Psychopharmacology
Psychopharmacology(1982) 78:141- 146
9 Springer-Verlag 1982
Chlorpromazine Hyperalgesia Antagonizes Clonidine Analgesia, but Enhances Morphine Analgesia in Rats Tested in a Hot-Water Tail-Flick Paradigm R. M. Gleeson and D. M. Atrens Department of Psychology,The University of Sydney, Sydney,NSW, Australia 2006
Abstract. Seventy-six male Sprague-Dawley rats were tested in a hot-water (55 ~ + 0.5~ tail-flick paradigm. Tail-flick latencies (TFL) were obtained at 30 and 15min before intraperitoneal injection of either morphine (2.5, 5.0 and 10.0 mg/kg) clonidine (25, 50, 100 and 200 ~tg/kg), chlorpromazine (CPZ, 2.5 and 5.0 mg/kg), dual injections of these drug combinations, or a saline control injection. Further TFL measures were taken immediately following drug administration and thereafter at 15 rain intervals. Tile mean of the pre-drug TFL's served as each rat's baseline. All other TFL's were calculated as percentage changes from that baseline. Mean changes were determined for each treatment group and differences between groups, at each test time, were analysed. Our results demonstrated morphine and clonidine analgesia but CPZ hyperalgesia. The drug interaction studies revealed that morphine analgesia is enhanced by coadministration of either clinidine or CPZ but that clonidine analgesia is antagonized by chlorpromazine. These data suggest that morphine and clonidine exert their analgesic effects through different neurochemical mechanisms. It is particularly interesting that the clonidine-CPZ combination should result in TFL's similar to baseline levels, even though both drugs are sedatives. The investigation emphasizes the value of chlorpromazine as a pharmacological tool in analgesic research because of its ability to induce hyperalgesia even though it is a sedating agent. Key words: Analgesia - Hyperalgesia - Chlorpromazine Clonidine - Morphine - Tail-flick
Physiological and pharmacological investigations have demonstrated cellular level interactions between opiate and aminergic systems (Meldrum and Isom 1981 ; Strahlendorfet al. 1980; Watson et al. 1980). There is also a growing body of evidence imPlicating serotonergic and c~-noradrenergic mechanisms in the analgesia produced by morphine (Berge et al. 1980; Hammond et al. 1980 a; Johansson et al. 1980; Satoh et al. 1980). The c~-noradrenergic agonist clonidine produces analgesia and suppresses certain opiate withdrawal signs (Crawley et al. 1979; G old et al. 1979, 1980 ; Lipman and Spencer 1980). That clonidine does not act directly on opiate receptors is indicated by receptor binding studies (Golembiowska-Nikitin et al. 1980) and the fact that clonidine-induced analgesia is not blocked by the opiate antagonist naloxone (Fielding et al. Offprint requests to D. M. Atrens
1978; Spaulding et al. 1979). However, an interaction between noradrenergic and serotonergic (5-HT) systems is shown by the fact that the 5-HT antagonist methysergide antagonizes the analgesia produced by intracerebral injections of phentolamine, a noradrenaline antagonist (Hammond et al. 1980a). This apparently coherent body of information can be interpreted on alternative bases. A primary concern is the characteristic sedating properties of most analgesic drugs (Marcais et al. 1981; Malec et al. 1978; Yaksh and Reddy 1981) and many of these drugs, even in low doses, also produce motor impairment (Anden and Grabowska-Anden 1980; Hammond et al. 1980b; Havemann et al. 1980). This is particularly obvious with the induction of the straub tail phenomenon in rats following high dose administration of morphine (Mayer and Liebeskind 1974) and intracranial injections of endorphin analogs (Walker et al. 1977) and ACTH (Walker et al. 1980). Sedative-ataxic effects should not be confused with analgesia. Nor should analgesia be inferred from analyses of responding that artificially accelerate the approach to maximum or minimum limits. Unfortunately, many behavioural studies use an "index of analgesia" which relies upon an arbitrarily chosen cutoff latency in the tail flick paradigm (Mayer and Hayes 1975). With constant test and baseline latencies, the value of the "analgesia index" can be made to vary from slightly above 0 % to 100 % simply by reassigning a cut-off latency (Jacquet and Lajtha 1976). The present experiment addresses the question of the sedating effects of analgesics by the use of chlorpromazine (CPZ), a drug with well known sedative-ataxic properties (Carlsson 1975; van Praag et al. 1975), and examines the analgesia produced by morphine, clonidine and chlorpromazine and the interactions of these drugs. The methodological difficulties of conventional analgesic indices may be avoided by referencing tail flick latency (TFL) changes to each animal's baseline latency and comparing these data with a saline-treated group. This group provides a control for the changes in responding produced by the stress, sensitization and learning which may occur in a repeated testing paradigm. By conducting TFL tests at 15rain intervals, before and following drug treatment, a more complete understanding of the time course of the analgesia induced by these drugs can be obtained. Materials and Methods Animals. A total of 76 male Sprague-Dawley rats (mean
weight 350 g) were used in these experiments. The rats were 0033-3158/82/0078/0141/$01.20
142 not tested on more than two occasions and a minimum of 2weeks elapsed between tests for ten of the rats and 4 6 weeks for the remainder. The rats were housed in groups of three until each test day. They were maintained on tap water and freely available lab chow. The animal colony temperature was kept at 20 ~ _+ 2~ throughout the experiment.
Drug Solutions. All drugs were mixed with sterile saline (0.9%) and injected intraperitoneally (IP) at volumes of 1 ml/kg body weight. The order of drug administration was fully randomized both within and between groups. Morphine sulphate (a gift from A b b o t t Laboratories) was given in doses of 2.5, 5.0 and 10mg/kg. Clonidine HC1 (a gift from Boehringer-Ingelheim) was given in doses of 25, 50, 100, and 200 gg/kg. Chlorpromazine HC1 (a gift from Poulenc Ltd.) was given in doses of 2.5 and 5.0 mg/kg.
Apparatus. A glass tank (45 x 2 6 x 2 5 c m ) containing 121 water was maintained at a temperature of 55 ~ _+ 0.5~ by a Thermomix (1420E) heater circulator. The water temperature was continually monitored by a digital readout thermometer (Bailey Inst., Model BAT8).
Procedure. On the test day the rats were weighed and transferred from their home cages to individual cages in the
test room. They were left for at least 1.5 h to allow them to adapt to the changed environment. The sequence of testing began with the experimenter bringing the rat's cage to the test bath, taking the rat from its cage and positioning it above the water bath. As soon as any tail movement or struggling responses had abated, the rat was rapidly lowered so that its tail was immersed in the water to a point 2 cm from the scrotum. The experimenter recorded the time from when the tail reached its full immersion depth to when a tail-flick response was observed. This provided a TFL. The rat was immediately removed from the water, its tail was gently dried with a soft towel and it was returned to its cage. Eight tests were conducted on each rat in each test session. Fifteen minutes elapsed between each test, with two being taken before drug treatment ( - 3 0 and - 1 5 r a i n ) , one immediately after drug administration (0rain), and five following treatment (15, 30, 45, 60, and 75 rain). A t time zero (0 min) each rat was injected (IP) with the appropriate drug (or drugs) and returned briefly to its cage.
Results The present experiment identified three distinct forms of tailflick responding to hot water immersion. Some exhibited a
Table 1. Differences between groups at each test time (i) Drugs and dosages
(a) Morphine 10 mg/kg 5.0 mg/kg 2.5 mg/kg (b) Clonidine 200 gg/kg 100 gg/kg 50 gg/kg (c) Clonidine and morphine interactions 200 gg/kg clonidine plus 2.5 mg/kg morphine 200 gg/kg clonidine plus 5.0 mg/kg morphine 200 Bg/kg clonidine plus 5.0 mg/kg morphine (d) CPZ and CPZ, clonidine and morphine interactions 5.0 mg/kg CPZ 2.5 mg/kg CPZ 2.5 mg/kg CPZ plus 200 ~g/kg clonidine 2.5 mg/kg CPZ plus 200 gg/kg clonidine 2.5 mg/kg CPZ plus 10 mg/kg morphine
(ii) Significantly greater TFL percentage change of (i) when compared to:
(iii) Level of significance (P<)
(iv) Test times (rain)
2.5 and 5.0 mg/kg Saline 2.5 mg/kg Saline
0.01 0.001 0.02 0.01
Saline
0.05
All post-drug test times + 45 All post-drug test times + 75
0.01 0.05 0.05 0.05 0.05
+30, + 60, +30, + 60, + 30,
0.05
+ 30, + 60
0.01 0.01
+ 15, +30, +45 + 60, + 75 + 45
Saline Saline 2.5 mg/kg CPZ plus 50 i-tg/kgclonidine
0.05 0.02 0.05
+ 60 + 60 + 30
Saline
0.05
+ 30
10 mg/kg morphine
0.05
+ 15
25 gg/kg and saline 50 gg/kg 100 Ixg/kg 25 gg/kg and saline 25 gg/kg and saline
50 gg/kg clonidine plus 2.5 mg/kg morphine 50 gg/kg clonidine plus 2.5 mg/kg morphine 200 gg/kg clonidine plus 2.5 mg/kg morphine '
+45, +60, +75 + 75 +45, +75 + 75 + 75
143 110' FIG. tb
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"straight tail-lift", particularly after high dose morphine administration, others displayed either a "corkscrew-lift" or a "whip-lift". All three forms of responding were observed under both baseline and drug conditions, and these responses were quite different from the rotating "tail-swing" often observed when a rat is picked up. A tail-flick response was defined as occurring when the tip of the rat's tail began to curl and lift, irrespective of the lifting pattern. This proved to be a very reliable index as tail-flicks provided by 137 rats (76 experimental and 61 pilot subjects) had a mean latency of 3.12 s and a standard error of the mean of 0.04 s. The mean of the first two T F L ' s ( - 30 and - 15 min) was taken as each animal's baseline. All subsequent TFL's we're calculated as a percentage change from that baseline. Means (2) and standard errors of the means (SEM) of the percentage changes were determined for each experimental group at the test times, zero (0) to + 7 5 m i n . The mean T F L ' s were elevated above baseline at time zero, for all treatment groups, and this general increase is likely due to the stress of the handling and injection procedure. Student's t-tests were used to analyse the differences between groups at each test time and the significant differences are presented in Table 1. Both morphine and clonidine produced dose and time ~ dependent analgesia which, however, differed in the time course of onset and peak effects (Fig. l a and b). As shown in Fig. 1 and 2 morphine and clonidine exhibited a positive analgesic interaction. The 50 pg/kg clonidine plus 2.5mg/kg morphine group attained a peak effect at 45 rain of 2 = 64.53 ~o ( + SEM, 10.59 ~o) whereas a simple additive effect at this test time would yield an increase of 23.49 ~. Similarly, 200 pg/kg clonidine plus 2.5 mg/kg of morphine at 30 min and 200 pg/kg clonidine plus 5.0 mg/kg morphine at 45min showed greater than additive effects ( 1 0 8 . 7 6 ~ + 1 6 . 9 1 ~ o compared to 57.16~ and 145.30~ _+14.46 ~ compared to 71.55 ~, respectively). An indication of a bimodal time course of action for 2.5 mg/kg of morphine was suggested by the results which showed this dosage to be significantly less effective than 5.0 mg/kg only at 45 rain (Table la). The drug interaction studies also supported this view by demonstrating an inability
75
a Mean percentage tail-flick latency changes from baseline following injection of either saline (N = 10) or morphine; 2.5 mg/kg (N = 14), 5.0 mg/kg (N = 9) and 10.0 mg/kg (N = 8). b Mean percentage tail-flick latency changes from baseline following injection of clonidine; 25 gg/kg (N = 10), 501ag/kg(N = t5), 100 ~tg/kg (N = 10) and 200 gg/kg (N = 9)
150" 140130" 120110" 100o
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00" E O ,.~ 70`
~
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Lu 40"
~- 3o2010"
0
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Fig. 2. Mean percentage tail-flick latency changes from baseline following dual injections of either; 50 gg/kg clonidine plus 2.5 mg/kg morphine (N= 10) (D D), 200gg/kg clonidine plus 2.5mg/kg morphine (N= 10) (V V) or 200gg/kg clonidine plus 5.0mg/kg morphine (N = 10) (0
O)
of the high dose of clonidine (200gg/kg) + 2 . 5 m g / k g of morphine to produce a greater analgesic effect at test time 45 rain than the low clonidine dose (50 gg/kg) + 2.5 mg/kg of morphine combination. In addition, 200 lag/kg of clonidine administered in conjunction with 5.0mg/kg of morphine resulted in a significant increase in TFL, compared to that clonidine dose + 2 . 5 m g / k g of morphine, only at 45rain (Table I c). If2.5 mg/kg of morphine does have a bimodal time course of action then clearly, "one-shot" sampling of analgesic
144 Although CPZ produces hyperalgesia and antagonizes clonidine-induced analgesia, it does not antagonize morphine analgesia. In fact, the reverse was the case with 2.5 mg/kg of CPZ, significantly increasing the analgesic effect of 10 mg/kg of morphine at 15rain (Fig. 3b).
150140130-
SALINE
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CHLOR PROMAZINE IcY'z) r ~ 2~ m g / k g
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Fig. 3. a Mean percentagetail-flicklatencychanges from baseline following injection of either saline (N= 10) or chlorpromazine (CPZ); 2.5 mg/kg (N = 16) and 5.0 mg/kg (N = 5). b Mean percentage tail-flick latency changes from baseline following dual injections of either; 50 ~tg/kgclonidine plus 2.5 mg/kg CPZ (N = 6), 200 gg/kg clonidine plus 2.5mg/kg CPZ (N= t0) or 10mg/kg morphine plus 2.5mg/kg CPZ (N = 6)
potency following administration of this dosage could result in quite misleading conclusions. Sedation or motoric deficits were assessed by measuring the latency for a rat to step down from a rod placed under its forepaws and elevated by 10cm (Yaksh et at. 1976). The assessments were made in CPZ (2.5 mg/kg)-treated (N = 6) and clonidine (50 gg/kg)-treated (N = 6) rats, 30 min before injection ( - 3 0 min), immediately after the injection (zero = 0), and at 30 and 60 min post-injection, and percentage changes from the - 30 min baseline were calculated for these test times. The two drug-treated groups showed no significant differences. The rod step-down latencies increased over the duration of the test, indicating some degree of sedation or motor impairment attributable to either drug and, in terms of their appearance and responsiveness to being handled, the rats seemed sedated. Even though CPZ produced a level of sedation similar to that produced by clonidine, the effects of these drugs on nociception were quite the opposite (Fig. 3a) with CPZ resulting in hyperalgesia and clonidine in analgesia. The hyperalgesic effect of CPZ was replicated on two separate occasions for the 2.5 mg/kg dosage (N = 11 and N = 5) and once for 5 mg/kg (N --- 5). The pooled (N = 16) 2.5 mg/kg and the 5.0 mg/kg CPZ data both showed a significant decrease in the TFL at 60 rain post-injection (Table 1 d). The analgesic effect of 200 gg/kg of clonidine at 30 min (Fig. 1 b) was not greatly affected by the concurrent injection of 2.5 mg/kg CPZ (Fig. 3b), however CPZ reduced the clonidine induced analgesia at the later test times (45, 60 and 75 min). This effect was particularly marked at 60 min where a purely additive effect would yield a mean T F L increase of 33.89 % but an increase of only 12.75 % ( _+ 11.41% SEM) was obtained.
Morphine analgesia has been well documented and in recent years clonidine analgesia has also received a good deal of interest (Spaulding et al. 1979). However, the present data demonstrate that the analgesia produced by clonidine differs from that of morphine in some respects. Morphine (10mg/kg) analgesia was apparent by 15 min post-injection, whereas the clonidine (200 I~g/kg) analgesic effect had a slower onset and, unlike morphine, maintained its peak level throughout the post-drug testing. The time course of the analgesic effects of the two intermediate doses of clonidine appear to be complementary. Since these doses are in the range of transition from the presynaptic to postsynaptic effects of clonidine (De Langen et al. 1979; Glossmann et at. 1980; Malec et at. 1978; Reddy et at. 1980), it is tempting to conclude that the early appearing analgesia represents a presynaptic effect. However, the fact that the very high dose (200 gg/kg) produces both early and late analgesic components is not readily accounted for by a presynapticpostsynaptic shift hypothesis, despite the fact that all of the peak TFL's cluster at either 30 or 60 min post-injection for this dosage. Clearly though, both morphine and clonidine analgesia are complex, and the temporal dissociations imply that they may be subserved by different neurochemical mechanisms and that even different doses of clonidine may produce their analgesic effects in quite different ways. The data presented in Fig. 2 support those of Spaulding et al. (1979), in showing that clonidine increases the analgesia produced by morphine. The peak effects in each case were approximately twice as great as the simple additive effects of the individual drugs at these times. It is premature to label the positive interaction between clonidine and morphine as "potentiation" in the true pharmacological sense (Dews 1976; Mitchell 1976) since the drugs may well have been operating on parallel or serial systems rather than being biochemically synergistic. There is, however, evidence that opiates modulate noradrenergic tone (Aghajanian 1978; Pepper and Henderson 1980). Since clonidine and morphine can both produce sedativeataxic effects an experiment was conducted to evaluate these potential confounding influences. It was found that sedative-ataxic doses of CPZ resulted not in analgesia but in a slow onset (60min) and brief duration hyperalgesia. This increased responsiveness to noxious stimulation was particularly impressive since the animals were unresponsive to hfindling, and in the rod step-down test they were as sedated as rats that received an analgesic dose of clonidine (50 gg/kg). In at least this instance, the degree of drug-induced sedation or ataxia does not correlate with the degree of induced analgesia. Furthermore, if the analgesia observed after clonidine administration was due to the sedating properties of that drug then the combined sedating effects of clonidine plus CPZ should have resulted in a greater degree of analgesia than that obtained from clonidine alone. In fact, the
145 low dose (2.5 mg/kg) of CPZ almost totally eliminated the analgesia produced by clonidine. As with CPZ hyperalgesia, the elimination of clonidine analgesia had a slow onset. However, it is interesting to note that CPZ also eliminated clonidine analgesia at 45 and 75 min, times at which CPZ did not, by itself, produce hyperalgesia. The CPZ hyperalgesia finding contrasts with that of Mitchell (1966) and of Paalzow and Paalzow (1975) who found that either CPZ was an analgesic equipotent with morphine or that it had no analgesic properties at all. However, meaningful comparisons are difficult because, in the first instance the latency to escape tooth-pulp stimulation in the cat was assessed, whereas in the second case, response threshold were determined after noxious electrical stimulation of the tail. Despite this, there is evidence of another (primarily) dopaminergic antagonist, haloperidol, inducing hyperalgesia in the hot-plate test (55~ when administered intracranially in rats ( L i n e t al. 1981). Whereas CPZ antagonized clonidine analgesia, it significantly increased the early phase of morphine analgesia. Mitchell (1966) and Paalzow and Paalzow (1975) also reported CPZ enhancement of morphine effects in their studies. Though it is possible that this represents a peripheral effect on morphine metabolism (Yeh and Mitchell 1971) rather than a central neurochemical interaction, our data argue for different neurochemica! mechanisms subserving morphine- and clonidine-induced analgesias and since CPZ is a neurochemically promiscuous drug, the basis of its modulation of nociception remains to be determined. It is clear, however, that the ability of CPZ to induce hyperalgesia even though it is a sedating agent makes it a valuable pharmacological tool for analgesic research.
Acknowledgements. This research was supported by a grant from the Australian Research Grants Committee (Grant No. 7.L29.207). We thank Dr. L. Storlien for his critical reading of this paper and for his invaluable comments, and Dr. G. Paxinos for the loan of apparatus.
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