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Systemic morphine blocks the seizures induced by intracerebroventricular (i.c.v.) injections of opiates and opioid peptides
Brain Research, 246 (1982) 121-126
121
Elsevier Biomedical Press
Systemic Morphine Blocks the Seizures Induced by Intracerebroventricular (i.c.v.) Injections o[ Opiates and Opioid Peptides GIDEON URCA and HANAN FRENK Department of Physiology and Pharmacology, Tel Aviv University School of Medicine and Department of Psychology, Tel Aviv University, Ramat Aviv (Israel)
(Accepted January 28th, 1982) Key words: opiates - - endogenous opioids - - convulsant action - - anticonvulsant action - - naloxone
lntracerebroventricular (i.c.v.) injections of the endorphins and of morphine in rats produce highly characteristic, naloxone sensitive, electrographic seizures. In contrast, systemic injections of morphine have been shown to exert a marked anticonvulsant effect. The present study demonstrates that systemic morphine pretreatment can prevent the occurrence of electrographic seizures induced by i.c.v, morphine, Leu-enkephalin and fl-endorphin and that the anti-epileptic effect of morphine can be reversed by naloxone. Male albino rats, previously prepared for chronic i.c.v, injections and EEG recording, were pretreated with 0-100 mg/kg of intraperitoneal (i.p.) morphine. Thirty five minutes later morphine (520 nmol), Leu-enkephalin (80 nmol) or fl-endorphin (5 nmol) were injected i.c.v. Pretreatment with i.p. morphine blocked the occurrence of seizures induced by morphine and both endogenous opioids. Lower doses of systemic morphine (50 mg/kg) were necessary to block i.c.v, morphine seizures than the dose (100 mg/kg) necessary to block seizures induced by i.c.v. Leu-enkephalin and fl-endorphin. Naloxone (1 mg/kg) administered 25 min following 50 mg/kg of i.p. morphine and preceding the injections of i.c.v, morphine reversed the antiepileptic effect of systemic morphine. These results demonstrate the possible existence of two opiate sensitive systems, one with excitatory-epileptogenic effects and the other possessing inhibitory-antiepileptic properties. The possible relationship between these findings and the known heterogeneity of opiate receptors and opiate actions is discussed. INTRODUCTION One o f the most striking effects of endogenous opioids is their ability to produce highly characteristic electrographic seizures when injected into the lateral ventricle of the brain13,14,24, 26. Such seizures can be seen following the intracerebroventricular (i.c.v.) injection o f the enkephalins13,z6 and fl-endorphin 14, injections of their more stable anaJogues 24 and also following morphine13,z6. Naloxone, the specific opiate antagonist, blocks the occurrence o f these seizures13,14. In contrast, systemic injections of most narcotic analgesics result in pronounced inhibitory effects in m a n y species including humans, with convulsions occurring only at extremely high concentrationsa6. Furthermore, a n u m b e r o f studies have shown that systemic injections o f opiates can produce anticonvulsant effects. Thus, morphine and related opiates have been shown to retard the onset of both chemically induced convulsions 1,25 and auditory seizures 7 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
and to attenuate the behavioral manifestations o f seizures induced by stimulation o f the amygdala 18. Naloxone reversed the antiepileptic effect o f most opiatesS,Z5. It would therefore appear that opiates possess opposing properties that are evident when different routes of administration are employed. If such is the case then it should be expected that systemic injections o f morphine block the epileptic effects o f i.c.v. opiates and opioids. In this study we show that morphine administered systemically can block the epileptic effects of i.c.v, morphine, Leu-enkephalin, and fl-endorphin and that this antiepileptic effect can be reversed by naloxone. MATERIALS AND METHODS Naive, male Wistar derived rats (180-240 g) were surgically prepared with a single cannula guide and 5 E E G electrodes. Guides were made from 21 gauge stainless steel tubing fitted with a 25 gauge stylet
122 aimed 2 mm above the right lateral ventricle. Injections were subsequently performed via a 25 gauge stainless steel cannula which extended 2 mm beyond the tip of the guide cannula. EEG electrodes were stainless steel jeweller's screws threaded into the skull over frontal and occipital areas. Insulated wires led from the electrodes to a connector plug which was cemented to the skull with dental acrylic. Testing began at least one week after surgery. Testing was then conducted in the following manner. Animals were pretreated with intraperitoneal (i.p.) morphine followed 25 rain later by an i.p. injection of either saline or naloxone. Animals were then restrained in a plexiglass tube and connected to a polygraph for 10 min of baseline EEG recording. Thirty five min following the beginning of pretreatment morphine (520 nmol), Leu-enkephalin (80 nmol) or /%endorphin (5 nmol) were infused automatically over a period of 30 s into the lateral ventricle. The dose of drug used for i.c.v, injections was determined on the basis of pilot studies showing that it was a minimal dose for obtaining consistent seizures. All drugs were dissolved in saline and administered in a volume of 10 #1. EEG was monitored for 15 min following i.c.v, injection. At the end of the experiment all animals were sacrificed and histology performed to determine cannula placements. The results reported are those obtained from animals with confirmed ventricular placements. The first experiment consisted of a dose-response investigation of the effect of i.p. morphine (0, 5, 15, and 50 mg/kg) pretreatment on i.c.v, induced morphine seizures. A second study compared the ability of systemic morphine (0, 50 and 100 mg/kg) to block seizures induced by i.c.v, morphine, Leu-enkephalin and/3-endorphin. Finally, a third study investigated the ability of naloxone to reverse the antiepileptic effect of morphine, when such an effect was obtained. In order to determine the dose of naloxone appropriate for the third study the minimal dose of naloxone sufficient to inhibit i.c.v, seizures was established with naloxone doses of 1, 5 and 10 mg/kg. Six animals were employed in each dose group. The lowest dose of naloxone which did not produce inhibition of opiate seizures was then administered to another group of animals following systemic morphine pretreatment and preceding the i.c.v, injection.
RESU LTS
Intracerebroventricular injection of" morphine, Leu-enkephalin or fl-endorphin produced highly characteristic electrographic seizures in most animals (Fig. I A, B) as described beforeZ~. The seizure started with a burst of rapidly repetitive spikes that lasted for 20-35 s and in general developed into slower high amplitude sharp waves. This pattern was followed by 30 s or less of low amplitude, desynchronized EEG sometimes with low amplitude spikes after which a second buildup of regularly repetitive sharp waves or spike and wave complexes (1-3 per second) lasting for about 30 s. These seizures appeared 0.5-3 rain following i.c.v, injections and were predominantly electrographic with wet dog shakes and in some animals myoclonic twitches of the tail or one of the limbs was also seen. Administration of 520 nmol of morphine i.c.v. produced epileptic activity in 7 of the 11 animals (Fig. 2), an effect which was completely abolished in all 9 animals pretreated with 50 mg/kg of morphine (P -< 0.05). Injection of this dose of morphine produced behavioral immobility and catalepsy with prominent changes in EEG. These changes consisted of an increase in amplitude and a reduction of frequency. Both spikes and sharp waves occurred often in spindle form bursts. Seizure~ induced by i.c.v. injections of all opioids could easily be distinguished from the changes of EEG induced by 50 mg/kg or even higher doses of morphine (.see Fig. 1B) by the highly consistent pattern and latency of occurrence of the i.c.v, induced seizures. Doses lower than 50 mg/kg of morphine did not effect morphine induced seizures. Additional animals pretreated with saline receiving i.c.v. Leu-enkephalin or fl-endorphin showed seizures in 11 of 11, and 7 of 9 cases, respectively. The systemic administration of 50 mg/kg of morphine which blocked seizures induced by i.c;v. morphine failed to block the incidence of seizures produced by i.c,v, injections of both opioids (Fig. 2) with all 4 animals receiving Leu-enkephalin and 4 of the 5 animals receiving /3-endorphin showing seizures. Increasing the dose of systemic morphine pretreatment to 100 mg/kg resulted in a blockade of opioid induced seizures (P ~ 0.05) with only 1 of 7 an imals receiving Leu-enkephalin and 2 of 9 animals receiving i.c.v. /%endorphin now showing seizures.
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C Fig. I. The effect of i.c.v, injection of fl-endorphin on the EEG of 3 different animals (A, B, C). The upper trace in each pair shows the EEG following either i.p. saline (A), or morphine 100 mg/kg (B and C) and prior to i.c.v, injection. The bottom trace in each pair shows the EEG following 5 nmol of i.c.v. /4-endorphin. Note the characteristic electrographic seizures induced by i.c.v, l/endorphin after saline (A) or unsuccessful blockade of seizures by i.p. morphine (B). In contrast, no change is visible in the EEG following i.c.v, injection when morphine effectively blocks the effects of i.c.v. /#endorphin (C). M O RPHINE(520 nmolel
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50 100 saline morphme(rng/kg)
PRETREATMENT
Fig. 2. The effects of systemic pretreatment with various doses of morphine on the incidence of seizures elicited by i.c.v. morphine, fl-endorphin and Leu-enkephalin. The number in parentheses denotes the number of animals employed in each group.
Naloxone at doses up to 5 mg/kg did not effect i.c.v, morphine induced seizures whereas 10 mg/kg of naloxone blocked opiate seizures in 5 of the 6 animals so tested. Injection o f the antagonist (1 mg/kg) 25 min following i.p. morphine (50 mg/kg) and 10 min prior to i.c.v, morphine resulted in a marked electrographic and behavioral effect. Thus, instead of the high amplitude low frequency E E G and behavioral sedation seon following the administration o f morphine, desynchronized E E G accompanied by high levels of motor activation were seen. lntracerebroventricular injeciion of morphine in these animals resulted in electrographic seizures in all 5 animals thus treated (P < 0.05). In contrast, the lowest dose o f naloxone tested in this study (I mg/ kg) prevented the appearance of seizures in all animals injected with either i.c.v. Leu-enkephalin or
124 fl-endorphin. In accordance with these findings administration of 1-5 mg/kg of naloxone following 100 mg/kg of systemic morphine and prior to i.c.v. injection of either endogenous opioid did not result in the reappearance of seizures following i.c.v, injection. DISCUSSION Systemic injections of morphine can block the electrographic seizures induced by i.c.v, morphine, Leu-enkephalin and fl-endorphin. These seizures can also be blocked by the opiate antagonist naloxonel~, 14. However, naloxone at doses which are not sufficient to block seizures induced by i.c.v, injections can reverse the antiepileptic effects of morphine as is the case when naloxone is administered following i.p. morphine and prior to i.c.v, injection of morphine. The data present what appear to be two paradoxical lines of evidence. The first is a demonstration of the ability of both an opiate agonist and an antagonist to block electrographic seizures produced by i.c.v, opioids and opiates. The second apparent paradox is the ability of the same opiate, morphine, to inhibit when injected systemically its own epileptogenic action induced by i.c.v, injection. This dual naloxone sensitive effect points toward the existence of two opiate systems, one excitatory and epileptogenic and the other possessing inhibitory-antiepileptic properties. The action of these systems may be explained using two complementary approaches. One approach relies on the fact that the distribution of systemically administered opiates differs from that of i.c.v, injected opiates. Indeed, systemically administered opiates are distributed within the CNS following the now well-known distribution of opiate receptor sites 2-4. In contrast, injection of radiolabeled opiates into the lateral ventricle of the brain i esults in a distribution of opiates shortly after injection, which is limited to periventricular sites 22. Given such a differential distribution it may be that the opiates must act at anatomically different brain sites to exert either an epileptic or an antiepileptic effect. It may also be that the inhibitory and excitatory effects of opiates are due not only to their action at
anatomically distinct brain sites but also to the existence of different opiate receptors within each site, differing in their affinities to the various opioid ligands, which mediate the opposing effects of opiates (ref. 12). The immediate onset of opioid seizures and the anatomical location of limbic brain structures in which opiates are known to exert excitatory effects and epileptogenic action 10,17 support the hypothesis that opioid seizures are a result of the action of opiates on sites at or near the ventricular system. In addition, the rank order of epileptogenic potency of fl-endorphin, Leu-enkephalin and morphine as compared to their reported analgesic potencyS,6,2a and their activity in different bioassay systems20 supports the previous suggestion 12 that the analgesic and epileptic effects of opioids are mediated via different receptors. Related to the antiepileptic effect of morphine observed in this study is its ability to retard the onset of chemically induced seizures 1,s,25. It has been proposed that the anticonvulsant effect of opiates is mediated via the # and the a receptor s. Naloxone can only block that componeilt which is mediated via the # receptor, a receptor category which has also been implicated as a mediator of morphine analgesial2, 20. Naloxone, in our study reversed the antiepileptic effect of systemic morphine, suggesting a parallel between its behavioral and electrographic antiepileptic effect. The relatively high doses of morphine necessary to exert both the anticonvatsant and the antiepileptic effect may be due not to a specific opiate effect but to some general depressant action of opiates. However, we have recently found (in preparation) that injection of low concentrations of morphine into the nucleus accumbens can block the seizures induced by i.c.v, enkephalin. These data taken together with the ability of naloxone to antagonize the epileptic action of opiates make it likely that the observed antiepileptic effect does indeed possess a specific opiate nature. Additional indirect support for the involvement of the # receptor in the antiepileptic effect of morphine has been provided by a recent study studying the effects of i.c.v, injection of levorphanol tartarate tg. Administration of this drug into the lateral ventricle produces potent analgesia but is without epileptic effects even at lethal doses. However, when potassium sulfate is
125 injected together with l e v o r p h a n o l n a l o x o n e reversible o p i o i d seizures a p p e a r in m o r e t h a n h a l f o f the animals. Interestingly, a m a r k e d a t t e n u a t i o n o f the analgesic potency o f l e v o r p h a n o l is also seen. Thus, a m a n i p u l a t i o n which results in the a t t e n u a t i o n o f a # receptor m e d i a t e d b e h a v i o r such as analgesia causes a c o n c o m i t a n t a p p e a r a n c e o f epileptic activity. It m a y therefore be that decreasing the efficacy o f l e v o r p h a n o l at the antiepileptic r e c e p t o r allows the manifestation o f its epileptogenic potential. R e p e a t e d d e m o n s t r a t i o n s have been p r o v i d e d showing that opiates possess b o t h excitatory a n d i n h i b i t o r y properties. F o r example, in the rat increases in m o t o r b e h a v i o r xl, core t e m p e r a t u r e 15 a n d fluid c o n s u m p t i o n 2a can be seen with either low doses o f m o r p h i n e or a n u m b e r o f hours following a d m i n i s t r a t i o n o f high doses o f the drug. In contrast, shortly after the a d m i n i s t r a t i o n o f intermediate or high doses o f m o r p h i n e the classical inhibitory p h e n o m e n a usually associated with the action o f narcotic analgesics is seen. However, rats ren-
dered cataleptic by m o r p h i n e m a y be highly activated when external s t i m u l a t i o n is a p p l i e d 9. These findings suggest that systemic injections o f opiates act simultaneously on b o t h i n h i b i t o r y a n d excitatory systems a n d that in most species, b u t not in all, the i n h i b i t o r y effect prevents the expression o f m o r phine's excitatory action. The ability o f opiates a n d o p i o i d s to elicit n a l o x o n e reversible excitation as evidenced by their ability to p r o d u c e seizures a n d the antiepileptic effect o f systemic m o r p h i n e d e m o n strates the existence o f such opposite, mutually a n t a gonistic, opiate systems.
REFERENCES
l0 Elazar, Z., Motles, E., Ely, Y. and Simantov, R., Acute tolerance to the excitatory effect of enkephalin microinject ions into hippocampus, Life Sci., 24 (1979) 541-548. II Fog, R., Behavioral effects in rats of morphine and amphetamine and a combination of the two drugs, Psychopharmacologia, 16 (1970) 305 312. 12 Frenk, H., McCarty, B. C. and Liebeskind, J. C., Different brain areas mediate the analgesic and epileptic properties of enkephalin, Science, 200 (1978) 335 337. 13 Frenk, H., Urca, G. and Liebeskind, J. C., Epileptic properties of leucine- and methionine-enkephalin: comparison with morphine and reversibility by naloxone, Brain Research, 147 (1978) 327-337. 14 Henriksen, S. J., Bloom, F. E., McCoy, F., Ling, N. and Guillemin, R., fl-Endorphin induces nonconvulsive limbic seizures, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 52215225. 15 Herman, J. B., The pyretic action on rats of small doses of morphine, J. Pharmae. exp. Ther., 76 (1942) 309-315. 16 Jaffe, J. H. and Martin, W. R., Opiate analgesics and antagonists. In A. G. Gilman, L. S. Goodman and A. Gilman (Eds.), The Pharmacological Basis o f Therapeutics, MacMillan, New York, 1980, pp. 494-534. 17 Kerr, F. W. L., Triplett, Jr., J. N. and Beeler, G. W., Reciprocal (push pull) effects of morphine on single units in the ventromedian and lateral hypotha[amus and influences on other nuclei: with a comment on methadone effects during withdrawal from morphine, Brain Research, 74 (1974) 81 103. 18 Le Gal La Salle, G., Calvino, B. and Ben-Ari, Y., Morphine enhances amygdaloid seizures and increases i nter-ictal spike frequency in kindled rats, Neurosci. Lett., 6 (1977) 255-260. 19 Lewis, J. W., Caldecott-Hazard, S., Cannon, J. T. and
1 Adler, M. W., Lin, C. H., Keinath, S. H., Braverman, S. and Geller, E. B., Anticonvulsant action of acute morphine administration in rats, J. Pharmacol. exp. Ther., 198 (1976) 655-660. 2 Atweh, S. F. and Kuhar, M. J., Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla, Brain Research, 124 (1977) 53-67. 3 Atweb, S. E. and Kuhar, M. J., Autoradiographic localization of opiate receptors in rat brain, lI. The brainstem, Brain Research, 129 (1977) 1-12. 4 Atweh, S. F. and Kuhar, M. J., Autoradiographic localization of opiate receptors in rat brain, lII. The telencepbalon, Brain Research, 134 (1977) 393-405. 5 Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. and Stein, L., Analgesia induced in vivo by central administration of enkephalin in the rat, Nature (Lond.), 260 (1976) 625-626. 6 Buscher, H. H., Hill, R. C., Romer, D., Cardinaux, F., Closse, A., Hauser, D. and Pless, J., Evidence for analgesic activity of enkephalin in the mouse, Nature (Lond.), 261 (1976) 423~125. 7 Chen, C. S., Gates, G. R. and Reynoldson, J. A., Effect of morphine and naloxone on priming-induced audiogenie seizures in Balb/c mice, Br#. J. PharmacoL, 58 (1976) 5 [ 7-520. 8 Cowan, A., Geller, E. B. and Adler, M. W., Classification of opioids on the basis of change in seizure threshold in rats, Science, 206 (1979) 465-467. 9 De Ryck, M., Schallert, T. and Teitelbaum, P., Morphine versus haloperidol catalepsy in the rat: a behavioral analysis of postural support mechanisms, Brain Research, 201 (1980) 143 172.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by a grant o f the Israel N a t i o n a l Council for Research a n d D e v e l o p m e n t to H.F., a n d by a grant f r o m the Israel N a t i o n a l A c a d e m y o f Sciences to G . U . The naloxone used was a gift from Endo L a b o r a t o r i e s , G a r d e n City, N.Y.
126 Liebeskind, J. C., Possible role of opioid peptides in pain inhibition and seizures. In J. B. Martin (Ed.), Neurosecretion and Brain Peptides : Implications ]br Brain Function and Neurological Disease, Raven, New York, in press. 20 Lord, J. A. H., Waterfield, A. A., Hughes, J. and Kosterlitz, H. W., Endogenous opioid peptides: multiple agonists and receptors, Nature (Lond.), 267 (1977) 495-499. 21 Maickel, R. P., Braude, M. C. and Zabik, J. E., The effects of various narcotic agonists and antagonists on deprivation-induced fluid consumption, Neuropharmacology, 16 (1977) 863-866. 22 Schubert, P., Teschemacher, H., Kreutzberg, G. W. and Herz, A., lntracerebral distribution pattern of radioactive morphine and morphine-like drugs after intraventricular and intrathecal injection, Histochemie, 22 (1970) 277-288.
23 Szekely, J. 1., Ronai, A. Z., Dunai-Kovacs, Z., Miglecz, E., Bajusz, S. and Graf, k., Cross tolerance between morphine and fl-endorphin in vivo, Life Sci., 20 (1977) 1259-1264. 24 Tortella, F. C., Moreton, J. E. and Khazan, N., EEG and behavioral effects of o-Ata2-methionine-enkephalin amide and morphine in the rat, J. PharmacoL e.rp. Ther., 206 (1978) 636-642. 25 Urca, G. and Frenk, H., Pro- and anticonvulsantaction of morphine in rats, Pharmacol. Biochem. Behav., 13 (i980) 343-347. 26 Urca, G., Frenk, H., Liebeskind, J. C. and Taylor, A. N., Morphine and enkephalin: analgesic and epileptic properties, Science, 197 (1977) 83--86.
121
Elsevier Biomedical Press
Systemic Morphine Blocks the Seizures Induced by Intracerebroventricular (i.c.v.) Injections o[ Opiates and Opioid Peptides GIDEON URCA and HANAN FRENK Department of Physiology and Pharmacology, Tel Aviv University School of Medicine and Department of Psychology, Tel Aviv University, Ramat Aviv (Israel)
(Accepted January 28th, 1982) Key words: opiates - - endogenous opioids - - convulsant action - - anticonvulsant action - - naloxone
lntracerebroventricular (i.c.v.) injections of the endorphins and of morphine in rats produce highly characteristic, naloxone sensitive, electrographic seizures. In contrast, systemic injections of morphine have been shown to exert a marked anticonvulsant effect. The present study demonstrates that systemic morphine pretreatment can prevent the occurrence of electrographic seizures induced by i.c.v, morphine, Leu-enkephalin and fl-endorphin and that the anti-epileptic effect of morphine can be reversed by naloxone. Male albino rats, previously prepared for chronic i.c.v, injections and EEG recording, were pretreated with 0-100 mg/kg of intraperitoneal (i.p.) morphine. Thirty five minutes later morphine (520 nmol), Leu-enkephalin (80 nmol) or fl-endorphin (5 nmol) were injected i.c.v. Pretreatment with i.p. morphine blocked the occurrence of seizures induced by morphine and both endogenous opioids. Lower doses of systemic morphine (50 mg/kg) were necessary to block i.c.v, morphine seizures than the dose (100 mg/kg) necessary to block seizures induced by i.c.v. Leu-enkephalin and fl-endorphin. Naloxone (1 mg/kg) administered 25 min following 50 mg/kg of i.p. morphine and preceding the injections of i.c.v, morphine reversed the antiepileptic effect of systemic morphine. These results demonstrate the possible existence of two opiate sensitive systems, one with excitatory-epileptogenic effects and the other possessing inhibitory-antiepileptic properties. The possible relationship between these findings and the known heterogeneity of opiate receptors and opiate actions is discussed. INTRODUCTION One o f the most striking effects of endogenous opioids is their ability to produce highly characteristic electrographic seizures when injected into the lateral ventricle of the brain13,14,24, 26. Such seizures can be seen following the intracerebroventricular (i.c.v.) injection o f the enkephalins13,z6 and fl-endorphin 14, injections of their more stable anaJogues 24 and also following morphine13,z6. Naloxone, the specific opiate antagonist, blocks the occurrence o f these seizures13,14. In contrast, systemic injections of most narcotic analgesics result in pronounced inhibitory effects in m a n y species including humans, with convulsions occurring only at extremely high concentrationsa6. Furthermore, a n u m b e r o f studies have shown that systemic injections o f opiates can produce anticonvulsant effects. Thus, morphine and related opiates have been shown to retard the onset of both chemically induced convulsions 1,25 and auditory seizures 7 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
and to attenuate the behavioral manifestations o f seizures induced by stimulation o f the amygdala 18. Naloxone reversed the antiepileptic effect o f most opiatesS,Z5. It would therefore appear that opiates possess opposing properties that are evident when different routes of administration are employed. If such is the case then it should be expected that systemic injections o f morphine block the epileptic effects o f i.c.v. opiates and opioids. In this study we show that morphine administered systemically can block the epileptic effects of i.c.v, morphine, Leu-enkephalin, and fl-endorphin and that this antiepileptic effect can be reversed by naloxone. MATERIALS AND METHODS Naive, male Wistar derived rats (180-240 g) were surgically prepared with a single cannula guide and 5 E E G electrodes. Guides were made from 21 gauge stainless steel tubing fitted with a 25 gauge stylet
122 aimed 2 mm above the right lateral ventricle. Injections were subsequently performed via a 25 gauge stainless steel cannula which extended 2 mm beyond the tip of the guide cannula. EEG electrodes were stainless steel jeweller's screws threaded into the skull over frontal and occipital areas. Insulated wires led from the electrodes to a connector plug which was cemented to the skull with dental acrylic. Testing began at least one week after surgery. Testing was then conducted in the following manner. Animals were pretreated with intraperitoneal (i.p.) morphine followed 25 rain later by an i.p. injection of either saline or naloxone. Animals were then restrained in a plexiglass tube and connected to a polygraph for 10 min of baseline EEG recording. Thirty five min following the beginning of pretreatment morphine (520 nmol), Leu-enkephalin (80 nmol) or /%endorphin (5 nmol) were infused automatically over a period of 30 s into the lateral ventricle. The dose of drug used for i.c.v, injections was determined on the basis of pilot studies showing that it was a minimal dose for obtaining consistent seizures. All drugs were dissolved in saline and administered in a volume of 10 #1. EEG was monitored for 15 min following i.c.v, injection. At the end of the experiment all animals were sacrificed and histology performed to determine cannula placements. The results reported are those obtained from animals with confirmed ventricular placements. The first experiment consisted of a dose-response investigation of the effect of i.p. morphine (0, 5, 15, and 50 mg/kg) pretreatment on i.c.v, induced morphine seizures. A second study compared the ability of systemic morphine (0, 50 and 100 mg/kg) to block seizures induced by i.c.v, morphine, Leu-enkephalin and/3-endorphin. Finally, a third study investigated the ability of naloxone to reverse the antiepileptic effect of morphine, when such an effect was obtained. In order to determine the dose of naloxone appropriate for the third study the minimal dose of naloxone sufficient to inhibit i.c.v, seizures was established with naloxone doses of 1, 5 and 10 mg/kg. Six animals were employed in each dose group. The lowest dose of naloxone which did not produce inhibition of opiate seizures was then administered to another group of animals following systemic morphine pretreatment and preceding the i.c.v, injection.
RESU LTS
Intracerebroventricular injection of" morphine, Leu-enkephalin or fl-endorphin produced highly characteristic electrographic seizures in most animals (Fig. I A, B) as described beforeZ~. The seizure started with a burst of rapidly repetitive spikes that lasted for 20-35 s and in general developed into slower high amplitude sharp waves. This pattern was followed by 30 s or less of low amplitude, desynchronized EEG sometimes with low amplitude spikes after which a second buildup of regularly repetitive sharp waves or spike and wave complexes (1-3 per second) lasting for about 30 s. These seizures appeared 0.5-3 rain following i.c.v, injections and were predominantly electrographic with wet dog shakes and in some animals myoclonic twitches of the tail or one of the limbs was also seen. Administration of 520 nmol of morphine i.c.v. produced epileptic activity in 7 of the 11 animals (Fig. 2), an effect which was completely abolished in all 9 animals pretreated with 50 mg/kg of morphine (P -< 0.05). Injection of this dose of morphine produced behavioral immobility and catalepsy with prominent changes in EEG. These changes consisted of an increase in amplitude and a reduction of frequency. Both spikes and sharp waves occurred often in spindle form bursts. Seizure~ induced by i.c.v. injections of all opioids could easily be distinguished from the changes of EEG induced by 50 mg/kg or even higher doses of morphine (.see Fig. 1B) by the highly consistent pattern and latency of occurrence of the i.c.v, induced seizures. Doses lower than 50 mg/kg of morphine did not effect morphine induced seizures. Additional animals pretreated with saline receiving i.c.v. Leu-enkephalin or fl-endorphin showed seizures in 11 of 11, and 7 of 9 cases, respectively. The systemic administration of 50 mg/kg of morphine which blocked seizures induced by i.c;v. morphine failed to block the incidence of seizures produced by i.c,v, injections of both opioids (Fig. 2) with all 4 animals receiving Leu-enkephalin and 4 of the 5 animals receiving /3-endorphin showing seizures. Increasing the dose of systemic morphine pretreatment to 100 mg/kg resulted in a blockade of opioid induced seizures (P ~ 0.05) with only 1 of 7 an imals receiving Leu-enkephalin and 2 of 9 animals receiving i.c.v. /%endorphin now showing seizures.
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B
6 sec
C Fig. I. The effect of i.c.v, injection of fl-endorphin on the EEG of 3 different animals (A, B, C). The upper trace in each pair shows the EEG following either i.p. saline (A), or morphine 100 mg/kg (B and C) and prior to i.c.v, injection. The bottom trace in each pair shows the EEG following 5 nmol of i.c.v. /4-endorphin. Note the characteristic electrographic seizures induced by i.c.v, l/endorphin after saline (A) or unsuccessful blockade of seizures by i.p. morphine (B). In contrast, no change is visible in the EEG following i.c.v, injection when morphine effectively blocks the effects of i.c.v. /#endorphin (C). M O RPHINE(520 nmolel
/~ ENDORPHIN(5 nmale)
LEU"ENKEPHALIN(80nmole)
?:
(I 1)
3>
t
g
5
saline
15
50
morphine long,kgJ
nt
50 100 saline morphmeIrn~'kg)
SYSTEMIC
50 100 saline morphme(rng/kg)
PRETREATMENT
Fig. 2. The effects of systemic pretreatment with various doses of morphine on the incidence of seizures elicited by i.c.v. morphine, fl-endorphin and Leu-enkephalin. The number in parentheses denotes the number of animals employed in each group.
Naloxone at doses up to 5 mg/kg did not effect i.c.v, morphine induced seizures whereas 10 mg/kg of naloxone blocked opiate seizures in 5 of the 6 animals so tested. Injection o f the antagonist (1 mg/kg) 25 min following i.p. morphine (50 mg/kg) and 10 min prior to i.c.v, morphine resulted in a marked electrographic and behavioral effect. Thus, instead of the high amplitude low frequency E E G and behavioral sedation seon following the administration o f morphine, desynchronized E E G accompanied by high levels of motor activation were seen. lntracerebroventricular injeciion of morphine in these animals resulted in electrographic seizures in all 5 animals thus treated (P < 0.05). In contrast, the lowest dose o f naloxone tested in this study (I mg/ kg) prevented the appearance of seizures in all animals injected with either i.c.v. Leu-enkephalin or
124 fl-endorphin. In accordance with these findings administration of 1-5 mg/kg of naloxone following 100 mg/kg of systemic morphine and prior to i.c.v. injection of either endogenous opioid did not result in the reappearance of seizures following i.c.v, injection. DISCUSSION Systemic injections of morphine can block the electrographic seizures induced by i.c.v, morphine, Leu-enkephalin and fl-endorphin. These seizures can also be blocked by the opiate antagonist naloxonel~, 14. However, naloxone at doses which are not sufficient to block seizures induced by i.c.v, injections can reverse the antiepileptic effects of morphine as is the case when naloxone is administered following i.p. morphine and prior to i.c.v, injection of morphine. The data present what appear to be two paradoxical lines of evidence. The first is a demonstration of the ability of both an opiate agonist and an antagonist to block electrographic seizures produced by i.c.v, opioids and opiates. The second apparent paradox is the ability of the same opiate, morphine, to inhibit when injected systemically its own epileptogenic action induced by i.c.v, injection. This dual naloxone sensitive effect points toward the existence of two opiate systems, one excitatory and epileptogenic and the other possessing inhibitory-antiepileptic properties. The action of these systems may be explained using two complementary approaches. One approach relies on the fact that the distribution of systemically administered opiates differs from that of i.c.v, injected opiates. Indeed, systemically administered opiates are distributed within the CNS following the now well-known distribution of opiate receptor sites 2-4. In contrast, injection of radiolabeled opiates into the lateral ventricle of the brain i esults in a distribution of opiates shortly after injection, which is limited to periventricular sites 22. Given such a differential distribution it may be that the opiates must act at anatomically different brain sites to exert either an epileptic or an antiepileptic effect. It may also be that the inhibitory and excitatory effects of opiates are due not only to their action at
anatomically distinct brain sites but also to the existence of different opiate receptors within each site, differing in their affinities to the various opioid ligands, which mediate the opposing effects of opiates (ref. 12). The immediate onset of opioid seizures and the anatomical location of limbic brain structures in which opiates are known to exert excitatory effects and epileptogenic action 10,17 support the hypothesis that opioid seizures are a result of the action of opiates on sites at or near the ventricular system. In addition, the rank order of epileptogenic potency of fl-endorphin, Leu-enkephalin and morphine as compared to their reported analgesic potencyS,6,2a and their activity in different bioassay systems20 supports the previous suggestion 12 that the analgesic and epileptic effects of opioids are mediated via different receptors. Related to the antiepileptic effect of morphine observed in this study is its ability to retard the onset of chemically induced seizures 1,s,25. It has been proposed that the anticonvulsant effect of opiates is mediated via the # and the a receptor s. Naloxone can only block that componeilt which is mediated via the # receptor, a receptor category which has also been implicated as a mediator of morphine analgesial2, 20. Naloxone, in our study reversed the antiepileptic effect of systemic morphine, suggesting a parallel between its behavioral and electrographic antiepileptic effect. The relatively high doses of morphine necessary to exert both the anticonvatsant and the antiepileptic effect may be due not to a specific opiate effect but to some general depressant action of opiates. However, we have recently found (in preparation) that injection of low concentrations of morphine into the nucleus accumbens can block the seizures induced by i.c.v, enkephalin. These data taken together with the ability of naloxone to antagonize the epileptic action of opiates make it likely that the observed antiepileptic effect does indeed possess a specific opiate nature. Additional indirect support for the involvement of the # receptor in the antiepileptic effect of morphine has been provided by a recent study studying the effects of i.c.v, injection of levorphanol tartarate tg. Administration of this drug into the lateral ventricle produces potent analgesia but is without epileptic effects even at lethal doses. However, when potassium sulfate is
125 injected together with l e v o r p h a n o l n a l o x o n e reversible o p i o i d seizures a p p e a r in m o r e t h a n h a l f o f the animals. Interestingly, a m a r k e d a t t e n u a t i o n o f the analgesic potency o f l e v o r p h a n o l is also seen. Thus, a m a n i p u l a t i o n which results in the a t t e n u a t i o n o f a # receptor m e d i a t e d b e h a v i o r such as analgesia causes a c o n c o m i t a n t a p p e a r a n c e o f epileptic activity. It m a y therefore be that decreasing the efficacy o f l e v o r p h a n o l at the antiepileptic r e c e p t o r allows the manifestation o f its epileptogenic potential. R e p e a t e d d e m o n s t r a t i o n s have been p r o v i d e d showing that opiates possess b o t h excitatory a n d i n h i b i t o r y properties. F o r example, in the rat increases in m o t o r b e h a v i o r xl, core t e m p e r a t u r e 15 a n d fluid c o n s u m p t i o n 2a can be seen with either low doses o f m o r p h i n e or a n u m b e r o f hours following a d m i n i s t r a t i o n o f high doses o f the drug. In contrast, shortly after the a d m i n i s t r a t i o n o f intermediate or high doses o f m o r p h i n e the classical inhibitory p h e n o m e n a usually associated with the action o f narcotic analgesics is seen. However, rats ren-
dered cataleptic by m o r p h i n e m a y be highly activated when external s t i m u l a t i o n is a p p l i e d 9. These findings suggest that systemic injections o f opiates act simultaneously on b o t h i n h i b i t o r y a n d excitatory systems a n d that in most species, b u t not in all, the i n h i b i t o r y effect prevents the expression o f m o r phine's excitatory action. The ability o f opiates a n d o p i o i d s to elicit n a l o x o n e reversible excitation as evidenced by their ability to p r o d u c e seizures a n d the antiepileptic effect o f systemic m o r p h i n e d e m o n strates the existence o f such opposite, mutually a n t a gonistic, opiate systems.
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ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by a grant o f the Israel N a t i o n a l Council for Research a n d D e v e l o p m e n t to H.F., a n d by a grant f r o m the Israel N a t i o n a l A c a d e m y o f Sciences to G . U . The naloxone used was a gift from Endo L a b o r a t o r i e s , G a r d e n City, N.Y.
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