196
Brain Research, 538 (1991) 196-202 Elsevier
BRES 16224
Kindling from stimulation of a highly sensitive locus in the posterior part of the piriform cortex. Comparison with amygdala kindling and effects of antiepileptic drugs Dagmar H6nack, Ulrich Wahnschaffe and Wolfgang L6scher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Hannover (F.R. G.) (Accepted 7 August 1990) Key words: Epilepsy; Kindling; Amygdala; Piriform cortex; Antiepileptic drug
The piriform cortex, especially its deep anterior part, has been recently suggested to be a crucial epileptogenic site in the rat brain. We investigated the susceptibility of different parts of the piriform cortex to the development of electrical kindling as compared to that of the basolateral amygdala. A locus in the deep cell layer (layer III) of the rostral portion of the posterior piriform cortex (PPC) is described, which is considerably more sensitive to electrical stimulation than adjacent areas of the PPC, including the deep prepiriform cortex or the amygdala. The sensitive locus in the PPC can be readily kindled, and focal seizure thresholds in fully kindled rats are 60-90% lower than respective thresholds in rats kindled from other loci. Treatment of fully kindled rats with the antiepileptic drugs diazepam, carbamazepine, phenobarbital, and valproate showed that anticonvuisant effects of these drugs in animals kindled from stimulation of the PPC were comparable to respective effects in animals kindled from stimulation of the basolateral amygdala, although the locus in the PPC tended to be more resistant. The data support the idea that the piriform area may contain the most sensitive neuronal tissue responsible for the generation of seizures during kindling. It remains to be determined if the described locus in the PPC is critical to the kindling process when kindling is induced from other structures within the olfactory-limbic system. INTRODUCTION The piriform (primary olfactory) cortex receives its primary afferent input from the olfactory bulb, but there are also inputs from the amygdala, hippocampal formation and neocortical areas 8-1°'16. Although the piriform cortex is not considered to be a part of the limbic system, its outputs directly and indirectly project to several limbic structures, such as the septal area, amygdala and hippocampal region 8-1°'16. Goddard et al. 7 were the first to demonstrate that structures of the olfactory-limbic system, including the piriform cortex, are highly susceptible to the development of seizures following local application of electrical stimuli. In recent years, several lines of evidence have suggested that the piriform cortex might be critically involved in generation and/or propagation of seizure discharge. Thus, (1) kindled seizures induced by repeated focal stimulation develop most quickly after electrical stimulation of the amygdala, piriform cortex or its primary afferent, the olfactory bulbT'2°'28; (2) regardless of the location of subcortical kindling sites, interictal discharges begin first in the piriform cortexll'26; (3) autoradiographic studies during amygdala kindling de-
velopment showed high metabolic activity in the piriform cortex1'5'19; and (4) following status epilepticus evoked by stimulation of a kindled amygdala, significant neuronal degeneration is observed in the entire ipsilateral piriform cortex iS. If the piriform cortex is involved in seizure generation, the question still remains as to whether there is a sole generator within that region, where within the rather large piriform region the generator resides, and how much tissue is involved. It was, therefore, of interest that Piredda and Gale 22 reported the existence of a specific area in the deep anterior part of the piriform cortex (deep prepiriform cortex (DPC)) in which microinjection of picomole amounts of bicuculline or other chemoconvulsants produced electrographic and convulsive responses resembling those induced by amygdala kindling. Piredda and Gale hypothetized that this tiny area within the D P C ('area tempestas'; anatomically the ventral endopiriform nucleus, which is deep to layer III of the prepiriform cortex 16) may be a crucial epileptogenic site in the brain and may also be responsible for the development of kindled seizures. However, several more recent studies suggest that the role of the D P C in epileptogenesis is limited. Thus, it was shown that the
Correspondence: W. LOscher, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, ER.G. 0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
197 t h r e s h o l d for i n d u c t i o n of a f t e r d i s c h a r g e s ( A D T ) in the D P C is n o t d i f f e r e n t f r o m t h e A D T o f the a m y g d a l a o r a r e a s i m m e d i a t e l y a d j a c e n t to D P C 3'3°. T h e p r e p i r i f o r m c o r t e x can be readily k i n d l e d , but n o t faster t h a n t h e a m y g d a l a 3'2°'3°. L e s i o n s o f t h e D P C , including the site d e s c r i b e d by P i r e d d a and G a l e 22, did n o t affect the r a t e of electrical k i n d l i n g o f the a m y g d a l a 3"2°'3° o r the convulsant
potency
of
systemically
administered
bicuculline 29. Similarly, knife-cuts, disrupting c o m m u n i c a t i o n b e t w e e n a m y g d a l a and a n t e r i o r p i r i f o r m structures, did n o t significantly i n c r e a s e the n u m b e r of s t i m u l a t i o n s r e q u i r e d for a m y g d a l a kindling 27. H o w e v e r , t h e r e is still t h e possibility that o t h e r parts o f the p i r i f o r m c o r t e x m a y s e r v e as a g e n e r a t o r
of limbic seizures.
I n d e e d , lesions o f the p i r i f o r m c o r t e x that i n v o l v e d the p o s t e r i o r p a r t o f this r e g i o n w e r e r e c e n t l y s h o w n to significantly i n c r e a s e t h e n u m b e r of stimulations r e q u i r e d to c o m p l e t e septal kindling 27. In the p r e s e n t study, we d e s c r i b e a locus in the d e e p p o s t e r i o r p i r i f o r m c o r t e x ( P P C ) which is substantially m o r e susceptible to electrical stimulation
t h a n the a m y g d a l a o r o t h e r parts of the
p i r i f o r m cortex. T h e p h a r m a c o l o g i c a l sensitivity o f this locus has b e e n c h a r a c t e r i z e d by e x p e r i m e n t s with clinically e s t a b l i s h e d a n t i e p i l e p t i c drugs.
MATERIALS AND METHODS
Preparation of animals All experiments were carried out in female Wistar rats, weighing 210-230 g. Sixty-five rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and received stereotaxic implantation of one bipolar electrode into the right hemisphere according to the surgery methods described in the atlas of Paxinos and Watson 21. Rats in the amygdala group (n = 23) received implantation of one electrode aimed at the basolateral amygdala at the following coordinates: AP -2.2, L -4.8, V -8.5. Rats in the DPC group (n = 10) received implantation of one electrode aimed at the region ('area tempestas') described by Piredda and Gale 22 at the following coordinates: AP +2.2, L -2.6, V -7.8. Rats in the PPC group (n = 32) were subdivided into a group (PPC1; n -- 22) with electrode implantation in the deep rostral portion of the PPC, and a group (PPC2; n = 10) in which the electrode was aimed at a more caudal locus in the PPC, according to the PPC locus previously examined by Racine 24 and Cain et al. 3. Coordinates for PPC 1 were AP -0.8, L-4.8, V -8.5, and for PPC 2 AP -3.3, L -5.9, V -9.0. All coordinates were measured from bregma. Skull screws served as the indifferent reference electrode. The electrode assembly was attached to the skull by dental acrylic.
Kindling After a postoperative period of 2 weeks, constant-current stimulations (500-/~A, 1-ms, monophasic square-wave pulses, 50/s for 1 s) were delivered to the piriform cortex or amygdala at intervals of 1 day until 10 stage 5 seizures were elicited. The electrical susceptibility of the stimulated region was recorded on the first day of the experiment (initial ADT) as well as after kindling acquisition (with an interval of at least 4 days after the 10th stage 5 seizure) using an ascending stairstep procedure 6. The initial current intensity was 10 aA, and the current intensity was increased in steps of about 20% of the previous current at intervals of 1 min until an afterdischarge of at least 3-s duration was elicited. Since
almost all fully kindled animals (except for 4) exhibited generalized seizures (stage 4-5) at the ADT current, it was not necessary to determine the threshold for generalized seizures (GST) separately. In fully kindled rats, the ADT determined with interstimulation intervals of 1 day was not different from ADT values determined with interstimulation intervals of 1 min, thus demonstrating that the short interstimulation interval did not bias ADT determinations. In addition to ADT, in fully kindled rats, the following parameters of kindled seizures were measured after stimulation with either the ADT current or suprathreshold stimulation with 500/zA. Seizure severity was classified according to Racine23: 1, immobility, eye closure, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 4, rearing, often accompanied by bilateral forelimb clonus; 5, rearing with loss of balance and falling accompanied by generalized clonic seizures. Seizure duration was the duration of limbic (stage 1-2) and/or motor seizures (stage 3-5); limbic seizure activity sometimes occurring after termination of stage 3-5 seizures was not included in seizure duration. Afterdischarge duration was the total time of spikes in the EEG recorded from the site of stimulation.
Drug testing The effects of diazepam (5 mg/kg i.p.), valproate (VPA; 200 mg/kg i.p.), carbamazepine (20 mg/kg i.p.) and phenobarbital (20 and 30 mg/kg i.p.) were compared in PPC 1- and amygdala-kindled rats. For anticonvulsant drug testing, all stimulations were carried out with 500/~A. The drugs were injected 15 min (VPA), 30 min (diazepam, carbamazepine) or 60 min (phenobarbital) prior to stimulation. Dosages and pretreatment times were chosen on the basis of previous dose-effect experiments with the drugs in kindled rats 14. Control readings were determined 2-3 days prior to and after each drug administration, and the next drug experiment was only undertaken if all rats showed reproducible stage 5 seizures. None of the drug vehicles (see below) had an effect on kindled seizure parameters. All rats received each treatment; the sequence of drug testing was diazepam, VPA, carbamazepine and phenobarbital; at least 4 days were interposed between two drug injections. In order to examine if this sequence of drug testing altered anticonvulsant drug efficacy, one of these drugs, i.e. diazepam, was retested at the end of the experiments. No difference in anticonvulsant efficacy was found between the two test sessions with diazepam.
Histology After termination of the last drug experiment, placement of stimulating electrodes was examined histologically in all animals. The rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and perfused with a fixative consisting of 4% phosphate buffered formaldehyde (pH 7.3). Two h later, the brains were removed, postfixed overnight and processed for paraffin embedding. Subsequently, serial sections of the entire brain were cut coronally at 7 gm, and every 20th section was mounted on a glass slide and stained with Cresyl violet or hematoxylin eosin.
Statistics All data are given as means + S.E.M. Significance of differences between seizure readings in the same group of rats was calculated by the Wilcoxon signed-rank test for paired replicates. Significance of differences between different groups was calculated by Student's t-test.
Drugs VPA, phenobarbital (both used as their sodium salts) and carbamazepine were obtained from Desitin (Hamburg, ER.G.), and diazepam from Hoffmann La Roche (Basle, Switzerland). VPA, phenobarbital and diazepam were dissolved in water (diazepam by means of dilute HCI), carbamazepine was dissolved in 30% polyethylene glycol 400 in water. All antiepileptic drugs were freshly dissolved prior to each experiment and injected i.p. at a volume of 2-3 mi/kg. Dosages refer to the free drug forms.
198 RESULTS
consisted of 14 rats which had electrode tips in the deep cell layer (layer III) of the rostral portion of the PPC near to the b o u n d a r y between PPC and the anterior piriform cortex s. The PPC2 group comprised 7 animals with electrode tips in the locus in the PPC previously examined by other groups 3'z4. E l e c t r o d e placements of the amygdala group are shown in Fig. 2. Sixteen animals had electrode tips within the basolaterai nucleus. M a r k e d differences between PPC 1 and the other loci were already found at the first electrical stimulation in
A c c o r d i n g to histological examination of the electrode placements, all animals with placements outside the a i m e d targets were d r o p p e d from the experiments. Fig. 1 shows the positions of electrode tips in the 3 groups of piriform cortex-kindled animals used for final evaluation of kindling data. Four animals had electrodes in the area described by Piredda and Gale 22, and these animals were used as D P C subgroup (cf. Table I). The PPC1 group
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Brain Research, 538 (1991) 196-202 Elsevier
BRES 16224
Kindling from stimulation of a highly sensitive locus in the posterior part of the piriform cortex. Comparison with amygdala kindling and effects of antiepileptic drugs Dagmar H6nack, Ulrich Wahnschaffe and Wolfgang L6scher Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Hannover (F.R. G.) (Accepted 7 August 1990) Key words: Epilepsy; Kindling; Amygdala; Piriform cortex; Antiepileptic drug
The piriform cortex, especially its deep anterior part, has been recently suggested to be a crucial epileptogenic site in the rat brain. We investigated the susceptibility of different parts of the piriform cortex to the development of electrical kindling as compared to that of the basolateral amygdala. A locus in the deep cell layer (layer III) of the rostral portion of the posterior piriform cortex (PPC) is described, which is considerably more sensitive to electrical stimulation than adjacent areas of the PPC, including the deep prepiriform cortex or the amygdala. The sensitive locus in the PPC can be readily kindled, and focal seizure thresholds in fully kindled rats are 60-90% lower than respective thresholds in rats kindled from other loci. Treatment of fully kindled rats with the antiepileptic drugs diazepam, carbamazepine, phenobarbital, and valproate showed that anticonvuisant effects of these drugs in animals kindled from stimulation of the PPC were comparable to respective effects in animals kindled from stimulation of the basolateral amygdala, although the locus in the PPC tended to be more resistant. The data support the idea that the piriform area may contain the most sensitive neuronal tissue responsible for the generation of seizures during kindling. It remains to be determined if the described locus in the PPC is critical to the kindling process when kindling is induced from other structures within the olfactory-limbic system. INTRODUCTION The piriform (primary olfactory) cortex receives its primary afferent input from the olfactory bulb, but there are also inputs from the amygdala, hippocampal formation and neocortical areas 8-1°'16. Although the piriform cortex is not considered to be a part of the limbic system, its outputs directly and indirectly project to several limbic structures, such as the septal area, amygdala and hippocampal region 8-1°'16. Goddard et al. 7 were the first to demonstrate that structures of the olfactory-limbic system, including the piriform cortex, are highly susceptible to the development of seizures following local application of electrical stimuli. In recent years, several lines of evidence have suggested that the piriform cortex might be critically involved in generation and/or propagation of seizure discharge. Thus, (1) kindled seizures induced by repeated focal stimulation develop most quickly after electrical stimulation of the amygdala, piriform cortex or its primary afferent, the olfactory bulbT'2°'28; (2) regardless of the location of subcortical kindling sites, interictal discharges begin first in the piriform cortexll'26; (3) autoradiographic studies during amygdala kindling de-
velopment showed high metabolic activity in the piriform cortex1'5'19; and (4) following status epilepticus evoked by stimulation of a kindled amygdala, significant neuronal degeneration is observed in the entire ipsilateral piriform cortex iS. If the piriform cortex is involved in seizure generation, the question still remains as to whether there is a sole generator within that region, where within the rather large piriform region the generator resides, and how much tissue is involved. It was, therefore, of interest that Piredda and Gale 22 reported the existence of a specific area in the deep anterior part of the piriform cortex (deep prepiriform cortex (DPC)) in which microinjection of picomole amounts of bicuculline or other chemoconvulsants produced electrographic and convulsive responses resembling those induced by amygdala kindling. Piredda and Gale hypothetized that this tiny area within the D P C ('area tempestas'; anatomically the ventral endopiriform nucleus, which is deep to layer III of the prepiriform cortex 16) may be a crucial epileptogenic site in the brain and may also be responsible for the development of kindled seizures. However, several more recent studies suggest that the role of the D P C in epileptogenesis is limited. Thus, it was shown that the
Correspondence: W. LOscher, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, ER.G. 0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
197 t h r e s h o l d for i n d u c t i o n of a f t e r d i s c h a r g e s ( A D T ) in the D P C is n o t d i f f e r e n t f r o m t h e A D T o f the a m y g d a l a o r a r e a s i m m e d i a t e l y a d j a c e n t to D P C 3'3°. T h e p r e p i r i f o r m c o r t e x can be readily k i n d l e d , but n o t faster t h a n t h e a m y g d a l a 3'2°'3°. L e s i o n s o f t h e D P C , including the site d e s c r i b e d by P i r e d d a and G a l e 22, did n o t affect the r a t e of electrical k i n d l i n g o f the a m y g d a l a 3"2°'3° o r the convulsant
potency
of
systemically
administered
bicuculline 29. Similarly, knife-cuts, disrupting c o m m u n i c a t i o n b e t w e e n a m y g d a l a and a n t e r i o r p i r i f o r m structures, did n o t significantly i n c r e a s e the n u m b e r of s t i m u l a t i o n s r e q u i r e d for a m y g d a l a kindling 27. H o w e v e r , t h e r e is still t h e possibility that o t h e r parts o f the p i r i f o r m c o r t e x m a y s e r v e as a g e n e r a t o r
of limbic seizures.
I n d e e d , lesions o f the p i r i f o r m c o r t e x that i n v o l v e d the p o s t e r i o r p a r t o f this r e g i o n w e r e r e c e n t l y s h o w n to significantly i n c r e a s e t h e n u m b e r of stimulations r e q u i r e d to c o m p l e t e septal kindling 27. In the p r e s e n t study, we d e s c r i b e a locus in the d e e p p o s t e r i o r p i r i f o r m c o r t e x ( P P C ) which is substantially m o r e susceptible to electrical stimulation
t h a n the a m y g d a l a o r o t h e r parts of the
p i r i f o r m cortex. T h e p h a r m a c o l o g i c a l sensitivity o f this locus has b e e n c h a r a c t e r i z e d by e x p e r i m e n t s with clinically e s t a b l i s h e d a n t i e p i l e p t i c drugs.
MATERIALS AND METHODS
Preparation of animals All experiments were carried out in female Wistar rats, weighing 210-230 g. Sixty-five rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and received stereotaxic implantation of one bipolar electrode into the right hemisphere according to the surgery methods described in the atlas of Paxinos and Watson 21. Rats in the amygdala group (n = 23) received implantation of one electrode aimed at the basolateral amygdala at the following coordinates: AP -2.2, L -4.8, V -8.5. Rats in the DPC group (n = 10) received implantation of one electrode aimed at the region ('area tempestas') described by Piredda and Gale 22 at the following coordinates: AP +2.2, L -2.6, V -7.8. Rats in the PPC group (n = 32) were subdivided into a group (PPC1; n -- 22) with electrode implantation in the deep rostral portion of the PPC, and a group (PPC2; n = 10) in which the electrode was aimed at a more caudal locus in the PPC, according to the PPC locus previously examined by Racine 24 and Cain et al. 3. Coordinates for PPC 1 were AP -0.8, L-4.8, V -8.5, and for PPC 2 AP -3.3, L -5.9, V -9.0. All coordinates were measured from bregma. Skull screws served as the indifferent reference electrode. The electrode assembly was attached to the skull by dental acrylic.
Kindling After a postoperative period of 2 weeks, constant-current stimulations (500-/~A, 1-ms, monophasic square-wave pulses, 50/s for 1 s) were delivered to the piriform cortex or amygdala at intervals of 1 day until 10 stage 5 seizures were elicited. The electrical susceptibility of the stimulated region was recorded on the first day of the experiment (initial ADT) as well as after kindling acquisition (with an interval of at least 4 days after the 10th stage 5 seizure) using an ascending stairstep procedure 6. The initial current intensity was 10 aA, and the current intensity was increased in steps of about 20% of the previous current at intervals of 1 min until an afterdischarge of at least 3-s duration was elicited. Since
almost all fully kindled animals (except for 4) exhibited generalized seizures (stage 4-5) at the ADT current, it was not necessary to determine the threshold for generalized seizures (GST) separately. In fully kindled rats, the ADT determined with interstimulation intervals of 1 day was not different from ADT values determined with interstimulation intervals of 1 min, thus demonstrating that the short interstimulation interval did not bias ADT determinations. In addition to ADT, in fully kindled rats, the following parameters of kindled seizures were measured after stimulation with either the ADT current or suprathreshold stimulation with 500/zA. Seizure severity was classified according to Racine23: 1, immobility, eye closure, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 4, rearing, often accompanied by bilateral forelimb clonus; 5, rearing with loss of balance and falling accompanied by generalized clonic seizures. Seizure duration was the duration of limbic (stage 1-2) and/or motor seizures (stage 3-5); limbic seizure activity sometimes occurring after termination of stage 3-5 seizures was not included in seizure duration. Afterdischarge duration was the total time of spikes in the EEG recorded from the site of stimulation.
Drug testing The effects of diazepam (5 mg/kg i.p.), valproate (VPA; 200 mg/kg i.p.), carbamazepine (20 mg/kg i.p.) and phenobarbital (20 and 30 mg/kg i.p.) were compared in PPC 1- and amygdala-kindled rats. For anticonvulsant drug testing, all stimulations were carried out with 500/~A. The drugs were injected 15 min (VPA), 30 min (diazepam, carbamazepine) or 60 min (phenobarbital) prior to stimulation. Dosages and pretreatment times were chosen on the basis of previous dose-effect experiments with the drugs in kindled rats 14. Control readings were determined 2-3 days prior to and after each drug administration, and the next drug experiment was only undertaken if all rats showed reproducible stage 5 seizures. None of the drug vehicles (see below) had an effect on kindled seizure parameters. All rats received each treatment; the sequence of drug testing was diazepam, VPA, carbamazepine and phenobarbital; at least 4 days were interposed between two drug injections. In order to examine if this sequence of drug testing altered anticonvulsant drug efficacy, one of these drugs, i.e. diazepam, was retested at the end of the experiments. No difference in anticonvulsant efficacy was found between the two test sessions with diazepam.
Histology After termination of the last drug experiment, placement of stimulating electrodes was examined histologically in all animals. The rats were anesthetized with chloral hydrate (360 mg/kg i.p.) and perfused with a fixative consisting of 4% phosphate buffered formaldehyde (pH 7.3). Two h later, the brains were removed, postfixed overnight and processed for paraffin embedding. Subsequently, serial sections of the entire brain were cut coronally at 7 gm, and every 20th section was mounted on a glass slide and stained with Cresyl violet or hematoxylin eosin.
Statistics All data are given as means + S.E.M. Significance of differences between seizure readings in the same group of rats was calculated by the Wilcoxon signed-rank test for paired replicates. Significance of differences between different groups was calculated by Student's t-test.
Drugs VPA, phenobarbital (both used as their sodium salts) and carbamazepine were obtained from Desitin (Hamburg, ER.G.), and diazepam from Hoffmann La Roche (Basle, Switzerland). VPA, phenobarbital and diazepam were dissolved in water (diazepam by means of dilute HCI), carbamazepine was dissolved in 30% polyethylene glycol 400 in water. All antiepileptic drugs were freshly dissolved prior to each experiment and injected i.p. at a volume of 2-3 mi/kg. Dosages refer to the free drug forms.
198 RESULTS
consisted of 14 rats which had electrode tips in the deep cell layer (layer III) of the rostral portion of the PPC near to the b o u n d a r y between PPC and the anterior piriform cortex s. The PPC2 group comprised 7 animals with electrode tips in the locus in the PPC previously examined by other groups 3'z4. E l e c t r o d e placements of the amygdala group are shown in Fig. 2. Sixteen animals had electrode tips within the basolaterai nucleus. M a r k e d differences between PPC 1 and the other loci were already found at the first electrical stimulation in
A c c o r d i n g to histological examination of the electrode placements, all animals with placements outside the a i m e d targets were d r o p p e d from the experiments. Fig. 1 shows the positions of electrode tips in the 3 groups of piriform cortex-kindled animals used for final evaluation of kindling data. Four animals had electrodes in the area described by Piredda and Gale 22, and these animals were used as D P C subgroup (cf. Table I). The PPC1 group
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t
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'~'i
