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Epileptogenesis in neocortical slices
Brain Research, 127 (1977) 191-196
191
© Elsevier/North-Holland Biomedical Press
Epileptogenesis in neocortical slices
KENNETH R. COURTNEY* and DAVID A. PRINCE** Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.)
(Accepted February 3rd, 1977)
A variety of "simple" model systems have been used to study epileptogenesis because of their advantages over the mammalian brain in experiments where detailed analysis of cellular events is required 2-4,6,11. The disadvantages of this approach are that the investigator must deal with a preparation which may be quite different anatomically, physiologically, and pharmacologically from mammalian cortex (the site involved in most studies of epilepsy in vivo, including those in man). Also, the abnormal electroencephalographic discharges, which are the hallmark of epileptogenesis in the mammalian brain, are absent in such preparations making it difficult to determine whether changes in neuronal activities detected after an experimental maneuver such as application of a convulsant drug are even related to epilepsy. An example of this problem is the observation that both convulsant (strychnine) and anticonvulsant (phenobarbital) drugs applied to a simple preparation by the same investigator16,17 and in the same laboratory may induce cellular depolarization shifts (DSs) in many ways similar to those occurring in neurons of acute epileptogenic foci of the mammalian brainS,V-9,15. Some of these problems have been eliminated by use of the in vitro hippocampal slice preparation to study epileptogenesis12,19,z0,2L Spontaneous and evoked epileptiform field potentials and neuronal DSs may be elicited when the slice is exposed to low concentrations of a convulsant agent such as penicillin 19,2°. At the same time many of the advantages of an in vitro system (e.g., absence of pulsations and ability to manipulate ionic composition of extracellular space) are retained. In the experiments reported here, we have extended the in vitro slice technique to the neocortex to determine whether such a preparation might be useful in the study of epileptogenesis. We were particularly interested in obtaining data for comparison with that available from experiments in neocortical epileptogenic foci in vivo and hippocampal slices in vitro. Experiments were done on neocortical slices obtained from 17 guinea pigs. Most * Present address: Department of Anesthesia, Stanford University School of Medicine, Stanford, Calif. 94305, and Stanford Research Institute, Menlo Park, Calif. 94025, U.S.A. ** Send reprint requests to: David A. Prince, Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305, U.S.A.
192 aspects of slice preparation were similar to those detailed for hippocampus is. Transverse sections were made from the neocortical region corresponding approximately to sensorimotor cortex of the guinea pig. One hemisphere was cut transversely 2-4 mm from its rostral pole. The ventral surface of the amputated piece was trimmed to reduce slice size for easier handling and to provide a flat surface on which the tissue rested during slicing. Transverse slices (350-400/~m thick) were cut, placed in an in vitro chamber where they were maintained during the recording session as previously described 1s,21. They were supported on a mesh platform and perfused with an oxygenated solution containing 124 m M NaCl, 5 m M KC1, 2 m M CaC12, 2 m M MgSO4, 1.25 m M NaH~PO4, 26 m M NaHCO3, and l0 m M glucose at pH 7.4 at a rate of 1.5 ml/min. Slices remained in good condition for several hours as judged by the quality of intracellular recordings. Potassium acetate-filled micropipettes with resistances of 30-50 M~) at 60 Hz were used to obtain neuronal recordings. Penetrations were made in cortex on the dorsal and lateral aspects of the slice. Bipolar stimuli were delivered to the subcortical
1
2
3
4 T2o mv
!
I
!
-J
I,
l
H
50 msec Fig. 1. Representative records obtained from normal neocortical slices (A and B) and those with added sodium penicillin (C). AI: antidromic spike and subsequent depolarizing-hyperpolarizing sequence evoked by white matter stimulation. As: same stimulus evokes isolated EPSP. A3 and A4: records from another cell in which stimuli evoke an EPSP which sometimes triggers a spike (Aa). Note faster sweep in A3-4. B: repetitive firing evoked by 200 msec intracellular current pulses of increasing intensity (0.1-1 hA) in B1 through B4. C1 : extracellular multipeaked potential elicited by white matter stimulation in slice exposed to 1000 U/ml penicillin. C~ and C~: alternation of field potential responses in another preparation exposed to 2200 U/ml penicillin. Every other stimulus (C3) evokes a long latency epileptiform event with multiple peaks. C4: intracellular record showing DS and post-DS inhibitory period of about 300 msec in another cell exposed to 2200 U/ml penicillin. Bracketed time calibrations represent 50 msec in As for A1-2; in A4 for A3-4 ; in C1 for C1-3. Solid bars in B mark 200 msec intracellular depolarizing current pulses. Voltage calibration in A4:20 mV for lines A and B; 4 mV for C1-C3; and 40 mV for C4. Spikes retouched for clarity. Dots in Figs. l and 2: white matter stimuli.
193 white matter, and, on occasion, to the "pial" surface, using pairs of 75/zm insulated stainless steel wires glued side by side and cut flush at their tips. Slices were exposed to penicillin by substituting a solution containing from 100 to 3300 U/ml of Na Penicillin G (0.17-5.6 m M ) for the normal perfusate. These solutions were not significantly different in p H from the control solution, and changes in osmolality for penicillin concentrations which consistently yielded significant alterations in neuronal activities were insignificant. A total of 72 neurons were sampled in 17 experiments including 30 intracellular recordings in which neurons generated spikes with amplitudes of 40-60 mV for periods of up to 30 min (Fig. 1A, B). Stable intracellular records were more difficult to obtain in the neocortex than in other experiments in which identical techniques were used to penetrate CA1 hippocampal neurons. This may be a sampling difference since cell bodies in the latter preparation are packed tightly in a visible layer which allows precise positioning of the microelectrode. Most successful penetrations were made about 800-1000 # m below the dorsal or lateral cortical surface of the slice. In contrast to neurons in the hippocampal slice, little spontaneous activity was present in neocortical cells. Bipolar stimulation of white matter often evoked both antidromic and orthodromic activity. For example, in Fig. 1A1 stimuli elicit an antidromic spike followed by a brief depolarizing-hyperpolarizing sequence in a neuron not exposed to penicillin. Excitatory and inhibitory synaptic potentials (EPSPs and IPSPs) were the usual orthodromic responses (Fig. 1A2-4) and few isolated EPSPs or IPSPs were seen
1 A;
2
.....
3
4 ;..
H
I---I
50 msec
Fig. 2. All records from slices exposed to 2200 U/ml sodium penicillin. A-B: intracellular recording from depolarized unit. In A every other stimulus at 1/sec evokes DS at latency of ca. 60 msec. In B increase in stimulus strength shortens latency to 40 msec and results in regular DS triggering at same frequency. C: short latency EPSP evoked by each stimulus, and longer latency DS by every second stimulus at 0.8/sec. Note faster sweeps in C3 4. Calibrations in C4 for all traces except slower time base in C1-2 shown in C1.
194 under these experimental conditions. In good penetrations depolarizing current pulses elicited expected depolarizations and repetitive spike generation (Fig. l B). Application of solutions containing penicillin to the slice produced changes in field potentials and cellular activities which were marked at concentrations of 1000 U/ml (1.7 mM) but definite when as little as 300 U/ml (0.5 mM) of the drug were present. At the higher concentrations of penicillin, stimuli evoked events which appeared identical to the DSs previously reported in neurons of acute loci in the cat s,9,15. These are illustrated in 3 neurons in Fig. 1C4 and in Fig. 2. In each, a large DS (amplitude approx. 20-25 mV) lasting about 50 msec is evoked by the stimulus. Such potentials were usually followed by a hyperpolarization lasting 300 msec or more 9,14. These hyperpolarizations were 2-5 × the amplitude of IPSPs normally recorded in slice cells and much longer in duration. The DS itself varied from cell to cell depending on the condition of the unit. In injured units with low amplitude spikes there might be complete spike inactivation at the peak of the DS (Fig. 2C4). DSs never occurred without stimulation in this preparation in contrast to the situation in the hippocampal slice where they occur spontaneously every 2-5 sec19,2°. They were never elicited by direct (intracellular) depolarizing pulses. Concurrent to these cellular changes, multiphasic complex field potentials were seen when the electrode was in an extracellular position (Fig. 1C1 and C3). Stimuli were not always effective in evoking DSs and at times there was an alternation of response pattern similar to that seen in neocorticai foci in vivo la. This is illustrated in the extracellular recording of Fig. 1C2 a. Here every stimulus elicited a short latency response but only every other stimulus evoked a long latency (ca. 50 msec) epileptiform field potential (Fig. l C3). Alternation of DS triggering in a deteriorated unit is also seen in Fig. 2A, and in another neuron in Fig. 2C. These alternating response patterns were sensitive to both stimulus rate and intensity. Triggered DSs could be reliably evoked when strong stimuli were delivered at 1-2/sec but at higher rates (5/sec) stimuli would fail to evoke DSs at all. Increases in stimulus intensity (Fig. 2B compared to 2A) would shorten the latency for the evoked DS and increase the likelihood that a DS could be triggered. DSs usually occurred 20-50 msec after stimulation of underlying white matter but latencies as long as 100 msec were observed. These types of interactions between stimulus parameters and the latency and frequency of DS responses are similar to those observed in vivo (e.g. ref. 13 and others). Our data indicate that events which in many respects resemble those seen in acute epileptiform loci of the intact brain may be generated in the penicillin-treated neocortical slice. Triggered epileptiform field potentials and large cellular depolarizations identical in appearance to those termed depolarization shifts in intact cortex are regularly produced. Other features of cellular activities in acute epileptiform loci in vivo are present in the slice including (l) a clear separation between short latency EPSPs and longer latency DSs; (2) alternation of responses in which DSs are triggered with those in which EPSPs alone are evoked; (3) relationships between stimulus intensity, DS latency, and the frequency with which DSs are evoked; (4) suppression of DS triggering at high stimulus frequencies; and (5) marked hyperpolarizations following each DS 14.
195 These features are also present in penicillin-treated h i p p o c a m p a l slices. H o w ever, s p o n t a n e o u s e p i l e p t i f o r m events, similar to those f o u n d in the h i p p o c a m p a l slice 19,20 were n o t r e c o r d e d in these experiments a n d we are n o t certain whether this reflects intrinsic differences in synaptic circuitry a n d o r g a n i z a t i o n o f these two structures o r o t h e r variables. F o r example, it is possible t h a t the slice m a d e transversely across h i p p o c a m p u s preserves sufficient connectivity 1 for s p o n t a n e o u s epileptogenesis, while a slice m a d e r a n d o m l y t h r o u g h n e o c o r t e x does not. A l t e r n a t i v e e x p l a n a t i o n s such as different degrees o f t r a u m a in the two p r e p a r a t i o n s are certainly possible. To date we have n o t r e c o r d e d electrical " i c t a l " events 10 in either h i p p o c a m p a l or neocortical slices, a l t h o u g h these occur readily in neocortical islands t r e a t e d with penicillin (Prince, u n p u b l i s h e d observations). One possible e x p l a n a t i o n for this is that a larger n e u r o n a l aggregate is required to generate electrographic ictal episodes t h a n exists in the slice. These p r e l i m i n a r y d a t a indicate that certain aspects o f basic cellular m e c h a n i s m s underlying neocortical epileptogenesis m a y be a m e n a b l e to analysis in vitro. These experiments were s u p p o r t e d by the M o r r i s Research F u n d a n d by R e s e a r c h G r a n t N S 06477 f r o m the N I N C D S , N I H (D.A.P.). W e t h a n k Professor Philip S c h w a r t z k r o i n for suggestions regarding the slice p r e p a r a t i o n a n d G e r a l d i n e Chase for secretarial assistance.
1 Andersen, P., Organization of hippocampal neurons and their interconnections. In R. L. lsaacson and K. H. Pribram (Eds.), The Hippocampus. 1Iol. 1. Structure and Development, Plenum Press, New York ,1975, pp. 155-175. 2 Ayala, G. F., Lin, S. and Vasconetto, C., Penicillin as epileptogenic agent: its effect on an isolated neuron, Science, 167 (1970) 1257-1260. 3 Ayala, G. F., Spencer, W. A. and Gummit, R. J., Penicillin as an epileptogenic agent: effect on an isolated synapse, Science, 171 (1971) 915-917. 4 Davidoff, R., Penicillin and presynaptic inhibition in the amphibian spinal cord, Brain Research, 36 (1972) 218-222. 5 Dichter, M. and Spencer, W. A., Penicillin-induced discharges from the cat hippocampus. I. Characteristics and topographical features, J. NeurophysioL, 32 (1969) 649-662. 6 Futamachi, K. J. and Prince, D. A., Effect of penicillin on an excitatory synapse, Brain Research, 100 (1975) 589-597. 7 Li, C. L., Cortical intracellular potentials and their responses to strychnine, J. Neurophysiok, 22 (1959) 436~50. 8 Matsumoto, H., Intracellular events during the activation of cortical epileptiform discharges, Electroenceph. clin. NeurophysioL, 17 (1964) 294-307. 9 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964) 286-304. 10 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestations, Exp. NeuroL, 9 (1964) 305-326. I 1 Meyer, H. and Prince, D. A., Convulsant actions of penicillin : effects on inhibitory mechanisms, Brain Research, 53 (1973) 477-482. 12 Ogata, N., Mechanism of the stereotyped high-frequency burst in hippocampal neurons in vitro, Brain Research, 103 (1976) 386-388. 13 Prince• D. A.• M•di•cati•n •f f•caI c•rtical epilept•genic discharge by a•erent in•uences• Epi•epsia• (Amst.), 7 (1966) 181-201. 14 Prince, D. A., Inhibition in "epileptic" neurons, Exp. Neurol., 21 (1968) 307-321.
196 15 16 17 18 19 20 21 22
Prince, D. A., The depolarization shift in "epileptic" neurons, Exp. Neurol., 21 (1968)467-485. Pritchard, J. W., Effect of strychnine on the leech Retzin's cell, Exp. Neurol., 32 (1971) 275-286. Pritchard, J. W., Effect of phenobarbital on a leech neuron, Neuropharmacology, 11 (1972) 585-590. Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436. Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced activity in hippocampal slices maintained in vitro, Neurosci. Abstr., 2 (1976) 266. Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced epileptiform activity in the hippocampal in vitro preparation, Ann. NeuroL, in press. Skrede, K. R. and Westgaard, R. H., The transverse hippocampal slice: a well-defined cortical structure maintained in vitro, Brain Research, 35 (1971) 589-593. Yamamoto, C. and Kawai, N., Generation of the seizure discharge in thin sections from the guinea pig brain in chloride-free medium in vitro, Jap. J. Physiol., 18 (1968) 620-631.
191
© Elsevier/North-Holland Biomedical Press
Epileptogenesis in neocortical slices
KENNETH R. COURTNEY* and DAVID A. PRINCE** Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.)
(Accepted February 3rd, 1977)
A variety of "simple" model systems have been used to study epileptogenesis because of their advantages over the mammalian brain in experiments where detailed analysis of cellular events is required 2-4,6,11. The disadvantages of this approach are that the investigator must deal with a preparation which may be quite different anatomically, physiologically, and pharmacologically from mammalian cortex (the site involved in most studies of epilepsy in vivo, including those in man). Also, the abnormal electroencephalographic discharges, which are the hallmark of epileptogenesis in the mammalian brain, are absent in such preparations making it difficult to determine whether changes in neuronal activities detected after an experimental maneuver such as application of a convulsant drug are even related to epilepsy. An example of this problem is the observation that both convulsant (strychnine) and anticonvulsant (phenobarbital) drugs applied to a simple preparation by the same investigator16,17 and in the same laboratory may induce cellular depolarization shifts (DSs) in many ways similar to those occurring in neurons of acute epileptogenic foci of the mammalian brainS,V-9,15. Some of these problems have been eliminated by use of the in vitro hippocampal slice preparation to study epileptogenesis12,19,z0,2L Spontaneous and evoked epileptiform field potentials and neuronal DSs may be elicited when the slice is exposed to low concentrations of a convulsant agent such as penicillin 19,2°. At the same time many of the advantages of an in vitro system (e.g., absence of pulsations and ability to manipulate ionic composition of extracellular space) are retained. In the experiments reported here, we have extended the in vitro slice technique to the neocortex to determine whether such a preparation might be useful in the study of epileptogenesis. We were particularly interested in obtaining data for comparison with that available from experiments in neocortical epileptogenic foci in vivo and hippocampal slices in vitro. Experiments were done on neocortical slices obtained from 17 guinea pigs. Most * Present address: Department of Anesthesia, Stanford University School of Medicine, Stanford, Calif. 94305, and Stanford Research Institute, Menlo Park, Calif. 94025, U.S.A. ** Send reprint requests to: David A. Prince, Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305, U.S.A.
192 aspects of slice preparation were similar to those detailed for hippocampus is. Transverse sections were made from the neocortical region corresponding approximately to sensorimotor cortex of the guinea pig. One hemisphere was cut transversely 2-4 mm from its rostral pole. The ventral surface of the amputated piece was trimmed to reduce slice size for easier handling and to provide a flat surface on which the tissue rested during slicing. Transverse slices (350-400/~m thick) were cut, placed in an in vitro chamber where they were maintained during the recording session as previously described 1s,21. They were supported on a mesh platform and perfused with an oxygenated solution containing 124 m M NaCl, 5 m M KC1, 2 m M CaC12, 2 m M MgSO4, 1.25 m M NaH~PO4, 26 m M NaHCO3, and l0 m M glucose at pH 7.4 at a rate of 1.5 ml/min. Slices remained in good condition for several hours as judged by the quality of intracellular recordings. Potassium acetate-filled micropipettes with resistances of 30-50 M~) at 60 Hz were used to obtain neuronal recordings. Penetrations were made in cortex on the dorsal and lateral aspects of the slice. Bipolar stimuli were delivered to the subcortical
1
2
3
4 T2o mv
!
I
!
-J
I,
l
H
50 msec Fig. 1. Representative records obtained from normal neocortical slices (A and B) and those with added sodium penicillin (C). AI: antidromic spike and subsequent depolarizing-hyperpolarizing sequence evoked by white matter stimulation. As: same stimulus evokes isolated EPSP. A3 and A4: records from another cell in which stimuli evoke an EPSP which sometimes triggers a spike (Aa). Note faster sweep in A3-4. B: repetitive firing evoked by 200 msec intracellular current pulses of increasing intensity (0.1-1 hA) in B1 through B4. C1 : extracellular multipeaked potential elicited by white matter stimulation in slice exposed to 1000 U/ml penicillin. C~ and C~: alternation of field potential responses in another preparation exposed to 2200 U/ml penicillin. Every other stimulus (C3) evokes a long latency epileptiform event with multiple peaks. C4: intracellular record showing DS and post-DS inhibitory period of about 300 msec in another cell exposed to 2200 U/ml penicillin. Bracketed time calibrations represent 50 msec in As for A1-2; in A4 for A3-4 ; in C1 for C1-3. Solid bars in B mark 200 msec intracellular depolarizing current pulses. Voltage calibration in A4:20 mV for lines A and B; 4 mV for C1-C3; and 40 mV for C4. Spikes retouched for clarity. Dots in Figs. l and 2: white matter stimuli.
193 white matter, and, on occasion, to the "pial" surface, using pairs of 75/zm insulated stainless steel wires glued side by side and cut flush at their tips. Slices were exposed to penicillin by substituting a solution containing from 100 to 3300 U/ml of Na Penicillin G (0.17-5.6 m M ) for the normal perfusate. These solutions were not significantly different in p H from the control solution, and changes in osmolality for penicillin concentrations which consistently yielded significant alterations in neuronal activities were insignificant. A total of 72 neurons were sampled in 17 experiments including 30 intracellular recordings in which neurons generated spikes with amplitudes of 40-60 mV for periods of up to 30 min (Fig. 1A, B). Stable intracellular records were more difficult to obtain in the neocortex than in other experiments in which identical techniques were used to penetrate CA1 hippocampal neurons. This may be a sampling difference since cell bodies in the latter preparation are packed tightly in a visible layer which allows precise positioning of the microelectrode. Most successful penetrations were made about 800-1000 # m below the dorsal or lateral cortical surface of the slice. In contrast to neurons in the hippocampal slice, little spontaneous activity was present in neocortical cells. Bipolar stimulation of white matter often evoked both antidromic and orthodromic activity. For example, in Fig. 1A1 stimuli elicit an antidromic spike followed by a brief depolarizing-hyperpolarizing sequence in a neuron not exposed to penicillin. Excitatory and inhibitory synaptic potentials (EPSPs and IPSPs) were the usual orthodromic responses (Fig. 1A2-4) and few isolated EPSPs or IPSPs were seen
1 A;
2
.....
3
4 ;..
H
I---I
50 msec
Fig. 2. All records from slices exposed to 2200 U/ml sodium penicillin. A-B: intracellular recording from depolarized unit. In A every other stimulus at 1/sec evokes DS at latency of ca. 60 msec. In B increase in stimulus strength shortens latency to 40 msec and results in regular DS triggering at same frequency. C: short latency EPSP evoked by each stimulus, and longer latency DS by every second stimulus at 0.8/sec. Note faster sweeps in C3 4. Calibrations in C4 for all traces except slower time base in C1-2 shown in C1.
194 under these experimental conditions. In good penetrations depolarizing current pulses elicited expected depolarizations and repetitive spike generation (Fig. l B). Application of solutions containing penicillin to the slice produced changes in field potentials and cellular activities which were marked at concentrations of 1000 U/ml (1.7 mM) but definite when as little as 300 U/ml (0.5 mM) of the drug were present. At the higher concentrations of penicillin, stimuli evoked events which appeared identical to the DSs previously reported in neurons of acute loci in the cat s,9,15. These are illustrated in 3 neurons in Fig. 1C4 and in Fig. 2. In each, a large DS (amplitude approx. 20-25 mV) lasting about 50 msec is evoked by the stimulus. Such potentials were usually followed by a hyperpolarization lasting 300 msec or more 9,14. These hyperpolarizations were 2-5 × the amplitude of IPSPs normally recorded in slice cells and much longer in duration. The DS itself varied from cell to cell depending on the condition of the unit. In injured units with low amplitude spikes there might be complete spike inactivation at the peak of the DS (Fig. 2C4). DSs never occurred without stimulation in this preparation in contrast to the situation in the hippocampal slice where they occur spontaneously every 2-5 sec19,2°. They were never elicited by direct (intracellular) depolarizing pulses. Concurrent to these cellular changes, multiphasic complex field potentials were seen when the electrode was in an extracellular position (Fig. 1C1 and C3). Stimuli were not always effective in evoking DSs and at times there was an alternation of response pattern similar to that seen in neocorticai foci in vivo la. This is illustrated in the extracellular recording of Fig. 1C2 a. Here every stimulus elicited a short latency response but only every other stimulus evoked a long latency (ca. 50 msec) epileptiform field potential (Fig. l C3). Alternation of DS triggering in a deteriorated unit is also seen in Fig. 2A, and in another neuron in Fig. 2C. These alternating response patterns were sensitive to both stimulus rate and intensity. Triggered DSs could be reliably evoked when strong stimuli were delivered at 1-2/sec but at higher rates (5/sec) stimuli would fail to evoke DSs at all. Increases in stimulus intensity (Fig. 2B compared to 2A) would shorten the latency for the evoked DS and increase the likelihood that a DS could be triggered. DSs usually occurred 20-50 msec after stimulation of underlying white matter but latencies as long as 100 msec were observed. These types of interactions between stimulus parameters and the latency and frequency of DS responses are similar to those observed in vivo (e.g. ref. 13 and others). Our data indicate that events which in many respects resemble those seen in acute epileptiform loci of the intact brain may be generated in the penicillin-treated neocortical slice. Triggered epileptiform field potentials and large cellular depolarizations identical in appearance to those termed depolarization shifts in intact cortex are regularly produced. Other features of cellular activities in acute epileptiform loci in vivo are present in the slice including (l) a clear separation between short latency EPSPs and longer latency DSs; (2) alternation of responses in which DSs are triggered with those in which EPSPs alone are evoked; (3) relationships between stimulus intensity, DS latency, and the frequency with which DSs are evoked; (4) suppression of DS triggering at high stimulus frequencies; and (5) marked hyperpolarizations following each DS 14.
195 These features are also present in penicillin-treated h i p p o c a m p a l slices. H o w ever, s p o n t a n e o u s e p i l e p t i f o r m events, similar to those f o u n d in the h i p p o c a m p a l slice 19,20 were n o t r e c o r d e d in these experiments a n d we are n o t certain whether this reflects intrinsic differences in synaptic circuitry a n d o r g a n i z a t i o n o f these two structures o r o t h e r variables. F o r example, it is possible t h a t the slice m a d e transversely across h i p p o c a m p u s preserves sufficient connectivity 1 for s p o n t a n e o u s epileptogenesis, while a slice m a d e r a n d o m l y t h r o u g h n e o c o r t e x does not. A l t e r n a t i v e e x p l a n a t i o n s such as different degrees o f t r a u m a in the two p r e p a r a t i o n s are certainly possible. To date we have n o t r e c o r d e d electrical " i c t a l " events 10 in either h i p p o c a m p a l or neocortical slices, a l t h o u g h these occur readily in neocortical islands t r e a t e d with penicillin (Prince, u n p u b l i s h e d observations). One possible e x p l a n a t i o n for this is that a larger n e u r o n a l aggregate is required to generate electrographic ictal episodes t h a n exists in the slice. These p r e l i m i n a r y d a t a indicate that certain aspects o f basic cellular m e c h a n i s m s underlying neocortical epileptogenesis m a y be a m e n a b l e to analysis in vitro. These experiments were s u p p o r t e d by the M o r r i s Research F u n d a n d by R e s e a r c h G r a n t N S 06477 f r o m the N I N C D S , N I H (D.A.P.). W e t h a n k Professor Philip S c h w a r t z k r o i n for suggestions regarding the slice p r e p a r a t i o n a n d G e r a l d i n e Chase for secretarial assistance.
1 Andersen, P., Organization of hippocampal neurons and their interconnections. In R. L. lsaacson and K. H. Pribram (Eds.), The Hippocampus. 1Iol. 1. Structure and Development, Plenum Press, New York ,1975, pp. 155-175. 2 Ayala, G. F., Lin, S. and Vasconetto, C., Penicillin as epileptogenic agent: its effect on an isolated neuron, Science, 167 (1970) 1257-1260. 3 Ayala, G. F., Spencer, W. A. and Gummit, R. J., Penicillin as an epileptogenic agent: effect on an isolated synapse, Science, 171 (1971) 915-917. 4 Davidoff, R., Penicillin and presynaptic inhibition in the amphibian spinal cord, Brain Research, 36 (1972) 218-222. 5 Dichter, M. and Spencer, W. A., Penicillin-induced discharges from the cat hippocampus. I. Characteristics and topographical features, J. NeurophysioL, 32 (1969) 649-662. 6 Futamachi, K. J. and Prince, D. A., Effect of penicillin on an excitatory synapse, Brain Research, 100 (1975) 589-597. 7 Li, C. L., Cortical intracellular potentials and their responses to strychnine, J. Neurophysiok, 22 (1959) 436~50. 8 Matsumoto, H., Intracellular events during the activation of cortical epileptiform discharges, Electroenceph. clin. NeurophysioL, 17 (1964) 294-307. 9 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964) 286-304. 10 Matsumoto, H. and Ajmone Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestations, Exp. NeuroL, 9 (1964) 305-326. I 1 Meyer, H. and Prince, D. A., Convulsant actions of penicillin : effects on inhibitory mechanisms, Brain Research, 53 (1973) 477-482. 12 Ogata, N., Mechanism of the stereotyped high-frequency burst in hippocampal neurons in vitro, Brain Research, 103 (1976) 386-388. 13 Prince• D. A.• M•di•cati•n •f f•caI c•rtical epilept•genic discharge by a•erent in•uences• Epi•epsia• (Amst.), 7 (1966) 181-201. 14 Prince, D. A., Inhibition in "epileptic" neurons, Exp. Neurol., 21 (1968) 307-321.
196 15 16 17 18 19 20 21 22
Prince, D. A., The depolarization shift in "epileptic" neurons, Exp. Neurol., 21 (1968)467-485. Pritchard, J. W., Effect of strychnine on the leech Retzin's cell, Exp. Neurol., 32 (1971) 275-286. Pritchard, J. W., Effect of phenobarbital on a leech neuron, Neuropharmacology, 11 (1972) 585-590. Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436. Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced activity in hippocampal slices maintained in vitro, Neurosci. Abstr., 2 (1976) 266. Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced epileptiform activity in the hippocampal in vitro preparation, Ann. NeuroL, in press. Skrede, K. R. and Westgaard, R. H., The transverse hippocampal slice: a well-defined cortical structure maintained in vitro, Brain Research, 35 (1971) 589-593. Yamamoto, C. and Kawai, N., Generation of the seizure discharge in thin sections from the guinea pig brain in chloride-free medium in vitro, Jap. J. Physiol., 18 (1968) 620-631.