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Effects of phenytoin on field bursts of rat hippocampal slices in low-calcium solutions
~28-3908/85 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
~europhQr~acaIogy Vol. 24, No. 9, pp. 915-917, 1985 Printed in Great Britain. All rights reserved
PRELIMINARY NOTES EFFECTS OF PH~NYTOIN ON FIELD BURSTS OF RAT HIPPOCAMPAL SLICES IN LOW-CALCIUM SOLUTIONS R W Snow' , Z Hasan*
and F E Dudek1'3
'Department of Physiology, Tulane University School of Medicine New Orleans, LA 70112, U.S.A. *Yarmouk University School of Medicine, Irbid, Jordan
Summary - Recurring bursts of population spikes, a simple model of epileptiform y+tivity, can be 9r+duced by exposing slices of rat hippocampus to saline containing 0.2 mM [Ca I and 4.0 mM [Mg I, at which concentration chemical svnaotic activity is blocked. Phenvtoin at 7.3-73uM shortened the duration of these bursts. At 73 UM the bursts were slowed ani often eliminated. This model appears to be more sensitive to the action of phenytoin than the penicillin model of epileptiform bursting.
Synchronous bursting of CA1 pyramidal neurons has been observed recently in low-[Ca2+J solutions that block chemical svnaoses. Electrical interactions and chanaes in ionic concentrations appear to mediate the synchrony and spread of this activiti (Jefferys and Haas, 1982; Konnerth, Heinemann and Yaari, 1984; Taylor and Dudek, 19821. The bursts are very sensitive to compounds that modify neuronal conductances. This preparation may therefore be a rapid and sensitive system for testing the postsynaptic action of drugs (Haas, Jefferys, Slater and Carpenter, 19841. Recent reports have noted phenytoin actions on such synchronous bursts (Heinemann, Franceschetti, Hamon, Konnerth and Yaari, 1985; Hood, Siegfried and Haas, 1983). The mechanism by which phenytoin suppresses e ileptic activity is unclear (MeClean and Macdonald, 1983). S~hneiderman and Schwart~kroin P19821 observed no effect of phenytoin on neuronal electrical characteristics even at high doses f73-146 uM). and thev concluded that phenytoin suppressed penicillin-inducedepi tiform activity by reducing synaptic activity. We have used hippocampal slices in low-CCa solution (i.e.with chemical synapses blocke;fl) to determine if phenytoin more effectively suppresses synchronous bursting induced by low-[Ca 3 solutions than that induced by penicllin, and if it acts in an acute preparation at concentrations equivalent to those measured clinically in cerebrospinal fluid (3.65-21.9iiM1. ~TERIALS -AND METHODS Hippocampal slices were prepared and stored submerged at 22-24' C in oxygenated artificial cerebrospinal fluid as previously described (Taylor and Dudek, 1982). After at least 1.hr incubation, slices were placed on a ramp in a recording chamber wah,medto 34' C and perfused with the incubation medium for 10 min before switching to low-[Ca I saline (Jefferys and Haas, 1982) containing 400 ~1 of drug vehicle/l medium (see below). A stream of warmed humidified 95% 02f5% CO2 was directed over the slices. Extracellular field potentials were recorded with WPI 707 DC amplifiers. Recording electrodes (IO-20 meqohm) were filled with 150 mM NaCl. Stimulatina electrodes were 50-urn_ teflon-insulated tungsten wires. Stimuli were 50-usec pulses. Da& were recorded on FM tape with a frequency response of 1250 Hz, and transcribed at a final frequency response of 240 Hz on a rectilinear chart recorder. This accurately reproduced the waveform of the population spikes and bursts. Phenytoin (5,5-Diphenylhydanto~n, Sigma) was dissolved at 182 mM in a vehicle of 40% propylene glycol, 10% ethanol, 50% distilled water (Schneiderman and Schwartzkroin, 1982). Drug was applied after the spontaneous bursts stabilized (20-30 min). Bursts were analysed far frequency (average time for 10 bursts) and duration (average of 10 bursts at half-maximum amplitude1 at the time of maximal response (30-70 min after drug application). 3To whom correspondense should be addressed. 415
Preliminary
916
Notes
RESULTS Figure 1 shows examples of phenytoin action at different doses. Changes in the bursts were first detected after 20 min exposure to phenytoin. Neurotransmitters act much faster in this preparation (Haas et al., 1984). Once steady bursting was established, the duration of the bursts in control medium (i.e., with drug vehicle alone) increased 18+7% (S.E.M., n=4) over a l-hr period. When phenytoin was applied $t 7.3 uM to a different set of slices, burst duration decreased from the pre-drug level to 67-8% (n=4). A dose of 73 uM further decreased burst duration to 57-15% (n=6) in a third set of slices. These effects of phenytoin were significantly different from control (pcO.05, two-tailed J-test). Burst frequency increased to 112+6% in control medium in these same experiments. Only tht high dose of phenytoin had a significant effect on burst frequency: this was a decrefse to 63+ 19% of the pre-drug level. After washing for 1 hr, burst frequency increased to 159-262 of the pre-application level, but burst duration continued to decrease to 4228%. In one experiment, when a second application of 73 uM phenytoin was made after 1 hr of wash, burst frequency was depressed further than during the first presentation (Fig. lC4), indicating a cumulative effeci on pyramidal cell excitability. Only in this one experiment did phenytoin completely and reversibly block bursting. _
Al 2 3
Control 45min.7.3pt.4 60 minwash
Figure 1. Effects of phenytoin (5,5-diphenylhydantoin) on spontaneous synchronous bursts produced by hippocampal CA1 pyramidah+neurons bathed in $r+tificial cerebrospinal fluid containing 0.2 mM [Ca 1 and 4.0 mM [Mg 1. The treatment is indicated above each trace. The second application of 73 PM (C4) completely suppressed the bursts after the last one shown. Some of the bursts in the presence of 73 uM phenytoin were larger than control (arrow).
DISCUSSION We did not test enough concentrations to determine the dose-dependency of phenytoin on field bursts. Nevertheless, the decrease in burst duration at both low and high doses of phenytoin, and the depression of burst frequency at the high dose were significantly different from control and are therefore probably actual drug effects. Our z$sults corroborate those of Heinemann et al. (1985) who found that phenytoin depressed low-CCa 1 bursts at doses of 25-501 and sometimes at doses of 5 PM. However, Hood et al. (1983) could find no effect of phenytoin at doses of l-10 uM, possibly because they did not measure burst duration. Schneiderman and Schwartzkroin (1982) only detected effects of phenytoin at 73-146 uM on synaptic transmission (and not excitability) using penicillin-treated hippocampal slices (but see Hershkowitz and Ayala, 1981). Therefore, our data and those of Heinemann et al. (1985) suggest that hippoy+mpal pyramidal neurons are more sensitive to phenytoin when they are exposed to low-CCa 1 solutions than when they are maintained in normal or penicillin-containir medium.
Preliminary
Notes
917
Our results indicate that phenytoin stabilizes the hyperexcitablity induced by lowered [Ca*+l in hippocampal slices, just as it does in other excitable tissues (Carnay and Grundfest, 19741. Because synaptic transmission is blocked in our preparation (Heinemann et al, 1985; Jefferys and Haas, 1982; Taylor and Dudek, 19821, the effects of phenytoin demonstrated here cannot be due to changes in synaptic transmission (cf. Schneiderman and Schwartzkroin, 1982). Our data are consistent with the hypothesis of McClean and Macdonald (1983) that repetitive firing is the parameter most sensitive to phenytoin. Reduction of repetitive firing would be expected to shorten burst duration, as we observed with a dose of 7.3 pM. A lack of reversibility of the effects of phenytoin was not noted in the work of Heinemann The long delay required et al. (19851, but is not surprising since phenytoin is lipid soluble. to detect phenytoin actions, plus the poor reversibility and cumulative effects, are consistent The with the clinical observations that phenytoin's anticonvulsant action develops slowly. tendency of phenytoin to bind to brain and blood tissue has complicated the interpretation of acute experiments, and the dose relevant to therapeutic mechanism has been debated (McClean and Macdonald, 19831. The preliminary datf+presented here corroborate other work (Heinemann et al., 19851 indicating that the low-CCa 1 model of epileptiform bursting is sensitive to doses of phenytoin similar to those measured clinically in cerebrospinal fluid.
This work was supported by National We thank Dr. Arnold Gerall for loaning equipment. Institutes of Health Grant NS 16683 to F.E.D. and a Fellowship from American Heart Association of Louisiana to R.W.S. REFERENCES Carnay L. and Grundfest S. (19741 Excitable membrane calcium. Neuropharmacology 13: 1097-1108.
stabilization
by diphenylhydantoin
and
Haas H. L., Jefferys J. G. R., Slater N. T. and Carpenter D. 0. (19841 Modulation of low calcium induced field bursts in the hippocampus by monoamines and cholinomimetics. Pflugers Arch. 400 2: 8-33. S., Hamon B., Konnerth A. and Yaari Y. (1985) Effects of Heinemann U., Franceschetti anticonvulsants on spontaneous epileptiform activity which develops in the absence chemical synaptic transmission in hippocampal slices. -Brain Res. 325: 349-352. Hershkowitz N. and Ayala G. F. (1981) Effects hippocampus. -Brain Res. 208: 487-492.
of phenytoin
on pyramidal
neurons
Hood T. W., Seigfried, J. and Haas H. L. (1983) Analysis of carbamazepine hippocampal slices of the rat. -Cell. Mol. Neurobiol. 3: 213-222.
of the rat
actions
Jefferys J. G. R. and Haas H. L. (19821 Synchronized bursting of CA1 hippocampal cells in the absence of synaptic transmission. Nature 300: 448-450.
of
in
pyramidal
Konnerth U., Heinemann, U. and Yaari, Y. (19841 Slow transmission of neural activity in hippocampal area CA1 in the absence of active chemical synapses. Nature 307: 69-71. McClean M. J. and Macdonald R. L. (1983) Multiple actions of phenytoin neurons in cell culture. J- Pharmacol. -~ Exp. Therap. 227: 779-789.
on mouse
spinal cord
Schneiderman J. H. and Schwartzkroin P. A. (1982) Effects of phenytoin on normal activity and on penicillin-induced bursting in the guinea pig hippocampal slice. Neurology 32: 730738. Taylor C. P. and Dudek F. E. (19821 Synchronous neural afterdischarges slices without active chemical synapses. Science 218 810-812.
in rat hippocampal
~europhQr~acaIogy Vol. 24, No. 9, pp. 915-917, 1985 Printed in Great Britain. All rights reserved
PRELIMINARY NOTES EFFECTS OF PH~NYTOIN ON FIELD BURSTS OF RAT HIPPOCAMPAL SLICES IN LOW-CALCIUM SOLUTIONS R W Snow' , Z Hasan*
and F E Dudek1'3
'Department of Physiology, Tulane University School of Medicine New Orleans, LA 70112, U.S.A. *Yarmouk University School of Medicine, Irbid, Jordan
Summary - Recurring bursts of population spikes, a simple model of epileptiform y+tivity, can be 9r+duced by exposing slices of rat hippocampus to saline containing 0.2 mM [Ca I and 4.0 mM [Mg I, at which concentration chemical svnaotic activity is blocked. Phenvtoin at 7.3-73uM shortened the duration of these bursts. At 73 UM the bursts were slowed ani often eliminated. This model appears to be more sensitive to the action of phenytoin than the penicillin model of epileptiform bursting.
Synchronous bursting of CA1 pyramidal neurons has been observed recently in low-[Ca2+J solutions that block chemical svnaoses. Electrical interactions and chanaes in ionic concentrations appear to mediate the synchrony and spread of this activiti (Jefferys and Haas, 1982; Konnerth, Heinemann and Yaari, 1984; Taylor and Dudek, 19821. The bursts are very sensitive to compounds that modify neuronal conductances. This preparation may therefore be a rapid and sensitive system for testing the postsynaptic action of drugs (Haas, Jefferys, Slater and Carpenter, 19841. Recent reports have noted phenytoin actions on such synchronous bursts (Heinemann, Franceschetti, Hamon, Konnerth and Yaari, 1985; Hood, Siegfried and Haas, 1983). The mechanism by which phenytoin suppresses e ileptic activity is unclear (MeClean and Macdonald, 1983). S~hneiderman and Schwart~kroin P19821 observed no effect of phenytoin on neuronal electrical characteristics even at high doses f73-146 uM). and thev concluded that phenytoin suppressed penicillin-inducedepi tiform activity by reducing synaptic activity. We have used hippocampal slices in low-CCa solution (i.e.with chemical synapses blocke;fl) to determine if phenytoin more effectively suppresses synchronous bursting induced by low-[Ca 3 solutions than that induced by penicllin, and if it acts in an acute preparation at concentrations equivalent to those measured clinically in cerebrospinal fluid (3.65-21.9iiM1. ~TERIALS -AND METHODS Hippocampal slices were prepared and stored submerged at 22-24' C in oxygenated artificial cerebrospinal fluid as previously described (Taylor and Dudek, 1982). After at least 1.hr incubation, slices were placed on a ramp in a recording chamber wah,medto 34' C and perfused with the incubation medium for 10 min before switching to low-[Ca I saline (Jefferys and Haas, 1982) containing 400 ~1 of drug vehicle/l medium (see below). A stream of warmed humidified 95% 02f5% CO2 was directed over the slices. Extracellular field potentials were recorded with WPI 707 DC amplifiers. Recording electrodes (IO-20 meqohm) were filled with 150 mM NaCl. Stimulatina electrodes were 50-urn_ teflon-insulated tungsten wires. Stimuli were 50-usec pulses. Da& were recorded on FM tape with a frequency response of 1250 Hz, and transcribed at a final frequency response of 240 Hz on a rectilinear chart recorder. This accurately reproduced the waveform of the population spikes and bursts. Phenytoin (5,5-Diphenylhydanto~n, Sigma) was dissolved at 182 mM in a vehicle of 40% propylene glycol, 10% ethanol, 50% distilled water (Schneiderman and Schwartzkroin, 1982). Drug was applied after the spontaneous bursts stabilized (20-30 min). Bursts were analysed far frequency (average time for 10 bursts) and duration (average of 10 bursts at half-maximum amplitude1 at the time of maximal response (30-70 min after drug application). 3To whom correspondense should be addressed. 415
Preliminary
916
Notes
RESULTS Figure 1 shows examples of phenytoin action at different doses. Changes in the bursts were first detected after 20 min exposure to phenytoin. Neurotransmitters act much faster in this preparation (Haas et al., 1984). Once steady bursting was established, the duration of the bursts in control medium (i.e., with drug vehicle alone) increased 18+7% (S.E.M., n=4) over a l-hr period. When phenytoin was applied $t 7.3 uM to a different set of slices, burst duration decreased from the pre-drug level to 67-8% (n=4). A dose of 73 uM further decreased burst duration to 57-15% (n=6) in a third set of slices. These effects of phenytoin were significantly different from control (pcO.05, two-tailed J-test). Burst frequency increased to 112+6% in control medium in these same experiments. Only tht high dose of phenytoin had a significant effect on burst frequency: this was a decrefse to 63+ 19% of the pre-drug level. After washing for 1 hr, burst frequency increased to 159-262 of the pre-application level, but burst duration continued to decrease to 4228%. In one experiment, when a second application of 73 uM phenytoin was made after 1 hr of wash, burst frequency was depressed further than during the first presentation (Fig. lC4), indicating a cumulative effeci on pyramidal cell excitability. Only in this one experiment did phenytoin completely and reversibly block bursting. _
Al 2 3
Control 45min.7.3pt.4 60 minwash
Figure 1. Effects of phenytoin (5,5-diphenylhydantoin) on spontaneous synchronous bursts produced by hippocampal CA1 pyramidah+neurons bathed in $r+tificial cerebrospinal fluid containing 0.2 mM [Ca 1 and 4.0 mM [Mg 1. The treatment is indicated above each trace. The second application of 73 PM (C4) completely suppressed the bursts after the last one shown. Some of the bursts in the presence of 73 uM phenytoin were larger than control (arrow).
DISCUSSION We did not test enough concentrations to determine the dose-dependency of phenytoin on field bursts. Nevertheless, the decrease in burst duration at both low and high doses of phenytoin, and the depression of burst frequency at the high dose were significantly different from control and are therefore probably actual drug effects. Our z$sults corroborate those of Heinemann et al. (1985) who found that phenytoin depressed low-CCa 1 bursts at doses of 25-501 and sometimes at doses of 5 PM. However, Hood et al. (1983) could find no effect of phenytoin at doses of l-10 uM, possibly because they did not measure burst duration. Schneiderman and Schwartzkroin (1982) only detected effects of phenytoin at 73-146 uM on synaptic transmission (and not excitability) using penicillin-treated hippocampal slices (but see Hershkowitz and Ayala, 1981). Therefore, our data and those of Heinemann et al. (1985) suggest that hippoy+mpal pyramidal neurons are more sensitive to phenytoin when they are exposed to low-CCa 1 solutions than when they are maintained in normal or penicillin-containir medium.
Preliminary
Notes
917
Our results indicate that phenytoin stabilizes the hyperexcitablity induced by lowered [Ca*+l in hippocampal slices, just as it does in other excitable tissues (Carnay and Grundfest, 19741. Because synaptic transmission is blocked in our preparation (Heinemann et al, 1985; Jefferys and Haas, 1982; Taylor and Dudek, 19821, the effects of phenytoin demonstrated here cannot be due to changes in synaptic transmission (cf. Schneiderman and Schwartzkroin, 1982). Our data are consistent with the hypothesis of McClean and Macdonald (1983) that repetitive firing is the parameter most sensitive to phenytoin. Reduction of repetitive firing would be expected to shorten burst duration, as we observed with a dose of 7.3 pM. A lack of reversibility of the effects of phenytoin was not noted in the work of Heinemann The long delay required et al. (19851, but is not surprising since phenytoin is lipid soluble. to detect phenytoin actions, plus the poor reversibility and cumulative effects, are consistent The with the clinical observations that phenytoin's anticonvulsant action develops slowly. tendency of phenytoin to bind to brain and blood tissue has complicated the interpretation of acute experiments, and the dose relevant to therapeutic mechanism has been debated (McClean and Macdonald, 19831. The preliminary datf+presented here corroborate other work (Heinemann et al., 19851 indicating that the low-CCa 1 model of epileptiform bursting is sensitive to doses of phenytoin similar to those measured clinically in cerebrospinal fluid.
This work was supported by National We thank Dr. Arnold Gerall for loaning equipment. Institutes of Health Grant NS 16683 to F.E.D. and a Fellowship from American Heart Association of Louisiana to R.W.S. REFERENCES Carnay L. and Grundfest S. (19741 Excitable membrane calcium. Neuropharmacology 13: 1097-1108.
stabilization
by diphenylhydantoin
and
Haas H. L., Jefferys J. G. R., Slater N. T. and Carpenter D. 0. (19841 Modulation of low calcium induced field bursts in the hippocampus by monoamines and cholinomimetics. Pflugers Arch. 400 2: 8-33. S., Hamon B., Konnerth A. and Yaari Y. (1985) Effects of Heinemann U., Franceschetti anticonvulsants on spontaneous epileptiform activity which develops in the absence chemical synaptic transmission in hippocampal slices. -Brain Res. 325: 349-352. Hershkowitz N. and Ayala G. F. (1981) Effects hippocampus. -Brain Res. 208: 487-492.
of phenytoin
on pyramidal
neurons
Hood T. W., Seigfried, J. and Haas H. L. (1983) Analysis of carbamazepine hippocampal slices of the rat. -Cell. Mol. Neurobiol. 3: 213-222.
of the rat
actions
Jefferys J. G. R. and Haas H. L. (19821 Synchronized bursting of CA1 hippocampal cells in the absence of synaptic transmission. Nature 300: 448-450.
of
in
pyramidal
Konnerth U., Heinemann, U. and Yaari, Y. (19841 Slow transmission of neural activity in hippocampal area CA1 in the absence of active chemical synapses. Nature 307: 69-71. McClean M. J. and Macdonald R. L. (1983) Multiple actions of phenytoin neurons in cell culture. J- Pharmacol. -~ Exp. Therap. 227: 779-789.
on mouse
spinal cord
Schneiderman J. H. and Schwartzkroin P. A. (1982) Effects of phenytoin on normal activity and on penicillin-induced bursting in the guinea pig hippocampal slice. Neurology 32: 730738. Taylor C. P. and Dudek F. E. (19821 Synchronous neural afterdischarges slices without active chemical synapses. Science 218 810-812.
in rat hippocampal