- Email: [email protected]
Pain modulation by adrenergic agents and morphine as measured by three pain tests
Life Sciences, Vol. 26, pp. 1247-1259 Printed in the U.S.A.
Pergamon Press
PAIN MODULATION BY ADRENERGIC AGENTS AND MORPHINE AS MEASURED BY THREE PAIN TESTS Stephen G. Dennis~ Ronald Melzack, Samuel Gutman, and Frangoise Boucher Department of Psychology, McGill University 1205 Avenue Docteur Penfield Montreal, Quebec, Canada H3A IBI (Received in final form February 11, 19"80) SUMMARY The effects of several adrenergic agents on pain and morphine analgesia were assessed using three pain tests in rats. These tests--Tail-Flick, Hot-Plate, and Formalin--allow comparison of the effects of different noxious stimuli and different motor responses. Each pain test yielded a unique constellation of adrenergic influences, suggesting that variation of stimulus and response parameters can change the functional expression of adrenergic systems related to pain processing. The salient drug effects include: i) a pronounced, relatively selective analgesic effect of yohimbine in the Hot-Plate test; 2) a selective analgesic effect of clonidine in the Formalin test; 3) a striking, but variable, antagonism of morphine analgesia by a combination of yohimbine and propranolol in the Formalin test; 4) a nonlinear dose-response curve for antagonism of morphine analgesia by p r o p r a n o l o l i n t h e Hot-Plate test; and 5) a generalized interference with pain responding and enhancement of morphine analgesia by most drugs in the Formalin test. The data suggest that the type of pain test is crucial in determining the pattern of drug influences that is revealed. Interactions of opiate analgesics with drugs known to affect certain neurochemical systems, notably the monoamines, have long been studied. In particular, a substantial amount of research has been done on the effects of adrenergic agents on morphine analgesia (cf. i). Unfortunately, the salient feature of this
aAddress reprint requests to Dr. Melzack. address is Neurosciences Research Program, Jamaica Plain Station, Boston, MA 02130
Dr. Dennis' current 165 Allandale Street,
0024-3205/80/151247-13502.00/0 Copyright (c) 1980 Pergamon Press Lid
1248
Adrenerglc Effects on Morphine Analgesia
Vol. 26, No. 15. 1980
literature, part of which is sun~arized in Table I, is its variability. Several experiments have demonstrated the antagonism of morphine analgesia by adrenergic systems, others have suggested enhancement, while still others have found no significant effect. On the basis of the available data, it appears that adrenergic systems do interact with morphine in the course of pain assessment procedures, but it is difficult to predict with confidence precisely how they will interact under any given set of circumstances. Clearly, it would be useful to know the sources of this variability. TABLE I Effects of Adrenergic Antagonists, Agonists, and Precursors on Morphine Analgesia a Drug Class
Enhance
No Effect
Antagonize
e-Antagonists b
2, 3, 4
5, 6, 7, 8, 9, I0
4, ii, 12, 13
8-Antagonists c
12, 14
2, 5, 6, 7, 9, I0, 15
3, 8, 16
Agonists & Precursors d
5, 8, 17, 18
19
20, 21, 22
aEntries are reference numbers. Experiments were predominantly on rats or mice. Various pain tests and drug dosages were used. bIncludes yohimbine, phentolamine, azapetine, dihydroergotamine. CIncludes
propranolol,
phenoxybenzamine,
dichloroisoproterenol,
dIncludes clonidine, isoproterenol, norepinephrine, L-tyrosine.
tolazoline,
pronethalol.
methozamine,
epinephrine,
Among the many potential sources of variation in this paradigm, the type of pain test frequently has been cited as critical. Certain pain tests are apparently more suitable than others for demonstrating particular drug effects. However, in general it is not known precisely what makes one pain test different from another with respect to assessing drug interactions. The present experiments approach this behavioral question by comparing three pain tests selected to emphasize two major variables: I) the physical and temporal properties of the noxious stimulus; and 2) the pattern of the required motor response. The former is assessed by comparing the Hot-Plate (23) and Formalin (24) tests, which record the same general movement pattern but in response to markedly different noxious stimuli. The latter is assessed by comparing the Hot-Plate and Tail-Flick (25) tests, both of which use noxious heat but which trigger quite different responses. Insofar as possible, all other factors are held constant. The purpose of this study,
therefore,
is to examine
the el-
Vol. 26, No. 15, 1 9 8 0
Adrenerglc Effects on Morphine Analgesia
1249
fects of adrenergic agents on pain and morphine analgesia, and to see whether the use of different pain tests leads to qualitatively different profiles of the neural substrates that signal or modulate pain.
METHODS Subjects. Male, Sprague-Dawley rats (Canadian Breeding Fazms & Laboratories) weighing 325-400 g were used throughout. Rats were housed three to a cage with food and water always present. A 14 hr light--10 hr dark schedule was imposed, and testing was done during the light hours. Behavioral Tests. For the Tail-Flick test, the rat was clceely restrained in a wire mesh cage, and radiant heat was applied to a blackened portion of the tail approximately 1.5-2.0 cm from the tip. The latency to the first movement of the tail (usually a sudden flicking or lashing) was recorded. The heat source was adjusted to give an average baseline latency of about 3.5 sec in a sample of untreated rats. In the absence of a response, the heat source was switched off automatically at 7.0 sec, and this m a x i m u m value was recorded. Four trials were run with 2-min intertrial intervals. For the Hot-Plate test, the rat was placed on a grid of stainless steel tubes 0.5 cm in outer diameter and spaced 1.2 cm apart. Heated distilled water was pumped continuously by a constant temperature circulator. Bath temperature was m a i n t a i n e d at 57.0"C, usually about 1.5°C higher than the grid surface temperature. The latency to lick either hind paw was taken as the primary response measure. Front paw-lick latencies were also estimated for the most rats. Two trials separated by a 3-min interval were run, and a 30 sec cutoff was imposed. The Formalin test has been described in detail elsewhere (24). Briefly, 0.05 ml of 5% formalin was injected subcutaneously in the dorsal surface of the forepaw. This provoked a series of favoring responses characterized by elevation and occasional licking of the paw. A weighted average of time spent in four basic categories was taken as the primary response measure. The categories and weighting factors were: 0) full weight is placed on the paw; I) the injected paw rests lightly on the floor and bears no weight; 2) the injected paw is selectively elevated and in contact with no other surface; and 3) the paw is licked or bitten. Cumulated data from the second half-hour postinjection were used in comparisons with the other pain tests, except in the naloxone experiments where the last 15 min was taken. In addition to the three pain tests, a modified Bar test for catalepsy (lack of spontaneous movement) was used (26). The r@t's forepaws were placed on a bar 0.5 cm in diameter at a height of 10 cm from the floor. The latency for the rat to remove both forepaws from the bar was recorded. Two trials were run with a 30 sec cutoff imposed. Except for possible discomfort during handling, this behavioral test was painless. The Tail-Flick, Hot-Plate, and Bar tests were run as a battery, each rat going through the entire sequence of tests. The
1250
Adrenerglc Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
sequence always started and ended with a Bar trial, and the order of pain testing was counterbalanced. Separate groups of rats were run in the Formalin test. Drug Tests. Table II shows the drugs and dosages used. Injection times were calculated relative to the time of the morphine or saline control injection. Taking this point as zero, prior events were given a negative sign, and subsequent events a positive sign. Thus, clonidine (CLN) was injected 30 min before morphine or normal saline. In the Formalin test, the formalin injection always occurred at +15 min, and data were analyzed for the +45 to +75 min period. The Tail-Flick, Hot-Plate, and Bar tests were all run from +50 to about +70 min. Since all drug injections were based on the same zero time point, comparisons of a single drug across all behavioral tests fall within the same average injection-test interval. All injections were done intraperitoneally. For about 80% of the animals, the observers were kept "blind" as to the drugs used, their putative effects, or the hypothesis under consideration. TABLE II D r u g Doses, Injection Times, and Major Effects Drug
Abbreviation
Doses (mg/kg) a
Injection Time (min)
Effect b
Clonidine
CLN
.03,.15
-30
e-Agonist
Isoproterenol HCI
ISO
.5, 4
-I0
8-Agonist
Yohimbine HCI
YOH
I, 4
-15
e-Antagonist
Propranolol HCI
PRO
i, 4, 8
-5
B-Antago~st
Naloxone HCI
NAL
5
+45
Opiate Antagonist
Morphine Sulfate
MOR
2.5, i0
0
Opiate Analgesic
aAll doses refer to the weight of the salt. bRefers to major effects at indicated-doses. Statistical Analysis. The method of overall analysis was Dunnett's procedure for multiple comparisons with a control (27). The otherwise untreated morphine or saline groups were taken as controls, and individual analyses were run on each behavioral test. A value of p<.05 was considered significant. Individual t-tests or analyses of variance were done as necessary. Group means, together with the standard error (M ± SEM), are expressed in seconds for the Hot-Plate, Tail-Flick, and Bar tests, and in arbitrary units for the Formalin test.
Vol. 26, No. 15, 1 9 8 0
Adrenetglc Effects on Morphine Analgesla
1251
RESULTS Morphine Analgesia and its Reversal by Naloxone. Morphine (10 mg/kg) significantly reduced pain responding in all three tests, and naloxone (5 mg/kg) antagonized this effect in each test (see Fig. 1). This dose of morphine produced sedation, as reflected in a small but significant elevation of Bar test scores (SAL: M = 3.2 ± 0.7; MORI0: M = 8.1 ± 1.7; t = 2.70, df = 30, p<.02), although the animals moved normally when lightly prodded. In the Formalin test, where the time course of naloxone's effect was continuously monitored, reversal of sedation usually preceded reversal of analgesia by 5-8 min. Effects of Adrener~ic Drugs A10ne. Figure 2 shows the baseline effects on pain responding of the adrenergic drugs used. Each pain test presents a different pattern of effects. In the Tail-Flick test, the only significant effect was a modest elevation of latencies produced by 4 mg/kg propranolol. Higher (8 mg/kg) and lower (I mg/kg) doses had no effect. In the Hot-Plate test, yohimbine (1 and 4 mg/kg) significantly prolonged response latencies. This apparent analgesia was comparable to or'exceeded a i0 mg/kg dose of morphine (cf. Fig. 1). However, naloxone did not antagonize yohimbine analgesia (YOH4-SAL: M = 28.7 ± 0.6; YOH4-NAL5: M = 30.0 ± 0.0; t 2.00, df = 14, p>.05). Forepaw-lick latencies were also elevated by yohimbine (see Table III). Propranolol showed a tendency • t the higher doses (4 and 8 mg/kg) toward reduced latencies
TAIL-FLICK
~
HOT-PLATE
250
FORMALJN
°I
6.0
>. 5.0.-
46 SAL MOR MORIO 0 NAL 5
SAt MOR MOR~0 10 NAL5
SAL MOR MOA~O t0 NAL5
FIG. 1 Effects of morphine and naloxone as measured by three pain tests. Note that the scale on the Formalin test ordinate has been inverted to conform to the "analgesia up" convention of the other graphs. Asterisks indicate statistically significant differences (p<.01) compared to the MORI0 group for each pain test. Standard errors are shown. Numbers on the bars indicate N per group.
1252
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
(hyperalgesia). This is not significant in the overall analysis, although individual post-hoc comparisons are significant (t[PR04 vs SAL] = 2.59, df = 30, p<.02; t[PRO8 vs SAL] = 3.69, df = 22, p<.002). When both propranolol and yohimbine were injected, no significant effects were observed. Thus, the apparent analgesia produced by yohimbine in this test was not m a n i f e s t e d in the presence of propranolol.
SAL
T.O ~
CLN .03 ,15
05
[SO 4.0
YOH ].0 4.0
tO
PRO 4.0 8.0
YOH| YOH4 PRO t 1 ~ 0 4
6.0
w
~'~
5.0
..1¢~
~'~
4.0
~
3.0
30.0
4=
25.0 u LU
,., ~ ZO.O
I
I,-
,
W
"I" ...1
5.0
0.0
+
0.'~ co
Z
~=~ ,0 t.5
n IIiNI m
8
181
8
FIG. 2 Effects of adrenergic agents on baseline responding in three pain tests. Conventions as In Fig. i, and all groups are compared to the SAL control at the left of each panel. Propranolol (4 mg/kg) increases latencies in the Tail-Flick test, while yohimbine (i and 4 mg/kg) increases latencies in the Hot-Plate test. In the Formalin test, all agents interfere somewhat with responding, the most powerful effect being that of clonidine (0.03 and 0.15 mg/kg). These data show that each behavioral test yields a different pharmacological profile of adrenergic influences.
Vol. 26, No. 15, 1 9 8 0
Adrenerglc Effects on Morphine Analgesia
1253
A strikingly different pattern of results was seen in the Formalin test. Powerful dose-dependent analgesia was observed with clonidine (0.03 and 0.15 mg/kg) and isoproterenol (4 mg/kg). Clonidine's effect rivaled morphine's in magnitude (cf. Fig. 1) but was unaffected by 5 mg/kg naloxone (CLN. 15-SAL: M = 0.i ± 0.1; CLN. 15-NAL5: M = 0.2 ± 0.1; t = 1.75, df = 14, p>.2). Yohimbine (1 mg/kg) partially reversed clonidine's effect (CLN.15SAL: M = 0.i ± 0.i; CLN.15-YOHI: M = 0.7 ± 0.i; t = 5.11, df = 14, p<.001). Yohimbine (i and 4 mg/kg) and propranolol (1 and 4 mg/kg) also reduced pain responding in this test, although the effects were not obviously dose-dependent. The combination of uand B-blockers showed the same effect as either agent alone. The overall analysis of Bar scores revealed no significant effects, indicating that at the doses employed, none of these drugs interfered with the execution of this simple dismounting response. Interaction of Adrenergic Dru@s with Morphine. Figure 3 shows the effects of combinations of morphine and the various adrenergic drugs in the Tail-Flick, Hot-Plate, and Formalin tests. In the Tail-Flick test, propranolol (1 mg/kg) significantly interfered with the analgesia produced by morphine. Neither of the higher doses of propranolol had a significant effect, although the difference between these two groups is significant ( t [ P R 0 4 v s P R O 8 ] - 2.28, df = 22, p<.05). No other effects were Bignificant in the overall analysis. In the Hot-Plate test, the antagonistic effect of propranolol (i mg/kg) was also observed, but in addition, the 8 mg/kg dose had a significant effect. The 4 mg/kg dose remained ineffective. Yohimbine (4 mg/kg) significantly enhanced latencies o v e r the morphine control levels. However, because of possible ceiling effects in these data, the strength of yohimbine's enhancement is uncertain. Comparable data on forepaw-lick latencies (Table III), where there is no ceiling effect, are consistent with an additive model of yohimbine and morphine analgesia in this test. In the Formalin test, the only measurable departure from control levels was a significant antagonism of analgesia produced by the combination of yohimbine (i mg/kg) and propranolol (i mg/ kg). This effect was highly variable, with four rats showing complete antagonism while the remaining four were less affected. Antagonism was usually characterized by prolonged periods of licking or biting the injected paw (rating 3). This striking antagonism was also seen in a separate experiment in which several of the Formalin test conditions were replicated in rats deprived of food for 18 hr prior to testing. A g a i n , however, there was substantial individual variability. In order to characterize better the interactions of adrenergic agents and morphine in the Formalin test, a study was carried out with a lower dose of morphine (2.5 mg/kg). The results, shown in Fig. 4, demonstrate the improvement of analgesia by each drug tested. Comparison with the data in Fig. 2 suggests that the improvement was at least additive in all cases, and possibly multiplicative for clonidine, isoproterenol, and the combination of yohimbine and propranolol. It is of interest that the com-
1254
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
bination of antagonists, which tended to antagonize the larger dose of morphine, had no such effect on the smaller dose. In the Bar test, a significant elevation of latencies over morphine control levels was seen with clonidine (0.15 mg/kg). This correlated with a powerful reduction of spontaneous activity produced by this drug combination. However, these rats generally
7.0
MOR I0
CLN .03 .15
YOH
1.0
4Q
1.0
PRO 4.0 +
8.0
YOHI PROt
s,ot
..,
f.o
,6
I
91 a
30.0
f
~25.0
÷
t~J
"9'20.0
J
I,,- I,,,-
-e. _j I 0 . 0
511
+
le
le
12
*+
+
j
"!-
0,5 Z
zF--
?,~ ,,o ~_ !
8
2.0
FIG.
:3
8
2
3
3
Interaction of adrenergic agents with morphine in three pain tests. Conventions as in Fig. i, with all groups compared to the MORI0 group at the left of each panel. Asterisks indicate p<.01; pluses indicate p<.05. Propranolol (1 mg/kg) reduces morphine analgesia in both the Tail-Flick and Hot-Plate tests but not in the Formalin test, while propranolol at 8 mg/kg is effective only in the Hot-Plate test. Yohimbine increases analgesia in the Hot-Plate test. In the Formalin test, the combination of yohimbine and propranolol significantly antagonizes morphine's effect. The different patterns of effects suggest that each pain test taps a different constellation of adrenergic-opiate interactions.
Vol. 26, No. 15, 1 9 8 0
Adrenergic Effects on Morphine Ana18esla
1255
tended to struggle when handled and they reacted well to p r o d d i n ~ Hot-Plate scores also indicate that the rats retained the capacity to make coordinated movements to certain noxious stimuli under the appropriate conditions. TABLE III Effects of Yohimbine and Morphine on Forepaw Latencies in the Hot-Plate Test Drug,
Dose
(mg/kg)
Mean Latency Saline
Saline Control
Yohimbine
1.0
Yohimbine
4.0
(in sec ± SEM) Morphine
4-+1
9±2
(n+8)
(n-8)
9±2 (n=8)
14±4 (n=8)
13 -+ 2
22 -+ 3
• (n=16)
MOR
0.0
(10 mg/kg)
CLN
t$O
.03
.5
(n=12)
YOH |.0
4.0
PRO tO 4 D
+,
(D Z 0.5
z~-
YOH 1.0 PRO t.0
I
~ l : ~ ~,o
¢I.
~.
~.
11.
8
$
8
6
i8
8
8
FIG. 4 Effects of adrenergic agents in combination with a lower dose of morphine (2.5 mg/kg) in the Formalin test. Conventions as in previous figures. Each agent is shown to produce analgesia in combination with this dose of morphine.
1256
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, !980
DISCUSSION These data show that the type of pain test is crucial in determining the pattern of analgesic or anti-analgesic effects of various adrenergic drugs either alone or in combination with morphine. Drug effects that are salient in one test may be absent or even reversed in another test. Since generalization of results from one test to another cannot be assumed, a single pain test is insufficient to characterize the adrenergic pharmacology of pain and analgesia. The variability of effects found in the present study resembles that reported in the literature (see Table I), except that in the present case, an effort has been made to isolate certain behavioral factors as the primary independent variables. Other factors such as species, strain, diet, diurnal cycle, housing, social factors, and prior stress have been held constant, insofar as possible. That the pattern of results still varies attests to the importance of the type of pain test as a source of the variability found in the literature. The data point to two factors that may contribute to the different pharmacological profiles. The first, stimulus differences, may be inferred from comparisons of the Hot-Plate and Formalin tests. These tests share at least one common motor sequence--elevation and licking of a limb--as an index of pain. However, the noxious stimuli used to evoke this response are markedly different: in the one case/ a non-damaging, escapable heat stimulus, and in the other, inescapable pain that undoubtedly arises from subcutaneous tissue damage. The salient pharmacological differences observed in the present experiments include: i) contrasting effects of adrenergic agonists, notably the uagonist clonidine; 2) differing efficacies of the e-antagonist yohimbine; 3) contrasting effects of propranolol, a 8-antagonist; and 4) contrasting effects of the combination of yohimbine and propranolol. Although the response measures for the Hot-Plate and Formalin tests share important features, they are not identical, and therefore one must avoid drawing too strong an inference regarding stimulus differences from these data. It is possible that certain of the observed pharmacological differences pertain to subtle motor differences. In any case, these experiments encourage further procedural refinements in order to produce more nearly identical behavior patterns in response to different stimyli. The physical and temporal differences between the stimuli used in the Hot-Plate and Formalin tests may be useful in evaluating recent proposals regarding the neural basis of pain-signalling (28,29). We have argued that long-lasting pain of pathological origin may be processed differently than transient pain arising from brief stimuli or at the onset of injury. It is well known that the analgesic efficacy of morphine differs with respect to these types of stimuli (30). Likewise, analgesia produced by central electrical stimulation (cf. 31), which may engage endogenous opioid systems (32,33), is more effective against long-lasting pathological pain than agains brief, experimental pain (34,35). The pharmacological differences observed in the present experiments are consistent with the notion
Vol. 26, No. 15, 1980
Adrenersic Effects on Morphine AnalEesla
1257
that such "tonic" and "phasic" pain stimuli are processed differently in the nervous system (28,29). Comparisons of the Hot-" Plate and Formalin tests might provide a useful animal model for further development of this hypothesis. Response differences appear to be the second major factor that contributes to the different pharmacological profiles. In the present study, this factor is inferred from comparisons of the Tail-Flick and Hot-Plate tests. In both tests, the noxious stimulus is heat, but in one case, it is applied to the tail and evokes a reflexive lashing or flicking response, while in the other case, the paws are heated evoking a series of responses culminating in elevation and licking. The main pharmacological differences observed under the present conditions are: i) contrasting effects of the u-antagonist yohimbine; and 2) differential effects of the higher doses of propranolol. The specific motor differences that contribute to the pharmacological profiles remain to be determined. One possibility that has been implicated in other studies (4,36) is the "level" of response integration along the neuraxis. Paalzow and Paalzow (4) have shown clear pharmacological differences in morphine analgesia in a tail-stimulation/vocalization test, depending on whether vocalization was recorded during stimulation, or following it. These two responses are thought to be integrated at brainstem and diencephalic levels respectively. The Tail-Flick response is thought to be a spinal reflex, while the Hot-Plate response clearly involves at least the brainstem level since coordination of the head and limbs is required. Thus, the level of response integration may be a determinant of the observed differences in the present data. Interestingly, Yaksh (37) has shown that the u-blocker phentolamine, injected spinally, blocked analgesia produced by the injection of morphine into the periaqueductal grey matter. The effect was observed both in the Tail-Flick and Hot-Plate tests. In the present study, no clear evidence of morphine antagonism by u-blockade was seen in either of these tests. Thus, the spinal mechanism that is revealed by local drug injections, or other treatments (38,39), is apparently obscured when drugs are administered systemically. The present data suggest that adrenergic systems, probably of both u and 8 types, are involved in pain modulation, and that manipulation of such systems can alter the analgesic effectiveness of morphine. However, more extensive pharmacological studies are needed before the profiles obtained here can be resolved into a coherent hypothesis on the interactions of adrenergic and opiate (endorphinergic) mechanisms in the natural course of pain processing. What seems clear from the present data is that a complete understanding of such interactions will require a systematic analysis of the behavioral variables that define the pain test.
ACKNOWLEDGEMENTS This research was supported by Grant A-7891 to Prof. Melzack from the Natural Sciences and Engineering Research Council of
1258
AdrenerEic Effects on Morphine AnalEesla
Vol. 26, No. 15, 1980
Canada, and by a National Research Service Award to Dr. Dennis from NINCDS. Naloxone hydrochloride and clonidine were generously provided by Endo Laboratories and B o e h r i n g e r - I n g e l h e i m respectively. The authors gratefully acknowledge helpful discussions with Prof. Keith Franklin and Dr. Fran Abbott. REFERENCES 1. 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.
A.E. TAKEMORI, Ann. N.Y. Acad. Sci. 281 262-272 (1976). T.J. CICERO, E.L. MEYER and B.R. SMITHLOFF, J. Pharmacol. Exp. Ther. 189 72-82 (1974). S.K. GUPTA and V.R. DESHPANDE, Indian J. Physiol. Pharmacol. 9 163-165 (1965). PAALZOW and L. PAALZOW, Psychopharmacologia 45 9-20 (1975). W.L. DEWEY, L.S. HARRIS, J.F. HOWES, and J.A. NUITE, J. Pharmacol. Exp. Ther. 175 435-442 (1970). M.R. FENNESSY and J.R. LEE, J. Pharm. Pharmacol. 22 930-935 (1970). B.-D. GORLITZ and H.-H. FREY, Eur. J. Pharmacol. 20 171-180 (1972). B. HELLER, J.M. SAAVEDRA, and E. FISHER, Experientia 24 804805 (1968). C.T. MAJOR and B.J. PLEUVRY, Brit. J. Pharmacol. 42 512-521 (1971). F.C. TULUNAY, I. YANO, and A.E. TAKEMORI, Eur. J. Pharmacol. 35 285-292 (1976). W.---T.CHANCE and M.D. SCHECHTER, Soc. Neurosci. Abstr. 5 607 (1979). V.H. SETHY, R.J. PRADHAM, S.S. MANDREKAR, and U.K. SHETH, Indian J. Med. Res. 58 1453-1458 (1970). S.F. SLIVKO and V.R. S---TETS, Farmakol. Toksikol. 41 544-548 (1978). H.N. BHARGAVA, S.L. CHAN, and E.L. WAY, Proc. West. Pharmacol. Soc. 15 4-7 (1972). R.E. CHIPKIN, W.L. DEWEY, L.S. HARRIS, and W. LOWENTHAL, Pharmacol. Biochem. Behav. 3 843-847 (1975). U.H. SHAH, M.N. JINDAL, and--V.K. PATEL, Arzneim. Forsch. 24 1581-1584 (1974). G. PAALZOW, Naunyn Schmiedebergs Arch. Pharmacol. 304 1-4 (1978). T.C. SPAULDING, S. FIELDING, J.J. VENAFRO, and H. LAL, Eur. J. Pharmacol. 58 19-25 (1979). L. SAARNIVAARA, Ann. Med. Exp. Biol. Fenn. 47 180-190 (1969). L. MUDGILL, A.J. FRIEDHOFF, and J. TOBEY, Ar-ch. Int. Pharmacodyn. Ther. 210 85-91 (1974). K. REINHOLD, J. BLASIG, and A. HERZ, Naunyn Schmiedebergs Arch. Pharm~col. 278 69-80 (1973). R.D.E. SEWELL and P.S.J. SPENCER, Psychopharmacologia 42 6771 (1975). H.G. WOOLFE and A.D. MACDONALD, J. Pharmacol. Exp. Ther. 80 300-307 (1944). D. DUBUISSON and S.G. DENNIS, Pain 4 161-174 (1977). F.E. D'AMOUR and D.L. SMITH, J. Pha~macol. Exp. Ther. 72 7479 (1941). B. COSTALL, D.H. FORTUNE, R.J. NAYLOR, C.D. MARSDEN, and C. PYCOCK, Neuropharmacology 14 859-868 (1975). C.W. DUNNETT, Biometrics 20--482-491 (1964). S.G. DENNIS and R. MELZACK--, Advances in Pain Research and
Vol. 26, No. 15, 1 9 8 0
29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
AdrenergicEffects on Morphine Analgesia
1259
Therapy, Vol. 3, J.J. Bonica, J.C. Liebeskind, and D. Albe-Fessard (Eds.), pp. 747-760, Raven Press, New York (1979). S.G. DENNIS and R. MELZACK, Pain 4 97-132 (1977). H.K. BEECHER, Pain: Henry Ford Hospital Symposium, R.S. Knighton and P.R. Dumke (Eds.), pp. 221-239, Little, Brown & Co., Boston (1966). D.J. MAYER and J.C. LIEBESKIND, Brain Res. 68 73-93 (1974). H. AKIL, D.J. MAYER, and J.C. LIEBESKIND, Sc-~ence 191 961962 (1976). J.L. OLIVERAS, Y. HOSOBUCHI, F. REDJEMI, G. GUILBAUD, and J.-M. BESSON, Brain Res. 120 221-229 (1977) Y. HOSOBUCHI, J.E. ADAMS, and R. LINCHITZ, Science 197 183186 (1977). D.E. RICHARDSON and H. AKIL, J. Neurosurg. 47 184-194 (1977). M.N. CARROL and R.K.S. LIM, Arch. Int. Pharmacodyn. Ther. 125 383-403 (1960). T.L. YAKSH, Brain Res. 160 180-185 (1979). Y. KURAISHI, Y. HARADA, M. SATOH, and H. TAKAGI, Neuropharmacology 18 107-110 (1979). T.C. SPAULDING, J.J. VENAFRO, M.G. MA and S. FIELDING, Neur~pharmacology 18 103-105 (1979).
Pergamon Press
PAIN MODULATION BY ADRENERGIC AGENTS AND MORPHINE AS MEASURED BY THREE PAIN TESTS Stephen G. Dennis~ Ronald Melzack, Samuel Gutman, and Frangoise Boucher Department of Psychology, McGill University 1205 Avenue Docteur Penfield Montreal, Quebec, Canada H3A IBI (Received in final form February 11, 19"80) SUMMARY The effects of several adrenergic agents on pain and morphine analgesia were assessed using three pain tests in rats. These tests--Tail-Flick, Hot-Plate, and Formalin--allow comparison of the effects of different noxious stimuli and different motor responses. Each pain test yielded a unique constellation of adrenergic influences, suggesting that variation of stimulus and response parameters can change the functional expression of adrenergic systems related to pain processing. The salient drug effects include: i) a pronounced, relatively selective analgesic effect of yohimbine in the Hot-Plate test; 2) a selective analgesic effect of clonidine in the Formalin test; 3) a striking, but variable, antagonism of morphine analgesia by a combination of yohimbine and propranolol in the Formalin test; 4) a nonlinear dose-response curve for antagonism of morphine analgesia by p r o p r a n o l o l i n t h e Hot-Plate test; and 5) a generalized interference with pain responding and enhancement of morphine analgesia by most drugs in the Formalin test. The data suggest that the type of pain test is crucial in determining the pattern of drug influences that is revealed. Interactions of opiate analgesics with drugs known to affect certain neurochemical systems, notably the monoamines, have long been studied. In particular, a substantial amount of research has been done on the effects of adrenergic agents on morphine analgesia (cf. i). Unfortunately, the salient feature of this
aAddress reprint requests to Dr. Melzack. address is Neurosciences Research Program, Jamaica Plain Station, Boston, MA 02130
Dr. Dennis' current 165 Allandale Street,
0024-3205/80/151247-13502.00/0 Copyright (c) 1980 Pergamon Press Lid
1248
Adrenerglc Effects on Morphine Analgesia
Vol. 26, No. 15. 1980
literature, part of which is sun~arized in Table I, is its variability. Several experiments have demonstrated the antagonism of morphine analgesia by adrenergic systems, others have suggested enhancement, while still others have found no significant effect. On the basis of the available data, it appears that adrenergic systems do interact with morphine in the course of pain assessment procedures, but it is difficult to predict with confidence precisely how they will interact under any given set of circumstances. Clearly, it would be useful to know the sources of this variability. TABLE I Effects of Adrenergic Antagonists, Agonists, and Precursors on Morphine Analgesia a Drug Class
Enhance
No Effect
Antagonize
e-Antagonists b
2, 3, 4
5, 6, 7, 8, 9, I0
4, ii, 12, 13
8-Antagonists c
12, 14
2, 5, 6, 7, 9, I0, 15
3, 8, 16
Agonists & Precursors d
5, 8, 17, 18
19
20, 21, 22
aEntries are reference numbers. Experiments were predominantly on rats or mice. Various pain tests and drug dosages were used. bIncludes yohimbine, phentolamine, azapetine, dihydroergotamine. CIncludes
propranolol,
phenoxybenzamine,
dichloroisoproterenol,
dIncludes clonidine, isoproterenol, norepinephrine, L-tyrosine.
tolazoline,
pronethalol.
methozamine,
epinephrine,
Among the many potential sources of variation in this paradigm, the type of pain test frequently has been cited as critical. Certain pain tests are apparently more suitable than others for demonstrating particular drug effects. However, in general it is not known precisely what makes one pain test different from another with respect to assessing drug interactions. The present experiments approach this behavioral question by comparing three pain tests selected to emphasize two major variables: I) the physical and temporal properties of the noxious stimulus; and 2) the pattern of the required motor response. The former is assessed by comparing the Hot-Plate (23) and Formalin (24) tests, which record the same general movement pattern but in response to markedly different noxious stimuli. The latter is assessed by comparing the Hot-Plate and Tail-Flick (25) tests, both of which use noxious heat but which trigger quite different responses. Insofar as possible, all other factors are held constant. The purpose of this study,
therefore,
is to examine
the el-
Vol. 26, No. 15, 1 9 8 0
Adrenerglc Effects on Morphine Analgesia
1249
fects of adrenergic agents on pain and morphine analgesia, and to see whether the use of different pain tests leads to qualitatively different profiles of the neural substrates that signal or modulate pain.
METHODS Subjects. Male, Sprague-Dawley rats (Canadian Breeding Fazms & Laboratories) weighing 325-400 g were used throughout. Rats were housed three to a cage with food and water always present. A 14 hr light--10 hr dark schedule was imposed, and testing was done during the light hours. Behavioral Tests. For the Tail-Flick test, the rat was clceely restrained in a wire mesh cage, and radiant heat was applied to a blackened portion of the tail approximately 1.5-2.0 cm from the tip. The latency to the first movement of the tail (usually a sudden flicking or lashing) was recorded. The heat source was adjusted to give an average baseline latency of about 3.5 sec in a sample of untreated rats. In the absence of a response, the heat source was switched off automatically at 7.0 sec, and this m a x i m u m value was recorded. Four trials were run with 2-min intertrial intervals. For the Hot-Plate test, the rat was placed on a grid of stainless steel tubes 0.5 cm in outer diameter and spaced 1.2 cm apart. Heated distilled water was pumped continuously by a constant temperature circulator. Bath temperature was m a i n t a i n e d at 57.0"C, usually about 1.5°C higher than the grid surface temperature. The latency to lick either hind paw was taken as the primary response measure. Front paw-lick latencies were also estimated for the most rats. Two trials separated by a 3-min interval were run, and a 30 sec cutoff was imposed. The Formalin test has been described in detail elsewhere (24). Briefly, 0.05 ml of 5% formalin was injected subcutaneously in the dorsal surface of the forepaw. This provoked a series of favoring responses characterized by elevation and occasional licking of the paw. A weighted average of time spent in four basic categories was taken as the primary response measure. The categories and weighting factors were: 0) full weight is placed on the paw; I) the injected paw rests lightly on the floor and bears no weight; 2) the injected paw is selectively elevated and in contact with no other surface; and 3) the paw is licked or bitten. Cumulated data from the second half-hour postinjection were used in comparisons with the other pain tests, except in the naloxone experiments where the last 15 min was taken. In addition to the three pain tests, a modified Bar test for catalepsy (lack of spontaneous movement) was used (26). The r@t's forepaws were placed on a bar 0.5 cm in diameter at a height of 10 cm from the floor. The latency for the rat to remove both forepaws from the bar was recorded. Two trials were run with a 30 sec cutoff imposed. Except for possible discomfort during handling, this behavioral test was painless. The Tail-Flick, Hot-Plate, and Bar tests were run as a battery, each rat going through the entire sequence of tests. The
1250
Adrenerglc Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
sequence always started and ended with a Bar trial, and the order of pain testing was counterbalanced. Separate groups of rats were run in the Formalin test. Drug Tests. Table II shows the drugs and dosages used. Injection times were calculated relative to the time of the morphine or saline control injection. Taking this point as zero, prior events were given a negative sign, and subsequent events a positive sign. Thus, clonidine (CLN) was injected 30 min before morphine or normal saline. In the Formalin test, the formalin injection always occurred at +15 min, and data were analyzed for the +45 to +75 min period. The Tail-Flick, Hot-Plate, and Bar tests were all run from +50 to about +70 min. Since all drug injections were based on the same zero time point, comparisons of a single drug across all behavioral tests fall within the same average injection-test interval. All injections were done intraperitoneally. For about 80% of the animals, the observers were kept "blind" as to the drugs used, their putative effects, or the hypothesis under consideration. TABLE II D r u g Doses, Injection Times, and Major Effects Drug
Abbreviation
Doses (mg/kg) a
Injection Time (min)
Effect b
Clonidine
CLN
.03,.15
-30
e-Agonist
Isoproterenol HCI
ISO
.5, 4
-I0
8-Agonist
Yohimbine HCI
YOH
I, 4
-15
e-Antagonist
Propranolol HCI
PRO
i, 4, 8
-5
B-Antago~st
Naloxone HCI
NAL
5
+45
Opiate Antagonist
Morphine Sulfate
MOR
2.5, i0
0
Opiate Analgesic
aAll doses refer to the weight of the salt. bRefers to major effects at indicated-doses. Statistical Analysis. The method of overall analysis was Dunnett's procedure for multiple comparisons with a control (27). The otherwise untreated morphine or saline groups were taken as controls, and individual analyses were run on each behavioral test. A value of p<.05 was considered significant. Individual t-tests or analyses of variance were done as necessary. Group means, together with the standard error (M ± SEM), are expressed in seconds for the Hot-Plate, Tail-Flick, and Bar tests, and in arbitrary units for the Formalin test.
Vol. 26, No. 15, 1 9 8 0
Adrenetglc Effects on Morphine Analgesla
1251
RESULTS Morphine Analgesia and its Reversal by Naloxone. Morphine (10 mg/kg) significantly reduced pain responding in all three tests, and naloxone (5 mg/kg) antagonized this effect in each test (see Fig. 1). This dose of morphine produced sedation, as reflected in a small but significant elevation of Bar test scores (SAL: M = 3.2 ± 0.7; MORI0: M = 8.1 ± 1.7; t = 2.70, df = 30, p<.02), although the animals moved normally when lightly prodded. In the Formalin test, where the time course of naloxone's effect was continuously monitored, reversal of sedation usually preceded reversal of analgesia by 5-8 min. Effects of Adrener~ic Drugs A10ne. Figure 2 shows the baseline effects on pain responding of the adrenergic drugs used. Each pain test presents a different pattern of effects. In the Tail-Flick test, the only significant effect was a modest elevation of latencies produced by 4 mg/kg propranolol. Higher (8 mg/kg) and lower (I mg/kg) doses had no effect. In the Hot-Plate test, yohimbine (1 and 4 mg/kg) significantly prolonged response latencies. This apparent analgesia was comparable to or'exceeded a i0 mg/kg dose of morphine (cf. Fig. 1). However, naloxone did not antagonize yohimbine analgesia (YOH4-SAL: M = 28.7 ± 0.6; YOH4-NAL5: M = 30.0 ± 0.0; t 2.00, df = 14, p>.05). Forepaw-lick latencies were also elevated by yohimbine (see Table III). Propranolol showed a tendency • t the higher doses (4 and 8 mg/kg) toward reduced latencies
TAIL-FLICK
~
HOT-PLATE
250
FORMALJN
°I
6.0
>. 5.0.-
46 SAL MOR MORIO 0 NAL 5
SAt MOR MOR~0 10 NAL5
SAL MOR MOA~O t0 NAL5
FIG. 1 Effects of morphine and naloxone as measured by three pain tests. Note that the scale on the Formalin test ordinate has been inverted to conform to the "analgesia up" convention of the other graphs. Asterisks indicate statistically significant differences (p<.01) compared to the MORI0 group for each pain test. Standard errors are shown. Numbers on the bars indicate N per group.
1252
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
(hyperalgesia). This is not significant in the overall analysis, although individual post-hoc comparisons are significant (t[PR04 vs SAL] = 2.59, df = 30, p<.02; t[PRO8 vs SAL] = 3.69, df = 22, p<.002). When both propranolol and yohimbine were injected, no significant effects were observed. Thus, the apparent analgesia produced by yohimbine in this test was not m a n i f e s t e d in the presence of propranolol.
SAL
T.O ~
CLN .03 ,15
05
[SO 4.0
YOH ].0 4.0
tO
PRO 4.0 8.0
YOH| YOH4 PRO t 1 ~ 0 4
6.0
w
~'~
5.0
..1¢~
~'~
4.0
~
3.0
30.0
4=
25.0 u LU
,., ~ ZO.O
I
I,-
,
W
"I" ...1
5.0
0.0
+
0.'~ co
Z
~=~ ,0 t.5
n IIiNI m
8
181
8
FIG. 2 Effects of adrenergic agents on baseline responding in three pain tests. Conventions as In Fig. i, and all groups are compared to the SAL control at the left of each panel. Propranolol (4 mg/kg) increases latencies in the Tail-Flick test, while yohimbine (i and 4 mg/kg) increases latencies in the Hot-Plate test. In the Formalin test, all agents interfere somewhat with responding, the most powerful effect being that of clonidine (0.03 and 0.15 mg/kg). These data show that each behavioral test yields a different pharmacological profile of adrenergic influences.
Vol. 26, No. 15, 1 9 8 0
Adrenerglc Effects on Morphine Analgesia
1253
A strikingly different pattern of results was seen in the Formalin test. Powerful dose-dependent analgesia was observed with clonidine (0.03 and 0.15 mg/kg) and isoproterenol (4 mg/kg). Clonidine's effect rivaled morphine's in magnitude (cf. Fig. 1) but was unaffected by 5 mg/kg naloxone (CLN. 15-SAL: M = 0.i ± 0.1; CLN. 15-NAL5: M = 0.2 ± 0.1; t = 1.75, df = 14, p>.2). Yohimbine (1 mg/kg) partially reversed clonidine's effect (CLN.15SAL: M = 0.i ± 0.i; CLN.15-YOHI: M = 0.7 ± 0.i; t = 5.11, df = 14, p<.001). Yohimbine (i and 4 mg/kg) and propranolol (1 and 4 mg/kg) also reduced pain responding in this test, although the effects were not obviously dose-dependent. The combination of uand B-blockers showed the same effect as either agent alone. The overall analysis of Bar scores revealed no significant effects, indicating that at the doses employed, none of these drugs interfered with the execution of this simple dismounting response. Interaction of Adrenergic Dru@s with Morphine. Figure 3 shows the effects of combinations of morphine and the various adrenergic drugs in the Tail-Flick, Hot-Plate, and Formalin tests. In the Tail-Flick test, propranolol (1 mg/kg) significantly interfered with the analgesia produced by morphine. Neither of the higher doses of propranolol had a significant effect, although the difference between these two groups is significant ( t [ P R 0 4 v s P R O 8 ] - 2.28, df = 22, p<.05). No other effects were Bignificant in the overall analysis. In the Hot-Plate test, the antagonistic effect of propranolol (i mg/kg) was also observed, but in addition, the 8 mg/kg dose had a significant effect. The 4 mg/kg dose remained ineffective. Yohimbine (4 mg/kg) significantly enhanced latencies o v e r the morphine control levels. However, because of possible ceiling effects in these data, the strength of yohimbine's enhancement is uncertain. Comparable data on forepaw-lick latencies (Table III), where there is no ceiling effect, are consistent with an additive model of yohimbine and morphine analgesia in this test. In the Formalin test, the only measurable departure from control levels was a significant antagonism of analgesia produced by the combination of yohimbine (i mg/kg) and propranolol (i mg/ kg). This effect was highly variable, with four rats showing complete antagonism while the remaining four were less affected. Antagonism was usually characterized by prolonged periods of licking or biting the injected paw (rating 3). This striking antagonism was also seen in a separate experiment in which several of the Formalin test conditions were replicated in rats deprived of food for 18 hr prior to testing. A g a i n , however, there was substantial individual variability. In order to characterize better the interactions of adrenergic agents and morphine in the Formalin test, a study was carried out with a lower dose of morphine (2.5 mg/kg). The results, shown in Fig. 4, demonstrate the improvement of analgesia by each drug tested. Comparison with the data in Fig. 2 suggests that the improvement was at least additive in all cases, and possibly multiplicative for clonidine, isoproterenol, and the combination of yohimbine and propranolol. It is of interest that the com-
1254
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, 1980
bination of antagonists, which tended to antagonize the larger dose of morphine, had no such effect on the smaller dose. In the Bar test, a significant elevation of latencies over morphine control levels was seen with clonidine (0.15 mg/kg). This correlated with a powerful reduction of spontaneous activity produced by this drug combination. However, these rats generally
7.0
MOR I0
CLN .03 .15
YOH
1.0
4Q
1.0
PRO 4.0 +
8.0
YOHI PROt
s,ot
..,
f.o
,6
I
91 a
30.0
f
~25.0
÷
t~J
"9'20.0
J
I,,- I,,,-
-e. _j I 0 . 0
511
+
le
le
12
*+
+
j
"!-
0,5 Z
zF--
?,~ ,,o ~_ !
8
2.0
FIG.
:3
8
2
3
3
Interaction of adrenergic agents with morphine in three pain tests. Conventions as in Fig. i, with all groups compared to the MORI0 group at the left of each panel. Asterisks indicate p<.01; pluses indicate p<.05. Propranolol (1 mg/kg) reduces morphine analgesia in both the Tail-Flick and Hot-Plate tests but not in the Formalin test, while propranolol at 8 mg/kg is effective only in the Hot-Plate test. Yohimbine increases analgesia in the Hot-Plate test. In the Formalin test, the combination of yohimbine and propranolol significantly antagonizes morphine's effect. The different patterns of effects suggest that each pain test taps a different constellation of adrenergic-opiate interactions.
Vol. 26, No. 15, 1 9 8 0
Adrenergic Effects on Morphine Ana18esla
1255
tended to struggle when handled and they reacted well to p r o d d i n ~ Hot-Plate scores also indicate that the rats retained the capacity to make coordinated movements to certain noxious stimuli under the appropriate conditions. TABLE III Effects of Yohimbine and Morphine on Forepaw Latencies in the Hot-Plate Test Drug,
Dose
(mg/kg)
Mean Latency Saline
Saline Control
Yohimbine
1.0
Yohimbine
4.0
(in sec ± SEM) Morphine
4-+1
9±2
(n+8)
(n-8)
9±2 (n=8)
14±4 (n=8)
13 -+ 2
22 -+ 3
• (n=16)
MOR
0.0
(10 mg/kg)
CLN
t$O
.03
.5
(n=12)
YOH |.0
4.0
PRO tO 4 D
+,
(D Z 0.5
z~-
YOH 1.0 PRO t.0
I
~ l : ~ ~,o
¢I.
~.
~.
11.
8
$
8
6
i8
8
8
FIG. 4 Effects of adrenergic agents in combination with a lower dose of morphine (2.5 mg/kg) in the Formalin test. Conventions as in previous figures. Each agent is shown to produce analgesia in combination with this dose of morphine.
1256
Adrenergic Effects on Morphine Analgesia
Vol. 26, No. 15, !980
DISCUSSION These data show that the type of pain test is crucial in determining the pattern of analgesic or anti-analgesic effects of various adrenergic drugs either alone or in combination with morphine. Drug effects that are salient in one test may be absent or even reversed in another test. Since generalization of results from one test to another cannot be assumed, a single pain test is insufficient to characterize the adrenergic pharmacology of pain and analgesia. The variability of effects found in the present study resembles that reported in the literature (see Table I), except that in the present case, an effort has been made to isolate certain behavioral factors as the primary independent variables. Other factors such as species, strain, diet, diurnal cycle, housing, social factors, and prior stress have been held constant, insofar as possible. That the pattern of results still varies attests to the importance of the type of pain test as a source of the variability found in the literature. The data point to two factors that may contribute to the different pharmacological profiles. The first, stimulus differences, may be inferred from comparisons of the Hot-Plate and Formalin tests. These tests share at least one common motor sequence--elevation and licking of a limb--as an index of pain. However, the noxious stimuli used to evoke this response are markedly different: in the one case/ a non-damaging, escapable heat stimulus, and in the other, inescapable pain that undoubtedly arises from subcutaneous tissue damage. The salient pharmacological differences observed in the present experiments include: i) contrasting effects of adrenergic agonists, notably the uagonist clonidine; 2) differing efficacies of the e-antagonist yohimbine; 3) contrasting effects of propranolol, a 8-antagonist; and 4) contrasting effects of the combination of yohimbine and propranolol. Although the response measures for the Hot-Plate and Formalin tests share important features, they are not identical, and therefore one must avoid drawing too strong an inference regarding stimulus differences from these data. It is possible that certain of the observed pharmacological differences pertain to subtle motor differences. In any case, these experiments encourage further procedural refinements in order to produce more nearly identical behavior patterns in response to different stimyli. The physical and temporal differences between the stimuli used in the Hot-Plate and Formalin tests may be useful in evaluating recent proposals regarding the neural basis of pain-signalling (28,29). We have argued that long-lasting pain of pathological origin may be processed differently than transient pain arising from brief stimuli or at the onset of injury. It is well known that the analgesic efficacy of morphine differs with respect to these types of stimuli (30). Likewise, analgesia produced by central electrical stimulation (cf. 31), which may engage endogenous opioid systems (32,33), is more effective against long-lasting pathological pain than agains brief, experimental pain (34,35). The pharmacological differences observed in the present experiments are consistent with the notion
Vol. 26, No. 15, 1980
Adrenersic Effects on Morphine AnalEesla
1257
that such "tonic" and "phasic" pain stimuli are processed differently in the nervous system (28,29). Comparisons of the Hot-" Plate and Formalin tests might provide a useful animal model for further development of this hypothesis. Response differences appear to be the second major factor that contributes to the different pharmacological profiles. In the present study, this factor is inferred from comparisons of the Tail-Flick and Hot-Plate tests. In both tests, the noxious stimulus is heat, but in one case, it is applied to the tail and evokes a reflexive lashing or flicking response, while in the other case, the paws are heated evoking a series of responses culminating in elevation and licking. The main pharmacological differences observed under the present conditions are: i) contrasting effects of the u-antagonist yohimbine; and 2) differential effects of the higher doses of propranolol. The specific motor differences that contribute to the pharmacological profiles remain to be determined. One possibility that has been implicated in other studies (4,36) is the "level" of response integration along the neuraxis. Paalzow and Paalzow (4) have shown clear pharmacological differences in morphine analgesia in a tail-stimulation/vocalization test, depending on whether vocalization was recorded during stimulation, or following it. These two responses are thought to be integrated at brainstem and diencephalic levels respectively. The Tail-Flick response is thought to be a spinal reflex, while the Hot-Plate response clearly involves at least the brainstem level since coordination of the head and limbs is required. Thus, the level of response integration may be a determinant of the observed differences in the present data. Interestingly, Yaksh (37) has shown that the u-blocker phentolamine, injected spinally, blocked analgesia produced by the injection of morphine into the periaqueductal grey matter. The effect was observed both in the Tail-Flick and Hot-Plate tests. In the present study, no clear evidence of morphine antagonism by u-blockade was seen in either of these tests. Thus, the spinal mechanism that is revealed by local drug injections, or other treatments (38,39), is apparently obscured when drugs are administered systemically. The present data suggest that adrenergic systems, probably of both u and 8 types, are involved in pain modulation, and that manipulation of such systems can alter the analgesic effectiveness of morphine. However, more extensive pharmacological studies are needed before the profiles obtained here can be resolved into a coherent hypothesis on the interactions of adrenergic and opiate (endorphinergic) mechanisms in the natural course of pain processing. What seems clear from the present data is that a complete understanding of such interactions will require a systematic analysis of the behavioral variables that define the pain test.
ACKNOWLEDGEMENTS This research was supported by Grant A-7891 to Prof. Melzack from the Natural Sciences and Engineering Research Council of
1258
AdrenerEic Effects on Morphine AnalEesla
Vol. 26, No. 15, 1980
Canada, and by a National Research Service Award to Dr. Dennis from NINCDS. Naloxone hydrochloride and clonidine were generously provided by Endo Laboratories and B o e h r i n g e r - I n g e l h e i m respectively. The authors gratefully acknowledge helpful discussions with Prof. Keith Franklin and Dr. Fran Abbott. REFERENCES 1. 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.
A.E. TAKEMORI, Ann. N.Y. Acad. Sci. 281 262-272 (1976). T.J. CICERO, E.L. MEYER and B.R. SMITHLOFF, J. Pharmacol. Exp. Ther. 189 72-82 (1974). S.K. GUPTA and V.R. DESHPANDE, Indian J. Physiol. Pharmacol. 9 163-165 (1965). PAALZOW and L. PAALZOW, Psychopharmacologia 45 9-20 (1975). W.L. DEWEY, L.S. HARRIS, J.F. HOWES, and J.A. NUITE, J. Pharmacol. Exp. Ther. 175 435-442 (1970). M.R. FENNESSY and J.R. LEE, J. Pharm. Pharmacol. 22 930-935 (1970). B.-D. GORLITZ and H.-H. FREY, Eur. J. Pharmacol. 20 171-180 (1972). B. HELLER, J.M. SAAVEDRA, and E. FISHER, Experientia 24 804805 (1968). C.T. MAJOR and B.J. PLEUVRY, Brit. J. Pharmacol. 42 512-521 (1971). F.C. TULUNAY, I. YANO, and A.E. TAKEMORI, Eur. J. Pharmacol. 35 285-292 (1976). W.---T.CHANCE and M.D. SCHECHTER, Soc. Neurosci. Abstr. 5 607 (1979). V.H. SETHY, R.J. PRADHAM, S.S. MANDREKAR, and U.K. SHETH, Indian J. Med. Res. 58 1453-1458 (1970). S.F. SLIVKO and V.R. S---TETS, Farmakol. Toksikol. 41 544-548 (1978). H.N. BHARGAVA, S.L. CHAN, and E.L. WAY, Proc. West. Pharmacol. Soc. 15 4-7 (1972). R.E. CHIPKIN, W.L. DEWEY, L.S. HARRIS, and W. LOWENTHAL, Pharmacol. Biochem. Behav. 3 843-847 (1975). U.H. SHAH, M.N. JINDAL, and--V.K. PATEL, Arzneim. Forsch. 24 1581-1584 (1974). G. PAALZOW, Naunyn Schmiedebergs Arch. Pharmacol. 304 1-4 (1978). T.C. SPAULDING, S. FIELDING, J.J. VENAFRO, and H. LAL, Eur. J. Pharmacol. 58 19-25 (1979). L. SAARNIVAARA, Ann. Med. Exp. Biol. Fenn. 47 180-190 (1969). L. MUDGILL, A.J. FRIEDHOFF, and J. TOBEY, Ar-ch. Int. Pharmacodyn. Ther. 210 85-91 (1974). K. REINHOLD, J. BLASIG, and A. HERZ, Naunyn Schmiedebergs Arch. Pharm~col. 278 69-80 (1973). R.D.E. SEWELL and P.S.J. SPENCER, Psychopharmacologia 42 6771 (1975). H.G. WOOLFE and A.D. MACDONALD, J. Pharmacol. Exp. Ther. 80 300-307 (1944). D. DUBUISSON and S.G. DENNIS, Pain 4 161-174 (1977). F.E. D'AMOUR and D.L. SMITH, J. Pha~macol. Exp. Ther. 72 7479 (1941). B. COSTALL, D.H. FORTUNE, R.J. NAYLOR, C.D. MARSDEN, and C. PYCOCK, Neuropharmacology 14 859-868 (1975). C.W. DUNNETT, Biometrics 20--482-491 (1964). S.G. DENNIS and R. MELZACK--, Advances in Pain Research and
Vol. 26, No. 15, 1 9 8 0
29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
AdrenergicEffects on Morphine Analgesia
1259
Therapy, Vol. 3, J.J. Bonica, J.C. Liebeskind, and D. Albe-Fessard (Eds.), pp. 747-760, Raven Press, New York (1979). S.G. DENNIS and R. MELZACK, Pain 4 97-132 (1977). H.K. BEECHER, Pain: Henry Ford Hospital Symposium, R.S. Knighton and P.R. Dumke (Eds.), pp. 221-239, Little, Brown & Co., Boston (1966). D.J. MAYER and J.C. LIEBESKIND, Brain Res. 68 73-93 (1974). H. AKIL, D.J. MAYER, and J.C. LIEBESKIND, Sc-~ence 191 961962 (1976). J.L. OLIVERAS, Y. HOSOBUCHI, F. REDJEMI, G. GUILBAUD, and J.-M. BESSON, Brain Res. 120 221-229 (1977) Y. HOSOBUCHI, J.E. ADAMS, and R. LINCHITZ, Science 197 183186 (1977). D.E. RICHARDSON and H. AKIL, J. Neurosurg. 47 184-194 (1977). M.N. CARROL and R.K.S. LIM, Arch. Int. Pharmacodyn. Ther. 125 383-403 (1960). T.L. YAKSH, Brain Res. 160 180-185 (1979). Y. KURAISHI, Y. HARADA, M. SATOH, and H. TAKAGI, Neuropharmacology 18 107-110 (1979). T.C. SPAULDING, J.J. VENAFRO, M.G. MA and S. FIELDING, Neur~pharmacology 18 103-105 (1979).