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Opiate analgesia: Evidence for circadian rhythms in mice
Brain Research, 249 (1982) 265-270
265
Elsevier Biomedical Press
Opiate Analgesia" Evidence for Circadian Rhythms in Mice ALBERTO OLIVERIO, CLAUDIO CASTELLANO and STEFANO PUGLISI-ALLEGRA (A.O., C.C. and S.P.A.) Istituto di Psicobiologia e Psicofarmacologia, CNR, via Reno, 1-00198 Rome, and (.4.0.) Istituto di Fisiologia Generale, University of Rome, Rome (Italy)
(Accepted March 25th, 1982) Key words: circadian rhythms - - pain - - endogenous opioids - - morphine - - pacemaker mechanisms - - mice
The effects of a 12-12 h light-dark (L-D) cycle and of constant light (L-L) on nociceptive thresholds and morphine-induced analgesia were studied in two strains of mice: C57BL/6 and SEC/1Re. Under the L-D condition, a diurnal rhythm was observed in the responsivity of mice to nociceptive stimuli, and in the analgesic effects of morphine. Under the L-L schedule clear patterns of daily rhythmicity were evident in both strains for both nociceptive thresholds and responsivity to morphine. Finally, under the L-L schedule, the overall responsivity to pain and the antinociceptive effects of morphine were clearly increased in comparison to the patterns evident in the L-D condition. The administration of naloxone decreased the nociceptive thresholds, thus indicating an involvement of the endogenous opioid peptides. These phenomena are discussed in terms of the different pacemaker mechanisms involved in the expression of various patterns of daily behavioral and neurochemical rhythmicity in these strains, and of the effects of light in controlling the release of endogenous opioids. INTRODUCTION A number of reports indicate that, in man, a different sensitivity to pain and responsiveness to analgesics depend on the time of day 2. In addition, Frederickson et al. 3 and Rosenfeld and Rice 11 have noticed that baseline levels in mouse pain sensitivity showed significant day-night variations. Diurnal rhythms in the analgesic effects of morphine and of the opiate antagonist naloxone were also demonstrated a. These findings were interpreted by suggesting that variations of nociception and of the power of analgesic drugs were related to different levels of endogenous opioids, and that these levels are controlled by the l i g h t , l a r k cycle. This type of explanation is supported by a number of findings indicating the existence of circadian rhythms in the synthesis of various pituitary hormones 4 and monoamines, whose function and release are controlled by endogenous opioids 5,7. The results of Frederickson et al. 3 prove that there are diurnal variations in nociceptive mechanisms, but they do not indicate whether they also present circadian fluctuations, e.g. whether pain res0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
ponsivity shows fluctuations in the absence of an external light synchronizer. In fact, endogenous fluctuations of pain thresholds, which is an important adaptive mechanism, are strongly supported by the existence of several other circadian rhythms connected to a number of relevant behavioral, neurophysiological and neurochemical mechanisms in mammals1,9,12. In order to ascertain this possibility we decided to test pain responsivity and its modifications by morphine under a 12-12 h light-dark cycle (L-D) or under constant light (L-L). Two strains of mice which are characterized by different patterns of wheel running and sleep rhythms were selected. In particular, C57BL/6 (C57) and SEC/1Re (SEC) mice are shown to present diurnal behavioral fluctuations in the presence of a light-dark schedule while, under continuous light, clear patterns of circadian rhythmicity were evident in the C57 strain only x°. A number of brain neurochemical fluctuations were also evident in C57 mice and in other strains characterized by the absence or presence of circadian rhythmicity in the absence of a luminous external synchronizer 6.
266 The goal of the present research was therefore to assess : ( 1) if there are circadian fluctuations of nociception in mice, and (2) if there is a single main pacemaker responsible for different behavioral and neurochemical patterns. In other words, we wanted to determine if C57 mice, which present clearcut circadian rhythmicity for a number of behavioral and neurochemical factors also present circadian fluctuations of nociception, and if SEC mice, which are characterized by the absence of rhythmicity under constant light, also present the absence of rhythmicity of nociceptive mechanisms. METHO DS
Animals C57 and SEC mice were studied beginning at 90-110 days of age. The subjects were maintained in groups of 8 in clear plastic pens.
Analgesia Analgesia was determined with the hot plate method. The endpoint used was the licking of the forepaws or hindpaws. A mouse was removed as soon as it reacted or if it failed to react after 30 s. The animals were injected intraperitoneally with saline or morphine-HCl (6 mg/kg). In some experiments naloxone-HCl, at a dose of 5 mg/kg, was also used. Saline and drugs were injected 15 rain before testing in a volume of 0.1 ml/10 g of body weight.
Environmental procedures and testing The animal pens were placed in two different light-tight and sound-insulated enclosures with 30 W lamps. In one enclosure the lamps were cycled by an external clock, thus obtaining a 12-12 h L-D schedule: lights were switched on at 08.00 h and switched off at 20.00 h. In the other enclosure a constant light cycle was provided. The temperature of the enclosures was constant. Before testing the animals were adapted to the L-D or the L-L schedule for 12 days. Testing of the animals occurred in a room lighted with artificial fluorescent lamps during the light hours, or in the presence of a shaded red lamp during the dark hours. Different groups of 8 mice were tested for hot plate response latencies at 2 h intervals throughout the day. All data were collected over a period of one week.
To obtain a quantitative measure for tl3c amplitude of the circadian rhythm of analgesia the 12 i7 period with the lowest arithmetic mean IXI2 rain: min : minimum) was determined following the method ofBorb61yetal. Z Thus the amplitude A was defined by the expression A :
100 (I
XI2 min -_ ) X24
(X24 is the arithmetic mean for 24 h). Therefore A represents the average amplitude of the circadian rhythm and may vary between 0 and 100. The average amplitude was given preference over the more usual amplitude of the acrophase, since the latter is more subjected to variations due to rhythms with small amplitude t". The Wilcoxon rank sum (r.s.) test and the Wilcoxon signed rank sum (s.r.s.) test were used to test for significant differences. Two-tailed tests were applied in all instances. RESULTS
L-D Schedule Fig. I presents the latencies o f C 5 7 and SEC mice injected with saline (controls) and morphine. Control mice of both strains are characterized by a circadian pattern when their paw-licking latencies are considered. Control latencies are highest, representing a period of decreased pain sensitivity, during the hours 08.00 to 14.00 h, and lowest, representing a period of increased sensitivity, during the hours 22.00 to 04.00 h. No strain differences were evident when the overall mean 24 h paw-licking latencies are considered, C57 mice showing mean latencies of 9.88 0.32 and SEC mice mean latencies of 9.79 ~: 0.25 s (Table I). An analysis of the amplitude of the diurnal rhythms in the two strains also indicated nonsignificant differences (P ~ 0.05, Wilcoxon r.s. test 1. Treatment with morphine significantly increased the overall latencies, the effects of the drug not being dependent on the time of day (Fig. 1). When the mean 24 h paw-licking latencies are considered (Table 1), a significant increase is evident in both strains, the effects being higher in SEC mice. Table I1 shows that the mean amplitudes of morphine-injected C57 mice were lower but not statistically diffe-
267
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Figs. I and 2. Rhythms in latencies to the paw-licking response (ins)of C57 and SEC mice subjected to the 12-12 h L-D schedule (Fig. 1) or to the L-L schedule (Fig. 2). Closed circles and triangles refer to mice treated with morphine at 6 mg/kg. The latencies (mean zL S.E., n = 8), in seconds, are plotted as a function of the time of the day. The data points are the means of values collected over 2 h periods. In the L-D condition lights were off during the time periods marked by a black bar. Computer-drawn extrapolated curves are shown in order to better illustrate the rhythms.
rent in relation to those of control mice, while a significant reduction was evident in the SEC strain. L-L schedule
Figure 2 presents the latencies of C57 and SEC mice injected with saline (controls) and morphine.
Control mice ot both strains are characterized by a circadian pattern. Their latencies are highest, representing a period of decreased pain sensitivity, during the hours 22.00 to 02.00 h, and lowest, representing a period of increased sensitivity, during the hours 08.00 to 20.00 h. An analysis of the overall
268 TABLE I Mean 24 h paw licking latencies ( in s) and S. E.M. under the 12 12 h L-D schedule and the L-L scheduh'
In the C57 strain morphine significantly increased paw licking latencies under the L-D (P 0.011 and L-L schedules (P . 0.01 ): the latencies o f the mice subjected to the L-L schedule and injected with morphine were significantly higher in comparison to those of morphine-injected mice under the L-D schedule (P , 0.01 ). In the SEC strain morphine significantly increased paw licking latencies under the L-D (P 0.011 and L-L schedules (P . 0.01 ); the latencies o f the mice subjected to the L-L schedule were significantly higher when saline-injected mice (P 0.01 ) or morphine-injected mice are considered (P 0.011. Schedule
Strains SEC
C57 L-D
Saline Morphine
9.88 ::1 0.32 11,61 ! 0.35
L-L
10.66 j 0.23 13.04 ~ 0.33
mean 24 h latencies (Table I) indicates that these were higher in control SEC mice (12.73 ~ 0.28 s) than in the C57 strain (10.60:5 0.23 s) (P ~- 0.01, Wilcoxon r.s. test). The circadian rhythms of control mice kept under constant light was clearly damped in relation to the rhythm evident under the L-D schedule. This reduction was particularly evident in C57 mice, the amplitude under the L-L schedule being almost 4 times lower in comparison to the values observed under the L-D schedule. Treatment with morphine significantly increased the overall latencies, the effect of the opiate being not dependent on the time of the day in both strains. The analgesic effects of morphine were particularly evident in the SEC strain, where the overall mean 24 h latencies reached a level of 17.43 ! 0.34 s from a control level of 12.73 ~: 0.28.
L-D
9.79 ~ 0.25 12.17 t 0.27
L-L
12.73 0.28 17.43 ! 0.34
Table 11 shows that, under the L-L schedule, the mean amplitudes of morphine-injected mice were not significantly reduced in comparison with control mice. A clearcut significant reduction was evident in C57 mice when the mean amplitudes of morphineinjected mice kept under the L-L schedule are compared to those of morphine-injected mice subjected to the L-D schedule. in order to assess whether analgesia reflected increased levels of endogenous opioids, two additional groups of 8 mice for each strain were tested at 02.00 h, an hour in which highest latencies were evident. For each strain one group was injected with saline and tested for paw-licking latencies, while the other was injected with naloxone and tested 15 rain later. The latencies of the C57 mice decreased from a level of 12.76 0.71 s (saline) to 8.13 ± 0.33 s (naloxone)
T A B L E II Mean amplitude (see Methods) and S.E.M. o f 24 h pan, licking latencies rhythm under the 12- 12 I1 L-D schedule and the L-L schedule
In the C57 strain the amplitudes o f saline-injected and morphine-injected mice were not statistically different under either L-D or L-L schedule. The amplitudes under the L-L schedule were reduced significantly in comparison to the values observed under the L-D schedule (P < 0.01). In the SEC strain the amplitude of morphine-injected mice was significantly reduced in comparison to that o f saline-injected mice under the L-D schedule only (P -: 0.011. The amplitudes under the L-L schedule were reduced significantly in comparison to the values observed under the L-D schedule in saline-injected mice ( P , 0.01 ). Schedule
Strains C57
Saline Morphine
SEC
L-D
L-L
L-D
L-L
16.90 .~ 0.91 15.51 ::i 0.58
4.30 :L 0.27 4.20 ~ 0.25
15.30 ~ 0.74 10.78 I 0.51
11.10 0.51 10.10 ~: 0.63
269 and those of SEC mice from 17.65 ~ 0.40 s (saline) to 7.65 :~ 0.64 s (naloxone). These effects were significant in both strains (P < 0.01). DISCUSSION Our data in C57 and SEC mice describe the temporal distribution throughout the day of pain sensitivity and of the analgesic effects of morphine under a 12-12 L-D schedule and under constant light. Inspection disclosed clear patterns of diurnal rhythmicity under the L-D condition, and of circadian rhythmicity in the absence of a luminous external synchronizer. The first finding, i.e. presence of day-night differences in sensitivity to nociceptive stimuli and to the effects of morphine is in agreement with the findings of Frederickson et al. a and of Rosenfeld and Rice 11. In addition, our data also show that a circadian rhythm is evident under the LL schedule in both strains, an effect which suggests that a 'biological clock' regulates the production of endogenous opioids. Under both the L-D and the L-L schedules the rhythms of pain sensitivity of C57 and SEC mice were paralleled by the rhythms of morphine-induced analgesia. In fact, when the L-D schedule is considered, the lowest latencies of saline-injected mice occurred at 02.00 h, a time of the day in which the mice injected with morphine also showed the lowest level of analgesia. Under the L-L schedule, where the lowest and the highest levels of analgesia do not occur at the same time as in the L-D group, the highest latencies of saline-injected mice occurred from 24.00 to 04.00 h, a time of the day in which the mice injected with morphine also showed the highest level of analgesia. The parallelism between the temporal patterns of the latencies of salineinjected and morphine-injected mice under both LD and L-L schedules supports the hypothesis that there are daily fluctuations in the release of endogenous opioids. Thus, the period of greater effect of
REFERENCES 1 Borb61y,A., Huston, J. and Waser, P., Control of sleep states in the rat by short light-dark cycles, Brain Research, 95 (1975) 89-101. 2 Dawes, C., Circadian and circannual maps for human saliva. In L. E. Scheving, F. Halberg and J. E. Pauly, (Eds.),
morphine may be due to a synergism with endogenous opioids whose activity differs depending on the time of the day. Further evidence in favor of this hypothesis is provided by the fact that naloxone clearly reduces the peak latencies of control C57 and SEC mice, an effect which may be interpreted in terms of its antagonism of opioids. Previous studies on the wheel-running behavior of C57 and SEC mice indicated that a daily rhythmicity persisted in a circadian fashion only in the C57 strain when the mice were tested unde~ conditions of constant light or darkness 9,1°. These findings, supported by various neurochemical correlates 6, were interpreted in terms of pacemaker mechanisms whose expression is modulated by the genetic factors. The present data, indicating that both strains are characterized by a circadian pattern of nociception suggest that different pacemaker mechanisms are involved in the control of different behavioral, neurochemical or neurophysiological mechanisms, as suggested by Mitler et al. s. In fact, SEC mice are not characterized by a circadian rhythm for other behavioral or neurochemical activities. Finally, the fact that under the L-L schedule the overall latencies of saline-injected and morphineinjected mice were higher than those of mice kept on the L-D schedule, suggests that higher levels of endorphins are released under constant light. The power of light in enhancing the levels of analgesia, i.e. the release of opioids, is particularly evident in control and morphine-injected mice belonging to the SEC strain. Ongoing studies will ascertain a possible relationship between the level of illumination and the release of opioids. Since endogenous opioids modulate a number of emotional and social patterns, the analysis of the interactions between the levels of environmental luminosity or the length of the photoperiodism, and the release of endorphins, might be relevant for its implications in a number of emotional patterns and behavioral disturbances.
Chronobiology, lgaku Shoin, Tokyo, 1974, pp. 224-227. 3 Frederickson, R. C. A., Burgis, V. and Edwards, J. D., Hyperalgesia induced by naloxone follows diurnal rhythm in responsivity to painful stimuli, Science, 198 (1977), 756-758. 4 Halberg, F., Chronobiology, Ann. Rev. Physiol., 31 (1969) 675-725.
270 5 Henderson, G. and Hughes, J., [ h e effects of morphine on the release of noradrenaline from the mouse vas deferens, Brit. J. Pharmacol., 57 (1976), 551 557. 6 Kempf, E., Mandel, P., Oliverio, A. and Pugli,i-Allegra, S., Circadian variations of noradrenaline, 5-hydroxytryptamine and dopamine in specific brain areas of C57BL/6 and BALB/c mice, Brain Research, 232 (1982) 472-478. 7 Loh, H. H., Brase, D. A., Sampath-Kanna, S., Mar, J. B. and Way, E. L., fl-Endorphin in vitro inhibition of striatal dopanfine release, Nature (Lomt.), 264 (1976), 567 568. 8 Mitler, M. M., Lund, R., Sokalove, P. G., Pittendrigh+ C. S., and Dement, W., Sleep and activity rhythms in mice: a description of circadian patterns and unexpected destructions in sleep, Brain Research, t31 (1977) 129 145.
9 0 l i v e r i o , A., Castellano, t. +. and Puglisi-Allegra, S.. A genetic approach to behavioral plasticity and rigidity. In J. R. Royce and L. P. Maas (Eds.), Theoretical Advam'es m Behm'i~r Genetics, Nato ASI Sijthoff and Noordhoff, A[phen aan den Rijn, 1979, pp. 139 165. 10 Oliverio, A. and Malorni, W., Wheel running alld sleep m two strain of mice: plasticity and rigidity in the expression of circadian rhythmicity, Brain Research. 163 (t979i+ 121 133. 11 Rosenfeld, J. P. and Rice, P. t:., Diurnal rhythms in nociceptive thresholds of rats, Physiol. Behav., 23 ~19791, 419 420. 12 Rusak, B. and Zucker, I., Neural regulation oi +circadian rhythms, Physiol. Rev., 59 (1979), 449. 526.
265
Elsevier Biomedical Press
Opiate Analgesia" Evidence for Circadian Rhythms in Mice ALBERTO OLIVERIO, CLAUDIO CASTELLANO and STEFANO PUGLISI-ALLEGRA (A.O., C.C. and S.P.A.) Istituto di Psicobiologia e Psicofarmacologia, CNR, via Reno, 1-00198 Rome, and (.4.0.) Istituto di Fisiologia Generale, University of Rome, Rome (Italy)
(Accepted March 25th, 1982) Key words: circadian rhythms - - pain - - endogenous opioids - - morphine - - pacemaker mechanisms - - mice
The effects of a 12-12 h light-dark (L-D) cycle and of constant light (L-L) on nociceptive thresholds and morphine-induced analgesia were studied in two strains of mice: C57BL/6 and SEC/1Re. Under the L-D condition, a diurnal rhythm was observed in the responsivity of mice to nociceptive stimuli, and in the analgesic effects of morphine. Under the L-L schedule clear patterns of daily rhythmicity were evident in both strains for both nociceptive thresholds and responsivity to morphine. Finally, under the L-L schedule, the overall responsivity to pain and the antinociceptive effects of morphine were clearly increased in comparison to the patterns evident in the L-D condition. The administration of naloxone decreased the nociceptive thresholds, thus indicating an involvement of the endogenous opioid peptides. These phenomena are discussed in terms of the different pacemaker mechanisms involved in the expression of various patterns of daily behavioral and neurochemical rhythmicity in these strains, and of the effects of light in controlling the release of endogenous opioids. INTRODUCTION A number of reports indicate that, in man, a different sensitivity to pain and responsiveness to analgesics depend on the time of day 2. In addition, Frederickson et al. 3 and Rosenfeld and Rice 11 have noticed that baseline levels in mouse pain sensitivity showed significant day-night variations. Diurnal rhythms in the analgesic effects of morphine and of the opiate antagonist naloxone were also demonstrated a. These findings were interpreted by suggesting that variations of nociception and of the power of analgesic drugs were related to different levels of endogenous opioids, and that these levels are controlled by the l i g h t , l a r k cycle. This type of explanation is supported by a number of findings indicating the existence of circadian rhythms in the synthesis of various pituitary hormones 4 and monoamines, whose function and release are controlled by endogenous opioids 5,7. The results of Frederickson et al. 3 prove that there are diurnal variations in nociceptive mechanisms, but they do not indicate whether they also present circadian fluctuations, e.g. whether pain res0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
ponsivity shows fluctuations in the absence of an external light synchronizer. In fact, endogenous fluctuations of pain thresholds, which is an important adaptive mechanism, are strongly supported by the existence of several other circadian rhythms connected to a number of relevant behavioral, neurophysiological and neurochemical mechanisms in mammals1,9,12. In order to ascertain this possibility we decided to test pain responsivity and its modifications by morphine under a 12-12 h light-dark cycle (L-D) or under constant light (L-L). Two strains of mice which are characterized by different patterns of wheel running and sleep rhythms were selected. In particular, C57BL/6 (C57) and SEC/1Re (SEC) mice are shown to present diurnal behavioral fluctuations in the presence of a light-dark schedule while, under continuous light, clear patterns of circadian rhythmicity were evident in the C57 strain only x°. A number of brain neurochemical fluctuations were also evident in C57 mice and in other strains characterized by the absence or presence of circadian rhythmicity in the absence of a luminous external synchronizer 6.
266 The goal of the present research was therefore to assess : ( 1) if there are circadian fluctuations of nociception in mice, and (2) if there is a single main pacemaker responsible for different behavioral and neurochemical patterns. In other words, we wanted to determine if C57 mice, which present clearcut circadian rhythmicity for a number of behavioral and neurochemical factors also present circadian fluctuations of nociception, and if SEC mice, which are characterized by the absence of rhythmicity under constant light, also present the absence of rhythmicity of nociceptive mechanisms. METHO DS
Animals C57 and SEC mice were studied beginning at 90-110 days of age. The subjects were maintained in groups of 8 in clear plastic pens.
Analgesia Analgesia was determined with the hot plate method. The endpoint used was the licking of the forepaws or hindpaws. A mouse was removed as soon as it reacted or if it failed to react after 30 s. The animals were injected intraperitoneally with saline or morphine-HCl (6 mg/kg). In some experiments naloxone-HCl, at a dose of 5 mg/kg, was also used. Saline and drugs were injected 15 rain before testing in a volume of 0.1 ml/10 g of body weight.
Environmental procedures and testing The animal pens were placed in two different light-tight and sound-insulated enclosures with 30 W lamps. In one enclosure the lamps were cycled by an external clock, thus obtaining a 12-12 h L-D schedule: lights were switched on at 08.00 h and switched off at 20.00 h. In the other enclosure a constant light cycle was provided. The temperature of the enclosures was constant. Before testing the animals were adapted to the L-D or the L-L schedule for 12 days. Testing of the animals occurred in a room lighted with artificial fluorescent lamps during the light hours, or in the presence of a shaded red lamp during the dark hours. Different groups of 8 mice were tested for hot plate response latencies at 2 h intervals throughout the day. All data were collected over a period of one week.
To obtain a quantitative measure for tl3c amplitude of the circadian rhythm of analgesia the 12 i7 period with the lowest arithmetic mean IXI2 rain: min : minimum) was determined following the method ofBorb61yetal. Z Thus the amplitude A was defined by the expression A :
100 (I
XI2 min -_ ) X24
(X24 is the arithmetic mean for 24 h). Therefore A represents the average amplitude of the circadian rhythm and may vary between 0 and 100. The average amplitude was given preference over the more usual amplitude of the acrophase, since the latter is more subjected to variations due to rhythms with small amplitude t". The Wilcoxon rank sum (r.s.) test and the Wilcoxon signed rank sum (s.r.s.) test were used to test for significant differences. Two-tailed tests were applied in all instances. RESULTS
L-D Schedule Fig. I presents the latencies o f C 5 7 and SEC mice injected with saline (controls) and morphine. Control mice of both strains are characterized by a circadian pattern when their paw-licking latencies are considered. Control latencies are highest, representing a period of decreased pain sensitivity, during the hours 08.00 to 14.00 h, and lowest, representing a period of increased sensitivity, during the hours 22.00 to 04.00 h. No strain differences were evident when the overall mean 24 h paw-licking latencies are considered, C57 mice showing mean latencies of 9.88 0.32 and SEC mice mean latencies of 9.79 ~: 0.25 s (Table I). An analysis of the amplitude of the diurnal rhythms in the two strains also indicated nonsignificant differences (P ~ 0.05, Wilcoxon r.s. test 1. Treatment with morphine significantly increased the overall latencies, the effects of the drug not being dependent on the time of day (Fig. 1). When the mean 24 h paw-licking latencies are considered (Table 1), a significant increase is evident in both strains, the effects being higher in SEC mice. Table I1 shows that the mean amplitudes of morphine-injected C57 mice were lower but not statistically diffe-
267
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Figs. I and 2. Rhythms in latencies to the paw-licking response (ins)of C57 and SEC mice subjected to the 12-12 h L-D schedule (Fig. 1) or to the L-L schedule (Fig. 2). Closed circles and triangles refer to mice treated with morphine at 6 mg/kg. The latencies (mean zL S.E., n = 8), in seconds, are plotted as a function of the time of the day. The data points are the means of values collected over 2 h periods. In the L-D condition lights were off during the time periods marked by a black bar. Computer-drawn extrapolated curves are shown in order to better illustrate the rhythms.
rent in relation to those of control mice, while a significant reduction was evident in the SEC strain. L-L schedule
Figure 2 presents the latencies of C57 and SEC mice injected with saline (controls) and morphine.
Control mice ot both strains are characterized by a circadian pattern. Their latencies are highest, representing a period of decreased pain sensitivity, during the hours 22.00 to 02.00 h, and lowest, representing a period of increased sensitivity, during the hours 08.00 to 20.00 h. An analysis of the overall
268 TABLE I Mean 24 h paw licking latencies ( in s) and S. E.M. under the 12 12 h L-D schedule and the L-L scheduh'
In the C57 strain morphine significantly increased paw licking latencies under the L-D (P 0.011 and L-L schedules (P . 0.01 ): the latencies o f the mice subjected to the L-L schedule and injected with morphine were significantly higher in comparison to those of morphine-injected mice under the L-D schedule (P , 0.01 ). In the SEC strain morphine significantly increased paw licking latencies under the L-D (P 0.011 and L-L schedules (P . 0.01 ); the latencies o f the mice subjected to the L-L schedule were significantly higher when saline-injected mice (P 0.01 ) or morphine-injected mice are considered (P 0.011. Schedule
Strains SEC
C57 L-D
Saline Morphine
9.88 ::1 0.32 11,61 ! 0.35
L-L
10.66 j 0.23 13.04 ~ 0.33
mean 24 h latencies (Table I) indicates that these were higher in control SEC mice (12.73 ~ 0.28 s) than in the C57 strain (10.60:5 0.23 s) (P ~- 0.01, Wilcoxon r.s. test). The circadian rhythms of control mice kept under constant light was clearly damped in relation to the rhythm evident under the L-D schedule. This reduction was particularly evident in C57 mice, the amplitude under the L-L schedule being almost 4 times lower in comparison to the values observed under the L-D schedule. Treatment with morphine significantly increased the overall latencies, the effect of the opiate being not dependent on the time of the day in both strains. The analgesic effects of morphine were particularly evident in the SEC strain, where the overall mean 24 h latencies reached a level of 17.43 ! 0.34 s from a control level of 12.73 ~: 0.28.
L-D
9.79 ~ 0.25 12.17 t 0.27
L-L
12.73 0.28 17.43 ! 0.34
Table 11 shows that, under the L-L schedule, the mean amplitudes of morphine-injected mice were not significantly reduced in comparison with control mice. A clearcut significant reduction was evident in C57 mice when the mean amplitudes of morphineinjected mice kept under the L-L schedule are compared to those of morphine-injected mice subjected to the L-D schedule. in order to assess whether analgesia reflected increased levels of endogenous opioids, two additional groups of 8 mice for each strain were tested at 02.00 h, an hour in which highest latencies were evident. For each strain one group was injected with saline and tested for paw-licking latencies, while the other was injected with naloxone and tested 15 rain later. The latencies of the C57 mice decreased from a level of 12.76 0.71 s (saline) to 8.13 ± 0.33 s (naloxone)
T A B L E II Mean amplitude (see Methods) and S.E.M. o f 24 h pan, licking latencies rhythm under the 12- 12 I1 L-D schedule and the L-L schedule
In the C57 strain the amplitudes o f saline-injected and morphine-injected mice were not statistically different under either L-D or L-L schedule. The amplitudes under the L-L schedule were reduced significantly in comparison to the values observed under the L-D schedule (P < 0.01). In the SEC strain the amplitude of morphine-injected mice was significantly reduced in comparison to that o f saline-injected mice under the L-D schedule only (P -: 0.011. The amplitudes under the L-L schedule were reduced significantly in comparison to the values observed under the L-D schedule in saline-injected mice ( P , 0.01 ). Schedule
Strains C57
Saline Morphine
SEC
L-D
L-L
L-D
L-L
16.90 .~ 0.91 15.51 ::i 0.58
4.30 :L 0.27 4.20 ~ 0.25
15.30 ~ 0.74 10.78 I 0.51
11.10 0.51 10.10 ~: 0.63
269 and those of SEC mice from 17.65 ~ 0.40 s (saline) to 7.65 :~ 0.64 s (naloxone). These effects were significant in both strains (P < 0.01). DISCUSSION Our data in C57 and SEC mice describe the temporal distribution throughout the day of pain sensitivity and of the analgesic effects of morphine under a 12-12 L-D schedule and under constant light. Inspection disclosed clear patterns of diurnal rhythmicity under the L-D condition, and of circadian rhythmicity in the absence of a luminous external synchronizer. The first finding, i.e. presence of day-night differences in sensitivity to nociceptive stimuli and to the effects of morphine is in agreement with the findings of Frederickson et al. a and of Rosenfeld and Rice 11. In addition, our data also show that a circadian rhythm is evident under the LL schedule in both strains, an effect which suggests that a 'biological clock' regulates the production of endogenous opioids. Under both the L-D and the L-L schedules the rhythms of pain sensitivity of C57 and SEC mice were paralleled by the rhythms of morphine-induced analgesia. In fact, when the L-D schedule is considered, the lowest latencies of saline-injected mice occurred at 02.00 h, a time of the day in which the mice injected with morphine also showed the lowest level of analgesia. Under the L-L schedule, where the lowest and the highest levels of analgesia do not occur at the same time as in the L-D group, the highest latencies of saline-injected mice occurred from 24.00 to 04.00 h, a time of the day in which the mice injected with morphine also showed the highest level of analgesia. The parallelism between the temporal patterns of the latencies of salineinjected and morphine-injected mice under both LD and L-L schedules supports the hypothesis that there are daily fluctuations in the release of endogenous opioids. Thus, the period of greater effect of
REFERENCES 1 Borb61y,A., Huston, J. and Waser, P., Control of sleep states in the rat by short light-dark cycles, Brain Research, 95 (1975) 89-101. 2 Dawes, C., Circadian and circannual maps for human saliva. In L. E. Scheving, F. Halberg and J. E. Pauly, (Eds.),
morphine may be due to a synergism with endogenous opioids whose activity differs depending on the time of the day. Further evidence in favor of this hypothesis is provided by the fact that naloxone clearly reduces the peak latencies of control C57 and SEC mice, an effect which may be interpreted in terms of its antagonism of opioids. Previous studies on the wheel-running behavior of C57 and SEC mice indicated that a daily rhythmicity persisted in a circadian fashion only in the C57 strain when the mice were tested unde~ conditions of constant light or darkness 9,1°. These findings, supported by various neurochemical correlates 6, were interpreted in terms of pacemaker mechanisms whose expression is modulated by the genetic factors. The present data, indicating that both strains are characterized by a circadian pattern of nociception suggest that different pacemaker mechanisms are involved in the control of different behavioral, neurochemical or neurophysiological mechanisms, as suggested by Mitler et al. s. In fact, SEC mice are not characterized by a circadian rhythm for other behavioral or neurochemical activities. Finally, the fact that under the L-L schedule the overall latencies of saline-injected and morphineinjected mice were higher than those of mice kept on the L-D schedule, suggests that higher levels of endorphins are released under constant light. The power of light in enhancing the levels of analgesia, i.e. the release of opioids, is particularly evident in control and morphine-injected mice belonging to the SEC strain. Ongoing studies will ascertain a possible relationship between the level of illumination and the release of opioids. Since endogenous opioids modulate a number of emotional and social patterns, the analysis of the interactions between the levels of environmental luminosity or the length of the photoperiodism, and the release of endorphins, might be relevant for its implications in a number of emotional patterns and behavioral disturbances.
Chronobiology, lgaku Shoin, Tokyo, 1974, pp. 224-227. 3 Frederickson, R. C. A., Burgis, V. and Edwards, J. D., Hyperalgesia induced by naloxone follows diurnal rhythm in responsivity to painful stimuli, Science, 198 (1977), 756-758. 4 Halberg, F., Chronobiology, Ann. Rev. Physiol., 31 (1969) 675-725.
270 5 Henderson, G. and Hughes, J., [ h e effects of morphine on the release of noradrenaline from the mouse vas deferens, Brit. J. Pharmacol., 57 (1976), 551 557. 6 Kempf, E., Mandel, P., Oliverio, A. and Pugli,i-Allegra, S., Circadian variations of noradrenaline, 5-hydroxytryptamine and dopamine in specific brain areas of C57BL/6 and BALB/c mice, Brain Research, 232 (1982) 472-478. 7 Loh, H. H., Brase, D. A., Sampath-Kanna, S., Mar, J. B. and Way, E. L., fl-Endorphin in vitro inhibition of striatal dopanfine release, Nature (Lomt.), 264 (1976), 567 568. 8 Mitler, M. M., Lund, R., Sokalove, P. G., Pittendrigh+ C. S., and Dement, W., Sleep and activity rhythms in mice: a description of circadian patterns and unexpected destructions in sleep, Brain Research, t31 (1977) 129 145.
9 0 l i v e r i o , A., Castellano, t. +. and Puglisi-Allegra, S.. A genetic approach to behavioral plasticity and rigidity. In J. R. Royce and L. P. Maas (Eds.), Theoretical Advam'es m Behm'i~r Genetics, Nato ASI Sijthoff and Noordhoff, A[phen aan den Rijn, 1979, pp. 139 165. 10 Oliverio, A. and Malorni, W., Wheel running alld sleep m two strain of mice: plasticity and rigidity in the expression of circadian rhythmicity, Brain Research. 163 (t979i+ 121 133. 11 Rosenfeld, J. P. and Rice, P. t:., Diurnal rhythms in nociceptive thresholds of rats, Physiol. Behav., 23 ~19791, 419 420. 12 Rusak, B. and Zucker, I., Neural regulation oi +circadian rhythms, Physiol. Rev., 59 (1979), 449. 526.