ESPEKI.\IEXTAL

NECROLOGY

54, 233-250

(1977)

Seizures and Related Epileptiform Transplanted to the Anterior I. Characterization BARRY

of Seizures,

J.

HOFFER,

Spikes,

AECE SEIGER, AXD

Laboratory

lnterictal

ROBERT

Activity Chamber

DAVID

and Synchronous

TAYLOR,

FREED~~AN

in Hippocampus of the Eye

LARS

OLSOK,

o

of ~‘z~rvophav~rlacolog~~, DisGion of Special Mental Health Natiorlal Iwtitrtte of Mental Health, St. Elizabeth Hospital, lb’askington, D.C. 20032; aud Department of Histology, Karolinska Institutct, Stockholm, Swcdcta Receizvd

July

Activity1

Research,

26, 1976

Fetal hippocampus, transplanted to the anterior chamber of the eye of an adult host, develops to a structure capable of responding to a variety of epileptogenic treatments. Seizures are obtained in response to electrical stimulation, penicillin superfusion, and cobalt iontophoresis. These seizures can be antagonized by systemic barbiturates or intraocular superfusion of diazepam or local anesthetics. Activity comparable to paroxysmal depolarizing shifts, interictal spikes, and hypersynchrony can also be elicited. No seizure-like activity can be seen in transplanted cerebellum.

INTRODUCTION Much physiologic research in epilepsy has attempted to answer two questions: First, what is the minimum local neuronal substrate necessary to produce normal and abnormal activity in the electroencephalogram (EEG) , and, second, are there modulatory roles played by afferent input from other brain structures? Because of its low seizure thresholds (l), the hippocampus has been extensively used in epilepsy research. In view of the 1 This work was supported in part by the Swedish Medical Research Council (04X-03185), Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. The authors thank Miss Ingrid Strijmberg and Miss Maud Eriksson for skillful technical assistance. 2Dr. Freedman’s present address is Department of Psychiatry, University of Chicago, Chicago, Illinois 60637; Dr. Seiger and Dr. Olson are at the Department of Histology, Karolinska Institutet S-10401, Stockholm, Sweden; and Dr. Hoffer’s present address is Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80220.

Copyright All rights

0 1977 by Academic Press, of reproduction in any form

Inc. reserved.

ISSN

0014-4886

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AL.

difficulties in distinguishing endogenous from transmitted epileptiform activity in sib, many investigators have attempted to use “isolated hippocampal systems” to define the site of the abnormal discharge. Afcr chronic transection of the fornix, electrical stimulation of the caudal portion readily elicits hippocampal seizures ( 14). In chronically isolated hippocampal islands, electrical stimulation or treatment with penicillin produces interictal spikes and overt seizures (6). Intracellular recordings both in situ and in these isolated systems have shown a large depolarization in pyramidal neurons, known as the paroxysmal depolarizing shift, to be associated with the seizure waves. The shift, believed to be an abnormally large excitatory postsynaptic potential (EPSP) (7, 23)) h as also been seen in explants of hippocampus in tissue culture after treatment with the convulsant bicuculline (29) and in hippocampal slice preparations (21, 28). Recently, it has been found that regions of fetal hippocampus grow and develop a laminar neuronal organization after transplantation into the anterior chamber of the eye of an adult host animal. The transplants also form the intrinsic excitatory and inhibitory circuitry typical of the hippocampus in situ (19). Although isolated from the rest of the central nervous system, the transplant receives cholinergic (11) and adrenergic fibers [Freedman et al., in preparation] (10) from the autonomic ground plexus of the iris. If the transplants could be shown to manifest abnormal electrical activities similar to those seen in situ, their well-developed local organization, defined autonomic inputs, and isolation from other brain regions would greatly facilitate delineation of factors regulating such electrical activities. In this paper we examine certain types of EEG activities recorded from the transplants : high-voltage hypersynchrony, interictal spikes, and overt seizures. The questions to be answered are: Are these activities generated within the transplant ; are they elicited and inhibited by the same treatments effective in situ; and are they dependent on the organization of the transplants ? In succeeding papers (Freedman et al., in preparation) we will treat, respectively, the effects of the cholinergic and adrenergic inputs from the autonomic ground plexus of the iris as models for the possible modulatory effects of central adrenergic and cholinergic inputs to the hippocampus in situ. METHODS Regio superior (CA1 and 2) of hippocampal anlage was removed from fetal rats with a crown-rump length of 21 to 40 mm, and placed in the anterior chamber of the eye of an adult rat through a slit in the cornea, as described earlier (19, 20). C are was taken to avoid including the regio inferior and the dentate gyrus. The transplants were allowed to develop

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for at least 1 month itz orzrlo before recording. Some of the mature transplants were fixed in formalin and processed for light microscopy. For some control experiments, cerebellar transplants were produced as described earlier (12). For electrophysiological experiments, the host rat was anesthetized with urethane (1.25 g/kg, intraperitoneally) and intubated. This level of anesthetic was sufficient to prevent eye movements and to block the cornea1 reflex. The cornea was incised and reflected. A plastic ring was fixed to the skin with agar to form a chamber around the transplant. The chamber was

lilt. 1. Mature hippucampal lran~plant. Several latnina of pyramidal cells (1’) arc visible. Also labelled is the stratum oriens (0) and the stratum radiatum (K). The fusion with the iris is not visible in this section. Crown-rump length of donor fetus: 40 mm. Montage of microphotographs of a 6-pm toluidine blue-stained section. X40.

FIG. 2. Seizure activity in a hippocampal transplant after electrical stimulation. A-Seizure produced the transplant (upper trace). In all figures, the stimulating electrode is a bipolar surface electrode and micropipet (1 to 3 MO) generally near the pyramidal cell layer. Unless otherwise noted, negative is and positive is up on all oscilloscope tracings. Seizure activity is not recorded if the recording electrode transplant (lower trace). B-Seizure produced by two single shocks in a transplant which was already as a result of previous seizures, also induced by electrical stimulation.

by pulse train stimulation of the recording electrode is a up on all inkwriter tracings, is in the bath above the discharging interictal spikes,

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superfused with Earle’s balanced salt solution (Grand Island Biological Co.) and warmed to 37”C, using a heating coil regulated by a thermistor in the bath. Drugs were either dissolved in the solution and superfused over the transplant or administered parenterally to the host animals. The pH of all superfusion solutions was measured and adjusted to 7.1 if necessary. Cobalt was delivered by microiontophoresis using a programmable 120-V solid-state constant-current source with automatic current neutralization. Techniques for the evaluation of iontophoretic responses and for the control of current, pH, and local anesthetic artifacts have been previously described (9, 13). Slow-wave and single-neuron electrical activity were recorded by single and multibarrel glass micropipets with recording barrels filled with 3 M I\‘aCl. Resistances were 1 to 2 MO. The activity was amplified by a unity gain dc-coupled preamplifier, subsequently filtered 3 tlb at 0.6 and 10,000 Hz and displayed on a polygraph recortler (Gould Instruments), photographed from an 0sciIloscope screen, or led to 3 computer (PDP-12. Digital Equipment Co.). lf a single-unit action potential was also recorded, the signal was filtered further (-3 dB at 300 and 5000 the output of which was inteHz) and led to a window discriminator, grated over l-s intervals to indicate discharge rate and led to the computer. Electrical stimulation of the transplant surface was provided through a bipolar stimulating electrode constructed from twisted wires having a tip separation of 0.1 mm. Monophasic square-wave pulses 0.05 to 0.1 ms in width were used. Slow-wave or field-potential responses to stimulation were averaged by the computer, using appropriate software (25). Singleunit action potential responses were similarly summed to form poststimulustime histograms, RESULTS The hippocampal grafts proliferated extensively ilz orl/lo, and the various strata found in normal hippocampus could be readily identified cytologically in the transplants (Fig. 1). Their growth and histological organization have been described in detail in a pre\ious communication (19) and will not be further dealt with here. In a healthy, unstimulated transplant, the “EEG” was usually of a very low voltage giving a flat appearance (Figs. 2, 3, 9). 1\Yth the appropriate electrical or chemical stimulation, however, various high-voltage phenomena were recorded from the transplants, resembling seizures, interictal spikes, and synchronous slowswave activity. Such activity in an unstimulated transplant was minimal, but these EEG phenomena were reliably elicited by electrical stimulation, penicillin superfusion, or cobalt iontophoresis. Figure 2R shows a seizure elicited by electrical stimulation. The parameters used, 16 V, 20 Hz, were typical of

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AL.

I. CONTROL EEG

2. INITIAL SEIZURE

ts

iv, 20/set, 5 *ec 3. 3 min POSTICTAL

4. 5 min POSTICTAL

FIG. 3. Postictal refractoriness to electrical stimulation after a seizure. l-control electroencephalogram (EEG) shows no spikes. Z-Prolonged spiking discharge following initial train of electrical stimuli (arrow). Seizure lasted about 2 min, then activity restored to control level. 3--Stimulation with the same parameters 3 min later produced only a few afterdischarges. 4-A more prolonged seizure is produced by stimulation 5 min after (3). The duration of spiking still does not equal the initial seizure.

those used to produce seizures, which were generally of 20- to 90-s duration. Because the activity could be recorded only in the transplant itself, not in the bath above (Fig. 2A), it was presumed to be generated in the transplant. Fifteen to twenty minutes after an initial electrical seizure, a long-term increase in excitability was often seen. This was manifested by spontaneous interictal spikes and seizures elicited by only one or two surface stimuli. The example in Fig. 2B shows a seizure established after two shocks in a transplant which already had interictal spiking following an initial seizure. On the other hand, immediately after an initial seizure, the transplant was generally completely refractory to repeated stimulation (Fig. 3). Recovery began after several minutes but was not complete for up to 15 to 30 min. Superfusion of penicillin at adequate doses (generally 5000 U/ml) for 20 min

also resulted

in seizures.

Lower

doses produced

either

interictal

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30

IO min after PCN

I3

30 -

AFTER PCN 1600u/ml -2

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2 2

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38

after superfusion of FIG. 4. Penicillin-induced interictal spikes. A-Immediately penicillin (PCN), 5000 U/ml, interictal spiking is rapid but quickly slows (upper trace). After several minutes a steady level of discharge is reached, which persists up to several hours without reapplication of penicillin. The inset shows an interictal spike with a faster time base. The notches on the initial depolarization may represent population spikes. B-Effects of temperature on penicillin interictal spike (IIS) interval. Average interictal spike time for 10 consecutive intervals is plotted against bath temperature. The linear relationship has a Q10 of 2.3. Inset shows the configuration of the interictal spike in this transplant. No interictal spikes were seen prior to infusion of penicillin in either transplant.

spikes firing spontaneously or markedly augmented field potentials, with surface stimulation, which probably represent the extracellular field of a paroxysmal depolarizing shift. Immediately following superfusion of penicillin, the interictal spikes were at a quite high frequency (Fig. 4A) but, after several minutes, reached a stable discharge level which was usually maintained for several hours without reapplication of penicillin. The interictal spikes and paroxysmal depolarizing shift field potentials had a biphasic appearance, with the characteristic wavelets representing population spikes found on the initial portion. Penicillin was always effective in producing either seizures or interictal spikes (Table 1). Doses as low as 1000 U/ml readily produced interictal spiking lasting for several hours. Evidence for

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TABLE Induction

ET

AL.

1

and Antagonism of Seizures and Epileptiform in Hippocampal Transplants Treatment

Activity

Number

of transplants

Induced seizure Electrical stimulation With normal autonomic innervation After superior cervical ganglionectomy After superior cervical and ciliary ganglionectomy Cobalt iontophoresis Penicillin superfusion Hippocampal transplants Cerebellar transplants

12 6 1 2

0 0 0 0

s 0

0 2

No block

Barbiturate injection Procaine superfusion Diazepam superfusion

No seizure

0 0 0

Antagonized or blocked seizure 2 4 3

local generation of these activities, in addition to the fact that they disappeared when the recording was in the bath (Fig. 7B), came from two experiments. First, local change in the temperature of the superfusion bath markedly altered the rate of interictal discharge (Fig. 4B). The QIo for this change was 2.3. Second, the amplitude of the interictal spikes or paroxysmal depolarizing shift fields were changed by moving up and down in the transplant as shown in Fig. 5. Prior to the application of penicillin in this experiment, surface stimulation elicited a small population spike (Fig. 5A) with almost no field potential. After penicillin, a large field potential was elicited which changed in amplitude markedly with movements within the transplant. The coincidence of the maximum point of the wave form with the pyramidal cell body layer suggests that the intrinsic hippocampal circuitry may be involved in its generation. Still further evidence for generation of interictal spikes by pyramidal cell intrinsic circuitry comes from the observation of their inhibitory interactions as shown in Fig. SE. When multifocal interictal spikes were produced, the largest one was able to inhibit the others for up to 2 s.

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Another type of EEG activity, in addition to seizures and interictal spikes, could be recorded from the transplants. Synchronous slow-wave activity, though also rarely present spontaneously, was easily elicited by cholinergic input or by mechanical stimulation of the transplant. Evidence for the involvement of specific muscarinic cholinergic receptors in generating this activity will be presented in a subsequent communication (Freedman et ~1.. in preparation). In the present study, we wish to show only the temporal and spatial characteristics of these waves and document that they are derived from intrinsic neuronal activity. Examples of slow-wave activity are shown in Figs. 6 and /‘A. The frequency was usually 4 to 6 Hz although waves as slow as 2 Hz were observed. \Vhen elicited, these lvaves were relatively stable in pattern and

A

3.

200pm down

4.

500pm down

I. EBSS

-‘2:

B

PCN5000u/ml

5.

600pm up

PCNlO,OOOu/ml

FIG. 5. A-Conversion of a normal driven response to a paroxysmal depolarizing shift by penicillin. 1-A control record in Earle’s balanced salt solution IEBSS) shows minimal field potential and population spike (dot) to threshold surface stimulation. Record is computer-generated average response to 30 single stimuli of 6 V at 0.2/s. Inset is a poststimulus-time histogram (PSTH.) of the response of a single unit at this recording site shelving a short latency excitation. This response also identifies the placement of the electrode in the pyramidal cell layer. Z-After superfusion of penicillin (PCN), 5000 U/ml, the driven response becomes a paroxysmal depolarizing shift. 3, 4, and S-Movement of the electrode below the pyramidal cell layer results in the loss of response amplitude (4), and movement back toward the layer restores it (5). B-Occasionally multiple foci of interictal spikes are:; induced by penicillin superfusion. In the example shown here, the foci producing the larg-e spikes inhibit the smaller ones.

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FIG. 6. Depth profile of hypersynchronous discharge induced by superfusion of lo-’ carbachol. Two micropipets, one fixed just under the surface and the other at various depths within the transplant, were used to show phase reversal of hypersynchrony. In the top tracing, left to right, the moving electrode penetrates more deeply into the transplant. Initially both tracings are in phase. As the moving pipet penetrates more deeply, it reaches a place where no wave is recorded, at approximately the level of the pyramidal cell layer itself. On the far right, phase reversal is seen at a point where the electrode is presumably below the pyramidal cell layer. The bottom traces show that this phase reversal is present regardless of whether the surface of the transplant is stimulated (arrow, 8 V at 0.2/s) or not. The successive tracings also show that the reversal is a stable finding. M

usually of 0.5 to 1.0 mV in amplitude. Occasionally, activity greater than 1 mV was found (Fig. 7A). When recorded simultaneously from superficial and deep regions of the transplant, the waves were 180” out of phase, with an interposed “null zone” of 50 to 75 pm (Fig. 6). In a further effort to prove that these EEG phenomena were generated locally at the recording site and were dependent on neuronal activity, procaine (4%) was superfused over the surface of three transplants, and it readily reduced synchronous slow-wave activity (Fig. 7A). In four other transplants, interictal spikes elicited by penicillin were also blocked by procaine (Fig. 7B). The interaction of interictal spikes induced by penicillin superfusion and anticonvulsant drugs was also studied. Parenteral administration of barbiturates (10 mg/kg) blocked seizures and interictal spikes in two transplants (Fig. 7C). Superfusion of 1O-5 M diazepam (Fig. S) similarly antagonized interictal spikes in all three transplants studied. Interestingly, in the transplant shown in Fig. 8, the interictal spikes were multiple with a prominent neuronal discharge on the late negativity and very little positivity (star). During diazepam superfusion, the interictal spikes, prior to their abolition, becamesingle, with little neuronal discharge and a markedly augmented positivity (star).

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FIG. 7. Effects of drugs on hypersynchrony and interictal spikes (IIS). (A), (B), and (C) are from three different transplants. A-Hypersynchronous waves induced by superfusion of IO-’ M acetylcholine are blocked by superfusion of 4% procaine. BInterictal spikes are similarly blocked by 4% procaine. Moreover, the spikes can be recorded only in the transplant, not in the bath above. C-Interictal spikes are abolished after intraperitoneal injection of pentobarbital. Spikes in (B) and (C) were induced by prior superfusion with penicillin, 1600 U/ml.

Cobalt, another classical epileptogenic agent, could also be used to elicit seizure activity. A multibarrel micropipet was used to apply cobalt at the site of recording. In the example shown (Fig. 9), placement of the electrode in the proximity of the pyramidal cell layer was indicated, prior to cobalt iontophoresis, by the excitatory and inhibitory responses to paired surface stimuli during the control period. The computer-generated averaged evoked responsesabove the flattened control EEG (Fig. 9-l) shows that the conditioning stimulus elicits a population spike (dot) and associated field potential. A second test stimulus, 50 ms later, evokes a much smaller response, suggesting an intact intrinsic inhibitory system (19). Cobalt application reproducibly triggered sustained interictal spiking and seizure activity. This phenomenon was repeatedly observed in two transplants. Cerebellar transplants were also treated with penicillin and subjected to electrical stimulation. Because the cerebellum does not generally exhibit seizure activity in situ (1)) it provides a suitable control tissue. Although electrical stimulation excited the Purkinje cells in the transplant and penicillin strongly increased their spontaneous discharge, neither synchronous slow-wave activity, interictal spikes, nor seizures were ever observed (Fig. 10).

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DISCUSSION The purpose of the present experiments was to determine if the transplanted hippocampus, grown in isolation from the rest of the central nervous system, could sustain seizures and other types of electroencephalographic activity. Several types of activity were observed in the transplants which would seem to correspond to the electrical phenomena associated with the hippocampus ill situ: full seizures, interictal spikes, paroxysmal depolarizing shifts, and hypersynchronous discharge. Several lines of evidence suggest, moreover, that these activities are generated discretely within the transplant and can be induced or antagonized by drugs eliciting similar responses in sita. Electrical seizures in the transplants have many features of seizure activity in the hippocampus in situ. A single shock was never effective in producing an initial seizure ; the optimal parameters, 20 to 100 Hz for 5 to 10 s, compare closely to those reported in situ (1). The seizures often seem to have a “tonic” and a “clonic” phase, i.e., a period of low-voltage activity preceding the large spiking waves, as in situ (23). Intrinsic generation of the seizure within the transplant itself was demonstrated by two methods. PCN 1600 U/cc CONTROL

DIAZEPAM 0.01 mM

RECOVERY

FIG. 8. Effects of diazepam on penicillin-induced interictal spikes in hippocampal markedly slows and eventually abolishes transplants. Superfusion of IO4 M diazepam (not shown) interictal spike discharge. The configuration of the interictal spike is also altered by diazepam. Note that in both specimen records the descending limb of the initial negativity was used to trigger the oscilloscope and is not shown. Before and after recovery from diazepam, the interictal spikes are multiple with a prominent discharge on the late negativity and very little positivity. During diazepam treatment, the late negativity is abolished and the positivity is augmented, producing a “single” interictal spike pattern. Stars denote positivity in both records.

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First, tltc activity was recorded only in the transplant and could not he detected at the same amplification in the bath above. Second, local anesthetics such as procaine superfused over the surface of the transplant abolished the activity. As in sitz~, parenteral barbiturates and cliazepam also blocked seizure activity. Electrically induced seizure activity has been previously recorded in hippocampus partly isolated from the rest of the nervous system. Transection of the fornix, for example, produces a hippocampus, with many afferents destroyed. Electrical stimulation, nevertheless, is still effective in producing seizures in this preparation (13). More-isolated preparations, such as tissue slices, do not sustain seizures after electrical stimulation, although they propagate afterdischarges (18, 28). Hippocampus grown in tissue culture forms synapses, and a paroxysmal depolarizing shift-like discharge has been seen after pharmacological treatment with convulsants (29). However, it does not show propagated seizure-like activity (3). Thus the transplanted preparation may have a unique role in the physiologist’s armamentarium, because it can sustain prolonged electrical seizures despite its isolation from the rest of the central nervous system.

FIG. 9. Cobalt induction of interictal spikes. (l), (2), and (3) continuously. The electroencephalographic tracing initially is rather cobalt iontophoresis, seizure-like activity and regularly occurring were induced. Placement of the recording electrode in the pyramidal suggested in the control period by observing the computer-generated sponses to 50 paired surface stimuli (arroM.s, 7 V at 0.2/s) in the control electroencephalogram. The evoked population spike (dot) and potential are diminished after a conditioning-testing interval of 50 ms, that intrinsic inhibition is also intact.

were recorded flat, but during interictal spikes cell layer was summed reinset above the associated field which indicates

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At.

I. EBSS EEG I. EBSS EEG p PSTH

RATE

2. PCN 5000u/ml Imv

I

2. PCN lO,OOOu/ml

3. PCN 20,00Ou/ml 500mr

FIG. 10. Effects of penicillin on a cerebellar transplant. Two different transplants are shown (left and right). In each case surface electroencephalographic records and ratemeter records from Purkinje neurons are shown during superfusion with Earle’s balanced salt solution (EBSS) alone and containing various concentrations of penicillin (PCN). A poststimulus-time histogram (PSTH, left) documents the response of the cell to surface parallel-fiber stimulation. Despite a large increase in the firing rate in response to penicillin in both cases, the electroencephalograms showed no signs of seizure activity.

One of the most popular preparations for the study of acute epileptic foci has been the penicillin-treated hippocampus (5-8, 22). In addition to overt seizures, the paroxysmal depolarizing shifts and the interictal spikes have been extensively studied. Both of these could be demonstrated in the transplant. The paroxysmal depolarizing shift, by definition, of course, must be recorded intracellularly. However, many of its characteristics are evident in extracellular recordings. The shift in situ has been described as a giant, stereotyped excitatory postsynaptic potential (7) caused by penicillin altering the synaptic mechanisms. This is followed by a prominent inhibitory postsynaptic potential (IPSP), especially at the border of the focus (8, 23). In a previous paper, the transplants were shown to have intrinsic

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excitatory and inhibitory circuits similar to those found in situ (19). Thus the appropriate synaptic substrata for generation of the paroxysmal depolarizing shift are present. The extracellular field of the shift in the transplant is maximal where the population spike and its associated field potential were recorded prior to the application of penicillin. Like the intracellular shift, the extracellular wave form is stereotyped and many times larger than the normal field potential. As ifz sitl~, the paroxysmal depolarizing shift could be evoked by stimulation of the local synaptic pathways (7). Penicillin, as ilz situ, also induced spontaneous interictal spikes, which consisted of a large stereotyped wave similar to the paroxysmal depolarizing shift. Population spikes were often seen as wavelets on the negative phase. Other similarities of the interictal spikes to the in situ phenomenon included the sensitivity to local temperature change (17) and the antagonism by barbiturates. When comparing the transplant with the hippocampal island, a principal difference may be sensitivity to penicillin. As little as 1000 U/ m 1 in the transplant produced seizures and interictal spikes, whereas 10,000 to 20,000 U are frequently needed in sifu (6-S). Otherwise, as in situ, the spikes started rapidly, then slowed to a regular rate. The Qr,, of 2.3 agrees closely with the value found in situ (17). Another form of discharge noted in the transplant was termed hypersynchrony. Like other forms of activity, the hypersynchronous discharge could not be recorded in the bath above the transplant and was abolished by local anesthesia, which suggests that it is generated within the transplant. Further evidence for intrinsic generation of this activity in the transplant comes from the experiments with two recording electrodes (Fig. 6)) which show a null zone several hundred micrometers below the surface of the transplants and phase reversal at greater depths. Although accurate depth measurements are difficult to obtain in the transplants because of displacement of the tissue caused by movements of the recording electrode, the reversal suggests generation of this activity near the pyramidal cell layer. The hypersynchronous activity has many of the characteristics of the theta rhythm, characteristic of hippocampus in situ. Winson (24) and Bland et al. (2) have shown that two separate theta generators exist in the hippocampus, one of which is in the CA1 zone. Depth studies within CA1 in situ show a null point and phase reversal similar to that reported here. Unlike true theta, the hypersynchronous activity in the transplants was extremely stable, was often greater than 0.5 mV in amplitude (see Fig. 7A), and was not abolished by the light anesthesia given to the host animal. It was also slower, usually 2 to 6 Hz, as compared to 4 to 7 Hz for theta in situ. Recently, however, a report of theta in a urethane-anesthetized animal has appeared (15) which is related to cholinergic input and has a slightly slower than normal periodicity. Significantly, hypersyn-

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chronous activity could be induced in tranplants by cholinergic stimulation (Freedman et al., in preparation). Experiments are now in progress with cholinomimetics and antagonists to determine the involvements of specific acetylcholine receptors in generating these waves. Such data are required to evaluate the relationship of the hypersynchronous discharge to the cholinergic input. In assaying the usefulness of the transplanted hippocampus for studies of epilepsy, it is important to show that the hippocampus retains its characteristic response to epileptogenic treatments and anticonvulsants. Responses to electrical stimulation and penicillin were described above. Cobalt, another classical epileptogenic agent (4, 22), when applied to the surface of the cat cerebral cortex, produces seizure activity within 30 min (26). In our experiments, cobalt applied directly within the pyramidal cell layer was effective in several seconds. Similarly, barbiturates and diazepam, widely used anticonvulsant drugs (27), powerfully antagonized seizure discharge in the transplants. These data indicate that these anticonvulsants may act directly on the hyperexcitable focus rather than indirectly at other brain loci as has been suggested by Julien (16). Finally, the question arises as to whether the marked seizure propensity of the hippocampal transplant is due, in part, to its initial surgical disruption and subsequent transplantation. Accordingly, both electrical stimulation and penicillin were used with cerebellar transplants to attempt to produce seizure activity. The cerebellum has been reported to show no seizures ipz sitz~ Although electrical stimulation caused parallel fiber-type excitation and penicillin significantly raised Purkinje cell spontaneous activity, seizure activity did not appear. Thus seizures in the hippocampal transplants cannot be explained simply as a response to transplantation. In conclusion, the properties of the hippocampal transplants evident from the experiments described above suggest that the hippocampal transplant is a useful model for studies on the mechanism of hippocampal epilepsy and EEG synchrony. The various electrical activities described in this study, seizures, paroxysmal depolarizing shifts, interictal spikes, and hypersynchrony, resemble phenomena seen in situ. The presence of epileptiform activity in the hippocamfial trabsplants and the sensitivity of that activity to anticonvulsants offer many phenomena which can be examined in unique ways with these preparations. For example, the isolation and accessability of the transplants would facilitate measurement and alteration of the ionic environment and direct administration of drugs by superfusion. In addition, the invasion of the transplants by adrenergic and cholinergic fibers from the autonomic ground plexus of the iris [ (11)) Freedman et al., in preparation] makes possible the assessment of the role of these transmitters in modulation of seizures, in a preparation unencumbered by other extrinsic inputs.

219

1. AJRIONE-MARSAN, C. 1972. Focal electrical stimulation. Pages 147-172 in D. PURPURA, J. PENRY, D. TOWER, D. WOODBURY, AND R. WALTER, Eds., Experimental Models Of Epilrpsy. Raven Press, New York. 2. BLAND, P. N., P. ANDERSEN, AND T. GAXES. 1975. Two generators of hippocampal theta activity in rabbits. Brain Rex 94: 199-218. 3. GRAIN, S. M., C. RAINE, AND hf. BORX~EIN. 1975. Early formation of synaptic networks in culture of fetal mouse cerebral cortex and hippocampus. J. iVe~robiol. 6: 329-336. 4. DAWSON, G. D., AND 0. HOLMES. 1966. Cobalt applied to the sensorimotor area of the cortex cerebri of the rat. J. Physiol. (Lo&.) 8.5: 455-470. 5. DICHTER, M., C. HER&IAN, AXD M. SELZER. 1972. Silent cells during interictal discharges and seizures in hippocampal penicillin foci: Evidence for the role of extracellular K+ in the transition from the interictal state to seizures. Braht Res. 48 : 173-183. 6. DICHTER, islands

M., C. HERMAN, of hippocampus.

AND M. SELTZER. Electroertc~phalogr.

1973.

Penicillin

epilepsy

Clirt. Ncwojltysiol.

34:

in isolated 631638.

7. DICHTER, M., AND W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features. J. ATeuro#hysiol. 32 : 649-662. 8. DICHTER, M., AND W. A. SPENCER. 1969. Penicillin-induced interictal discharges from the cat hippocampus. II. Mechanisms underlying origin and restriction. J. Neuropltysiol. 32 : 663-W. 9. FREED&IAN, R., B. J. HOFFER, ASD D. J. \~TOOD\I.ARD. 1975. A quantitative microiontophoretic analysis of the responses of central neurones to noradrenaline : Interactions with cobalt, manganese, verapamil and dichloroisoprenaline. Brit. J, Pharmacol. 54 : 529-W. 10. FREEDIIAN, R., D. TAYLOR, A. SEIGER, L. OLSON, ASD B. HOFFER. Seizures and related epileptiform activity in hippocampus transplanted to the eye. II. Modulation by cholinergic and adrenergic inputs. In preparation. 11. HOFFER, B., A. SEIGER, R. FREEDMAN, L. OLSOIC, AND D. TAYLOR. 1976. Electrophysiology and cytology of hippocampal formation transplants in the anterior chamber of the eye. II. Cholinergic mechanisms. Brais Res., 119: 107-132. 12. HOFFER, B., A. SEIGER, T. LJUNGBERG, AXD L. OLSON. 1974. Electrophysiological and cytological studies of brain homografts in the anterior chamber of the eye: Maturation of cerebellar cortex in orulo. Brabt Rcs. 79: 165-184. 13. HOFFER, B. J., G. R. SIGGIXS, AND F. E. BLOOX 1971. Studies on norepinephrinecontaining afferents to Purkinje cells of rat cerebellum. Bvaiu Rcs. 25: 523-534. 14. KANDEL, E. R., AND W. A. SPENCER. 1961. Excitation and inhibition of single pyramidal cells during hippocampal seizure. Bsp. i\‘c!rrol. 4: 162-179. 15. KRAMIS, R., C. H. VANDER~OLF, AND B. H. BLAND. 1975. Two types of hippocampal rhythmic slow activity in the rat: Relations to behavior and effects of atropine, diethylether, urethane and pentobarbital. Bxp. Nenrol. 49 : 58-85. 16. JULIEN, R. M. 1972. Cerebellar involement in the antiepileptic action of diazepam. Neurophnr,llacologJIro~~larFllacolog~l 11 : 683-691. 17. LEBOVITZ, R. M. 1975. Effects of temperature on interictal discharge at penicillin epileptogenic foci. Epiltps,ia 16 : 21 S-222, 18. MCILWAIN, I-I. M. 1972. Electrical stimulation of specified subsystems of the mammalian brain as isolated tissue preparations. Pages 269-290 irz 1). Pcrarua~,

250

HOFFER

J. PENKY,

ET

AND R. WALTER, Eds., Experi~~zer~tal York. 19. L., B. HOFFER. 1976. Electrophysiology and cytology of hippocampal formation transplants in the anterior chamber of the eye. I. Intrinsic organization. Brain Res., 119: 87-106. 20. OLSON, L., AND T. MALMFORS. 1970. Growth characteristics of adrenergic nerves in the adult rat. Fluorescence histochemical and ‘H-noradrenaline uptake studies using tissue transplantations to the anterior chamber of the eye. Acto Physiol. Sca&. [Suppl.] 348 : 1-112. 21. OGATA, N. 1975. Ionic mechanisms of the depolarization shift in thin hippocampal slices. Exp. Newel. 46 : 147-155. 22. PRINCE, D. A. 1972. Topical convulsant drugs and metabolic antagonists. Pages 51-84 in D. PURPURA, D. J. PENRY, D. TOWER, D. WOODBURY, AND R. WALTER, Eds., Erperiww~ttal Models of Epilepsy. Raven Press, New York. 23. PRINCE, D. A. 1974. Neuronal correlates of epileptiform discharge and cortical DC potentials. Pages 1056-2070 iti A. REMOND, Ed., Handbook of Electroencephalography and Clinical Neurophysiology, Vol. 2, Part C. Elsevier, Amsterdam. 24. WINSON, J. 1973. Patterns of hippocampal theta rhythm in the freely moving rat. Electroencephalogr. Clifl. Newophysiol. 36 : 291-301. 25. WOODWARD, D. J. 1973. Poststinzulzts Time and Interspike Interval Histogram, Decus Library, 12-65. Digital Equipment Co., Maynard, Massachusetts. 26. WILLMORE, L., P. FULLER, A. BUTLER, AND N. BASS. 1975. Neuronal compartmentation of ionic cobalt in rat cerebral cortex during initiation of epileptiform activity. Exp. Ncwol. 47 : 280-289. 27. WOODBURY, D. M., J. K. PENRY, AND R. SCHMIDT. 1972. Antiepileptic Drugs. Raven Press, New York. 28. YAMAMOTO, C. 1972. Intracellular study of seizure-like afterdischarges elicited in thin hippocampal sections in vitro. Exp. Neural. 35 : 154-164. 29. ZIPSER, B., S. CRAIN, AND M. BORNSTEIN. 1973. Directly evoked. paroxysmal depolarization of mouse hippocampal neurons in synaptically organized explants in long term culture. Brain Res. 60: 489-495. Models OLSON,

D. TO~EK, D. WOODBURY, of Ep,ilepsy. Raven Press, New R. FREEDMAN, A. SEIGER, AND

AL.