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Effects of clonidine and yohimbine, alone and in combination with morphine, on supraspinal analgesia
Neuropharmacolog~Vol. 29,No. I,pp.25-29,1990 Printed in Great Britain. All rights reserved
0028-3908/90$3.00+ 0.00 Copyright 0 1990Pergsmon Press plc
EFFECTS OF CLONIDINE AND YOHIMBINE, ALONE AND IN COMBINATION WITH MORPHINE, ON SUPRASP~NAL ANALGESIA S. IZENWASSER*and C. KORNETSKY Laboratory of Behavioral Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118, U.S.A. (Accepted 9 August 1989)
Summary-Morphine raised the threshold for escape from aversive electrical stimulation, delivered to the mesencephalic reticular formation. Clonidine, given alone, had no effect; however, when administered with morphine it blocked the analgesic effect of morphine. Conversely, clonidine, but not morphine, increased the latency to respond to the aversive stimulation, suggesting that clonidine may not have analgesic properties but may merely impair the ability of the animal to respond to the nociceptive stimuIation. Yohimbine produced hyperalgesia and also blocked the effect of morphine. These findings are similar to those seen with dopamine agonists and may be related to the effects of yohimbine on the release of dopamine. I(ey words-analgesia,
clonidine, morphine, norepinephrine,
Although there have been many studies suggesting that norepinephrine plays an important role in modulating the analgesia produced by opiates, its function is still unclear. It has been shown, for example, that stimulation of the locus coeruleus produces analgesia (Margalit and Segal, 1979; Sandberg and Segal, 1978) and that it potentiates antinoci~ption induced by morphine (Segal and Sandberg, 1977). However, contrary evidence, that noxious stimulation increases the firing of these cells, has also been reported (Korf, Bunney, and Aghajanian, 1974). Further, although it has been reported that destruction of the locus coeruleus attenuates the analgesia produced by morphine (Kostowski, Jerlicz, Bidzinski and Hauptmann 1978; Sasa, Munekiyo, Osumi and Takaori, 1977) there have also been reports of no change in antinociception induced by morphine after such lesions (Nakamura, Kuntzman, Maggio, Augulis and Conney, 1973). Pharmacological manipulations of norepineph~ne have also suggested that norepinephrine may play a role in the analgesic effect of morphine. It has previously been reported that nisoxetine, a selective inhibitor of the uptake of norepinephrine, potentiated morphine-induced analgesia, using a supraspinal model of pain (Izenwasser and Kornetsky, 1988). Intracerebroventricular administration of norepinephrine has also been reported to produce analgesia (Handley and Spencer, 1969). Conversely, inhibition of dopamine-eta-hydroxyla~, the enzyme which
converts dopamine to norepinephrine, by U-14,264 (I-phenyl-3-(2-thiazolyl)2-thiourea), also leads to a prolonged analgesic effect of morphine (Watanabe, Matsui and Iwata, 1969; Buxbaum, Yarbrough and Carter, 1973; Bhargava and Way, 1974; Cicero, 1974). Yoh~mbine, an alphas-noradrenergic receptor antagonist, has been reported tc produce analgesia in a hot-plate test (Dennis, Melzack, Gutman and Boucher, 1980), as have phenoxybenzamine and phentolamine, which are also alpha-receptor blockers (Cicero, 1974). Further, systemic administration of clonidine, an alpha,-noradrenergic agonist, has also been shown to produce analgesia in the rat, mouse and dog (Paalzow, 1974; Skingle, Hayes and Tyers, 1982) in such tests of analgesia as the tail-flick (Spadding, Venafro, Ma and Fielding, 1979), the phenylquinone-induced writhing test (Fielding, Wilker, Hynes, Szewczak, Novick and Lal, 1977) and the hot-plate test (Skingle et al., 1982). Thus, there are conflicting results about the role of both norepinephrine itself and of the alpha,-receptor in the mediation of analgesia, with both agonists and antagonists reported to produce analgesia. The present study was conducted to determine the effects of clonidine and yohimbine on the escape threshold and on morphine-induced analgesia, using a supraspinal model of pain. Morphine (Marcus and Kornetsky, 1974; Wheeling, Sasson and Kornetsky, 1981; Sasson, Unterwald and Kornetsky, 1986) and other opioid analgesics, such as heroin (Hubner and Kornetsky, 1985), the mixed agonist/antagonists ~ntaz~ine and cyclazocine (Sasson and Kornetsky, 1986) and ethylketocycl~o~ne (Unterwald, Sasson and Kornetsky, 1987) have been shown to raise
*Address for correspondence: Department of Pharmacology, Uniformed Services University, 4301 Jones Bridge Rd, Bethesda, Maryland 20814-4799, U.S.A. NP 29,‘tb-c
yohimbine.
L3
S.
26
IZENWASSERand
dose-dependently the threshold for escape from electrical stimulation, delivered to the mesencephalic reticular formation, while having no effect on the latency to respond. METHODS
Animal preparation
Four male albino rats (F344 strain, Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts), weighing approximately 300 g, were used in each experiment. Only one of the rats was used in both experiments. Prior to surgery, they were anesthetized with xylazine (13 mg/kg) and ketamine (87 mg/kg). Stainless steel bipolar electrodes, which were 0.13 mm in diameter and insulated except at the tips, were stereotaxically implanted in the mesencephalic reticular formation (MRF), utilizing the following coordinates with the skull levelled between bregma and lambda: - 7.0 mm from brcgma, 2.5 mm lateral to the midline suture, and - 7.0 mm from the skull surface.
C.
KORNETSKY
delayed by the remaining available response time. Responding during intertrial intervals had no scheduled consequences. A modi~cation of the classical psychophysics! method of limits was used to determine the escape threshold. Stimuli were presented in alternating ascending and descending intensities with a step size of 1or 2 PA, depending on the sensitivity of the animal. An ascending series was initiated at a previouslydetermined subthreshold intensity. Three trials were given in succession at each intensity. Two or 3 escape responses were scored as a plus, while less than two were scored as a minus. An ascending series was conducted until plus scores were observed in 2 successive intensity levels. A descending series was then initiated at this intensity level and the current intensities continued to decrease until 2 successive minus scores were observed. The threshold for a particular ascending or descending series was defined as the midpoint between those intensities that delimited the transition from plus to minus scores. Four series, 2 ascending and 2 descending, comprised a session. A session threshold was computed as the mean of the 4 series thresholds. The response latency was defined as the time between the onset of the aversive stimulation and the occurrence of an escape response. This latency was determined at each intensity presented to which the animal escaped. A stimulus intensity vs latency regression line was used to determine the latency at the calculated escape threshold for each session.
Animals were trained and tested in a plastic chamber (21 x 21 x 3Scm), which was enclosed within a light- and sound-attenuating cabinet. Circulation of air and illumination (15 W bulb) were provided. A cyljndrical manipulandum (15 cm in length and 7.5 cm in diameter). mounted within one wall of the chamber, had four equally spaced cams positioned on one of the end plates. Rotating the manipulandum caused the cam to close a microswitch. The occurrence of closures of the microswitch was conveyed to an on-line microprocessor (Sunrise Systems, Pembroke, Massachusetts), which controlled stimulus and response contingencies. A constant current stimulator was used to deliver the stimuli, which consisted of biphasic symmetrical rectangular pulse pairs. Pulse pairs occurred at a frequency of 160 Hz, with a pulse width of 0.2 msec and a delay of 0.2 msec between the initia1 positive pulse and the subsequent negative pulse.
After the first session was completed, the animals were injected su~utaneously with either clonidine or saline, foilowed 20 min later by a subcutaneous injection of either morphine or saline. Both clonidine and morphine were dissolved in isotonic saline and all injections were made in volumes of 1 ml/kg body weight. On control test days, both injections were saline. The second test session began 10 min after the second injection and the duration of each testing session averaged 60 min.
Procedure
Experiment 2
A discrete trial escape task was employed in which each trial was initiated by the onset of stimulation. A response, defined as one half-turn of the manipuiandum, equivalent to 2 microswitch ciosures, was shaped by successive approximations. Animals usually learned the escape response by the end of two or three 45 min training sessions, at intensity levels ranging from 40 to 70 PA, depending on the animal. Responding was reinforced by the immediate termination of the electrical stimulus. If no response was elicited before 7.5 set, the stimulation was automatically terminated and the onset of the next trial occurred approximately 15 set later. If a response occurred prior to termination of the 7.5 set available response time, the onset of the next trial was further
Experiment 2 was conducted exactly as Experiment 1, except that the animals were injected intraperitoneally with either yoh~mbine or vehicle, followed 10 min later by a subcutaneous injection of either morphine or saline. Yohimbine was dissolved in a 10% propylene glycol/distilled water solution, which was used as a control vehicle on non-drug days.
Espwimen t I
Analysis of data
The differences in threshold between the preinjection and post-injection session, for drug days were converted to z-scores based on the mean and standard deviation of changes in threshold for all saline days (z-score of 0 equals mean of all saline days). A z-score is a standardized score, derived from
Norepinephrine
and morphine analgesia
a comparison of the threshold on a given drug day, to the mean threshold of all saline days for an individual animal. A z-score of 1 would represent a value which is one standard deviation away from the mean of all saline days and a z-score of 2 would be two standard deviations away from the mean, etc. Thus, any score which is greater than + 2 (22 SD from the saline mean) would be outside the 95% confidence limits for all saline or vehicle days and would be considered to be significantly different from control data. The latency at threshold calculations were analyzed in a similar fashion. Paired comparison t-tests were used to compare the effect of morphine alone on the threshold to morphine administered together with either clonidine or yohimbine.
4
I
-4
Histology
At the ~mpietion of the experiment, the animals were sacrificed with an overdose of anesthetic. After intracardial perfusion with saline, followed by a 10% formaldehyde solution, the brains were removed and examined histologically to verify the electrode placements.
27
Latency
I
I
I
I
I
0.02
0.04
0.08
0.16
0.32
Dose
of
I mg
clonidine
/ kg
I
Fig. i . Mean f SEM standard score {z-score) of changes in the escape threshold (0) and response latency (0) from pre- to post-drug for clonidine for 4 animals. Saline is reqesented as a z-score of 0 and --- indicates the 95% confidence limits (z-score off 2). Points outside these lines are significantly different from saline.
RESULTS
The mean pre-saline or pre-drug threshold was 29.46 & 3.11 PA and the mean post-saline threshold was 29.32 f 2.91 PA, with the average change in the threshold after saline or vehicle (post minus pre) being -0.14 2 0.26 @A. The mean change in latency (post minus pre) with saline was 0.02 sec. These values were used in calculating the z-scores for changes in threshold or latency after the administration of drug and are represented as a z-score of 0 in the figures. E.yperiment
1
Figure I shows the mean effect of clonidine on the escape threshold and on the latency to respond to the electrical stimulation. Clonidine had no effect on the escape threshold at any of the doses tested in any of the animals tested. In all of the animals tested however, clonidine, in some of the larger doses, increased the latency to respond to the aversive stimulation. Morphine, as previously reported, raised the escape threshold. When administered concomitantly with morphine, 0.04 mg/kg clonidine blocked the analgesic effect of morphine (Fig. 3). Morphine, given alone, had no effect on the response latency and the combination of clonidine and morphine produced no significant increases in latency. Experiment
2
Figure 2 shows the mean effect of yohimbine on the escape threshold and on response latency. Yohimbine led to a significant dose-de~ndent lowering of the escape threshold. It did not, however, have any significant effect on the latency to respond to the stimulation.
Latency
Threshold
-61
I
,
I
1
0.75
1.5
3.0
6.0
Dose
of
yohimbine
I
(mg/kg)
Fig. 2. Mean f SEM standard score (z-score) of changes in the escape threshold (a) and response latency (0) from pre- to post-drug for yohimbine for 4 animals. Saline is represented as a z-score of 0 and --- indicates the 95% confidence fimits (z-score of $2). Points outside these lines are significantly different from saline.
Figure 3 shows the effect of morphine alone and in combination with yohimbine, on the escape threshold. Morphine significantly raised the escape threshold and this effect was blocked by the prior injection of 1.5 mg/kg of yohimbine. Neither morphine alone, nor the combination of morphine and yohimbine, had a significant effect on the latency to respond to the aversive stimulation. DISCUSSION
The findings that both clonidine and yohimbine antagonized the analgesic effect of morphine seems
S.
28
and
IZENWASSER
Morphine
Morphine
+ 0.04
mglkg
Morphine
+ 1.5 mg/kg
Yohimbine
4.0
2.0
Dose
Clonidine
of
morphine
Fig. 3. Mean k SEM standard
score
8.0
(mg/kg) (i-score)
of changes
in
from pre- to post-drug for morphine alone (n = 8) (0) and in combination with either 0.04 mg/kg clonidine (n = 4) (0) or 1.5 mg/kg yohimbine (n = 4) (m). Saline is represented as a z-score of 0 and ~~~ indicates the 95% confidence limits (z-score of + 2). Points outside these lines are significantly different from saline. (*P < 0.01 as compared to morphine alone as determined by a paired comparison r-test.) escape
threshold
since one is an agonist and the other an antagonist at the alpha, -noradrenergic receptor. It has previously been reported that yohimbine antagonizes the duration and efficacy of etorphine on analgesia, as measured with the tail-flick test (Ossipov, Malseed, Eisenman and Goldstein, 1984). Yohimbine, however, while having great affinity for alphaz-noradrenergic receptors, does have effects on other neurotransmitter systems. For example, it has been shown that yohimbine increases the release of dopamine (Van Oene, De Vries and Horn, 1984; Papeschi, 1974; Waldmeier, Ortmann and Bischoff, 1982) and that it does so by blockade of dopamine autoreceptors (Van Oene et al., 1984). It has previously been reported that amfonelic acid, an indirect dopamine agonist, which acts by blocking uptake and increasing the release of dopamine, produced hyperalgesia and blocked morphine-induced analgesia, using this model of pain (Izenwasser and Kornetsky, 1988). The hyperalgesia and blockade of morphine-induced analgesia, seen with yohimbine, are characteristic of dopaminergic modulation and are likely not to be due to interactions with the alpha,-receptor. That clonidine, given alone, did not have an effect on the escape threshold in any of the animals tested, however, is contrary to the numerous published reports of the analgesic effect of clonidine, as measured by other pain tests. For example, it has been shown that clonidine increased the response latency in the radiant heat tail-flick test and in the hot water tail-withdrawal test (Fielding, Wilker, Hynes, Szewczak, Novick and La], 1978; Skingle ef al., 1982). It should be noted, however, that in large doses, clonidine may no longer be paradoxical,
C.
KORNETSKY
selective for alpha,-receptors (Anden, Grabowska and Strombom, 1976; Svensson, Bunney and Aghajanian, 1975; Morpugo, 1968) and that the doses needed to produce analgesia in these studies were quite large. Thus, it is impossible to determine what the underlying mechanism of this putative analgesic effect is. It does appear to be independent of opiate receptors, since the effect is not reversed by naloxone, even at doses as large as 5 mg/kg (Fielding et al., 1978). Further, it has been suggested by Spaulding et al. (I 979) that the antinociceptive effect of clonidine may be mediated entirely at a spinal level. They reported that, in spinally transected mice, morphine-induced analgesia was greatly attenuated, yet there was no effect on the antinociceptive action of clonidine. It has also been shown that, although analgesia produced by etorphine is potentiated by systemicallyadministered clonidine, it is antagonized by clonidine administered centrally into the locus coeruleus (Ossipov et al., 1984). If, as these findings suggest, clonidine-induced analgesia is mediated entirely at a spinal level, it is not surprising that clonidine did not produce analgesia when the brain-stimulation escape model, in which the stimulation is delivered to a supraspinal site, was used. Furthermore, there is evidence that a certain level of norepinephrine in the synapse may play an important role in producing opioid-induced analgesia. Increasing synaptic norepinephrine with nisoxetine. a selective inhibitor of the reuptake of norepinephrine, potentiated morphine-induced analgesia using this model of pain (Izenwasser and Kornetsky, 1988) as well as the tail-flick method (Kellstein, Malseed and Goldstein, 1988) and the writhing test in the mouse (Hynes and Henderson, 1984). This may explain why clonidine, which leads to a decrease in synaptic norepinephrine, did not produce analgesia in the present study. As in the present study, previous studies in this laboratory have shown that prototypical analgesic drugs, such as morphine (Marcus and Kornetsky, 1974; Wheeling et al., 1981) and heroin (Hubner and Kornetsky, 1985) will raise the threshold for escape from intracranial nociceptive stimulation, while having no effect on the response latency. Clanidine had the opposite effect, in that it led to an increase in response latency, while the escape threshold remained unchanged. Additionally, at the larger doses tested, the animals were clearly sedated. Since these other experiments, which suggested that clonidine does have analgesic properties, employed methods which used response latency as the dependent variable, the findings may reflect an action of the drug on a response system and not the perception of pain. Thus, it seems that this model may be able to differentiate between motor or sedative effects and the true analgesic effects of drugs. Acknowledgements-Supported and NIDA
Research
Scientist
by NIDA Grant DA 02326 Award DA 00099 to C.K.
Norepinephrine
and morphine analgesia
REFERENCES
Anden N. E., Grabowska M. and Strombom U. (1976) Different alpha-adrenoceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents. NaunynSchmiedebergs
Arch. Pharmac. 292: 43-52.
Ehargava H. N. and Way E. L. (1974) Effect of l-phenyl3-(2-thiazay~)-2-thiour~~, a dopamine-~-hydroxylase inhibitor, on morphine analgesia, tolerance and -physical dependence. J. Pharmac. exv. Ther. 190: 1655175. Buxdaum D. M., Yarbrough 6. G. and Carter M. E. (1973) Biogenic amines and narcotic effects. 1. Modification of morphine-induced anaIgesia and motor activity after alteration of cerebral amine levels. J. Pharmac. exp. Ther. 185: 317-327.
Cicero T. J. (1974) Effects of alpha-adrenergic blocking agents on narcotic-induced analgesia. Arch. ht. Pharmacodyn. 208: 5-13.
Dennis S. G., Melzack R., Gutman S. and Boucher F. (1980) Pain modulation by adrenergic agents and morphine as measured by three pain tests. tife Sci. 26: 1247-1259.
Fielding S., Wilker J., Hynes M., Szewczak M., Novick W. J. and La1 H. (1977) Antin~i~ptive and antiwithdrawal actions of cionidine: A comparison with morphine. Fed. Proc. 36: 1024. Fielding S., Wilker J., Hynes M., Szewczak M., Novick W. J. and Lal. H. (1978) A comoarison of cionidine with _ morphine for antinociceptive and antiwithdrawal actions. J. Pharmac. exp. Ther. 207: 899-905.
Handley S. L. and Spencer P. S. J. (1969) Analgesic activity after intracerebral injection in the mouse. Br. J. Pharmac. 35: 361-362.
Hubner C. B. and Kornetsky C. (1985) A comparison of the relative potency of heroin and morphine on brainstimulation escape and reward. Sue. Neurosci. Abstr. 11: 1281. Hynes M. D. and Henderson J. K. (1984) Modi~cation of alpha-adrenoceptor agonist antinociceptive activity by nisoxetine: A selective inhibitor of noradrenergic uptake. Pharmac. Biochem. Behav. 20: 463-466.
Izenwasser S. and Kornetsky C. (1988) Potentiation of morphine analgesia by d-amphetamine is mediated by norepinephrine-and not dopamine. Pain 33: 363-368. Kellstein D. E.. Malseed R. T. and Goldstein F. J. (1988) EiIect of chronic tricyclic antidepressant treatment‘ upon intrathecal morphine and monoamine antinociception, Neuropharmacology
27: l-14.
Korf J., Bunney B. S. and Aghajanian G. K. (1974) Noradrenergic neurons: morphine inhibition of spontaneous activity. Eur. J. Pharmac. 25: 165-169. Kostowski W., Jerlicz M., Bidzinski A. and Hauptmann M. (1978) Reduced analgesic effects of morphine after bilateral lesions of the locus coeruleus in rats. Pal. J. Pharmac. Pharm. 30: 49-53.
Marcus R. and Kornetsky C. (1974) Negative and positive intracranial reinforcement threshoids: Effects of morphine. Psychopharmacology 38: I-13. Margalit D. and Segal M. (1979) A pharmacologic study of analgesia produced by stimulation of the nucleus locus coeruleus. Psychopharmacology 62: 169-183. Morpugo C. (1968) Aggressive behavior induced by large doses of 2-(2,6-dichlorophenylamino)-2-imidazoline
hydrochloride
29
(ST 155) in mice. Eur. J. Pharmac. 3:
374-371.
Nakamura K., Kuntzman R., Maggio A. C., Augulis V., Connev A. H. (19731 Influence of 6-hvdroxvdonamine . . . on the effect of morphine on the tail-flick latency. Psychopharmacology 31: 177-189. Ossipov M. H., Malseed R. T., Eisenman L. M. and Goldstein F. J. (1984) Effect of q adrenergic agents upon central etorphine antino~iception in the cat. Brain Res. 309: 135-142.
Paalzow L. (1974)Analgesia produced by clonidine in mice and rats. J. Pharmac. Pharm. 26: 361-363. Papeschi R. (1974) An investigation on the behavioral and hypothermic effects of yohimbine:interaction with drugs affecting central and peripheral monoamines. Archs inf. Pharmacodyn. ThPr. 208: 69.
Sandberg D. E. and Segal M. (1978) Pharmacological analysis of analgesia and self-stimulation elicited by electrical stimulation of catecholamine nuclei in rat brain. Brain Res. 152: 529-542.
Sasa M., Munekiyo K., Osumi Y. and Takaori S. (1977) Attenuation of morphine analgesia in rats with lesions of the locus coeruleus and dorsal raphe nucleus. Eur. f. Pharma&. 42: 53-62.
Sasson S. and Kornetsky C. (1986) Evidence for a supraspinal anaIgesic effect with cyciazocine and pentazocine. Lzje Sci. 38: 21-26. Sasson S., Unterwald E. and Kometsky C. (1986) Potentiation of morphine analgesia by d-amphetamine. Psychopharmac., Berl. 90: 163-165.
Segal M. and Sandberg D. (1977) Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain. Brain Res. 123: 369-372. Skingle M., Hayes A. G. and Tyers M. B. (1982) Antinociceptive activity of clonidine in the mouse, rat and dog. Life Sci. 31: 1123-1132. Spaulding T. C., Venafro J. J., Ma M. G. and Fielding S. (1979) The dissociation of the antinociceptive effect of clonidine from supraspinal structures. ~~ropharmaeulogy 18: 103-105.
Svensson T. H., Bunney 8. S. and Aghajanian G. K. (1975) Inhibition of both noradrenergic and serotonergic neurons in brain by the alpha-adrenergic agonist clonidine. Brain Res. 92: 291-306. Unterwald E. M., Sasson S. and Kornetsky C. (1987) Evaluation of the supraspinal analgesic activity and abuse liability of ethylketocyclazocine. Eur. J. Pharmac. 133: 275-28 1. Van Oene J. C., De Vries J. B. and Horn A. S. (1984) The effectiveness of yohimbine in blocking rat central dopamine autoreceptors in viva. IVaunyn-Schmeidebergs Arch. Pharmac. 327: 3043 11.
Waldmeier P. C., Ortmann R. and Bischoff S. (1982) Modulation of dopaminergic transmission by alphanoradrenergic agonists and antagonists. E~perjeniia 38: 1168-l 176.
Watanabe K., Matsui Y. and Iwata H. (1969) Enhancement of the analgesic effect of morphine by sodium diethyldithiocarbamate in rats. Experientia 25: 950-951. Wheeling H. S., Sasson S. and Kornetsky C. (1981) Tolerance to the effect of morphine on escape from reticular formation stimulation. Subst. Alcoh. Actions/Misuse 2: 107-I 14.
0028-3908/90$3.00+ 0.00 Copyright 0 1990Pergsmon Press plc
EFFECTS OF CLONIDINE AND YOHIMBINE, ALONE AND IN COMBINATION WITH MORPHINE, ON SUPRASP~NAL ANALGESIA S. IZENWASSER*and C. KORNETSKY Laboratory of Behavioral Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118, U.S.A. (Accepted 9 August 1989)
Summary-Morphine raised the threshold for escape from aversive electrical stimulation, delivered to the mesencephalic reticular formation. Clonidine, given alone, had no effect; however, when administered with morphine it blocked the analgesic effect of morphine. Conversely, clonidine, but not morphine, increased the latency to respond to the aversive stimulation, suggesting that clonidine may not have analgesic properties but may merely impair the ability of the animal to respond to the nociceptive stimuIation. Yohimbine produced hyperalgesia and also blocked the effect of morphine. These findings are similar to those seen with dopamine agonists and may be related to the effects of yohimbine on the release of dopamine. I(ey words-analgesia,
clonidine, morphine, norepinephrine,
Although there have been many studies suggesting that norepinephrine plays an important role in modulating the analgesia produced by opiates, its function is still unclear. It has been shown, for example, that stimulation of the locus coeruleus produces analgesia (Margalit and Segal, 1979; Sandberg and Segal, 1978) and that it potentiates antinoci~ption induced by morphine (Segal and Sandberg, 1977). However, contrary evidence, that noxious stimulation increases the firing of these cells, has also been reported (Korf, Bunney, and Aghajanian, 1974). Further, although it has been reported that destruction of the locus coeruleus attenuates the analgesia produced by morphine (Kostowski, Jerlicz, Bidzinski and Hauptmann 1978; Sasa, Munekiyo, Osumi and Takaori, 1977) there have also been reports of no change in antinociception induced by morphine after such lesions (Nakamura, Kuntzman, Maggio, Augulis and Conney, 1973). Pharmacological manipulations of norepineph~ne have also suggested that norepinephrine may play a role in the analgesic effect of morphine. It has previously been reported that nisoxetine, a selective inhibitor of the uptake of norepinephrine, potentiated morphine-induced analgesia, using a supraspinal model of pain (Izenwasser and Kornetsky, 1988). Intracerebroventricular administration of norepinephrine has also been reported to produce analgesia (Handley and Spencer, 1969). Conversely, inhibition of dopamine-eta-hydroxyla~, the enzyme which
converts dopamine to norepinephrine, by U-14,264 (I-phenyl-3-(2-thiazolyl)2-thiourea), also leads to a prolonged analgesic effect of morphine (Watanabe, Matsui and Iwata, 1969; Buxbaum, Yarbrough and Carter, 1973; Bhargava and Way, 1974; Cicero, 1974). Yoh~mbine, an alphas-noradrenergic receptor antagonist, has been reported tc produce analgesia in a hot-plate test (Dennis, Melzack, Gutman and Boucher, 1980), as have phenoxybenzamine and phentolamine, which are also alpha-receptor blockers (Cicero, 1974). Further, systemic administration of clonidine, an alpha,-noradrenergic agonist, has also been shown to produce analgesia in the rat, mouse and dog (Paalzow, 1974; Skingle, Hayes and Tyers, 1982) in such tests of analgesia as the tail-flick (Spadding, Venafro, Ma and Fielding, 1979), the phenylquinone-induced writhing test (Fielding, Wilker, Hynes, Szewczak, Novick and Lal, 1977) and the hot-plate test (Skingle et al., 1982). Thus, there are conflicting results about the role of both norepinephrine itself and of the alpha,-receptor in the mediation of analgesia, with both agonists and antagonists reported to produce analgesia. The present study was conducted to determine the effects of clonidine and yohimbine on the escape threshold and on morphine-induced analgesia, using a supraspinal model of pain. Morphine (Marcus and Kornetsky, 1974; Wheeling, Sasson and Kornetsky, 1981; Sasson, Unterwald and Kornetsky, 1986) and other opioid analgesics, such as heroin (Hubner and Kornetsky, 1985), the mixed agonist/antagonists ~ntaz~ine and cyclazocine (Sasson and Kornetsky, 1986) and ethylketocycl~o~ne (Unterwald, Sasson and Kornetsky, 1987) have been shown to raise
*Address for correspondence: Department of Pharmacology, Uniformed Services University, 4301 Jones Bridge Rd, Bethesda, Maryland 20814-4799, U.S.A. NP 29,‘tb-c
yohimbine.
L3
S.
26
IZENWASSERand
dose-dependently the threshold for escape from electrical stimulation, delivered to the mesencephalic reticular formation, while having no effect on the latency to respond. METHODS
Animal preparation
Four male albino rats (F344 strain, Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts), weighing approximately 300 g, were used in each experiment. Only one of the rats was used in both experiments. Prior to surgery, they were anesthetized with xylazine (13 mg/kg) and ketamine (87 mg/kg). Stainless steel bipolar electrodes, which were 0.13 mm in diameter and insulated except at the tips, were stereotaxically implanted in the mesencephalic reticular formation (MRF), utilizing the following coordinates with the skull levelled between bregma and lambda: - 7.0 mm from brcgma, 2.5 mm lateral to the midline suture, and - 7.0 mm from the skull surface.
C.
KORNETSKY
delayed by the remaining available response time. Responding during intertrial intervals had no scheduled consequences. A modi~cation of the classical psychophysics! method of limits was used to determine the escape threshold. Stimuli were presented in alternating ascending and descending intensities with a step size of 1or 2 PA, depending on the sensitivity of the animal. An ascending series was initiated at a previouslydetermined subthreshold intensity. Three trials were given in succession at each intensity. Two or 3 escape responses were scored as a plus, while less than two were scored as a minus. An ascending series was conducted until plus scores were observed in 2 successive intensity levels. A descending series was then initiated at this intensity level and the current intensities continued to decrease until 2 successive minus scores were observed. The threshold for a particular ascending or descending series was defined as the midpoint between those intensities that delimited the transition from plus to minus scores. Four series, 2 ascending and 2 descending, comprised a session. A session threshold was computed as the mean of the 4 series thresholds. The response latency was defined as the time between the onset of the aversive stimulation and the occurrence of an escape response. This latency was determined at each intensity presented to which the animal escaped. A stimulus intensity vs latency regression line was used to determine the latency at the calculated escape threshold for each session.
Animals were trained and tested in a plastic chamber (21 x 21 x 3Scm), which was enclosed within a light- and sound-attenuating cabinet. Circulation of air and illumination (15 W bulb) were provided. A cyljndrical manipulandum (15 cm in length and 7.5 cm in diameter). mounted within one wall of the chamber, had four equally spaced cams positioned on one of the end plates. Rotating the manipulandum caused the cam to close a microswitch. The occurrence of closures of the microswitch was conveyed to an on-line microprocessor (Sunrise Systems, Pembroke, Massachusetts), which controlled stimulus and response contingencies. A constant current stimulator was used to deliver the stimuli, which consisted of biphasic symmetrical rectangular pulse pairs. Pulse pairs occurred at a frequency of 160 Hz, with a pulse width of 0.2 msec and a delay of 0.2 msec between the initia1 positive pulse and the subsequent negative pulse.
After the first session was completed, the animals were injected su~utaneously with either clonidine or saline, foilowed 20 min later by a subcutaneous injection of either morphine or saline. Both clonidine and morphine were dissolved in isotonic saline and all injections were made in volumes of 1 ml/kg body weight. On control test days, both injections were saline. The second test session began 10 min after the second injection and the duration of each testing session averaged 60 min.
Procedure
Experiment 2
A discrete trial escape task was employed in which each trial was initiated by the onset of stimulation. A response, defined as one half-turn of the manipuiandum, equivalent to 2 microswitch ciosures, was shaped by successive approximations. Animals usually learned the escape response by the end of two or three 45 min training sessions, at intensity levels ranging from 40 to 70 PA, depending on the animal. Responding was reinforced by the immediate termination of the electrical stimulus. If no response was elicited before 7.5 set, the stimulation was automatically terminated and the onset of the next trial occurred approximately 15 set later. If a response occurred prior to termination of the 7.5 set available response time, the onset of the next trial was further
Experiment 2 was conducted exactly as Experiment 1, except that the animals were injected intraperitoneally with either yoh~mbine or vehicle, followed 10 min later by a subcutaneous injection of either morphine or saline. Yohimbine was dissolved in a 10% propylene glycol/distilled water solution, which was used as a control vehicle on non-drug days.
Espwimen t I
Analysis of data
The differences in threshold between the preinjection and post-injection session, for drug days were converted to z-scores based on the mean and standard deviation of changes in threshold for all saline days (z-score of 0 equals mean of all saline days). A z-score is a standardized score, derived from
Norepinephrine
and morphine analgesia
a comparison of the threshold on a given drug day, to the mean threshold of all saline days for an individual animal. A z-score of 1 would represent a value which is one standard deviation away from the mean of all saline days and a z-score of 2 would be two standard deviations away from the mean, etc. Thus, any score which is greater than + 2 (22 SD from the saline mean) would be outside the 95% confidence limits for all saline or vehicle days and would be considered to be significantly different from control data. The latency at threshold calculations were analyzed in a similar fashion. Paired comparison t-tests were used to compare the effect of morphine alone on the threshold to morphine administered together with either clonidine or yohimbine.
4
I
-4
Histology
At the ~mpietion of the experiment, the animals were sacrificed with an overdose of anesthetic. After intracardial perfusion with saline, followed by a 10% formaldehyde solution, the brains were removed and examined histologically to verify the electrode placements.
27
Latency
I
I
I
I
I
0.02
0.04
0.08
0.16
0.32
Dose
of
I mg
clonidine
/ kg
I
Fig. i . Mean f SEM standard score {z-score) of changes in the escape threshold (0) and response latency (0) from pre- to post-drug for clonidine for 4 animals. Saline is reqesented as a z-score of 0 and --- indicates the 95% confidence limits (z-score off 2). Points outside these lines are significantly different from saline.
RESULTS
The mean pre-saline or pre-drug threshold was 29.46 & 3.11 PA and the mean post-saline threshold was 29.32 f 2.91 PA, with the average change in the threshold after saline or vehicle (post minus pre) being -0.14 2 0.26 @A. The mean change in latency (post minus pre) with saline was 0.02 sec. These values were used in calculating the z-scores for changes in threshold or latency after the administration of drug and are represented as a z-score of 0 in the figures. E.yperiment
1
Figure I shows the mean effect of clonidine on the escape threshold and on the latency to respond to the electrical stimulation. Clonidine had no effect on the escape threshold at any of the doses tested in any of the animals tested. In all of the animals tested however, clonidine, in some of the larger doses, increased the latency to respond to the aversive stimulation. Morphine, as previously reported, raised the escape threshold. When administered concomitantly with morphine, 0.04 mg/kg clonidine blocked the analgesic effect of morphine (Fig. 3). Morphine, given alone, had no effect on the response latency and the combination of clonidine and morphine produced no significant increases in latency. Experiment
2
Figure 2 shows the mean effect of yohimbine on the escape threshold and on response latency. Yohimbine led to a significant dose-de~ndent lowering of the escape threshold. It did not, however, have any significant effect on the latency to respond to the stimulation.
Latency
Threshold
-61
I
,
I
1
0.75
1.5
3.0
6.0
Dose
of
yohimbine
I
(mg/kg)
Fig. 2. Mean f SEM standard score (z-score) of changes in the escape threshold (a) and response latency (0) from pre- to post-drug for yohimbine for 4 animals. Saline is represented as a z-score of 0 and --- indicates the 95% confidence fimits (z-score of $2). Points outside these lines are significantly different from saline.
Figure 3 shows the effect of morphine alone and in combination with yohimbine, on the escape threshold. Morphine significantly raised the escape threshold and this effect was blocked by the prior injection of 1.5 mg/kg of yohimbine. Neither morphine alone, nor the combination of morphine and yohimbine, had a significant effect on the latency to respond to the aversive stimulation. DISCUSSION
The findings that both clonidine and yohimbine antagonized the analgesic effect of morphine seems
S.
28
and
IZENWASSER
Morphine
Morphine
+ 0.04
mglkg
Morphine
+ 1.5 mg/kg
Yohimbine
4.0
2.0
Dose
Clonidine
of
morphine
Fig. 3. Mean k SEM standard
score
8.0
(mg/kg) (i-score)
of changes
in
from pre- to post-drug for morphine alone (n = 8) (0) and in combination with either 0.04 mg/kg clonidine (n = 4) (0) or 1.5 mg/kg yohimbine (n = 4) (m). Saline is represented as a z-score of 0 and ~~~ indicates the 95% confidence limits (z-score of + 2). Points outside these lines are significantly different from saline. (*P < 0.01 as compared to morphine alone as determined by a paired comparison r-test.) escape
threshold
since one is an agonist and the other an antagonist at the alpha, -noradrenergic receptor. It has previously been reported that yohimbine antagonizes the duration and efficacy of etorphine on analgesia, as measured with the tail-flick test (Ossipov, Malseed, Eisenman and Goldstein, 1984). Yohimbine, however, while having great affinity for alphaz-noradrenergic receptors, does have effects on other neurotransmitter systems. For example, it has been shown that yohimbine increases the release of dopamine (Van Oene, De Vries and Horn, 1984; Papeschi, 1974; Waldmeier, Ortmann and Bischoff, 1982) and that it does so by blockade of dopamine autoreceptors (Van Oene et al., 1984). It has previously been reported that amfonelic acid, an indirect dopamine agonist, which acts by blocking uptake and increasing the release of dopamine, produced hyperalgesia and blocked morphine-induced analgesia, using this model of pain (Izenwasser and Kornetsky, 1988). The hyperalgesia and blockade of morphine-induced analgesia, seen with yohimbine, are characteristic of dopaminergic modulation and are likely not to be due to interactions with the alpha,-receptor. That clonidine, given alone, did not have an effect on the escape threshold in any of the animals tested, however, is contrary to the numerous published reports of the analgesic effect of clonidine, as measured by other pain tests. For example, it has been shown that clonidine increased the response latency in the radiant heat tail-flick test and in the hot water tail-withdrawal test (Fielding, Wilker, Hynes, Szewczak, Novick and La], 1978; Skingle ef al., 1982). It should be noted, however, that in large doses, clonidine may no longer be paradoxical,
C.
KORNETSKY
selective for alpha,-receptors (Anden, Grabowska and Strombom, 1976; Svensson, Bunney and Aghajanian, 1975; Morpugo, 1968) and that the doses needed to produce analgesia in these studies were quite large. Thus, it is impossible to determine what the underlying mechanism of this putative analgesic effect is. It does appear to be independent of opiate receptors, since the effect is not reversed by naloxone, even at doses as large as 5 mg/kg (Fielding et al., 1978). Further, it has been suggested by Spaulding et al. (I 979) that the antinociceptive effect of clonidine may be mediated entirely at a spinal level. They reported that, in spinally transected mice, morphine-induced analgesia was greatly attenuated, yet there was no effect on the antinociceptive action of clonidine. It has also been shown that, although analgesia produced by etorphine is potentiated by systemicallyadministered clonidine, it is antagonized by clonidine administered centrally into the locus coeruleus (Ossipov et al., 1984). If, as these findings suggest, clonidine-induced analgesia is mediated entirely at a spinal level, it is not surprising that clonidine did not produce analgesia when the brain-stimulation escape model, in which the stimulation is delivered to a supraspinal site, was used. Furthermore, there is evidence that a certain level of norepinephrine in the synapse may play an important role in producing opioid-induced analgesia. Increasing synaptic norepinephrine with nisoxetine. a selective inhibitor of the reuptake of norepinephrine, potentiated morphine-induced analgesia using this model of pain (Izenwasser and Kornetsky, 1988) as well as the tail-flick method (Kellstein, Malseed and Goldstein, 1988) and the writhing test in the mouse (Hynes and Henderson, 1984). This may explain why clonidine, which leads to a decrease in synaptic norepinephrine, did not produce analgesia in the present study. As in the present study, previous studies in this laboratory have shown that prototypical analgesic drugs, such as morphine (Marcus and Kornetsky, 1974; Wheeling et al., 1981) and heroin (Hubner and Kornetsky, 1985) will raise the threshold for escape from intracranial nociceptive stimulation, while having no effect on the response latency. Clanidine had the opposite effect, in that it led to an increase in response latency, while the escape threshold remained unchanged. Additionally, at the larger doses tested, the animals were clearly sedated. Since these other experiments, which suggested that clonidine does have analgesic properties, employed methods which used response latency as the dependent variable, the findings may reflect an action of the drug on a response system and not the perception of pain. Thus, it seems that this model may be able to differentiate between motor or sedative effects and the true analgesic effects of drugs. Acknowledgements-Supported and NIDA
Research
Scientist
by NIDA Grant DA 02326 Award DA 00099 to C.K.
Norepinephrine
and morphine analgesia
REFERENCES
Anden N. E., Grabowska M. and Strombom U. (1976) Different alpha-adrenoceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents. NaunynSchmiedebergs
Arch. Pharmac. 292: 43-52.
Ehargava H. N. and Way E. L. (1974) Effect of l-phenyl3-(2-thiazay~)-2-thiour~~, a dopamine-~-hydroxylase inhibitor, on morphine analgesia, tolerance and -physical dependence. J. Pharmac. exv. Ther. 190: 1655175. Buxdaum D. M., Yarbrough 6. G. and Carter M. E. (1973) Biogenic amines and narcotic effects. 1. Modification of morphine-induced anaIgesia and motor activity after alteration of cerebral amine levels. J. Pharmac. exp. Ther. 185: 317-327.
Cicero T. J. (1974) Effects of alpha-adrenergic blocking agents on narcotic-induced analgesia. Arch. ht. Pharmacodyn. 208: 5-13.
Dennis S. G., Melzack R., Gutman S. and Boucher F. (1980) Pain modulation by adrenergic agents and morphine as measured by three pain tests. tife Sci. 26: 1247-1259.
Fielding S., Wilker J., Hynes M., Szewczak M., Novick W. J. and La1 H. (1977) Antin~i~ptive and antiwithdrawal actions of cionidine: A comparison with morphine. Fed. Proc. 36: 1024. Fielding S., Wilker J., Hynes M., Szewczak M., Novick W. J. and Lal. H. (1978) A comoarison of cionidine with _ morphine for antinociceptive and antiwithdrawal actions. J. Pharmac. exp. Ther. 207: 899-905.
Handley S. L. and Spencer P. S. J. (1969) Analgesic activity after intracerebral injection in the mouse. Br. J. Pharmac. 35: 361-362.
Hubner C. B. and Kornetsky C. (1985) A comparison of the relative potency of heroin and morphine on brainstimulation escape and reward. Sue. Neurosci. Abstr. 11: 1281. Hynes M. D. and Henderson J. K. (1984) Modi~cation of alpha-adrenoceptor agonist antinociceptive activity by nisoxetine: A selective inhibitor of noradrenergic uptake. Pharmac. Biochem. Behav. 20: 463-466.
Izenwasser S. and Kornetsky C. (1988) Potentiation of morphine analgesia by d-amphetamine is mediated by norepinephrine-and not dopamine. Pain 33: 363-368. Kellstein D. E.. Malseed R. T. and Goldstein F. J. (1988) EiIect of chronic tricyclic antidepressant treatment‘ upon intrathecal morphine and monoamine antinociception, Neuropharmacology
27: l-14.
Korf J., Bunney B. S. and Aghajanian G. K. (1974) Noradrenergic neurons: morphine inhibition of spontaneous activity. Eur. J. Pharmac. 25: 165-169. Kostowski W., Jerlicz M., Bidzinski A. and Hauptmann M. (1978) Reduced analgesic effects of morphine after bilateral lesions of the locus coeruleus in rats. Pal. J. Pharmac. Pharm. 30: 49-53.
Marcus R. and Kornetsky C. (1974) Negative and positive intracranial reinforcement threshoids: Effects of morphine. Psychopharmacology 38: I-13. Margalit D. and Segal M. (1979) A pharmacologic study of analgesia produced by stimulation of the nucleus locus coeruleus. Psychopharmacology 62: 169-183. Morpugo C. (1968) Aggressive behavior induced by large doses of 2-(2,6-dichlorophenylamino)-2-imidazoline
hydrochloride
29
(ST 155) in mice. Eur. J. Pharmac. 3:
374-371.
Nakamura K., Kuntzman R., Maggio A. C., Augulis V., Connev A. H. (19731 Influence of 6-hvdroxvdonamine . . . on the effect of morphine on the tail-flick latency. Psychopharmacology 31: 177-189. Ossipov M. H., Malseed R. T., Eisenman L. M. and Goldstein F. J. (1984) Effect of q adrenergic agents upon central etorphine antino~iception in the cat. Brain Res. 309: 135-142.
Paalzow L. (1974)Analgesia produced by clonidine in mice and rats. J. Pharmac. Pharm. 26: 361-363. Papeschi R. (1974) An investigation on the behavioral and hypothermic effects of yohimbine:interaction with drugs affecting central and peripheral monoamines. Archs inf. Pharmacodyn. ThPr. 208: 69.
Sandberg D. E. and Segal M. (1978) Pharmacological analysis of analgesia and self-stimulation elicited by electrical stimulation of catecholamine nuclei in rat brain. Brain Res. 152: 529-542.
Sasa M., Munekiyo K., Osumi Y. and Takaori S. (1977) Attenuation of morphine analgesia in rats with lesions of the locus coeruleus and dorsal raphe nucleus. Eur. f. Pharma&. 42: 53-62.
Sasson S. and Kornetsky C. (1986) Evidence for a supraspinal anaIgesic effect with cyciazocine and pentazocine. Lzje Sci. 38: 21-26. Sasson S., Unterwald E. and Kometsky C. (1986) Potentiation of morphine analgesia by d-amphetamine. Psychopharmac., Berl. 90: 163-165.
Segal M. and Sandberg D. (1977) Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain. Brain Res. 123: 369-372. Skingle M., Hayes A. G. and Tyers M. B. (1982) Antinociceptive activity of clonidine in the mouse, rat and dog. Life Sci. 31: 1123-1132. Spaulding T. C., Venafro J. J., Ma M. G. and Fielding S. (1979) The dissociation of the antinociceptive effect of clonidine from supraspinal structures. ~~ropharmaeulogy 18: 103-105.
Svensson T. H., Bunney 8. S. and Aghajanian G. K. (1975) Inhibition of both noradrenergic and serotonergic neurons in brain by the alpha-adrenergic agonist clonidine. Brain Res. 92: 291-306. Unterwald E. M., Sasson S. and Kornetsky C. (1987) Evaluation of the supraspinal analgesic activity and abuse liability of ethylketocyclazocine. Eur. J. Pharmac. 133: 275-28 1. Van Oene J. C., De Vries J. B. and Horn A. S. (1984) The effectiveness of yohimbine in blocking rat central dopamine autoreceptors in viva. IVaunyn-Schmeidebergs Arch. Pharmac. 327: 3043 11.
Waldmeier P. C., Ortmann R. and Bischoff S. (1982) Modulation of dopaminergic transmission by alphanoradrenergic agonists and antagonists. E~perjeniia 38: 1168-l 176.
Watanabe K., Matsui Y. and Iwata H. (1969) Enhancement of the analgesic effect of morphine by sodium diethyldithiocarbamate in rats. Experientia 25: 950-951. Wheeling H. S., Sasson S. and Kornetsky C. (1981) Tolerance to the effect of morphine on escape from reticular formation stimulation. Subst. Alcoh. Actions/Misuse 2: 107-I 14.