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In vivo electrochemical studies of rat striatal dopamine and serotonin release after morphine
Life Sciences, Vol. 36, pp. 2269-2275 Printed in the U.S.A.
Pergamon Press
V~VO ELECTROCHEMICAL STUDIES OF RAT STRIATAL DOPAMINE AND SEROTONIN RELEASE AFTER MORPHINE
ZN
Patricia A. Broderick Department of Psychiatry [F-G-39N] The Albert Einstein College of Medicine 1300 Morris Park Avenue, Bronx, New York 10461 (Received in final form March 29, 1985) Summary The effect of the reference opiate, morphine (d-morphine-sulfate), on endogenously released striatal dopamine and serotonin was studied in male, adult, anesthetized Sprague-Dawley rats. The intraperitoneal administration of morphine produced a biphasic effect on striatal dopamine release. A significant increase in the dopamine signal was seen in the first hour after drug administration; a significant decrease in the dopamine signal was seen in the second and third hour after drug administration. On the other hand, the effect of morphine on striatal serotonin release was monophasic. Morphine significantly increased serotonin release from rat striatum. The effect lasted three hours after morphine administration, i.e., the effect persisted significantly throughout the study. These data show a simultaneous opiate-dopaminergic and opiate-serotonergic interaction in rat striatum. These data further extend studies which have suggested that the pharmacological mechanism of action of morphine may have its etiology in the concurrent modulation of more than one neurotransmitter. Ever since the report that morphine produced central nervous system depletion of noradrenalin concomitantly with the elicitation of behavioral excitation (i), the neurochemistry underlying the pharmacological effects of morphine has been vigorously investigated. Subsequent reports extended the studies of morphine treatment, tolerance and dependence to studies of the brain neurotransmitters, dopamine and serotonin (2-5). Morphine was shown to produce a dose-dependent biphasic effect on dopamine levels in rat striatum (6) and to increase dopamine levels in rat striatum during naloxone-precipitated withdrawal (7). Morphine increased dopamine turnover (8) and increased dopamine synthesis in rat striatum (9). Morphine increased dopamine release from cat caudate nucleus (i0) and decreased potassium-induced release of dopamine from slices of rat striatum (11,12). Specifically, striatal dopamine has been shown in the behavioral manifestations of morphine, i.e., in stereotypy (13), circling behavior (7), locomotor activity (14), catalepsy (15) and muscular rigidity
(16). The role of serotonin in analgesia has become increasingly interesting as evidence has been presented to link serotonin to the neurophysiology of pain (17). Although the role of serotonin in morphine analgesia remains controversial (18), there is evidence that serotonin receptor blockers reduced morphineinduced antinociception in the rat (19). In addition, morphine increased the metabolite of serotonin, 5-hydroxyindoleacetic acid in rat brain (20) and increased serotonin turnover in rat brain (21). Specifically, the nigrostriatal region, the caudate, and serotonin, have previously been shown to be involved 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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in morphine and stimulation-induced analgesia (22). Morphine caused an increase both in serotonin synthesis and metabolism in rat striatum (23). The purpose of the present study was to demonstrate the effect of morphine on endogenously released dopamine and serotonin from rat striatum by in vivo electrochemistry. By studying another parameter of dopaminergic and serotonergic activity, i.e., release, one can more accurately elucidate the role of these neurotransmitters in the pharmacological action of morphine. Materials and Methods Chloral hydrate anesthetized, male, adult, Sprague-Dawley rats (body temperature maintained at 37 ° C) underwent stereotaxic surgery for positioning of a teflon coated working electrode (150-175 D) in anterior striatum. Stereotaxic coordinates were: 2.6 mm anterior to Bregma, 2.5 mm lateral to midline and 4.0 mm dorsoventral to the rat's skull surface (24). The placement of the working electrode [stearate modification] (25) in anterior striatum was histologically confirmed. A Ag/AgCI reference electrode and a stainless steel auxiliary electrode were placed in contact with the rat cortex. Semiderivative voltammograms were recorded every ten minutes, between 0 and 500 mv, at a scan rate of i0 mv sec -I. The observed electrochemical signals are directly related to dopamine and serotonin release and are referred to as dopamine and serotonin signals. Calibration of the working electrode took place in physiological phosphate buffer solution containing either dopamine or serotonin. D-morphine sulfate was made fresh daily, in distilled water solution and injected intraperitoneally (5 mg/kg) approximately one hour after reproducible basal dopamine and serotonin signals were recorded from rat anterior striatum. The effect of morphine on striatal dopamine and serotonin signals was studied for a period of three hours. Alterations in the dopamine and serotonin signals after morphine were measured by comparing the mean of pre-morphine injection values with both the mean and maximum of post-morphine injection values. Results are expressed in terms of percent change. Statistically significant values were determined by Analysis of Differences. Results The results from in vivo semiderivative electroanalysis showed that the opiate, morphine, produced a biphasic effect on dopamine release from striatum. These results are seen in histogram form in Figure i. The first phase, which lasted from thirty to seventy minutes, was characterized by a statistically significant (p<0.05) 30% increase in the dopamine signal. The second phase, which lasted up to two hours, was characterized by a statistically significant (p<0.05) 32% decrease in dopamine signal. The maximum percent increase in dopamine seen was 68% and the maximum percent decrease in dopamine seen was 50%. On the other hand, there was a statistically significant (p<0.01) 121% increase in the serotonin signal, seen after morphine administration. These results are seen in histogram form in Figure 2. The increase in serotonin release began shortly after the intraperitoneal injection of morphine and remained constant throughout the three hour period studied. The intraperitoneal administration of saline alone had no appreciable effect on the signals produced by either dopamine or serotonin. Semiderivative voltammograms are seen in Figure 3. The dopamine peak, which is seen at approximately 125 mv, when the working electrode has been modified with stearate, is at first enhanced, then depressed, becoming the most depressed in the third hour after morphine administration. The serotonin peak, seen at 275 mv with the stearate modified electrode, is greater than that signal seen in the control voltammogram (upper left quadrant) after mor-
Vol. 36, No. 24, 1985
Morphine and Biogenic Amine Release
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phine treatment, an effect which lasted throughout the study. The semiderivative voltammograms seen in Figure 3, represent original data from one animal studied for morphine's effects on basal dopamine and serotonin release.
7°I
60
50
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40 a:~
**p
20
I
~E
Saline
~ change1es~terhr)m°rphine
T
~-~--'~ Maximum change after morphine (lSlhr) Y, change after morphine (2 nd and 3 rd hr) Maximum change after morphine (2ndand 3 rd hr)
mg
~: ~ -40 4o f -50
FIG. I A histogram showing the effect of the intraperitoneal administration of morphine (5 mg/kg) on dopamine release from striatum of male, Sprague-Dawley rats. Vertical bars represent the mean ± SEM for seven rats.
i
I
Saline
[ ~ ] Mean chonge after Morphine
g g 220 -g
°~180~,=.
~ .
Maximum change after Morphine
* p
20
FIG. 2 A histogram showing the effect of the intraperitoneal administration of morphine (5 mg/kg) on serotonin release from striatum of male, Sprague-Dawley rat& Vertical bars represent the mean ± SEM for seven rats.
2272
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BEFORE TREATMENTWITH MORPHINE
]
0
.100
.200
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0
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.400
hAI5'2
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.200
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]HR AFTER TREATMENT WITH MORPHJNE
O.2nAs ,~z
2 HR AFTER TREATMENT WITH MORPHINE
~ ~
Vol. 36, No. 24, 1985
.200
.300
.400
3 HR AFTER TREATMENT WITH MORPHINE
0
.400
E (Volts)
I00
2OO
3OO
4OO
E (rolls)
STRIATUM
FIG. 3 Semiderivative voltammograms showing the effect of morphine on dopamine and serotonin release from striatum of a male, Sprague-Dawley rat. The recording seen in the upper left quadrant represents basal dopamine and serotonin release from rat striatum. Discussion The results show that morphine, at a pharmacologically effective dose, significantly alters rat striatal dopamine release and does so in a biphasic fashion, i.e., an initial excitation occurs which is then followed by a longer inhibitory action. These biochemical data follow the current theories of a biphasic overlapping of dose-related dopaminergic agonist-antagonist effects for morphine. This was shown, for example, by behavioral data in which morphineinduced locomotion and rearing was very characteristic, and seen in phases as bursts of activity followed by phases of sedation (26), and by behavioral data concomitant with electrophysiological data (27). Interestingly, these data may lend an explanatory note for the previously discrepant reports of an increased dopamine release after morphine (i0), a decreased dopamine release after morphine (11,12), and no change in dopamine release after morphine (28); although the species difference in the previous studies is particularly relevant in the interpretation of the data, since opiates have been shown to have different effects dependent on species (29). The second and third hour data presented in this paper would appear not to be entirely consistent with the report that morphine's action is via an inhibition of dopamine reuptake into striatal nerve terminals (30), unless this type of mechanism might lead to the biphasic effect seen. These data do not appear to be consistent with the report that opiates acting on only delta opiate receptors may presynaptically regulate the release of striatal dopamine (31). Particularly relevant to the data presented in this paper, though, is the report that systemic administration of morphine exerted a naloxone-reversible suppression of spontaneously discharging neurons in the substantia nigra reticulata, which resulted in the morphine suppression of caudate neuronal activities (32); the caudate suppression was time-dependent on the nigral suppression.
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The effect of morphine has been attributed to a variety of manipulations within the dopaminergic nigrostriatal circuitry, such as presynaptic sites in striatum (33), presynaptic striatal autoreceptors (34), and a blockade of dopaminergic transmission other than receptor blockade (35,36). In addition, since the report of the existence of enkephalin receptors on dopaminergic neurons in rat striatum (37), studies of dopaminergic-enkephalinergic interactions in rat nigrostriatal systems have been shown both behaviorally and biochemically (3841). Both pre- and postsynaptic innervation of dopaminergic neurons by enkephalinergic nigrostriatal interneurons has been reported (42). Because the in vivo electrochemical methodology is thought to measure release of neurotransmitters in the extracellular fluid, a presynaptic site of action for morphine is likely. Nonetheless, it should not be unremarked that postsynaptic activity of dopaminergic agonists can be detected with in vivo electrochemistry (43). In addition, because morphine is the reference opiate, an action at dopamine receptor sites is unlikely, although excitation- mediating and inhibit i o n - m e d i a t i n g dopamine receptors in striatum have been demonstrated (44). However, in the absence of lesioning studies, a striatonigral feedback pathway to complement a nigrostriatal feedback pathway cannot be ruled out, especially when one considers the time course of morphine's excitatory and inhibitory actions on dopamine release reported in the present paper. Perhaps the significance of the data presented in this paper, lies in previously reported data which show that tolerance, dependence and withdrawal can be manifested after acute systemic administration of morphine, within three hours after injection (45,46). The biphasic modality of the morphine effect on dopamine release may be a reflection of phenomena related to addiction processes, either by dopaminergic - enkephalinergic/opiate mechanisms per se (47), or by a modulatory influence by serotonin, also known to be involved in the processes of acute morphine withdrawal (48). That a time- dependent modulatory role is played by the neurotransmitter, serotonin, in the opiate actions on catecholamines has been advocated by others (49,50) and is consistent with the purported lack of interaction between serotonin and stereospecific receptor sites in homogenates and subcellular fractions of rat brain (51). Indeed, the development of tolerance, specifically in striatum, has been demonstrated (52). The ability of morphine to increase serotonin release from rat striatum is a new finding. Whether or not the effect is opiate- receptor mediated remains to be studied. Previous data have shown that, although degeneration Qf ascending serotonergic pathways after lesioning of the midbrain raph~ nuclei did not affect stereospecific opiate binding in the serotonergically innervated forebrain (53), the increase in serotonin synthesis and metabolism in rat striatum after morphine administration, was found to be naloxone reversible (23). Increases in serotonin metabolism produced by morphine, however, may not be related to the enzyme, tryptophan hydroxylase, activity, since morphine did not produce significant changes in that enzyme (19). Moreover, morphine did not affect serotonin levels in rat striatum (23) and morphine was a poor inhibitor of serotonin uptake in rat striatal slices (54). Taken together, it would appear that the increased rat striatal serotonin release, seen in the data presented in this paper, would be more directly related to morphine's effects on serotonin synthesis and metabolism, suggesting a functional relationship between serotonin release, synthesis and metabolism, after morphine in rat striatum.
the act nal and the
The results provide further evidence for a nigrostriatal involvement in pharmacological effects of morphine, since unlike kappa compounds, which predominantly on the spinal cord, morphine has both spinal and s u p r a - s p i sites of action (55). Indeed, the simultaneous measurements of dopamine serotonin in the alive animal after morphine, may well be contributive to basic mechanism of action studies, particularly to further elucidate the
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development of tolerance and physical dependence and in the neurophysiology of pain. Although a role for mu type opiate receptor compounds has been implicated in the regulation of nigrostriatal dopaminergic function (56), whether or not the neurotransmitter pharmacological profile presented here will be true of other mu receptor type opiate compounds, remains to be studied. The mu type opiate receptor compound, morphine, has been shown to regulate serotonin release in mesolimbic areas of rat brain (57). Acknowledgements The author wishes to thank Bridget O'Sullivan, O.P., for secretarial assistance and Marie Buschke, for histological preparations. References i. 2. 3. 4. 5. 6.
7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
M. VOGT, J. Physiol. 123 451-458 (1954). L.-M. GUNNE, Acta Physiol. Scand. 58 1-91 (1963). E.L. WAY, H.H. LOH and F.-H. SHEN, Science 162 1290-1292 (1968). A. HERZ, J. BL~SIG, E. LASCHKA and CHR. GRAMSCH, Adv. Biochem. Psychopharmacol. 16 597-599 (1977). S.B. SPARBER, V.F. GELLERT and L. FOSSUM, Adv. Biochem. Psychopharmacol. 20 453-491 (1979). D.H. CLOUET, J.C. JOHNSON, M. RATNER, N. WILLIAMS and G.J. GOLD, Frontiers in Catecholamine Research. (eds. E. USDIN and S. SNYDER) 1039-1042 (Pergamon Press, London, 1973). E.T. IWAMOTO, H.H. LOH and E.L. WAY, J. Pharmacol. exp. Ther. 197 503-516 (1976). L.-M. GUNNE, J. JONSSON and K. FUXE, Eur. J. Pharmacol. 5 338-342 (1969). D.H. CLOUET and M. RATNER, Science 168 854-856 (1970). M.F. CHESSELET, A. CH~RAMY, T.D. REISINE and J. GLOWINSKI, Nature (Lond.) 291 320-322 (1981). B. CELSEN and K. KUSCHINSKY, Naunyn-Schmiedeberg's Arch. Pharmacol. 284 159-165 (1974). H.H. LOH, D.A. BRASE, S. SAMPATH-KHANNA, J.B. MAR and E.L. WAY, Nature (Lond.) 264 567-568 (1976). R. FOG, Psychopharmacol. 16 305-312 (1970). K. KUSCHINSKY and O. HORNYKIEWITZ, Eur. J. Pharmacol. 26 41-50 (1974). K. KUSCHINSKY and O. HORNYKIEWITZ, Eur. J. Pharmacol. 19 119-122 (1972). U. HAVEMANN and K. KUSCHINSKY, Neurochem. International. 4 (4) 199-215 (1982). H. AKIL and D.J. MAYER, Brain Res. 44 692-697 (1972). R.B. MESSING and L.D. LYTLE, Pain 4 1-21 (1977). G.G. YARBROUGH, D.M. BUXBAUM and E. SANDERS-BUSH, J. Pharmacol. exp. Ther. 185 328-335 (1973). I. GOODLET and M.F. SUGRUE, Eur. J. Pharmacol. 29 241-248 (1974). D.R. HAUBRICH and D.E. BLAKE, Biochem. Pharmacol. 22 2753-2759 (1973). C.G. LINEBERRY and C.J. VIERCK, Brain Res. 98 119-134 (1975). C.A. JOHNSTON and K.E. MOORE, J. Neural Transm. 57 65-73 (1983). L.J. PELLIGRINO and A.J. CUSHMAN, A Stereotaxic Atlas of the Rat Brain, p. 19, Appleton-Century-Crofts, New York (1967). C.D. BLAHA and R.F. LANE, Brain Res. Bull. i0 861-864 (1983). J. SCHEEL-KR~GER, Frontiers in Catecholamine Research. (eds. E. USDIN and S. SNYDER) 1027-1029 (Pergamon Press, London, 1973). N.L. OSTROWSKI, I. PAUL, M. DRNACH and A.R. CAGGIULA, Soc. Neurosci. Abstm 9 280 (1983). S ARBILLA and S.Z. LANGER, Nature (Lond.) 271 559-560 (1978). R.G. BROWNE and D.S. SEGAL, Biol. Psychiat. 15 (I) 77-86 (1980). C.M. LEE and P.C.L. WONG, Biochem. Pharmacol. 28 163-170 (1979). C. LUBETZKI, M.F. CHESSELET and J. GLOWINSKI, J. Pharmacol. exp. Ther. 222
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435-440 (1982). E.P. FINNERTY and S.H.H. CHAN, Eur. J. Pharmacol. 59 307-310 (1979). G. DICHIARA, M.L. PORCEDDU, W. FRATTA and G.L. GESSA, Nature (Lond.) 267 270-272 (1977). M. GARCIA-MUNOZ, N.M. NICOLAOU, I.F. TULLOCH, A.K. WRIGHT and G.W. ARBUTHNOTT , Nature (Lond.) 265 363-365 (1977). K. IWATSUBO and D.H. CLOUET, Biochem. Pharmacol. 24 1499-1503 (1975). K. IWATSUBO and D.H. CLOUET, J. Pharmacol. exp. Ther. 202 429-436 (1977). H. POLLARD, C. LLORENS-CORTES and J.C. SCHWARTZ, Nature (Lond.) 268 745747 (1977). G. BIGGIO, M. CASU, M.G. CORDA, C. DIBELLO and G.L. GESSA, Science 200 552-554 (1978). A.E. KELLEY, L. STINUS and S.D. IVERSEN, Behav. Br. Res. 1 3-24 (1980). P.A. BRODERICK, C.D. BLAHA and R.F. LANE, Brain Res. 269 ~78-381 (1983). P.A. BRODERICK, E.L. GARDNER and H.M. VAN PRAAG, Biol. Psychiat. 19 4553 (1984). B.I. DIAMOND and R.L. BORISON, Neurology 28 1085-1088 (1978). P.A. BRODERICK, Annals of the N.Y. Acad. of Sci. in press (1985). A.R. COOLS and J.M. VAN ROSSUM, Psychopharmacol. (Berl.) 45 243-254 (1976). G.S.F. LING, J.M. MACLEOD, S. LEE, S.H. LOCKHART and G.W. PASTERNAK, Science 226 462-464 (1984). S.B. SPARBER, V.F. GELLERT, L. LICHTBLAU and R. EISENBERG, Factors Affecting the Action of Narcotics. (eds. M.L. ADLER, L, MANERA and R. SAMANIN) 63-91 (Raven Press, New York, 1978). M.P. MARTRES, J. COSTENTIN, M. BAUDRY, H. MARCAIS, P. PROTAIS and J.C. SCHWARTZ, Brain Res. 136 319-337 (1977). S.B. SPARBER, M.S. KLEVEN and B.S. NEAL, International Narcotics Research Conference, Cambridge, England, July, 1984. A. HERZ and J.BL~SIG, Serotonin in Health and Disease, Vol, II. Physiological Resulation and Pharmacological Action. (ed. W.B. ESSMAN) 341-347 (Spectrum, New York, 1978). D.A. BRASE, Adv. Biochem. Psychopharmacol. 20 409-428 (1979). C. PERT and S. SNYDER, Proc. Natl. Acad. Sci. 70 2243-2247 (1973). R.H. ROTH, N.G. BACOPOULOS, G. BUSTOS and D.E. REDMOND, JR., Adv. BiochemPsychopharmacol. 24 513-520 (1980). M.J. KUHAR, C.B. PERT and S.H. SNYDER, Nature (Lond.) 245 447-450 (1973). J.E. THORNBURG and D.E. BLAKE, Fed. Proc. 30 501 (1971). P.L. WOOD, A. RACKHAM and J.W. RICHARD, Life Sci. 28 2119-2125 (1981). P.L. WOOD, M. STOTLAND, J.W. RICHARD and A. RACKHAM, J. Pharmacol. exp. Ther. 215 (3) 697-703 (1980). P.A. BRODERICK, Neuropeptides, ~ (4-6) 587-590 (1985).
Pergamon Press
V~VO ELECTROCHEMICAL STUDIES OF RAT STRIATAL DOPAMINE AND SEROTONIN RELEASE AFTER MORPHINE
ZN
Patricia A. Broderick Department of Psychiatry [F-G-39N] The Albert Einstein College of Medicine 1300 Morris Park Avenue, Bronx, New York 10461 (Received in final form March 29, 1985) Summary The effect of the reference opiate, morphine (d-morphine-sulfate), on endogenously released striatal dopamine and serotonin was studied in male, adult, anesthetized Sprague-Dawley rats. The intraperitoneal administration of morphine produced a biphasic effect on striatal dopamine release. A significant increase in the dopamine signal was seen in the first hour after drug administration; a significant decrease in the dopamine signal was seen in the second and third hour after drug administration. On the other hand, the effect of morphine on striatal serotonin release was monophasic. Morphine significantly increased serotonin release from rat striatum. The effect lasted three hours after morphine administration, i.e., the effect persisted significantly throughout the study. These data show a simultaneous opiate-dopaminergic and opiate-serotonergic interaction in rat striatum. These data further extend studies which have suggested that the pharmacological mechanism of action of morphine may have its etiology in the concurrent modulation of more than one neurotransmitter. Ever since the report that morphine produced central nervous system depletion of noradrenalin concomitantly with the elicitation of behavioral excitation (i), the neurochemistry underlying the pharmacological effects of morphine has been vigorously investigated. Subsequent reports extended the studies of morphine treatment, tolerance and dependence to studies of the brain neurotransmitters, dopamine and serotonin (2-5). Morphine was shown to produce a dose-dependent biphasic effect on dopamine levels in rat striatum (6) and to increase dopamine levels in rat striatum during naloxone-precipitated withdrawal (7). Morphine increased dopamine turnover (8) and increased dopamine synthesis in rat striatum (9). Morphine increased dopamine release from cat caudate nucleus (i0) and decreased potassium-induced release of dopamine from slices of rat striatum (11,12). Specifically, striatal dopamine has been shown in the behavioral manifestations of morphine, i.e., in stereotypy (13), circling behavior (7), locomotor activity (14), catalepsy (15) and muscular rigidity
(16). The role of serotonin in analgesia has become increasingly interesting as evidence has been presented to link serotonin to the neurophysiology of pain (17). Although the role of serotonin in morphine analgesia remains controversial (18), there is evidence that serotonin receptor blockers reduced morphineinduced antinociception in the rat (19). In addition, morphine increased the metabolite of serotonin, 5-hydroxyindoleacetic acid in rat brain (20) and increased serotonin turnover in rat brain (21). Specifically, the nigrostriatal region, the caudate, and serotonin, have previously been shown to be involved 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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in morphine and stimulation-induced analgesia (22). Morphine caused an increase both in serotonin synthesis and metabolism in rat striatum (23). The purpose of the present study was to demonstrate the effect of morphine on endogenously released dopamine and serotonin from rat striatum by in vivo electrochemistry. By studying another parameter of dopaminergic and serotonergic activity, i.e., release, one can more accurately elucidate the role of these neurotransmitters in the pharmacological action of morphine. Materials and Methods Chloral hydrate anesthetized, male, adult, Sprague-Dawley rats (body temperature maintained at 37 ° C) underwent stereotaxic surgery for positioning of a teflon coated working electrode (150-175 D) in anterior striatum. Stereotaxic coordinates were: 2.6 mm anterior to Bregma, 2.5 mm lateral to midline and 4.0 mm dorsoventral to the rat's skull surface (24). The placement of the working electrode [stearate modification] (25) in anterior striatum was histologically confirmed. A Ag/AgCI reference electrode and a stainless steel auxiliary electrode were placed in contact with the rat cortex. Semiderivative voltammograms were recorded every ten minutes, between 0 and 500 mv, at a scan rate of i0 mv sec -I. The observed electrochemical signals are directly related to dopamine and serotonin release and are referred to as dopamine and serotonin signals. Calibration of the working electrode took place in physiological phosphate buffer solution containing either dopamine or serotonin. D-morphine sulfate was made fresh daily, in distilled water solution and injected intraperitoneally (5 mg/kg) approximately one hour after reproducible basal dopamine and serotonin signals were recorded from rat anterior striatum. The effect of morphine on striatal dopamine and serotonin signals was studied for a period of three hours. Alterations in the dopamine and serotonin signals after morphine were measured by comparing the mean of pre-morphine injection values with both the mean and maximum of post-morphine injection values. Results are expressed in terms of percent change. Statistically significant values were determined by Analysis of Differences. Results The results from in vivo semiderivative electroanalysis showed that the opiate, morphine, produced a biphasic effect on dopamine release from striatum. These results are seen in histogram form in Figure i. The first phase, which lasted from thirty to seventy minutes, was characterized by a statistically significant (p<0.05) 30% increase in the dopamine signal. The second phase, which lasted up to two hours, was characterized by a statistically significant (p<0.05) 32% decrease in dopamine signal. The maximum percent increase in dopamine seen was 68% and the maximum percent decrease in dopamine seen was 50%. On the other hand, there was a statistically significant (p<0.01) 121% increase in the serotonin signal, seen after morphine administration. These results are seen in histogram form in Figure 2. The increase in serotonin release began shortly after the intraperitoneal injection of morphine and remained constant throughout the three hour period studied. The intraperitoneal administration of saline alone had no appreciable effect on the signals produced by either dopamine or serotonin. Semiderivative voltammograms are seen in Figure 3. The dopamine peak, which is seen at approximately 125 mv, when the working electrode has been modified with stearate, is at first enhanced, then depressed, becoming the most depressed in the third hour after morphine administration. The serotonin peak, seen at 275 mv with the stearate modified electrode, is greater than that signal seen in the control voltammogram (upper left quadrant) after mor-
Vol. 36, No. 24, 1985
Morphine and Biogenic Amine Release
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phine treatment, an effect which lasted throughout the study. The semiderivative voltammograms seen in Figure 3, represent original data from one animal studied for morphine's effects on basal dopamine and serotonin release.
7°I
60
50
*p
40 a:~
**p
20
I
~E
Saline
~ change1es~terhr)m°rphine
T
~-~--'~ Maximum change after morphine (lSlhr) Y, change after morphine (2 nd and 3 rd hr) Maximum change after morphine (2ndand 3 rd hr)
mg
~: ~ -40 4o f -50
FIG. I A histogram showing the effect of the intraperitoneal administration of morphine (5 mg/kg) on dopamine release from striatum of male, Sprague-Dawley rats. Vertical bars represent the mean ± SEM for seven rats.
i
I
Saline
[ ~ ] Mean chonge after Morphine
g g 220 -g
°~180~,=.
~ .
Maximum change after Morphine
* p
20
FIG. 2 A histogram showing the effect of the intraperitoneal administration of morphine (5 mg/kg) on serotonin release from striatum of male, Sprague-Dawley rat& Vertical bars represent the mean ± SEM for seven rats.
2272
Morphine and Biogenic Amine Release
BEFORE TREATMENTWITH MORPHINE
]
0
.100
.200
.300
0
.K)O
.400
hAI5'2
.100
.200
.300
]HR AFTER TREATMENT WITH MORPHJNE
O.2nAs ,~z
2 HR AFTER TREATMENT WITH MORPHINE
~ ~
Vol. 36, No. 24, 1985
.200
.300
.400
3 HR AFTER TREATMENT WITH MORPHINE
0
.400
E (Volts)
I00
2OO
3OO
4OO
E (rolls)
STRIATUM
FIG. 3 Semiderivative voltammograms showing the effect of morphine on dopamine and serotonin release from striatum of a male, Sprague-Dawley rat. The recording seen in the upper left quadrant represents basal dopamine and serotonin release from rat striatum. Discussion The results show that morphine, at a pharmacologically effective dose, significantly alters rat striatal dopamine release and does so in a biphasic fashion, i.e., an initial excitation occurs which is then followed by a longer inhibitory action. These biochemical data follow the current theories of a biphasic overlapping of dose-related dopaminergic agonist-antagonist effects for morphine. This was shown, for example, by behavioral data in which morphineinduced locomotion and rearing was very characteristic, and seen in phases as bursts of activity followed by phases of sedation (26), and by behavioral data concomitant with electrophysiological data (27). Interestingly, these data may lend an explanatory note for the previously discrepant reports of an increased dopamine release after morphine (i0), a decreased dopamine release after morphine (11,12), and no change in dopamine release after morphine (28); although the species difference in the previous studies is particularly relevant in the interpretation of the data, since opiates have been shown to have different effects dependent on species (29). The second and third hour data presented in this paper would appear not to be entirely consistent with the report that morphine's action is via an inhibition of dopamine reuptake into striatal nerve terminals (30), unless this type of mechanism might lead to the biphasic effect seen. These data do not appear to be consistent with the report that opiates acting on only delta opiate receptors may presynaptically regulate the release of striatal dopamine (31). Particularly relevant to the data presented in this paper, though, is the report that systemic administration of morphine exerted a naloxone-reversible suppression of spontaneously discharging neurons in the substantia nigra reticulata, which resulted in the morphine suppression of caudate neuronal activities (32); the caudate suppression was time-dependent on the nigral suppression.
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The effect of morphine has been attributed to a variety of manipulations within the dopaminergic nigrostriatal circuitry, such as presynaptic sites in striatum (33), presynaptic striatal autoreceptors (34), and a blockade of dopaminergic transmission other than receptor blockade (35,36). In addition, since the report of the existence of enkephalin receptors on dopaminergic neurons in rat striatum (37), studies of dopaminergic-enkephalinergic interactions in rat nigrostriatal systems have been shown both behaviorally and biochemically (3841). Both pre- and postsynaptic innervation of dopaminergic neurons by enkephalinergic nigrostriatal interneurons has been reported (42). Because the in vivo electrochemical methodology is thought to measure release of neurotransmitters in the extracellular fluid, a presynaptic site of action for morphine is likely. Nonetheless, it should not be unremarked that postsynaptic activity of dopaminergic agonists can be detected with in vivo electrochemistry (43). In addition, because morphine is the reference opiate, an action at dopamine receptor sites is unlikely, although excitation- mediating and inhibit i o n - m e d i a t i n g dopamine receptors in striatum have been demonstrated (44). However, in the absence of lesioning studies, a striatonigral feedback pathway to complement a nigrostriatal feedback pathway cannot be ruled out, especially when one considers the time course of morphine's excitatory and inhibitory actions on dopamine release reported in the present paper. Perhaps the significance of the data presented in this paper, lies in previously reported data which show that tolerance, dependence and withdrawal can be manifested after acute systemic administration of morphine, within three hours after injection (45,46). The biphasic modality of the morphine effect on dopamine release may be a reflection of phenomena related to addiction processes, either by dopaminergic - enkephalinergic/opiate mechanisms per se (47), or by a modulatory influence by serotonin, also known to be involved in the processes of acute morphine withdrawal (48). That a time- dependent modulatory role is played by the neurotransmitter, serotonin, in the opiate actions on catecholamines has been advocated by others (49,50) and is consistent with the purported lack of interaction between serotonin and stereospecific receptor sites in homogenates and subcellular fractions of rat brain (51). Indeed, the development of tolerance, specifically in striatum, has been demonstrated (52). The ability of morphine to increase serotonin release from rat striatum is a new finding. Whether or not the effect is opiate- receptor mediated remains to be studied. Previous data have shown that, although degeneration Qf ascending serotonergic pathways after lesioning of the midbrain raph~ nuclei did not affect stereospecific opiate binding in the serotonergically innervated forebrain (53), the increase in serotonin synthesis and metabolism in rat striatum after morphine administration, was found to be naloxone reversible (23). Increases in serotonin metabolism produced by morphine, however, may not be related to the enzyme, tryptophan hydroxylase, activity, since morphine did not produce significant changes in that enzyme (19). Moreover, morphine did not affect serotonin levels in rat striatum (23) and morphine was a poor inhibitor of serotonin uptake in rat striatal slices (54). Taken together, it would appear that the increased rat striatal serotonin release, seen in the data presented in this paper, would be more directly related to morphine's effects on serotonin synthesis and metabolism, suggesting a functional relationship between serotonin release, synthesis and metabolism, after morphine in rat striatum.
the act nal and the
The results provide further evidence for a nigrostriatal involvement in pharmacological effects of morphine, since unlike kappa compounds, which predominantly on the spinal cord, morphine has both spinal and s u p r a - s p i sites of action (55). Indeed, the simultaneous measurements of dopamine serotonin in the alive animal after morphine, may well be contributive to basic mechanism of action studies, particularly to further elucidate the
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development of tolerance and physical dependence and in the neurophysiology of pain. Although a role for mu type opiate receptor compounds has been implicated in the regulation of nigrostriatal dopaminergic function (56), whether or not the neurotransmitter pharmacological profile presented here will be true of other mu receptor type opiate compounds, remains to be studied. The mu type opiate receptor compound, morphine, has been shown to regulate serotonin release in mesolimbic areas of rat brain (57). Acknowledgements The author wishes to thank Bridget O'Sullivan, O.P., for secretarial assistance and Marie Buschke, for histological preparations. References i. 2. 3. 4. 5. 6.
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