Characterization of Audiogenic-Like Seizures in Naive Rats Evoked by Activation of AMPA and NMDA Receptors in the Inferior Colliculus

Experimental Neurology 164, 396 – 406 (2000) doi:10.1006/exnr.2000.7401, available online at http://www.idealibrary.com on

Characterization of Audiogenic-Like Seizures in Naive Rats Evoked by Activation of AMPA and NMDA Receptors in the Inferior Colliculus Shin Yasuda,* ,1 Nobuya Ishida,* ,† Akiko Higashiyama,‡ Shigeru Morinobu,* and Nobumasa Kato* ,§ *Department of Psychiatry, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520-2192, Japan; †Health Administration Center, Shiga University of Medical Science, Otsu 520-2192, Japan; ‡School of Human Courses, The University of Shiga Prefecture, Hikone 522-8553, Japan; and §Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan Received October 21, 1999; accepted March 14, 2000

The role of glutamate receptors in the inferior colliculus (IC) in audiogenic and audiogenic-like seizures was investigated in adult rats with transient neonatal hypothyroidism by 0.02% propylthiouracil (PTU) treatment through mother’s milk (PTU rats) and in naive rats treated intracisternally with N-methylD-aspartate (NMDA), ␣-amino-3-hydroxy-5-methyl-4isoxazole-proprionic acid (AMPA), or cyclothiazide, an inhibitor of rapid AMPA receptor desensitization. All rats showed audiogenic or audiogenic-like seizures characterized by running fit (RF) and generalized tonic– clonic seizures (GTCS). While systemically administered MK-801 inhibited GTCS, intracisternally administered NBQX inhibited RF and GTCS in both audiogenic and audiogenic-like seizures. Auditory stimulation shortened the latency to GTCS induced by AMPA, but not NMDA, at a subclinical dose and further elongated the shortened duration of RF, but not GTCS, induced by MK-801 pretreatment. Furthermore, Northern blot analysis was used to evaluate the expression of the immediate-early gene c-fos in the IC following induction of audiogenic or audiogenic-like seizures. The significant induction of c-fos mRNA by audiogenic seizures in PTU rats or by AMPA- or cyclothiazide-induced seizures in naive rats was prominent in the IC. MK-801 suppressed c-fos mRNA expression in the IC induced by audiogenic seizures in PTU rats or by AMPA-induced seizures in naive rats. NBQX suppressed the expression of c-fos mRNA in the IC induced by AMPA-induced seizures but did not suppress c-fos mRNA in PTU rats or rats with cyclothiazide-induced seizures. Auditory stimuli failed to affect c-fos mRNA induction by AMPA. The present study suggests that audiogenic-like seizures can be reproduced by glutamate receptor agonists in which AMPA receptors are primarily linked to the initiation of audiogenic seizures 1

To whom correspondence should be addressed. Present address: Department of Neuropharmacology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. 0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

(RF) while NMDA receptors presumably located within the IC are involved in the propagation of GTCS in audiogenic seizures. © 2000 Academic Press Key Words: audiogenic seizure; NMDA receptors; AMPA receptors; inferior colliculus; cyclothiazide; cfos mRNA; propylthiouracil.

INTRODUCTION

Several animal species, such as mouse, rat, chicken, and rabbit (10, 20, 22, 25), are genetically predisposed to audiogenic seizures. Audiogenic seizures in rodents, particularly in DBA/2 mice (30) and genetically epilepsy-prone rats (GEPR) (25), have been extensively studied. In DBA/2 mice sensitivity to noise develops between the 12th and 17th postnatal days, reaches its maximum toward the 22nd day, and progressively declines thereafter (30). In GEPR, sensitivity to auditory stimulation increases between the 17th and 20th postnatal days (8). Both animals show wild running (running fit, RF) followed by generalized tonic-clonic seizures (GTCS) after auditory stimulation. Seizures in DBA/2 mice are often lethal, while seizures in GEPR are not. Taken together, these genetic models of audiogenic seizures have been considered to be appropriate models of generalized epilepsy or reflex epilepsy in humans, and the previous findings indicate Postnatal Days 12 to 22 to be a critical period in the development of audiogenic seizure susceptibility in rodents. Lactating rats that receive 0.02% propylthiouracil (PTU) in their drinking water transfer the goitrogenic effects to their offspring through their milk. This treatment induces a temporary mild hypothyroid condition in the pups. Van Middlesworth and Norris (48) reported that rats treated with PTU for 19 days postnatally exhibited a marked susceptibility to audiogenic seizures, starting from the age 7 weeks and persisting into adulthood. Kato et al. (27) studied the expression of c-fos mRNA after audiogenic seizure in PTU rats and found that the significant induction of c-fos mRNA by

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audiogenic seizure was prominent in several brain areas including the inferior colliculus (IC). It is known that glutamate receptors mediate major transmission at excitatory synapses in the central nervous system, acting at both NMDA (N-methyl-D-aspartate) and non-NMDA receptors (7). The non-NMDA receptors were classified into AMPA (␣-amino-3hydroxy-5-methyl-4-isoxazole-propionic acid)-selective and kainate-selective receptors (44). The AMPA receptors are characterized by rapid desensitization (or inactivation) (47), which can be strongly blocked by cyclothiazide (50). It has been noted that systemic administration of NMDA receptor antagonists significantly decreases the severity of audiogenic seizure, and the IC is the most critical nucleus for audiogenic seizure development in GEPR (12). Higashiyama et al. (21) have recently reported that NMDA receptors in the IC, presumably in the external cortex of the IC, play a critical role in the initiation of audiogenic seizures in PTU rats. Meanwhile, AMPA receptor antagonists have been reported to suppress audiogenic seizures in both GEPR and DBA/2 mice (6, 12, 43, 45). The IC is a processing center that receives and integrates information from all parts of the auditory system (24, 38), and there is strong evidence suggesting that auditory transmission is mediated by glutamate. Significant amounts of endogenous glutamate (1, 18), glutamate-immunoreactive neurons (39), and glutamate binding sites (9, 19) are observed in the mammalian IC. Iontophoretic studies indicate that glutamate increases neuronal firing in the IC (11). Recent intensive studies of auditory transmission in the barn owl IC revealed that NMDA and AMPA receptors contributed differently to auditory responses depending on the region within the IC (15–17). Caicedo and Eybalin recently reported age-dependent changes in the expression of AMPA and NMDA receptor subunits in the IC and other parts of the auditory system (4). The purpose of this study is to clarify the role of NMDA and AMPA receptors in the IC and related networks in audiogenic seizures. We examined the effects of NMDA and AMPA receptor antagonists on seizures in PTU-treated rats. Subsequently seizures caused by the infusion of NMDA, AMPA, or cyclothiazide into the cisterna ambiens or the IC were characterized in naive rats with or without antagonist pretreatment. Northern blot analysis was used to evaluate the expression of the immediate-early gene c-fos mRNA in the IC of PTU-treated rats or AMPA- or cyclothiazide-treated naive rats. MATERIALS AND METHODS

Animal Preparation PTU-treated rats were prepared as previously described (21). In brief, five pregnant Sprague–Dawley

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(SD) rats, obtained from Clea Japan, Inc., were housed individually and allowed to litter normally. On the day of delivery (Day 0), all pups were collected, randomized, and then redelivered to each dam. Four dams received 0.02% propylthiouracil (2-thio-4-hydroxy-6-npropylpyrimidine, Sigma) in their drinking water from Day 0 through Day 19 of postparturition. One dam served as a control and received water only. The pups were then reared until the age of 28 days (weaning). At the age of 3 months, all rats in both the PTUtreated and the control group were tested for audiogenic seizure susceptibility. PTU-treated and control rats at the age of 5– 6 months were subjected to the experiments. For naive rats, male SD rats were used at the age of 8 weeks. For topical drug administration in the rats, cannulas were implanted in the cisterna ambiens or the bilateral IC. Animals were anesthetized with pentobarbital (50 mg/kg). Polyethylene (PE) tubes made by fusion of the outer part (PE-50, Beckton–Dickinson) and the infracranial part (PE-10) were stereotaxically placed over the skull and cemented with dental acrylic at the following coordinates: AP ⫽ ⫺8.8 mm from bregma, L ⫽ ⫾0 mm, and V ⫽ ⫺2 mm (cisterna ambiens) or AP ⫽ ⫺8.8 mm from bregma, L ⫽ ⫾2 mm, V ⫽ ⫺3.5 mm (IC). Implanted animals were allowed at least a week for recovery. The locations of cannula tips were verified histologically. Drugs To induce audiogenic-like seizures, NMDA (Research Biochemicals International) and AMPA (Tocris Cookson) dissolved in phosphate buffer or cyclothiazide (Tocris Cookson) dissolved in dimethyl sulfoxide (DMSO, Sigma) were used as agonists. MK-801 (dizocilpine, Tocris Cookson) dissolved in saline was used in this study as an NMDA receptor antagonist. NBQX (6-nitro-7-sulphamoylbenzo( f )quinoxaline-2,3-dione, Tocris Cookson) dissolved in DMSO and 1-naphthylacetyl spermine (1-NA-Spm) (26, 46), an analog of Joro spider toxin (JSTX), dissolved in phosphate buffer (pH 7.4) were used as AMPA receptor antagonists. In the preliminary experiment, the dose of each antagonist was optimized. MK-801 at doses of 0.1 and 0.5 mg/kg or NBQX at doses of 5 and 25 nmol/brain was used against audiogenic seizure in PTU rats. MK-801 at doses of 0.5 and 1 mg/kg or NBQX at doses of 0.5 and 5 nmol/brain was used against NMDA-induced seizures. MK-801 at doses of 0.1 and 0.5 mg/kg or NBQX at doses of 5 and 50 nmol/brain was used against AMPA-induced seizures. MK-801 at a dose of 0.5 mg/kg or NBQX at a dose of 25 nmol/brain was used against cyclothiazide-induced seizures. 1-NA-Spm at a dose of

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50 ␮g/brain was used for PTU rats or cyclothiazideinduced seizures. MK-801 was administered intraperitoneally (ip) and NBQX or 1-NA-Spm was injected into the cisterna ambiens. Treatment Procedure for PTU-Treated Rats For PTU rats, 30 min after treatment with MK-801 or 5 min after treatment with NBQX or 1-NA-Spm, 80-s acoustic stimulus was provided. The same volume of vehicle was administered to the control group. PTU rats were placed in a new cage and then audiogenic stimulation was applied by ringing the emergency bell (112dB) for 80 s. The behavioral changes that included latency to running fit (RF) and GTCS and the duration of RF and GTCS were observed in each animal tested. Treatment Procedure for Naive Rats NMDA at doses of 3, 5, 10, and 20 ␮g, AMPA at doses of 3 and 6 ␮g, and cyclothiazide at doses of 0.1, 0.5, and 1 ␮mol were injected into the cisterna ambiens, and cyclothiazide at doses of 0.05, 0.1, and 0.5 ␮mol was also bilaterally injected into the IC. Thirty minutes after treatment with MK-801 or 5 min after treatment with NBQX or 1-NA-Spm, NMDA (20 ␮g), AMPA (6 ␮g), or cyclothiazide (1 ␮mol) was administered into the cisterna ambiens. Immediately after treatment with NMDA (5 ␮g) or AMPA (3 ␮g), auditory stimulation was applied by ringing the emergency bell for 80 s. Furthermore, 30 min after treatment with MK-801 or 5 min after treatment with NBQX, AMPA (3 ␮g) was administered into the cisterna ambiens, and auditory stimulation was applied by ringing the emergency bell for 80 s. Behavioral changes, including latency to RF and GTCS, and duration of RF and GTCS were observed in each animal tested for 80 s (with auditory stimuli) or 30 min (without auditory stimuli). Northern Blot Analysis PTU-treated rats and naive rats treated with AMPA or cyclothiazide, with or without agonist pretreatment, were examined for c-fos mRNA expression. Thirty minutes after auditory stimulation or drug administration of either agonist, animals were sacrificed by decapitation. The sections of the IC were dissected and immediately frozen for RNA extraction and Northern blot analysis as described below. Total RNA was isolated from sections of the IC by the guanizine/cesium chloride centrifugation method (37). Levels of c-fos mRNA were determined by Northern blot analysis using random-primed, 32P-labeled cDNA as previously described (37). Briefly, 15 ␮g of total RNA was electrophoresed on 1% agarose gel and RNA was transferred to nitrocellulose filters. The resulting fil-

ters were then incubated with the 32P-labeled probes for 18 h at 42°C and washed twice in 2⫻ SSC/0.1% sodium dodecyl sulfate (SDS) at 52°C for 20 min and then in 0.3⫻ SSC/0.1% SDS at 52°C for 20 min. Levels of total RNA for each lane were determined to be approximately equal by reprobing the nitrocellulose filters with a 32P-labeled cyclophilin cDNA probe. The radiolabeled mRNA bands were visualized by autography. Levels of c-fos mRNA were determined by outlining the band on Northern blots and then quantitated on a Macintosh-based ATTO Image analysis program, Version 3.01 (Tokyo, Japan). Data Analysis Statistical analysis was performed using ANOVA followed by a Tukey test and a Student’s t test. Rates of incidence following the injection with each drug were analyzed using the ␹ 2 test. RESULTS

The Development of Audiogenic Seizure in PTU Rats In PTU rats, the appearance rate of RF was 100%, the mean latency of RF was 15.0 ⫾ 11.6 s, and the mean duration of RF was 11.5 ⫾ 2.75 s. The appearance rates of GTCS was also 100%, the latency to GTCS was 42.3 ⫾ 9.86 s, and the duration of GTCS 19.3 ⫾ 3.59 s. No behavioral difference of audiogenic seizure was observed between male and female rats. In control rats, no behavioral change was observed. No animal died after the seizure. Effects of MK-801 and NBQX for PTU-Treated Rats Figure 1 shows the rates of incidence of RF and GTCS (left), the latency to RF and GTCS (top right), and the duration of RF and GTCS (bottom right) after ip administration of MK-801 or intracisternal administration of NBQX. MK-801 significantly suppressed only GTCS with prolonged latency and shortened duration at a dose of 0.5 mg/kg, whereas NBQX at a dose of 25 nmol suppressed both RF and GTCS with prolonged latency and shortened duration. Audiogenic seizures caused a marked induction of c-fos mRNA in the IC in PTU rats (Fig. 2). Quantitative analysis of optical density showed that a robust seizure-induced expression of c-fos mRNA after auditory stimulation in the IC was significantly reduced by MK801 pretreatment at a dose of 0.5 mg/kg. In contrast, NBQX failed to reduce the expression of c-fos mRNA in the IC despite the potent suppression of behavioral seizure (Fig. 2, top). Pretreatment with 1-NA-Spm showed no effect on either seizure manifestation (data not shown) or c-fos mRNA expression (Fig. 2).

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FIG. 1. (Left) Rates of incidence of RF alone (stippled bars) and RF ⫹ GTCS (solid bars). (Top right) Latency to RF (open circles) and GTCS (closed circles) (Bottom right) Duration of RF and GTCS following ip administration with MK-801 at doses of 0.1 and 0.5 mg/kg or intracisternal administration with NBQX at doses of 5 and 25 nmol/brain to PTU rats. (a) % RF, P ⬍ 0.05 vs control (100%). (b) % RF⫹ GTCS, P ⬍ 0.01 vs control (100%) (␹ 2 test). The number of animals in each group is shown at the top of each bar. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs vehicle (ANOVA followed by a Tukey test).

NMDA, AMPA, and Cyclothiazide Elicited AGS-Like Seizures The intracisternal administration of NMDA, AMPA, or cyclothiazide in naive rats dose dependently elicited RF followed by GTCS, similar to AGS found in PTU rats (Fig. 3). Administration of cyclothiazide into the IC also elicited RF and GTCS (Fig. 3). The latency and duration of RF and GTCS observed in NMDA-, AMPA-, or cyclothiazide-induced seizures are shown in Fig. 4. Administration of cyclothiazide into the IC was almost as equipotent as in intracisternal administration. Effects of MK-801 and NBQX on NMDA- and AMPA-Induced Seizures Effects of MK-801 or NBQX pretreatment on NMDA (20 ␮g)- or AMPA (6 ␮g)-induced seizures were shown as functions of the rates of incidence of RF and GTCS (Fig. 5) and the latency and duration of RF and GTCS (Fig. 6). GTCS, but not RF, induced by both NMDA and AMPA treatment was significantly blocked by MK-801 pretreatment, whereas NBQX blocked both RF and GTCS induced by NMDA and AMPA treatment (Fig. 5). Irrespective of either NMDA- or AMPA-induced seizures, MK-801 prolonged the latency and shortened

the duration of GTCS without effect on RF. In contrast, NBQX significantly suppressed both RF and GTCS with prolonged latency and shortened duration in both NMDA- and AMPA-induced seizures (Fig. 6). Effects of MK-801 and NBQX on CyclothiazideInduced Seizures In the case of cyclothiazide (1 ␮mol)-induced seizures, the effects of MK-801 and NBQX pretreatment are summarized in Figs. 5 and 6. Figure 5 shows the rates of incidence of RF and GTCS and Figure 6 shows the latency and duration of RF and GTCS. GTCS, but not RF, was significantly blocked by MK-801 pretreatment, whereas NBQX failed to block both RF and GTCS induced by cyclothiazide (Fig. 5). Both MK-801 and NBQX prolonged the latency to GTCS in cyclothiazide-induced seizures. The duration of GTCS was shortened by MK-801, while the duration of RF was shortened by NBQX in cyclothiazide-induced seizures (Fig. 6). Cyclothiazide-induced seizures caused a marked induction of c-fos mRNA in the IC (Fig. 7). MK-801 (0.5 mg/kg) or NBQX (25 nmol/brain) pretreatment did not reduce the level of c-fos mRNA expression (Fig. 7). The pretreatment of 1-NA-Spm showed no effect on either seizure manifestation (data not shown) or c-fos mRNA expression (Fig. 7).

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icant levels of both latency and duration of AMPAinduced GTCS (Fig. 8, right). AMPA (3 ␮g/brain)-induced seizure caused a marked induction of c-fos mRNA in the IC (Fig. 9). Auditory stimulation did not augment c-fos mRNA expression induced by AMPA (Fig. 9), though it behaviorally augmented seizures (Fig. 8). MK-801 (0.5 mg/kg) or NBQX (25 nmol/brain) pretreatment significantly reduced the level of c-fos mRNA expression, irrespective of the presence of auditory stimuli (Fig. 9). DISCUSSION

Both NMDA and AMPA Receptor Antagonists Blocked Audiogenic Seizures

FIG. 2. The levels of c-fos mRNA are determined by Northern blot using a 32P-labeled c-fos cDNA probe, and radiolabeled mRNA is visualized by autography (bottom) and quantification of radiolabeled bands by densitometric scanning (top). The number of animals in each condition is shown at the bottom of each lane. The filters were reprobed with 32P-labeled cyclophilin cDNA probe and standardized. Each column shows the mean ⫾ SEM. ***P ⬍ 0.001 (ANOVA followed by a Tukey test).

Auditory Stimulation Augmented AMPA-Induced Seizures Auditory stimulation shortened the latency to GTCS induced by AMPA at a lower dose of 3 ␮g/brain (Fig. 8), but failed to further augment the proconvulsant action of NMDA (data not shown). The effects of auditory stimulation on the anticonvulsant action of MK-801 or NBQX on AMPA (3 ␮g/ brain)-induced RF/GTCS are shown in Fig. 8. The prolonged latency and shortened duration of AMPA-induced GTCS by MK-801 (0.5 mg/kg) pretreatment were not altered with auditory stimuli; however, the shortened RF duration induced by MK-801 was abolished with auditory stimuli (Fig. 8, center). While NBQX (25 nmol/brain) completely blocked AMPA-induced seizure, auditory stimulation partially abolished the anticonvulsant action of NBQX as evidenced by insignif-

Our previous and present studies indicate that MK801 and NBQX dose dependently blocked audiogenic seizures in PTU rats (Fig. 1). Audiogenic seizure caused a marked induction of c-fos mRNA in the IC, which was significantly reduced by MK-801, but not NBQX, in PTU rats (Fig. 2). Behaviorally, while NBQX significantly blocked both RF and GTCS, MK-801 blocked GTCS without significant effect on RF (Fig. 1). The present results on NBQX appear contradictory to our previous results (21). In our previous study, NBQX administered intraperitoneally at doses of 3 to 30 mg/kg failed to block audiogenic seizures in PTU rats, whereas, in this study, NBQX showed a potent anticonvulsant action at doses of 5 or 25 nmol/brain administered into the cisterna ambiens over doses administered in the IC. Swedberg et al. (45) reported that NBQX administered intraperitoneally was effective against audiogenic seizures in DBA/2 mouse at a dose of 3.62 mg/kg. In GEPR, CNQX administered into the IC (15–20 nmol/side) (12) and intraperitoneal injection of NBQX (7.6 –30 ␮mol/kg) (43) were found to be effective against audiogenic seizure. This discrepancy was not readily explained, and the effects of systemic injections of NBQX, in contrast to intracerebral NBQX, have been ambiguous in previous studies (28, 29, 36, 43). Neuroprotective, anti-ischemic, or anticonvulsant actions of systemic NBQX have been reported to be short-lived (28, 43) or very limited (29, 36), which might be a reflection of a lower intracerebral concentration of NBQX administered systemically, supposedly due to low penetration rate through the blood– brain barrier and/or rapid clearance from the brain. NMDA, AMPA, or Cyclothiazide Reproduces Audiogenic-Like Seizures NMDA or AMPA administered into the cisterna ambiens reproduced audiogenic-like seizures (Figs. 3 and 4). Furthermore, cyclothiazide, known a potent agent known to inhibit the desensitization of AMPA receptors (50), administered into the cisterna ambiens or the

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FIG. 3. Rates of incidence of RF alone (stippled bars) and RF ⫹ GTCS (solid bars) following intracisternal administration with NMDA at doses of 3, 5, 10, and 20 ␮g/brain; AMPA at doses of 3 and 6 ␮g/brain; and cyclothiazide at doses of 0.1, 0.5, and 1 ␮mol/brain or bilateral microinjection into the IC at doses of 0.05, 0.1, and 0.5 ␮mol/side. The number of animals in each condition is shown at the top of each bar.

IC also successfully reproduced audiogenic-like seizures (Figs. 3 and 4). AMPA-induced seizures at a dose of 6 ␮g are characterized by durations of RF and GTCS shorter than those of NMDA-induced seizures (Fig. 4), which obviously reflects a rapid desensitization of AMPA receptors in naive rats. Cyclothiazide-induced seizures with longer durations of RF and GTCS at a dose of 1 ␮mol

were similar to that NMDA-induced seizures (Fig. 4). Fedele et al. (13, 14) showed that the cyclothiazideinduced response was prevented not only by NBQX or DNQX, another AMPA receptor antagonist, but also by MK-801. Intracellular recordings in the IC have shown that the time courses of NMDA and AMPA receptor currents are quite different: AMPA currents have rapid rise and decay times, whereas NMDA receptor

FIG. 4. Latency (top) and duration (bottom) of RF (open circles) and GTCS (closed circles) following each drug administration as shown in Fig. 3.

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FIG. 5. Rates of incidence of RF and GTCS after pretreatment with 0.5 and 1 mg/kg MK-801 (IP) or 0.05 and 0.5 nmol/brain NBQX (intracisternal) in NMDA (20 ␮g/brain)-induced seizures, after pretreatment with 0.1 and 0.5 mg/kg MK-801 or 5 and 50 nmol/brain NBQX in AMPA (6 ␮g/brain)-induced seizures and after pretreatment with 0.5 mg/kg MK-801 and 25 nmol/brain NBQX in cyclothiazide (1 ␮mol/brain)-induced seizures. The number of animals in each condition is shown at the top of each bar. Otherwise same as Fig. 1.

currents generally have slow rise and decay times (31). In the barn owl IC, auditory inputs to the external nucleus IC have been found to activate both slow NMDA and fast AMPA receptor currents, indicating that early spikes are primarily generated by AMPA

receptors and that later spikes are supported by both NMDA and AMPA receptors with the NMDA component increasing over the course of the response (16). Taken together, it is speculated that continuous stimulation of the endogenous AMPA receptors elicited by

FIG. 6. Latency (top) and duration (bottom) of RF (open circles) and GTCS (closed circles) in NMDA-, AMPA-, or cyclothiazide-induced seizures with or without pretreatment with MK-801 and NBQX, as shown in Fig. 5. *P ⬍ 0.05, **P ⬍ 0.01 vs vehicle-treated group.

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NBQX to affect cyclothiazide-induced seizures might be explained by the following speculation: cyclothiazide may continuously stimulate endogenous AMPA receptors, which results in retarded and prolonged activation of NMDA receptors (13). To our knowledge, the present result is the first evidence suggesting different roles of NMDA receptors and AMPA receptors within the IC and its vicinity in terms of the components (RF and GTCS) of audiogenic seizures. In the present study, auditory stimulation immediately after the subclinical dose of AMPA administered into the cisterna ambiens augmented AMPA-induced seizures but not NMDA-induced seizures and partially abolished the anticonvulsant effects of NBQX. The same auditory stimulation was found to antagonize MK-801’s effects on RF, but not on GTCS. Therefore it is suggested that the auditory stimulation itself is primarily linked to AMPA receptors. Browning et al. (2) have investigated the effect of NMDA infusions into the IC of normal and lesioned rats and have reported some augmenting effects of auditory stimulation on NMDA-induced seizures when

FIG. 7. (Bottom) Levels of c-fos mRNA are determined by Northern blot using a 32P-labeled c-fos cDNA probe and radiolabeled mRNA is visualized by autography. (Top) Quantification of radiolabeled bands by densitometric scanning. Lane 1, 30 min after cyclothiazide-induced seizures without pretreatment; lane 2, pretreatment with MK-801 (0.5 mg/kg, ip); lane 3, pretreatment with NBQX (25 nmol/brain, intracisternal); lane 4, pretreatment with 1-NA-Spm (50 ␮g/brain, intracisternal). Otherwise same as in Fig. 2.

cyclothiazide permits prolonged activation of NMDA receptors. The Involvement of Glutamate Receptors in Audiogenic or Audiogenic-Like Seizures Both MK-801 and NBQX blocked audiogenic-like seizures induced by NMDA and AMPA in naive rats. However, these antagonists exhibited different behavioral actions; while NBQX significantly blocked RF and GTCS, MK-801 blocked GTCS only in both NMDA- and AMPA-induced seizures as shown in Figs. 5 and 6. This pattern is strikingly similar to the findings in PTU rats. It is thus suggested that NMDA receptors and AMPA receptors are closely related within the neuronal network linking the IC and that the initiation of RF is primarily triggered by AMPA receptors, while the relay mechanism from RF to GTCS is almost completely monopolized by NMDA receptors. The failure of

FIG. 8. Latency (top) and duration (bottom) of RF (open circles) and GTCS (closed circles) in six groups: from left to right (corresponding to lanes 1– 6 in Fig. 9); rats with AMPA (3 ␮g/brain, intracisternal, control condition)-induced seizures (lane 1, Fig. 9), AMPA-induced seizures with auditory stimuli (lane 2), AMPA-induced seizures following pretreatment with MK-801 (0.5 mg/kg, ip) (lane 3), AMPA-induced seizures with auditory stimuli following pretreatment with MK-801 (lane 4), AMPA-induced seizures following pretreatment with NBQX (25 nmol/brain, intracisternal) (lane 5), AMPA-induced seizures with auditory stimuli following pretreatment with NBQX (lane 6). The number of animals in each condition is shown at the bottom of each condition. *P ⬍ 0.05 vs control condition (ANOVA followed by a Tukey test) indicated by shaded bands.

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FIG. 9. The levels of c-fos mRNA were determined by Northern blot as described in Figs. 2 and 7. Shown are representative autoradiograms of c-fos mRNA (bottom) and quantification of radiolabeled bands by densitometric scanning (top) in six groups of rats as shown in Fig. 8. The number of animals in each condition is shown at the bottom of each lane. Each column shows the mean ⫾ SEM. **P ⬍ 0.01 vs control condition (ANOVA followed by a Tukey test).

auditory stimuli were applied 10 min after NMDA infusion. This discrepancy may be due to the different time point of auditory stimulation after the injection of excitatory amino acids. In the present study, the immediate effect of auditory stimulation on AMPA-induced seizures, presumed to be rapidly attenuated, is primarily investigated, since the early response to auditory stimulation is known to be generated by AMPA receptors in the barn owl (16). The Pathway of Audiogenic Seizure Mechanism—Implication of c-fos Expression The immediate-early gene c-fos is thought to be a useful tool for mapping neuronal activity and seizures represent one of the most conspicuous stimuli that induce c-fos expression. Furthermore, c-fos expression may directly be linked to the intracellular seizuregenerating mechanism, since null mutation of c-fos is reported to attenuate pathological plastic changes induced by seizures (49). Our previous in situ hybridiza-

tion (21) and present Northern blot analysis clearly demonstrate the robust increase of c-fos mRNA in the IC following audiogenic or audiogenic-like seizures, implying the critical role of this structure. Expression c-fos does not necessarily indicate the presence of a seizure itself, as shown in the present study where NBQX failed to abolish a marked c-fos expression in the IC despite its potent anticonvulsant action. Audiogenic seizures in PTU rats or AMPA- or cyclothiazide-induced seizures in naive rats caused a marked induction of c-fos mRNA in the IC. In PTU rats, c-fos mRNA was significantly reduced by MK-801, but not NBQX. In naive rats, cyclothiazide-induced seizures caused a similar marked induction of c-fos mRNA in the IC, which was reduced to 85% of control by MK-801 pretreatment though not significant. NBQX again failed to affect c-fos mRNA expression. In contrast to cyclothiazide treatment, a robust c-fos mRNA expression induced by AMPA was strongly inhibited by both MK-801 and NBQX. This indicates that audiogenic seizures in PTU rats strikingly resemble cyclothiazide-induced seizures in terms of behavioral manifestations as well as c-fos mRNA expression in the IC. One can speculate that AMPA receptors are overexpressed in the IC of PTU rats, resulting in audiogenic susceptibility. Fedele et al. (13) have reported that cyclothiazide administered into the rat hippocampus activates AMPA receptors and the rat displays preconvulsive behavior characterized by frequent “wet dog shakes” (WDS) and increases in cGMP levels. They also reported that MK-801 suppressed WDS and cGMP levels, whereas NBQX suppressed WDS but not cGMP levels, which is known to be an activator of c-fos mRNA expression (41). The present semiquantitative method of Northern blot derived from the IC extract indicates that a considerable amount of c-fos mRNA is still preserved even after the application of MK-801, which, we postulate, is due to the overexpressed AMPA receptors. The present study provides no information on the origin of this overexpressed c-fos mRNA, though our previous in situ hybridization study (21) demonstrated that NMDA receptor-sensitive c-fos was expressed in the external cortex IC. According to the study in the barn owl IC (16), auditory information inputs into the central nucleus IC and then propagates to the external cortex IC. It remains to be clarified by further investigations whether this strong expression of c-fos due to the pathological activation of AMPA receptors resides in the central nucleus IC or in other neighboring structures. It has been reported that the anatomical circuitry required for GTCS evoked by sound stimulation in GEPR resides within the brain stem (3) and that the IC is the most critical site in audiogenic seizure propagation (5). In the present study, every drug except cyclothiazide was infused into the cisterna ambiens over the IC in

NMDA AND AMPA RECEPTORS ACT DIFFERENTLY IN AGS

order to leave the IC intact for Northern blot analysis. Although the significance of the IC in the audiogenic seizure generation mechanism is obvious, the implication of neighboring structures can not be excluded. McCown et al. (33, 34) have proposed two distinct pathways for the generation of seizure activity from the IC to the forebrain; one is for RF and the other for GTCS. Likewise, the specific property of drugs tested should be taken into account especially considering some conflicting results on NBQX. It has been reported that NBQX action prefers initial physiological activation to prolonged pathological activation of AMPA receptors (40) and that NBQX is highly selective in vitro, but inhibits both AMPA- and NMDA-evoked spike activity in vivo (32). Our results showed that 1-NA-Spm failed to suppress both audiogenic seizures in PTU rats and audiogenic-like seizure induced by cyclothiazide in naive rats. We have reported a potent anticonvulsant action of 1-NA-Spm against quisqualate-induced seizures (26) and amygdaloid kindled seizures (46). Previous reports show that JSTX specifically blocks Ca 2⫹-permeable AMPA receptor channels lacking the GluR2 subunit (23, 35, 42), which might be independent from audiogenic seizure mechanisms.

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