low-dose PHT (Fig. 4D). The cortical spike–wave discharge was the same regardless of the whether forebrain seizure behavior was expressed. Such findings are consistent with the hypothesis that severe brainstem seizures mask the expression of forebrain seizure behavior.

image

Figure 4. EEG recordings from genetically epilepsy-prone rat (GEPR)-9s during audiogenic seizure testing throughout audiogenic kindling. A: Record from initial seizure test in untreated animal that displayed all-limb tonic extension [audiogenic response score (ARS), 9]. No seizure discharge occurred during or after the tonic extension. B: EEG recording from an untreated GEPR-9 (ARS, 9) during ninth seizure test. Rhythmic discharge begins to be present in the EEG during the tonic extension. C: EEG recording taken during seizure test 20 in an untreated animal that displayed all-limb tonic extension. During the extension, spike–wave discharge was indicative of forebrain seizure activity. After the extension, the animal displayed facial clonus, a mild forebrain seizure behavior. D: EEG recording during seizure test 18 in a GEPR-9 pretreated with phenytoin (PHT). The PHT selectively suppressed the brainstem seizure, and the animal displayed only running behavior (ARS, 1). After the run, the rat displayed facial and forelimb clonus followed by a loss of posture and clonus of all four limbs.

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The effect of widespread 5-HT depletion on audiogenic kindling in GEPR-3s

GEPR-3s were depleted of brain 5-HT before subjecting them to 24 daily audiogenic seizure tests. Two weeks after 5,7-DHT administration, rats underwent 24 consecutive daily audiogenic seizure tests to examine the effect of 5-HT depletion on the rate and severity of audiogenic kindling. As previously reported, GEPR-3s displayed significantly more severe audiogenic seizures after 5,7-DHT treatment (Fig. 5A). The majority of treated animals (six of 10) displayed tonic seizures during the first audiogenic seizure test; whereas none of the vehicle-treated animals displayed tonic seizures (none of 12). Although the audiogenic seizure-severity scores fluctuated across the 24 audiogenic seizure tests, 5,7-DHT–treated animals tended to display more severe audiogenic seizures. The increase in seizure severity reached significance on bell tests 1, 5, 6, 7, 10, 11, 12, 14, and 23 (p < 0.05 with a Mann–Whitney U nonparametric test). Forebrain seizure behavior was not present before repeated audiogenic seizure tests. Animals treated with 5,7-DHT displayed significantly fewer severe forebrain kindled seizures on bell tests 14,15, 18,19, and 23 (p < 0.05 with a Mann–Whitney U nonparametric test; Fig. 5B). Furthermore, treated animals displayed significantly fewer F&F clonic seizures. Across the 24 bell tests, vehicle-treated animals displayed an average of 5.1 ± 0.69 F&F clonic seizures, whereas 5,7-DHT–treated animals displayed on average 2.3 ± 0.45 (p < 0.05 with a Student's t test) forebrain seizures which included F&F clonus.

image

Figure 5. The effect of intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) on brainstem and forebrain seizure severity in genetically epilepsy-prone rat (GEPR)-3s. A: Brainstem seizure severity. Treatment with 5,7-DHT increased the brainstem seizure severity across many of the audiogenic seizure tests. B: Forebrain seizure severity. Treatment with 5,7-DHT decreased the forebrain seizure severity evoked by audiogenic kindling. *p < 0.05 compared with vehicle-treated controls with a Mann–Whitney U nonparametric test (n = 12, vehicle; 10, 5,7-DHT). The mean seizure-severity scores ± SEM are presented in the graph.

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As can be seen in Table 2, 5,7-DHT treatment caused a marked depletion of brain 5-HT. The cortex showed a 93% reduction in 5-HT, which was the region with the greatest depletion of 5-HT. The midbrain showed a 64% reduction, and the pons/medulla showed a 45% reduction in 5-HT compared with vehicle-treated controls. Protriptyline pretreatment was effective in protecting the noradrenergic neurons from the neurotoxic effects of 5,7-DHT because 5,7-DHT treatment did not significantly reduce NE content in the cortex.

Table 2. The regional brain serotonin (5-HT) and norepinephrine concentration in GEPR-3s after intracerebroventricular treatment with 5,7-DHT
RegionVehicle treated (n = 12)5,7-DHT treated (n = 10) % Depletion
  1. Rats received 5,7-DHT (150 μg/30 μl) or saline-ascorbate (vehicle) 2 h after pretreatment with protriptyline (20 mg/kg, i.p.). 5,7-DHT produced a marked depletion of 5-HT in the cortex, midbrain, and pons/medulla without significantly reducing norepinephrine content in the cortex.

  2. ap < 0.05 compared with vehicle-treated controls with an unpaired Student's t test. The numbers reported are the mean concentration ± SEM.

Serotonin (ng/g tissue)
 Cortex274.2 ± 7.9 20.2 ± 7.2a*−93
 Midbrain683.4 ± 18.1244.7 ± 29.5a−64
 Pons/medulla672.2 ± 43.4363.2 ± 51.3a−46
Norepinephrine (ng/g tissue)
 Cortex212.5 ± 11.4189.9 ± 5.6  −11

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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).

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Browning RA, Wang C, Nelson DK, et al. Effect of precollicular transection on audiogenic seizures in genetically epilepsy-prone rats. Exp Neurol 1999;155: 295301.
  • 2
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