Functional anatomy of limbic seizures: Focal discharges from medial entorhinal cortex in rat

Brain Research, 280 (1983) 25-40

25

Elsevier

Functional Anatomy of Limbic Seizures: Focal Discharges from Medial Entorhinal Cortex in Rat ROBERT C. COLLINS, ROBERT G. TEARSE and ERIC W. LOTHMAN Department of Neurology, Washington University School of Medicine, St. Louis, MO 63I lO ( U.S.A. )

(Accepted March 22nd, 1983) Key words': epilepsy - - limbic system - - functional anatomy - - entorhinal cortex

Focal seizure discharges were induced in the ventral aspect of the medial entorhinal cortex of awake, freely moving rats, either with cannula injections of penicillin or picrotoxin (0.02/*1 every 10--15 min) or by repetitive tetanic electrical stimulation. [~~CJDeoxyglucose autoradiography (DG) was performed when animals were in a "steady-state' with respect to electrographic discharges and/or behavioral changes. During simple interictal spikes behavior remained normal and DG labeling was increased only in the entorhinal focus and stratum moleculare of the ventral dentate gyrus. With complex spikes and short seizures animals exhibited staring, decreased responsiveness, and occasional wet dog shakes. DG labeling was increased in all layers of the dentate gyrus, Ammon's horn (ipsilateral > contralateral) and, to a lesser degree, in ipsilateral amygdala, and the accumbens-ventral pallidum area. During strong seizures, rearing and forelimb clonus occurred and metabolism was strongly activated bilaterally in the hippocampal formation, amygdala, accumbens, substantia nigra, and the anterior and periventricular thalamic nuclei. These studies indicate that the dentate gyrus initially restricts the entry of seizures from entorhinal cortex into the rest of hippocampus. As this is overcome there is rapid bilateral spread through the hippocampal formation with passive interruption of normal behavior. With prolonged seizure discharges there is further capture of amygdala and subcortical extrapyramidal and thalamic nuclei associated with behavioral convulsions. INTRODUCTION T e m p o r a l l o b e - l i m b i c system seizures are the most common form of epilepsy in man. In part this is thought to reflect the interplay of 3 factors. First, the hippocampus is unusually vulnerable to a great variety of epileptogenic insults, from hypoxia-ischemia in the perinatal period to head t r a u m a in the adult. Second, recent anatomical studies emphasize the high degree of interconnectedness between hippocampus, entorhinal cortex and amygdala, such that seizures originating in one part of the system potentially have access to all others 36,37,54,67-72. Third, physiological studies emphasize that several types of synaptic potentiation are p r o m i n e n t at excitatory synapses within h i p p o c a m p a l circuits 1,8,9,23,24.28,41 - a factor implicated in the proclivity of the limbic system to develop seizures 30,5S,60. The present study was u n d e r t a k e n to examine changes in functional a n a t o m y during the expression of acute graded limbic seizures in rat. In particular we wanted to know how seizure discharges spread 0006-8993/83/$03.00 © 1983 Elscvier Science Publishers B.V.

from a single focus in entorhinal cortex throughout anatomical pathways of the limbic system to cause behavioral changes. O u r research strategy was directed at answering 3 questions. (1) What is the relationship between the intensity of a seizure focus in entorhinal cortex and the spread of activity measured electrographically and metabolically with the [14C]deoxyglucose autoradiographic technique? (2) What are the preferred anatomical pathways used during the spread of seizures? (3) What is the relationship between the site and intensity of seizure activity and behavioral changes'? Previous studies combining electrographic recording of seizures with metabolic 2-deoxyglucose mapping have proved useful in determining the functional anatomy of focal neocortical seizuresl J.J3->,3~ as well as limbic system seizuresl~,32,~7,<. Part of this present work has appeared in abstract form 7~. METHODS Experiments were p e r f o r m e d on adult male Spra-

26 gue-Dawley rats weighing 270-325 g that were acclimated to small cages and fasted overnight, using halothane for anesthesia we inserted PE-50 catheters into the femoral artery and vein and routed these subcutaneously to exit from the back of the neck. For experiments with chemical convulsants we used a 30gauge prefilled steel cannula insulated to 0.5-1.0 mm of the tip connected by PE-10 tubing to a 1.0~1 Hamilton syringe. The recordings at the focus were obtained from the cannula and referred to an indifferent electrode over the frontal nasal sinus. For electrically induced convulsions we used a bipolar coaxial electrode (tip diameter 100 j~m, tip to shaft separation 750 ktm, shaft diameter 200 ~m, Rhodes Electronics, Duarte, CA) connected to an electronic switch that allowed stimulation and recording from the same site. The cannula, or electrode, was inserted into the ventral aspect of the medial entorhinal cortex using the coordinates of Pellegrino et al. (nasal bar +5.0 mm; posterior from bregma 6.0 ram, lateral 4.0-5.6 mm, deep 5.5-6.0 mm). In some animals bipolar electrodes were also inserted unilaterally or bilaterally into dorsal hippocampus, amygdala and contralateral entorhinal cortex to monitor electroencephalographic (EEG) activity at sites distant from the focus. Discharges were amplified and monitored on a Grass 7P polygraph. Electrode and cannula positions were verified histologically. Experiments were begun two or more hours after anesthesia when animals were alert and freely mobile. Forty-nine animals were studied using either different convulsants or electrical paradigms in order to avoid results and conclusions unique to one method of stimulation. Gradations of seizures from mild to severe were induced by varying the amount of convulsant delivered, or by repetitively applying closely spaced tetanic stimuli. For chemical seizures we injected either penicillin (n = 9; 150 mg/ml) or picrotoxin (n = 22; 0.3-0.5 mg/ml) balanced isosmotically with NaCl and containing 3.0 retool/1 KCI. In preliminary experiments 6 animals were studied using [x4C]penicillin to determine the spread of convulsant from the injection site. These animals were sacrificed from 1 to 60 min after cannula injections ranging from 0.1 to 0.5/~1 of [14C]penicillin (150 mg/ml; 80 gCi/ml, Amersham Searle). Dissected brains were frozen immediately and processed for autoradiography ~5. Based upon results from these animals we sub-

sequently used injection schedules of 0.02-41.05 ul over 1-2 min every 10-30 rain to reduce a restricted seizure focus. Total amounts varied from 0.05 to 0.6 ~tl for 1-4 h, for mild to strong seizure states, respectively. For electrical stimulation, the bipolar electrode in the entorhinal cortex was electronically switched to a constant current isolated stimulation while the recording amplifier was grounded. Following a tetanic stimulus (100-1 ms, biphasic pulses at 10 Hz, 800-2000 ~A) the electrode was switched to a record mode and the amplitude was adjusted to faithfully monitor afterdischarges. The stimulus was delivered every 5 min. The initial stimuli produced mild seizures; later stimuli produced marked seizures as described below TM. We studied animals with the [~4C]deoxyglucose (DG) autoradiographic technique 65 once electrographic discharges or behavioral convulsions became stable, usually 3-5 h after anesthesia. During the 45 rain of D G circulation the animals' behavioral activity was scored in one of 4 categories: (1) normal --animals intermittently slept, explored the cage, or groomed; (2) mild convulsions - - the animals' active behavior was interrupted by staring, sniffing and occasional chewing movements: (3) moderate convulsions - - immobile staring, occasional wet dog shakes (< 1/5 rain), occasional reared immobile 'praying' posture; (4) marked convulsions - - frequent wet dog shakes (usually > 5/5 rain), rearing with forelimb clonus, tonic head or torso movements, loss of posture. For D G control animals (n = 7) we used rats with electrodes or cannula who were not stimulate& At the end of the DG experiments the animals were killed with barbiturate and then their brains were perfused and fixed with fresh phosphate-buffered 3.3% paraformaldehyde and cut frozen at 20 ~m m either a horizontal or coronal axis for subsequent autoradiographic analysis. Optical densities (OD) were measured on triplicate sections using a microscope photometer with an adjustable aperture. Brain structures in experimental groups were compared against areas in controls using the method of ratios (OD of experimental area/OD experimental corpus callosum; OD of control animal's area/OD control corpus callosum), All animals received the same dose of [t4C]DG (7 pCi/100 g) and were processed similarly, conditions found necessary for the use of internal controls and the method of ratios ~7.7a. In addition, in

27 this study we report only major changes where the ratio was greater than 1.3, findings readily appreciated by direct visual inspection of the autoradiograms. Some of the sections used for autoradiography were subsequently stained with thionin to determine details within the histological sites of seizure spread. RESULTS Seizure f)~eus

All animals were examined histologically to localize the track of the electrode or cannula, and individual animals were excluded when the tip was not found in the ventral aspect of the medial entorhinal cortex. We used two factors to estimate the boundaries of the focus. First, DG autoradiograms usually revealed a well-defined area of increased metabolism confined to the tissue immediately surrounding or adjacent to the tip of the cannula track. This was due to local stimulation as electrode sites in unstimulated control animals showed normal metabolism or a slight depression. This feature could not be used in animals having strong seizures, however, since metabolism was markedly increased throughout the entire histological field and no discrete focal autoradiographic features could be appreciated (see below). Second, autoradiograms of animals injected with [HC]penicillin revealed that our methods of infusion resulted in a well-confined stimulus site. Animals frozen 1 rain after large volume, fast injections (0.1 ul/10 s) were found Io have an autoradiographic focus greater than 3 mm 3 with spread of convulsant beyond entorhinal cortex. Lower rates of infusion (< 0.05 ul/min) resulted in discrete foci with volumes < 2 mm ~, Although small amounts of convulsant may have escaped into intraventricular or subarachnoid space in individual animals we think that our slow rate of injections (average 0.02 l~l/min every 15 rain) and long period of study (average 2 h) would have allowed for sufficient dilution and clearance so that disrant areas would not have been influenced by the convulsant. In our deoxyglucose studies we waited until animals reached a steady state of electrographic discharges or showed consistent pattern of abnormal behavior before injecting radioactive DG intravenously, usually 20 rain to an hour following the final iniection of convulsant.

Mild con vulsive state

It was not possible to distinguish mild convulsive states from normal behavior in most cases. Even animals showing spike discharge rates up to 30/rain at the recording cannula exhibited only intermittent staring and sniffing - - activity that was present but less frequent or prolonged in control animals. Electrographic records from these animals showed high amplitude repetitive spikes of simple configuration at the entorhinal focus with distant sites remaining normal (Fig. 1A). Deoxyglucose studies in these animals (n = 7) showed a restricted pattern of metabolic activation (Figs. 2 and 5B). In animals with discharge rates below 10/rain there were no DG changes apparent at the focus or distant sites. Above this rate the entorhinal cortex (especially deep layers) and ipsilateral ventral dentate gyrus (especially stratum moleculare) showed prominent activation, between 50 and 170% above control. There was no metabolic activation in successive terminal fields beyond dentate gyrus.

Moderate con vulsive state

Individual spike discharges rarely exceeded 40/rain. Above this rate grouped polyspikes and short seizures (Fig. 2B) appeared followed later by long seizures (> 10 s). The transition from spikes to electrographic seizures was dependent on the total amouni of convulsant and the duration of the study. Seizure discharges typically emerged after two or more hours of intermittent injections of low dose picrotoxin, 0.01 ug/0.02 ul every 10-15 rain. Seizure discharges were not linked with any one single convulsive behavior. Wet dog shakes were the clearest behavioral abnormality, but occasional sniffing and head nodding also occurred. Most often rats remained motionless during a seizure discharge and would not respond to tactile stimuli or even presentation of food. The metabolic pattern in animals who remained in this state (n = 4) showed increased DG uptake in pathways beyond the ipsilateral dentate gyrus (Fig. 3). There was prominent activation in Ammon's horn (ipsilateral > contralateral: CA3 > CA1) and in the ipsilateral lateral septal nucleus. Two of the 4 animals in this group also showed > 309~ increased uptake in the ipsilateral lateral and basolateral amygdala, nucleus accumbens and ventral pallidum-lateral preoptic area (Fig. 3A).

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Fig. 1. Specimen records from animals in different seizure states. A: mild seizures - - normal behavior. Large amplitude intcrictal spikes were seen in the focus at rates up to 40/rain. Distant sites, such as the ipsilateral dorsal hippocampus remained normal. B: moderate seizures - - wet dog shakes. Complex polyphasic spikes and short electrographic seizures were seen in the focus and at distant sites. Not all of the spikes were propagated. C: severe seizures - - rearing and forelimb clonus. Prolonged etectrographic discharges were seen in the focus and distant sites. Tracings from two or more sites were often dissociated with seizures out of phase and OCCasionally appearing to begin in areas distant from the entorhinal focus, as in the dorsal bippocampus in this animal (arrow, upper time trace). Behavioral features such as wet dog shakes (arrows, lower trace), rearing, clonus, or staring did not correlate with any particular electrographic feature when all animals were examined and compared.

29

Fig. 2. DG metaholisn3 - - mild behavioral convulsions (staring). A: the only change in metabolism occurred in the entorhinal focus Idark area. lower right) and ipsilaleral ventral dentatc gyrus. B: normal DG metabolism in hippocampus with relativc accentuation along the terminal ticld of the pcrforant pathway on either side of the hippocampal fissure. A slight accentuation is also commonly seen oxcr ('A3. Notc rclative pallor in dentate gyrus. (': histological stain of thc section used for the autoradiogram in B. D: higher magnification of hippocampus from section A. The cannula track is marked by the arrow. Metabolism in the R)cus is particularly active m deep layers of cntorhinal cortcx. Ad.iacent subicular fields are normal. There is a marked increase in metabolism in stratum moleculate of the inner and outer bladcs of the dentate gyrus while Amm{m's horn remains normal. E: histological stain of section used in D. F: circuit diagram showing input from entorhinal cortex into its terminal field ( 1) along dendrites of granule cells and dendritic tips of CA 1 and CA3 pyramidal neurons. Subsequent feed forward excitation through the circuit would increase metabolism in the terminal field of the dentate gyrus into CA3 (2) and CA3 into CA1 (3); discharges from CA3 and CA1 woulc~ feed back into subicular fields and cntorhinal cortex (4). Sec Figs. ?, and 4 for comparison. Abbreviations: CAI, CA3. histological subfields of cornu ammonis; ECm, entorhinal cortcx pars mcdialis; IB, OB, inner and outer blade of dentatc gyrus: Pro, prestibiculum: Par. parasubiculum: Sub, subicuI U nl,

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Fig. 3. DG metabolism - - moderate behavior convulsions (wet dog shakes, nodding). Sections are ventral to dt~rsai in A-I2. A: metabolism is increased in ipsilateral medial and lateral entorhinal cortex, hippocampus, amygdala and accumbel~s-ventralpallidum (see Fig. 4B for labeling of histological fields). B: note accentuation in Ammon's horn as well as dentate gyrus. The pallor in the cntorhinal focus reflects local edema associated with the cannula placement. C: metabolism is increased in distant fields, including ipsilateral lateral septum, anterior and midline periventricular thalamus, and contralateral hippocampus. D: increased metabolism in dorsal hippocampus. E and F: higher power view of metabolism and histological fields of a horizontal section of dorsal hippocampus. The greatest accentuation occurs in neuropil, primarily, but not exclusively, in stratum moleculare surrounding dentate gyrus

complex spike discharges were less frequent and often suppressed following a prolonged seizure. Tonic high frequency discharges were often seen in animals who were immobile and staring, and grouped high amplitude clonic discharges were often present during rearing and forelimb clonus. It should be emphasized, however, that each of these electrographic events was also seen on occasion in individual animals without behavioral abnormalities. There was no absolute correlation between particular electrographic discharges and individual behavioral manifestations. In addition, on several occasions low amplitude high frequency discharges were seen to begin at electrode sites distant from the focus (e.g. Fig. lC, upper arrow).

"Fhe metabolic pattern in this group of animals (n = 4) showed p r o m i n e n t bilateral activation of entorhinal cortex (5(I-18/)%), A m m o n ' s horn (5(1-118%), amygdala (5(1-93%), n. accumbens and ventral pallidum (3(1-6(1%) and substantia nigra (43-79%) (Fig. 4). Particular features within each site were of interest. Metabolism in medial and lateral e m o r h i n a l cortex was increased up to and including perirhinal cortex, but stopped abruptly at neocortex. In hippocampus the pattern of activation was dentate gyrus > CA3 > C A 1 . In the dentate gyrus and CA3 the greatest activation occurred over the cell body layers - stratum granulare and stratum pyramidale, in contradistinction to animals with mild convulsions where increased activity remained localized to stratum molec-

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Fig. 4. DG metabolism - - severe convulsions (rearing with forelimb clonus). Sections are ventral to dorsal in A-H. A: metabolism is increased bilaterally in entorhinal cortex, hippocampus, amygdala, basal forebrain, and substantia nigra, There is no increase in hypothalamus or neocortex. B: histological stain of section used in A. Abbreviations: A, amygdala - - basolateral and lateral nuclei: AC, accumbens; H, hypothalamus; PO, lateral preoptic ventral pallidum area; SN. substantia nigra, C: metabolism is increased bilaterally in hippocampus and allocortex up to the rhinal fissures (small arrows). Edema and pallor surround cannula tip (large arrow). D: histological stain of section used for C. Abbreviations: C, caudate; S, septum. E: higher horizontal section showing bilateral changes in septurn and anterior periventricular thalamus (compare with C). F and G: high power views of metabolism and histological fields of hippocampus. Thc zones of greatest metabolism overlie stratum granulare of dentate gyrus, and stratum pyramidale of CA3 (compare with Fig. 2D-E). H: metabolism is increased bilaterally in dorsal hippocampus. Pallor on right marks recording electrode.

ulare of the dentate gyrus (compare Fig. 2 D - E with 4F-G). The greatest increase in activity in the amygdala occurred in the lateral and basolateral nuclei, with a mild increase above background in other nuclei. Nucleus accumbens and the lateral preoptic area showed increased metabolic activity as a unit (see unilateral activation, Fig. 3A, and bilateral activation in Fig. 4A). Anterior and midline periventricular thalamic nuclei were found to be activated in several animals. By contrast, the main body of caudate-putamen in the extrapyramidal system, neocortex, and the entire hypothalamus were never above control level.

Pathways of seizure spread The route and timing of seizure spread can be par-

tially inferred from examining the metabolic pattern of activity in seizure states of increasing severity. In order to visualize preferential pathways of spread we mapped changes in metabolism on the 'unfolded hippocampus' - - a topographic method devised by Swanson et al. 72 for mapping orthograde connections. Representative animals are shown in Figs. 5 and 6. During interictal spike discharges and mild convulsions animals showed activation at the entorhihal focus and first order synaptic beds, most prominent along the perforant pathway at the outer blade of the dentate gyrus. A mild but inconsistent increase in activity was also found in contralateral hippocampus (Fig. 5), With stronger convulsions a prominent increase in metabolism was found beyond dentate gyrus into Ammon's horn, ipsilateral > contralateral

32

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Fig, 5. Metabolic change in hippocampus during interictal spike discharges in medial entorhinal cortex. Sections are rostral to caudal in A-C. The seizure focus is seen in the right entorhinal cortex in C. The small density seen in neocortex probably represents activation from penicillin around the cannula in this animal. Metabolism is increased in ipsilateral ventral dentate gyrus, B: metabolism in dorsal hippocampus, A, is normal. Optical density measurements were made in the subfields of the hippocampus in 15 coronal sections of control animals and in a representative animal having sustained interictal discharges (25/rain). The relative changes compared to control are indicated on the unfolded hippocampal map below 72.

(Fig. 6). This was always more prominent in CA4 and CA3 subfields than in CA1, and in the ventral or temporal sectors of the hippocampus compared to the dorsal portion. Metabolic activity also became increased in subicular fields and lateral entorhinal cortex as seizures became stronger. Animals showing this incomplete bilateral pattern also showed activa-

tion of subcortical structures, particularly the ipsilateral lateral septum, and the lateral and basolateral amygdala nuclei. Once very strong convulsions were achieved the entire hippocampal formation showed intense activity, and subcortical spread became equal bilaterally (map not shown; see Fig. 4).

33

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Fig. 6. Metabolic change in hippocampus seen during electrographic seizures from entorhinal cortex and moderate convulsive behavior. Sections are rostral to caudal in A-C. By comparison with metabolic changes during mild seizures (Fig. 5), there is now activation throughout most of hippocampus. This remains greatest in the temporal (ventral) pole on the ipsilateral side. Seizure sprcad through sequential synapses has activated sites from ipsilateral to contralateral sides, and from ventral to dorsal sectors. In general, the relative increase in metabolism is dependent on synaptic proximity to the focus in entorhinal cortex, i.e. dentate gyrus > CA3 > CAl. A representative animal from a group of 4 is illustrated.

Correlations between electrographic discharges', convulsive behavior and metabolism

al points. First, it was possible for animals to h a v e up to 10 spikes/min w i t h o u t any definite increase in me-

T h e data f r o m 26 of the e x p e r i m e n t a l animals w e r e

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34 DEOXYGLUCOSE METABOLIC PATTERNS .

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Fig. 7. Relationship between seizure intensity in the focus (abscissa) and changes in deoxyglucose metabolic pattern (ordinate). Electrographic discharges were counted during the period of DG circulation for each animal using both simple spikes and individual deflections during a seizure discharge. The transition from individual spikes to seizures is indicated by hatched lines on the abscissa. Twenty-six individual animals are plotted with respect to stimulus (P, penicillin; Px, picrotoxin; E, electrical) and behavioral rank (0, mild - - staring (n = 7); [2]. moderate - - wet dog shakes, nodding (n = 2); A, severe - rearing with clonus (n = 4)). Unenclosed symbols represent animals who were judged to be normal.

tent staring appeared abnormal. Metabolism in these animals was increased only in the entorhinal focus and dentate gyrus. Third, with increasing number and duration of seizure discharges in entorhinal cortex there was increasing convulsive behavior and increasing metabolism in distant sites. In general, positive, convulsive manifestations such as wet dog shakes, rearing and forelimb clonus only occurred after complex polyphasic discharges or electrographic seizures became established and spread through bilateral limbic circuits, subcortical thalamic and extrapyramidal sites. There were two exceptions: one animal in the mild behavioral category (staring) was found to have bilateral metabolic changes in hippocampus, while another with moderate convulsions (wet dog shakes) showed only ipsilateral change. DISCUSSION There are 3 principal conclusions from this study. First, the transition from simple interictal spike discharges to more fully developed paroxysms is necessary for activation of metabolism in successive synaptic relays and the appearance of clear behavioral

manifestations. Second, the sequential changes in DG metabolism suggest that the dentate gyrus acts as a restrictive gateway for seizure spread from entorhihal cortex to the rest of the limbic system, while the amygdala acts as a principal exit for spread of limbic seizures to subcortical extrapyramidal pathways. Third, the functional anatomy of limbic seizures can be divided into two groups of circuits. Seizures confined to bilateral hippocampus cause negative symptoms - - interruption of behavioL, staring and decreased response to stimuli. Seizure spread that includes bilateral amygdala and its pathways causes positive symptoms - - head nodding, rearing, and forelimb clonus. These conclusions arc reached primarily by comparing the deoxyglucose metabolic pattern of animals in different convulsive states with anatomical and electrophysiological studies of entorhinal cortex in particular, and the limbic system in general.

The importance of the interictal-ictat transition A major finding of this study was the seeming impotence of interictal spikes in the entorhinal cortex for causing metabolic change or behavioral manifestations. Technical as well as physiological factors probably account for the metabolic finding. First, the D G autoradiographic method integrates all metabolic events over the 50 rain of study. Paroxysmal discharges in neurons are followed by hyperpotarization and decreased firing so that metabolic demands in these two periods may cancel out. In addition, one must consider activity in local inhibitory circuits as well as ion pumping in adjacent gila as contributing to the metabolic activity within a focus. Put simply, the [t4C]DG method cannot resolve the metabolic labeling of these different physiological events with respect to time or space in this type of study. The results of these experiments, as well as metabolic studies of interictal spikes in neocortex ts, suggest that low frequencies of discharge ( < 10/min) result in a net balance of glucose metabolism over time as seen within the histological field. An additional consideration in each case is that cannula and electrodes cause a variable, albeit small amount of trauma and edema which diminish the autoradiographic image and give a falsely low metabolic value. High rates of interictai spikes caused an increase in metabolism in the focus and its projection into the first synaptic bed in most cases. This would represent

35 glucose utilization in neuropil in response to ion pumping and synaptic transmission associated with the axonal endings of layer 1I entorhinal neurons firing onto the dendrites of granule cells39-~'s-~% Metabolic activity within entorhinal terminals along dendritic tips of CA1 and CA3 neurons would also contribute to the autoradiographic picture of activation% Although each seizure focus was largely confined to the medial, compared to the lateral entorhinal cortex we could not appreciate any gradation of metabolic change either within stratum moleculare of the inner or outer blade of the dentate gyrus or within the hmgitudinal extent of the alvear path as might be expected from findings of anatomical studies (e.g., Fig. 21 of ref. 67). Such observations may be beyond the limits of resolution for carbon-14 film autoradiography. The lack of metabolic activation in neuronal chains beyond this first synapse - - in particular the dentate hilum (CA4), and stratum radiatum, pyramidale, or oriens of CA3 or CA1 - - is a major finding of this study. We believe this containment of seizure spread might reflect 3 physiological processes. First, the granule cells of the dentate gyrus have both a relativelv high activation potential as well as a relatively long posl-actixation 1PSP ~,as,49. The first factor would limit the response of granule cells to synchronous input from a population of entorhinal cells (estimated at > 400 afferent fibers). The second factor would limit its ability to follow frequency stimulation much abme 10/s. Second, of particular importance for seizure spread is that granule cells are resistant to developing burst discharge even during strong epileptogenic stimuli 5
were greatly dampened as they projected through the first synaptic relay. This is not to say that n o discharges were relayed forward through polysynaptic pathways. Electrophysiological studies have demonstrated that it is possible to evoke potentials in many distant sites from single or short train stimuli of entorhinal cortex or perforant pathway 3,1L~,42. In this study we did not use a sufficient number of distant electrodes to assess this possibility in every case. From this and other studies ~5.j7 we think the DG method would be less sensitive than electrode mapping for identifying isolated or infrequent events. Physiological studies emphasize that the success of evoking distant polysynaptic potentials in the limbic system is dependent upon stimulus parameters, with isolated or low frequency stimuli (10-12 Hz) being optimal. We conclude that high frequency discharges associated with spikes probably 'jam the circuits" at the first relay. In this regard an important question is whether disruption of n o r m a l activity at this synapse might change behavior. The rodent hippocampus is thought to play an active role during environmental exploration 5~. or during tasks requiring 'working memory "53. Stimulation of entorhinal cortex has been found to disrupt active avoidance responses 45. We did not use tests of learning or memory in these experiments and may have missed alterations in these functions during periods of interictal spikes. Once electrographic seizures occur in the focus then metabolism increases in Ammon's horn and overt behavioral abnormalities begin. We think the appearance of these distant changes would reflect in part the cummulative effect of prolonged (> 10 s) tetanic stimuli passing through successive hippocampal synapses. Many investigators have found a potentiation of synaptic effect in hippocampal pathways with afferent stimuli of low frequency, 10-12 Hz, and long duration, ~> 10 s ~,s,y--'3,e4,2s. In separate experiments we found that recurrent (q. 5 rain) tetanic stimulation of this nature results in generalized, permanent stage 5 kindled seizures in rat 3s. Once seizures spread successfully beyond the denrate gyrus then many mechanisms could come into play that would aggravate the seizure process further. Discharges in CA3 and CA I would feed back into entorhinal cortex and cause further potentiation t'~.25. Local hippocampal inhibitory interneurons lose potency as seizures progress 4.~s,44. Antidromic

36 firing could also play a roledL Finally, as recurrent seizures continue over several hours, excessive activity in CA3 might likely accentuate the bursting property of these pyramidal neurons either through intrinsic changes in the dendrites, the immediate ionic milieu, or through other mechanisms3L56,63,6z. This area could potentially initiate an 'independent' seizure discharge and lead to the type of limbic 'status epilepticus' seen during our state of severe focal seizures. The complexity of these events was previewed succinctly by Green and Adey in 195631: 'The properties of the hippocampal system when stimulated repetitively indicate that the potentiation produced by single shocks or volleys are cumulative. A slow decay of excitability, seemingly exponential in form, follows each shock, and the period of increased excitability increases with the number of pulses in the conditioningvolley. The fact that repetitive stimuli can apparently act as a kind of pacemaker suggests that following each response a refractory period occurs during which spontaneous activity fails to appear, but as the train is gradually lengthened a point is reached when the level of excitability or post-tetanic potentiation becomes high enough to overcome the pacemaker effect. This may have some possible general significance in the origin of seizure discharges . . .

Anatomical spread of seizures The autoradiographic images of DG metabolism during interictal spikes are similar to images published from experiments using the autoradiographic method for tracing efferent pathways from entorhinal cortex 67,68,71,77. With infrequent discharges the metabolic activity in the dentate gyrus was more prominent in the ventral than the dorsal hippocampus on the ipsilateral side (see Fig. 3a in ref. 77). As spikes become more frequent, images of the ventral projections become darker at the same time metabolism begins to increase in dentate gyrus of the dorsal hippocampus. This probably reflects discharges from a growing focus as it spreads from ventral towards dorsal entorhinal cortex (see Fig. 4 in ref. 67 and Fig. 2 in ref. 77). There are no longitudinal association pathways within the dentate gyrus itself that would explain ventral to dorsal spread. These studies did not reveal clear evidence for a lamellar organization of hippocampus 5. Our results do not disprove such a scheme, however, since our seizure stimuli would include a large output from entorhinal cortex compared to the restricted focal stimuli of perforant pathways used by Anderson. Our stud-

ies do confirm the relative separation of hippocampus into ventral (or temporal) and dorsal (or septal) components as found by previous electrophysiological studies z5 and more recent anatomical 7~.7:,77 as well as DG 35 experiments. Contralateral homotopic projections to entorhinal cortex or heterotopic projections to subicular fields were only weakly activated metabolically in two of 12 animals having interictal spikes, consistent with relatively weak commissural systems from ventral entorhinal cortex 7r'. If behavioral convulsions occurred during DG circulation, then metabolism was found to be increased bilaterally through Ammon's horn. With mild convulsions the magnitude of change in different sites was proportional to the synaptic distance from the entorhinal cortex, dentate gyrus :> CA3 > CA1. In addition, once convulsions began the accentuation in metabolism became more equal in dorsal and ventral hippocampus, perhaps reflecting in part the spread ol activity through the longitudinal association bundle of Lorente de N6 (see discussion in ref. 72). The contralateral increase of activity could reflect spread through contra[ateral dentate gyrus, as well as spread through the extensive commissural system from CA4 and CA3. The dense autoradiographic changes seen with very strong seizures offers only inferential evidence with respect to pathways of seizure spread and we emphasize our caution in interpreting this pattern. Two points merit discussion. First, metabolism m amygdala was increased only when the ipsilateral ventral subicular fields, lateral cntorhinal cortex and perirhinal cortex were also activated. Since these latter sites became involved only when activity in Ammon's horn was very intense, and only after several hours of seizures, we suspect that the sequence of seizure spread to amygdala followed upon posterior spread from CA3 and CA1 to allocortex rather than direct spread of convulsant or stimulating current beyond the confines of medial entorhinal cortex 54,~'2,::'. As indicated, our material does not allow a definitive answer in this point. Nevertheless, the amygdala was never involved earlier or to a greater extent than Ammon's horn. Second, whereas metabolic activity increased in septal nuclei (ipsilateral > contralateral) concomitant with activity in Ammon's horn, subcortical extrapyramidal nuclei showed activation very late, when amygdala were very dense. Bilateral acti-

37 vation of substantia nigra became particularly intense late in the course, perhaps reflecting activity relayed both from the central amygdaloid nucleus and nucleus accumbensUL22,37.70. The explanation for lack of any dramatic change in hypothalamus and ventromedial caudate is not apparent since these areas receive limbic input s3,71. Seizure spread into ventral subiculum should have been projected further into ventral medial hypothalamus. A strict reading of our autoradiograms suggests that amygdalohypothalamic circuits would not have contributed to changes here, however, since the medial amygdaloid nucleus was not greatly activated. The lack of clear change in the body of the caudate is more problematical considering the strong input from the basolateral amygdala33. Our results indicate that the main effect of seizures in these circuits is, instead, in n. accumbens and the ventral pallidum area. Activity here could represent input from subiculum and amygdala 22. These considerations are summarized in an anatomical schema illustrated in Fig. 8. Increased metabolism found in hypothalamus 35, neocortex 26 and

FUNCTIONAL

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Fig. 8. Summary of major neuroanatomical sites where glucose utilization becomes increased during strong IAmbic seizures. The anatomical schema reflects major neuroanatomical connections in IAmbic pathwaysT,llL22,33.3~,37,54,67 72.77. Findings in the present study suggest focal discharges beginning in medial entorhinal cortex go through 3 phases in becoming generalized. (1) lnterictal spikes activate dentate gyrus. (2) Short seizures spread further into Ammon's hum and its projections contralateral to CA3 and CA4, into septum, and back to subiculum and cntorhinal cortex. Normal behavior is interrupted with staring during this phase. In addition, ipsilateral amygdala and accumbens begin to become involved. (3) Bilateral cortical and subcortical activation occurs during prolonged recurrent seizures, when electrographic records indicate strong interaction between multiple sites within the system. Involvement of subcortical sites occurs with behavioral convulsions.

caudate 7s during focal limbic seizures by others may reflect their use of stronger stimuli or the spread of convulsant from larger volumes of injection.

Functional anatomy of limbic seizures In general, the behavior changes observed here are similar to those observed by others during experimental limbic seizures in cap.20,2-~ and, to a lesser degree, in monkey46, 66. With increasing number and severity of seizures from the posterior hippocampus in the cat 20 the sequence of phenomena is nearly identical: staring, face and head movements, then generalized motor phenomena and postural disturbances. In addition, the behavioral changes seen here are st foreshortened picture of the successive convulsive responses induced over days or weeks by kindling t h e hippocampus in cat0~ or raP0.-% or over hours by rapid electrical kindling in raft s . Finally, the intravenous use of systemic convulsants - - either kainic acid (12 mg/kg) or dipiperdinoethane (400 mg/kg) - - also causes an identical behavioral sequence which develops over several hoursl~. 4j. DG autoradiographic images and depth recordings during different stages of these IAmbic seizures indicate that discharges and metabolism are accentuated primarily in hippocampus during early phases of staring and behavioral arrest. Once seizures become strong, with rearing and forelimb clonus, there is bilateral activation of all allocortex, amygdala, nucleus accumbens and substantia nigra. Taken together these studies indicate that although subtle differences exist in early phases of discharges from different IAmbic foci. once strong seizures occur the system behaves as a unit with stereotyped convulsive manifestations. The relationship of these experimental results in animals to limbic seizures in man is speculativeS~. Temporal lobe epileptic phenomena often start with a blank stare, interruption of behavior and loss of conscious response to environmental stimuli:;. Depth electrode studies in isolated cases have given some clues with respect to functional anatomy. Several investigators have found that consciousness is lost and memory functions are suspended once discharges spread bilaterally through the hippocampus4,.;~. Unilateral IAmbic discharges or stimulation can evoke experiential phenomena in man without loss of consciousness:~. Motor phenomena - - particularly involving muscles of facial expression and mastication

38

-

can be directly a c t i v a t e d with r e l a t i v e l y w e a k tet-

r e p o r t e d as an e x p e r i e n t i a l a u r a in m a n ; that bilateral

anic stimulation of a m y g d a l a , but not of the h i p p o -

h i p p o c a m p a l s p r e a d results in i m m o b i l i t y , staring

c a m p u s or e n t o r h i n a l c o r t e x 59,60. Finally, seizure dis-

and unconsciousness; and bilateral activity in amygda-

charges f r o m a m y g d a l a are p r o b a b l y n e c e s s a r y for

la and its subcortical p a t h w a y s results in the e m e r -

autonomic

g e n c e of positive m o t o r and a u t o n o m i c p h e n o m e n a .

-

manifestations,

seen p r i m a r i l y as sali-

v a t i o n late in e x p e r i m e n t a l limbic seizures, In summ a r y , o u r results s u p p o r t the g e n e r a l c o n c l u s i o n s of o t h e r s that u n i l a t e r a l limbic seizures m a y be silent, or

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