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Deep prepiriform cortex kindling and amygdala interactions
94
EpilepsyRes., 1 (1987) 94-1(11 Elsevier
ERS 00122
R
ch Reports
Deep prepiriform cortex kindling and amygdala interactions D ayao Y. Zhao* and Solomon L. Mosh6*'** Departments of *Neurologyand **Pediatrics,Albert Einstein Collegeof Medicine, Bronx, NY10461 (U.S.A.) (Received 10 October 1986; accepted 15 October 1986)
Key words: Seizures; Convulsions; Kindling; Amygdala; Prepiriform cortex; Rat
The deep prepiriform cortex (DPC) has been recently suggested to be a crucial epileptogenic site in the rat brain. We investigated the susceptibility of the DPC to the development of electrical kindling as compared to that of the superficial prepiriform cortex (SPC) and amygdala as well as the transfer interactions between the two prepiriform sites and amygdala. Adult rats with electrodes implanted in the right prepiriform cortex (DPC or SPC) and left amygdala were divided into a DPC-amygdala and SPC-amygdala group while a third group consisted of rats with electrodes implanted in the ipsilateral DPC and amygdala. Within each group the rats were initially kindled from one site selected randomly and then rekindled from the other site. Both DPC and SPC were as sensitive to the development of kindling as the amygdala. The behavioral seizures elicited with DPC or SPC primary kindling were identical to those induced by amygdala kindling. Initial DPC kindling facilitated the development of kindling from either ipsilateral or contralateral amygdala with the ipsilateral transfer being significantly more potent than the contralateral. SPC kindling also facilitated the development of contralateral amygdala kindling but was less effective than DPC kindling. On the other hand, amygdala kindling did not facilitate contralateral SPC or DPC kindling although it transferred to the ipsilateral DPC. These results indicate that the prepiriform cortex can be readily kindled but not faster than the amygdala and that there are unequal kindling transfer interactions between prepiriform cortex and amygdala.
INTRODUCTION The ability of different brain structures to respond to epileptogenic stimuli varies from site to site. Overall there is abundant evidence that neocortical and limbic structures are highly susceptible to the development of seizures following local application of chemoconvulsants or electrical stimuli2~11|6-19"24"3°-33. Recently, Piredda and Gale 25-27 reported the existence of a specific area in the deep prepiriform cortex (DPC) with the leaCorrespondenceto: Dr. S.L. Mosh6, Albert Einstein College of Medicine, Department of Neurology, Building F, Room G-9, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A. (212) 430-2833.
ture of having both a high epileptogenic potential and the capability to attenuate seizures induced by systemic administration of chemoconvulsants. Thus unilateral application of picomole amounts of bicuculline, kainic acid or carbachol consistently produced generalized clonic seizures in rats, while microinfusions of muscimol (a GABA agonist) or 2-amino-phosphoheptanoic acid (an antagonist of the aspartate receptor) prevented the development of seizures induced by the systemic administration of bicuculline. Piredda and Gale hypothesized that this tiny area (less than 1 mm in diameter) within the DPC may be a crucial epileptogenic site of the brain and a site mediating the anticonvulsant action of GABA agonists and aspartate antagonists. Furthermore, these authors
0920-1211/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
95 proposed that the DPC may also be responsible for the development of kindled seizures since the seizures induced by the local drug application are behaviorally similar to the secondarily generalized kindled seizures induced from various limbic structures 25-27. Within the limbic system the amygdala appear to be most susceptible to the development of kindling H. The present study was designed to investigate the susceptibility of the DPC to kindling as compared to that of the amygdala and the superficial prepiriform cortex (SPC), and to determine the kindling interactions between the amygdala and the two sites within the prepiriform cortex (DPC and SPC). MATERIALS AND M E T H O D S
Surgery Thirty-four male Sprague-Dawley albino rats weighing 300 + 25 g were separated into 3 groups. The first group included rats in which bipolar electrodes (Plastic Products, MS 303/3) were stereotaxically implanted in the right DPC and the left amygdala (n = 12); the second group contained rats with electrodes in the right SPC and left amygdala respectively (n = 8); the third group included rats with electrodes implanted in the right DPC and ipsilateral amygdala (n = 14). For the surgery, all rats were anesthetized with a mixture of ketamine (66 mg/kg) and xylazine (10 mg/kg) injected i.m. The following coordinates were used: DPC, 3.8 mm anterior and 2.5 mm lateral from bregma, depth, 7.3 mm from skull; SPC, anterior 3.8 mm, lateral: 2.5 mm, depth: 7.8 mm; the incisor bar was set 5 mm above the interaural line for both DPC and SPC; amygdala, 1.8 mm posterior and 3.7 mm lateral from bregma, depth, 9.0 mm, with the incisor bar 3.5 mm below the interaural line 2~'22.
charge (AD). Each rat was stimulated initially with 1 sec long 30 pA, peak-to-peak, 60 Hz sinusoidal current generated from a sine wave stimulator (Lafayette Instrument Co.). The intensity of the current was increased by 30 pA steps every 2 min until an AD was elicited which was arbitrarily construed to be the ADT. Once the A D T of the primary site was determined, the rat was kept stimulation free for 24 h and then the A D T of the other site was measured as described above. The choice of the primary site was made randomly. Nine rats were initially kindled from the amygdala, 7 from the DPC and 4 from the SPC. The kindling stimulation consisted of a 1 sec train of 400 p A peak-to-peak 60 Hz sine wave current. The rats were stimulated once per hour, 8-10 times per day, until they developed generalized seizures or a total of 20 stimulations was delivered. During each trial, the behavioral changes following the stimulation were observed and classified according to Racine's scoring scale31: stage 1 consisted of motionless staring, stage 2 of head nodding, stage 3 of unilateral forelimb clonus, stage 4 of rearing and bilateral forelimbs clonus, and stage 5 of the above plus loss of balance. During each stimulation trial 2-channel simultaneous EEG recordings were obtained using a Grass Model 79D polygraph. Subsequently, after a 24 h interval all rats were kindled from their respective secondary sites using the same paradigm as before. In the second experiment, the rats in the third group, with the ipsilateral amygdala electrodes, were kindled using a 1 sec train of 400pA peak-topeak 60 Hz sine wave current. Seven rats were kindled initially from the DPC and 7 rats from the ipsilateral amygdala. After the completion of primary kindling, the rats were kindled from their respective secondary site as described above.
Histology Kindling After a 48 h recovery period, the rats in the first 2 groups (with electrodes implanted in the contralateral amygdala) were subjected to the kindling procedure from the respective primary site (amygdala, DPC, SPC). First, the afterdischarge threshold (ADT) was determined defined as the lowest current intensity which produced an afterdis-
Upon the completion of the experiments, all rats were decapitated. The brains were removed and frozen, then sectioned in 20 ~tm slices and stained with thionine. The placements of the electrode tips were verified microscopically by another person blind to the kindling data.
96
Statistics The data were statistically analyzed using the one-way ANOVA with appropriate post-hoc comparisons (Neuman-Keuls).
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Behavioral observations The development of behavioral kindled seizures induced by stimulations of either the DPC or the SPC were identical to that observed with amygdala kindling. Therefore, the seizures were classified, as described by Racine for amygdala kindled seizures3t. When the behavioral seizures reached stage 3, the end of the convulsion was heralded by the onset of wet dog shakes irrespective of the site of stimulation.
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Primary site kindling In the first two groups, the AD threshold, the AD duration at the AD threshold stimulation, the number of stimulations required for the development of a stage 5 seizure, and the AD duration of the first stage 5 seizure were used as the indices depicting the susceptibility to kindling as a function of site (Table I). There were no statistical differences in all these kindling parameters among the 3 sites. These results were reproduced in the 3rd group where there were no differences in the number of stimulations required for the development of a stage 5 seizure between the rats stimulated initially from the DPC (~ + S.E. = 8.6 + 0.6) or the ipsilateral amygdala (8.9 + 0.6).
Fig. 1. Continuous E E G recording during D P C kindling. The stimulation (no. 5) was delivered to the DPC. Black arrows indicate the independent A D s from the amygdala in B and C. The black bars on the time marker indicate the onset of wet dog shakes. DPC, deep prepiriform cortex; A M G , amygdala.
TABLE I The development o f primary kindling induced either f r o m the deep prepiriform cortex (DPC), superficial prepiriform cortex (SPC), or contralateral amygdala ( A M G ) Values are m e a n s _+ S.E. ; A D , afterdischarge; the A D threshold is defined as the lowest current intensity which produced an A D ; ns, not significant.
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A D threshold (I~A)
A D duration at threshold (sec)
No. o f stimulations f o r 1st stage 5 seizure
A D duration at 1st stage 5 seizure (sec)
154.3 + 31.0 125.0 + 26.0 130.0 + 16.0 0.3 ns
13.9 + 1.4 13.0 + 3.0 21.2 + 3.8 2.0 ns
12.6 + 1.2 13.2 +_ 1.7 12.9 + 1.3 0.04 ns
48.0 +_ 6.3 91.0 ___25.3 69.8 _+ 8.6 2.8 ns
97
Electroencephalography Two-channel simultaneous EEG recordings from the prepiriform cortex (either DPC or SPC) and the amygdala demonstrated a unique electrical seizure pattern during DPC, SPC or amygdala kindling. Even when the first stimulation was applied to the DPC or SPC, the ADs immediately spread to the contralateral amygdala (Fig. 1A). With the repetitive prepiriform cortex stimulations long-lasting ADs were observed in the amygdala, which were completely independent from the DPC or SPC ADs (Fig. 1B). These independent ADs usually emerged after stage 2 or 3 seizures had occurred and increased in frequency and amplitude in line with the development of kindled seizures. After the amygdala ADs stopped, the rats remained motionless, staring, and the EEGs from both leads recovered to the normal level for 10 sec. Subsequently, there was another episode of
amygdala ADs which lasted for about 30 sec (Fig. 1C). Rats in which the stimulations were initially applied to the amygdala did not exhibit any independent ADs from the DPC or SPC. However, 20 sec after the end of a generalized seizure, secondary spontaneous ADs occasionally occurred localized in the amygdala (Fig. 2B, C).
Secondary site kindling Previous kindling induced a unidirectional transfer from the prepiriform cortex (both DPC and SPC) to the contralateral amygdala (Table II). The development of primary amygdala kindling required (~ + S.E.) 12.9 + 3.10 stimulations, however, rats kindled initially from the DPC required only 3.4 + 1.5 amygdala stimulations to develop generalized seizures (P < 0.001) (Table IIA). SPC
TABLE I1 Transfer effects between the contralateral amygdala and DPC or SPC
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c Fig. 2. Continuous E E G recording following amygdala stimulation. The stimulation (no. 11) was applied to the left amygdala. Black arrows indicate two clearly separated in time, wellmodulated ADs which occurred in the amygdala only (B, C). The black bars on the time marker after the motor seizure indicate the occurrence of a cluster of wet dog shakes. DPC, deep prepiriform cortex; AMG, amygdala.
C. From SPC Primary kindling (n = 4) Secondary kindling after AMG kindling (n = 4) Fl. 6
13.3 _+ 1.8 13.5 + 2.4 0.007 (ns)
Values are means _+ S.E. a Significantly different from rats initially kindled from the amygdala (post-hoc Neuman-Keuls, P < 0.01) and from rats initially kindled from the SPC (P < 0.05). b p < 0.01 from rats kindled initially from amygdala. c Two additional rats in this group did not develop any generalized seizures even though they received 20 stimulations and were not included in this analysis, ns = not significant.
98 kindling also facilitated the establishment of contralateral amygdala kindling (P < 0.01), although more stimulations (7.2 + 1.3) were required than for the D P C to contralateral amygdala transfer (post hoc P < 0.05). Table liB and C demonstrate that contralateral amygdala kindling did not transfer to either the D P C or the SPC. In fact, in the amygdala/DPC group, 2 rats did not develop generalized kindled seizures from the D P C within 20 stimulations after previous contralateral amygdala kindling while in all other transfer experiments the rats developed generalized seizures. Thus the mean kindling stage was 3.6 + 1.4 for D P C kindling in this group, while it was 5 + 0 in the group kindled initially from the D P C (P < 0.001). This indicates the existence of a negative transfer effect from the contralateral amygdala to the DPC. Such a negative transfer was not observed between contralateral amygdala and SPC. In contrast to the above findings, a bidirectional transfer was observed between D P C and ipsilateral amygdala, albeit of different degrees. D P C kindling readily transferred to the ipsilateral amygdala since all rats required only 1 stimulation to develop a stage 5 seizure accounting for a net reduction in the n u m b e r of stimulations of 89%. Rats initially kindled from the ipsilateral amygdala
required 4.6 + 0.5 stimulations to reach stage 5 when secondarily stimulated from the DPC; this represents a reduction of 47% (Table III). Oneway analysis of variance revealed that secondary amygdala kindling was established significantly faster following D P C kindling (F1, 2 = 55.0, P < 0.001) than secondary D P C kindling preceded by ipsilateral amygdala kindling. Histology Fig. 3 depicts the histological placements of the electrode tips. The D P C group included the rats in which the electrode tips were in the small area, l m m in diameter, reported by Piredda and Gale 26'27 as a crucial site for epileptogenesis. The SPC group included the rats in which the electrode tips were in the superficial layers of prepiriform cortex 21'22. All amygdala electrode tips were found within the basolateral nucleus 21'22 DISCUSSION The prepiriform cortex, also known as the lateral olfactory gyrus, constitutes a m a j o r part of the
TABLE III Transfer effects between the ipsilateral amygdala and DPC Number of stimulations required for the development of stage 5 seizures. A. From amygdala Primary kindling (n = 7) Secondary kindling after DPC kindling (n = 7) F1,12 B. From DPC Primary kindling (n = 7) Secondary kindling after AMG kindling (n = 7) FI,12
8.9 + 0.6 1.0 + 0.0a 13.2 (P < 0.005) 8.6 + 0.6 4.6 + 0.5 6.1 (e < 0.05)
Values are means ___S.E. a Secondary amygdala kindling was established significantly faster following DPC kindling (Fl. 12= 55.0, P < 0.001) than secondary DPC kindling preceded by amygdala kindling.
Fig. 3. Section through the rat brain 9.8 mm anterior to the interaural line redrawn from Pellegrino and Cushman22depicting the location of the electrodes in the DPC (the same site as shown in Piredda and Gale 26"27) and SPC. CC, corpus callosum; CP, caudate-putamen; NA, nucleus accumbens; OT, lateral olfactory tract.
99 oldest cortical derivatives 7. Anatomically Haberly and Price 12 have divided the prepiriform cortex into a superficial plexiform layer (layer I), superficial compact cell layer (layer II) and deep cell layer (layer III). Deep to layer III there is a well-developed group of polymorphous cells called endopiriform nucleus 12'14'15. Our DPC electrodes were within the latter nucleus and our locations were identical to those shown by Piredda and Gale. The SPC electrodes were located within the first 3 layers. In mammals, the prepiriform cortex is anatomically and functionally related to olfactory structures and to many limbic structures. Since its afferent fibers are derived from the lateral olfactory stria, the prepiriform cortex is regarded as an olfactory relay center 7. Furthermore, although the prepiriform cortex is not considered to be part of the limbic system, its outputs directly and indirectly (via the entorhinal cortex) project to several limbic structures such as the septal area, amygdala, and entorhinal cortex 7'13'15'35. These output projections seem to use excitatory amino acids as transmitters a. Although the fibers from the prepiriform cortex mainly project to the ipsilateral limbic structures, a sparse contralateral projection has been identified 12. Limbic sites do not directly project back to the prepiriform cortex. However, there is a polysynaptic pathway from the amygdala feeding back to the prepiriform cortex, via the accessory olfactory bulb 28'29'34. Kindling has been induced from many olfactory structures such as the olfactory bulb, tract and nucleus, piriform cortex and entorhinal cortex 3'11'30'31'33. It has been suggested that these structures are less sensitive to kindling than the amygdala 2'11'13'23'24'32'33,although there is one report indicating that the olfactory bulb may require fewer stimulations to develop generalized seizures than the amygdala 3. To date, there has been no report on the kindling of the prepiriform cortex. Our study shows that both parts of the prepiriform cortex, DPC and SPC, are as sensitive to kindling as the amygdala, as determined by the local AD threshold and the number of stimulations required for the development of generalized kindled seizures. Therefore, it can be concluded that the DPC is not only sensitive to chemical convulsants,
but also to electrical kindling. However, the DPC does not differ from the SPC in terms of primary kindling. This result must be contrasted with the discrete pharmacological localization reported by Piredda and Gale using chemical convulsants 25-27. The seizure behaviors induced by DPC and SPC kindling are identical to those produced by amygdala kindling and are characterized by gradually potentiated limbic symptoms such as staring, head nodding, and finally the development of generalized seizures. These observations suggest that the development of DPC and SPC kindling depends on the gradual activation of other sites including the amygdala. The EEG data support this hypothesis. The amygdala were very susceptible to the generation of independent ADs even when the DPC or SPC were stimulated; independent ADs did not occur from either DPC or SPC irrespective of the site of stimulation. Previous studies have demonstrated transfer effects between a number of brain structures during kindlinf '4-6'1°'18'31. By definition, 'transfer' indicates a significant acceleration of the development of kindling from a secondary site. Nevertheless, transfer also occurs between different kindling agents such as flurothyl, pentylenetetrazol, carbachol, opiates and electrical kindling 4'5'2°'36. Thus, transfer seems to be a widespread phenomenon throughout the brain. However, our data show that transfer did not occur from contralateral amygdala to DPC or SPC. Instead in two rats there was interference (negative transfer) since amygdala kindling retarded the development of contralateral DPC kindling. Since the lack of transfer may be related to the presence of only indirect connections between amygdala and prepiriform cortex 28'29'34, we studied the transfer relationships between ipsilaterally stimulated sites. In this case a bilateral interaction occurred albeit of different strengths. DPC kindling readily transferred to the ipsilateral amygdala while the transfer from the amygdala was less potent. The savings in secondary kindling of ipsilateral DPC to amygdala kindling may be due to the presence of direct excitatory inputs to the amygdala from the prepiriform cortex. Based on this finding, the anatomical existence of predominantly unilateral connections between DPC and amygda-
100 la 7'9'12 and rich intra-amygdala connections ~4'~5, we suggest that DPC kindling may transfer to the contralateral amygdala through the ipsilateral amygdala.
REFERENCES 1 Burnham, W.M., Primary and 'transfer' seizure development in the kindled rat, Canad. J. neuroL Sci., 2 (19751 417-428. 2 Burnham, W.M., Cortical and limbic kindling: similarities and differences. In: K.E. Livingston and O. Hornykiewicz (Eds,), Limbic Mechanisms, The Continuing Evolution of the Limbic System Concept, Plenum Press, New York, 1978, pp. 507-519. 3 Cain, D.P., Seizure development following repeated electrical stimulation of central olfactory structures, Ann. N.Y. Acad. Sci., 290 (1977)200-216. 4 Cain, D.P,, Bidirectional transfer of electrical and carbachol kindling, Brain Res., 260 (19831 135-138. 5 Cain, D.P., Transfer of kindling: clinical relevance and a hypothesis of its mechanism, Prog. Neuro-Psychopharmacol, biol. Psychiat., 9 (1985) 467-472. 6 Cain, D.P. and Corcoran, M.E., Kindling with low-frequency stimulation: generality, transfer, and recruiting effects, Exp. NeuroL, 73 (1981)219-232, 7 Carpenter, M.B., Human Neuroanatomy, 7th edn. Williams and Wilkins, Baltimore, MD, 1976, pp. 521-546, 8 Collins, G.G.S., Amino acid transmitter candidates in various regions of the primary olfactory cortex following bulbectomy, Brain Res., 296 (1984) 145-147, 9 Cowan, W.M., Raisman, G. and Powell, T.P.S., The connections of the amygdala, J. Neurol. Neurosurg. Psychiat., 28 (1965) 137-151. 10 Cullen, N. and Goddard, G.V., Kindling in the hypothalamus and transfer to the ipsilateral amygdala, Behav. Biol., 15 (1975) 119-131. t 1 Goddard, G.V., Mclntyre, D.C. and Leech, C~K., A permanent change in brain function resulting from daily electrical stimulation, Exp. NeuroL, 25 (1969) 295-330, 12 Haberly, L,B. and Price, J.L., Association and commissural fiber systems of the olfactory cortex of the rat, J, comp. Neurol., 178 (19781 711-740. 13 Kairiss, E.W., Racine, R.J. and Smith, G.K.. The development of the interictal spike during kindling in the rat, Brain Res., 322 (1984) 101-110. 14 Krettek, J.E. and Price, J.L., Projections from the amygdaIoid complex to the cerebral cortex and thalamus in the rat and cat, J. cornp. Neurol., 172 (1977) 687-722. 15 Krettek, J.E, and Price, J.L., A description of the amygdaloid complex in the rat and cat with observations on intra-
ACKNOWLEDGEMENTS Supported in part by Grant NS 20253 from the NINCDS. Dr. D.Y. Zhao is the recipient of the 1985 Beijing-Einstein College of Medicine Visiting Fellowship. We thank Ms. Donna Platyan for her help in the preparation of this manuscript.
amygdaloid axonal connections, J. comp. NeuroL, 178 (1978) 255-280, 16 Le Gal la Salle, G,, Kindling of motor seizures from the bed nucleus of the stria terminalis, Exp. Neurol., 66 (19791 309-318. 17 Le Gal la Salle, G,, Amygdaloid organization related to the kindling effect. In: P.A. Buser, W.A. Cobb and T. Okuma (Eds.), Kyoto Symposia (EEG SuppL No. 36). Elsevier Biomedical Press, Amsterdam, t982, pp. 239-248. 18 McIntyre, D.C~ and Stuckey, G.N., Dorsal hippocampat kindling and transfer in split-brain rats, Exp. Neurol., 87 (19851 86-95. 19 MoriUo, L.E,, Ebner, T.J. and Bloedel, J.R., The early involvement of subcortical structures during the development of a cortical seizure focus, Epilepsia, 23 (1982) 571-585. 20 Okada, R,, Mosh6, S.L., Ono, K. and Albala, B,J,, Unidirectional interaction between flurothyl seizures and amygdala kindling, Brain Res., 344 (1985) 103-108. 21 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 22 Pellegrino, L.J. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, PLenum Press, New York, 1979. 23 Pinel, J.P.J., Kindling-induced experimental epilepsy in rats: cortical stimulation, Exp. NeuroL, 72 (1981) 559-569. 24 Pinel, J.P,J. and Rovner, L.I., Electrode placement and kindling-induced experimental epilepsy, Exp. Neurol.. 58 ( 19781 335-346. 25 Piredda, S. and Gale, K., A crucial epileptogenic site in the deep prepiriform cortex. Nature (Lond.). 317 (19851 623-625. 26 Piredda, S. and Gale, K., Anticonvulsant action of 2-amino-7-phosphonoheptanoic acid and muscimol in the deep prepiriform cortex, Europ. J. PharmacoL, 120 (19861 1t5-118. 27 Piredda, S. and Gale, K., Role of excitatory amino acid transmission in the genesis of seizures elicited from the deep prepiriform cortex, Brain Res., 377 (1986) 205-210. 28 Powell, T.P.S., Cowan, W.M. and Raisman, G,. The central olfactory connections, J. Anat. (Lond.). 99 (19651 791-813. 29 Price, J.L. and Powell, T.P.S., An experimental study of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat, J. Anat. (Lond.). t(17 (1970) 215-237. 30 Racine, R.J., Modification of seizure activity by electrical stimulation, t. After-discharge threshold, Electroenceph.
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clin. Neurophysiol., 32 (1972) 269-279. 31 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281-294. 32 Racine, R.J., Modification of seizure activity by electrical stimulation: cortical areas, Electroenceph. clin. Neurophysiol., 38 (1975) 1-12. 33 Racine, R.J., Okujava, V. and Chipashvili, S., Modification of seizure activity by electrical stimulation. III, Mechanisms, Electroenceph. clin. Neurophysiol., 32 (1972) 295-299.
34 Raisman, G., An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system, Exp. Brain Res., 14 (1982) 395-408. 35 Watanabe, S., Nakanishi, H., Shibata, S. and Ueki, S., Changes in amygdaloid afterdischarges and kindling effect following olfactory bulbectomy in the rat, Physiol. Behav., 28 (1982) 687-692. 36 Wong, B., MosM, S.L. and Okada, R., Flurothyl seizures facilitate the development of cortical kindling, Epilepsia, 26 (1985) 518.
EpilepsyRes., 1 (1987) 94-1(11 Elsevier
ERS 00122
R
ch Reports
Deep prepiriform cortex kindling and amygdala interactions D ayao Y. Zhao* and Solomon L. Mosh6*'** Departments of *Neurologyand **Pediatrics,Albert Einstein Collegeof Medicine, Bronx, NY10461 (U.S.A.) (Received 10 October 1986; accepted 15 October 1986)
Key words: Seizures; Convulsions; Kindling; Amygdala; Prepiriform cortex; Rat
The deep prepiriform cortex (DPC) has been recently suggested to be a crucial epileptogenic site in the rat brain. We investigated the susceptibility of the DPC to the development of electrical kindling as compared to that of the superficial prepiriform cortex (SPC) and amygdala as well as the transfer interactions between the two prepiriform sites and amygdala. Adult rats with electrodes implanted in the right prepiriform cortex (DPC or SPC) and left amygdala were divided into a DPC-amygdala and SPC-amygdala group while a third group consisted of rats with electrodes implanted in the ipsilateral DPC and amygdala. Within each group the rats were initially kindled from one site selected randomly and then rekindled from the other site. Both DPC and SPC were as sensitive to the development of kindling as the amygdala. The behavioral seizures elicited with DPC or SPC primary kindling were identical to those induced by amygdala kindling. Initial DPC kindling facilitated the development of kindling from either ipsilateral or contralateral amygdala with the ipsilateral transfer being significantly more potent than the contralateral. SPC kindling also facilitated the development of contralateral amygdala kindling but was less effective than DPC kindling. On the other hand, amygdala kindling did not facilitate contralateral SPC or DPC kindling although it transferred to the ipsilateral DPC. These results indicate that the prepiriform cortex can be readily kindled but not faster than the amygdala and that there are unequal kindling transfer interactions between prepiriform cortex and amygdala.
INTRODUCTION The ability of different brain structures to respond to epileptogenic stimuli varies from site to site. Overall there is abundant evidence that neocortical and limbic structures are highly susceptible to the development of seizures following local application of chemoconvulsants or electrical stimuli2~11|6-19"24"3°-33. Recently, Piredda and Gale 25-27 reported the existence of a specific area in the deep prepiriform cortex (DPC) with the leaCorrespondenceto: Dr. S.L. Mosh6, Albert Einstein College of Medicine, Department of Neurology, Building F, Room G-9, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A. (212) 430-2833.
ture of having both a high epileptogenic potential and the capability to attenuate seizures induced by systemic administration of chemoconvulsants. Thus unilateral application of picomole amounts of bicuculline, kainic acid or carbachol consistently produced generalized clonic seizures in rats, while microinfusions of muscimol (a GABA agonist) or 2-amino-phosphoheptanoic acid (an antagonist of the aspartate receptor) prevented the development of seizures induced by the systemic administration of bicuculline. Piredda and Gale hypothesized that this tiny area (less than 1 mm in diameter) within the DPC may be a crucial epileptogenic site of the brain and a site mediating the anticonvulsant action of GABA agonists and aspartate antagonists. Furthermore, these authors
0920-1211/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
95 proposed that the DPC may also be responsible for the development of kindled seizures since the seizures induced by the local drug application are behaviorally similar to the secondarily generalized kindled seizures induced from various limbic structures 25-27. Within the limbic system the amygdala appear to be most susceptible to the development of kindling H. The present study was designed to investigate the susceptibility of the DPC to kindling as compared to that of the amygdala and the superficial prepiriform cortex (SPC), and to determine the kindling interactions between the amygdala and the two sites within the prepiriform cortex (DPC and SPC). MATERIALS AND M E T H O D S
Surgery Thirty-four male Sprague-Dawley albino rats weighing 300 + 25 g were separated into 3 groups. The first group included rats in which bipolar electrodes (Plastic Products, MS 303/3) were stereotaxically implanted in the right DPC and the left amygdala (n = 12); the second group contained rats with electrodes in the right SPC and left amygdala respectively (n = 8); the third group included rats with electrodes implanted in the right DPC and ipsilateral amygdala (n = 14). For the surgery, all rats were anesthetized with a mixture of ketamine (66 mg/kg) and xylazine (10 mg/kg) injected i.m. The following coordinates were used: DPC, 3.8 mm anterior and 2.5 mm lateral from bregma, depth, 7.3 mm from skull; SPC, anterior 3.8 mm, lateral: 2.5 mm, depth: 7.8 mm; the incisor bar was set 5 mm above the interaural line for both DPC and SPC; amygdala, 1.8 mm posterior and 3.7 mm lateral from bregma, depth, 9.0 mm, with the incisor bar 3.5 mm below the interaural line 2~'22.
charge (AD). Each rat was stimulated initially with 1 sec long 30 pA, peak-to-peak, 60 Hz sinusoidal current generated from a sine wave stimulator (Lafayette Instrument Co.). The intensity of the current was increased by 30 pA steps every 2 min until an AD was elicited which was arbitrarily construed to be the ADT. Once the A D T of the primary site was determined, the rat was kept stimulation free for 24 h and then the A D T of the other site was measured as described above. The choice of the primary site was made randomly. Nine rats were initially kindled from the amygdala, 7 from the DPC and 4 from the SPC. The kindling stimulation consisted of a 1 sec train of 400 p A peak-to-peak 60 Hz sine wave current. The rats were stimulated once per hour, 8-10 times per day, until they developed generalized seizures or a total of 20 stimulations was delivered. During each trial, the behavioral changes following the stimulation were observed and classified according to Racine's scoring scale31: stage 1 consisted of motionless staring, stage 2 of head nodding, stage 3 of unilateral forelimb clonus, stage 4 of rearing and bilateral forelimbs clonus, and stage 5 of the above plus loss of balance. During each stimulation trial 2-channel simultaneous EEG recordings were obtained using a Grass Model 79D polygraph. Subsequently, after a 24 h interval all rats were kindled from their respective secondary sites using the same paradigm as before. In the second experiment, the rats in the third group, with the ipsilateral amygdala electrodes, were kindled using a 1 sec train of 400pA peak-topeak 60 Hz sine wave current. Seven rats were kindled initially from the DPC and 7 rats from the ipsilateral amygdala. After the completion of primary kindling, the rats were kindled from their respective secondary site as described above.
Histology Kindling After a 48 h recovery period, the rats in the first 2 groups (with electrodes implanted in the contralateral amygdala) were subjected to the kindling procedure from the respective primary site (amygdala, DPC, SPC). First, the afterdischarge threshold (ADT) was determined defined as the lowest current intensity which produced an afterdis-
Upon the completion of the experiments, all rats were decapitated. The brains were removed and frozen, then sectioned in 20 ~tm slices and stained with thionine. The placements of the electrode tips were verified microscopically by another person blind to the kindling data.
96
Statistics The data were statistically analyzed using the one-way ANOVA with appropriate post-hoc comparisons (Neuman-Keuls).
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Behavioral observations The development of behavioral kindled seizures induced by stimulations of either the DPC or the SPC were identical to that observed with amygdala kindling. Therefore, the seizures were classified, as described by Racine for amygdala kindled seizures3t. When the behavioral seizures reached stage 3, the end of the convulsion was heralded by the onset of wet dog shakes irrespective of the site of stimulation.
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Primary site kindling In the first two groups, the AD threshold, the AD duration at the AD threshold stimulation, the number of stimulations required for the development of a stage 5 seizure, and the AD duration of the first stage 5 seizure were used as the indices depicting the susceptibility to kindling as a function of site (Table I). There were no statistical differences in all these kindling parameters among the 3 sites. These results were reproduced in the 3rd group where there were no differences in the number of stimulations required for the development of a stage 5 seizure between the rats stimulated initially from the DPC (~ + S.E. = 8.6 + 0.6) or the ipsilateral amygdala (8.9 + 0.6).
Fig. 1. Continuous E E G recording during D P C kindling. The stimulation (no. 5) was delivered to the DPC. Black arrows indicate the independent A D s from the amygdala in B and C. The black bars on the time marker indicate the onset of wet dog shakes. DPC, deep prepiriform cortex; A M G , amygdala.
TABLE I The development o f primary kindling induced either f r o m the deep prepiriform cortex (DPC), superficial prepiriform cortex (SPC), or contralateral amygdala ( A M G ) Values are m e a n s _+ S.E. ; A D , afterdischarge; the A D threshold is defined as the lowest current intensity which produced an A D ; ns, not significant.
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No. o f stimulations f o r 1st stage 5 seizure
A D duration at 1st stage 5 seizure (sec)
154.3 + 31.0 125.0 + 26.0 130.0 + 16.0 0.3 ns
13.9 + 1.4 13.0 + 3.0 21.2 + 3.8 2.0 ns
12.6 + 1.2 13.2 +_ 1.7 12.9 + 1.3 0.04 ns
48.0 +_ 6.3 91.0 ___25.3 69.8 _+ 8.6 2.8 ns
97
Electroencephalography Two-channel simultaneous EEG recordings from the prepiriform cortex (either DPC or SPC) and the amygdala demonstrated a unique electrical seizure pattern during DPC, SPC or amygdala kindling. Even when the first stimulation was applied to the DPC or SPC, the ADs immediately spread to the contralateral amygdala (Fig. 1A). With the repetitive prepiriform cortex stimulations long-lasting ADs were observed in the amygdala, which were completely independent from the DPC or SPC ADs (Fig. 1B). These independent ADs usually emerged after stage 2 or 3 seizures had occurred and increased in frequency and amplitude in line with the development of kindled seizures. After the amygdala ADs stopped, the rats remained motionless, staring, and the EEGs from both leads recovered to the normal level for 10 sec. Subsequently, there was another episode of
amygdala ADs which lasted for about 30 sec (Fig. 1C). Rats in which the stimulations were initially applied to the amygdala did not exhibit any independent ADs from the DPC or SPC. However, 20 sec after the end of a generalized seizure, secondary spontaneous ADs occasionally occurred localized in the amygdala (Fig. 2B, C).
Secondary site kindling Previous kindling induced a unidirectional transfer from the prepiriform cortex (both DPC and SPC) to the contralateral amygdala (Table II). The development of primary amygdala kindling required (~ + S.E.) 12.9 + 3.10 stimulations, however, rats kindled initially from the DPC required only 3.4 + 1.5 amygdala stimulations to develop generalized seizures (P < 0.001) (Table IIA). SPC
TABLE I1 Transfer effects between the contralateral amygdala and DPC or SPC
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c Fig. 2. Continuous E E G recording following amygdala stimulation. The stimulation (no. 11) was applied to the left amygdala. Black arrows indicate two clearly separated in time, wellmodulated ADs which occurred in the amygdala only (B, C). The black bars on the time marker after the motor seizure indicate the occurrence of a cluster of wet dog shakes. DPC, deep prepiriform cortex; AMG, amygdala.
C. From SPC Primary kindling (n = 4) Secondary kindling after AMG kindling (n = 4) Fl. 6
13.3 _+ 1.8 13.5 + 2.4 0.007 (ns)
Values are means _+ S.E. a Significantly different from rats initially kindled from the amygdala (post-hoc Neuman-Keuls, P < 0.01) and from rats initially kindled from the SPC (P < 0.05). b p < 0.01 from rats kindled initially from amygdala. c Two additional rats in this group did not develop any generalized seizures even though they received 20 stimulations and were not included in this analysis, ns = not significant.
98 kindling also facilitated the establishment of contralateral amygdala kindling (P < 0.01), although more stimulations (7.2 + 1.3) were required than for the D P C to contralateral amygdala transfer (post hoc P < 0.05). Table liB and C demonstrate that contralateral amygdala kindling did not transfer to either the D P C or the SPC. In fact, in the amygdala/DPC group, 2 rats did not develop generalized kindled seizures from the D P C within 20 stimulations after previous contralateral amygdala kindling while in all other transfer experiments the rats developed generalized seizures. Thus the mean kindling stage was 3.6 + 1.4 for D P C kindling in this group, while it was 5 + 0 in the group kindled initially from the D P C (P < 0.001). This indicates the existence of a negative transfer effect from the contralateral amygdala to the DPC. Such a negative transfer was not observed between contralateral amygdala and SPC. In contrast to the above findings, a bidirectional transfer was observed between D P C and ipsilateral amygdala, albeit of different degrees. D P C kindling readily transferred to the ipsilateral amygdala since all rats required only 1 stimulation to develop a stage 5 seizure accounting for a net reduction in the n u m b e r of stimulations of 89%. Rats initially kindled from the ipsilateral amygdala
required 4.6 + 0.5 stimulations to reach stage 5 when secondarily stimulated from the DPC; this represents a reduction of 47% (Table III). Oneway analysis of variance revealed that secondary amygdala kindling was established significantly faster following D P C kindling (F1, 2 = 55.0, P < 0.001) than secondary D P C kindling preceded by ipsilateral amygdala kindling. Histology Fig. 3 depicts the histological placements of the electrode tips. The D P C group included the rats in which the electrode tips were in the small area, l m m in diameter, reported by Piredda and Gale 26'27 as a crucial site for epileptogenesis. The SPC group included the rats in which the electrode tips were in the superficial layers of prepiriform cortex 21'22. All amygdala electrode tips were found within the basolateral nucleus 21'22 DISCUSSION The prepiriform cortex, also known as the lateral olfactory gyrus, constitutes a m a j o r part of the
TABLE III Transfer effects between the ipsilateral amygdala and DPC Number of stimulations required for the development of stage 5 seizures. A. From amygdala Primary kindling (n = 7) Secondary kindling after DPC kindling (n = 7) F1,12 B. From DPC Primary kindling (n = 7) Secondary kindling after AMG kindling (n = 7) FI,12
8.9 + 0.6 1.0 + 0.0a 13.2 (P < 0.005) 8.6 + 0.6 4.6 + 0.5 6.1 (e < 0.05)
Values are means ___S.E. a Secondary amygdala kindling was established significantly faster following DPC kindling (Fl. 12= 55.0, P < 0.001) than secondary DPC kindling preceded by amygdala kindling.
Fig. 3. Section through the rat brain 9.8 mm anterior to the interaural line redrawn from Pellegrino and Cushman22depicting the location of the electrodes in the DPC (the same site as shown in Piredda and Gale 26"27) and SPC. CC, corpus callosum; CP, caudate-putamen; NA, nucleus accumbens; OT, lateral olfactory tract.
99 oldest cortical derivatives 7. Anatomically Haberly and Price 12 have divided the prepiriform cortex into a superficial plexiform layer (layer I), superficial compact cell layer (layer II) and deep cell layer (layer III). Deep to layer III there is a well-developed group of polymorphous cells called endopiriform nucleus 12'14'15. Our DPC electrodes were within the latter nucleus and our locations were identical to those shown by Piredda and Gale. The SPC electrodes were located within the first 3 layers. In mammals, the prepiriform cortex is anatomically and functionally related to olfactory structures and to many limbic structures. Since its afferent fibers are derived from the lateral olfactory stria, the prepiriform cortex is regarded as an olfactory relay center 7. Furthermore, although the prepiriform cortex is not considered to be part of the limbic system, its outputs directly and indirectly (via the entorhinal cortex) project to several limbic structures such as the septal area, amygdala, and entorhinal cortex 7'13'15'35. These output projections seem to use excitatory amino acids as transmitters a. Although the fibers from the prepiriform cortex mainly project to the ipsilateral limbic structures, a sparse contralateral projection has been identified 12. Limbic sites do not directly project back to the prepiriform cortex. However, there is a polysynaptic pathway from the amygdala feeding back to the prepiriform cortex, via the accessory olfactory bulb 28'29'34. Kindling has been induced from many olfactory structures such as the olfactory bulb, tract and nucleus, piriform cortex and entorhinal cortex 3'11'30'31'33. It has been suggested that these structures are less sensitive to kindling than the amygdala 2'11'13'23'24'32'33,although there is one report indicating that the olfactory bulb may require fewer stimulations to develop generalized seizures than the amygdala 3. To date, there has been no report on the kindling of the prepiriform cortex. Our study shows that both parts of the prepiriform cortex, DPC and SPC, are as sensitive to kindling as the amygdala, as determined by the local AD threshold and the number of stimulations required for the development of generalized kindled seizures. Therefore, it can be concluded that the DPC is not only sensitive to chemical convulsants,
but also to electrical kindling. However, the DPC does not differ from the SPC in terms of primary kindling. This result must be contrasted with the discrete pharmacological localization reported by Piredda and Gale using chemical convulsants 25-27. The seizure behaviors induced by DPC and SPC kindling are identical to those produced by amygdala kindling and are characterized by gradually potentiated limbic symptoms such as staring, head nodding, and finally the development of generalized seizures. These observations suggest that the development of DPC and SPC kindling depends on the gradual activation of other sites including the amygdala. The EEG data support this hypothesis. The amygdala were very susceptible to the generation of independent ADs even when the DPC or SPC were stimulated; independent ADs did not occur from either DPC or SPC irrespective of the site of stimulation. Previous studies have demonstrated transfer effects between a number of brain structures during kindlinf '4-6'1°'18'31. By definition, 'transfer' indicates a significant acceleration of the development of kindling from a secondary site. Nevertheless, transfer also occurs between different kindling agents such as flurothyl, pentylenetetrazol, carbachol, opiates and electrical kindling 4'5'2°'36. Thus, transfer seems to be a widespread phenomenon throughout the brain. However, our data show that transfer did not occur from contralateral amygdala to DPC or SPC. Instead in two rats there was interference (negative transfer) since amygdala kindling retarded the development of contralateral DPC kindling. Since the lack of transfer may be related to the presence of only indirect connections between amygdala and prepiriform cortex 28'29'34, we studied the transfer relationships between ipsilaterally stimulated sites. In this case a bilateral interaction occurred albeit of different strengths. DPC kindling readily transferred to the ipsilateral amygdala while the transfer from the amygdala was less potent. The savings in secondary kindling of ipsilateral DPC to amygdala kindling may be due to the presence of direct excitatory inputs to the amygdala from the prepiriform cortex. Based on this finding, the anatomical existence of predominantly unilateral connections between DPC and amygda-
100 la 7'9'12 and rich intra-amygdala connections ~4'~5, we suggest that DPC kindling may transfer to the contralateral amygdala through the ipsilateral amygdala.
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ACKNOWLEDGEMENTS Supported in part by Grant NS 20253 from the NINCDS. Dr. D.Y. Zhao is the recipient of the 1985 Beijing-Einstein College of Medicine Visiting Fellowship. We thank Ms. Donna Platyan for her help in the preparation of this manuscript.
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