Carbamazepine suppresses synchronized afterdischarging in disinhibited immature rat hippocampus in vitro

Brain Research, 400 (1987) 371-376

371

Elsevier BRE 21954

Carbamazepine suppresses synchronized afterdischarging in disinhibited immature rat hippocampus in vitro Karen L. Smith and John W. Swann Wad.~worth ('enter lbr Laboratories and Research, New York State Department of Health, Albany, N Y 12201 (U.S.A.)

(Accepted 19 August 1986) Key words.' Carbamazepine; Anticonvulsant: Epilepsy: Aftcrdischarge: Hippocampus

Bath application of therapeutic concentrations of the anticonvulsant carbamazepine suppressed penicillin-induced synchronized afterdischargmg in immature rat CA 3 hippocampal pyramidal cells. Afterdischarging was completely abolished in all preparations at a concentration of 30 uM (IC,~ -- 8.5 +_ 1.4 uM: mean +_ S.E.M.). The duration of the preceding epileptiform burst was not altered at this concentration and was diminished by only 24.4 _+_1.2% at a supratherapeutic concentration of 100 uM. These results suggest that a carbamazepine-sensitivc neurophysiological mechanism distinct from those responsible for cpileptiform burst generation plays a key role in the generation of afterdischarges in developing hippocampus.

In recent years a concerted effort has been made to determine the neurophysiological origins of interictal epileptiform events m neocortex and hippocampus. Comparatively few attenrpts have been made to stud}' the mechanisms which underlie ictal events. Since the anticonvulsant c a r b a m a z e p i n e (CBZ) is often the drug of first choice in treating children with seizure disorders that can originate in hippocampal formation a'll'l~22, we have examined its effects on ictal events in developing hippocampus. O u r hope is that an understanding of how C B Z prevents seizures will elucidate the physiological origins of the seizure discharges themselves. Bath application of penicillin to slices from mature hippocampus results m an extracellularly recorded interictal event, often referred to as the epileptiform burst >>'~t. This event is composed of a synchronized burst of action potentials in hippocampal pyramidal cells. The underlying intracellular potential is the paroxysmal depolarization shift ~. In slices from hippocampus of 1- and 2-week-old rats, penicillin also induces an cpileptiform burst, but the burst is followed by rhythmic, repetitive synchronized discharges which often approach 30 s in duration 25. The

results of our present study show that these afterdischarges are eliminated by therapeutic concentrations of C B Z in a highly d o s e - d e p e n d e n t manner. The epileptiform burst is relatively unaffected by much higher concentrations of this drug. Transverse hippocampal slices were p r e p a r e d by methods previously described :5. Briefly, brains were quickly removed from ether anesthetized rat pups 9 - 1 6 days of age, and the hippocampus was dissected free of surrounding tissue and placed on a tissue slicer. Several slices, each 500;um thick, were taken and placed in a dish of oxygenated artificial cerebrospinal fluid ( A C S F ) at room temperature. Prior to transfer to the experimental chamber the slices were viewed individually bv stereomicroscopy, and a wedge of the CAt~, subfield was dissected from each slice with a razor blade shard. Removing this wedge prevented invasion of the CAs region by spreading depression episodes, which occur in the C A t region of immature h i p p o c a m p a l slices (ref. 9 and our unpublished observations), and p e r m i t t e d pharmacological experiments of several hours duration under stable recording conditions. The C A 3 subfield remained intact in these experiments and recordings

('orre~po~Mence. ,I.W. Swann. Wadsworth Center for Laboratories and Research. New York State Department of l teahh. Empire State Plaza, Albany. NY 122(/I. U.S.A.

IIl/(t~>S993+S7:$(13,5{)@ 1987 [ Isevier Science Publishers B.V. (Biomedical Division)

372 from this region were identical to those from intact slices.

graphy. The volume of the re~~+tdillg chmnber ~ : ~ approximately 150 !fl, and the pcrt:usiot~ rate was i ml/min. Experiments were conducted al 32~ 33 "C

The microdissected slices were immediately transferred to the experimental chamber, where the?,'

Recordings were made cxttaccllularlv h ~ m the

rested at the interface of the circulating A C S F and overlying humidified 95% O2/5'7/c CO,. The A C S F

CA 3 pyramidal cell body laver and ~ere DC coupled, Glass micropipettes pulled from fiber-filled capillary

was composed of the following (in mM): NaCI

tubes and filled with 2 M Na('l had resistances of

122.75, KC1 5.0, CaCI2 2.(I. MgSO 4 2,0, NaHPO4 1.25, NaHCO3 26, and glucose 10.0. Epileptiform

5 - 2 0 M£]. When epileptiform cxcnts wcrc evoked, a rectangular constant current pulse i 1()0 z¢~ in dura-

discharges were produced by adding penicillin (1.7

tion) was delivered by a monopo!a~r stimulating elec-

mM) to the bathing medium. Carbamazepine (5-100 u M ) was added to this penicillin-containingm e d i u m .

trode placed in stratum radiatum Conventional techniques wcrc employed [o dis-

and the mixture was held in a separate reservoir be-

play the neurophysiological events,

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stored on FM magnetic tape. Data were played back

the drug a solution was warmed and sonicated before being added to the medium, The concentration of

and quantitated from a storage oscilloscope or bx a PDP 11/23 computer and were transferred to a D E C PRO-350, where thev were mmlvzed and plotted

C B Z in the perfusate was verified by gas chromato-

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Fig. 1. Effects of CBZ on spontaneous epileptiform events. The upper trace of panel l is a slow time base recording and at the arrow the perfusatc was switched to a CBZ-containing solution (30~uM). Lower trace in panel 1 shows the prolonged afterdischarge outlined by the frame in the upper trace but at a faster time base. Events labeled A and B in upper trace are also shown in panel 2, trace A and B at a faster time base. One event in each of these traces is further extended in time in traces C and D. In trace C the arrow points to the epileptiform burst, and the circles designate 4 subsequent synchronized discharges comprising that afterdischargc

373 with an integrated data analysis system (RS/I). For purposes of illustration, selected portions of experiments were played back from tape onto a rectilinear pen recorder. This was done at 25% of the original tape speed in order to faithfully reproduce the amplitude and time course of the epileptiform events under study. Results presented here are based on recordings from 53 slices taken from 48 rats. Bath application of CBZ at concentrations of 5-2(I /+M decreased the duration of synchronized afterdischarges. At 30 uM, aherdischarges were completely suppressed in virtually every slice tested, but the preceding epileptiform burst was unaffected. A typical experiment in which epileptiform events occurred spontaneously in the C A 3 subfield of a slice bathed in a medium containing penicillin is shown in Fig. 1. In the initial part of the upper trace of panel 1, CA 3 neurons discharged synchronously, and the epileptiform events cycled between brief and prolonged events. In the lower trace of this panel the prolonged event outlined by a frame in the upper trace is shown at a faster

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time base. in panel 2, trace A and C are selected events, recorded during the control period, are shown at faster time bases. Each of these events consisted of an initital epileptiform burst (trace C, arrow) which was followed by an afterdischarge. Each afterdischarge consisted of one or more synchronized discharges of CA~ pyramidal cells. In the event shown in trace C, 4 such discharges occurred (solid circles). Switching to a medium containing 30 #M CBZ abolished the afterdischarges (panel 1, upper trace). However, epileptiform burst generation continued, as is clearly seen at the faster time bases in trace B and D. To examine more thoroughly the ability of CBZ to selectively eliminate penicillin-induced afterdischarging, we elicited these events by orthodromic stimulation. Such events are virtually identical to those that occur spontaneously 25. At a frequency of stimulation similar to that of the endogenous 'pacemaker', they cycle, as spontaneous events do, between brief and prolonged discharges. However, we

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Fig. 2. Effects of C B Z on epileptiform events evoked by repeated (0.14 Hz) orthodromic stimulation. Panel 1 shows responses evoked at 4 times during the experiment. In trace A the arrows denote the beginning and end of an event evoked during the control period. The intervening interval was the duration of the event. Panel 2 displays the m e a n and S.E.M. of the duration of each succeeding 6 responses. Panel 3 shows the changes in the n u m b e r of synchronized discharges in each of the afterdischarges that occurred during this same period. Arrows in panels 2 and 3 show when the perfusate was switched to the CBZ-containing solution.

374 found that this cycling in afterdischarge duration was highly d e p e n d e n t on stimulus frequency. W h e n the frequency of o r t h o d r o m i c stimulation was 0 . 1 - 0 . 2 Hz, prolonged afterdischarges did not occur in most slices: and in many of these slices the duration of the epileptiform event was relatively constant and reproducible over periods of several hours. The duration of discharging varied among the slices from 200 to 800 msec, but within each slice it was relatively invariant. A typical experiment with repetitive o r t h o d r o m i c stimulation is shown in Fig. 2. Panel 1 confirms that the effects of C B Z on o r t h o d r o m i c a l l y evoked epileptiform events are identical to those observed on spontaneous activity. Before C B Z was a d d e d to the recording c h a m b e r (trace A ) , the event consisted of an epileptiform burst and a brief afterdischarge. A p proximately 26 min after addition of C B Z (trace D), the afterdischarge was blocked but the burst remained unaltered. The time course of C B Z ' s effects on the duration

tion was approximately i50 his As ;he duraIl~,n el the afterdischarge decreased, lhe synchronized discharges, comprising the afterdischargc, were clinlinated one by olle, until at ~(i mm none remained (panel 3). In all instances the tasi discharge was ~he next It) be eliminated (panel t, it:ices I~. (')= From results such ;is these ~ll<)~ll in Fig, 2, it seemed plausible that the gradual diminution irl afterdischarge duration evoked b~ ( ' B Z ~ a s a re-, flection of the gradual increase m the concentration of C B Z in the slice. Based on tins idea, we thought anticonvulsant d o s e - r e s p o n s e relationships could then be d e v e l o p e d by systematically varying the C B Z content of the perfusatc durine the course of our recordings. In 7 such experiments the C B Z content of the perfusate was varied from 5 to l(10 uM, As expected, C B Z limited the aftcrdischarge duration in a highly d o s e - d e p e n d e n t manner (Fig. 3, panel 1). The IC> for C B Z was 8,5 _+. 1.4uM (mean _+ S . E . M . ) . But as before, the cpileptiform burst was resistant to CBZ. This was true even when the concentration of the drug was increased lo l{t0 uM. As seen m representative traces from one of these experiments (panel 2), the duration of the afterdischarge decreased steadily with increasing C B Z concentration. At 3 0 ! t M C B Z the afterdischarge was blocked in each o r t h o d r o m i c response, but the epileptiform burst was still recorded mid very similar m

of the epileptiform discharges and on the n u m b e r of synchronized discharges in each afterdischargc arc shown in panels 2 and 3. Perfusion with C B Z (30!~M) decreased the duration of the afterdischarge gradually over the next 2 5 - 3 0 min until, as occurred during spontaneous activity, only the epileptiform burst remained (panel 2). In this e x p e r i m e n t the burst dura-

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Fig. 3. CBZ-induced dose-dependent decrease in the duration of epileptiform discharges. Events were evoked by orthodromic stimulation. Panel 1 is a semilog plot of the duration of discharges as a percentage of that during the control period. The average duration before CBZ in the 7 experiments analyzed was 588 + 17.5 ms (mean _+ S.E.M.). In each experiment the average duration was computed from 40 responses at each CBZ concentration. Once normalized, these values were in turn averaged and the mean and S.E.M. graphed. Panel 2 shows representative traces from one experiment. Calibrations for the inset in panel H are 2.5 mV/12.5 ms,

375 appearance to control recordings. With each further increase in CBZ content of the perfusate, the duration of the burst decreased slightly. At 100 #M there was approximately a 20% reduction in its duration. In 4 experiments comparing burst durations at 30 and 100 uM CBZ, the duration decreased 24.4 +_ 1.2%. This decrease is reflected in the dose-response curve in panel I. The inset above trace H in panel 2 shows the burst recorded at a faster time base. Even at supratherapeutic concentrations of CBZ, the threshold stimulation strength for burst generation was unaltered, and the burst maintained its characteristic all-or-none properties. We have made similar observations in slices bathed in media containing 200.uM CBZ. Several neurophysiological studies with in vivo experimental models of epilepsy have shown that afterdischarges are susceptible to the actions of CBZ -'
tion >. Instead, it is mediated by chemical synaptic transmission. In recent studies excitatory amino acid antagonists suppressed afterdischarges in both immature and mature CA 3 neurons 3~. As would be expected, these same drugs have no effect on low-Ca 2+induced afterdischarging 1°. In both experimental models CBZ may act by limiting neuronal excitability14.311. Previous recordings from penicillin foci have shown that a prolonged negative field potential is recorded after an epileptiform burst is generated 1'3. When the amplitude and duration of this field is increased experimentally, afterdischarges often occur. It has been suggested that these slow fields are measures of neuronal currents that play an important role in the onset of afterdischarges 5. The recordings in Figs. 1-3 clearly show that whenever CBZ decreased the duration of afterdischarges, the prolonged negative field on which the afterdischarges rode decreased concomitantly in duration and amplitude. Thus it is possible that the physiological events eliminated by CBZ and those which underlie slow field generation are one and the same. Recent current source-density analyses in our laboratory suggest that this slow potential is generated in the dendritic trees at sites distinct from those at which burst generation occurs >. Such a finding would further support the contention that mechanisms different from those which underlie the burst may contribute to the onset of an afterdischarge. In conclusion, we believe that the results reported here extend the use of in vitro preparations for study of the neuropharmacology of anticonvulsants. In addition, our results suggest that CBZ will be an important experimental tool in unraveling the physiological mechanisms that are responsible for synchronized afterdischarge generation and the unusual susceptibility of immature hippocampus for seizures. This work was supported bv NIH Grant NS18309. We thank Dr. David Martin for his helpful discussions, Patricia Normand for software development, Carolyn Wieland for secretarial assistance and Dr. Richard Jennv for gas chromatographic assays of carbamazepine. Carbamazepine was generously supplied by Ciba-Geigy Corporation (Summit, New Jersey).

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