Neur¢~science Letters, 108 (1990) 76 80 Elsevier Scientific Publishers Ireland Ltd.

76

NSL 06547

Epileptiform activity in hippocampal slice cultures with normal inhibitory synaptic drive Alfred T. M a l o u f l, Carol A. Robbins I and Philip A. Schwartzkroin I'2 Departments ~[" 1Neurological Surgery, and :Physiology and Biophysics University o[ Washington, Seattle, WA 98195 (U.S.A.) tReceived 26 May 1989; Accepted 22 August 1989)

Key word~." Hippocampus; Slice culture; Epileptiform activity; Paroxysmal depolarization shift: Inhibitory postsynaptic potential: Kynurenic acid; CA3 pyramidal neuron The synaptic events responsible for epileptiform burst discharge are often difficult to define. Blockade of inhibition has been used to produce epileptiform events, but it is unclear whether increased excitatory activity in the presence of normal inhibition can also result in burst discharge. In the hippocampal slice culture preparation, a small percentage of cultures exhibit spontaneous bursts. To determine whether the absence of inhibitory postsynaptic potentials (IPSPs) is responsible for these spontaneous bursts, we applied the glutamate antagonist, kynurenic acid (KYN) to block burst activity, and unmask any underlying IPSPs. KYN (10 mM) quickly reduced synaptic activity with concomitant loss of burst discharge. Washout of KYN resulted in a gradual return of synaptic activity, during which time both fast and slow IPSPs were clearly observed. As burst activity returned to control levels, excitatory postsynaptic potentials (EPSPs) were increasingly superimposed within the inhibitory events, obscuring (but not eliminating) the IPSPs. In these hippocampal slice cultures, therefore, epileptiform bursts appear to be the result of an abnormally high level of excitatory synaptic drive, not a reduction in inhibition.

The maintenance of normal (non-bursting) physiology probably depends on a balance of inhibitory and excitatory synaptic activity in the hippocampus. Most investigators studying epileptiform activity have emphasized the loss of inhibitory drive as a basis for epileptogenesis [3, 14]. Normal hippocampal tissue can be induced to exhibit epileptiform activity by blocking 7-aminobutyric acid (GABA)-mediated synaptic events. It is possible, however, that epileptiform bursts occur in tissue with normal inhibitory synaptic activity when excitatory drive becomes exaggerated. Indeed, early studies of the paroxysmal depolarization shift (PDS) described this event as a 'giant EPSP' [2], a concept that was de-emphasized as interest grew in the regulatory role of inhibition. The occurrence of normal IPSPs in bursting tissue is difficult to determine since the bursts often overlap in time with expected inhibitory events, and thus obscure them. We have been able to circumvent this problem using a hippoCorre.spondence: A.T. Malouf, Dept. of Neurological Surgery, RI-20, Univ. of Washington, Seattle, WA 98195, U.S.A.

77

campal slice culture preparation and demonstrate that IPSPs are present in tissue with epileptiform properties. Hippocampal slice cultures were prepared from 4 day old rat pups by the method of G/ihwiler [5]. Slices (400/tm) were attached to 12 x 24 glass coverslips by applying a drop of chicken plasma (Cocalico Biologicals, Reamstown, PA, or collected in our laboratory) and thrombin (Sigma, cat. no. T4265) to form a plasma clot over the slice. The coverslips were placed inside 15 ml conical tissue culture tubes containing 1.5 ml growth medium (50% BME, 25% Earl's balanced salt solution, 25% horse serum, Gibco), and incubated for 2-12 weeks at 36°C, while constantly rotating at 1/5 RPM (New Brunswick Scientific Rollordrum). On day 4 after culture, the slices were treated with a cocktail of 10 -7 M mitotic inhibitors (uridine, cytosine-fl-D-arabinofuranoside, and 5-fluorodeoxyuridine) for 20 h. Recordings were made from 18 to 31-day-old cultures. Electrophysiology was performed using standard intracellular techniques. Recording electrodes (50-100 MI2) were filled with 4 M potassium acetate, and visually located in the CA3 pyramidal cell layer using Nomarski optics (Olympus IMT-2). The stimulating electrode, a glass micropipette (tip diameter 1-3/lm) filled with 1 M NaC1, was placed in the hilus or in area CA3. The recording chamber (0.5 ml) maintained the recording buffer (Hank's balance salt solution, HBSS) at 34°C, at a flow rate of about 0.5 ml/min; HBSS was not oxygenated. Kynurenic acid (KYN; Sigma) was dissolved in HBSS and introduced to the recording chamber via a 3-way valve. Data were digitized and recorded on tape (Neuro Data model DR-484) for subsequent analysis; figures were photographed from oscilloscope traces. In most slice cultures, synaptic activity (spontaneous and evoked) resembles the pattern of events seen in acutely prepared hippocampal slices [5, 10]. Triphasic synaptic events, which include both early and late component IPSPs, are characteristic. However, in a small percentage of organotypic cultures (5-10%), there is little evi-

l

A

i

!

~

! ~!, ~ ,,IV,,~!~.IIILI

i~~,:!~II!,~,li,I ~I,iI~,~i

, ,

~II,I ,,l,,,

il~

,

, B~

~

C

"i

,

Fig. 1. Kynurenic acid blocks spontaneous epileptiform bursts. A: spontaneous bursts observed in a small percentage of slice cultures resemble prolonged paroxysmal depolarization shifts (PDSs), with an abrupt and prolonged depolarization and a high rate of action potential firing. Bursts are followed by a long hyperpolarization. Negative voltage deflections (stars) are responses to - 0 . 6 nA/I00 ms intracellular current injections. B: prior to bath application of KYN (10 mM), a 50 p A stimulus applied to the mossy fiber afferents elicited a burst like that shown in A. C: 3 min after starting KYN, a 50/tA stimulus elicits little or no response. Asterisk marks the stimulus artifact and dashed line marks the resting membrane potential in this and subsequent figures. Calibrations= 10 mV, 5 s (A); I0 mV, 2 s (B); 10 mV, 100 ms

(c).

78

dence of synaptic inhibition, and cells discharge in bursts resembling prolonged paroxysmal depolarization shifts (PDSs) [2, 13]. Spontaneous bursts are initiated by an abrupt 15 20 mV depolarization, characterized by high frequency action potential discharge (4-20 Hz), and last up to 30 s (Fig. I A). Stimulation of the mossy fiber pathway (50/~A) produces an identical response (Fig. 1B); burst duration varies with stimulus intensity (not shown). Bursts are terminated (or followed) by a long hyperpolarizing event that appears similar to afterhyperpolarizations (AHPs) described in acute hippocampal slices [6]. To determine whether IPSPs were present in spontaneously bursting cultures, but obscured by the depolarizing events, we blocked burst activity by bath applying 10 mM KYN, a competitive N-methyI-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonist. In 4 cultures, KYN quickly blocked spontaneous burst activity and significantly reduced the occurrence of all spontaneous PSPs within 3 10 rain. With KYN application, mossy fiber stimulation also produced little or no response (Fig. I C). The loss of EPSPs is attributable to the direct blockade of excitatory amino acid receptors by KYN. IPSPs were observed in two cultures during wash-in of KYN, during the brief period following elimination of burst activity but prior to complete loss of synaptic drive. Diminution of IPSPs is apparently due to the decrease of excitatory drive onto inhibitory interneurons. Spontaneous inhibitory synoptic events in CA3 pyramidal cells were seen consistently during washout of KYN, with gradual recovery of synaptic activity over the course of 60 min. Mossy fiber stimulation (100/~A) produced a hyperpolarizing PSP 2 rain after the start of washout (Fig. 2A). This evoked IPSP was initially brief, with a duration of about 80 ms. The return of inhibitory PSPs preceded EPSPs in all cultures treated with KYN. This observation confirms previous findings in acute slices that a very low level of EPSP drive is needed to activate inhibitory interneurons a level below that detectable in recordings from pyramidal cells [8]. One characteristic feature of normal (i.e. non-bursting) hippocampal slice cultures is that they exhibit spontaneous slow IPSPs, usually as part of a triphasic PSP [5, 10]. These triphasic PSPs are similar to those seen following mossy fiber stimulation

A ~

B , , . ........... ~~

C ~

Fig. 2. Fast and slow IPSPs appear during washout of KYN. A: 2 min after starting washout of KYN, a 100/~A stimulus to the mossy fibers elicits a small IPSP. B: between 6 and 7 min, spontaneous triphasic PSPs begin to appear; a depolarizing PSP is followed sequentially by a fast IPSP (solid arrow in this and subsequent figs.) and a slow IPSP (open arrow in this and subsequent figs.). C: at 9 rain, the initial EPSP has doubled its amplitude and duration from 6 mV/40 ms (2B) to 12 mV/80 ms, and elicits multiple action potentials. Additional EPSPs appear during the IPSP. Calibrations= 10 mV, 100 ms (A); 10 mV, t00 ms (B); 10 mV, 200 ms (C1

79

iI



I' C

__1 Fig. 3. ExcitatoryPSPs and PDS-likebursts return with washout of KYN. Between 14 and 20 min following the start of washout, multiple EPSPs occur within the spontaneous IPSP (A); alternatively,the IPSP is completelyobscured by small PDS-like bursts (B). The IPSP "disappears' as burst duration increases. Fifty to 60 min after start of washout, burst duration has returned to control levelsand no IPSP is evident (C). Calibrations= 10 mV, 500 ms (A); 10 mV, 2 s (B); 10 mV, 5 s (C).

in acute hippocampal slices (EPSP followed sequentially by fast and slow IPSPs) [1]. After 6-7 min of K Y N washout, spontaneous triphasic PSPs appeared in formerly bursting cultures (Fig. 2B). Over the next 2-3 min, the initial EPSP increased in amplitude and duration, and elicited multiple action potentials; additional EPSPs appeared during the slow IPSP (Fig. 2C). None of these depolarizing events were eliminated when negative current was applied through the recording electrode to hyperpolarize the membrane potential, confirming their synaptic nature. From 14 to 20 min following onset of washout, the IPSP was obscured by an increasing number of superimposed EPSPs (Fig. 3A), or by a growing PDS-like burst (Fig. 3B). The total burst discharge increased in magnitude until it matched that seen under control conditions (Fig. 3C). These results suggest that an abnormally high level of excitatory synaptic drive is responsible for the spontaneous burst activity observed in a small percent of the slice cultures. Although IPSPs were not observed initially in these cultures, both fast and slow IPSPs were clearly visible when excitatory synaptic 'gain' was reduced using the competitive glutamate receptor antagonist, kynurenic acid. During K Y N washout, increasingly stronger and more frequent EPSPs appeared to overwhelm the apparently normal inhibitory drive, resulting in burst activity. A morphological basis for the high level of synaptic drive in slice cultures is suggested by recent anatomical findings. Electron microscopic analysis reveals that cultured neurons receive comparable numbers of synapses to normal hippocampus [4]. Given the loss of afferent projections to the hippocampus in the culture preparation, a significant number of these synaptic contacts must be from neighboring pyramidal cells. The increase in the number of recurrent collaterals among pyramidal cells could account for the burst activity [15, 16] (and for the high frequency of spontaneous PSPs observed in non-bursting slice cultures). Most studies investigating the mechanisms responsible for the production of epiieptiform activity have focused on the loss or reduction of inhibitory synaptic drive. In part, this bias has been due to the relative ease of blocking inhibition, as opposed to increasing excitation, in experimental preparations. Hippocampal slice cultures

80 have provided a n ideal experimental system for observing the role of excitatory synaptic drive in the f o r m a t i o n of epileptiform activity since they m a y show exaggerated levels o f excitatory synaptic drive. The data collected in this study of slice cultures supports the view that the m a i n t e n a n c e of n o r m a l , n o n - b u r s t i n g , physiology depends o n a balance of excitatory a n d i n h i b i t o r y synaptic drive, n o t simply o n the integrity of the i n h i b i t o r y c o m p o n e n t . The data presented here, recent results describing the complex m o d u l a t i o n of N M D A receptors [7, 12, 17], a n d i n f o r m a t i o n a b o u t the capacity of m a n y synaptic systems for p o t e n t i a t i o n [1 1] a n d sprouting [9], highlight the i m p o r t a n t role o f excitatory synaptic drive in the etiology of epileptiform activity. We t h a n k Professor Beat H. G~ihwiler for his help with the slice culture technique a n d Paul J. Foecke a n d Paul R. Schwartz for their help in preparing this manuscript. This work was supported by N I H , N I N C D S G r a n t NS 15317 (P.A.S.) a n d a g r a n t from the Epilepsy F o u n d a t i o n of A m e r i c a (A.T.M.). 1 Alger, B.E. and Nicoll, R.A., Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro, J. Physiol. (Lond.), 328 (1982) 105--123. 2 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. 3 Dingledine, R. and Gjerstad, L., Reduced inhibition during epileptiform activity in the in vitro hippocampal slice, J. Physiol. (Lond.), 305 (1980) 297-313. 4 Frotscher, M. and G/ihwiler, B.H., Synaptic organization of the intracellularly stained CA3 pyramidal neurons in slicecultures of rat hippocampus, Neuroscience, 24 (1988) 541 551. 5 Gfihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 6 Hotson, J.R. and Prince, D.A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons, J. Neurophysiol., 43 (1980) 409-419. 7 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature (Lond.), 325 (1987) 529- 531. 8 Lacaille, J.-C., Mueller, A.L., Kunkel, D.D. and Schwartzkroin, P.A., Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology, J. Neurosci., 7 (1987) 1979 1993. 9 Laurberg, S. and Zimmer, J., Lesion-induced sprouting of hippocampal mossy fiber collaterals to the fascia dentata in developing and adult rats, J. Comp. Neurol., 200 (1981) 433-459. 10 Malouf, A.T., Robbins, C.A. and Schwartzkroin, P.A., Phaclofen inhibition of the slow IPSP in hippocampal slice cultures: A possible role for the GABAB-mediatedIPSP, Neuroscience, in press. l 1 Nicoll,R.A., Kauer, J.A. and Malenka, R.C., The current excitement in long-term potentiation, Neuron, 1 (1988)97-103. 12 Nowak, C., Bregestovski, P., Ascher, P., Herbert, A. and Prochiantz, A., Magnesium gates glutamateactivated channels in mouse central neurons, Nature (Lond.), 307 (1984) 462-465. 13 Prince, D.A., Neurophysiology of epilepsy, Annu. Rev. Neurosci., 1 (1978) 395~415. 14 Schwartzkroin, P.A. and Prince, D.A., Changes in excitatory and inhibitory synaptic potentials leading to epileptogenicactivity, Brain Res., 183 (1980) 61-76. 15 Traub, R.D., Miles, R. and Wong, R.K.S., Models of synchronizedhippocampal bursts in the presence of inhibition. I. Single population events. J. Neurophysiol. 58 (1987) 739-751. 16 Traub, R.D., Miles, R., Wong, R.K.S., Schulman, L.S. and Schneiderman, J.H., Models of synchronized hippocampal bursts in the presence of inhibition. II. Ongoing spontaneous population events, J. Neurophysiol., 58 (1987) 752 764. 17 Westbrook, G.L. and Mayer, M.L., Micromolar concentrations of Zn ++ antagonize NMDA and GABA responses in hippocampal neurons, Nature (Lond.), 328 (1987) 640-643.

Epileptiform activity in hippocampal slice cultures with normal inhibitory synaptic drive.

The synaptic events responsible for epileptiform burst discharge are often difficult to define. Blockade of inhibition has been used to produce epilep...
316KB Sizes 0 Downloads 0 Views