Seizure-like spreading depression in immature rabbit hippocampus in vitro

Developmental Brain Research, 14 (1984) 51-59 Elsevier

51

BRD 50043

Seizure-Like Spreading Depression in Immature Rabbit Hippocampus In Vitro MICHAEL M. HAGLUND and PHILIP A. SCHWARTZKROIN

Departments of Neurological Surgery and Physiology and Biophysics, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted January 10th, 1984)

Key words: spreading depression - - seizure - - hippocampus - - in vitro slice - - immature CNS - - inhibition

Study of immature rabbit hippocampus, using the in vitro slice preparation, has revealed seizure-like spreading depression (SD) episodes in tissue from 8-12-day-old animals. These SDs occur both spontaneously and in response to stimulation, and are seen in both extracellular and intracellular recordings. At the cellular level, SDs are similar to epileptiform ictal phenomena in their onset bursts of action potentials, prolonged membrane depolarizations, and afterdischarge bursts. Glial recordings indicate that a large rise in [K+]o occurs during these SD episodes. Low chloride concentration in the bathing medium facilitates SD occurrence. The immature CA1 region, where inhibition is slow to develop, is more susceptible to SDs than the CA3 region, where inhibitory post-synaptic potentials are potent early in development. These observations suggest that the slice preparation of immature rabbit hippocampus may provide a useful model in which to study epileptiform mechanisms, such as inhibitory efficacy and extracellular potassium clearance, which might be responsible for ictal onset and control.

INTRODUCTION Spreading depression (SD) was first described by Le~io 13, who found that repetitive stimulation of rabbit cerebral cortex evoked a depression of spontaneous cortical activity. Further investigationT. 13 led to a more complete characterization of the SD phenomenon, which includes the following prominent features: an initial burst of neuronal activity at the advancing front of the SD wave; a long-lasting depression of neuronal firing; and afterdischarge bursts as the SD episode endsI6, t9. Several characteristics of SD onset, propagation, and termination are similar to the activity that occurs during seizure episodes. In particular, both p h e n o m e n a share the onset burst of action potentials, a prolonged period of depolarization, and afterdischarge activity which occurs as neurons repolarize. Further, the SD waves propagate across cortex with a speed similar to that of the Jacksonian march of clinical epilepsy 15. Finally, cellular activity between SD episodes and between ictal events appears quite 'normal'. These similarities suggest that while SD may be primarily an experimental

'artifact', the study of mechanisms underlying SD may provide insights into mechanisms involved in seizure generation. Indeed, a number of studies employing ion-selective microelectrodes have shown that seizure activity and spreading depression are both correlated with a significant increase in extracellular potassium concentration ([K+]o) 6. During seizures the increase in [K+]o is 'clamped' at a ceiling of 10--12 mMS.6,17, whereas [K+]o may rise to 36-40 mM during spreading depression6. 9. Epileptiform activity, and the effects of extracellular potassium concentration on cellular excitability, have been studied both in vivo and in vitro2.5. 20. Recent studies using the in vitro slice preparation have been especially fruitful in providing information about the mechanisms underlying burst discharges 24. These discharges appear to be analogous to the interictal electroencephalographic 'spike', and similar to the afterdischarge bursts seen at the end of SD and ictal episodes. Since ictal activity is rarely seen in healthy in vitro preparations, the in vitro studies have been focused exclusively on interictal burst phenomena, and have consequently added little information

Correspondence: P. A. Schwartzkroin, Department of Neurological Surgery RI-20, University of Washington, Seattle, WA 98195, U.S.A. (206) 543-9125 0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.

52 about the mechanisms underlying generation of ictal episodes. In the course of in vitro studies of rabbit hippocampus, we found that immature rabbit hippocampus generates spontaneous seizure-like SD episodes at a certain stage of development. Experiments were subsequently carried out to examine this SD phenomenon as a possible model for spontaneous seizure activity. In the present report, we describe the SD events generated in immature rabbit hippocampus in vitro, and examine changes in the characteristics of spontaneous and stimulus-evoked SD episodes resulting from alterations in extracellular ion concentrations (potassium, chloride) or addition of an epileptogenic agent (penicillin) to the bathing medium. MATERIALS AND METHODS The in vitro hippocampal slice preparation used for the present experiments has been described previously21. Briefly, the experimental procedure was as follows: New Zealand white rabbits, ages 8-12 days, were decapitated, the brain rapidly removed, and the hippocampus isolated. The hippocampus was then sliced perpendicular to its longitudinal axis into 500 ~m thick sections. Within 5 min of decapitation, the slices were placed in an incubation chamber where they were maintained on a nylon mesh at an interface between the bathing medium and warmed, humidified gas (95% 02, 5% CO2). The normal bathing medium was maintained at 35 + 0.5 °C, and consisted of 124 mM NaC1, 5 mM KCI, 1.25 mM NaHzPO 4, 26 mM NaHCO 3, 2.0 mM CaCI 2, 2.0 mM MgSO4, and 10 mM glucose. Recording electrodes were made from capillary tubes with intraluminal glass fibers, and were filled with 4 M potassium acetate (80-120 MQ). The electrode signals were amplified by high input impedance DC amplifiers, displayed on an oscilloscope, and stored on FM magnetic tape for later analysis. Stimulation of hippocampal slices to trigger SD episodes was carried out using two techniques: repetitive electrical stimuli (0.3-10 Hz, 0.05 ms pulse, 0.1-0.5 mA) were delivered through bipolar electrodes (sharpened tungsten electrodes insulated to their tips) placed in stratum radiatum or in the dentate hilus; alternatively, pressure ejection of micro-drops of 2 M KC1 (40 psi applied to a 30 M ~ electrode with tip di-

ameter of 2/~m) was used to obtain a controlled, focal SD onset. Intracellular penetrations of both CA1 and CA3 pyramidal cells were made at the level of stratum pyramidale. The criteria for healthy intracellular penetrations were: membrane resting potential of greater than --50 mV, input resistance of greater than 25 Mr2, action potentials of at least 55 mV, and a spike train response to depolarizing current injection (characteristic of the type of pyramidal cell penetrated) 22. In some experiments, simultaneous intracellular recordings from two neurons were obtained; in other experiments, an extracellular field potential in stratum pyramidale was monitored simultaneously with a single intracellular recording. In 3 experiments, the bathing medium was altered by raising the potassium concentration from 5 to 10 raM; in two other experiments, chloride concentration was lowered from 133 to 22 mM (NaCI replaced by Na isethionate). Penicillin (sodium penicillin G) was added to the bathing medium in concentrations of 3.4 mM (3 experiments) or 6.8 mM (1 experiment). RESULTS

Spontaneous~stimulus-evoked S D episodes Extracellular recordings were made from the level of stratum pyramidale in both CA1 and CA3 regions. Spontaneous SD events occurred in 33 of 58 slices. Extracellularly, the initial event in the SD episodes was a large negative DC shift of 15-35 mV (which accompanied the intracellular depolarization - - see below). There was then a 'relaxation' of this negativity, only to be followed by a further field potential shift, 3-8 mV, in the negative direction (Fig. 1A). Negative shifts of the field potential were maintained for 30-100 s; the field potential then started a slow return to its original level. During repolarization, field potential 'spikes' often occurred in the pattern of epileptiform aflerdischarge. Similar field potential shifts were observed whether the SD event was spontaneous or stimulus-elicited (Fig. 1). The waveform was relatively stereotyped from one episode to another in a given slice. In preparations showing SD activity, 120 intracellular CA1 recordings were obtained during spontaneous events, and 45 intracellular recordings obtained during stimulus-elicited (1-10 Hz for 5-10 s)

53

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Fig. 1. Field (extracellular) potential recordings from stratum pyramidale of the CA1 region during SD episodes. Note the large amplitude of the shifts in the field potentials, and the similarity of all 3 waveforms. The field potential electrode remained in the same location during all 3 SD events. A: spontaneous SD episode. B- stimulus-evoked SD; stimulation (0.3 Hz) of the mossy fibers at the dentate hilus triggered a SD episode in the CA1 region. C: stimulus-evoked SD; stimulation (10 Hz) in the fimbria elicited a CA1 SD event. In this and the following figures, action potentials have been truncated by the pen-writer. In field recordings, upward deflections from baseline represent positive DC shifts and downward deflections represent negative DC shifts.

SDs. In many cases, intracellular recordings were made simultaneously with extracellular field recordings, with the two electrodes separated by 50-150 /~m. There was a characteristic sequence of membrane potential changes seen in intracellular recordings, as illustrated in the CA1 records of Fig. 2A. First, there was a flurry of synaptic activity, riding on a gradual depolarization (2-5 mV) of the cell's resting potential (Fig. 2B). Second, the cell discharged in a burst of action potentials and then rapidly depolarized by 50-60 mV; this depolarization was concomitant with the field potential negative shift. Since this intracellular depolarization occurs concurrently with the negative shift in field potential, the magnitude of the transmembrane (Vin-Vout)potential shift is even greater than reflected by the intracellular measure. The cell m e m b r a n e potential remained depolarized (near 0 mV) for 50-100 s; during this period, m e m b r a n e input resistance was extremely low (near zero) (see Fig. 6B). Third, the cell began a slow repolarization, the initial phase of which was accompanied by the further negative shift in the field potential. Continuing cell repolarization was associated with a shift in field potential toward baseline and often accompanied by afterdischarge bursts. Afterdischarge bursts occurred simultaneously in neu-

Fig. 2. Simultaneous intracellular and field recordings in the CA1 region illustrating salient features of SD episodes. A: immature CA1 pyramidal neuron (bottom trace) during a spontaneous SD episode (for description see text). The field potential shifts (upper trace) occurred with a characteristic relationship to the intracellular changes. B: expanded trace of the onset of the spontaneous SD episode. Note the flurry of synaptic activity riding on the slow depolarizing wave in the intracellular recording (lower trace). A negative shift of the field potential (upper trace) occurred as the neuron depolarized. C: expanded trace illustrating the recovery phase of this SD event. The spontaneous afterdischarge bursts seen intracellularly (lower trace) were synchronized with the population bursts seen in the field electrode recording. Note the different calibration bars for A versus B and C.

ron and field recordings (Fig. 2C). Repolarization of the cell m e m b r a n e often continued beyond initial (i.e., pre-SD) resting potential, producing a prolonged period (2-5 min) of cell hyperpolarization (2-8 mV). This SD sequence could be repeated every 4-10 minutes during the course of an experiment (5-7 h). Similar results were obtained from recordings in the CA3 region, as shown in the simultaneous recordings of pyramidal cell and field potential in Fig. 3. Although characteristics of CA3 region SD events were basically the same as those observed in the CA1 region, spontaneous events occurred with much lower frequency in the CA3 region; furthermore, higher stimulus intensity and frequency were necessary to evoke CA3 region SD episodes. On a few occasions, the intracellular recording electrode penetrated an apparent glial celleS. Since SD events have long been associated with changes in extracellular potassium concentration3.7, and since the glial cell m e m b r a n e potential appears to be a sensitive indicator of [K÷]o, those cells were monitored during spontaneous SD episodes (Fig. 4). Glial potentials, like those of neurons, showed initial gradual depolarizations followed by rapid depolarizations of

54

A

Fig. 3. Simultaneous recording from a CA3 pyramidal neuron and an extracellular field potential electrode located in stratum pyramidale of the CA3 region during a spontaneous SD event. The major characteristics of this CA3 SD episode were similar to those occurring in the CA1 region. The prolonged hyperpolarization which follows SD episodes was particularly pronounced in this neuron.

30--40 mV (Fig. 4B). As is the case for neuronal depolarizations, the magnitude of the glial transmembrane potential shift is even greater than seen in the intracellular glial depolarization. The glial membrane remained depolarized for the duration of the field potential negative shift but started to repolarize well before the field potential began its return toward baseline. During the afterdischarge bursts of the recovery phase, glia produced slow depolarizing shifts of 3-9 mV in synchrony with field potential bursts (Fig. 4C). Simultaneous intracellular recordings were made from 8 pairs of CA1 pyramidal cells within 50-150/~m of each other. Both cells always participated in the spontaneous or stimulus-evoked SD episodes, but their large depolarizations were not precisely synchronized (Fig. 5). In all cases, the onset phase of the SD episode differed both in timing and rate of rise

20 sec

B J 2 0 mV sec

Fig. 4. Simultaneous recordings of a glial cell and field potential during SD. A: the glial cell depolarization coincided with a negative shift of the field potential at the onset of a spontaneous SD episode. B: faster sweep speed of SD onset to highlight glial cell depolarization. C: expanded trace illustrating one of the afterdischarge bursts, showing the synchrony between field potential bursts and glial cell depolarization. Note the different calibration bars for A, B and C.

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~ mv 1sec

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Fig, 5. Simultaneous recording from two CA1 pyramidal neurons during a SD episode. A: spontaneous SD occurred with approximately the same time course in these two CA1 cells, located 100/~m apart. B: faster sweep speed demonstrates the asynchronous onset and rate of rise of the depolarization in these two cells at the beginning of the SD event. C: expanded trace of the SD recovery, demonstrating the precise synchrony of the two cells' afterdischarge bursts.

(Fig. 5B). However, during the repolarization phase, afterdischarge activity was precisely synchronized in all CA1 pairs (Fig. 5C). This synchrony was in marked contrast to the independent firing that occurred prior to the SD episodes.

Response of CA1 region to bathing medium manipulations Extracellular potassium concentration in the bathing medium was elevated in 3 experiments. From a normal [K+]o of 5 mM, [K+]o was raised to 10 raM. This change caused an increase in spontaneous cell burst discharges which were synchronous with field potential spikes in all 8 cell/field pairs. In CA1 pyramidal cells which exhibited after hyperpolarizations (AHPs) (3 of 8)10,23, the A H P was in all cases decreased by the elevated [K+]o . Elevated [K+]o did not, however, cause an increase in frequency of spontaneous SD episodes or in the characteristics of SD events that did occur. In those slices where SD episodes were elicted only by electrical stimulation in normal medium, higher [K+]o in the bathing medium did not lead to any spontaneous SD events. During recordings from 15 CA1 pyramidal cells, the normal bathing medium was exchanged with a medium that included penicillin concentrations of 3.4 or 6.8 mM, The addition of penicillin triggered burst discharges in 12 of 15 cells, but did not change the frequency of occurrence of spontaneous SD episodes. Penicillin, at the higher concentration of 6.8 m M did, however, make it possible to trigger SDs electrically

55 with fewer and/or less intense stimulation. In some cases, a single stimulus elicited afterdischarge bursts which gave rise to a gradual depolarization of the cell; this depolarization evolved into a SD event. In 7 cases in which simultaneous recordings of a CA1 cell and field potential were obtained, the bathing medium was altered by lowering the [C1-] from 133 to 22 mM (Fig. 6). This change led to an increase in the frequency of occurrence of SD episodes. The size and duration of the negative field potential shift was increased during SDs in the low chloride solutions (cf. Fig. 6A, B), and the usual afterdischarge activity was lost. In contrast to the results of the experiments involving increased [K+]o, lowering [C1-] resulted in spontaneous SD episodes in slices which had previously exhibited only stimulus-triggered SD episodes. Spontaneous SD episodes have not been observed in the healthy, mature hippocampal slices bathed in the normal medium. Change of bathing medium to one containing low chloride concentration, however,

40

see

Fig. 6. Illustration of SD episodes in normal and low chloride bathing media. A: spontaneous SD event, as seen in simultaneous field and intraceUular recordings from the CA1 region of the immature hippocampus in normal bathing medium. B: recordings of the same cell and field during a subsequent spontaneous SD episode that occurred 10 min after chloride concentration in the bathing medium was lowered from 133 to 22 mM. A 0.5 nA hyperpolarizing current was injected every 3 s during the SD episode in order to monitor cell input resistance (lower trace). Note the membrane shunting (decrease in voltage deflection produced by the constant current pulse) during cell depolarization, and the eventual recovery of input resistance as the cell repolarized. C: intracellular recording from a CA3 pyramidal neuron from mature guinea pig hippocampal slice during a spontaneous SD event. This event occurred after the bath [CI-] was lowered to 22 mM. Note the brief duration of the SD episode in mature tissue compared to SDs in immature tissue (A and B).

does cause spontaneous SD events in mature tissue (Fig. 6C). As in the immature recordings, the mature SD episodes triggered in low chloride solutions exhibit no afterdischarge activity during recovery.

Comparison of the CA1 and CA3 regions In 10 experiments, 2 M KCI was pressure ejected from microelectrodes positioned in stratum pyramidale in order to trigger SD episodes. These local applications were carried out in the CA1, CA2 and CA3 regions during simultaneous recordings from CA1 cell and field, CA3 cell and field, and CA1 and CA3 cells. The local application of KCI, approximately 50--150/~m away from a CA1 cell/field pair, reliably triggered SD episodes. These episodes were of similar duration and had similar features to those SDs that occurred spontaneously or were electricallyevoked. Such KCl-elicited SD events were also observed in the CA3 region. As was the case with electrical stimulation, the major difference between CA1 and CA3 KCl-elicited SDs was the higher threshold needed to elicit SD episodes in CA3. The CA3 cells required longer pulses of KCi (150--300 ms) than did the CA1 cells (50--100 ms) to trigger SDs (from the same KCI electrode). The differential sensitivities of the two pyramidal cell regions were studied further in intracellular recordings from 20 pairs of CA3 and CA1 pyramidal cells. When application of KCI was made approximately equidistant between the two cells (in stratum pyramidale of the CA2 region), a SD event could be evoked in the CA1 cell but not in the CA3 cell (Fig. 7C). The CA1 SD episode occurred at a latency of 20-50 ms after the KC1 pulse. CA3 cells exhibited a 5-10 mV hyperpolarization concomitant with the peak depolarization in the CA1 cell (Fig. 7C). During CA1 cell repolarization, CA1 afterdischarge bursts were synchronized with small (2-3 mV) hyperpolarizations in the CA3 cell although no synchrony between the two cells was noted prior to the CA1 SD episode. With KC1 application near (50-200/~m) the CA1 electrode (Fig. 7B), there was very rapid onset of a CA1 SD episode. In the CA3 cell, a hyperpolarization again occurred at the peak of the CA1 SD event; the hyperpolarization was smaller, however, than when KC1 was applied between the two cells (in CA2). On rare occasions, a SD episode occurred in

56

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electrodes averaged 1600 p m ; conduction velocity for the spread of the SD from CA1 to CA3 was therefore calculated to be approximately 3.2 mm/min. When KC1 was applied near (50-200 p m ) the CA3 cell, and sufficient KCI was ejected to trigger a CA3 SD event, a SD episode was also recorded in the CA1 cell beginning 15-20 s after onset of SD in the CA3 cell (Fig. 7D). There was no observed synchrony between cells prior to the SD episodes, but afterdischarge bursts were synchronous in CA1 and CA3 neurons during recovery.

,tJl DISCUSSION

B CA1 ce

|20 mv 20 sec

Spreading depression in the in vitro hippocampus of immature rabbit has been characterized in the present study. These episodes, spontaneous or evoked, were observed primarily in slices from animals 8-12 days old. This form of neuronal hyperexcitability was rarely seen in tissue from mature animals or from newborn rabbits.

SD in the immature rabbit hippocampus

~i~'S- - ~ ~ D Fig. 7. Spreading depression episodes in a CA1 and a CA3 neuron evoked by local microinjection of 2 M KCI. A: diagrammatic representation of the locations of the CA1 and CA3 recording electrodes, and of the KC1 electrode as it was moved across the slice. B, C and D illustrate results of KC1 ejection in regions CA1, CA2, and CA3, respectively. B: when the potassium was ejected near the CA1 recording electrode, a large abrupt CAI cell depolarization occurred; the CA3 neuron hyperpolarized. C: potassium application in the CA2 region resuited in a response similar to that seen when KCI was ejected in CA1 (B). However, the CA3 neuron hyperpolarized further, and then exhibited a period of prolonged excitation. D: ejection of larger amounts of potassium near the CA3 electrode led to a CA3 SD episode. Several seconds after onset of the CA3 SD event, the CA1 neuron also depolarized in a SD episode,

the CA3 region, with a latency of approximately 30 s after onset of a SD event in the CA1 cell population. The distance between the CA1 and CA3 recording

The spreading depression episodes observed in immature rabbit hippocampal slices are phenomenologically identical to SD events recorded in in vivo preparations of rabbit cortex 3.13 and rat cerebellum llA2, and in in vitro rat hippocampal tissue 27. Cellular similarities between the in vivo and in vitro SD events include an intense burst of neuronal discharge at the onset of SD, a prolonged depolarization associated with low input resistance, afterdischarge bursts during repolarization, and a subsequent period of depressed neuronal activity and cell hyperpolarization. Previous studies of SD using in vivo preparations showed that an intense burst of neuronal activity occurred at the onset of a SD episodeS, 14. This burst of activity was foreshadowed by an increase in synaptic noise in the baseline of neurons in immature rabbit hippocampus just prior to the SD episode. Increased synaptic activity rode on a slowly depolarizing membrane potential, perhaps reflecting an activity-related increase in [K+]o . Such a possibility is supported by in vivo observations of Nicholson and Kraig18 who reported rises of [K+]o to 10-12 mM just prior to the large depolarization characteristic of the SD event.

57 In vitro, the duration of cellular SD depolarizations and the correlated negative field shift was approximately 50--100 s, similar to the duration of in vivo SD episodes which range from 40 to 300 s3. The large increases in the membrane conductance during these depolarizations are consistent with measurements made with ion-sensitive microelectrodes; such studies have shown large decreases in [Na+]o, [C1-]o, and [Ca2+]o, as well as a large increase in [K+]o, during the SD event 9'n. These results would be expected if there was a shunt in the membrane, allowing ions to follow their concentration gradients. At the end of the long period of SD depolarization, cells began a gradual repolarization during which they produced afterdischarge bursts. This period of afterdischarge appears similar to the clonic phase of ictal seizures 1,4. In recordings from CA1 cell pairs, these bursts were found to be synchronous, even though there was no evidence of neuronal synchrony before the SD episode. There was less precise synchrony in the activity (latency and rate of rise) of CA1 cell pairs observed during the onset of SD depolarization. Stratum pyramidale field potential spikes, which represent the synchronous discharge of a population of neurons, were observed only during cell repolarization. Phase-locked activity was also observed between the CA3 and CA1 regions during the repolarizationrecovery phase of SD. When a SD episode began in the CA3 region and was subsequently reflected in CA1, simultaneous recordings showed the CA3 cell afterdischarge bursts consistently occurred just prior to burst discharges in the CA1 cell. This discharge relationship suggests that an excitatory drive from the CA3 region was projected to the CA1 region, probably via the Schaffer collaterals. Clear evidence for the spread of SD events came from recordings of SD episodes which originated in the CA1 region and appeared 30 s later in the CA3 region. The calculated conduction velocity of 3.2 ram~rain is similar to that observed for the SD velocity of in vivo preparations3,15. When SD episodes were localized to just the CA1 region, afterdischarge activity of CA1 cells occurred synchronously with hyperpolarization of the CA3 cells. Such a discharge relationship could be mediated by antidromic activation ('backfiring') of the Schaffer collaterals in CA1; a consequent CA3 hyperpolarization would then be due to activation of

CA3 inhibitory recurrent collateral circuitry. The major difference between our in vitro studies and those carried out in vivo, and perhaps one of the most interesting features of these depolarization episodes in the immature hippocampus, was that SD occurred spontaneously in vitro. Since spontaneous SDs have not been described as a reliable phenomenon in other CNS preparations, the in vitro preparation of immature hippocampus provides a unique opportunity to examine the mechanisms underlying this event. More importantly, the similarities between SD and seizure activity, and the absence of ictal activity in healthy hippocampal slice tissue, make this SD model appealing for studying mechanisms underlying seizure discharge. Both repetitive stimulation and local KCI application resulted in SD episodes that were quite similar to those which occur spontaneously, thus providing consistent means for evoking SD phenomena. In many experiments, the immature hippocampal tissue proved to be mechanosensitive so that movement of recording or stimulating electrodes could trigger SD episodes. This mechanosensitivity is also a feature of SDs studied experimentally in vivo and of seizure activity in epileptic foci.

Effects of penicillin and of Cl- and K + ions on SD episodes In slice preparations of mature hippocampus, penicillin and bicuculline produce synchronous burst discharges 26 similar to those seen during the SD recovery period in our present studies. Since those drugs are thought to block chloride-mediated inhibitory postsynaptic potentials (IPSPs) produced by GABA on hippocampal pyramidal cells, one might hypothesize that a change in membrane chloride flux is involved in the generation of SD episodes. Indeed, the reduction of [Cl-]o in our preparations led to an increased frequency of SD episodes in the immature rabbit hippocampal slices. Even in mature hippocampus, where spontaneous SDs have not been observed, reduction of [C1-]o led to spontaneous SD events. Given these effects of chloride alterations, it was somewhat surprising that the addition of penicillin (a GABA/chloride antagonist) did not increase the frequency of spontaneous SD episodes. Penicillin did, however, increase the sensitivity of the tissue to electrically-elicted SD episodes. The chloride sensitivity of this tissue may be partic-

58 ularly acute in immature hippocampus, where IPSP mechanisms are poorly developed 23. We hypothesize that it is precisely the absence of mature IPSPs that renders immature tissue so prone to SD (and epileptiform) activity. This hypothesis is supported by our observations that the CA1 region has a much lower threshold for SD episodes than the CA3 region. The CA3 region, with its rare spontaneous SD events and higher thresholds to electrically- and potassiumevoked SDs, has a more 'mature' IPSP system at the developmental ages we studied 23. It is important to note that SD episodes occurred during a discrete window of development (8-12 days old) when inhibition is weak. SDs were not frequently observed at younger ages when inhibitory activity is even less well developed. These findings suggest that the SDs were not simply artifacts of the preparation (e.g., hypoxiarelated) and must depend on factors other than absence of inhibition. For example, further development of interconnections that mediate synchrony, shortening of cell refractory period (to allow repetitive action potential discharge), and/or maturation of potassium uptake mechanisms could also determine the time at which SD generation could occur in hippocampus. Previous studies3 have postulated that a rise in [K+]o "triggers' SD episodes 6. Although there is certainly a large increase in the [K+]o during the SD event, our studies using bath concentrations of 10-12 mM potassium showed no increase in frequency of occurrence or change in the characteristics of SD episodes. There was simply an increase in synchronous burst discharges in K+-treated slices as has been reported for slices from mature hippocampus 20. Other studies have shown an increase in tissue [K+]o to 10-12 mM precedes onset of the explosive SD even in

REFERENCES 1 Ayala, G. F., Dichter, M., Gumnit, R. J., Matsumoto, H. and Spencer, W. A., Genesis of epileptic interictal spikes. New knowledge of cortical feedback systems suggests a neurophysiological explanation of brief paroxysms, Brain Res., 52 (1973) 1-17. 2 Benninger, C., Kadis, J. and Prince, D. A., Extracellular calcium and potassium changes in hippocampal slices, Brain Res., 187 (1980) 165-182. 3 Bureg, J., Buregovfi, O. and Krivanek, J., The Mechanisms and Applications of Le~o's Spreading Depression of Electroencephalographic Activity, Academia, Prague, 1974.

vivo 11. Our data suggest, however, that this rise in [K+]o is, in itself, not sufficient to trigger the events involved in spreading depression or even seizure activity. An additional deficit, for instance, in the metabolic pumping mechanism (to handle the increased [K+]o load) is indicated by in vivo demonstrations that local injection of concentrated potassium solution does not always induce SD unless the brain is 'conditioned 'is. Conditioning procedures such as reducing [C1-]o, or blocking the N a - K pump render the increase in extracellular potassium concentration a much more potent trigger of SD episodes. In summary, we have been able to study spontaneous spreading depression episodes using the in vitro slice preparation of immature hippocampus. These SD events have many similarities to ictal episodes and may provide a model for studying the basic mechanisms underlying onset and control of seizure activity. Initial investigations suggest that the efficacy of the IPSP may play an important role in the control of SD episodes. Further studies of the mechanisms involved in conditioning and triggering SD episodes may provide key insights into the processes involved in generating seizure episodes. ACKNOWLEDGEMENTS This research was supported by NINCDS (NIH) Grants NS 15317, NS 00413, and NS 17111. M.M.H. is a predoctoral trainee in the Medical Scientist Training Program, University of Washington (GM 07266), and received a stipend from the Medical Student Research Training Program of the University of Washington. P.A.S. is an affiliate of the Child Development and Mental Retardation Center, University of Washington.

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Function, Vol. 1, Cortical Excitability and Steady State Potentials, UCLA Press, Los Angeles, CA, 1963, pp. 87-124. 9 Hansen, A. J. and Zeuthen, T., Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex, Acta physiol, scand., 113 (1981) 437-445. 10 Hotson, J. R. and Prince, D. A., Calcium activated afterhyperpolarization in hippocampal slices maintained in vitro, J. Neurophysiol., 43 (1980) 409--417. 11 Kraig, R. P. and Nicholson, C., Extracellular ionic variations during spreading depression, Neuroscience, 3 (1978) 1045-1049. 12 Kraig, R. P., Nicholson, C. and Philips, J. M., Sodium and chloride ion changes during spreading depression and anoxia in rat cerebellum, Soc. Neurosci. Abstr., 4 (1978) 66. 13 Le~o, A. A. P., Spreading depression activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359--390. 14 Le~o, A. A. P., On the spread of spreading depression. In M. A. B. Brazier (Ed.), Brain Function, Vol. 1, Cortical Excitability and Steady State Potentials, UCLA Press, Lo~ Angeles, CA, 1963, pp. 73-85. 15 Le~io, A. A. P., Spreading depression. In D. P. Purpura, J. K. Penry, D. B. Tower, D. M. Woodbury and R. D. Walter (Eds.), Experimental Models of Epilepsy -- A Manual for the Laboratory Worker, Raven Press, New York, 1972, pp. 174--196. 16 Marshall, W. H., Spreading cortical depression of Leho, Physiol. Rev., 39 (1959) 239-279. 17 Moody, Jr., W. J., Futamachi, K. J. and Prince, D. A., Extracellular potassium activity during epileptogenesis, Exp. Neurol., 42 (1964) 248-263.

18 Nicholson, C. and Kraig, R. P., The behavior of extracellular ions during spreading depression. In T. Zeuthen (Ed.), The Application of Ion-Selective Microelectrodes, Elsevier/ North Holland Biomedical Press, Amsterdam, 1981, pp. 217-238. 19 Ochs, S., The nature of spreading depression in neural networks, Int. Rev. Neurobiol., 4 (1962) 1-69. 20 Prince, D. A., Neurophysiology of epilepsy, Ann. Rev. Neurosci., 1 (1978) 395-415. 21 Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro preparation, Brain Res., 85 (1975) 423-436. 22 Schwartzkroin, P. A., Further characteristics of hippocampal CA1 cells in vitro, Brain Res., 128 (1977) 53--68. 23 Schwartzkroin, P. A., Development of rabbit hippocampus: physiology, Develop. Brain Res., 2 (1982) 469-486. 24 Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced epileptiform activity in the hippocampal in vitro preparation, Ann. Neurol., 1 (1977) 463--469. 25 Schwartzkroin, P. A. and Prince, D. A., Recordings from presumed glial cells in the hippocampal slice, Brain Res., 161 (1979) 533--538. 26 Schwartzkroin, P. A. and Prince, D. A., Changes in excitatory and inhibitory synaptic potentials leading to epileptogenic activity, Brain Res., 183 (1980) 61-76. 27 Snow, R. A., Taylor, C. P. and Dudek, F. E., Electrophysiological and optical changes in slices of rat hippocampus during spreading depression, J. Neurophysiol., 50 (1983) 561-572.