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An electrophysiological study on the common antitussive drug dextromethorphan in hippocampal slices in the rat
Neuropharmacology Vol. 28, No. 1I, pp. 1199-1202, 1989 Printedin Great Britain.All rights reserved
002%3908/89 $3.00 + 0.00 Copyright (Q 1989 PergamonPress plc
AN ELECTROPHYSIOLOGICAL STUDY ON THE COMMON ANTITUSSIVE DRUG DEXTROMETHORPHAN IN HIPPOCAMPAL SLICES IN THE RAT C. FRANK, S. SAGRATELLA and A. SCOTTI DE CAROLIS Laboratorio di Farmacologia, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy (Accepted 22 June 1989) Summary-Dextromethorphan, a common antitussive drug derived from the opioid class of morphinans, has been reported to have antiepileptic properties in some experimental tests. The effects of dextromethorphan were tested on the synaptic responses of the interconnections between Schaffer collaterals and CA, pyramidal cells in hippocampal slices of the rat. Dextromethorphan, up to 100-200 JoM, did not affect the basal field potentials resulting from electrical stimulation of the radiatum (0.1 Hz, lOO-2OOpA, 75 psec) of CA, pyramidal cells. Larger concentrations of the drug (3O&IOO/rM) depressed the amplitude and increased the duration of the CA, basal population spikes. Dextromethorphan, up to 200-3OOpM, affected neither the duration nor the incidence of the additional population spikes of the epileptiform bursting, resulting from perfusion of the slice with 1 mM penicillin or in the absence of magnesium ions. The data indicate that: (1) dextromethorphan has an influence on CA, pyramidal cells, different from that of other opioids; (2) the reported antiepileptic effects of dextromethorphan do not involve the hippocampal area. Key nlord.r4extromethorphan,
field potentials, hippocampal slices, epilepsy.
Dextromethorphan is a common antitussive drug that is derived from the opioid class of morphinans.
The drug has been reported to bind to high affinity binding sites in the central nervous system (Craviso and Musacchio, 1983). It is noteworthy that the prototypical anticonvulsant drug diphenylhydantoin enhances the binding of dextromethorphan in guinea pig brain (Craviso and Musacchio, 1983). Moreover, dextromethorphan protects against maximal electroshock-induced seizures (MES) (Tortella and Musacchio, 1986) and reduces kindled amygdaloid seizures in the rat (Freeser, Kadis and Prince, 1988) Dextrorphan, the 0-demethylated metabolite of dextromethorphan, has been recently found to antagonize the excitatory action of the convulsant Nmethyl-D-aspartate (NMDA) in spinal neurones in the rat (Church, Lodge and Berry, 1985). Therefore, it is possible to hypothesize an influence of the drugs on one of the subtypes of receptor of the excitatory amino acid neurotransmitter, glutamate. In addition, dextromethorphan and dextrorphan attenuate the neurotoxicity of glutamate in murine neocortical cultures (Choi, 1987). In the present study, the effect of dextromethorphan in slices of hippocampus of the rat, an area involved in the generation of epileptic phenomena, was examined. First, the effect of dextromethorphan on basal synaptic response of the Schaffer collateral-CA, pyramidal cell interconnections was examined and also the influence of the drug in two models of experimental epilepsy was investigated. In particular, the effects of dextroNP 28,1 I-0
methorphan on the epileptiform bursting induced was studied in hippocampal slices of the rat, when y-amino butyric acid (GABA) antagonist, penicillin, and in the absence of magnesium ions. This latter model of experimental seizures was chosen to assess the influence of the drug on NMDA receptors (Mayer, Westhook and Guthrie, 1984). METHODS 1.
Slice preparation
Male Wistar rats (20&250 g) were killed by decapitation, the skull was opened and the hippocampus rapidly removed. Slices of hippocampus (450pm thick) were cut with a tissue chopper (McIlwain) and immediately placed in the recording chamber, where they were constantly perfused (at a rate of 2-3 ml/min) with an artificial cerebral spinal fluid (CSF) saturated with 95% 02-5% C02. The composition of the artificial CSF was the following: 122 mM NaCl, 0.4 mM KH,PO,, 3 mM KCl, 1.2 mM MgSO,, 25 mM NaHCO,, 1.3 mM CaCl, 10 mM glucose (pH 7.3). The temperature of the perfusion chamber was maintained at 33°C f 1. An interval of 60-90 min elapsed between when the slices were cut and the start of the recording session. 2. Recording session Field potentials were recorded in the CA, area of the stratum pyramidale, after electrical stimulation (0.1 Hz, 70 set, 100-200 PA) of the Schaffer collaterals (struturn radiatum) through 3 M NaCl-filled
1199
1200
C. FRANK et al.
glass microelectrodes (10-20 MR). Electrical potentials were amplified, monitored on oscilloscopes and recorded on tape (Racal 4DS) by conventional techniques. Before beginning the application of drugs, a stable basal field potential was achieved within 30-60 min. 3. Drug session The drugs were added directly to the perfusion solutions. In the “magnesium free” epileptiform model 1.2 mM NaSO, replaced 1.2 mM MgSO, in composition of the artificial CSF. The slices were perfused with the epileptogenic agents (1 mM penicillin or “magnesium free” solution) until a stable epileptiform response was obtained within 3&40min. Then the perfusion with the epileptogenic agent plus dextromethorphan was started. The effects of the drug on the occurrence of additional epileptiform population spikes and on the duration of the epileptiform bursting were assessed after 60 min of perfusion. Statistical analysis (Paired Student’s r-test) was performed on the mean values (+ SEM) before and after the drug. Dextromethorphan HBr was a gift from Hoffman La Roche (Basel), Na penicillin was obtained from Farmitalia (Milano). RESULTS
Effects of dextromethorphan on basal synaptic transmission
The basal field potential consisted of a positivegoing excitatory postsynaptic potential and a single superimposed population spike (2-5 mV, 4-6 msec). Dextromethorphan was tested at concentrations ranging from 10 to 400 PM in a total of 12 experiments. Up to the concentration of 200 p M, the drug did not significantly affect the amplitude or the duration of the basal field potential (Table 1). Larger concentrations of the drug (30&400 PM) decreased the amplitude by almost 50% and increased the duration of basal population spikes (Table 1, Fig. 1) within 60 min of perfusion of the drug. Efects of dextromethorphan transmission
on stimulated synaptic
Within 3&40 min perfusion of the slice with the epileptogenic agent (either a 1 mM penicillin solution or a “magnesium free” solution) elicited the development of an epileptiform bursting characterized by: (1) and increase of amplitude by basal field potential (from 2-5 mV to 610 mV), (2) the appearance of several (2-5) additional population spikes, and (3) a bursting duration of 15-30 msec. A total number of 20 experiments was carried out in order to assess the effects of dextromethorphan at concentrations ranging from 50 to 300pM, against “magnesium free” and penicillin-induced epileptiform activity. Up to concentrations of 200-300pM, dextromethorphan did not affect the epileptiform activity elicited by the two epileptogenic agents.
Electrophysiology
A
B
-lrI
I 2mV
Fig. 1. Effects of dextromethorphan on CA, basal field potentials. A = control field potentials, B = 60 min after perfusion of drug, a reduction of the amplitude and an increase of the duration of the basal population spike were recorded.
Neither epileptiform population spikes nor the duration of epileptiform bursting were modified by the drug within 60 min of perfusion of the drug (Table 2). Larger concentrations of dextromethorphan (500600 p M) decreased epileptiform population spikes and the duration of bursting by almost 25%. However, these depressant effects were still present 60 min after withdrawal of dextromethorphan from the perfusion solution, indicating that the effects of these large concentrations of the drug were not specific. DISCUSSION
Opiates and opioids, such as morphine and enkephalins, acting at mu or delta opiate receptors, present a typical effect on basal synaptic transmission in hippocampal slices of the rat. Starting from small concentrations, they increased the amplitude and reduced the duration of basal population spikes in the CA, area of the pyramidal cell layer (Valentino and Dingledine, 1982). In addition, in large concentrations, the drugs elicited an epileptiform bursting, characterized by the appearance of additional population spikes (Valentino and Dingledine, 1982). In the present study dextromethorphan was devoid of this typical effect of opioids, up to the largest concentration tested (400 PM). The drug did not induce any significant increase in the amplitude of CA, population spikes, while at a concentration of 400 p M it elicited a reduction in the amplitude of basal CA, population spikes. The data indicate that, although dextromethorphan is derived from the opiate class of morphinans,
of dextromethorphan
1201
it did not show mu or delta opioid effects. On the contrary, the inhibitory effects that large concentrations of dextromethorphan had on CA, pyramidal cells might be related to an interaction of the drug with kappa opiate receptors. In fact, opiate kappa receptor agonists, such as dynorphin or ethylketocyclazocine, have a depressant effect on the firing of pyramidal neurones in hippocampal slices in the rat (Brookes and Bradley, 1984, Moises and Walker, 1985). Moreover, opiates and opioids have been reported to have an influence on experimental seizures: they have both convulsant and anticonvulsant effects (Sagratella and Massotti, 1982). Conversely, dextromethorphan had only anticovulsant effects, which appeared to depend upon and interaction of the drug with the NMDA receptor, a subtype of receptor for excitatory amino acids (see introduction). In this study, however, the drug did not affect the epileptiform activity in two in vitro models of experimental epilepsy in the hippocampus. Dextromethorphan, up to 300 PM, did not influence the epileptiform bursting resulting from perfusion of the slice with penicillin or in the absence of magnesium ions, which indicates that the anti-epileptic effect of dextromethorphan did not involve the hippocampal area. The antiepileptic effect of dextromethorphan is probably mainly exerted in the area of the cerebral cortex. In fact, dextromethorphan blocks the epileptiform activity elicited by a “magnesium free” solution or by NMDA in slices of cortex (Wong, Coulter, Choi and Prince, 1988).
REFERENCES
Brookes A. and Bradley P. B. (1984) Electrophysiological evidence for a K-agonist activity of dynorphin in rat brain. Neuropharmacology 23: 207-2 10. Choi D. W. (1987) Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity. Brain Res. 403: 333-336. Church J., Lodge D. and Berry S (1985) Differential effects of dextrorphan and levorphanol on the excitation of rat spinal neurons by amino acids. Eur. J. Pharmac. 111: 185-190. Craviso M. and Musacchio J. (1983) High affinity dextromethorphan binding sites in guinea pig brain, Molec. Pharmac. 23: 629-640. Freeser H. R., Kadis J. L. and Prince D. A. (1988) Dextromethorphan a common antitussive reduces kindled amygdala seizures in the rat. Neurosci. Leti. 86: 340-345. Mayer L. M., Westhook G. L. and Guthrie P. B. (1984) Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nafure 309: 261-263. Moises H. C. and Walker M. J. (1985) Electrophysiological effects of dynorphin peptides on hippocampal pyramidal cells in rat. Eur. J. Pharmac. 108: 85-88. Sagratella S. and Massotti M. (1982) Convulsantanticonvulsant effects of opioids. Relationship of GABA mediated neurotransmission. Neuropharmacology 21: 991-1000. Tortella F. and Musacchio J. (1986) Dextromethorphan and carbepentane: centrally acting non-opioid antitussive agents with novel anticonvulsant properties. Brain Res. 383: 314-318.
1202
C. FRANKet al.
Valentino R. J. and Dingledine R. (1982) Pharmacological characterization of opioid effects in the rat hippocampal slice. J. Pharmac. exp. Ther. 223: 502-509. Wong B. Y., Coulter D. A., Choi D. W. and Prince
D. A. (1988) Dextrorphan and dextromethorphan common antitussive and antiepileptic and antagonize N-methyl-D-aspartate in brain slices. Neurosci. Left. 85: 261-266.
002%3908/89 $3.00 + 0.00 Copyright (Q 1989 PergamonPress plc
AN ELECTROPHYSIOLOGICAL STUDY ON THE COMMON ANTITUSSIVE DRUG DEXTROMETHORPHAN IN HIPPOCAMPAL SLICES IN THE RAT C. FRANK, S. SAGRATELLA and A. SCOTTI DE CAROLIS Laboratorio di Farmacologia, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy (Accepted 22 June 1989) Summary-Dextromethorphan, a common antitussive drug derived from the opioid class of morphinans, has been reported to have antiepileptic properties in some experimental tests. The effects of dextromethorphan were tested on the synaptic responses of the interconnections between Schaffer collaterals and CA, pyramidal cells in hippocampal slices of the rat. Dextromethorphan, up to 100-200 JoM, did not affect the basal field potentials resulting from electrical stimulation of the radiatum (0.1 Hz, lOO-2OOpA, 75 psec) of CA, pyramidal cells. Larger concentrations of the drug (3O&IOO/rM) depressed the amplitude and increased the duration of the CA, basal population spikes. Dextromethorphan, up to 200-3OOpM, affected neither the duration nor the incidence of the additional population spikes of the epileptiform bursting, resulting from perfusion of the slice with 1 mM penicillin or in the absence of magnesium ions. The data indicate that: (1) dextromethorphan has an influence on CA, pyramidal cells, different from that of other opioids; (2) the reported antiepileptic effects of dextromethorphan do not involve the hippocampal area. Key nlord.r4extromethorphan,
field potentials, hippocampal slices, epilepsy.
Dextromethorphan is a common antitussive drug that is derived from the opioid class of morphinans.
The drug has been reported to bind to high affinity binding sites in the central nervous system (Craviso and Musacchio, 1983). It is noteworthy that the prototypical anticonvulsant drug diphenylhydantoin enhances the binding of dextromethorphan in guinea pig brain (Craviso and Musacchio, 1983). Moreover, dextromethorphan protects against maximal electroshock-induced seizures (MES) (Tortella and Musacchio, 1986) and reduces kindled amygdaloid seizures in the rat (Freeser, Kadis and Prince, 1988) Dextrorphan, the 0-demethylated metabolite of dextromethorphan, has been recently found to antagonize the excitatory action of the convulsant Nmethyl-D-aspartate (NMDA) in spinal neurones in the rat (Church, Lodge and Berry, 1985). Therefore, it is possible to hypothesize an influence of the drugs on one of the subtypes of receptor of the excitatory amino acid neurotransmitter, glutamate. In addition, dextromethorphan and dextrorphan attenuate the neurotoxicity of glutamate in murine neocortical cultures (Choi, 1987). In the present study, the effect of dextromethorphan in slices of hippocampus of the rat, an area involved in the generation of epileptic phenomena, was examined. First, the effect of dextromethorphan on basal synaptic response of the Schaffer collateral-CA, pyramidal cell interconnections was examined and also the influence of the drug in two models of experimental epilepsy was investigated. In particular, the effects of dextroNP 28,1 I-0
methorphan on the epileptiform bursting induced was studied in hippocampal slices of the rat, when y-amino butyric acid (GABA) antagonist, penicillin, and in the absence of magnesium ions. This latter model of experimental seizures was chosen to assess the influence of the drug on NMDA receptors (Mayer, Westhook and Guthrie, 1984). METHODS 1.
Slice preparation
Male Wistar rats (20&250 g) were killed by decapitation, the skull was opened and the hippocampus rapidly removed. Slices of hippocampus (450pm thick) were cut with a tissue chopper (McIlwain) and immediately placed in the recording chamber, where they were constantly perfused (at a rate of 2-3 ml/min) with an artificial cerebral spinal fluid (CSF) saturated with 95% 02-5% C02. The composition of the artificial CSF was the following: 122 mM NaCl, 0.4 mM KH,PO,, 3 mM KCl, 1.2 mM MgSO,, 25 mM NaHCO,, 1.3 mM CaCl, 10 mM glucose (pH 7.3). The temperature of the perfusion chamber was maintained at 33°C f 1. An interval of 60-90 min elapsed between when the slices were cut and the start of the recording session. 2. Recording session Field potentials were recorded in the CA, area of the stratum pyramidale, after electrical stimulation (0.1 Hz, 70 set, 100-200 PA) of the Schaffer collaterals (struturn radiatum) through 3 M NaCl-filled
1199
1200
C. FRANK et al.
glass microelectrodes (10-20 MR). Electrical potentials were amplified, monitored on oscilloscopes and recorded on tape (Racal 4DS) by conventional techniques. Before beginning the application of drugs, a stable basal field potential was achieved within 30-60 min. 3. Drug session The drugs were added directly to the perfusion solutions. In the “magnesium free” epileptiform model 1.2 mM NaSO, replaced 1.2 mM MgSO, in composition of the artificial CSF. The slices were perfused with the epileptogenic agents (1 mM penicillin or “magnesium free” solution) until a stable epileptiform response was obtained within 3&40min. Then the perfusion with the epileptogenic agent plus dextromethorphan was started. The effects of the drug on the occurrence of additional epileptiform population spikes and on the duration of the epileptiform bursting were assessed after 60 min of perfusion. Statistical analysis (Paired Student’s r-test) was performed on the mean values (+ SEM) before and after the drug. Dextromethorphan HBr was a gift from Hoffman La Roche (Basel), Na penicillin was obtained from Farmitalia (Milano). RESULTS
Effects of dextromethorphan on basal synaptic transmission
The basal field potential consisted of a positivegoing excitatory postsynaptic potential and a single superimposed population spike (2-5 mV, 4-6 msec). Dextromethorphan was tested at concentrations ranging from 10 to 400 PM in a total of 12 experiments. Up to the concentration of 200 p M, the drug did not significantly affect the amplitude or the duration of the basal field potential (Table 1). Larger concentrations of the drug (30&400 PM) decreased the amplitude by almost 50% and increased the duration of basal population spikes (Table 1, Fig. 1) within 60 min of perfusion of the drug. Efects of dextromethorphan transmission
on stimulated synaptic
Within 3&40 min perfusion of the slice with the epileptogenic agent (either a 1 mM penicillin solution or a “magnesium free” solution) elicited the development of an epileptiform bursting characterized by: (1) and increase of amplitude by basal field potential (from 2-5 mV to 610 mV), (2) the appearance of several (2-5) additional population spikes, and (3) a bursting duration of 15-30 msec. A total number of 20 experiments was carried out in order to assess the effects of dextromethorphan at concentrations ranging from 50 to 300pM, against “magnesium free” and penicillin-induced epileptiform activity. Up to concentrations of 200-300pM, dextromethorphan did not affect the epileptiform activity elicited by the two epileptogenic agents.
Electrophysiology
A
B
-lrI
I 2mV
Fig. 1. Effects of dextromethorphan on CA, basal field potentials. A = control field potentials, B = 60 min after perfusion of drug, a reduction of the amplitude and an increase of the duration of the basal population spike were recorded.
Neither epileptiform population spikes nor the duration of epileptiform bursting were modified by the drug within 60 min of perfusion of the drug (Table 2). Larger concentrations of dextromethorphan (500600 p M) decreased epileptiform population spikes and the duration of bursting by almost 25%. However, these depressant effects were still present 60 min after withdrawal of dextromethorphan from the perfusion solution, indicating that the effects of these large concentrations of the drug were not specific. DISCUSSION
Opiates and opioids, such as morphine and enkephalins, acting at mu or delta opiate receptors, present a typical effect on basal synaptic transmission in hippocampal slices of the rat. Starting from small concentrations, they increased the amplitude and reduced the duration of basal population spikes in the CA, area of the pyramidal cell layer (Valentino and Dingledine, 1982). In addition, in large concentrations, the drugs elicited an epileptiform bursting, characterized by the appearance of additional population spikes (Valentino and Dingledine, 1982). In the present study dextromethorphan was devoid of this typical effect of opioids, up to the largest concentration tested (400 PM). The drug did not induce any significant increase in the amplitude of CA, population spikes, while at a concentration of 400 p M it elicited a reduction in the amplitude of basal CA, population spikes. The data indicate that, although dextromethorphan is derived from the opiate class of morphinans,
of dextromethorphan
1201
it did not show mu or delta opioid effects. On the contrary, the inhibitory effects that large concentrations of dextromethorphan had on CA, pyramidal cells might be related to an interaction of the drug with kappa opiate receptors. In fact, opiate kappa receptor agonists, such as dynorphin or ethylketocyclazocine, have a depressant effect on the firing of pyramidal neurones in hippocampal slices in the rat (Brookes and Bradley, 1984, Moises and Walker, 1985). Moreover, opiates and opioids have been reported to have an influence on experimental seizures: they have both convulsant and anticonvulsant effects (Sagratella and Massotti, 1982). Conversely, dextromethorphan had only anticovulsant effects, which appeared to depend upon and interaction of the drug with the NMDA receptor, a subtype of receptor for excitatory amino acids (see introduction). In this study, however, the drug did not affect the epileptiform activity in two in vitro models of experimental epilepsy in the hippocampus. Dextromethorphan, up to 300 PM, did not influence the epileptiform bursting resulting from perfusion of the slice with penicillin or in the absence of magnesium ions, which indicates that the anti-epileptic effect of dextromethorphan did not involve the hippocampal area. The antiepileptic effect of dextromethorphan is probably mainly exerted in the area of the cerebral cortex. In fact, dextromethorphan blocks the epileptiform activity elicited by a “magnesium free” solution or by NMDA in slices of cortex (Wong, Coulter, Choi and Prince, 1988).
REFERENCES
Brookes A. and Bradley P. B. (1984) Electrophysiological evidence for a K-agonist activity of dynorphin in rat brain. Neuropharmacology 23: 207-2 10. Choi D. W. (1987) Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity. Brain Res. 403: 333-336. Church J., Lodge D. and Berry S (1985) Differential effects of dextrorphan and levorphanol on the excitation of rat spinal neurons by amino acids. Eur. J. Pharmac. 111: 185-190. Craviso M. and Musacchio J. (1983) High affinity dextromethorphan binding sites in guinea pig brain, Molec. Pharmac. 23: 629-640. Freeser H. R., Kadis J. L. and Prince D. A. (1988) Dextromethorphan a common antitussive reduces kindled amygdala seizures in the rat. Neurosci. Leti. 86: 340-345. Mayer L. M., Westhook G. L. and Guthrie P. B. (1984) Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nafure 309: 261-263. Moises H. C. and Walker M. J. (1985) Electrophysiological effects of dynorphin peptides on hippocampal pyramidal cells in rat. Eur. J. Pharmac. 108: 85-88. Sagratella S. and Massotti M. (1982) Convulsantanticonvulsant effects of opioids. Relationship of GABA mediated neurotransmission. Neuropharmacology 21: 991-1000. Tortella F. and Musacchio J. (1986) Dextromethorphan and carbepentane: centrally acting non-opioid antitussive agents with novel anticonvulsant properties. Brain Res. 383: 314-318.
1202
C. FRANKet al.
Valentino R. J. and Dingledine R. (1982) Pharmacological characterization of opioid effects in the rat hippocampal slice. J. Pharmac. exp. Ther. 223: 502-509. Wong B. Y., Coulter D. A., Choi D. W. and Prince
D. A. (1988) Dextrorphan and dextromethorphan common antitussive and antiepileptic and antagonize N-methyl-D-aspartate in brain slices. Neurosci. Left. 85: 261-266.