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Morphine-induced analgesia and explosive motor behavior in an amphibian
Brain Research, 273 (1983)297-305 Elsevier
297
Morphine-Induced Analgesia and Explosive Motor Behavior in an Amphibian PAUL D. PEZALLA Department of Biological Sciences, University of lllinois at Chicago, Box 4348, Chicago, IL, 60680 (U.S.A.)
(Accepted January llth, 1983) Key words: morphine-- naloxone-- amphibians-- analgesia-- explosivemotor behavior
Morphine sulfate is a weak analgesic in the frog Rana pipiens pipiens, causing a slight increase in nocieeptivethreshold at a dose of 10 mg/kg and a pronounced increase at 100 mg/kg. Morphine-inducedanalgesia persists for at least 165 rain and is significantlyattenuated by naloxone. The analgesicdoses of morphine are well below the lethal dose and are without noticeable effect on the behavior of the frogs or their responses to non-painful stimuli. Higher doses of morphine (320and 640 mg/kg) induced a state of hyper-responsiveness to sensory stimuli similar to the explosivemotor behavior induced in rats by microinjectionof morphine into the periaqueduetal gray.
INTRODUCTION Since the discovery of the opioid peptides, Met-enkephalin and Leu-enkephalin, in 197510, considerable effort has been expended in attempting to define the physiological functions of these and other endogenous opioids (generically referred to as endorphins). Much of this work has been based on previous research on the pharmacology of morphine and related narcotic analgesics. The reasoning behind this approach is that morphine is presumed to exert its actions by interacting with a receptor whose physiologically relevant ligand is one or another of the endogenous opioid peptides. Thus the actions of morphine are taken to be predictive of the functions of endorphins. Because one of the most striking, and clinically important, actions of morphine is its ability to suppress pain responsiveness, there has been extensive research on the possibility of endorphins functioning as neurotransmitters or neuromodulations in pain pathways. It is well established that endorphins have potent antinociceptive actions in mammals when administered intracerebrally and that this action is blocked by naloxone6,19, 35. Endorphins are also thought to mediate, at least in part, analgesia induced by acupuncture, electrical stimulation of the brain, and certain forms of stress2,3,7,8,15,17,18,20,22,31,36,37,39. Further support for the involvement of endorphins in mod0006-8993/83/$03.00© 1983Elsevier SciencePublishers B.V.
ulating pain sensitivity is provided by studies on the distribution of opioid receptors and peptides in the central nervous system. High densities of both are found in regions sensitive to stimulation-produced analgesia and to microinjections of morphine and endorphins19, 35. The fact that stimulation-produced analgesia is accompanied by increased levels of endorphins in cerebrospinal fluid is also indicative of a role of endorphins in pain pathwaysl,2,9,19. Thus, a variety of experimental approaches have provided evidence for the involvement of endorphins in mediating pain sensitivity in mammals. In nonmammalian vertebrates, however, our knowledge of the opioid systems is quite rudimentary. Opioid peptides and binding sites appear to be ubiquitous among the vertebrates26,33 but little is known about their possible functions. In particular it is often difficult to determine if opiates or opioids are analgesic in nonmammals because of the lack of appropriate tests for nociception and the problems of distinguishing pain reactivity from general motor deficits. The little data available suggests that morphine is, at best, a weak analgesic in goldfish 14, lizzards21, and chickens s. While these studies at least suggested an antinociceptire action of morphine in three vertebrate classes, the single report concerned with an amphibian failed to demonstrate such an effect on the frog Rana esculenta24. In this study, morphine at very high doses
298 (20-100 mg/kg) had no effect on the frogs' response to either electrical stimuli or to a hot plate 24. This failure to demonstrate an effect of morphine may have been due to the use of inappropriate tests for analgesia (vide infra). Because frogs, and other amphibians, do produce opioid peptides11,27.33 and do have opiate binding sites in their central nervous systems4, 33 and because previous work has suggested a possible antinociceptive action of morphine in representatives of other major vertebrate groups, I have reinvestigated the effect of morphine on pain responsiveness in a frog. Additionally, since naloxone blockage of morphine analgesia is a necessary criterion for the involvement of opiate receptors (one which has not been fulfilled for any non-mammalian species), I have shown the effect of naloxone on morphine analgesia. The data in this report are derived from experiments in which a new test for analgesia was used.
MATERIALS AND METHODS
Animals Northern grass frogs (Rana pipiens pipiens) weighing between 11 and 34 g were obtained from Nasco (Fort Atkinson, WI) and were kept at 20-22 °C in stainless steel terrestrial/aquatic enclosures with continuous water flow and a 12 h photoperiod (lights on at 07.00 h). The frogs were fed live crickets thrice weekly and were acclimated to laboratory conditions for at least 3 weeks before being used. They were not fed on the day they were used. The results reported here were obtained between September and February.
Drug administration Naloxone HCI (kindly supplied by Endo Laboratories) and morphine sulfate (a generous gift from Merck Sharp and Dohme) were dissolved in 0.7% NaCI at a concentration such that an injection of 10 /A/g body weight (20 #l/g for animals receiving 640 mg/kg) yielded the desired dose. Controls received 10 or 20/~l/g of 0.7% NaCI. The solutions were coded and the experimenter was uninformed of their identity until completion of the experiment. Injections were made into the dorsal lymph sac with a Hamilton microliter syringe fitted with a 23-gauge needle.
Nociceptive testing The nociceptive threshold of frogs was determined by observing their response to a drop of acetic acid applied to a hind limb. One hour before the start of an experiment, frogs were randomly assigned to a treatment group, weighed, and placed in individual plastic pans (20 × 27 x 15 cm high) containing water to a depth of 0.5 cm. Serial dilutions of acetic acid were made (2 vols. acetic acid: 1 vol. distilled water) beginning with a 15 M solution and proceeding to 0.39 M. This yields 10 dilutions of acid with concentrations equally spaced on a logarithmic scale. The solutions were coded, in order of increasing concentration, from 1 to 10. Nociceptive testing was done by placing, with a Pasteur pipet, a single drop of acetic acid on the dorsal surface of the thigh. Testing began with the lowest concentration of acetic acid and continued with increasing concentrations until the nociceptive threshold is reached. The nociceptive threshold is defined as the lowest concentration of acetic acid which causes the frog to vigorously wipe the treated leg. In order to prevent tissue damage, the acetic acid was rinsed off with water after 4 s if the animal failed to respond or immediately if the animal responded. The acid is applied to each leg alternately.
Experimental design To insure that the doses of morphine used were sub-lethal and without effect on general motor activity, frogs were injected with saline or morphine sulfate at 20, 40, 80, 160,320, and 640 mg/kg. The animals were observed periodically over a period of 48 h and their responses to tactile (a light touch), auditory (tap on the cage lid), and visual (turning the room lights off and on, passing a hand over the cage) stimuli were noted. The stability of the nociceptive threshold over time was determined by testing drug-free frogs at 90 min or 2-3 day intervals. The effect of morphine on nociceptive threshold was determined by testing animals 1 h before and twice after injection of saline or morphine sulfate (0.1-100 mg/kg). Groups of frogs were tested before and at either 15 and 105 min, 45 and 135 min, or 75 and 165 min after injection. By testing at 3, rather widely spaced, intervals it was possible to avoid the pronounced increase in threshold which oc-
299 curred when frogs were repeatedly tested at 15 or 30 min intervals. Data analysis
The nociceptive threshold (NT) was defined as the code number of the lowest concentration of acetic acid which elicited a vigorous wipe. The code numbers are related to the molar concentration (M) of acetic acid according to the expression log M = 0.1761 (code number) --0.5849. Data are displayed as either NT, change in NT ( A N T = post-injection NT minus pre-injection NT), or total change in NT (sum of ANT for each animal). In a few cases animals failed to respond to the highest concentration of acetic acid. The impact of this 'ceiling effect' was lessened by making the conservative assumption that, if it were possible to use the next highest concentration in the progression (22.5 M), it would have produced a response. Thus, animals not responding to solution number 10 were assigned an NT of 11. Data analysis was done using non-parametric tests because the conditions necessary for parametric tests were not met. The significance of treatment effects was assessed using Friedman two-way analysis of variance for related samples or Kruskal-Wallis one-way analysis of variance for unrelated samples 32. If significant treatment effects obtained, additional comparisons were made using the Randomization test 32. RESULTS Prior to studies on the possible nociceptive action of morphine, it was necessary to determine the lethal and toxic doses of morphine. Morphine was not le-
thai to frogs when injected into the dorsal lymph sac at doses ranging from 20 mg/kg to 320 mg/kg (Table I). A single death occurred 21 h after 640 mg/kg morphine. Only the two highest doses (320 and 640 mg/kg) of morphine caused any obvious behavioral changes. Three of 6 frogs receiving 320 mg/kg and all frogs receiving 640 mg/kg became excitable and hyper-responsive to sensory stimuli. Approaching the cage, gently tapping the cage, or touching the frogs lead to explosive jumping. Frogs receiving the highest dose responded similarly to changes in illumination and also croaked vigorously after these stimuli. Additionally, 640 mg/kg morphine induced an uncoordinated twitching and kicking of the hind limbs which progressed into a cataleptic-like state characterized by extreme muscular rigidity, fully extended limbs, and complete loss of muscular control. These gross effects of morphine were manifested 8-16 h after injection and, in the most extreme cases, persisted for more than 48 h. Animals treated with morphine at doses from 20 to 160 mg/kg were indistinguishable from saline injected controls with respect to locomotor ability, response to tactile or auditory stimuli, and righting ability. None appeared to be either sedated or excited. A necessary prerequisite for research on the actions of analgesics is the availability of a suitable test for nociceptive threshold. A number of methods commonly used on mammals were tried on frogs but were found to be unsatisfactory. Electric shock applied to the hind limbs caused muscular contractions at voltages that did not appear to be unpleasant to the frogs. Despite the rather vigorous twitching the frogs did not usually attempt to escape or show any other
TABLE I Mortality and overt behavioral changes caused by morphine Treatment
n
Number of animals 48 h post-injection
24 h post-injection Alive
Excitable
Rigid
Alive
Excitable
Rigid
Saline
3
3
0
0
3
0
0
Morphine 80 mg/kg 160 mg/kg 320 mg/kg 640 mg/kg
6 6 6 6
6 6 6 5
0 0 3 5
0 0 0 5
6 6 6 5
0 0 0 5
0 0 0 5
300 obvious signs of distress. It appears that electric shock activates elements other than nociceptors and, thus, is an inappropriate method for determining nociceptive threshold in frogs. Latency to escape from either a hot plate or a shallow hot water bath (40-60 °C) was also unsuitable because frogs frequently immediately attempt to escape from any novel environment. Those few frogs which remained calm would not attempt to escape even as their body temperatures rose to levels incompatible with life. Intraperitoneal injections of either acetic acid (concentrations ranging up to 15 M) or phenyl-p-benzoquinone (0.02% in 5% aqueous ethanol) also failed to induce any sign of discomfort. These two agents are commonly used to activate deep pain receptors in mammals and our preparations were capable of inducing writhing in rats. In contrast to the above methods, acetic acid applied to the hind limbs is suitable test for nociception in frogs. Subthreshold concentrations of acetic acid generally do not induce any reaction by the frog although in some cases the animal will draw its hind limbs in close to its torso, This reaction is also fre-
quently seen when the acid is rinsed off with a gentle stream of water. Higher concentrations of acetic acid cause the frog to vigorously wipe the spot of application with its foot and, in most cases, to jump away. The concentration causing this response was defined as the nociceptive threshold. As shown in Fig. 1, the nociceptive threshold is reasonably stable over a time course of hours or days. Frogs (n=24) tested 3 times at 90 min intervals showed a very slight and non-significant increase in pain threshold (Fig. 1A). Similarly, a group of frogs (n=7) tested on 3 different days showed no significant changes in threshold (Fig. 1B). Although we have not specifically investigated the possibility of seasonal variations in pain threshold, post hoc analysis of the pre-treatment thresholds gave no indication of seasonal trends over the months September through February. Fig. 2 shows the effect of morphine on nociceptive threshold. Kruskal-Wallis one-way analysis of variance of the data for all morphine and saline injected animals revealed a significant treatment effect (P < 0.001). Therefore a dose by dose comparison of the total change in threshold was made. As shown in
B
41A
2
TI
0
1
90
MINUTES
I
180
I
1
I
3
1
6
DAYS
Fig. 1. Stability of the nociceptive threshold (NT) over time. A: NT determined on a group of 24 frogs at 90 rain intervals. B: NT determined on a group of 7 frogs on 3 days. Data expressed as mean + S.E.M. No significant changes in NT occurred (Friedman two-way analysis of variance).
301
A 5
]
xxx
4 F- 3 Z
X~xxx x
B
xx
8
x
xxx
tZ
<3
<3
XXXX
6 4 X
0
15 45 75 105 135 165 Time after injection(min)
0 0.11.0 10 100 Dose(mg/kg)
Fig. 2. Morphine induced analgesia. Frogs were injected at t=0 with either saline or morphine sulfate (0.1, 1.0, 10 or 100 mg/kg). The NT of each animal was determined 1 h before and at two times (either 15 and 105 min, 45 and 135 rain, or 75 and 165 min) after injection. A: time course for change in NT after saline (o) or 10 mg/kg (e) or 100 mg/kg (n) morphine sulfate. Data are plotted as the post-injection NT minus the pre-injection NT. (mean +_S.E.M., n=6 animals per point for morphine, n = 12 for saline). B: dose effect of morphine on change in NT. Data obtained as in (A) and plotted as the sum of the change in NT for each animal (mean + S.E.M., n = 18 for each dose of morphine, n = 69 for saline), x, P < 0.05; xx, P < 0.01; xxx, P < 0.005; and xxxx, P < 0.0005 for morphine versus saline (Randomization test for two independent variables, one-tailed),
Fig. 2B, only the two highest doses of morphine, 10 and 100 mg/kg, produced significant analgesia (Randomization test). The time course for analgesia induced by these doses is shown in Fig. 2A. Morphine at 100 mg/kg caused a pronounced and significant analgesia which was apparent at 15 min and persisted for the duration of the experiment. At 10 mg/kg, significant analgesia was obtained only at 165 min. Analgesia induced by 100 mg/kg morphine was attenuated by pre-treatment with 100 mg/kg naloxone (Fig. 3). The total change in threshold for animals receiving naloxone plus morphine was significantly lower than that for animals receiving morphine alone (P < 0.0005, Randomization test, Fig. 3B) but was not as low as for animals receiving saline (compare Fig. 2B and Fig. 3B). A significant reduction in nociceptive threshold obtained at 45 rain and at each subsequent time (Fig. 3A). The thresholds did not differ significantly at 15 rain.
DISCUSSION I have not extensively documented the behavioral actions of morphine in the frog since my primary purpose was to investigate its analgesic actions. Observations on the behavior of frogs treated with high doses of morphine were made to ensure that I was working with doses which did not induce behavioral changes which would compromise my ability to assess analgesia and which were substantially below the lethal dose. My observations confirm those made over a century ago on two related species of frogs, Rana temporaria and Rana esculenta, by Witkoski as. He noted that morphine at 20-50 mg per animal induced increased sensitivity, 'stretch cramps' of the hind limbs, and an 'epileptic fit' type of condition and that frogs are remarkably tolerant to the lethal effects of morphine, surviving even 50 mg injections 38. Although the weights of the animals were not given, this
302
B XXX
41
xxx
<13
xx
8
XXXX
I I
I
15
45
I
75
ii
I
I
105 135 165
Time after injection(min)
Nal Mor + Mor
Fig. 3. Attenuation by naloxone of morphine induced analgesia. Data obtained and presented as in Fig. 2. A: change in NT after 100 mg/kg morphine sulfate (o, n = 16) or 100 mg/kg naloxone HC1 and 100 mg/kg morphine sulfate (o, n = 10). B: sum of the change in NT after morphine (Mor) or naloxone (Nal) plus morphine, x, P < 0.05; xx P < 0.025; xxx, P < 0.005; and xxxx, P < 0.0005 for morphine versus naloxone plus morphine.
was probably on the order of 500-1000 mg/kg, similar to my highest dose. My finding that 5 of 6 flogs survived a 640 mg/kg dose of morphine was not unexpected because morphine's toxicity is, at least in mammals, due to its depressant action on respiratory rate 13 and, while morphine does depress the respiratory rate of flogs (unpublished observations), they are capable of significant transcutaneous respiration2S. The hyper-responsivity observed in frogs after 320 or 640 mg/kg of morphine is remarkably similar to the explosive motor behavior (EMB) observed in rats microinjected with morphine into the periaqueductal gray (PAG)12,16. Both ratsl2,16 and frogs (this study) exhibit extreme excitability and hyper-responsivity to very mild visual, tactile, or auditory stimuli. Additionally, the EMB of rats is 'accompanied by shrill distress vocalizations'12 and that of frogs by excessive croaking. While it appears that croaking may be a 'distress vocalization' (it can, for example, be elicited by rough handling) it may be premature to classify
the croaking evoked by morphine as a sign of distress. There are two important differences between the EMB of rats and frogs. First, the syndrome becomes increasingly severe in frogs, leading eventually to rigid immobility. Second, the syndrome can be evoked in rats only by direct microinjection of morphine into the PAG, the putative substrate for this behavior 12, while in frogs systemic injections are effective. Jacquet et a1.12 and LaBella et al.16 have proposed that the EMB elicited morphine is evidence for a new class of opiate receptor, one differing from the classical opiate receptor in that it is insensitive to naloxone, endorphins, and enkephalins and is less stereospecific. They suggest that the classical receptor mediates analgesia and the mild hyperactive state and that it inhibits the putative 'excitatory', natoxone-insensitive receptor which mediates EMB. Thus, EMB can be evoked only by selective activation of the excitatory receptor, either by direct injection of morphine into the PAG or by blocking the classical receptor with naloxone. That EMB occurs in frogs af-
303 ter systemic injection suggests that the proposed inhibitory coupling between the two receptor types is less pronounced than in rats. Our behavioral observations are quite preliminary but do demonstrate that EMB is not limited to murine species and that the frog may be well suited for studies of this phenomenon. Studies on the drug specificity, stereospecificity, and naloxone sensitivity are clearly needed and are in progress. Morphine is a weak analgesic in frogs, producing a very slight increase in nociceptive threshold at 10 mg/kg (26.4/~mol/kg), a dose 2-5 times greater than EDs0 for intravenous morphine in rats29,40. Pronounced analgesia obtained only at 100 mg/kg, a very high dose (Fig. 2). This raises the possibility that the apparent analgesic action was non-specific. However, this possibility seems to be unlikely for a number of reasons. First, the doses of morphine effective in raising the nociceptive threshold were substantially lower than those causing motor deficits or other noticeable changes in behavior. Frogs receiving 10 or 100 mg/kg of morphine were indistinguishable from controls in their responses to non-painful stimuli. Second the effect was blocked by naloxone. Although a rather high dose of naloxone was used, it is unlikely that its action was non-specific because naloxone alone has no effect on nociceptive thresholds at doses up to 100 mg/kg and at an extremely high dose (640 mg/kg) induces flaccidity and unresponsiveness to stimuli (manuscript in preparation). Thus, the dominant non-specific action of naloxone would appear to be analgesia rather than hyper-responsiveness to stimulation. Finally, the acetic acid test is a true test for nociception since the nociceptors in frog skin have been shown by electrophysiological means to respond to weak acid solutions as well as to pinch and pin prick ~. The reason for the low potency of morphine in frogs is not known but several possibilities can be considered. The drug was administered via the dorsal lymph sac, a route which may not provide for expeditious delivery to the CNS. In mice, for example,
morphine is more effective after intravenous than subcutaneous 29 injection. Possibly it would also be more potent if given intravenously to frogs. It is also possible that morphine is a poor ligand for the frog receptor mediating analgesia or that it is of low efficacy once bound. Since fl-endorphins from nonmammalian vertebrates differ considerably in structure and binding activity from fl-endorphins from mammals23, it is likely that the receptors also differ. Finally, it may be that opioid systems have no role, or a minor one, in modulating nociception in frogs. There is little evidence that opioids have a significant role in pain pathways in any non-mammalian vertebrate. Goldfish submerged in morphine-containing water apparently exhibit an increase in threshold to electrical stimulation 14 but it is difficult to assess the significance of this because of uncertainties about the dose of morphine absorbed and because of the lack of documentation that the electrical shock was activating nociceptors. The apparent analgesia may in fact be a motor deficit since it has been shown that endorphin and enkephalin analogs reduce the general activity level of goldfish25. There are isolated reports on morphine-induced analgesia in reptiles and birds. Morphine at a fairly low dose (5 mg/kg) produces a slight increase in tail-flick latency in the lizzard Ano//s21 and is alsoanalgesic in birds when the painful stimulus is electrical wing-shock5 but not when it is toe-pinch 3°. In none of these cases was the effect of morphine shown to be blocked by naloxone nor was dose-dependency demonstrated. To my knowledge, the present paper is the first documenting naloxoneattenuation of morphine-induced analgesia in a nonmammalian vertebrate. ACKNOWLEDGEMENTS I thank Harriet Bielawski for technical assistance, Craig Stevens for comments on the manuscript, Joanne Graves for manuscript preparation and the Research Board of the University of Illinois at Chicago for support.
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297
Morphine-Induced Analgesia and Explosive Motor Behavior in an Amphibian PAUL D. PEZALLA Department of Biological Sciences, University of lllinois at Chicago, Box 4348, Chicago, IL, 60680 (U.S.A.)
(Accepted January llth, 1983) Key words: morphine-- naloxone-- amphibians-- analgesia-- explosivemotor behavior
Morphine sulfate is a weak analgesic in the frog Rana pipiens pipiens, causing a slight increase in nocieeptivethreshold at a dose of 10 mg/kg and a pronounced increase at 100 mg/kg. Morphine-inducedanalgesia persists for at least 165 rain and is significantlyattenuated by naloxone. The analgesicdoses of morphine are well below the lethal dose and are without noticeable effect on the behavior of the frogs or their responses to non-painful stimuli. Higher doses of morphine (320and 640 mg/kg) induced a state of hyper-responsiveness to sensory stimuli similar to the explosivemotor behavior induced in rats by microinjectionof morphine into the periaqueduetal gray.
INTRODUCTION Since the discovery of the opioid peptides, Met-enkephalin and Leu-enkephalin, in 197510, considerable effort has been expended in attempting to define the physiological functions of these and other endogenous opioids (generically referred to as endorphins). Much of this work has been based on previous research on the pharmacology of morphine and related narcotic analgesics. The reasoning behind this approach is that morphine is presumed to exert its actions by interacting with a receptor whose physiologically relevant ligand is one or another of the endogenous opioid peptides. Thus the actions of morphine are taken to be predictive of the functions of endorphins. Because one of the most striking, and clinically important, actions of morphine is its ability to suppress pain responsiveness, there has been extensive research on the possibility of endorphins functioning as neurotransmitters or neuromodulations in pain pathways. It is well established that endorphins have potent antinociceptive actions in mammals when administered intracerebrally and that this action is blocked by naloxone6,19, 35. Endorphins are also thought to mediate, at least in part, analgesia induced by acupuncture, electrical stimulation of the brain, and certain forms of stress2,3,7,8,15,17,18,20,22,31,36,37,39. Further support for the involvement of endorphins in mod0006-8993/83/$03.00© 1983Elsevier SciencePublishers B.V.
ulating pain sensitivity is provided by studies on the distribution of opioid receptors and peptides in the central nervous system. High densities of both are found in regions sensitive to stimulation-produced analgesia and to microinjections of morphine and endorphins19, 35. The fact that stimulation-produced analgesia is accompanied by increased levels of endorphins in cerebrospinal fluid is also indicative of a role of endorphins in pain pathwaysl,2,9,19. Thus, a variety of experimental approaches have provided evidence for the involvement of endorphins in mediating pain sensitivity in mammals. In nonmammalian vertebrates, however, our knowledge of the opioid systems is quite rudimentary. Opioid peptides and binding sites appear to be ubiquitous among the vertebrates26,33 but little is known about their possible functions. In particular it is often difficult to determine if opiates or opioids are analgesic in nonmammals because of the lack of appropriate tests for nociception and the problems of distinguishing pain reactivity from general motor deficits. The little data available suggests that morphine is, at best, a weak analgesic in goldfish 14, lizzards21, and chickens s. While these studies at least suggested an antinociceptire action of morphine in three vertebrate classes, the single report concerned with an amphibian failed to demonstrate such an effect on the frog Rana esculenta24. In this study, morphine at very high doses
298 (20-100 mg/kg) had no effect on the frogs' response to either electrical stimuli or to a hot plate 24. This failure to demonstrate an effect of morphine may have been due to the use of inappropriate tests for analgesia (vide infra). Because frogs, and other amphibians, do produce opioid peptides11,27.33 and do have opiate binding sites in their central nervous systems4, 33 and because previous work has suggested a possible antinociceptive action of morphine in representatives of other major vertebrate groups, I have reinvestigated the effect of morphine on pain responsiveness in a frog. Additionally, since naloxone blockage of morphine analgesia is a necessary criterion for the involvement of opiate receptors (one which has not been fulfilled for any non-mammalian species), I have shown the effect of naloxone on morphine analgesia. The data in this report are derived from experiments in which a new test for analgesia was used.
MATERIALS AND METHODS
Animals Northern grass frogs (Rana pipiens pipiens) weighing between 11 and 34 g were obtained from Nasco (Fort Atkinson, WI) and were kept at 20-22 °C in stainless steel terrestrial/aquatic enclosures with continuous water flow and a 12 h photoperiod (lights on at 07.00 h). The frogs were fed live crickets thrice weekly and were acclimated to laboratory conditions for at least 3 weeks before being used. They were not fed on the day they were used. The results reported here were obtained between September and February.
Drug administration Naloxone HCI (kindly supplied by Endo Laboratories) and morphine sulfate (a generous gift from Merck Sharp and Dohme) were dissolved in 0.7% NaCI at a concentration such that an injection of 10 /A/g body weight (20 #l/g for animals receiving 640 mg/kg) yielded the desired dose. Controls received 10 or 20/~l/g of 0.7% NaCI. The solutions were coded and the experimenter was uninformed of their identity until completion of the experiment. Injections were made into the dorsal lymph sac with a Hamilton microliter syringe fitted with a 23-gauge needle.
Nociceptive testing The nociceptive threshold of frogs was determined by observing their response to a drop of acetic acid applied to a hind limb. One hour before the start of an experiment, frogs were randomly assigned to a treatment group, weighed, and placed in individual plastic pans (20 × 27 x 15 cm high) containing water to a depth of 0.5 cm. Serial dilutions of acetic acid were made (2 vols. acetic acid: 1 vol. distilled water) beginning with a 15 M solution and proceeding to 0.39 M. This yields 10 dilutions of acid with concentrations equally spaced on a logarithmic scale. The solutions were coded, in order of increasing concentration, from 1 to 10. Nociceptive testing was done by placing, with a Pasteur pipet, a single drop of acetic acid on the dorsal surface of the thigh. Testing began with the lowest concentration of acetic acid and continued with increasing concentrations until the nociceptive threshold is reached. The nociceptive threshold is defined as the lowest concentration of acetic acid which causes the frog to vigorously wipe the treated leg. In order to prevent tissue damage, the acetic acid was rinsed off with water after 4 s if the animal failed to respond or immediately if the animal responded. The acid is applied to each leg alternately.
Experimental design To insure that the doses of morphine used were sub-lethal and without effect on general motor activity, frogs were injected with saline or morphine sulfate at 20, 40, 80, 160,320, and 640 mg/kg. The animals were observed periodically over a period of 48 h and their responses to tactile (a light touch), auditory (tap on the cage lid), and visual (turning the room lights off and on, passing a hand over the cage) stimuli were noted. The stability of the nociceptive threshold over time was determined by testing drug-free frogs at 90 min or 2-3 day intervals. The effect of morphine on nociceptive threshold was determined by testing animals 1 h before and twice after injection of saline or morphine sulfate (0.1-100 mg/kg). Groups of frogs were tested before and at either 15 and 105 min, 45 and 135 min, or 75 and 165 min after injection. By testing at 3, rather widely spaced, intervals it was possible to avoid the pronounced increase in threshold which oc-
299 curred when frogs were repeatedly tested at 15 or 30 min intervals. Data analysis
The nociceptive threshold (NT) was defined as the code number of the lowest concentration of acetic acid which elicited a vigorous wipe. The code numbers are related to the molar concentration (M) of acetic acid according to the expression log M = 0.1761 (code number) --0.5849. Data are displayed as either NT, change in NT ( A N T = post-injection NT minus pre-injection NT), or total change in NT (sum of ANT for each animal). In a few cases animals failed to respond to the highest concentration of acetic acid. The impact of this 'ceiling effect' was lessened by making the conservative assumption that, if it were possible to use the next highest concentration in the progression (22.5 M), it would have produced a response. Thus, animals not responding to solution number 10 were assigned an NT of 11. Data analysis was done using non-parametric tests because the conditions necessary for parametric tests were not met. The significance of treatment effects was assessed using Friedman two-way analysis of variance for related samples or Kruskal-Wallis one-way analysis of variance for unrelated samples 32. If significant treatment effects obtained, additional comparisons were made using the Randomization test 32. RESULTS Prior to studies on the possible nociceptive action of morphine, it was necessary to determine the lethal and toxic doses of morphine. Morphine was not le-
thai to frogs when injected into the dorsal lymph sac at doses ranging from 20 mg/kg to 320 mg/kg (Table I). A single death occurred 21 h after 640 mg/kg morphine. Only the two highest doses (320 and 640 mg/kg) of morphine caused any obvious behavioral changes. Three of 6 frogs receiving 320 mg/kg and all frogs receiving 640 mg/kg became excitable and hyper-responsive to sensory stimuli. Approaching the cage, gently tapping the cage, or touching the frogs lead to explosive jumping. Frogs receiving the highest dose responded similarly to changes in illumination and also croaked vigorously after these stimuli. Additionally, 640 mg/kg morphine induced an uncoordinated twitching and kicking of the hind limbs which progressed into a cataleptic-like state characterized by extreme muscular rigidity, fully extended limbs, and complete loss of muscular control. These gross effects of morphine were manifested 8-16 h after injection and, in the most extreme cases, persisted for more than 48 h. Animals treated with morphine at doses from 20 to 160 mg/kg were indistinguishable from saline injected controls with respect to locomotor ability, response to tactile or auditory stimuli, and righting ability. None appeared to be either sedated or excited. A necessary prerequisite for research on the actions of analgesics is the availability of a suitable test for nociceptive threshold. A number of methods commonly used on mammals were tried on frogs but were found to be unsatisfactory. Electric shock applied to the hind limbs caused muscular contractions at voltages that did not appear to be unpleasant to the frogs. Despite the rather vigorous twitching the frogs did not usually attempt to escape or show any other
TABLE I Mortality and overt behavioral changes caused by morphine Treatment
n
Number of animals 48 h post-injection
24 h post-injection Alive
Excitable
Rigid
Alive
Excitable
Rigid
Saline
3
3
0
0
3
0
0
Morphine 80 mg/kg 160 mg/kg 320 mg/kg 640 mg/kg
6 6 6 6
6 6 6 5
0 0 3 5
0 0 0 5
6 6 6 5
0 0 0 5
0 0 0 5
300 obvious signs of distress. It appears that electric shock activates elements other than nociceptors and, thus, is an inappropriate method for determining nociceptive threshold in frogs. Latency to escape from either a hot plate or a shallow hot water bath (40-60 °C) was also unsuitable because frogs frequently immediately attempt to escape from any novel environment. Those few frogs which remained calm would not attempt to escape even as their body temperatures rose to levels incompatible with life. Intraperitoneal injections of either acetic acid (concentrations ranging up to 15 M) or phenyl-p-benzoquinone (0.02% in 5% aqueous ethanol) also failed to induce any sign of discomfort. These two agents are commonly used to activate deep pain receptors in mammals and our preparations were capable of inducing writhing in rats. In contrast to the above methods, acetic acid applied to the hind limbs is suitable test for nociception in frogs. Subthreshold concentrations of acetic acid generally do not induce any reaction by the frog although in some cases the animal will draw its hind limbs in close to its torso, This reaction is also fre-
quently seen when the acid is rinsed off with a gentle stream of water. Higher concentrations of acetic acid cause the frog to vigorously wipe the spot of application with its foot and, in most cases, to jump away. The concentration causing this response was defined as the nociceptive threshold. As shown in Fig. 1, the nociceptive threshold is reasonably stable over a time course of hours or days. Frogs (n=24) tested 3 times at 90 min intervals showed a very slight and non-significant increase in pain threshold (Fig. 1A). Similarly, a group of frogs (n=7) tested on 3 different days showed no significant changes in threshold (Fig. 1B). Although we have not specifically investigated the possibility of seasonal variations in pain threshold, post hoc analysis of the pre-treatment thresholds gave no indication of seasonal trends over the months September through February. Fig. 2 shows the effect of morphine on nociceptive threshold. Kruskal-Wallis one-way analysis of variance of the data for all morphine and saline injected animals revealed a significant treatment effect (P < 0.001). Therefore a dose by dose comparison of the total change in threshold was made. As shown in
B
41A
2
TI
0
1
90
MINUTES
I
180
I
1
I
3
1
6
DAYS
Fig. 1. Stability of the nociceptive threshold (NT) over time. A: NT determined on a group of 24 frogs at 90 rain intervals. B: NT determined on a group of 7 frogs on 3 days. Data expressed as mean + S.E.M. No significant changes in NT occurred (Friedman two-way analysis of variance).
301
A 5
]
xxx
4 F- 3 Z
X~xxx x
B
xx
8
x
xxx
tZ
<3
<3
XXXX
6 4 X
0
15 45 75 105 135 165 Time after injection(min)
0 0.11.0 10 100 Dose(mg/kg)
Fig. 2. Morphine induced analgesia. Frogs were injected at t=0 with either saline or morphine sulfate (0.1, 1.0, 10 or 100 mg/kg). The NT of each animal was determined 1 h before and at two times (either 15 and 105 min, 45 and 135 rain, or 75 and 165 min) after injection. A: time course for change in NT after saline (o) or 10 mg/kg (e) or 100 mg/kg (n) morphine sulfate. Data are plotted as the post-injection NT minus the pre-injection NT. (mean +_S.E.M., n=6 animals per point for morphine, n = 12 for saline). B: dose effect of morphine on change in NT. Data obtained as in (A) and plotted as the sum of the change in NT for each animal (mean + S.E.M., n = 18 for each dose of morphine, n = 69 for saline), x, P < 0.05; xx, P < 0.01; xxx, P < 0.005; and xxxx, P < 0.0005 for morphine versus saline (Randomization test for two independent variables, one-tailed),
Fig. 2B, only the two highest doses of morphine, 10 and 100 mg/kg, produced significant analgesia (Randomization test). The time course for analgesia induced by these doses is shown in Fig. 2A. Morphine at 100 mg/kg caused a pronounced and significant analgesia which was apparent at 15 min and persisted for the duration of the experiment. At 10 mg/kg, significant analgesia was obtained only at 165 min. Analgesia induced by 100 mg/kg morphine was attenuated by pre-treatment with 100 mg/kg naloxone (Fig. 3). The total change in threshold for animals receiving naloxone plus morphine was significantly lower than that for animals receiving morphine alone (P < 0.0005, Randomization test, Fig. 3B) but was not as low as for animals receiving saline (compare Fig. 2B and Fig. 3B). A significant reduction in nociceptive threshold obtained at 45 rain and at each subsequent time (Fig. 3A). The thresholds did not differ significantly at 15 rain.
DISCUSSION I have not extensively documented the behavioral actions of morphine in the frog since my primary purpose was to investigate its analgesic actions. Observations on the behavior of frogs treated with high doses of morphine were made to ensure that I was working with doses which did not induce behavioral changes which would compromise my ability to assess analgesia and which were substantially below the lethal dose. My observations confirm those made over a century ago on two related species of frogs, Rana temporaria and Rana esculenta, by Witkoski as. He noted that morphine at 20-50 mg per animal induced increased sensitivity, 'stretch cramps' of the hind limbs, and an 'epileptic fit' type of condition and that frogs are remarkably tolerant to the lethal effects of morphine, surviving even 50 mg injections 38. Although the weights of the animals were not given, this
302
B XXX
41
xxx
<13
xx
8
XXXX
I I
I
15
45
I
75
ii
I
I
105 135 165
Time after injection(min)
Nal Mor + Mor
Fig. 3. Attenuation by naloxone of morphine induced analgesia. Data obtained and presented as in Fig. 2. A: change in NT after 100 mg/kg morphine sulfate (o, n = 16) or 100 mg/kg naloxone HC1 and 100 mg/kg morphine sulfate (o, n = 10). B: sum of the change in NT after morphine (Mor) or naloxone (Nal) plus morphine, x, P < 0.05; xx P < 0.025; xxx, P < 0.005; and xxxx, P < 0.0005 for morphine versus naloxone plus morphine.
was probably on the order of 500-1000 mg/kg, similar to my highest dose. My finding that 5 of 6 flogs survived a 640 mg/kg dose of morphine was not unexpected because morphine's toxicity is, at least in mammals, due to its depressant action on respiratory rate 13 and, while morphine does depress the respiratory rate of flogs (unpublished observations), they are capable of significant transcutaneous respiration2S. The hyper-responsivity observed in frogs after 320 or 640 mg/kg of morphine is remarkably similar to the explosive motor behavior (EMB) observed in rats microinjected with morphine into the periaqueductal gray (PAG)12,16. Both ratsl2,16 and frogs (this study) exhibit extreme excitability and hyper-responsivity to very mild visual, tactile, or auditory stimuli. Additionally, the EMB of rats is 'accompanied by shrill distress vocalizations'12 and that of frogs by excessive croaking. While it appears that croaking may be a 'distress vocalization' (it can, for example, be elicited by rough handling) it may be premature to classify
the croaking evoked by morphine as a sign of distress. There are two important differences between the EMB of rats and frogs. First, the syndrome becomes increasingly severe in frogs, leading eventually to rigid immobility. Second, the syndrome can be evoked in rats only by direct microinjection of morphine into the PAG, the putative substrate for this behavior 12, while in frogs systemic injections are effective. Jacquet et a1.12 and LaBella et al.16 have proposed that the EMB elicited morphine is evidence for a new class of opiate receptor, one differing from the classical opiate receptor in that it is insensitive to naloxone, endorphins, and enkephalins and is less stereospecific. They suggest that the classical receptor mediates analgesia and the mild hyperactive state and that it inhibits the putative 'excitatory', natoxone-insensitive receptor which mediates EMB. Thus, EMB can be evoked only by selective activation of the excitatory receptor, either by direct injection of morphine into the PAG or by blocking the classical receptor with naloxone. That EMB occurs in frogs af-
303 ter systemic injection suggests that the proposed inhibitory coupling between the two receptor types is less pronounced than in rats. Our behavioral observations are quite preliminary but do demonstrate that EMB is not limited to murine species and that the frog may be well suited for studies of this phenomenon. Studies on the drug specificity, stereospecificity, and naloxone sensitivity are clearly needed and are in progress. Morphine is a weak analgesic in frogs, producing a very slight increase in nociceptive threshold at 10 mg/kg (26.4/~mol/kg), a dose 2-5 times greater than EDs0 for intravenous morphine in rats29,40. Pronounced analgesia obtained only at 100 mg/kg, a very high dose (Fig. 2). This raises the possibility that the apparent analgesic action was non-specific. However, this possibility seems to be unlikely for a number of reasons. First, the doses of morphine effective in raising the nociceptive threshold were substantially lower than those causing motor deficits or other noticeable changes in behavior. Frogs receiving 10 or 100 mg/kg of morphine were indistinguishable from controls in their responses to non-painful stimuli. Second the effect was blocked by naloxone. Although a rather high dose of naloxone was used, it is unlikely that its action was non-specific because naloxone alone has no effect on nociceptive thresholds at doses up to 100 mg/kg and at an extremely high dose (640 mg/kg) induces flaccidity and unresponsiveness to stimuli (manuscript in preparation). Thus, the dominant non-specific action of naloxone would appear to be analgesia rather than hyper-responsiveness to stimulation. Finally, the acetic acid test is a true test for nociception since the nociceptors in frog skin have been shown by electrophysiological means to respond to weak acid solutions as well as to pinch and pin prick ~. The reason for the low potency of morphine in frogs is not known but several possibilities can be considered. The drug was administered via the dorsal lymph sac, a route which may not provide for expeditious delivery to the CNS. In mice, for example,
morphine is more effective after intravenous than subcutaneous 29 injection. Possibly it would also be more potent if given intravenously to frogs. It is also possible that morphine is a poor ligand for the frog receptor mediating analgesia or that it is of low efficacy once bound. Since fl-endorphins from nonmammalian vertebrates differ considerably in structure and binding activity from fl-endorphins from mammals23, it is likely that the receptors also differ. Finally, it may be that opioid systems have no role, or a minor one, in modulating nociception in frogs. There is little evidence that opioids have a significant role in pain pathways in any non-mammalian vertebrate. Goldfish submerged in morphine-containing water apparently exhibit an increase in threshold to electrical stimulation 14 but it is difficult to assess the significance of this because of uncertainties about the dose of morphine absorbed and because of the lack of documentation that the electrical shock was activating nociceptors. The apparent analgesia may in fact be a motor deficit since it has been shown that endorphin and enkephalin analogs reduce the general activity level of goldfish25. There are isolated reports on morphine-induced analgesia in reptiles and birds. Morphine at a fairly low dose (5 mg/kg) produces a slight increase in tail-flick latency in the lizzard Ano//s21 and is alsoanalgesic in birds when the painful stimulus is electrical wing-shock5 but not when it is toe-pinch 3°. In none of these cases was the effect of morphine shown to be blocked by naloxone nor was dose-dependency demonstrated. To my knowledge, the present paper is the first documenting naloxoneattenuation of morphine-induced analgesia in a nonmammalian vertebrate. ACKNOWLEDGEMENTS I thank Harriet Bielawski for technical assistance, Craig Stevens for comments on the manuscript, Joanne Graves for manuscript preparation and the Research Board of the University of Illinois at Chicago for support.
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