Comparative fos immunoreactivity in the brain after forebrain, brainstem, or combined seizures induced by electroshock, pentylenetetrazol, focally induced and audiogenic seizures in rats

Neuroscience 123 (2004) 279 –292

COMPARATIVE FOS IMMUNOREACTIVITY IN THE BRAIN AFTER FOREBRAIN, BRAINSTEM, OR COMBINED SEIZURES INDUCED BY ELECTROSHOCK, PENTYLENETETRAZOL, FOCALLY INDUCED AND AUDIOGENIC SEIZURES IN RATS J. B. EELLS,a R. W. CLOUGH,b* R. A. BROWNINGa AND P. C. JOBEc

to be highly activated during both forebrain and brainstem seizures; however, facial and forelimb clonus characteristic of forebrain seizures are not observable during a brainstem seizure. This observation suggests that forebrain-seizure behaviors may be behaviorally masked during the more severe tonic brainstem seizures induced either by MES, PTZ or AGS in GEPRs. This suggestion was corroborated using the sequential seizure paradigm. Similar to findings using MES and PTZ, forebrain regions activated by AT bicuculline were similar to those activated by AGS in the GEPR. However, in the combination seizure group, those areas that showed increased FND in the forebrain showed even greater FND in the combination trial. Likewise, those areas of the brainstem showing FI in the AGS model, showed an even greater effect in the combination paradigm. Finally, the medial amygdala, ventral hypothalamus and cortices of the inferior colliculi showed markedly increased FND that appeared dependent upon activation of both forebrain and brainstem seizure activity in the same animal. These findings suggest these latter areas may be transitional areas between forebrain and brainstem seizure interactions. Collectively, these data illustrate a generally consistent pattern of forebrain Fos staining associated with forebrain-type seizures and a consistent pattern of brainstem Fos staining associated with brainstem-type seizures. Additionally, these data are consistent with a notion that separate seizure circuitries in the forebrain and brainstem mutually interact to facilitate one another, possibly through involvement of specific “transition mediating” nuclei. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Physiology, Southern Illinois University School of Medicine-Carbondale, Carbondale, IL 62901-6503, USA b Department of Anatomy, Southern Illinois University School of Medicine-Carbondale, Carbondale, IL 62901-6503, USA c

Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, IL, USA

Abstract—To help discern sites of focal activation during seizures of different phenotype, the numbers of Fos immunoreactive (FI) neurons in specific brain regions were analyzed following “brainstem-evoked,” “forebrain-evoked” and forebrain/brainstem combination seizures induced by a variety of methods. First, pentylenetetrazol (PTZ, 50 mg/kg) induced forebrain-type seizures in some rats, or forebrain seizures that progressed to tonic/clonic brainstem-type seizures in other rats. Second, minimal electroshock induced forebrain seizures whereas maximal electroshock (MES) induced tonic brainstem-type seizures in rats. Third, forebrain seizures were induced in genetically epilepsy-prone rats (GEPRs) by microinfusion of bicuculline into the area tempestas (AT), while brainstem seizures in GEPRs were induced by audiogenic stimulation. A final set was included in which AT bicuculline-induced forebrain seizures in GEPRs were transiently interrupted by audiogenic seizures (AGS) in the same animals. These animals exhibited a sequence combination of forebrain clonic seizure, brainstem tonic seizure and back to forebrain clonic seizures. Irrespective of the methods of induction, clonic forebrain- and tonic/clonic brainstem-type seizures were associated with considerable Fos immunoreactivity in several forebrain structures. Tonic/ clonic brainstem seizures, irrespective of the methods of induction, were also associated with FI in consistent brainstem regions. Thus, based on Fos numerical densities (FND, numbers of Fos-stained profiles), forebrain structures appear

Key words: epilepsy, immediate early gene, seizure models, rats.

Apart from models of absence epilepsy, two general types of motor convulsions are usually observed in experimental animals induced to have generalized seizures: 1) facial and forelimb (F&F) clonic seizures with or without rearing and falling, and; 2) wild running-bouncing (R/B) episodes followed by clonic/tonic convulsion that may culminate in whole body tonus with complete hindlimb extension (Browning, 1994). Behavioral display of F&F clonic seizures requires the integrity of forebrain structures and these seizures are often referred to as “forebrain” or “limbic” seizures. Complete brainstem transection at the precollicular level separating the prosencephalon from the brainstem, although abolishing F&F clonus due to interruption of seizure propagation to lower motor neurons of the neuraxis, does not alter cortical seizure discharge as measured by electroencephalography (Browning and Nelson, 1986; Magistratis et al., 1988; Browning et al., 1993). In

*Corresponding author. Tel: ⫹1-618-453-1578; fax: ⫹1-618-4531527. E-mail address: [email protected] (R. W. Clough). Abbreviations: AGS, audiogenic seizure; AT, area tempestas; CG, central gray; CnF, cuneiform nucleus; DAB, 3–3⬘ diaminobenzidine-HCI; DCIC, dorsal cortex inferior colliculus; ECIC, external cortex inferior colliculus; F&F, facial and forelimb; FI, Fos immunoreactivity/immunoreactive; FND, Fos numerical density; GEPR, genetically epilepsy-prone rat; GiA, gigantocellularis anterior; LC, locus coeruleus; LPBS, lateral parabrachial nucleus superior; MES, maximal electroshock seizure; mES, minimal electroshock seizure; PBS, phosphate-buffered saline; PBS-Tx-Az, 0.1 M PBS with 0.1% Triton X-100 and 0.1% sodium azide; PL, paralemniscal area; PP, peripeduncular area; PTZ, pentylenetetrazol; R/B, runningbouncing; Rt, reticular thalamic nucleus; SC, superior colliculus; S.D., Sprague–Dawley; SG, supragenicular area; SNpr, substantia nigra pars reticularis; SPF, subparafasicular thalamic nucleus; STh, subthalamic nucleus; VMH, ventromedial nucleus of the hypothalamus.

0306-4522/04$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.08.015

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contrast to F&F clonic seizures, R/B clonic/tonic convulsions require brainstem components at initiation and are often referred to as “brainstem” seizures. Complete precollicular transection does not abolish brainstem seizures showing that these seizures do not require forebrain components for their display. Several animal models of brainstem and forebrain seizures exist including both genetic and experimentally induced models. Genetic rodent-models of brainstem-mediated, generalized seizures include genetically epilepsy-prone rats (GEPRs), audiogenic-susceptible Wistar rats and genetically audiogenic-susceptible mice (reviewed by Buchhalter, 1993; Jobe et al., 1995). Indeed, genetically engineered mouse models of generalized seizure have even been developed (Szot et al., 2001). GEPRs, originally inbred from the Sprague–Dawley strain, represent a naturally occurring model of generalized tonic/ clonic seizures in which intense acoustic stimulation elicits a generalized seizure with no apparent (initial) seizure activation in the forebrain (Jobe et al., 1994; Naritoku et al., 1992). In addition to a marked propensity for audiogenic seizures (AGS), GEPRs show a decreased threshold to a wide variety of other seizure-inducing methods including chemoconvulsants, hyperthermia, electroshock and kindling (Reigel et al., 1986; Faingold, 1988). Models of forebrain seizures are quite extensive and include such treatments as low dose pentylenetetrazol (PTZ), focally applied bicuculline, minimal electroshock, systemic or focally applied excitant amino acids, electrogenic kindling and a number of additional methods (reviewed by Sarkisian, 2001). Interestingly, although forebrain and brainstem seizure circuits can maintain seizure discharge independent of connections with each other, activation of either seizure circuit can eventually modify the other seizure circuit. For example, sequential daily audiogenic seizure (AGS) of brainstem origin can gradually recruit forebrain structures and result in the eventual expression of F&F clonic convulsions and electrographic seizure discharge characteristic of forebrain seizure (Marescaux et al., 1987; Naritoku et al., 1992; Hirsch et al., 1992). In the Wistar audiogenic susceptible rat, the phenomenon of kindling transfer from brainstem to forebrain appears to require the amygdala (Hirsch et al., 1997). Also, previous AGS in GEPR-9s markedly facilitates amygdala kindling in these animals (Savage et al., 1986; Coffey et al., 1996). Thus, brainstemevoked seizures clearly appear to facilitate the kindling of forebrain seizures and may do so through precise and regionally specific neuroanatomical structures. The effects of forebrain seizure activity on brainstem circuits are also apparent but are subtle to observation. For example, repeated forebrain-seizures do not typically induce the motor display of brainstem seizures. Rather, once forebrain seizures are achieved, by kindling for example, the motor pattern of F&F clonus with rearing is maintained. However, in brainstem-seizure susceptible rats (i.e. GEPR-9s), electrical kindling from the amygdala that initially causes F&F clonic forebrain seizures can result in generalization to tonic brainstem seizures (Coffey et al., 1996). Moreover, amygdala kindling that does not elicit

brainstem convulsive behavior in otherwise normal rats is known to reduce the threshold of electroshock induced brainstem seizure (Applegate et al., 1991). Thus, in reciprocity to “kindling” of forebrain seizures by repeated brainstem seizures, there appears to be a facilitating transfer of forebrain seizure circuit activity into what is believed to be a separate brainstem seizure circuitry. Indeed, an elegant study by Ferland and Applegate (1998) examined Fosexpression profiles and found that repeated fluorothyl-induced forebrain seizures result in the eventual induction of brainstem seizure behavior and that an important neuroanatomical locus in this transfer may be the ventromedial nucleus of the hypothalamus (VMH). Our previous studies in GEPRs and other models have also utilized Fos immunocytochemistry to identify potential seizure-related brain nuclei (Clough et al., 1997; Eells et al., 1997, 1998, 2000). Although not without limitations (Dragunow and Faull, 1989; Applegate et al., 1995), expression of c-Fos mRNA or the Fos protein has been a useful marker of neuronal activation associated with a number of physiological and pathophysiological processes (Morgan et al., 1987; Bullit, 1990; Sheng and Greenberg, 1990; Ehret and Fischer, 1991; Honkaniemi 1992) including seizure (Sagar et al., 1988; Dragunow and Robertson, 1987; Daval et al., 1989). The present study sought to directly compare Fos numerical densities (FND) in several rat-brain areas following forebrain seizures or brainstem seizures, each induced by a variety of methods. Additionally, to identify potential sites of interaction between brainstem and forebrain seizure circuitry, a unique combination-seizure paradigm was used. Forebrain or brainstem seizures in rats were induced by a number of different methods. First, low current minimal electroshock (mES) stimuli applied though corneal electrodes cause forebrain seizures (F&F clonic convulsions) whereas higher current maximal electroshock (MES) results in tonic brainstem-type convulsions. Second, administration of a low dose of the chemoconvulsant PTZ in rats induces forebrain-type F&F clonic convulsions whereas high dose PTZ induces initial forebrain-type seizures that are quickly followed by brainstem-type tonic convulsions. Moderate doses of PTZ induce some animals to have typical forebrain seizures with F&F clonus, whereas, other rats at the same dose will show a progression beginning with forebrain type seizure behavior and culminating in tonic convulsion. A moderate single dose was used in the present studies in order to better correlate findings to seizure phenotype rather than dose of the chemoconvulsant. Another model was used in which forebrain seizures were induced in GEPR-9s by microinfusion of bicuculline into an area of the deep prepiriform cortex termed the area tempestas (AT; Piredda and Gale, 1985; Gale, 1992). Brains of these animals were compared with GEPR-9s following a typical brainstem-evoked AGS. Finally, a set of animals was included in which bicuculline was first infused into the AT of the GEPR to induce a forebrain seizure. Once the F&F clonic seizure was clearly under way, animals were exposed to an audiogenic stimulus resulting in a tonic AGS in the GEPR-9. We hypothesized that this combination paradigm (i.e. sequential in-

J. B. Eells et al. / Neuroscience 123 (2004) 279 –292

duction) of AT- bicuculline induced forebrain seizure in the GEPR-9 concomitant with AGS may reveal forebrain, brainstem and perhaps transitional-site patterns of seizure related Fos expression.

EXPERIMENTAL PROCEDURES All experimentation on rats in these studies has been reviewed and approved by the institutional Animal Care and Use Committee of Southern Illinois University. Sprague–Dawley (S.D.) rats (Harlan Laboratories, Indianapolis, IN, USA) or age-matched GEPR-9s obtained from the University of Illinois College of Medicine at Peoria (250 –350 g) were used in this study. All rats were housed in a vivarium on a 12-h light/dark cycle with food and water ad libitum. Rats were monitored for seizure behavior in a cylindrical Plexiglass chamber (40 cm diameter⫻50 cm height). Forebrain seizure behavior was assessed and scored based on a severity rating system developed by Racine (1972). Brainstem seizures were assessed and scored based on a severity rating system developed by Jobe et al. (1973). The numbers of animals in each group are indicated in the tabled results. Electroshock-evoked seizures were induced in S.D. rats via transcorneal stimulation using a Whalquist stimulator and a current duration of 0.2 s (60 Hz, AC). F&F clonic seizures were induced with 22–24 mA of current. Only rats that showed stage 4 or stage 5 F&F clonus, using the scoring method of Racine (1972), were used in the study. Animals that displayed escape or explosive running behavior were not included in the analysis (these represent potential brainstem-evoked activity). Tonic electroshock seizures were induced using the same stimulator and electrodes as that for F&F clonic seizures except that a 150 mA of current was applied. Only rats that showed convulsive behavior consisting of tonic seizures were included in the analysis. PTZ seizures were induced using a dose of 50 mg/kg (i.p.) of PTZ. At this dose of PTZ, a portion of the rats show F&F clonic convulsions only; whereas other rats show an the initial F&F clonic seizure that temporally progresses to a tonic convulsion with forelimb extension and hindlimb flexion. This single dose method was use so that possible differences in Fos patterning in the brains could be attributed to seizure phenotype rather than dosage of PTZ. In all studies, efforts were made to minimize the numbers of animals used and the likelihood of pain and suffering.

Cannula implantation GEPR-9s in which forebrain seizures were to be induced had a stainless steel cannula implanted into the AT. GEPRs were anesthetized with chloral hydrate (350 mg/kg, i.p., Sigma Chemical Co., St. Louis, MO, USA) and placed in a Kopf stereotaxic apparatus. After surgical preparation, an incision was made in the scalp and the skin was reflected. A small hole was drilled in the skull and a single 22-gauge stainless steel guide cannula was lowered into the designated coordinates (1 mm dorsal to the AT). Coordinates for guide cannula placement were modified from a rat brain atlas (Paxinos and Watson, 1986) and were 4 mm anterior to Bregma, 3.1 mm lateral from midline, and 5.4 mm ventral from dura with the incisor bar at ⫹5.0 mm. Coordinate modification was necessary because the GEPR has slightly different skull dimensions then the rats used to develop the Paxinos and Watson (1986) atlas. Appropriate AT cannula placement was histologically confirmed in pilot studies. Three stainless-steel anchoring screws were placed in the skull and the cannula was then permanently secured in place with dental acrylic. Animals were allowed 1 week to recover from guide cannula placement.

AT seizure induction in GEPR-9s F&F clonic seizures were triggered from the AT using the methods of Gale and co-workers (Gale, 1988; Pierrada and Gale, 1985;

281

Maggio et al., 1993). An infusion needle attached to a length of polyethylene (PE-10) tubing was inserted into the guide cannula such that it extended 1 mm below the guide cannula. After placement, 0.5 ␮l bicuculline (200 ng/␮l; Sigma Chemical, St. Louis, MO, USA) was infused into the AT using a Hamilton syringe (attached to the other end of the PE tubing) in a Harvard Apparatus infusion pump. The rats were immediately placed in a Plexiglas chamber and monitored continuously for seizure behavior. Seizures were scored using the rating scale of Racine (1972), and those rats that displayed a stage 4 or 5 seizure following infusion of bicuculline into the AT were designated as the “forebrain seizure” group. GEPR-9s, designated the “brainstem seizure” group, were placed in a Plexiglas chamber and exposed to a 110 dB bell to induce a tonic AGS that was not preceded by F&F clonus. Fos expression in the AT bicuculline-infused animals was compared with GEPR-9s following AGS seizures of severity score 9 (tonic seizure with complete hindlimb extension). In the AT-infused GEPR-9 group undergoing a sequential forebrain seizure interrupted by a brainstem seizure, the animals were observed until they developed F&F clonic convulsions (at least stage 4). Once generalized F&F clonic convulsions were observed, the GEPRs were exposed to a 110 dB bell to induce a tonic AGS. The GEPR-9s used in the brainstem seizure group were seizure naive animals.

Immunocytochemistry Following seizure induction (2.5 h from the beginning of the onset of generalized seizure), animals were processed for Fos immunocytochemistry. Animals were deeply anesthetized with pentobarbital and were transcardially perfused with 50 ml 0. 1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS (200 ml) at 4.0 °C. Brains were removed, post-fixed for 2 h in the same fixative and transferred to 25% sucrose in PBS for cryoprotection. After equilibration in sucrose for 48 h, brain sections were cut on a cryostat-microtome at 40 ␮m thickness and processed free-floating according to conventional ABC (Vector Laboratories, Burlingame, CA, USA) methods (as modified by Berghorn et al., 1994). All rats of each seizure pairing were batch-processed such that comparisons between like brain areas between rats were performed on sections that had been simultaneously processed. Fos immunocytochemistry was performed using a rabbit polyclonal anti-Fos antiserum raised against amino acid residues 4 –17 (Ab2; Oncogene Sci., Uniondale, NY, USA). Briefly, sections were rinsed three times in 0.1 M PBS with 0.1% Triton X-100 and 0. 1% sodium azide (PBS-Tx-Az) followed by incubation in blocking-serum (diluted in PBS-Tx-Az) for 30 min and primary antiserum (diluted 1:1000 in PBS-Tx-Az with 0.1% BSA) for 2 h at room temperature followed by 48 h at 40 °C. The sections were rinsed 10 times in PBS-Tx-Az, incubated in biotinylated anti-rabbit immunoglobulin (diluted in PBS-Tx-Az) for 2 h and rinsed five additional times in PBS-Tx (no azide). The avidin and biotinylated horseradish peroxidase macromolecule complex (ABC) was mixed in PBS-Tx and allowed to equilibrate for 30 min before incubation. Sections were transferred to ABC reagent and incubated for 2 h. The sections were then rinsed three times in 0.1 M PBS and twice in Tris-buffered saline (Tris; 0.2 M). The reaction product was visualized using the chromagen 3–3⬘ diaminobenzidine-HCI (DAB) at 0.44 mg/ml activated with 0.02% hydrogen peroxide. Sections were incubated in activated DAB solution for 5–7 min at room temperature then washed three times in Tris. Sections were mounted on gelatin-dipped slides, dehydrated in a graded alcohol series and coverslipped with clearium. Brain FND was examined on an Olympus BH-2 microscope with a drawing tube attachment. Immunocytochemical controls consisted of sections processed without primary or secondary antibodies as well as a negative control monoclonal antibody provided by Oncogene Science. Fos immunoreactivity was absent in the control sections.

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Quantitative morphometry

A

Brain areas were analyzed for the presence of Fos immunoreactivity (FI) using the following procedure. Processed and mounted sections were first coded for blind analysis and then were examined using a single-blind design to identify brain nuclei that contained FI neurons. Fos immunoreactive regions were mapped on each section according to the Paxinos and Watson atlas (1986) and drawn in camera lucida using a drawing tube attached to the Olympus BH-2 microscope. A semi-quantitative analysis was performed to quantify FND. Nearly all regions showing FI showed a homogeneous spatial distribution of labeled profiles that were numerically quantified by projecting the region under study onto a paper grid of known magnification-corrected dimensions. The grid area represented a minimum of 250 ␮m2 of brain area under study (different brain areas are different size). The number of Fos immunoreactive nuclei were then counted within the grid area to determine the number of nuclei per unit area (⫽numerical area density). Cell nuclei that intersected with the inferior and right lateral lines of the gird area were included; those intersecting with the superior and left lateral were excluded according to standard grid counting stereology. The number of sections/area/rat analyzed was between four and eight. The numbers of nuclei counted in the grid counts were then normalized to 1 mm3 in order make statistical comparisons on standardized norms. From the numerical area density, the numerical volume density (NV⫽number of neuron nuclei/volume of tissue in mm3) was calculated according to standard stereological method (Elias, 1980) as previously described in more detail (Clough et al., 1997). The results are reported as FND.

VMH

ArN

Minimal electroshock

ME

B 3rd V

Data analysis

ArN

Comparisons of FND between paired brain nuclei following high or low current ES were made by two-tailed Student’s t-test. Comparisons of FND between paired brain nuclei following F&F clonic seizure and R/B tonic seizures induced by PTZ were made by Student’s t-test. One-way ANOVAs with Tukey-Kramer post-hoc comparisons were used to compare FND in brain areas following AT seizures, tonic AGS and combination AT seizure with tonic AGS. Probabilities of less than or equal to 0.05 were considered significant in all statistical tests.

RESULTS Seizure behaviors Low-current electroshock (22–24 mA) induced F&F clonic seizures in all four rats examined. MES (150 mA) induced tonic brainstem seizures consisting of tonic extension of the forelimbs or hindlimbs in all four rats examined. PTZ administration (50 mg/kg; i.p.) resulted in stage 5 F&F clonic seizures in three rats, whereas in three other rats, PTZ caused convulsions that began with F&F clonus but progressed to brainstem seizures with tonic extension of the forelimbs (PTZ given i.p. rarely induces tonic extension of the hindlimbs). The tonic PTZ seizures usually subsided to be followed by F&F clonic convulsions that persisted for 8 –10 min. Bicuculline infusion into the AT of GEPR-9s resulted in prolonged F&F clonic seizures in three rats. F&F clonus was observed within 5 min after the end of the infusion and persisted for a duration ranging from 20 to 40 min. Audiogenic stimulation in GEPR-9s resulted in the typical tonic extensor seizures in four rats that were not followed by any forebrain-type seizure activity. In three AT bicuculline-infused GEPRs displaying limbic motor sei-

Maximal electroshock

ME

Fig. 1. Representative bright field photomicrographs of Fos immunolabeling in the ventromedial nuclei of the hypothalamus (VMH). Panel A shows limited Fos immunoreactive profiles in an animal subjected to minimal electroshock and showing facial and forelimb clonic seizures. Panel B shows extensive Fos labeling in the VMH following maximal electroshock winduced running & bouncing tonic seizure. Fos immunoreactive profiles were counted using standard grid counting stereology. ArN⫽arcuate nucleus of the hypothalamus; ME⫽median eminence; the space of the third ventricle is indicated.

zures, audiogenic stimuli during the seizures resulted in transient R/B wild-running episodes culminating in full tonic extension of the fore- and hindlimbs. During tonic AGS, F&F clonic convulsions were not observed; however, about 3–5 min following recovery from the tonic AGS, each rat regained postural control and F&F clonic convulsions re-emerged. FI was observed in numerous brain areas following each seizure type induced by each method. FI Brain FI in these studies are exampled in Fig. 1. Fos labeling in a variety of brain regions, subsequent to seizures, was characterized by punctate, well circumscribed, dark reaction product against a homogeneously gray opaque background. FND and not the intensity of Fos staining per se, was analyzed in these studies. The criterion for inclusion of any labeled profile in the quantitative

J. B. Eells et al. / Neuroscience 123 (2004) 279 –292

283

Table 1. FI neuron-numerical densities (mean⫾S.E.M.) in the forebrain (A.) and thalamus (B.) after F&F clonic or tonic electroshock (ES) A.

F&F clonic ES

Forebrain Bed nucleus of stria terminalis Cortex Dorsal endopiriform cortex Piriform cortex

6333⫾995 16593⫾5783 18145⫾9556 78189⫾29336

4824⫾536 17075⫾2727 2309⫾121 68745⫾10446

Medial amygdala Lateral amygdala Cortical amygdala Amygdalo-hippocampal area Dentate gyrus CA1

73657⫾29647 10601⫾4873 21222⫾8614 10095⫾8614 1919⫾157 468⫾160

32288⫾8828 9189⫾1563 13525⫾1298 18891⫾2608 108190⫾11051* 3550⫾809*

540⫾238 936⫾279

7632⫾1438* 4688⫾681*

CA2 CA3

Tonic ES

B. Thalamus Paraventricular thalamic nucleus Centromedial thalamic nucleus Reunions Lateral posterior mediorostral thalamic nucleus Rt STh SPF PP SG Lateral posterior mediocaudal thalamic nucleus Medial geniculate

F&F clonic ES

Tonic ES

12644⫾2972 11305⫾3596 4565⫾1235 15704⫾3146

11665⫾1226 7311⫾1797 2855⫾224 18361⫾1472

1817⫾557 390⫾42 15680⫾2701 4681⫾1578 2470⫾244 9748⫾3937

1692⫾472 329⫾89 39165⫾3496* 20530⫾2134* 9747⫾934* 11038⫾1887

326⫾167

144⫾97

* Significantly greater using two-tailed Student’s t-test (P⬍0.05) (n⫽4 animals per group).

morphometry was the presence of staining which was clearly evident when viewed through the microscope. Random comparisons between two independent observers revealed an inter-observer reliability of greater than 98%. All clearly labeled cells were included in the grid counts despite subtle but graded differences in staining darkness due to depth location within the section or to the absolute amount of Fos expression in any given cell. Fos expression in clonic vs. tonic electroshock seizures Quantitative FND throughout the telencephalon following either mES induced F&F clonic, or MES induced tonic seizures are shown in Table 1. Generally, although the behaviors were remarkably different, FND in telencephalic areas were similar and independent of the electroshock seizure current. One exception to this was the hippocampus in which MES-induced tonic seizure was related to substantial Fos expression throughout the hippocampus including Ammon’s horn and the dentate gyrus. This labeling was not observed after F&F clonic seizures induced by mES. In contrast to the hippocampus, electroshock-induced seizures of both types produced extremely high FND in the piriform cortex and the medial and cortical amygdala. Electroshock-induced seizures also resulted in increased FND in many of the midline and intralaminar thalamic nuclei; however, no significant differences in FND were found in these areas between rats undergoing clonic and tonic seizures. Several structures in the caudal thalamus, including the subparafasicular thalamic nucleus (SPF), supragenicular area (SG) and peripeduncular area (PP) showed greater FND after tonic seizure compared with the same areas following F&F clonic seizures. Marked FND was evident in the VMH after a MES-induced tonic seizure but was not present after a single F&F clonic seizure. In addition to the VMH, a significantly greater FND was seen in the lateral hypothalamus after MES although the overall labeling in this region was quite small. As shown in Table 2, a number of structures in the tectum and brainstem tegmentum also showed a greater FND after

tonic seizures compared with F&F clonic seizures. These areas included the central gray (CG), deep and superficial layers of the superior colliculus (SC), external cortex of the inferior colliculus (ECIC), cuneiform nucleus (CnF), superior lateral subnucleus of the lateral parabrachial complex (LPBS), paralemniscal area (PL), locus coeruleus (LC), and the nucleus gigantocellularis anterior (GiA). Fos expression in clonic vs. tonic PTZ-induced seizures FND in the forebrain and thalamus following either F&F clonic or tonic seizures induced with PTZ are shown in Table 3. Overall, the most abundant FND labeling in all of these studies was apparent in the PTZ-treated animals even though the seizures were comparatively mild. Fos expression in cortical and limbic structures following PTZ-induced seizures was similar to the electroshockinduced seizure findings in that Fos expression was generally independent of seizure phenotype. Notably, however, the overall amount of cortical and limbic Fos labeling in the PTZ-treated animals was invariably greater than that seen in the electroshock-treated rats. Areas of the telencephalon that showed differences between the F&F clonic seizures and tonic seizure types induced by PTZ included the cerebral cortex and dentate gyrus, both of which showed a significantly greater FND after tonic PTZ-induced compared with F&F clonic PTZ-induced seizures. Other cortical/limbic structures that showed remarkable Fos labeling included the bed nucleus of the stria terminalis, prepiriform cortex and the medial and cortical amygdala. PTZ induced seizures were also related to Fos expression in the thalamus although the only structures that displayed differences related to the types of convulsions included the reticular thalamic nucleus (Rt), SPF and SG region where greater FND was found after tonic PTZ-induced seizures. The PP also showed elevated FND labeling in the tonicseizure group; however, this was not significantly different between groups. Fos labeling in hypothalamus and brainstem following PTZ is shown in Table 4. As with

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Table 2. FI neuron-numerical densities (mean⫾S.E.M.) in the hypothalamus and tectum (A.) and pons/medulla (B.) after F&F clonic or tonic electroshock (ES) A.

F&F clonic

Hypothalamus Anterior hypothalamus VMH Lateral hypothalamus Dorsomedial hypothalamus Tectum Superior Colliculus-superficial layers (SCs) Superior Colliculus-deep layers (SCd) Inferior Colliculus-Dorsal Cortex (DCIC) Inferior Colliculus-External Cortex (ECIC) Inferior Colliculus-Central Nucleus (CIC) Dorsal Nucleus of Lateral Lemniscus (DNLL)

Tonic ES

5768⫾1615 4500⫾898 1044⫾215 5455⫾1580

5267⫾855 43590⫾6992* 2990⫾542* 9487⫾1311

2157⫾449

15421⫾2887*

4136⫾1033

7378⫾655*

10058⫾1449

14245⫾1130

3689⫾1543

B. Pons/Medulla CG dorsal CG medial CG ventral Trapezoid nucleus Periolivary area Superior lateral subnucleus of lateral parabrachial area CnF

F&F clonic ES

Tonic ES

5898⫾617 5153⫾347 2348⫾670 2261⫾953 1784⫾536 6968⫾2765

24329⫾1423* 19976⫾1922* 7257⫾1139* 3053⫾1391 2419⫾753 21496⫾3004*

4133⫾1102

13662⫾1946*

PL

5364⫾1089

13125⫾1367*

15233⫾1916*

LC

15694⫾3817

37337⫾3970*

3697⫾1865

4264⫾1102

GiA

706⫾164

1695⫾178*

108⫾69

107⫾44

* Significantly greater using two-tailed Student’s t-test (P⬍0.05) (n⫽4 animals per group).

tonic electroshock-induced seizures, tonic PTZ seizures resulted in much higher FND in the hypothalami compared with F&F clonic seizures. Significantly greater FND was observed in the anterior hypothalamus, VMH, and dorsomedial hypothalamus of the tonic seizure group. Throughout the tectum and pons/medulla, the areas that showed greater FND following tonic PTZinduced compared with F&F clonic PTZ induced seizures included the CG, SC, dorsal cortex inferior colliculus (DCIC), ECIC, LPBS, CnF, PL, and GiA. In contrast to the other seizure models, no difference in FND in the LC was observed between the different seizure types induced with PTZ.

Fos expression in clonic AT, tonic audiogenic and combination seizures in GEPRs Quantitative FI in GEPR-9 forebrain and thalamus after AT bicuculline-induced F&F clonic convulsions, audiogenicinduced tonic seizures and combination of AT-induced F&F clonic and tonic AGS are shown in Table 5. Forebrain seizures in GEPR-9s, induced by infusion of bicuculline into the AT, were associated with abundant Fos labeling in forebrain nuclei that were similar in most respects, though quantitatively lower in overall numerical density, to Fos patterns after forebrain seizures induced by mES and PTZ that induced F&F seizures in normal rats. FND in forebrain

Table 3. FI neuron-numerical densities (mean⫾S.E.M.) in forebrain (A.) and thalamus (B.) after F&F clonic seizures or tonic seizures induced with PTZ A. Forebrain Bed nucleus of stria terminalis Cortex Dorsal endopiriform cortex Piriform cortex Medial amygdala Lateral amygdala Cortical amygdala Amygdalohippocampal area Dentate gyrus CA1 CA2 CA3

F&F clonic PTZ

Tonic PTZ

24442⫾6664

39339⫾6275

41715⫾3774

60670⫾4395*

10513⫾2707

17339⫾7582

80060⫾7047

90826⫾18467

89889⫾4476 9034⫾1921 53987⫾3426 45876⫾5156

85964⫾8040 12193⫾4286 58621⫾5918 67909⫾9248

167880⫾18206 5024⫾369

254420⫾22092* 8128⫾1584

6160⫾992 6240⫾786

12494⫾3837 15756⫾7732

B. Thalamus Paraventricular thalamic nucleus Centromedial thalamic nucleus Reunions Lateral posterior mediorostral thalamic nucleus Rt STh SPF PP SG Lateral posterior mediocaudal thalamic nucleus Medial geniculate

* Significantly greater using two-tailed Student’s t-test (P⬍0.05) (n⫽3 animals per group).

F&F clonic PTZ

Tonic PTZ

40856⫾13275

56525⫾4855

23368⫾5813

53475⫾9187

21371⫾4460

35426⫾6750

30372⫾4832

35811⫾2032

21428⫾1537 5101⫾2202 27394⫾1469 3258⫾508

38443⫾5456* 19253⫾8447 75390⫾5546* 18657⫾7137

8372⫾2460 17801⫾3409

21005⫾1672* 19931⫾2330

2809⫾1501

2254⫾973

J. B. Eells et al. / Neuroscience 123 (2004) 279 –292

285

Table 4. FI neuron-numerical densities (mean⫾S.E.M.) in hypothalamus, tectum (A.) and pons/medulla (B.) after F&F clonic seizures or tonic seizures induced with PTZ A. Hypothalamus Anterior hypothalamus VMH Lateral hypothalamus Dorsomedial hypothalamus Tectum SC superficial layers SC deep layers DCIC ECIC Inferior colliculus-central nucleus Dorsal nucleus of lateral lemniscus

F&F clonic PTZ

Tonic PTZ

B.

F&F clonic PTZ

6321⫾781 4768⫾1134 2021⫾591 9899⫾1450

26690⫾3742* 33175⫾9719* 15698⫾6111 32907⫾7924*

Pons/Medulla CG-dorsal CG-medial CG ventral Trapezoid nucleus

2165⫾943

10517⫾4570

5203⫾1665 8858⫾1526 3104⫾1032 7387⫾3351

30507⫾1452* 35918⫾5035* 15436⫾639* 9675⫾4262

264⫾134

307⫾19

Periolivary area Superior lateral subnucleus of lateral parabrachial area CnF PL LC GiA

Tonic PTZ

6068⫾1813 8017⫾2129 4983⫾1195 6125⫾2682

49318⫾2264* 39027⫾2028* 38691⫾7613* 5814⫾1484

4109⫾1335 7584⫾2863

6213⫾438 36582⫾5131*

2752⫾927 5276⫾465 41167⫾4888 1225⫾230

28299⫾6821* 23305⫾937* 47043⫾2976 5914⫾660*

* Significantly greater using two-tailed Student’s t-test (P⬍0.05) (n⫽3 animals per group).

whereas the combination group showed marked FND in these areas. There was, however, some variability in Fos expression following F&F clonic AT seizures; certain brain areas showed asymmetrical labeling. For example, the medial amygdala and piriform cortex in one rat of the three showed only ipsilateral Fos labeling compared with strong bilateral labeling in the other two. In contrast, the ipsilateral hippocampus showed strong FI in only one of three rats. In the thalamus, high FND was observed in the Rt after AT

regions in the AT infused group were generally higher, though not statistically, than that in GEPRs undergoing AGS only. The combination of AT-induced F&F clonic and tonic AGS resulted in a dramatic increase in FND in several brain areas over and above that induced by either seizure type alone. This was particularly evident in the medial amygdala and the thalamic nucleus reunions. In these two areas, no differences in FND were seen between the F&F clonic AT seizure and tonic AGS groups

Table 5. FI neuron-numerical densities (mean⫾S.E.M.) in the forebrain (A.) and thalamus (B.) after F&F clonic seizures induced from the AT, tonic AGS or the combination of a F&F clonic seizure and tonic AGS in GEPR-9s A.

F&F clonic AT Tonic AGS

Forebrain Bed nucleus of stria 9537⫾2259 terminalis Cortex 13273⫾4232 Dorsal endopiriform cortex Piriform cortex Medial amygdala Lateral amygdala Cortical amygdala Amygdalohippocampal area Dentate gyrus CA1 CA2 CA3 a

2352⫾701

6576⫾812

F&F clonic AT with tonic AGS

B.

15193⫾1627b b

1499⫾394

36466⫾11334

4757⫾2427

5603⫾1772

71765⫾22332 59644⫾4915 126421⫾9496b 75377⫾11741a,b 19934⫾4218a 46908⫾10912 52396⫾12659

22360⫾11352 8050⫾4021 16590⫾11071 20401⫾14077

7712⫾863 4536⫾1989 7469⫾2035 8161⫾1747

75909⫾72645 1579⫾1336

288⫾184 77⫾45

120334⫾46853 3960⫾1166

3571⫾3255 6634⫾5908

142⫾10 334⫾148

4896⫾1420 11953⫾5980

F&F clonic AT Tonic AGS

Thalamus Paraventricular thalamic 13873⫾5297 nuc Centromedial thalamic 10926⫾4040 nuc Reunions 6528⫾1226

15217⫾2300

29041⫾9631

10163⫾1443

20929⫾5790

8713⫾919

21298⫾4817 11714⫾2194 Lateral posterior mediorostral thalamic nuc Rt 43611⫾4200b 221⫾76 STh 24971⫾7547b 101⫾68 SPF 6129⫾856 30290⫾2685a PP 1095⫾220 22630⫾1350a SG 2088⫾589 16966⫾3264 Lateral posterior mediocaudal thalamic nuc Medial geniculate 1560⫾1560

F&F clonic AT with tonic AGS

6308⫾196a 20132⫾736 72⫾42

25137⫾6375a,b 25850⫾5577 54153⫾5638b 27259⫾8819b 34274⫾1770a 23066⫾4298a 5616⫾1087a 21070⫾2114 214⫾104

Significantly greater than F&F clonic AT seizure group. Significantly greater than tonic AGS group. Superscripts represent a significantly greater Fos immunoreactive numerical density after a one way ANOVA with Tukey-Kramer post-hoc comparisons (n⫽4 Animals per group except F&F clonic seizure group that had three). b

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Table 6. FI neuron-numerical densities (mean⫾S.E.M.) in the hypothalamus, tectum (A.) and pons/medulla (B.) after F&F clonic seizures induced from the AT, tonic AGS or the combination of a F&F clonic seizure and tonic AGS in GEPR-9s A. Hypothalamus Anterior hypothalamus VMH Lateral hypothalamus Dorsomedial hypothalamus Tectum SC superficial layers

F&F clonic AT

Tonic AGS

F&F clonic AT with tonic AGS

2511⫾1111 4875⫾2575 1435⫾336 4584⫾1020

7177⫾2531 13560⫾765 2507⫾392 9994⫾1607

15282⫾5582 28133⫾3905a,b 6744⫾862a,b 15112⫾3139a

326⫾134

12709⫾3337a

11032⫾345a

SC deep layers 1786⫾823 DCIC 1647⫾625 ECIC 571⫾258 Inferior colliculus-central 526⫾78 nucleus Dorsal nucleus of lateral 64⫾64 lemniscus

7118⫾715a 6032⫾378a 5313⫾223a 1514⫾78

6824⫾1053a 9639⫾812a,b 11152⫾225a,b 467⫾180

0⫾0

0⫾0

B. Pons/Medulla CG dorsal CG medial CG ventral Trapezoid nucleus

F&F clonic AT Tonic AGS

3581⫾950 5093⫾191 1867⫾563 144⫾110

Periolivary area 498⫾165 Superior lateral-lat. 1623⫾726 parabrachial area CnF 906⫾382 PL 2745⫾911 LC 12012⫾2698 GiA 675⫾113

F&F clonic AT with tonic AGS

20685⫾2899a 11882⫾1201 5850⫾996 452⫾266

25090⫾3021a 19209⫾3677a 8305⫾2394 517⫾282

2913⫾1000 52370⫾5727a

1642⫾572 44358⫾7619a

15478⫾567a 17789⫾434a 33433⫾3002a 2397⫾205a

14697⫾2612a 14682⫾1195a 40605⫾392a 1829⫾353a

a

Significantly greater than F&F clonic AT seizure group. Significantly greater than tonic AGS group. Superscripts represent a significantly greater Fos immunoreactive numerical density after a one way ANOVA with Tukey-Kramer post-hoc comparisons (n⫽4 Animals per group except F&F clonic seizure group that had three). b

seizures with or without tonic AGS while Fos expression after tonic AGS was virtually absent in the Rt. A similar pattern was observed in the subthalamic nucleus (STh) such that the FND was significantly greater following AT seizures compared with tonic AGS alone. Other thalamic structures including the SPF, PP and SG all showed significantly greater FND after a tonic AGS (with or without F&F clonic convulsions) compared with that following AT induced F&F clonus alone. Shown in Table 6, labeling in the hypothalamus and brainstem of the AT-infused rats was minimal although the LC displayed an appreciable number of Fos-labeled neurons. In the AGS only group, the patterns of labeling were quite similar to that of the MES rat group and the PTZ-treated rats whose seizures culminated in tonic brainstem seizures. Fos labeling in the brainstem was greater in the AGS only group compared with the AT infused group. Comparisons between AT induced F&F clonic and tonic AGS individually showed no significant differences in FND in any area of the hypothalamus analyzed. Notably however, the combination F&F clonic AT seizures and tonic AGS, resulted in significant increases in FND in the VMH and lateral hypothalamus compared with either seizure type alone. FND in the dorsomedial areas of the hypothalamus was also increased in the combination seizure group as compared with the F&F clonic seizure but not to the tonic AGS group alone. Throughout the rest of the tectum and brainstem, patterns of Fos expression observed after tonic AGS were similar to those seen in the other two tonic seizure models. Significantly greater FND was observed in the dorsal portion of the CG, superficial and deep layers of the SC, LPBS, CnF, PL, LC and GiA after tonic AGS compared with F&F clonic seizures alone. Within the IC, the Fos labeling in the ECIC and DCIC were significantly greater in

the tonic AGS group compared with the F&F clonic seizure group. Additionally, FND in the ECIC and DCIC after combination of tonic AGS and F&F clonic seizures were significantly greater then the two seizure types alone.

DISCUSSION F&F clonic seizures and R/B tonic/clonic seizures are two distinct phenotypes of motor convulsions and it is generally accepted that the former represent forebrain seizures and the latter brainstem evoked seizures (Browning, 1994). It was reasoned that inferences about brain areas participant in distinct motor convulsions could be made by paired analyses of FI between brainstem and forebrain type seizures induced by several different methods. Thus, the present study compared FI between brain areas following F&F clonic forebrain and R/B tonic brainstem seizures induced by three different methods. FI has been useful in determining potential involvement of specific neuronal populations in a number of different experimental seizure models (Morgan et al., 1987; Dragunow and Robertson, 1987; Daval et al., 1989; Shehab et al., 1992; Maggio et al., 1993). Notably however, the use of Fos expression to identify seizure-relevant brain areas is not without limitations (Dragunow and Faull, 1989; Applegate et al., 1995). For example, seizure activity per se might cause Fos activation in brain areas surrounding the seizure pathways that are not components of the seizure. However, in this context, one may expect to see a more generalized and perhaps uniform collection of Fos-labeled areas rather than the regionally restricted profiles that have usually been described in association with seizures and other physiological brain activations (Morgan et al., 1987; Bullit, 1990; Sheng and Greenberg, 1990; Ehret and Fischer,

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1991; Honkaniemi 1992; Clough et al., 1997; Eells et al., 1997; Ferland and Applegate, 1998). Additionally, brain areas involved in the seizure per se, may not show increased Fos expression in response to seizures, as for example occurs in AGS where the central nucleus of the IC shows increased 2-deoxyglucose utilization, but not Fos expression following intense AGS (Eells et al., 2000). Thus, although fos expression patterns have certainly advanced our knowledge of brain sites involved in seizures, there are confounding examples as with many other methodologies. Nevertheless, quantitative analysis and regional localization revealed considerable and site-specific differences in FI profiles between forebrain or brainstem seizure phenotypes. Forebrain FI subsequent to seizures Forebrain type seizures were induced by systemic PTZ, mES or microinfusion of bicuculline into the AT according to the general methods of Maggio et al., (1993). Several forebrain structures showed appreciable Fos labeling subsequent to these seizures. Notably, with few exceptions, the regional labeling of forebrain structures was similar between each of the three seizure-evoking stimuli. Additionally, the number of cells labeled in any given brain area appeared generally similar between the three different treatment groups although the highest apparent levels of Fos labeling, with few exceptions, were observed in the PTZ-treated animals. Forebrain-seizure related Fos expression was observed in the cerebral cortex, limbic and paralimbic structures, basal ganglia and certain parts of the diencephalon. Prosencephalic FI was observed in the cerebral cortex per se, bed nucleus of the stria terminalis, inferior temporal structures including the endopiriform and piriform cortex, the medial and cortical amygdala and the hippocampus. Of all the forebrain structures showing high Fos labeling, the piriform cortex and the amygdala were the most consistent between the three methods of forebrain seizure induction. In the hippocampal formation, labeling of the dentate gyrus was very high in the PTZ and the AT bicucullinetreated groups, the latter being similar to that reported by Maggio et al. (1993). In contrast, Fos labeling was nearly non-existent in mES-treated rats despite the display of seizures similar in phenotype to those induced by PTZ or AT-bicuculline. This observation suggests that the hippocampal formation may not be obligatory in the expression of the forebrain-type seizure despite its heavy Fos labeling in the PTZ and AT infused animals. This finding is not without precedence as Barton et al. (2001) have shown that the hippocampal formation exhibits little fos staining in mice undergoing 6 Hz mES-induced forebrain-type seizures. Similar differential activation of the hippocampus was reported following seizures induced with i.v. picrotoxin in which F&F clonic convulsions showed no Fos induction in the hippocampus, while tonic brainstem seizures induced strong Fos expression in the hippocampus of mice (Willoughby et al., 1995). In contrast, Samoriski et al. (1997) reported similar Fos expression in the hippocampus following either electroshock induced F&F clonic or tonic

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seizures in mice. This apparent discrepancy in regards to electroshock and hippocampal Fos labeling in mice may be due subtle differences in intensity currents, electrode placements or other variables that contribute to F&F clonic or tonic convulsion in mice. Although the hippocampus is commonly implicated in forebrain seizures, these data suggest that seizure spread to the hippocampus during mES may not be obligatory for the expression of F&F clonic convulsions or at least of sufficient intensity to induce expression of Fos. Apparently, activation of the hippocampus may depend upon the method of seizure induction rather than the phenotype of the seizure displayed. Brainstem-evoked seizures were induced with MES, audiogenic stimulation in GEPRs and in select animals following PTZ administration. In each seizure paradigm, Fos induction was strong throughout the forebrain. In general, forebrain Fos-labeling in any given area in the tonic seizure groups was similar in magnitude to the labeling following the forebrain type F&F seizures. This finding was expected for PTZ and MES, which provide a global stimulation of the brain, but was unexpected for audiogenic stimulation of GEPRs which is believed to trigger brainstem seizures selectively. Exceptions to this finding were observed in the hippocampal formation in which the AGS in the GEPR was not associated with labeling of Fos in the dentate gyrus or the cornu ammonis nor in the amygdala where AGS alone in the GEPR induced comparatively less Fos induction than in the other brainstem seizure models. It is noted that these GEPRs were seizure naive prior to the study. However, similar to the other brainstem seizure models, heavy Fos labeling was observed in the piriform cortex following AGS in the GEPR. This was not expected, insofar as the forebrain is not required for acute expression of tonic seizures in GEPRs and the significance of this finding, thus, is purely unknown. However, this observation is consistent with the notion that brainstem seizure activity is propagated into and may be related to kindling of the forebrain by brainstem seizures, as is known to occur in the GEPR (Naritoku et al., 1992) and the audiogenic Wistar rat (Hirsch et al., 1992). Some structures in the posterior thalamus were uniquely activated following tonic seizures as compared with F&F clonic seizures whereas other nuclei were uniquely activated following F&F clonic seizures but not tonic seizures. Interestingly, the Rt nuclei were intensely activated with F&F clonic AT seizures, AT seizures in combination with tonic AGS or with PTZ induced seizures. However, the Rt was virtually devoid of staining following tonic AGS in the GEPR-9s of the present study. The Rt is a collection of predominately GABAergic neurons that receive input from terminal collaterals of corticothalamic and thalamocortical axons (Houser et al., 1980; Mitrofanis and Guillery, 1993). The Rt in turn sends projections to nonspecific intralaminar and midline thalamic nuclei as well as sensory-specific thalamic nuclei (Kolmac and Mitrofanis, 1997). Absence seizures have been shown to accompany spike-wave seizure discharge involving the Rt and thalamo-cortical circuits (Avanzini et al., 1993) and a commonly used model for human absence seizures is low dose

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PTZ that results in spike-wave seizure discharge (Marescaux et al., 1984). Thus, the activation of the Rt as suggested by Fos expression after AT bicuculline or PTZ induced convulsions may reflect this spike-wave seizure activity; however, is was not possible to witness absence seizures in these animals owing to ongoing F&F clonus. In contrast to the Rt thalamic nuclei, the SPF and SG nuclei showed strong activation that was unique to tonic seizures, regardless of the inducing stimuli. In this regard, the SPF and SG thalamic nuclei were similar in response to several of the brainstem nuclei (next section). Several basal ganglia-related structures have been implicated as modulatory areas in seizure propagation and/or expression. In the present GEPR-9 study, the STh showed a response that was similar to the Rt. The STh was intensely activated in AT-bicuculline-induced seizures and combined AT-AGS in GEPRs, whereas it was devoid of staining in the AGS alone. The STh modulates excitability of the substantia nigra pars reticulata (SNpr) via an excitatory projection (Kita and Kitai, 1987; Smith and Parent, 1988; Smith et al., 1990; Albin et al., 1989). Inhibition of the STh or blockade of excitatory input to the SNpr has been reported to be anticonvulsant in a number of forebrain and brainstem seizure models including AT seizures (Maggio and Gale, 1989; reviewed by Gale, 1992), amygdala kindling (McNamara et al., 1984), tonic electroshock (Iadarola and Gale, 1982) and tonic AGS (Millan et al., 1988). Additionally, stimulation of the striatum, a basal ganglia structure with an inhibitory GABAergic projection to the SNpr, also has anticonvulsant effects in some seizure models (Cavalheiro et al., 1987; Turski et al., 1987). Based on these data, activation of STh neurons may activate the SNpr and result in a proconvulsant effect during AT-bicuculline seizures in GEPRs, but not in AGS alone. The high levels of Fos expression in the STh as a result of AT seizures suggest that activation of STh certainly occurs during AT seizures. Indeed, recent studies have shown that inhibition of the STh nucleus with muscimol attenuates F&F clonic AT seizures (Dybdal and Gale, 2000) as well as fluorthyl-induced limbic-type seizures (Veliskova et al., 1996). A possible differential role of the basal ganglia, particularly the STh nucleus, in forebrain versus brainstem seizures requires further study. Brainstem FI subsequent to seizures Considerable differences in the distributions of Fos were observed in midbrain, pontine and medullary structures after tonic seizures compared with F&F clonic seizures in all models analyzed. Tonic seizure resulted in a significantly greater density of FI neurons in PP, CG, portions of the SC and IC, LPBS, CnF, PL and the nucleus GiA. Each of these areas consistently showed greater Fos activation following generalized tonic/clonic seizures compared with F&F clonic seizures regardless of the method of seizure induction. Activation of brainstem nuclei during R/B tonic/ clonic seizures appears to result in a similar distribution of FI in S.D. rats and GEPR-9s no matter the inducing stimuli. Several other studies have analyzed Fos expression after brainstem seizures and have obtained results similar to the

present findings (Samoriski et al., 1996; Snyder-Keller and Pierson, 1992; Shehab et al., 1992; Simler et al., 1994). The effects of forebrain seizures on brainstem Fos expression were comparatively marginal with the notable exception of the mesencephalic tectum, particularly the cortices of the IC (see below). Brain areas of seizure interaction The interactive potential of forebrain and brainstem seizure activity upon one another provides an intriguing area of investigation. Two brain regions involved in the interactions between forebrain and brainstem seizure circuitry appear to be the VMH (Ferland and Applegate, 1998) and portions of the amygdala (Hirsch et al., 1997). Tonic seizures induced with either electroshock or PTZ in normal S.D. rats or audiogenic stimulation in GEPR-9s resulted in significantly greater Fos induction in the VMH and amygdala then did F&F clonic seizures alone. These data suggest that brainstem seizure activity may acutely propagate toward the forebrain via the VMH and/or directly on to the temporal lobe (piriform cortex and amygdala). Anatomical evidence supports the possibility of interactions of forebrain and brainstem structures though the VMH. Tracing studies have shown input to the VMH from forebrain structures including the medial amygdala, amygdalo-hippocampal area and PP (Luiten and Room, 1980; Fahrbach et al., 1989) as well as from several brainstem nuclei including the CG and LPBS (Arnault and Roger, 1987; Fulwiler and Saper, 1985). Our combined tract-tracing and Fos immunocytochemistry studies have shown that tonic-seizureinduced FI neurons in the medial amygdala, PP and LPBS have efferent projections to the VMH (Eells et al., 1998; and unpublished observations). Therefore, a number of seizure-activated areas in the forebrain and brainstem provide input to the VMH. Although a physiological role for the VMH in interactions between forebrain and brainstem seizure circuits remains to be established, fluorothyl-induced epileptogenesis has persistent effects on activation of the VMH neurons that are related to brainstem kindling by forebrain seizure circuits (Ferland and Applegate, 1998). Moreover, repeated F&F clonic flurothyl-induced seizures result in increasing levels of Fos in the VMH compared with an initial seizure suggesting that repeated seizures result in progressive alterations in the input and/or responsiveness of the VMH (Samoriski et al., 1996, 1997). Simler et al. (1994) also reported increased Fos expression in the “medial hypothalamus” after audiogenic kindling, however, whether this represents the VMH proper was not clear. Activation of the VMH is clearly induced by seizure activity. What remains to be determined is whether the VMH has a facilitating role in the presumed kindling interactions between forebrain and brainstem seizure circuitries or whether VMH activation is coincidental. Nonetheless, in addition to brainstem seizures facilitating forebrain seizures, it is evident that forebrain seizures can facilitate development of brainstem type seizures and that both phenomena may involve the VMH and amygdala (Samoriski et al., 1996; Ferland and Applegate, 1998).

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Tonic AGS, when displayed during the course of a forebrain seizure induced by AT bicuculline, appeared to markedly increase Fos expression in those areas of the forebrain already showing Fos induction in the forebrain seizure models. In nearly every forebrain area, the combination of seizures appeared to induce Fos expression over and above that induced by either seizure alone. This finding also corroborates a propagating communication between the brainstem and the forebrain. In particular, examination of the amygdala showed that Fos labeling in the combination paradigm was significantly greater than that observed after forebrain-induced seizures and the brainstem-evoked AGS. Other structures in the vicinity, including the piriform cortex and the amygdalo-hippocampal transition area also showed remarkable Fos induction in the combination-seizures paradigm although these differences did not reach statistical significance (perhaps owing to variability between ipsilateral and contralateral FI). Nonetheless, this observation suggests that this general area of the brain receives propagated signals from the brainstem that, theoretically over time, could transfer seizure propensity to forebrain circuitry as occurs with repeated AGS in GEPRs (Naritoku et al., 1992). Tonic AGS resulted in consistent and strong Fos expression in several cortical and sub-cortical structures even though the animals used in this study were seizure naive GEPRs. This is interesting in that previous reports have shown that initial AGS in GEPRs cause no cortical EEG activity (Naritoku et al., 1992). Nonetheless, the present findings using Fos expression suggest that AGS can profoundly activate certain forebrain structures, the hippocampus excepting. In contrast, AGS susceptible Wistar rats show very limited Fos expression in cortical and limbic regions after their first AGS but show increased telencephalic Fos expression with repetition of seizures (Simler et al., 1994). The slight difference in the Wistar and GEPR in forebrain Fos expression after initial AGS may relate to the intensity of seizure. For example, the seizure discharge in the GEPR brainstem may exceed that of the Wistar AGS susceptible rat insofar as the Wistar AGS does not culminate in complete tonic hindlimb extension. Alternatively, increased Fos expression in forebrain regions subsequent to AGS in the GEPR-9 may reflect a more generalized seizure predisposition that renders the GEPR brain more susceptible to both forebrain and brainstem seizures (Browning et al., 1990; Coffey et al., 1996). Indeed, GEPR-9s display enhanced susceptibility to a number of convulsive manipulations including electroshock, PTZ, flurothyl, and forebrain kindling (Browning et al., 1990; Franck et al., 1989; Savage et al., 1986). Repetitive AGS eventually results in forebrain seizure discharge that is absent following an initial AGS (Marescaux et al., 1987; Naritoku et al., 1992). Moreover, repetitive brainstem seizures eventually induce F&F clonic seizures that become behaviorally manifest after the brainstem seizures wane. Additionally, kindled AGS results in increased Fos expression in a number of forebrain areas including amygdala, hippocampus, piriform cortex and medial hypothalamus (Simler et al., 1994). Thus, repeated

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AGS appears to cause epileptogenic changes in forebrain areas that include amygdala, hippocampus, piriform cortex and medial hypothalamus. Both the piriform cortex and amygdala have been shown to be important components of the forebrain seizure circuitry using other experimental methods. For instance, the deep prepiriform cortex containing the AT is an extremely sensitive site of seizure initiation, and inhibition or inactivation of this area can attenuate convulsions in a number of forebrain clonic seizure models (Millan et al., 1986; Piredda and Gale, 1986; Stevens et al., 1988; Gale, 1992). One primary output pathway from AT may include the piriform cortex since blockade of excitatory amino acid transmission in piriform cortex prevents AT-initiated seizures (Halonen et al., 1991). Therefore, activation of medial amygdala and the piriform cortex may represent areas in which repeated AGS acts to induced F&F clonic seizure behavior. In contrast, the majority of caudal FI areas activated after tonic seizures were unaffected by F&F clonic convulsions. A notable exception was observed in the DCIC and ECIC respectively. These brainstem areas showed graded Fos expression such that low levels were expressed following F&F clonic seizure, significantly higher levels were seen with tonic seizures, and even greater induction of Fos was observed after a combination of F&F clonic and tonic AGS. As noted previously, forebrain seizures can have a facilitating effect on brainstem seizure propensity (Applegate et al., 1991); thus, a possible “brainstem-kindling” target of forebrain seizure activity may include the cortices of the IC. The IC is a seizure susceptible area of the brainstem and is believed to be the site of AGS initiation since lesions or focal inhibition of the IC abolish AGS (Willot and Lu, 1980; Frye et al., 1986; Faingold et al., 1988). The IC cortex is also a site highly susceptible to induction of brainstem seizure behavior using electrical stimulation or microinfusion of various excitatory agents and is an area previously shown to have an increased expression of Fos after AGS (Millan et al., 1986; Faingold et al., 1989; McCown et al., 1984; Clough et al., 1997). AGS is thought to require propagation from the central nucleus of the IC to the ECIC since transections separating these structures abolish AGS (Ribak et al., 1994). In the present study, a single AT seizure in the GEPR-9 did not generalize to tonic seizures without the audiogenic stimulation; however, amygdalakindled seizures can generalize to tonic seizures (Coffey et al., 1996). It is not known if repeated AT seizures in the GEPR would eventually trigger tonic seizures; however, if a similar “graded” activation of the DCIC or ECIC occurs after amygdala-kindling as it does after AT seizures, then amygdala-kindled seizures in GEPR-9s may generalize to tonic seizures as a result of activation of the DCIC and ECIC. Certainly, forebrain seizure activity had comparatively little effect on Fos expression in brainstem structures other than the cortical portions of the IC. This suggests that forebrain seizures do not potentiate brainstem seizures or Fos activation by way of the PP, CG, PBls, Cn, PL or the GiA. Although there is strong evidence that forebrain seizure activity potentiates brainstem seizure susceptibility, it has yet to be determined whether this occurs through the

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IC. Additionally, the physiological roles of the PP, CG, PBLs, Cn, PL and the GiA structures in seizures remain to be determined. Nonetheless, unique Fos expression in brainstem areas following brainstem-evoked seizures, as compared with forebrain seizure activation, is consistent with a theory of separate forebrain and brainstem seizure circuits mediating different convulsive seizure patterns. In summary, classification of phenotypically distinct convulsions as either forebrain or brainstem mediated appear to coincide with Fos expression in the appropriate levels of the neuraxis. Several previous studies have also shown that Fos expression is linked more closely to the seizure behavior rather than to the dose of the chemoconvulsant or the intensity of electrical stimulation (Willoughby et al., 1995; Samoriski et al., 1997). Additionally, Shehab et al. (1992) reported considerable anatomical differences in Fos localization between tonic ES and F&F clonic PTZ seizures in which convulsions were matched based on duration of seizure behavior and not the seizure phenotype. Thus, these data collectively are consistent with the notion of separate neural circuitries for brainstem and forebrain seizures. Finally, the anatomical interactions between forebrain and brainstem circuitries, as related to “circuit-facilitating” transfer of seizure activity between the two regions remains to be further explored. However, there appears to be a role for the VMH and perhaps the amygdaloid area in transfer of seizure activity between the brainstem and forebrain and vice versa. Acknowledgements—This work was supported originally by the Epilepsy Foundation of America (RWC) and by two internal SIU research initiative CRC awards (RWC, RAB). J. B. Eells was a recipient of a Predoctoral Dissertation Research Award from the Graduate School of SIU.

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(Accepted 14 August 2003)