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The effects of phenytoin on the penicillin-induced spike focus
90
Electroencephalography and Clinical Neurophysiology, 1980, 48:90--97
© Elsevier/North-Holland Scientific Publishers, Ltd.
THE E F F E C T S OF P H E N Y T O I N O N T H E PENICILLIN-INDUCED SPIKE FOCUS
L. BUSTAMANTE i H. LUEDERS, C, PIPPENGER and E.S. GOLDENSOHN Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 (U.S.A.)
(Accepted for publication : April 26, 1979)
In the 4 decades since its introduction by Merritt and Putnam in 1938, phenytoin (diphenylhydantoin, Dilantin) has been widely used as an effective c o m p o u n d for the treatment of focal epilepsy. The mechanisms of action of phenytoin are only partially understood and its effects on experimental epileptogenic spike foci have not been clearly defined. In experimental alumina cream spike foci, phenytoin given intravenously in doses equivalent to therapeutic range in humans produced no significant changes according to Rand et al. {1966). They noticed that spike frequency increased in some cases but did not interpret this finding other than to consider it paradoxical. In penicillin-induced spike foci, Louis et al. (1968) reported that phenytoin produced either no change or a slight decrease in spike frequency. In spite of the persistence of cortical epileptiform spiking at a rather constant rate in their animals, phenytoin administration resulted in a total cessation of actual convulsions. Julien and Halpern {1972) observed a significant decrease in the frequency and duration of after-discharges following the injection of phenytoin, but Edmonds et al. (1974) found no significant change of either spike frequency or the number of after-discharges following the injection of phenytoin. The study reported here analz Present address: Departamento de Fisiologia, Facultad de Medicina, Universidad de Zulia, Apartado 15158, Maracaibo, Venezuela.
yzes quantitatively the effects of phenytoin on epileptogenesis using the penicillin-induced spike focus model described in the preceding paper (Lueders et al. 1980).
M e t h o d and material
The methods of creating the spike foci and the system of analysis are identical to those described in more detail in the preceding paper (Lueders et al. 1980). Penicillin concentrations of 20,000 U/ml (weak foci) were used in 6 cats and concentrations of 100,000 U/ml {strong foci) in additional 6. First injections of phenytoin were given approximately 90 min after the appearance of the first spike exceeding 500 pV which was a time when spiking was relatively stable. Additional injections were given at 30 and 60 min intervals. Each phenytoin injection contained 20--30 mg/kg b o d y weight. Blood samples for serum levels of phenytoin were drawn every 15--30 min and analyzed either with gas liquid chromatography or with the enzyme multiple immunoassay technique (EMIT) (Pippenger et al. 1975). Graphs were made for each experiment and the results were compared to the control series described previously (Lueders et al. 1980). The graphs include: (1) spike frequency versus time; (2) spike duration versus spike frequency as a function of time; (3) spike amplitude versus spike frequency as a
PHENYTOIN
ON PENICILLIN-INDUCED
SPIKES
function of time; and (4) amplitude of prepositivity versus time. In these experiments the point in time immediately before the first injection of phenytoin is considered equivalent to the 90 min reference point in the control experiments and 20 min after each of the 3 injections of phenytoin is considered equivalent to the 1 1 0 , 1 5 5 and 200 min reference points of the control and are so expressed in the graphs. Blood pressure, heart rate and rectal temperature were monitored continuously in all experiments. Phenytoin injections were performed slowly over 3--6 min to avoid falls in blood pressure and bradycardia. Some early experiments were discarded following the rapid injection of phenytoin and all experiments in which the blood pressure decreased below 100/50 mm Hg were discarded.
91
Results
The effects of phenytoin on spike frequency in weak and strong penicillin-induced foci are shown in Figs. 1 and 2. After each of the first 2 injections, marked increases of spike frequency are noted, b u t after the third injection, in some instances (at equivalent toxic human serum levels), the spike is totally suppressed (Fig. 1). More often the third injection produced either no change (Fig. 2) or a further increase in spike frequency. The mean serum level of phenytoin after the first injection was 20.3 #g/ml, after the second was 29.3 pg/ml, and after the third injection was 42.9 pg/ml respectively. Increase of spike frequency after the first 2 injections occurred in all experiments (11--206% at 20 min after the first injection), in clear contrast to the control
EFFECT OF DPH ON FREQUENCY OF PRIMARY SPIKES Using 20,000 Units PNC/ml DPH 20 mg/Kg
DPH 2Omg/Kg
11
50 45
11
OPH 20 mg/Kg
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z 35 PNC uJ Q. m w ~ m
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. . 40
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. . . . . . . . . . . . //-v-//, 120 140 160 180 200 220 240 290 350 MINUTES Fig. 1. E f f e c t o f phenytoin on spike frequency in a 2 0 , 0 0 0 U / m ] penicillin-induced spike focus. Dotted lines indicate phenytoin serum concentration. Systolic and diastolic b l o o d pressure are shown in the lower traces. 80
.
. I00
.
92
L. BUSTAMANTE ET AL.
EFFECT OF DPH ON FREQUENCY OF BURSTS Using I00,000 Units PNC/ml Solvent 1.4 C.C.
,,35I-~30-
Solvent 1.4~c.
Solvent 1.4 c.c.
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40
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80
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120
140
160
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200
220
MINUTES
Fig. 2. Effect of phenytoin on spike frequency in a 100,000 U/ml penicillin-induced spike focus is shown in the upper part of the graph. Dotted lines indicate phenytoin serum concentrations. In the lower graph the effect on after-discharges is shown. After-discharges were abolished by phenytoin. After-discharges are defined as prolonged bursts of sequences of multiple secondary spikes and alternating primary and secondary spikes which have a cumulative duration exceeding 8 sec.
experiments in which the spike frequency decreases with time. Total abolition of the spike occurred in only 2 experiments. In both cases it was with a weak focus (20,000 U/ml) after the third injection and at blood levels of phenytoin toxic for humans. After-discharges Complete abolition of after-discharges occurred in all experiments following the first injection of phenytoin at serum levels between 15 and 26 pg/ml (Fig. 2). A doserelated progressive diminution in the number of secondary spikes was also noted following the second and third injections of phenytoin. Spike duration versus spike frequency as a function o f time Spike duration versus spike frequency as a
function of time for all controls and all phenytoin experiments excepting those which produced after
PHENYTOIN ON PENICILLIN-INDUCED SPIKES i1.--I O 13 H 0 0
50 40 50
z 0 :D a ft. (/)
93
CONTROL (n=4), 20,000 CONTROL (n = 2), I00,000 PHENYTOIN (n = 5), 20,000 PHENYTOIN (n=2), I00,000
Unit= PNC/cJ~ SPIKE FOCI Units PNC/c.c. SPIKE FOCI Units PNC/c.c. SPIKE FOCI Units PNC/c.c. SPIKE FOCI
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Fig. 3. Spike duration versus spike frequency as a function of time graph of 20,000 and I00,000 U/ml spike foci (controls and phenytoin experiments) which did not produce after-discharges.For each reference point in time the average of each of the 4 groups is represented. The spike duration and spike frequency immediately before the firstinjection of phenytoin (or at 90 rain for the control studies) are taken as reference 0 % of change). The change of spike duration and spike frequency (expressed in percentage) 20 rain after 3 injections of phenytoin (or 110,155 and 200 min in the control studies) determines the position of points number 1, 2 and 3).
spike later completely disappeared (at 46 and 27 min). These 2 experiments were the only ones in which the spike frequency decreased below the control level.
Spike amplitude The effect of phenytoin on spike amplitude was negligible in the strong (100,000 U/ml) penicillin spike foci. In the weak (20,000 U/ml) penicillin spike foci, the amplitude had a tendency to decrease progressively with the 3 injections of phenytoin.
Amplitude of prepositivity In 4 of the 12 animals prepositivity exceeded 10% of the total spike amplitude at time zero. In all 4 cases, phenytoin produced marked increases in the amplitudes of prepos-
itivities (Fig. 4). In the 2 instances in which spiking was abolished after the third injection of phenytoin, the initially predominantly negative spikes were transformed into completely positive spikes just before disappearing (Fig. 5). In the other 2 cases the.amplitudes of the prepositivity were increased by 76% and 87%. In 7 of the 8 animals which showed either no prepositivity or one of less than 10% of the total spike amplitude, phenytoin produced no change (Fig. 4). In 6 animals which were otherwise discarded from this study for a variety of reasons, the spiking disappeared during periods of observation before any phenytoin was administered. No significant increase in prepositivity occurred prior to the cessation of spiking.
94
L. BUSTAMANTE ET AL. Discussion
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REFERENCE POINTS IN TIME
Fig. 4. Amplitude of prepositivity as a function of time graph of I2 phenytoin experiments. See Method for definition of reference point in time number, 0, 1, 2 and 3.
B~
-
-
z
~
-
~
~
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Fig. 5. Typical spike (20,000 U/ml penicillin) immediately before (A) injection of phenytoin and 35 rain after (B) the third injection of phenytoin. The corresponding spike frequency versus time graph of this experiment is shown in Fig. 1.
These experiments demonstrate that the penicillin spike focus model modified for the purpose of testing anticonvulsant actions is (see Lueders et al. 1980), as predicted theoretically, extremely responsive to the action of phenytoin. Phenytoin modified all spike parameters measured and consistently suppressed after-discharges. Phenytoin abolished after-discharges consistently at blood levels within the equivalent of the therapeutic range for humans. It was found that higher phenytoin blood levels were needed to abolish secondary spikes than to abolish after-discharges. If we accept the hypothesis that bursts of after-discharges are dependent on activation of long polysynaptic pathways which continuously reactivate the primary spikes, then we can conclude that phenytoin is particularly effective in depressing these polysynaptic pathways. The abolition of bursts of after
P H E N Y T O I N ON P E N I C I L L I N - I N D U C E D S P I K E S
found that the frequency of primary spikes was inversely related to the degree of epileptogenicity of the spike focus. The present results show that phenytoin consistently increases the frequencies of primary spikes. This increase of primary spike frequency by phenytoin is consistent with its anticonvulsant effect. It is worthwhile to note in this context that Edmonds and Stark (1974), who failed to notice a significant change in spike frequency with phenytoin, reported a significant increase with another anticonvulsant, SC-13504 (Edmonds et al. 1974). The increase of spike frequency implies that phenytoin decreases the amount or duration of post-spike inhibition. If we assume that the amount of post-spike inhibition is directly related to the intensity of the spike and that the duration of the inhibition depends on prolonged polysynaptic pathways, the increase in spike frequency can be ascribed to the known action of phenytoin on prolonged polysynaptic pathways. The fact that phenytoin affects polysynaptic pathways, whether or not the ultimate effect is inhibitory or excitatory, has been demonstrated at the spinal level (Tuttle and Preston 1963). Another effect of phenytoin was to decrease primary spike duration. If spike duration is an expression of prolonged polysynaptic pathways, the effect of phenytoin on decreasing spike duration can be explained by the same mechanisms mentioned above explaining the frequency change. Thus, the effect of phenytoin on all 3 parameters discussed (after-discharges, spike frequency and spike duration) can be explained by a single mechanism of prolonged polysynaptic pathways which are blocked by phenytoin. Each of the parameters, however, is modified by a number of different influences as is evident by the fact that each of the spike parameters can change independently during control recordings. Phenytoin affected spike amplitude only at the weak spike focus. This was different from the effects on frequency, duration and afterdischarges which were affected at both the
95
strong and weak foci. The constancy of spike amplitude in the strong spike focus is consistent with the all-or-none nature of the spike at the center of the spike focus under the penicillin electrode. In other words, the number of neurons which generate PDSs in the center of the spike focus is essentially independent of the degree of epileptogenicity of the focus. Only at the limit, when the degree of epileptogenicity is only slightly above threshold, there is a graded spike amplitude. This was also noticed when extremely low concentrations of penicillin were used. This decrease of spike amplitude could be due to a decrease of a number of neurons generating PDSs and/or due to a decreased amplitude of the PDS. Evidence indicating that PDSs can be graded under special circumstances (contradicting, therefore, the all-or-none hypothesis of Dichter and Spencer 1969) has been presented by Matsumoto and Ajmone Marsan (1964), Humphrey (in Ajmone Marsan 1969) and Salazar et al. (1980), who demonstrated 3 grades of PDSs: sustained, truncated and larval. An increase of the amplitude of the prepositivity was noticed in 4 foci after two or more injections of phenytoin (corresponding to a blood level of 30--45 pg/ml). The most marked effect was seen in the two cases in which the spikes ultimately disappeared. This prepositivity is possibly an expression of short chain recurrent inhibitory postsynaptic potentials (IPSPs), but excitation deep in the cortex is also possible. The increase in amplitude of this prepositivity may be related to facilitation of recurrent IPSPs. It is more likely that phenytoin blocks PDS generation, and this effect unmasks recurrent IPSPs. The fact that phenytoin has its least effect on the shortest latency event, namely the prepositivity, suggests again that the greatest effects of phenytoin are on the longer polysynaptic pathways. We hypothesize that phenytoin acts mainly depressing long chain pathways whether or not the ultimate effects of these pathway~ are excitatory (after-discharges and spike duration) or inhibitor (spike frequency). We speculate that with relatively higher
96 p h e n y t o i n c o n c e n t r a t i o n s t h e r e is a selective decrease o f the n u m b e r a n d / o r a m p l i t u d e o f t h e PDSs at the c e n t e r o f t h e spike focus (spike a m p l i t u d e ) w i t h o u t a f f e c t i n g t h e s h o r t l a t e n c y r e c u r r e n t IPSPs ( a m p l i t u d e or prepositivity). Finally, w i t h high c o n c e n t r a t i o n s o f p h e n y t o i n , a w e a k spike f o c u s m a y b e c o m p l e t e l y abolished. T h e e f f e c t s o f p h e n y t o i n on field p o t e n t i a l s were i n t e r p r e t e d in t e r m s o f intraceUular events w i t h o u t a t t e m p t i n g d i r e c t m e a s u r e m e n t o f t h e intracellular p h e n o m e n a . Direct m e a s u r e m e n t o f t h e intracellular events is curr e n t l y p e r f o r m e d to test these h y p o t h e s e s directly.
Summary A quantitative evaluation of the effects of p h e n y t o i n on penicillin-induced spike foci is r e p o r t e d . P h e n y t o i n at b l o o d levels considered within t h e r a p e u t i c range in h u m a n s increased spike f r e q u e n c y , d e c r e a s e d spike d u r a t i o n and a b o l i s h e d after-discharges. Spiking at w e a k foci was c o m p l e t e l y abolished w h e n high c o n c e n t r a t i o n s o f p h e n y t o i n were used. I t is c o n c l u d e d t h a t p h e n y t o i n a f f e c t s m a i n l y t h e l o n g chain p a t h w a y s w h e t h e r the u l t i m a t e e f f e c t s o f t h e s e p a t h w a y s are excitat o r y or i n h i b i t o r y .
Rdsumd Effets de la phdnyto'fne sur les foyers de poin tes induits par pdnicilline Les a u t e u r s r a p p o r t e n t l ' ~ v a l u a t i o n quantitative des e f f e t s de la p h ~ n y t o ' / n e sur les f o y e r s de p o i n t e s induits p a r p~nicilline. La p h d n y t o ' / n e ~ des t a u x sanguins considdr~s l'int~rieur des valeurs t h ~ r a p e u t i q u e s chez l ' h o m m e a u g m e n t e la f r ~ q u e n c e des p o i n t e s , d i m i n u e la durde des p o i n t e s et a b o l i t les postddcharges. Les d~charges de p o i n t e s au niveau de f o y e r s faibles s o n t c o m p l ~ t e m e n t abolies lorsque des c o n c e n t r a t i o n s ~lev~es de p h o n y -
L. BUSTAMANTE ET AL. t o / n e s o n t utilis~es. Les a u t e u r s c o n c l u e n t q u e la p h d n y t o ' / n e a f f e c t e e s s e n t i e l l e m e n t les voies longues c h a i n e s , q u e les effets u l t i m e s de ces voies s o i e n t e x c i t a t e u r s ou inhibiteurs. References Ajmone Marsan, C. Acute effects of topical epileptogenic agents. In: H.H. Jasper, A.A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies. Little, Brown and Co., Boston, Mass., 1969: 299-319. Ayala, G.F., Matsumoto, H. and Gumnit, R.J. Excitability changes and inhibitory mechanisms in neocortical neurons during seizures. J. Neurophysiol., 1970, 33: 73--85. Dichter, M. and Spencer, W.A. Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographic features. J. Neurophysiol., 1969, 32: 649--662. Edmonds, H.L. and Stark, L.G. Penicillin-induced epileptogenic foci. II. The anticonvulsant and neuropharmacological effects of SC-13504 in the cat. Neuropharmacology, 1974, 13: 269--277. Edmonds, H.L., Stark, L.G. and Hollinger, M.A. The effects of diphenylhydantoin, phenobarbital and diazepam on the penicillin-induced epileptogenic focus in the rat. Exp. Neurol., 1974, 45: 377-386. Esplin, D.W. Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmacol. exp. Ther., 1957, 120: 301-323. Fromm, G.H. and Landgren, S. Effect of diphenylhydantojn on single cells in the spinal trigeminal nucleus. Neurology (Minneap.), 1963, 13: 34--37. Julien, R.M. and Halpern, L.M. Effects of diphenylhydantoin and other antiepileptic drugs on epileptiform activity and Purkinje cell discharge rates. Epilepsia (Amst.), 1972, 13 : 387--400. Louis, S., Kutt, H. and McDowell, F. Intravenous diphenylhydantoin in experimental seizures. II. Effect on penicillin-induced seizures in the cat. Arch. Neurol. (Chic.), 1968, 18: 472--477. Lueders, H., Bustamante, L., Zablow, L., Krinsky, A. and Goldensohn, E.S. Quantitative studies of spike loci induced by minimal concentrations of penicillin. Electroenceph. clin. Neurophysiol., 1980, 48:80--89. Matsumoto, H. and Ajmone Marsan, C. Cortical cellular phenomena in experimental epilepsy: ictal manifestations. Exp. Neurol., 1964, 9 : 305--326. Merritt, H.H. and Putnam, T.J. A new series of anticonvulsant drugs tested by experiments on animals. Arch. Neurol. Psychiat. (Chic.), 1938, 39: 1003.
PHENYTOIN ON PENICILLIN-INDUCED SPIKES Pippenger, Ch.E., Bastiani, R.J. and Schneider, R.S. Evaluation of an experimental homogenous enzyme immuno-assay for the quantitation of phenytoin and phenobarbital in serum or plasma. In: H. Schneider, D. Janz, C. Gardner-Thorpe, H. Meinardi and A.L. Sherwin (Eds.), Clinical Pharmacology of Anti-epileptic Drugs. Springer, New York, 1975: 331--336. Raines, A. and Standaert, F.G. An effect of diphenylhydantoin on posttetanic hyperpolarization of intramedullary nerve terminals. J. Pharmacol. exp. Ther., 1967, 156: 591--597.
97 Rand, B.O., Kelly, W.A. and Ward, Jr., A.A. Electrophysiological studies of the action of intravenous diphenylhydantoin (Dilantin). Neurology (Minneap.), 1966, 16: 1022--1032. Salazar, Jr., A.M., Goldensohn, E.S. and Zablow, L. The penicillin focus. II. Distribution of neuronal generators. 1980, In preparation. Turtle, R.S. and Preston, J.B. The effect of diphenylhydantoin (Dilantin) on segmental and suprasegmental facilitation and inhibition of segmental motoneurons in the cat. J. Pharmacol. exp. Ther., 1963, 141 : 84--91.
Electroencephalography and Clinical Neurophysiology, 1980, 48:90--97
© Elsevier/North-Holland Scientific Publishers, Ltd.
THE E F F E C T S OF P H E N Y T O I N O N T H E PENICILLIN-INDUCED SPIKE FOCUS
L. BUSTAMANTE i H. LUEDERS, C, PIPPENGER and E.S. GOLDENSOHN Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 (U.S.A.)
(Accepted for publication : April 26, 1979)
In the 4 decades since its introduction by Merritt and Putnam in 1938, phenytoin (diphenylhydantoin, Dilantin) has been widely used as an effective c o m p o u n d for the treatment of focal epilepsy. The mechanisms of action of phenytoin are only partially understood and its effects on experimental epileptogenic spike foci have not been clearly defined. In experimental alumina cream spike foci, phenytoin given intravenously in doses equivalent to therapeutic range in humans produced no significant changes according to Rand et al. {1966). They noticed that spike frequency increased in some cases but did not interpret this finding other than to consider it paradoxical. In penicillin-induced spike foci, Louis et al. (1968) reported that phenytoin produced either no change or a slight decrease in spike frequency. In spite of the persistence of cortical epileptiform spiking at a rather constant rate in their animals, phenytoin administration resulted in a total cessation of actual convulsions. Julien and Halpern {1972) observed a significant decrease in the frequency and duration of after-discharges following the injection of phenytoin, but Edmonds et al. (1974) found no significant change of either spike frequency or the number of after-discharges following the injection of phenytoin. The study reported here analz Present address: Departamento de Fisiologia, Facultad de Medicina, Universidad de Zulia, Apartado 15158, Maracaibo, Venezuela.
yzes quantitatively the effects of phenytoin on epileptogenesis using the penicillin-induced spike focus model described in the preceding paper (Lueders et al. 1980).
M e t h o d and material
The methods of creating the spike foci and the system of analysis are identical to those described in more detail in the preceding paper (Lueders et al. 1980). Penicillin concentrations of 20,000 U/ml (weak foci) were used in 6 cats and concentrations of 100,000 U/ml {strong foci) in additional 6. First injections of phenytoin were given approximately 90 min after the appearance of the first spike exceeding 500 pV which was a time when spiking was relatively stable. Additional injections were given at 30 and 60 min intervals. Each phenytoin injection contained 20--30 mg/kg b o d y weight. Blood samples for serum levels of phenytoin were drawn every 15--30 min and analyzed either with gas liquid chromatography or with the enzyme multiple immunoassay technique (EMIT) (Pippenger et al. 1975). Graphs were made for each experiment and the results were compared to the control series described previously (Lueders et al. 1980). The graphs include: (1) spike frequency versus time; (2) spike duration versus spike frequency as a function of time; (3) spike amplitude versus spike frequency as a
PHENYTOIN
ON PENICILLIN-INDUCED
SPIKES
function of time; and (4) amplitude of prepositivity versus time. In these experiments the point in time immediately before the first injection of phenytoin is considered equivalent to the 90 min reference point in the control experiments and 20 min after each of the 3 injections of phenytoin is considered equivalent to the 1 1 0 , 1 5 5 and 200 min reference points of the control and are so expressed in the graphs. Blood pressure, heart rate and rectal temperature were monitored continuously in all experiments. Phenytoin injections were performed slowly over 3--6 min to avoid falls in blood pressure and bradycardia. Some early experiments were discarded following the rapid injection of phenytoin and all experiments in which the blood pressure decreased below 100/50 mm Hg were discarded.
91
Results
The effects of phenytoin on spike frequency in weak and strong penicillin-induced foci are shown in Figs. 1 and 2. After each of the first 2 injections, marked increases of spike frequency are noted, b u t after the third injection, in some instances (at equivalent toxic human serum levels), the spike is totally suppressed (Fig. 1). More often the third injection produced either no change (Fig. 2) or a further increase in spike frequency. The mean serum level of phenytoin after the first injection was 20.3 #g/ml, after the second was 29.3 pg/ml, and after the third injection was 42.9 pg/ml respectively. Increase of spike frequency after the first 2 injections occurred in all experiments (11--206% at 20 min after the first injection), in clear contrast to the control
EFFECT OF DPH ON FREQUENCY OF PRIMARY SPIKES Using 20,000 Units PNC/ml DPH 20 mg/Kg
DPH 2Omg/Kg
11
50 45
11
OPH 20 mg/Kg
1
40' 2~
z 35 PNC uJ Q. m w ~ m
rso~
25.
t40, t3o
20 15. I0.
5-
I
13.
bJ ¢r
0 o _1 (z)
lO =E
04. , -I0 0
. . . I0 20
. . 40
. . 60
.
.
. . . . . . . . . . . . //-v-//, 120 140 160 180 200 220 240 290 350 MINUTES Fig. 1. E f f e c t o f phenytoin on spike frequency in a 2 0 , 0 0 0 U / m ] penicillin-induced spike focus. Dotted lines indicate phenytoin serum concentration. Systolic and diastolic b l o o d pressure are shown in the lower traces. 80
.
. I00
.
92
L. BUSTAMANTE ET AL.
EFFECT OF DPH ON FREQUENCY OF BURSTS Using I00,000 Units PNC/ml Solvent 1.4 C.C.
,,35I-~30-
Solvent 1.4~c.
Solvent 1.4 c.c.
DPH 20mg/Kg
H HH
z i2s-
DPH 20mg/Kg
DPH
30mg/Kg
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5432I0 -I0
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I0 20
40
60
80
I00
120
140
160
180
200
220
MINUTES
Fig. 2. Effect of phenytoin on spike frequency in a 100,000 U/ml penicillin-induced spike focus is shown in the upper part of the graph. Dotted lines indicate phenytoin serum concentrations. In the lower graph the effect on after-discharges is shown. After-discharges were abolished by phenytoin. After-discharges are defined as prolonged bursts of sequences of multiple secondary spikes and alternating primary and secondary spikes which have a cumulative duration exceeding 8 sec.
experiments in which the spike frequency decreases with time. Total abolition of the spike occurred in only 2 experiments. In both cases it was with a weak focus (20,000 U/ml) after the third injection and at blood levels of phenytoin toxic for humans. After-discharges Complete abolition of after-discharges occurred in all experiments following the first injection of phenytoin at serum levels between 15 and 26 pg/ml (Fig. 2). A doserelated progressive diminution in the number of secondary spikes was also noted following the second and third injections of phenytoin. Spike duration versus spike frequency as a function o f time Spike duration versus spike frequency as a
function of time for all controls and all phenytoin experiments excepting those which produced after
PHENYTOIN ON PENICILLIN-INDUCED SPIKES i1.--I O 13 H 0 0
50 40 50
z 0 :D a ft. (/)
93
CONTROL (n=4), 20,000 CONTROL (n = 2), I00,000 PHENYTOIN (n = 5), 20,000 PHENYTOIN (n=2), I00,000
Unit= PNC/cJ~ SPIKE FOCI Units PNC/c.c. SPIKE FOCI Units PNC/c.c. SPIKE FOCI Units PNC/c.c. SPIKE FOCI
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% O F CHANGE O F S P I K E - F R E Q U E N C Y
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Fig. 3. Spike duration versus spike frequency as a function of time graph of 20,000 and I00,000 U/ml spike foci (controls and phenytoin experiments) which did not produce after-discharges.For each reference point in time the average of each of the 4 groups is represented. The spike duration and spike frequency immediately before the firstinjection of phenytoin (or at 90 rain for the control studies) are taken as reference 0 % of change). The change of spike duration and spike frequency (expressed in percentage) 20 rain after 3 injections of phenytoin (or 110,155 and 200 min in the control studies) determines the position of points number 1, 2 and 3).
spike later completely disappeared (at 46 and 27 min). These 2 experiments were the only ones in which the spike frequency decreased below the control level.
Spike amplitude The effect of phenytoin on spike amplitude was negligible in the strong (100,000 U/ml) penicillin spike foci. In the weak (20,000 U/ml) penicillin spike foci, the amplitude had a tendency to decrease progressively with the 3 injections of phenytoin.
Amplitude of prepositivity In 4 of the 12 animals prepositivity exceeded 10% of the total spike amplitude at time zero. In all 4 cases, phenytoin produced marked increases in the amplitudes of prepos-
itivities (Fig. 4). In the 2 instances in which spiking was abolished after the third injection of phenytoin, the initially predominantly negative spikes were transformed into completely positive spikes just before disappearing (Fig. 5). In the other 2 cases the.amplitudes of the prepositivity were increased by 76% and 87%. In 7 of the 8 animals which showed either no prepositivity or one of less than 10% of the total spike amplitude, phenytoin produced no change (Fig. 4). In 6 animals which were otherwise discarded from this study for a variety of reasons, the spiking disappeared during periods of observation before any phenytoin was administered. No significant increase in prepositivity occurred prior to the cessation of spiking.
94
L. BUSTAMANTE ET AL. Discussion
PHENYTOIN
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REFERENCE POINTS IN TIME
Fig. 4. Amplitude of prepositivity as a function of time graph of I2 phenytoin experiments. See Method for definition of reference point in time number, 0, 1, 2 and 3.
B~
-
-
z
~
-
~
~
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Fig. 5. Typical spike (20,000 U/ml penicillin) immediately before (A) injection of phenytoin and 35 rain after (B) the third injection of phenytoin. The corresponding spike frequency versus time graph of this experiment is shown in Fig. 1.
These experiments demonstrate that the penicillin spike focus model modified for the purpose of testing anticonvulsant actions is (see Lueders et al. 1980), as predicted theoretically, extremely responsive to the action of phenytoin. Phenytoin modified all spike parameters measured and consistently suppressed after-discharges. Phenytoin abolished after-discharges consistently at blood levels within the equivalent of the therapeutic range for humans. It was found that higher phenytoin blood levels were needed to abolish secondary spikes than to abolish after-discharges. If we accept the hypothesis that bursts of after-discharges are dependent on activation of long polysynaptic pathways which continuously reactivate the primary spikes, then we can conclude that phenytoin is particularly effective in depressing these polysynaptic pathways. The abolition of bursts of after
P H E N Y T O I N ON P E N I C I L L I N - I N D U C E D S P I K E S
found that the frequency of primary spikes was inversely related to the degree of epileptogenicity of the spike focus. The present results show that phenytoin consistently increases the frequencies of primary spikes. This increase of primary spike frequency by phenytoin is consistent with its anticonvulsant effect. It is worthwhile to note in this context that Edmonds and Stark (1974), who failed to notice a significant change in spike frequency with phenytoin, reported a significant increase with another anticonvulsant, SC-13504 (Edmonds et al. 1974). The increase of spike frequency implies that phenytoin decreases the amount or duration of post-spike inhibition. If we assume that the amount of post-spike inhibition is directly related to the intensity of the spike and that the duration of the inhibition depends on prolonged polysynaptic pathways, the increase in spike frequency can be ascribed to the known action of phenytoin on prolonged polysynaptic pathways. The fact that phenytoin affects polysynaptic pathways, whether or not the ultimate effect is inhibitory or excitatory, has been demonstrated at the spinal level (Tuttle and Preston 1963). Another effect of phenytoin was to decrease primary spike duration. If spike duration is an expression of prolonged polysynaptic pathways, the effect of phenytoin on decreasing spike duration can be explained by the same mechanisms mentioned above explaining the frequency change. Thus, the effect of phenytoin on all 3 parameters discussed (after-discharges, spike frequency and spike duration) can be explained by a single mechanism of prolonged polysynaptic pathways which are blocked by phenytoin. Each of the parameters, however, is modified by a number of different influences as is evident by the fact that each of the spike parameters can change independently during control recordings. Phenytoin affected spike amplitude only at the weak spike focus. This was different from the effects on frequency, duration and afterdischarges which were affected at both the
95
strong and weak foci. The constancy of spike amplitude in the strong spike focus is consistent with the all-or-none nature of the spike at the center of the spike focus under the penicillin electrode. In other words, the number of neurons which generate PDSs in the center of the spike focus is essentially independent of the degree of epileptogenicity of the focus. Only at the limit, when the degree of epileptogenicity is only slightly above threshold, there is a graded spike amplitude. This was also noticed when extremely low concentrations of penicillin were used. This decrease of spike amplitude could be due to a decrease of a number of neurons generating PDSs and/or due to a decreased amplitude of the PDS. Evidence indicating that PDSs can be graded under special circumstances (contradicting, therefore, the all-or-none hypothesis of Dichter and Spencer 1969) has been presented by Matsumoto and Ajmone Marsan (1964), Humphrey (in Ajmone Marsan 1969) and Salazar et al. (1980), who demonstrated 3 grades of PDSs: sustained, truncated and larval. An increase of the amplitude of the prepositivity was noticed in 4 foci after two or more injections of phenytoin (corresponding to a blood level of 30--45 pg/ml). The most marked effect was seen in the two cases in which the spikes ultimately disappeared. This prepositivity is possibly an expression of short chain recurrent inhibitory postsynaptic potentials (IPSPs), but excitation deep in the cortex is also possible. The increase in amplitude of this prepositivity may be related to facilitation of recurrent IPSPs. It is more likely that phenytoin blocks PDS generation, and this effect unmasks recurrent IPSPs. The fact that phenytoin has its least effect on the shortest latency event, namely the prepositivity, suggests again that the greatest effects of phenytoin are on the longer polysynaptic pathways. We hypothesize that phenytoin acts mainly depressing long chain pathways whether or not the ultimate effects of these pathway~ are excitatory (after-discharges and spike duration) or inhibitor (spike frequency). We speculate that with relatively higher
96 p h e n y t o i n c o n c e n t r a t i o n s t h e r e is a selective decrease o f the n u m b e r a n d / o r a m p l i t u d e o f t h e PDSs at the c e n t e r o f t h e spike focus (spike a m p l i t u d e ) w i t h o u t a f f e c t i n g t h e s h o r t l a t e n c y r e c u r r e n t IPSPs ( a m p l i t u d e or prepositivity). Finally, w i t h high c o n c e n t r a t i o n s o f p h e n y t o i n , a w e a k spike f o c u s m a y b e c o m p l e t e l y abolished. T h e e f f e c t s o f p h e n y t o i n on field p o t e n t i a l s were i n t e r p r e t e d in t e r m s o f intraceUular events w i t h o u t a t t e m p t i n g d i r e c t m e a s u r e m e n t o f t h e intracellular p h e n o m e n a . Direct m e a s u r e m e n t o f t h e intracellular events is curr e n t l y p e r f o r m e d to test these h y p o t h e s e s directly.
Summary A quantitative evaluation of the effects of p h e n y t o i n on penicillin-induced spike foci is r e p o r t e d . P h e n y t o i n at b l o o d levels considered within t h e r a p e u t i c range in h u m a n s increased spike f r e q u e n c y , d e c r e a s e d spike d u r a t i o n and a b o l i s h e d after-discharges. Spiking at w e a k foci was c o m p l e t e l y abolished w h e n high c o n c e n t r a t i o n s o f p h e n y t o i n were used. I t is c o n c l u d e d t h a t p h e n y t o i n a f f e c t s m a i n l y t h e l o n g chain p a t h w a y s w h e t h e r the u l t i m a t e e f f e c t s o f t h e s e p a t h w a y s are excitat o r y or i n h i b i t o r y .
Rdsumd Effets de la phdnyto'fne sur les foyers de poin tes induits par pdnicilline Les a u t e u r s r a p p o r t e n t l ' ~ v a l u a t i o n quantitative des e f f e t s de la p h ~ n y t o ' / n e sur les f o y e r s de p o i n t e s induits p a r p~nicilline. La p h d n y t o ' / n e ~ des t a u x sanguins considdr~s l'int~rieur des valeurs t h ~ r a p e u t i q u e s chez l ' h o m m e a u g m e n t e la f r ~ q u e n c e des p o i n t e s , d i m i n u e la durde des p o i n t e s et a b o l i t les postddcharges. Les d~charges de p o i n t e s au niveau de f o y e r s faibles s o n t c o m p l ~ t e m e n t abolies lorsque des c o n c e n t r a t i o n s ~lev~es de p h o n y -
L. BUSTAMANTE ET AL. t o / n e s o n t utilis~es. Les a u t e u r s c o n c l u e n t q u e la p h d n y t o ' / n e a f f e c t e e s s e n t i e l l e m e n t les voies longues c h a i n e s , q u e les effets u l t i m e s de ces voies s o i e n t e x c i t a t e u r s ou inhibiteurs. References Ajmone Marsan, C. Acute effects of topical epileptogenic agents. In: H.H. Jasper, A.A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies. Little, Brown and Co., Boston, Mass., 1969: 299-319. Ayala, G.F., Matsumoto, H. and Gumnit, R.J. Excitability changes and inhibitory mechanisms in neocortical neurons during seizures. J. Neurophysiol., 1970, 33: 73--85. Dichter, M. and Spencer, W.A. Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographic features. J. Neurophysiol., 1969, 32: 649--662. Edmonds, H.L. and Stark, L.G. Penicillin-induced epileptogenic foci. II. The anticonvulsant and neuropharmacological effects of SC-13504 in the cat. Neuropharmacology, 1974, 13: 269--277. Edmonds, H.L., Stark, L.G. and Hollinger, M.A. The effects of diphenylhydantoin, phenobarbital and diazepam on the penicillin-induced epileptogenic focus in the rat. Exp. Neurol., 1974, 45: 377-386. Esplin, D.W. Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmacol. exp. Ther., 1957, 120: 301-323. Fromm, G.H. and Landgren, S. Effect of diphenylhydantojn on single cells in the spinal trigeminal nucleus. Neurology (Minneap.), 1963, 13: 34--37. Julien, R.M. and Halpern, L.M. Effects of diphenylhydantoin and other antiepileptic drugs on epileptiform activity and Purkinje cell discharge rates. Epilepsia (Amst.), 1972, 13 : 387--400. Louis, S., Kutt, H. and McDowell, F. Intravenous diphenylhydantoin in experimental seizures. II. Effect on penicillin-induced seizures in the cat. Arch. Neurol. (Chic.), 1968, 18: 472--477. Lueders, H., Bustamante, L., Zablow, L., Krinsky, A. and Goldensohn, E.S. Quantitative studies of spike loci induced by minimal concentrations of penicillin. Electroenceph. clin. Neurophysiol., 1980, 48:80--89. Matsumoto, H. and Ajmone Marsan, C. Cortical cellular phenomena in experimental epilepsy: ictal manifestations. Exp. Neurol., 1964, 9 : 305--326. Merritt, H.H. and Putnam, T.J. A new series of anticonvulsant drugs tested by experiments on animals. Arch. Neurol. Psychiat. (Chic.), 1938, 39: 1003.
PHENYTOIN ON PENICILLIN-INDUCED SPIKES Pippenger, Ch.E., Bastiani, R.J. and Schneider, R.S. Evaluation of an experimental homogenous enzyme immuno-assay for the quantitation of phenytoin and phenobarbital in serum or plasma. In: H. Schneider, D. Janz, C. Gardner-Thorpe, H. Meinardi and A.L. Sherwin (Eds.), Clinical Pharmacology of Anti-epileptic Drugs. Springer, New York, 1975: 331--336. Raines, A. and Standaert, F.G. An effect of diphenylhydantoin on posttetanic hyperpolarization of intramedullary nerve terminals. J. Pharmacol. exp. Ther., 1967, 156: 591--597.
97 Rand, B.O., Kelly, W.A. and Ward, Jr., A.A. Electrophysiological studies of the action of intravenous diphenylhydantoin (Dilantin). Neurology (Minneap.), 1966, 16: 1022--1032. Salazar, Jr., A.M., Goldensohn, E.S. and Zablow, L. The penicillin focus. II. Distribution of neuronal generators. 1980, In preparation. Turtle, R.S. and Preston, J.B. The effect of diphenylhydantoin (Dilantin) on segmental and suprasegmental facilitation and inhibition of segmental motoneurons in the cat. J. Pharmacol. exp. Ther., 1963, 141 : 84--91.