- Email: [email protected]
Hippocampal epileptiform activity induced by magnesium-free medium: differences between areas CA1 and CA2–3
Epilepsy Res., 6 (1990) 95-101
95
Elsevier EPIRES 00318
Hippocampal epileptiform activity induced by magnesium-free medium: differences between areas CA1 and CA2-3 Darrell V. Lewis a, Leslie Sargent Jones b'd and David D. M o t t c Departments of aPediatrics, bNeurobiologyand Cpharmacolosy, Duke University Medical Center, Durham, NC 27710 (U.S.A.), and dDepartment of Anatomy, School of Medicine, Universityof South Carolina, Columbia, SC 29208 (U.S.A.) (Received 28 June 1989; revision received and accepted 17 October 1989)
Key words: Interictai; Seizure; Hippocampal slice; Magnesium; CA1; CA3
Hippocampal slices, from which the entorhinal cortex had been removed, were exposed to artificial cerebrospinal fluid containing no magnesium (0-Mg ACSF) to elicit interictal bursts (IIBs) and electrographic seizures (EOSs). In 0-Mg ACSF, IIBs and EGSs occurred in both area CA1 and area CA3. The IIBs in CA3 led the fiBs in CA1 by several milliseconds. The epileptiform bursts occurring during the EGSs seemed to have the opposite relationship, with bursts in CA1 leading those in CA3 by several milliseconds. When the connections between CA1 and CA2-3 were cut, the IIBs ceased in CA1 and continued in CA3. To further characterize the local differences in epileptiform activity, totally separate minislices of area CA1 and area CA2-3 were prepared. In the CA2-3 minislices, a few EGSs occurred and thereafter only persistent IIBs prevailed. Conversely, in the CA1 minislices, many spontaneous EGSs occurred for long periods of time and no IIBs were seen. Periodic stimulation of the CA1 minislices triggered lIBs that suppressed the recurrent EGSs. In the hippocampal slice exposed to low magnesium, IIBs or ~,inate in CA2-3 and are propagated to CA1, where they can have a suppressant effect on EGSs. Furthermore, unlike IIBs, the bu, ~s making up ~he Et3Ss seem to start ~a CA1 and invade CA2-3.
INTRODUCTION It is now possible to study both interictal bursts (IIBs) and electrographic seizures (EGSs) in the in vitro hippocampal slice. EGSs have been observed in slices bathed in magnesium-free medium 1'8'~5,in high potassium medium4'7'14 and in calcium-free medium 6. Also, slices from immature animals treated with convulsants often produce EGSs 3,n. Investigations with these models have suggested that IIBs and EGSs may have anatomically sepaCorrespondence to: Darrell V. Lewis, M.D., Box 3430, Duke University Medical Center, Durham, NC 27710, U.S.A.
rate generators in the hippocampal slice. In high potassium, the IIBs appear to originate from the CA2-3 region, whereas the EGSs seem to be limited to the CA1 region4'7'14.In low calcium bathing medium, there is no synaptic transmission and only the tonic phases of EGSs are seen, but these occur in region CA16. We have previously described the complex pattern of epileptiform activity resulting when hippocampa! slices are bathed in artificial cerebrospinal fluid containing no added magnesium (0-Mg ACSF) 1. Both long EGSs and IIBs are generated in 0-Mg ACSF. However, the relationship between the IIBs and the EGSs in this model is enig-
0920-12111901503.50© 1990 Elsevier Science Publishers B.V. (Biomedical Division)
96 matic. Often the IIBs build up before an EGS and seem to trigger the EGS, but EGSs can also occur without any preceding IIBss. Furthermore, the IIBs appear, if triggered repetitively by external stimulation, to suppress the EGSs 12. The results of this study demonstrate both similarities and differences between the 0-Mg ACSF model and other methods of eliciting EGSs. In 0Mg ACSF, as in penicillin and bicuculline 16or high potassium 4'~'~4,the IIBs originate in area CA3 and area CA1 is more prone to generate EGSs than area CA3. However, unlike most other models, EGSs in 0-Mg ACSF involve both areas CA1 and CA3 simultaneously, resembling the EGSs seen in slices from immature rats treated with 4-aminopyridine 3. Finally, during the EGSs, activity in CA1 seems to drive the activity in CA3, the opposite of the relationship seen during IIBs.
formed using a monopolar tungsten stimulating electrode with rectangular 100-800 gA, constant current, monophasic pulses of 0.1 msec duration. 0-Mg ACSF was made by simply omitting the MgSO 4 from the ACSF. Before recording from the slices, the entorhinal cortex was trimmed from each slice using a razor blade because we wished to examine the interactions of areas CA1 and CA2-3 without potential interference from epileptiform activity in the entorhinal area. We have previously shown that spontaneous EGSs in the entorhinal area can trigger EGSs in the hippocampus is. The recording electrodes were placed in the stratum pyramidale of CAI and CA3 (Fig. 1A), and responses to stimula-
A
CAI Rec,--~,
METHODS Male Sprague-Dawley rats, 22-32 days old, were anesthetized with chloroform and sacrificed by decapitation. Young rats were used for this study because we have found 1, as have others it, that EGSs occur more consistently in slices from young rats than in slices from older rats. The hippocampi were dissected free from the rest of the brain and 625 #m thick hippocampal slices were prepared using a Mciiwain tissue chopper. After 1 h in a holding chamber, slices were pla~~ ~ in a submersion chamber of 2.5 ml volume and pop-fused at 6 ml/min with artificial cerebrospinal fluid (ACSF) containing in mM; NaC! 120; KCI 3.3, CaCI2 1.8, MgSO 4 1.2, NaHCO 3 25, NaH2PO 4 1.23, and dextrose 10 at pH 7.4 and 33.5 °C. The ACSF was constantly bubbled with a gas mixture of 95% 0 2 and 5% CO 2. Ventral slices (3rd-6th slice from the temporal end of each hippocampus) were used for these experiments to remain consistent with our previous studies of the effects of 0-Mg ACSF ~'s'~s. In addition to maintaining consistency with our other studies, we prefer to use ventral slices because they may be more prone to generate epileptiform activity than dorsal slices2. Only one slice was used from each animal. Extracellular recordings were inade with 2-5 Mf~ glass microelectrodes containing 2 M NaCl. Stimulation was per-
~CA3 Ree.
intact Slice-.E,lectrode Placement
8 CAIRec.--,~, CAI
"
S
t
CA I Fragment
~
" Portion Slice Cut Into Separate Pieces
Fig, l. Placement of electrodes and razor cuts. A: in the intact slices both CA1 and CA3 were monitored with recording electrodes in stratum pyramidale. When razor cuts were made, they were placed as indicated. Note that the entorhinal cortex is not included in the slices. B: shown here are the minislices (CA1 fragment and CA2-3 fragment) and the recording and stimulating arrangements for the minislices.
97 tion of the Schaffer collaterals in stratum radiatum of CA3 were examined. Stimulation in this area produces orthodromic activation cl area CA1 and an antidromic spike followed by an orthodromic spike and extracellular postsynaptic potential (EPSP) in area CA3s. Slices were rejected if the orthodromic population spikes were less than 0.5 mV amplitude or if more than one orthodromic population spike occurred after a single stimulus. Next, the bathing fluid was changed to 0-Mg ACSF and the ensuing epileptiform activity was recorded. When we examined in detail the relative timing ot the IIBs in CA1 and CA3, we used the IIBs to trigger a digitalization device (Tecmar, Inc.) which stored the llBs and the preceding 20 msec of baseline activity in a computer. They were then relayed to the chart recorder at reduced speed for faithful reproduction. In some slices, we severed the projections ot CA2-3 to CA1. A cut was made between CA1 and CA2-3 that extended from the surface of the alveus to the stratum lacunosum-moleculare and completely through the thickness of the slice (Fig. 1A). In other slices we wished to record the epileptiform activity in areas CA1 and CA2-3 when these areas were completely physically isolated from the rest of the slice. Therefore, these slices were cut into minislices and the stratum pyramidale of each minislice was monitored with a recording electrode (Fig. 1B). The CA1 minislices actually may have contained some of the prosubiculum as well, but will be referred to as CA1 minislices. All cuts were made under the microscope using a razor blade chip in a holder while the slices were being perfused in the submersion chamber. Field potentials in CA1 and CA2-3 were checked before and after cutting the slices and in all cases responses were preserved after the cuts. RESULTS Bathing intact slices in 0-Mg ACSF produced epileptiform activity that appeared to involve both areas CA1 and CA3 as previously reported ~. IIBs, recorded in these experiments extracellularly in the pyramidal cell body layer, are here defined as brief (50-100 msec) events consisting of a positive
slow wave with a variable number of superimposed sharp negative population spikes (as in Fig. 2B1.2). EGSs, also recorded extracellularly in the cell body layer, are defined as long duration (many seconds) events consisting of repetitive individual bursts (as in Fig. 2A). The bursts during the EGS usually consist of a leading negative population spike followed by a positive slow wave, often with other superimposed population spikes (as in Fig. 2B3.4). EGSs also have a typical temporal evolution wherein the bursts occur at a more rapid rate (usually 4-10 Hz) during the first part of the EGS than during the last portion of the EGS. We will refer to the initial, rapidly bursting phase as the tonic phase and to the later, slowly bursting phase as the clonic phase. Recording at slow chart recorder speeds, the UBs, as well as the bursts during the EGSs, appeared to occur simultaneous-
A CA3-!-I.
CA 1~
1
[ ! ! ~ 1 0 ~ s e :
.
mn~pl'" '"' ........ 2 f'"..', ","" .....
B
mV
.,o,.
1
CA3 CA1 3
20mWe v __l.j0'SmV 20 msec
Fig. 2. Timing of epileptiform activity in CA1 and CA3. A: an EGS recorded simultaneously in CA3 (top trace) and CA1 (bottom trace). The activity appears quite synchronous. B: bursts were captured and written out on an expanded time scale at various points during pre-ictal and ictal activity to show the latency differences. 1: the first burst of the interictal period where CA3 seems to lead CA1.2: a burst at EGS onset where CA1 is now leading CA3. 3: a burst captured during the tonic phase where the lead of CA1 has increased. 4: the last burst of the clonic phase where CA3 again leads slightly. Note that the gain in the CA1 traces is twice that of the CA3 traces.
98
ly in CA1 and CA3 (Fig. 2A). The IIBs increased in amplitude and duratiop before the EGSs and after each EGS there was a silent period with no IIBs. When the individual IIBs as well as the bursts comprising the EGSs were captured and analyzed at high speed, subtle differences in latency between CA1 and CA3 became apparent (Fig. 2B). In 7 of the 8 slices studied in this way, IIBs beginning soon after the post-ictal silence began 4-8 msec earlier in CA3 than in CA1 (Fig. 2B1). In one slice a clear latency difference could not be discerned in these early IIBs. The delay between areas CA3 and CA1 became less as the onset of the EGS approached. Close to the onset of the EGS, the latency difference was reversed and CA1 bursts began to precede the CA3 bursts by 4-8 msec (Fig. 2B2.3). The tendency for CA1 to lead CA3 during the EGSs was quite consistent, being seen in 8 out of 8 slices. In the clonic phase of the EGSs, the latency differences became less consistent and often CA3 again led CA1 in the last few bursts of the clonic phase (Fig. 2B4). These observations on latency differences suggested that CA3 could be the pacemaker for IIBs, and CA1 the pacemaker for the tonic phase of the EGSs. To test whether the IIBs originated from CA2-3, a large cut was made between CA2-3 and CA1 in 5 of the above described slices (Fig. 1A). To make the cut, the perfusion with 0-Mg ACSF was replaced temporarily by perfusion in normal ACSF. After the cut, stimulation in stratum radiaturn of CA1 elicited a normal field potential in stratum pyramidale of CA1, but no response in CA2-3. Likewise, stimulation in stratum radiatum of CA2-3 produced a normal field potential in CA3 with no spread to CA1. Prior to the cuts, the IIBs and the EGSs had been occurring in both areas CA1 and CA3 (Fig. 3A). After the cut, when the slices were again perfused with 0-Mg ACSF, the IIBs resumed in CA2-3 alone, with no propagation to CA1 (Fig. 3B). EGSs occurred asynchronously in both areas CA1 and CA3 after the cuts, but continued to recur in CA1 for longer periods than in CA3. After the cuts, the EGSs in CA1 had faster bursting rates than those in CA3 (Fig. 3C). When EGSs ceased in CA3, they were replaced by continuous IIBs,
~A1~
l
m
V
Dse(:
B CA3 CA l - ;
-~--,,.-
~ , ~ ~ ,
'-
~-
TI.v 10sec
c C~3
~
5 sec
Fig. 3. Effects of a razor cut between CA1 and CA2-3. A: simultaneous recording of EGS and preceding IIBs in CA1 and CA3 prior to cut. B: after the cut, CA3 generated continuous IIBs while CA1 generated recurrent spontaneous EGSs and no IIBs. C: this record is from a different slice showing a EGS in CA1 and one in CA3 after the cut. Note that the EGS in CA1 shows more rapid firing than the EGS in CA3.
while EGSs still continued in CA1. EGSs in CA1 occurred without extraneous stimulation except for the initial EGSs in two slices that needed to be triggered by single pulse stimuli to the stratum radiatum of CA1 and thereafter occurred spontaneously. These experiments supported the hypothesis that the IIBs were generated in CA2-3 and propagated to CA1. Furthermore, the persistence of EGSs in CA1 after only IIBs continued in CA3, suggested that CA1 might generate EGSs for longer periods of time if isolated from the IIBs propagated from CA2-3. To confirm this observation, slices from 6 more animals were used to create minislices (Fig. 1B) to completely physically isolate area CA1 from areas CA2-3 before the slices had any exposure to 0-Mg ACSF. Field potentials evoked by stimuli in the stratum radiatum were examined in both areas prior to and after dividing the slices. Cutting apart the tissue did not reduce the amplitude of the field
99 potentials nor did it induce epileptiform activity. The bathing fluid was switched to 0-Mg ACSF while recording from the strata pyramidale of both the CA1 and CA2-3 minislices. IIBs consistently (in 6 of 6 experiments) began in the CA2-3 minislice several minutes before any epileptiform activity was seen in the CA1 minislices. Spontaneous EGSs eventually began in most of the minislices, being seen in 4 out of 6 CA2-3 minislices and in all 6 CA1 minislices (Fig. 4A). The 2 CA2-3 minislices that did not exhibit EGSs showed only continuous IIBs. The CA2-3 EGSs had higher voltage bursts and were preceded by a build-up of IIBs (Fig. 4A). In the CA1 minislices no IIBs preceded the EGSs and the repetition rate of the bursts during the tonic phase of the EGSs was greater than that of the EGSs in the CA2-3 minislices (Fig. 4A). Continuous recordings were made from each pair of minislices for 2-3 h to observe the types of epileptiform activity generated in 0-Mg ACSF. In the CA2-3 minislices, the total number of EGSs was low (mean number of EGSs = 4 + 4, n = 6). EGSs ceased after 7-25 rain (mean time 11 rain) in 0-Mg ACSF and thereafter only IIBs were seen (Fig. 4B). In contrast, recurrent EGSs continued to occur every 1-2 rain for 15-210 rain (mean time 130 rain) in the CA1 minislices (mean number of EGSs = 43 + 19, n = 6). This difference between
numbers of EGSs in the CA1 vs. the CA2-3 minislices is significant at the P < 0.01 level using the t test for paired samples. In spite of the abundance of EGSs in the CA1 minislices, none of them exhibited any IIBs unless they were stimulated electrically (see below). In 5 of the above CA1 minislices that had been generating EGSs for 1-2 h, we began to deliver periodic single pulse stimuli to the stratum radiaturn at 0.1-0.2 Hz (Fig. 5). In intact slices, periodic electrical triggering of IIBs has been shown to inhibit EGSs 12. In the CA1 minislices, each stimulus triggered a brief field potential resembling an IIB but much lower in amplitude. EGSs were completely inhibited during the 25-45 min that stimulation was continued. When the stimulation was stopped, the EGSs began again within 1-2 rain in 3 of the 5 minislices. In the 2 other minislices, the EGSs did not recover and spontaneous IIBs appeared after the stimuli were stopped. DISCUSSION These results confirm that, as in other models4'7']4']6, the IIBs in 0-Mg ACSF originate in area CA2-3 and are propagated to area CA1. Also, in accord with other models 4J4, area CA1 did not generate any IIBs when isolated from CA2-3 and produced EGSs more readily than CA2-3. These aspects of the 0-Mg model are similar to the behav-
A
<23_t i, !
,mv
C A l ~ l l
IOllf
-_ ......
SpontaneouSei s zures
I__.~
- _ _~
~-~ ~
-
~
-
_
-
t Begun01Hz L.... ~
20 Iol
10 sic
CAI
~
l
m
~
[IIIIINIIIIIIIIIIII_IWIiLILIIIIIIlllllllllllllllllllllllllll{l{l
V
i r l l ] l l l H W l l l l , i , l l•~ l l f l l l l l , f l"' l'l W l l l l v I-l l r l , l l l l l i-' l l i l l l l-Shmulgl,on for 25 m~n - No Seizures
IOil¢
v I--r 11 "r I l l
~A2-3 | End Stimulation
CA
I~
2
m
I__.~
v
1oII(
30
tic
Fig. 4. Epileptiform activityin the minislices. A: examplesof typical EGSs occurring in CA2-3 (top) and CA1 (bottom) minislices. B: after a short time, the CA2-3 minisliceshowed only IIBs, whereas the CA1 minislice continues to generate spontaneous EGSs.
Fig. 5. Effect of periodic stimulation on the EGSs in a CA1 minislice. This is a recording from one CA1 minislice. Beginning at the upper left hand corner, spontaneous EGSs are occurring and had been occurring for 1,5 h. At the arrow, stimulation of the stratum radiatum at 0.1 Hz was begun for 25 rain and no EGSs occurred. After the cessation of the stimuli, the EGSs reappeared,
100 ior of other models of EGS activity in the hippocampal slice wher~ comparisons between CA1 and CA2-3 have been made. There are, however, differences between the 0Mg ACSF model and other models. In 0-Mg ACSF, EGSs occur simultaneously in both CA1 and CA2-3 when the slice is intact. In the high potassium and low calcium models, EGSs occur in CA1 without involving CA34'14. However, Chesnut and Swann 3 did observe simultaneous EGSs in CA1 and CA3 in slices from immature (10-15 days) rats exposed to 4-aminopyridine. In our experiments, the individual bursts making up the EGSs in CA1 led the bursts making up the EGSs in CA3 by several milliseconds. The latency differences s-ggest that the EGSs in CA1 could be driving the EGSs in CA3, although we are not aware of any orthodromic projections from CA1 to CA3 to account for this. Perhaps elevated extracellular potassium levels during the EGSs cause depolarization and backfiring of the Schaffer collateral axon terminals triggering bursts in CA2-3 9. Alternatively, enhanced ephaptic interactions may occur during EGSs and enable the extracellular currents of the bursts in CA1 to trigger bursts in CA2-31°A3.
During the clonic phase of EGSs in intact slices, CA1 no longer leads CA2-3 and in fact CA2-3 often again takes over the lead. Traynelis and Dingledine ~4and Jensen and Yaari 4 observed a similar phenomenon in high potassium. During the clonic phase of EGSs in CA1, the CA1 bursts seemed to be triggered by the IIBs in CA2-3. In 0-Mg ACSF, EGSs in CA1 can clearly occur in the absence of llBs. In addition, spontaneous EGSs occur readily in 0-Mg ACSF where IIBs are totally suppressed by baclofens'12. Traynelis and Dingledine ~4 found that in high potassium, CA1
REFERENCES 1 Anderson, W.W., Lewis, D.V., Swartzweider, H.S. and Wilson, W.A., Magnesium-free medium activates seizurelike events in the rat hippocampal slice, Brain Res., 398 (1986) 215-219. 2 Bragdon, A.C., Taylor, D.M. and Wilson, W.W., Potassium-induced epileptiform activity in area CA3 varies
could not generate EGSs when isolated from interictal input from CA2-3, although Jensen and Yaari4 reported that high potassium induced tonic EGSs do occur in CA1 in the absence of burst input from CA2-3. Finally, not only are IIBs unnecessary for triggering EGSs in 0-Mg ACSF, but continuous IIBs triggered by electrical stimulation can suppress EGSs 12. Since CA2-3 is the IIB generator, EGS activity may have continued for long periods in CA1 minislices because they were isolated from the constant IIBs in CA2-3. When we attempted to mimic interictal input from CA2-3 by periodically triggering bursts in the CA1 minislices, the EGSs were suppressed. These results support our hypothesis that in the intact slice, in 0-Mg ACSF, the lIBs from CA2-3 may suppress EGSs in other areas of the slice. We have previously observed that, in slices with retained entorhinal cortex, triggered IIBs in CA2-3 can suppress entorhinal cortex EGSs is. We conclude that CA2-3 is the generator of IIBs in 0-Mg ACSF. Although both CA1 and CA2-3 can generate EGSs, CA1 is the pacemaker for the tonic phase of the EGS in CA2-3. Furthermore, CA1 minislices produce EGSs for longer periods than CA2-3 minislices, perhaps because the IIBs in CA2-3 minislices suppress EGS activity. By studying minislices, the interactions of larger networks in generating epileptiform activity might be more easily understood. ACKNOWLEDGEMENTS This work was supported by NIH Grant NS22170 and D.D.M. was supported by the Chemical Industry Institute of Toxicology Predoctoral Fellowship.
markedly along the septotemporal axis of the rat hippocampus, Brain Res., 378 (1986) 169-173. 3 Chesnut, T.J. and Swann, J.W., Epileptiform activity induced by 4-aminopyridine in immature hippocampus, Epilepsy Res., 2 (1988) 187-193. 4 Jensen, M.S. and Yaari, Y., The relationship between interictal and ictal paroxysms in an in vitro model of focal hippocampal epilepsy, Ann. Neurol., 24 (1989) 591-598.
101 5 Jones, L.S., Mott, D.D. and Lewis, D.V., CA3 to CA1 delay reversed during interictal to ictal transition in 0-Mg induced ictaform events in the rat hippocampal slice, Soc. Neurosci. Abst., 14 (1988) 571. 6 Konnerth, A., Heinemann, U. and Yaari, Y., Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. I. Development of seizure-like activity in low extracellular calcium, J. Neurophysiol., 56 (1986) 409-423. 7 Korn, S.J., Giacchino, J.L., Chamberlain, N.L. and Dingledine, R., Epileptiform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition, J. Neurophysiol., 57 (1987) 325-340. 8 Lewis, D.V., Jones, L.S. and Swartzwelder, H.S., The effects of baclofen and pertussis toxin on epileptiform activity induced in the hippocampal slice by magnesium depletion, Epilepsy Res., 4 (1989) 109-118. 9 Prince, D.A., Neurophysiology of epilepsy, Annu. Rev. Neurosci., 1 (1978) 395-415. 10 Somjen, G.G., Aitken, P.G., Giacchino, J.L. and McNamara, J.O., Sustained potential shifts and paroxysmal dis-
charges in hippocampal formation, Y. Neurophysiol., 53 (1985) 1079-1097. 11 Swann, J.W. and Bady, R.J., Penicillin-induced epileptogenesis in immature rat CA3 hippocampal pyramidal cells, Dev. Brain Res., 12 (1984) 243-254. 12 Swartzwelder, H.S., Lewis, D.V., Anderson, W.W. and Wilson, W.A., Seizure-like events in brain slices: suppression by interictal activity, Brain Res., 410 (1987) 362-366. 13 Taylor, C.P. and Dudek, E.F., Excitation of hippocampal pyramidal cells by an electrical field effect, J. Neurophys. iol., 52 (1984) 126-142. 14 Traynelis, S.F. and Dingledine, R., Potassium-induced spontaneous electrographic seizures in the rat hippocampal slice, J. Neurophysiol., 59 (1988) 259- 276. 15 Wilson, W.A., Swartzwelder, H.S., Anderson, W.W. and Lewis, D.V., Seizure activity in vitro: a dual focus model, Epilepsy Res., 2 (1988) 289-293. 16 Wong, R.K.S. and Traub, R.D., Synchronized burst discharge in disinhibited hippocampal slice. I. Initiation in CA2-CA3 region, J. NeurophysioL, 49 (1983) 442-458.
95
Elsevier EPIRES 00318
Hippocampal epileptiform activity induced by magnesium-free medium: differences between areas CA1 and CA2-3 Darrell V. Lewis a, Leslie Sargent Jones b'd and David D. M o t t c Departments of aPediatrics, bNeurobiologyand Cpharmacolosy, Duke University Medical Center, Durham, NC 27710 (U.S.A.), and dDepartment of Anatomy, School of Medicine, Universityof South Carolina, Columbia, SC 29208 (U.S.A.) (Received 28 June 1989; revision received and accepted 17 October 1989)
Key words: Interictai; Seizure; Hippocampal slice; Magnesium; CA1; CA3
Hippocampal slices, from which the entorhinal cortex had been removed, were exposed to artificial cerebrospinal fluid containing no magnesium (0-Mg ACSF) to elicit interictal bursts (IIBs) and electrographic seizures (EOSs). In 0-Mg ACSF, IIBs and EGSs occurred in both area CA1 and area CA3. The IIBs in CA3 led the fiBs in CA1 by several milliseconds. The epileptiform bursts occurring during the EGSs seemed to have the opposite relationship, with bursts in CA1 leading those in CA3 by several milliseconds. When the connections between CA1 and CA2-3 were cut, the IIBs ceased in CA1 and continued in CA3. To further characterize the local differences in epileptiform activity, totally separate minislices of area CA1 and area CA2-3 were prepared. In the CA2-3 minislices, a few EGSs occurred and thereafter only persistent IIBs prevailed. Conversely, in the CA1 minislices, many spontaneous EGSs occurred for long periods of time and no IIBs were seen. Periodic stimulation of the CA1 minislices triggered lIBs that suppressed the recurrent EGSs. In the hippocampal slice exposed to low magnesium, IIBs or ~,inate in CA2-3 and are propagated to CA1, where they can have a suppressant effect on EGSs. Furthermore, unlike IIBs, the bu, ~s making up ~he Et3Ss seem to start ~a CA1 and invade CA2-3.
INTRODUCTION It is now possible to study both interictal bursts (IIBs) and electrographic seizures (EGSs) in the in vitro hippocampal slice. EGSs have been observed in slices bathed in magnesium-free medium 1'8'~5,in high potassium medium4'7'14 and in calcium-free medium 6. Also, slices from immature animals treated with convulsants often produce EGSs 3,n. Investigations with these models have suggested that IIBs and EGSs may have anatomically sepaCorrespondence to: Darrell V. Lewis, M.D., Box 3430, Duke University Medical Center, Durham, NC 27710, U.S.A.
rate generators in the hippocampal slice. In high potassium, the IIBs appear to originate from the CA2-3 region, whereas the EGSs seem to be limited to the CA1 region4'7'14.In low calcium bathing medium, there is no synaptic transmission and only the tonic phases of EGSs are seen, but these occur in region CA16. We have previously described the complex pattern of epileptiform activity resulting when hippocampa! slices are bathed in artificial cerebrospinal fluid containing no added magnesium (0-Mg ACSF) 1. Both long EGSs and IIBs are generated in 0-Mg ACSF. However, the relationship between the IIBs and the EGSs in this model is enig-
0920-12111901503.50© 1990 Elsevier Science Publishers B.V. (Biomedical Division)
96 matic. Often the IIBs build up before an EGS and seem to trigger the EGS, but EGSs can also occur without any preceding IIBss. Furthermore, the IIBs appear, if triggered repetitively by external stimulation, to suppress the EGSs 12. The results of this study demonstrate both similarities and differences between the 0-Mg ACSF model and other methods of eliciting EGSs. In 0Mg ACSF, as in penicillin and bicuculline 16or high potassium 4'~'~4,the IIBs originate in area CA3 and area CA1 is more prone to generate EGSs than area CA3. However, unlike most other models, EGSs in 0-Mg ACSF involve both areas CA1 and CA3 simultaneously, resembling the EGSs seen in slices from immature rats treated with 4-aminopyridine 3. Finally, during the EGSs, activity in CA1 seems to drive the activity in CA3, the opposite of the relationship seen during IIBs.
formed using a monopolar tungsten stimulating electrode with rectangular 100-800 gA, constant current, monophasic pulses of 0.1 msec duration. 0-Mg ACSF was made by simply omitting the MgSO 4 from the ACSF. Before recording from the slices, the entorhinal cortex was trimmed from each slice using a razor blade because we wished to examine the interactions of areas CA1 and CA2-3 without potential interference from epileptiform activity in the entorhinal area. We have previously shown that spontaneous EGSs in the entorhinal area can trigger EGSs in the hippocampus is. The recording electrodes were placed in the stratum pyramidale of CAI and CA3 (Fig. 1A), and responses to stimula-
A
CAI Rec,--~,
METHODS Male Sprague-Dawley rats, 22-32 days old, were anesthetized with chloroform and sacrificed by decapitation. Young rats were used for this study because we have found 1, as have others it, that EGSs occur more consistently in slices from young rats than in slices from older rats. The hippocampi were dissected free from the rest of the brain and 625 #m thick hippocampal slices were prepared using a Mciiwain tissue chopper. After 1 h in a holding chamber, slices were pla~~ ~ in a submersion chamber of 2.5 ml volume and pop-fused at 6 ml/min with artificial cerebrospinal fluid (ACSF) containing in mM; NaC! 120; KCI 3.3, CaCI2 1.8, MgSO 4 1.2, NaHCO 3 25, NaH2PO 4 1.23, and dextrose 10 at pH 7.4 and 33.5 °C. The ACSF was constantly bubbled with a gas mixture of 95% 0 2 and 5% CO 2. Ventral slices (3rd-6th slice from the temporal end of each hippocampus) were used for these experiments to remain consistent with our previous studies of the effects of 0-Mg ACSF ~'s'~s. In addition to maintaining consistency with our other studies, we prefer to use ventral slices because they may be more prone to generate epileptiform activity than dorsal slices2. Only one slice was used from each animal. Extracellular recordings were inade with 2-5 Mf~ glass microelectrodes containing 2 M NaCl. Stimulation was per-
~CA3 Ree.
intact Slice-.E,lectrode Placement
8 CAIRec.--,~, CAI
"
S
t
CA I Fragment
~
" Portion Slice Cut Into Separate Pieces
Fig, l. Placement of electrodes and razor cuts. A: in the intact slices both CA1 and CA3 were monitored with recording electrodes in stratum pyramidale. When razor cuts were made, they were placed as indicated. Note that the entorhinal cortex is not included in the slices. B: shown here are the minislices (CA1 fragment and CA2-3 fragment) and the recording and stimulating arrangements for the minislices.
97 tion of the Schaffer collaterals in stratum radiatum of CA3 were examined. Stimulation in this area produces orthodromic activation cl area CA1 and an antidromic spike followed by an orthodromic spike and extracellular postsynaptic potential (EPSP) in area CA3s. Slices were rejected if the orthodromic population spikes were less than 0.5 mV amplitude or if more than one orthodromic population spike occurred after a single stimulus. Next, the bathing fluid was changed to 0-Mg ACSF and the ensuing epileptiform activity was recorded. When we examined in detail the relative timing ot the IIBs in CA1 and CA3, we used the IIBs to trigger a digitalization device (Tecmar, Inc.) which stored the llBs and the preceding 20 msec of baseline activity in a computer. They were then relayed to the chart recorder at reduced speed for faithful reproduction. In some slices, we severed the projections ot CA2-3 to CA1. A cut was made between CA1 and CA2-3 that extended from the surface of the alveus to the stratum lacunosum-moleculare and completely through the thickness of the slice (Fig. 1A). In other slices we wished to record the epileptiform activity in areas CA1 and CA2-3 when these areas were completely physically isolated from the rest of the slice. Therefore, these slices were cut into minislices and the stratum pyramidale of each minislice was monitored with a recording electrode (Fig. 1B). The CA1 minislices actually may have contained some of the prosubiculum as well, but will be referred to as CA1 minislices. All cuts were made under the microscope using a razor blade chip in a holder while the slices were being perfused in the submersion chamber. Field potentials in CA1 and CA2-3 were checked before and after cutting the slices and in all cases responses were preserved after the cuts. RESULTS Bathing intact slices in 0-Mg ACSF produced epileptiform activity that appeared to involve both areas CA1 and CA3 as previously reported ~. IIBs, recorded in these experiments extracellularly in the pyramidal cell body layer, are here defined as brief (50-100 msec) events consisting of a positive
slow wave with a variable number of superimposed sharp negative population spikes (as in Fig. 2B1.2). EGSs, also recorded extracellularly in the cell body layer, are defined as long duration (many seconds) events consisting of repetitive individual bursts (as in Fig. 2A). The bursts during the EGS usually consist of a leading negative population spike followed by a positive slow wave, often with other superimposed population spikes (as in Fig. 2B3.4). EGSs also have a typical temporal evolution wherein the bursts occur at a more rapid rate (usually 4-10 Hz) during the first part of the EGS than during the last portion of the EGS. We will refer to the initial, rapidly bursting phase as the tonic phase and to the later, slowly bursting phase as the clonic phase. Recording at slow chart recorder speeds, the UBs, as well as the bursts during the EGSs, appeared to occur simultaneous-
A CA3-!-I.
CA 1~
1
[ ! ! ~ 1 0 ~ s e :
.
mn~pl'" '"' ........ 2 f'"..', ","" .....
B
mV
.,o,.
1
CA3 CA1 3
20mWe v __l.j0'SmV 20 msec
Fig. 2. Timing of epileptiform activity in CA1 and CA3. A: an EGS recorded simultaneously in CA3 (top trace) and CA1 (bottom trace). The activity appears quite synchronous. B: bursts were captured and written out on an expanded time scale at various points during pre-ictal and ictal activity to show the latency differences. 1: the first burst of the interictal period where CA3 seems to lead CA1.2: a burst at EGS onset where CA1 is now leading CA3. 3: a burst captured during the tonic phase where the lead of CA1 has increased. 4: the last burst of the clonic phase where CA3 again leads slightly. Note that the gain in the CA1 traces is twice that of the CA3 traces.
98
ly in CA1 and CA3 (Fig. 2A). The IIBs increased in amplitude and duratiop before the EGSs and after each EGS there was a silent period with no IIBs. When the individual IIBs as well as the bursts comprising the EGSs were captured and analyzed at high speed, subtle differences in latency between CA1 and CA3 became apparent (Fig. 2B). In 7 of the 8 slices studied in this way, IIBs beginning soon after the post-ictal silence began 4-8 msec earlier in CA3 than in CA1 (Fig. 2B1). In one slice a clear latency difference could not be discerned in these early IIBs. The delay between areas CA3 and CA1 became less as the onset of the EGS approached. Close to the onset of the EGS, the latency difference was reversed and CA1 bursts began to precede the CA3 bursts by 4-8 msec (Fig. 2B2.3). The tendency for CA1 to lead CA3 during the EGSs was quite consistent, being seen in 8 out of 8 slices. In the clonic phase of the EGSs, the latency differences became less consistent and often CA3 again led CA1 in the last few bursts of the clonic phase (Fig. 2B4). These observations on latency differences suggested that CA3 could be the pacemaker for IIBs, and CA1 the pacemaker for the tonic phase of the EGSs. To test whether the IIBs originated from CA2-3, a large cut was made between CA2-3 and CA1 in 5 of the above described slices (Fig. 1A). To make the cut, the perfusion with 0-Mg ACSF was replaced temporarily by perfusion in normal ACSF. After the cut, stimulation in stratum radiaturn of CA1 elicited a normal field potential in stratum pyramidale of CA1, but no response in CA2-3. Likewise, stimulation in stratum radiatum of CA2-3 produced a normal field potential in CA3 with no spread to CA1. Prior to the cuts, the IIBs and the EGSs had been occurring in both areas CA1 and CA3 (Fig. 3A). After the cut, when the slices were again perfused with 0-Mg ACSF, the IIBs resumed in CA2-3 alone, with no propagation to CA1 (Fig. 3B). EGSs occurred asynchronously in both areas CA1 and CA3 after the cuts, but continued to recur in CA1 for longer periods than in CA3. After the cuts, the EGSs in CA1 had faster bursting rates than those in CA3 (Fig. 3C). When EGSs ceased in CA3, they were replaced by continuous IIBs,
~A1~
l
m
V
Dse(:
B CA3 CA l - ;
-~--,,.-
~ , ~ ~ ,
'-
~-
TI.v 10sec
c C~3
~
5 sec
Fig. 3. Effects of a razor cut between CA1 and CA2-3. A: simultaneous recording of EGS and preceding IIBs in CA1 and CA3 prior to cut. B: after the cut, CA3 generated continuous IIBs while CA1 generated recurrent spontaneous EGSs and no IIBs. C: this record is from a different slice showing a EGS in CA1 and one in CA3 after the cut. Note that the EGS in CA1 shows more rapid firing than the EGS in CA3.
while EGSs still continued in CA1. EGSs in CA1 occurred without extraneous stimulation except for the initial EGSs in two slices that needed to be triggered by single pulse stimuli to the stratum radiatum of CA1 and thereafter occurred spontaneously. These experiments supported the hypothesis that the IIBs were generated in CA2-3 and propagated to CA1. Furthermore, the persistence of EGSs in CA1 after only IIBs continued in CA3, suggested that CA1 might generate EGSs for longer periods of time if isolated from the IIBs propagated from CA2-3. To confirm this observation, slices from 6 more animals were used to create minislices (Fig. 1B) to completely physically isolate area CA1 from areas CA2-3 before the slices had any exposure to 0-Mg ACSF. Field potentials evoked by stimuli in the stratum radiatum were examined in both areas prior to and after dividing the slices. Cutting apart the tissue did not reduce the amplitude of the field
99 potentials nor did it induce epileptiform activity. The bathing fluid was switched to 0-Mg ACSF while recording from the strata pyramidale of both the CA1 and CA2-3 minislices. IIBs consistently (in 6 of 6 experiments) began in the CA2-3 minislice several minutes before any epileptiform activity was seen in the CA1 minislices. Spontaneous EGSs eventually began in most of the minislices, being seen in 4 out of 6 CA2-3 minislices and in all 6 CA1 minislices (Fig. 4A). The 2 CA2-3 minislices that did not exhibit EGSs showed only continuous IIBs. The CA2-3 EGSs had higher voltage bursts and were preceded by a build-up of IIBs (Fig. 4A). In the CA1 minislices no IIBs preceded the EGSs and the repetition rate of the bursts during the tonic phase of the EGSs was greater than that of the EGSs in the CA2-3 minislices (Fig. 4A). Continuous recordings were made from each pair of minislices for 2-3 h to observe the types of epileptiform activity generated in 0-Mg ACSF. In the CA2-3 minislices, the total number of EGSs was low (mean number of EGSs = 4 + 4, n = 6). EGSs ceased after 7-25 rain (mean time 11 rain) in 0-Mg ACSF and thereafter only IIBs were seen (Fig. 4B). In contrast, recurrent EGSs continued to occur every 1-2 rain for 15-210 rain (mean time 130 rain) in the CA1 minislices (mean number of EGSs = 43 + 19, n = 6). This difference between
numbers of EGSs in the CA1 vs. the CA2-3 minislices is significant at the P < 0.01 level using the t test for paired samples. In spite of the abundance of EGSs in the CA1 minislices, none of them exhibited any IIBs unless they were stimulated electrically (see below). In 5 of the above CA1 minislices that had been generating EGSs for 1-2 h, we began to deliver periodic single pulse stimuli to the stratum radiaturn at 0.1-0.2 Hz (Fig. 5). In intact slices, periodic electrical triggering of IIBs has been shown to inhibit EGSs 12. In the CA1 minislices, each stimulus triggered a brief field potential resembling an IIB but much lower in amplitude. EGSs were completely inhibited during the 25-45 min that stimulation was continued. When the stimulation was stopped, the EGSs began again within 1-2 rain in 3 of the 5 minislices. In the 2 other minislices, the EGSs did not recover and spontaneous IIBs appeared after the stimuli were stopped. DISCUSSION These results confirm that, as in other models4'7']4']6, the IIBs in 0-Mg ACSF originate in area CA2-3 and are propagated to area CA1. Also, in accord with other models 4J4, area CA1 did not generate any IIBs when isolated from CA2-3 and produced EGSs more readily than CA2-3. These aspects of the 0-Mg model are similar to the behav-
A
<23_t i, !
,mv
C A l ~ l l
IOllf
-_ ......
SpontaneouSei s zures
I__.~
- _ _~
~-~ ~
-
~
-
_
-
t Begun01Hz L.... ~
20 Iol
10 sic
CAI
~
l
m
~
[IIIIINIIIIIIIIIIII_IWIiLILIIIIIIlllllllllllllllllllllllllll{l{l
V
i r l l ] l l l H W l l l l , i , l l•~ l l f l l l l l , f l"' l'l W l l l l v I-l l r l , l l l l l i-' l l i l l l l-Shmulgl,on for 25 m~n - No Seizures
IOil¢
v I--r 11 "r I l l
~A2-3 | End Stimulation
CA
I~
2
m
I__.~
v
1oII(
30
tic
Fig. 4. Epileptiform activityin the minislices. A: examplesof typical EGSs occurring in CA2-3 (top) and CA1 (bottom) minislices. B: after a short time, the CA2-3 minisliceshowed only IIBs, whereas the CA1 minislice continues to generate spontaneous EGSs.
Fig. 5. Effect of periodic stimulation on the EGSs in a CA1 minislice. This is a recording from one CA1 minislice. Beginning at the upper left hand corner, spontaneous EGSs are occurring and had been occurring for 1,5 h. At the arrow, stimulation of the stratum radiatum at 0.1 Hz was begun for 25 rain and no EGSs occurred. After the cessation of the stimuli, the EGSs reappeared,
100 ior of other models of EGS activity in the hippocampal slice wher~ comparisons between CA1 and CA2-3 have been made. There are, however, differences between the 0Mg ACSF model and other models. In 0-Mg ACSF, EGSs occur simultaneously in both CA1 and CA2-3 when the slice is intact. In the high potassium and low calcium models, EGSs occur in CA1 without involving CA34'14. However, Chesnut and Swann 3 did observe simultaneous EGSs in CA1 and CA3 in slices from immature (10-15 days) rats exposed to 4-aminopyridine. In our experiments, the individual bursts making up the EGSs in CA1 led the bursts making up the EGSs in CA3 by several milliseconds. The latency differences s-ggest that the EGSs in CA1 could be driving the EGSs in CA3, although we are not aware of any orthodromic projections from CA1 to CA3 to account for this. Perhaps elevated extracellular potassium levels during the EGSs cause depolarization and backfiring of the Schaffer collateral axon terminals triggering bursts in CA2-3 9. Alternatively, enhanced ephaptic interactions may occur during EGSs and enable the extracellular currents of the bursts in CA1 to trigger bursts in CA2-31°A3.
During the clonic phase of EGSs in intact slices, CA1 no longer leads CA2-3 and in fact CA2-3 often again takes over the lead. Traynelis and Dingledine ~4and Jensen and Yaari 4 observed a similar phenomenon in high potassium. During the clonic phase of EGSs in CA1, the CA1 bursts seemed to be triggered by the IIBs in CA2-3. In 0-Mg ACSF, EGSs in CA1 can clearly occur in the absence of llBs. In addition, spontaneous EGSs occur readily in 0-Mg ACSF where IIBs are totally suppressed by baclofens'12. Traynelis and Dingledine ~4 found that in high potassium, CA1
REFERENCES 1 Anderson, W.W., Lewis, D.V., Swartzweider, H.S. and Wilson, W.A., Magnesium-free medium activates seizurelike events in the rat hippocampal slice, Brain Res., 398 (1986) 215-219. 2 Bragdon, A.C., Taylor, D.M. and Wilson, W.W., Potassium-induced epileptiform activity in area CA3 varies
could not generate EGSs when isolated from interictal input from CA2-3, although Jensen and Yaari4 reported that high potassium induced tonic EGSs do occur in CA1 in the absence of burst input from CA2-3. Finally, not only are IIBs unnecessary for triggering EGSs in 0-Mg ACSF, but continuous IIBs triggered by electrical stimulation can suppress EGSs 12. Since CA2-3 is the IIB generator, EGS activity may have continued for long periods in CA1 minislices because they were isolated from the constant IIBs in CA2-3. When we attempted to mimic interictal input from CA2-3 by periodically triggering bursts in the CA1 minislices, the EGSs were suppressed. These results support our hypothesis that in the intact slice, in 0-Mg ACSF, the lIBs from CA2-3 may suppress EGSs in other areas of the slice. We have previously observed that, in slices with retained entorhinal cortex, triggered IIBs in CA2-3 can suppress entorhinal cortex EGSs is. We conclude that CA2-3 is the generator of IIBs in 0-Mg ACSF. Although both CA1 and CA2-3 can generate EGSs, CA1 is the pacemaker for the tonic phase of the EGS in CA2-3. Furthermore, CA1 minislices produce EGSs for longer periods than CA2-3 minislices, perhaps because the IIBs in CA2-3 minislices suppress EGS activity. By studying minislices, the interactions of larger networks in generating epileptiform activity might be more easily understood. ACKNOWLEDGEMENTS This work was supported by NIH Grant NS22170 and D.D.M. was supported by the Chemical Industry Institute of Toxicology Predoctoral Fellowship.
markedly along the septotemporal axis of the rat hippocampus, Brain Res., 378 (1986) 169-173. 3 Chesnut, T.J. and Swann, J.W., Epileptiform activity induced by 4-aminopyridine in immature hippocampus, Epilepsy Res., 2 (1988) 187-193. 4 Jensen, M.S. and Yaari, Y., The relationship between interictal and ictal paroxysms in an in vitro model of focal hippocampal epilepsy, Ann. Neurol., 24 (1989) 591-598.
101 5 Jones, L.S., Mott, D.D. and Lewis, D.V., CA3 to CA1 delay reversed during interictal to ictal transition in 0-Mg induced ictaform events in the rat hippocampal slice, Soc. Neurosci. Abst., 14 (1988) 571. 6 Konnerth, A., Heinemann, U. and Yaari, Y., Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. I. Development of seizure-like activity in low extracellular calcium, J. Neurophysiol., 56 (1986) 409-423. 7 Korn, S.J., Giacchino, J.L., Chamberlain, N.L. and Dingledine, R., Epileptiform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition, J. Neurophysiol., 57 (1987) 325-340. 8 Lewis, D.V., Jones, L.S. and Swartzwelder, H.S., The effects of baclofen and pertussis toxin on epileptiform activity induced in the hippocampal slice by magnesium depletion, Epilepsy Res., 4 (1989) 109-118. 9 Prince, D.A., Neurophysiology of epilepsy, Annu. Rev. Neurosci., 1 (1978) 395-415. 10 Somjen, G.G., Aitken, P.G., Giacchino, J.L. and McNamara, J.O., Sustained potential shifts and paroxysmal dis-
charges in hippocampal formation, Y. Neurophysiol., 53 (1985) 1079-1097. 11 Swann, J.W. and Bady, R.J., Penicillin-induced epileptogenesis in immature rat CA3 hippocampal pyramidal cells, Dev. Brain Res., 12 (1984) 243-254. 12 Swartzwelder, H.S., Lewis, D.V., Anderson, W.W. and Wilson, W.A., Seizure-like events in brain slices: suppression by interictal activity, Brain Res., 410 (1987) 362-366. 13 Taylor, C.P. and Dudek, E.F., Excitation of hippocampal pyramidal cells by an electrical field effect, J. Neurophys. iol., 52 (1984) 126-142. 14 Traynelis, S.F. and Dingledine, R., Potassium-induced spontaneous electrographic seizures in the rat hippocampal slice, J. Neurophysiol., 59 (1988) 259- 276. 15 Wilson, W.A., Swartzwelder, H.S., Anderson, W.W. and Lewis, D.V., Seizure activity in vitro: a dual focus model, Epilepsy Res., 2 (1988) 289-293. 16 Wong, R.K.S. and Traub, R.D., Synchronized burst discharge in disinhibited hippocampal slice. I. Initiation in CA2-CA3 region, J. NeurophysioL, 49 (1983) 442-458.