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The effect of neonatal exposure to chronic footshock on pain-responsiveness and sensitivity to morphine after maturation in the rat
105
Behavioural Brain Research, 36 (1990) 105-111 Elsevier BBR 00981
The effect of neonatal exposure to chronic footshock on pain-responsiveness and sensitivity to morphine after maturation in the rat C. Shimada, S. Kurumiya, Y. Noguchi and M. Umemoto Division of Psychology, Faculty of Letters, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka (Japan) (Received 16 November 1988) (Revised version received 10 May 1989) (Accepted 10 May 1989)
Key words: Stress-induced analgesia; Preweanling period; Hot-plate; Opiate receptor; Morphine
Rat pups in 3 groups respectively were given daily footshock, exposure to a footshock apparatus without shock, or no handling from birth to 21 days of age and reared with no manipulation afterwards. After maturation (90-100 days of age), they were assessed for hot-plate paw-lick latency, morphine-induced analgesia and opiate receptor binding assay. In footshocked animals, a significant increase was found in paw-lick latency and in antinociceptive effects of morphine (1.25, 2.5, and 5.0 mg/kg) in comparison with two control groups. The antinociceptive effect of morphine in all 3 groups was antagonized by pretreatment with naloxone (2.0 mg/kg). No significant difference was found in binding activities (Bmax and Kd) for both [3H]naloxone and [3H]Dala2,D-LeuS-enkephalin between the 3 groups. These results suggest that exposure to footshock stress in the preweanling period has a long-term effect on the sensitivity of rats to painful events, probably due to chronic functional changes in endogeneous opiate systems at presynaptic level rather than in postsynaptic opiate receptor binding activity.
INTRODUCTION
It is well documented that a variety of stressful manipulations induces an analgesic reaction, which has been termed stress-induced analgesia2"4'6"23. Since cross tolerance develops between morphine and stress-induced analgesia8'18"22'25, and analgesia induced by stressors can be antagonized by pretreatment with opioid antagonists 14"27, the involvement of endogenous opioids in mediation of stress-induced analgesia has been suggested. Furthermore, it has been reported that stress-induced analgesia is accompanied by increases in endogenous opioid level in the brain 2° and with decreased binding of exo-
genously added enkephalin to brain homogenates 7,9. Stress-induced analgesia is known to dissipate within 1-2 h after the exposure to stressors 1'5. It has also been reported that chronic exposure to stress decreases pain threshold with concomitant decreases in brain opioid levels 18,20,21.These findings obtained from adults may suggest that, in the matured central nervous systems, exposure to acute stress transiently activates an adaptation mechanism in which endogenous opioid systems possibly play an important role in producing analgesic reaction, but with chronic stress the matured system is not able to keep coping with extended stress resulting in a decrease in pain
Correspondence: M. Umemoto, Division of Psychology, Faculty of Letters, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
106 threshold, presumably due to the depletion of endogenous substances such as opioid peptides. Exposure to chronic stress during the preweanling period may influence the ontogenic development of the endogenous opioid system and may also produce a different effect on the pain sensitivity. In behavior theory, it is well known that environmental stimulation in the preweanling period results in a reduced emotionality and an enhancement of learning in adults, and is termed 'early experience'12'13"17. The effect of early experience has been suggested to be mediated by the facilitation of pituitaryadrenocortical responses 15. In these early studies, of course, the involvement of the endogenous opioid system in the effect of early experience has not been suggested. Now, as we have a knowledge of the presence of the endogenous opioid system which is known to play a role in controlling the pain, it is of interest to consider the effect of early experience on adult behavior from the view of a possible permanent change in pain sensitivity which might be attributed to a permanent change in the endogenous opioid systems. It has been shown that the development of the opioid system, such as the levels of opioid peptides and the number of opioid receptors in the brain, occurred rapidly during the preweanling period1°'11'22"26.It has been reported that in the rat chronically exposed to hot-plate noxious stimulation during early life, the endogenous opioid peptide level and opioid receptor binding in the brain were increased 24. On the other hand, chronic footshock treatment on the rat from birth to 21 days of age, does not affect the ontogeny of opioid receptors and reduces the efficacy of morphine in producing antinociceptive effects when tested on the day following the last footshock3. In the present study, we investigated whether chronic footshock treatment of rats during the preweanling period brings about the influences in pain sensitivity and in the analgesic effect of morphine at the time of full growth, by testing hotplate paw-lick latency, since it has been reported that the full development of the opioid receptorendogenous ligand system in the brain requires about one postnatal month 11. We also examined opioid receptor binding in the brain using
[3H]D-Ala2-D-LeuS-enkephalin and [ q t ]naloxone, to label b- and/~-receptors respectively, since Bardo etal. 3 used only [3H]naloxone, which examined presumably #-receptor ontogeny, and suggested the possibility that chronic preweanling footshock treatment altered the other type of opioid receptors such as the b-receptor. Besides, Wohltman et al. 28 reported the delayed development of the b-receptor during the postnatal period in comparison with the #-receptor. MATERIALS AND METHODS Animals Male Wistar rat pups were randomly assigned to mixed-fostered litters of 8-10 pups each on the day after parturition. After ablactation (on day 21), 3-4 pups from each litter were housed together with no treatment until they reached 90-100 days of age. Food and water were always available on demand throughout the experiment. Footshock treatment Pups were placed in a footshock apparatus with a grid floor for 30 min and administered inescapable electric shock (6 s duration, at an interval of 1 shock/rain) through the grid connected to a shock generator (LVS Co., SGS-004). Starting from 2 mA which is a value of calibration, the intensity was adjusted in descending order to the lowest which was enough to induce a writhing response. The actual current intensity given to pups was in a range between 0.3 and 0.4 mA measured by Delgado's method using an osciloscope. Pups received two daily treatments at an 8-h interval for 21 days after birth. Shamcontrol pups were treated the same except that the shock was not delivered. Control pups were left untreated during the preweanling period. Paw-lick latency At 90-100 days after birth, all animals were assessed for pain sensitivity and for changes in antinociceptive effect of morphine. Each rat was placed on the hot-plate at 56 °C, and hind-paw lick latency was measured, When the response was not observed within 60 s, the ~ t i n g was terminated and 60 s was recorded as the latency.
107 Each rat was tested 3 times and the mean latency from the last two testings was used as the baseline latency. After the baseline latency was determined, each rat was injected subcutaneously with morphine hydrochloride (Shionogi Co.) and assessed for paw-lick latency at 15, 30, 60, 90, 120 and 150 min after the drug administration. The dosages of morphine were 1.25, 2.5 and 5.0 mg/kg. Each animal was injected with one of the three doses of morphine in a different order, and tested with 10 days of interval between the injections. Morphine-induced changes in pawlick latency were expressed as the percent of the maximum possible effect (MPE) as follows: ~oMPE = postdrug latency - baseline latency
x 100
cut-off time (60 s) - baseline latency Naloxone hydrochloride (Endo Lab) (2.0 mg/kg) was injected 15 min before morphine administration. In addition, adjusting the temperature of hot-plate by steps of 1 °C so that 8-10 s of baseline paw-lick latency was obtained for each animal, effects of three doses of morphine, that is, 2.5, 5.0, 10.0 mg/kg, were also examined in control and footshocked animals.
Opioid receptor binding assay Twelve rats from footshock, sham-control and control groups (4 from each group) were sacrificed by decapitation and their brains were removed. The whole brain except for cellebellum and olfactory bulb was weighed and homogenized in 20 vols. of ice-cold 50 ml Tris-HCl buffer (pH 7.4). After centrifugation at 20000 g for 30 min, the pellet was resuspended in Tris-buffer and incubated at 32 °C for 30 min to remove endogenous opioid. Following recentrifugation at 20 000 g for 30 min, the pellet was resuspended in Tris-buffer to contain 0.5-0.6mg protein in 480 #1 which was determined by the method of Lowry et al. 19. Binding assays were performed in a final volume of 500/~1 with 8 concentrations (0.18-22.58 nM) of [3H]naloxone (New England Nuclear, 50.0 Ci/mmol) in the presence of 1 #M
naloxone, and with 7 concentrations of [3H]DAlaZ-D-LeuS-enkephalin (DADL; New England Nuclear, 43.6 Ci/mmol) in the presence or absence of 1 #1 D-Ala2-MetS-enkephalin (Peptide Lab.). Samples were incubated at 0 ° C for 3 h and diluted with 2.5 ml Tris-buffer, followed by filtration through Whatman GF/C glass fiber filter. After two 2.5-ml rinses with cold buffer, the filters were transferred to vials and radio-counted by liquid scintillation spectrometry 12 h after addition of 10 ml Univergel (Nakarai Chem). Specific binding of labeled ligands was calculated by subtracting the radioactivity which was obtained in the presence of non-labeled ligands from that obtained in the absence of non-labeled ligands.
Statistical analysis Analysis of variance (ANOVA) and post-hoc test by Tukey's method or Student's t-test were used to analyze overall effects and to test the differences between the means, respectively. RESULTS
Decreased pain sensitivity in neonatally footshocked rats Neonatal footshock treatment did not affect the increase in body weights in footshocked animals. Mean body weight (+ S.E.M.) of rats in footshock, sham-control and control groups were 399 + 9, 393 + 12 and 394 + 10 g, respectively. There were no significant differences among the 3 groups. Fig. 1 shows the mean paw-lick latencies obtained from 3 groups of rats which were exposed chronically to footshock (n = 8), exposed to the grid apparatus without shock (n = 8), and left unhandled (n = 8) during the preweanling period. Footshocked rats exhibited significantly longer paw-lick latencies than the two control groups (P < 0.01). There was no significant difference in paw-lick latencies between shamcontrol and control groups. Increased sensitivity to morphine in neonatally footshocked rats Morphine produced dose-dependent analgesic effects in all 3 groups. In footshocked animals,
108
morphine had stronger effects on both magnitude and duration of antinociception than in two control animals (Fig. 2). ANOVA for each dose of morphine revealed statistically significant effects among the groups (1.25mg/kg: F2,2, = 22.95, P < 0 . 0 1 ; 2.5mg/kg: F2,21=22.03, P < 0 . 0 1 : 5.0 mg/kg: Fz.21 = 14.90, P < 0.01); 2 mg/kg of naloxone antagonized the analgesic effect of morphine (5mg/kg) in all 3 groups, whereas footshocked animals were resistant to the antagonizing effect of naloxone (/72.2, = 10.24, P < 0.01), as shown in Fig. 2. The mean temperatures of the hot-plate which
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Groups Fig. 1. Mean +S.E.M. paw-lick latency at 56°C in footshock, sham-control and control animals, tested 90-100 days after birth. Footshock animals were exposed to chronic footshock for 21 days after birth. Sham-controls were exposed to footshock apparatus without shock during the same period. Control animals received no treatment. Number of animals were 8 for each group. *P < 0.01 as compared with sham-control and control groups (by Student's t-test).
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Fig. 2. Percent of the maximum possible analgesic effect (%MPE) of morphine (mean 4. S.E.M.) on paw-lick latency at 56 °C in 3 groups of rats (8 each) same as groups mentioned in Fig. 1. Naloxone (2 mg/kg) was administered 15 min before morphine (5 mg/kg). *P < 0.05 as compared with the controls; *P < 0.05, *P < 0.01 as compared with sham-controls (by Tukey's post hoc test).
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Fig. 3. Percent of the Maximum Possible Effect (% MPE) of morphine on paw-lick latency in footshock amt control animals (8 each). Hot-plate temperature was adjusted to produce 8-10 s paw-lick latency in each animal. Mean temperature were 59.4 + 0.2 °C and 5712 4. 03 °C for footshock and control animals respectively. *P < 0.05, **P < 0.01 as compared with the controls (by Tukey's post-hoe test).
109 produced 8-10 s baseline latencies were 59.4 + 0.2 °C and 57.2 + 0.3 °C in footshocked and control groups respectively, and a significant difference was found between them (P < 0.01). Fig. 3 shows significant increases in paw-lick latencies of footshocked animals compared with control animals, whose baseline latencies were similar between the two groups, after injection of morphine (2.5mg/kg: F I , 1 4 = 26.86, P < 0.01; 5.0 mg/kg: F I , 1 4 = 38.62, P < 0.01; 10 mg/kg: El,14 = 60.14, P < 0.01). Particularly after 10 mg/kg of morphine, paw-lick latencies in most of footshocked animals reached cut-off time 60 min later and remained much longer than in control animals even 150 min after the injection.
Opioid receptor binding assay Table I shows means + S.E.M. of the dissociation constant (Kd) and maximum binding capacity (Bmax) from independent Scatchard analysis of each group. There was no significant difference in both K d and B m a x for the [ 3 H ] naloxone and [3H]DADL binding sites between the 3 groups. Slight increases in B m a x for high affinity [3H]naloxone and [3H]DADL were observed in footshocked animals compared with two control groups of animals, though the differences were not statistically significant among them.
TABLE I
Receptor numbers (Bmax)and dissociation constants (Ke,) in each group using [~H]naloxone or [JH]o-Ala2,D-LeuS-enkephalin (DADL) as the labelled ligand Each value is expressed as m e a n + S.E.M.
[ 3H] naloxone High affinity B . . . . (fmol/mg protein) Control 170 _+ 3 Sham-control 179 + 13 Footshock 185 + 15 K~ (nM) Control 1.69 + 0.12 Sham-control 1.45 + 0.06 Footshock 1.61 + 0.16
[ 3H]D A D L Low affinity
4 6 3 + 18 510 + 32 509 + 43
147__+8 152 + 11 153 + 14
8.05 + 0.26 7.79 + 0.28 7.96 __+0.27
7.29 __+0.38 6.96 __+0.27 7.33 __+0.55
DISCUSSION
Behavioral results obtained in this study demonstrated that chronic footshock treatment during the preweanling period in the rat decreased pain sensitivity and increased the efficacy of morphine for producing analgesia, when tested in adulthood by the hot-plate method. Footshocked animals exhibited 1.5- to 2-fold longer paw-lick latency on the hot-plate at 56 °C than shamcontrol and control animals. On the other hand, no significant difference was found in paw-lick latency between the two control groups. The hotplate temperature necessary to produce equal baseline paw-lick latency (8-10 s) was significantly higher in footshocked animals than that in the controls. Furthermore, morphine-induced analgesia was significantly enhanced in footshocked animals in comparison with the controis at all doses tested. These findings are not consistent with a previous study 3 reporting that in rat pups exposed to chronic footshock for 21 days after birth the efficacy of morphine in producing antinociceptive effect was reduced. This inconsistency may be due to the difference in the time of testing for morphine. The previous study examined morphine-induced analgesia immediately following the last footshock, whereas in the present study we examined it in neonatally footshocked rats when they reached 90-100 days of age. Chronic stress of saline injection during maturation enhanced morphine efficacy when tested at maturity in mice, while saline injection for 4 weeks in adult mice had no effect on the analgesic effect of morphine 16. These results are in line with our results. It is likely that the exposure to chronic stress specifically during maturation results in a decrease in the pain sensitivity and an increase in the sensitivity to the analgesic effect of morphine, when tested at full maturity. However, the effects of neonatal chronic stress on changes in pain responsiveness and morphine efficacy observed in adulthood could not be attributed to the changes in opioid receptor binding to brain homogenates. No significant difference was found in the Bma x and Ka of [3H]DADL between footshocked and control animals. In contrast, Torda 26 found an increase in [3H]naloxone bind-
110 ing in rats chronically exposed to hot-plate stress during early postnatal life. On the other hand, it has also been reported that chronic footshock stress in the preweanling period did not alter #-type opioid receptor ontogeny 3. These results may indicate that different stressful stimulation brings about different influences on the endogenous ligand-receptor system. The present results obtained by using [ 3 H ] D A D L indicate that the chronic footshock treatment during the preweanling period does not alter b-type opioid receptor activity as well. Thus, it may be suggested that the development of the endogenous opioid system at the postsynaptic level is not affected by a class of stressor such as the footshock. Rather, at the presynaptic level, functional changes in the endogenous opioid system may occur. These cause the changes in pain responsiveness and morphine efficacy as observed in the present study. In adults, chronic footshock stress has been reported to deplete the central opioid leveP 9'2°, probably because of the disturbance in turnover, that is, the synthesis of opioid peptides cannot keep up with the excessive release of the peptides. However, when a developing endogenous opioid system was forced to synthesize and release endogenous opioid substances frequently by chronic footshock, the ability of the system to synthesize endogenous opioids in response to nociceptive stimuli might be increased, since it is well known that in the preweanling period protein synthesis such as an increase in enzymatic activity develops rapidly. The present findings that neonatally footshocked animals displayed decreased pain sensitivity and were more responsive to the analgesic effect of morphine, might be interpreted as that more abundant endogenous opioid peptides are synthesized and released in response to nociceptive situation such as the hot-plate in footshocked rats compared with the controls, since the hot-plate itself is a stressor which is known to increase the blood level of fl-endorphin 15, so that enhanced release of endogenous opioid peptides caused the elevation of the threshold for pain stimuli, and consequently analgesia is much more enhanced by increasing the number of receptors to be occupied by morphine after the injection of it. How-
ever, such an interpretation might not be limited to the opioid system, since even a large dose of naloxone (2 mg/kg) could not completely antagonize the antinociceptive effect of morphine in footshocked animals (Fig. 2), which may indicate the involvement ofnon-opioid neuropeptides. For example, it has been reported that stress-induced analgesia was reduced by hypophysectomy 18. So, in addition to assaying the turnover rate of endogenous opioid peptides, further studies which investigate the changes in the non-opioid system are needed. REFERENCES 1 Akil, H., Madden, J., Patrick, R.L. and Barchas, J.D., Stress-induced increase in endogenous opioid peptide: concurrent analgesia and its partial reversal by naloxon. In H. Kosteritz (Ed.), Opiate and Endogenous Opiate Peptides. Elsevier, Amsterdam, 1976, pp. 63-70. 2 Amir, S., Brown, Z.W. and Amit, Z., The role of endorphins in stress: evidence and speculations, Neurosci. Biobehav. Rev., 4 (1980) 44-86. 3 Bardo, M.T., Bhatnager, R.K. and Gebhart, G.F., Opiate receptor ontogeny and morphine-induced effects: influence of chronic footshock stress in preweanling rats, Dev. Brain Res., 1 (1981) 487-495. 4 Bodnar, R.J., Kelly, D.D., Brutus, M. and Glusman, M., Stress-induced analgesia: neural and hormonal determinants, Neurosci. Biobehav. Rev. 4 (1980) 87-100. 5 Bodnar, R.J., Kelly, D.D., Spiaggia, A., Ehrenberg, C. and Glusman, M., Dose-dependent reduction by naloxone of analgesia induced by cold-water stress, Pharmacol. Biochem. Behav., 8 (1978) 667-672. 6 Chance, W.T. Autoanalgesia: opiate and non-opiate mechanism, Neurosci. Biobehav. Rev., 4 (1980) 55-67. 7 Chance, W.T., White, A.C., Krynock, G:M. and Rosecrans, J.A., Conditioned fear-induced antinociception and decreased binding of [3H]-N-Leu-enkephalin to rat brain, Brain Res., 141 (1978) 371-374. 8 Chesher, G.B. and Chan, B., Footshock-induced analgesia in mice. Its reversal by and cross-tolerance with morphine, Life Sci., 21 (1977) 1569-1574. 9 Christie, M.J., Chesher, G.B. and Bird, K.D., The correlation between swim-stress induced antinociception and [3H]Leu-enkephalin binding to brain homogenates in mice, Pharmacol. Biochem. Behav., 15 (t981) 853-857. 10 Clendeninn, N.J., Petrait, M. and Simon, E.J., Ontological development of opiate receptors in rodent brain, Brain Res., 118 (1976) 157-160. 11 Coyle, J.T. and Pert, C.B., Ontogenic development of [3H]naloxonebinding in rat brain, Neuropharmacology, 15 (1976) 555-560. 12 Denenberg, V.H., Early experience and emotional development, Sci. Am., 208 (1963) 138-146.
111 13 Denenberg, V.H. and Bell, R.W., Critical periods for the effects of infantile experience on adult learning, Science, 131 (1960) 227-228. 14 Drugan, R.C. and Maier, S.F., Analgesic and opioid involvement in the shock-elicited activity and escape deficits produced by inescapable shock, Learn. Motiv., 14 (1983) 30-47. 15 Galina, Z.H., Sutherland, C.J. and Amit, Z., Effects of heat-stress on behavior and pituitary adrenal axis in rats, Pharmacol. Biochem. Behav., 19 (1983) 251-256. 16 Larson, A., Nociception in mice after chronic stress and chronic narcotic antagonists during maturation, Brain Res., 243 (1982) 323-328. 17 Levine, S., Stimulation in infancy, Sci. Am., 202 (1960) 80-86. 18 Lewis, J.W., Sherman, J.E. and Liebeskind, J.C., Opioid and non-opioid stress analgesia: assessment of tolerance and cross-tolerance with morphine, J. Neurosci., 1 (1981) 358-363. 19 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 20 Madden, J., Akil, H., Patrick, R.L. and Barchas, J.D., Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat, Nature (Lond.), 265 (1977) 358-360. 21 McGivern, R.F., Mousa, S., Couri, D. and Berntson,
22
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G.G., Prolonged intermittent footshock stress decreases Met- and Leu-enkephalin levels in brain with concomitant decreases in pain threshold, Life Sci., 33 (1983) 47-54. Miczek, K.A., Thompson, M.L. and Shuster, L., Opioidlike analgesia in defeated mice, Science, 215 (1982) 1520-1522. Millan, M.J. Stress and endogenous opioid peptides: a review. Mod. Probl. Pharmacopsychiat., 17 (1981) 49-67. Patey, G., De la Baume, S., Gros, C. and Schwartz, J., Ontogenesis of enkephalinergic systems in rat brain: postnatal changes in enkephalin levels, receptors and degrading enzyme activities, Life Sci., 27 (1980) 245-252. Spiaggia, A., Bodnar, R.J., Kelly, D.D. and Glusman, M., Opiate and non-opiate mechanisms of stress-induced analgesia: cross-tolerance between stressors, Pharmacol. Biochem. Behav., 10 (1979) 761-765. Torda, C., Effects of recurrent postnatal pain-related stressful events on opiate receptor-endogenous ligand system, Psyehoneuroendocrinology, 3 (1978) 85-92. Watkins, L. and Mayer, D.J., Organization of endogenous opiates and non-opiate pain control systems, Science, 216 (1982) 761-765. Wohltman, M., Roth, B.L. and Coscia, C., Differential postnatal development of mu and delta opiate receptors, Dev. Brain Res., 3 (1982) 679-684.
Behavioural Brain Research, 36 (1990) 105-111 Elsevier BBR 00981
The effect of neonatal exposure to chronic footshock on pain-responsiveness and sensitivity to morphine after maturation in the rat C. Shimada, S. Kurumiya, Y. Noguchi and M. Umemoto Division of Psychology, Faculty of Letters, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka (Japan) (Received 16 November 1988) (Revised version received 10 May 1989) (Accepted 10 May 1989)
Key words: Stress-induced analgesia; Preweanling period; Hot-plate; Opiate receptor; Morphine
Rat pups in 3 groups respectively were given daily footshock, exposure to a footshock apparatus without shock, or no handling from birth to 21 days of age and reared with no manipulation afterwards. After maturation (90-100 days of age), they were assessed for hot-plate paw-lick latency, morphine-induced analgesia and opiate receptor binding assay. In footshocked animals, a significant increase was found in paw-lick latency and in antinociceptive effects of morphine (1.25, 2.5, and 5.0 mg/kg) in comparison with two control groups. The antinociceptive effect of morphine in all 3 groups was antagonized by pretreatment with naloxone (2.0 mg/kg). No significant difference was found in binding activities (Bmax and Kd) for both [3H]naloxone and [3H]Dala2,D-LeuS-enkephalin between the 3 groups. These results suggest that exposure to footshock stress in the preweanling period has a long-term effect on the sensitivity of rats to painful events, probably due to chronic functional changes in endogeneous opiate systems at presynaptic level rather than in postsynaptic opiate receptor binding activity.
INTRODUCTION
It is well documented that a variety of stressful manipulations induces an analgesic reaction, which has been termed stress-induced analgesia2"4'6"23. Since cross tolerance develops between morphine and stress-induced analgesia8'18"22'25, and analgesia induced by stressors can be antagonized by pretreatment with opioid antagonists 14"27, the involvement of endogenous opioids in mediation of stress-induced analgesia has been suggested. Furthermore, it has been reported that stress-induced analgesia is accompanied by increases in endogenous opioid level in the brain 2° and with decreased binding of exo-
genously added enkephalin to brain homogenates 7,9. Stress-induced analgesia is known to dissipate within 1-2 h after the exposure to stressors 1'5. It has also been reported that chronic exposure to stress decreases pain threshold with concomitant decreases in brain opioid levels 18,20,21.These findings obtained from adults may suggest that, in the matured central nervous systems, exposure to acute stress transiently activates an adaptation mechanism in which endogenous opioid systems possibly play an important role in producing analgesic reaction, but with chronic stress the matured system is not able to keep coping with extended stress resulting in a decrease in pain
Correspondence: M. Umemoto, Division of Psychology, Faculty of Letters, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
106 threshold, presumably due to the depletion of endogenous substances such as opioid peptides. Exposure to chronic stress during the preweanling period may influence the ontogenic development of the endogenous opioid system and may also produce a different effect on the pain sensitivity. In behavior theory, it is well known that environmental stimulation in the preweanling period results in a reduced emotionality and an enhancement of learning in adults, and is termed 'early experience'12'13"17. The effect of early experience has been suggested to be mediated by the facilitation of pituitaryadrenocortical responses 15. In these early studies, of course, the involvement of the endogenous opioid system in the effect of early experience has not been suggested. Now, as we have a knowledge of the presence of the endogenous opioid system which is known to play a role in controlling the pain, it is of interest to consider the effect of early experience on adult behavior from the view of a possible permanent change in pain sensitivity which might be attributed to a permanent change in the endogenous opioid systems. It has been shown that the development of the opioid system, such as the levels of opioid peptides and the number of opioid receptors in the brain, occurred rapidly during the preweanling period1°'11'22"26.It has been reported that in the rat chronically exposed to hot-plate noxious stimulation during early life, the endogenous opioid peptide level and opioid receptor binding in the brain were increased 24. On the other hand, chronic footshock treatment on the rat from birth to 21 days of age, does not affect the ontogeny of opioid receptors and reduces the efficacy of morphine in producing antinociceptive effects when tested on the day following the last footshock3. In the present study, we investigated whether chronic footshock treatment of rats during the preweanling period brings about the influences in pain sensitivity and in the analgesic effect of morphine at the time of full growth, by testing hotplate paw-lick latency, since it has been reported that the full development of the opioid receptorendogenous ligand system in the brain requires about one postnatal month 11. We also examined opioid receptor binding in the brain using
[3H]D-Ala2-D-LeuS-enkephalin and [ q t ]naloxone, to label b- and/~-receptors respectively, since Bardo etal. 3 used only [3H]naloxone, which examined presumably #-receptor ontogeny, and suggested the possibility that chronic preweanling footshock treatment altered the other type of opioid receptors such as the b-receptor. Besides, Wohltman et al. 28 reported the delayed development of the b-receptor during the postnatal period in comparison with the #-receptor. MATERIALS AND METHODS Animals Male Wistar rat pups were randomly assigned to mixed-fostered litters of 8-10 pups each on the day after parturition. After ablactation (on day 21), 3-4 pups from each litter were housed together with no treatment until they reached 90-100 days of age. Food and water were always available on demand throughout the experiment. Footshock treatment Pups were placed in a footshock apparatus with a grid floor for 30 min and administered inescapable electric shock (6 s duration, at an interval of 1 shock/rain) through the grid connected to a shock generator (LVS Co., SGS-004). Starting from 2 mA which is a value of calibration, the intensity was adjusted in descending order to the lowest which was enough to induce a writhing response. The actual current intensity given to pups was in a range between 0.3 and 0.4 mA measured by Delgado's method using an osciloscope. Pups received two daily treatments at an 8-h interval for 21 days after birth. Shamcontrol pups were treated the same except that the shock was not delivered. Control pups were left untreated during the preweanling period. Paw-lick latency At 90-100 days after birth, all animals were assessed for pain sensitivity and for changes in antinociceptive effect of morphine. Each rat was placed on the hot-plate at 56 °C, and hind-paw lick latency was measured, When the response was not observed within 60 s, the ~ t i n g was terminated and 60 s was recorded as the latency.
107 Each rat was tested 3 times and the mean latency from the last two testings was used as the baseline latency. After the baseline latency was determined, each rat was injected subcutaneously with morphine hydrochloride (Shionogi Co.) and assessed for paw-lick latency at 15, 30, 60, 90, 120 and 150 min after the drug administration. The dosages of morphine were 1.25, 2.5 and 5.0 mg/kg. Each animal was injected with one of the three doses of morphine in a different order, and tested with 10 days of interval between the injections. Morphine-induced changes in pawlick latency were expressed as the percent of the maximum possible effect (MPE) as follows: ~oMPE = postdrug latency - baseline latency
x 100
cut-off time (60 s) - baseline latency Naloxone hydrochloride (Endo Lab) (2.0 mg/kg) was injected 15 min before morphine administration. In addition, adjusting the temperature of hot-plate by steps of 1 °C so that 8-10 s of baseline paw-lick latency was obtained for each animal, effects of three doses of morphine, that is, 2.5, 5.0, 10.0 mg/kg, were also examined in control and footshocked animals.
Opioid receptor binding assay Twelve rats from footshock, sham-control and control groups (4 from each group) were sacrificed by decapitation and their brains were removed. The whole brain except for cellebellum and olfactory bulb was weighed and homogenized in 20 vols. of ice-cold 50 ml Tris-HCl buffer (pH 7.4). After centrifugation at 20000 g for 30 min, the pellet was resuspended in Tris-buffer and incubated at 32 °C for 30 min to remove endogenous opioid. Following recentrifugation at 20 000 g for 30 min, the pellet was resuspended in Tris-buffer to contain 0.5-0.6mg protein in 480 #1 which was determined by the method of Lowry et al. 19. Binding assays were performed in a final volume of 500/~1 with 8 concentrations (0.18-22.58 nM) of [3H]naloxone (New England Nuclear, 50.0 Ci/mmol) in the presence of 1 #M
naloxone, and with 7 concentrations of [3H]DAlaZ-D-LeuS-enkephalin (DADL; New England Nuclear, 43.6 Ci/mmol) in the presence or absence of 1 #1 D-Ala2-MetS-enkephalin (Peptide Lab.). Samples were incubated at 0 ° C for 3 h and diluted with 2.5 ml Tris-buffer, followed by filtration through Whatman GF/C glass fiber filter. After two 2.5-ml rinses with cold buffer, the filters were transferred to vials and radio-counted by liquid scintillation spectrometry 12 h after addition of 10 ml Univergel (Nakarai Chem). Specific binding of labeled ligands was calculated by subtracting the radioactivity which was obtained in the presence of non-labeled ligands from that obtained in the absence of non-labeled ligands.
Statistical analysis Analysis of variance (ANOVA) and post-hoc test by Tukey's method or Student's t-test were used to analyze overall effects and to test the differences between the means, respectively. RESULTS
Decreased pain sensitivity in neonatally footshocked rats Neonatal footshock treatment did not affect the increase in body weights in footshocked animals. Mean body weight (+ S.E.M.) of rats in footshock, sham-control and control groups were 399 + 9, 393 + 12 and 394 + 10 g, respectively. There were no significant differences among the 3 groups. Fig. 1 shows the mean paw-lick latencies obtained from 3 groups of rats which were exposed chronically to footshock (n = 8), exposed to the grid apparatus without shock (n = 8), and left unhandled (n = 8) during the preweanling period. Footshocked rats exhibited significantly longer paw-lick latencies than the two control groups (P < 0.01). There was no significant difference in paw-lick latencies between shamcontrol and control groups. Increased sensitivity to morphine in neonatally footshocked rats Morphine produced dose-dependent analgesic effects in all 3 groups. In footshocked animals,
108
morphine had stronger effects on both magnitude and duration of antinociception than in two control animals (Fig. 2). ANOVA for each dose of morphine revealed statistically significant effects among the groups (1.25mg/kg: F2,2, = 22.95, P < 0 . 0 1 ; 2.5mg/kg: F2,21=22.03, P < 0 . 0 1 : 5.0 mg/kg: Fz.21 = 14.90, P < 0.01); 2 mg/kg of naloxone antagonized the analgesic effect of morphine (5mg/kg) in all 3 groups, whereas footshocked animals were resistant to the antagonizing effect of naloxone (/72.2, = 10.24, P < 0.01), as shown in Fig. 2. The mean temperatures of the hot-plate which
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Fig. 3. Percent of the Maximum Possible Effect (% MPE) of morphine on paw-lick latency in footshock amt control animals (8 each). Hot-plate temperature was adjusted to produce 8-10 s paw-lick latency in each animal. Mean temperature were 59.4 + 0.2 °C and 5712 4. 03 °C for footshock and control animals respectively. *P < 0.05, **P < 0.01 as compared with the controls (by Tukey's post-hoe test).
109 produced 8-10 s baseline latencies were 59.4 + 0.2 °C and 57.2 + 0.3 °C in footshocked and control groups respectively, and a significant difference was found between them (P < 0.01). Fig. 3 shows significant increases in paw-lick latencies of footshocked animals compared with control animals, whose baseline latencies were similar between the two groups, after injection of morphine (2.5mg/kg: F I , 1 4 = 26.86, P < 0.01; 5.0 mg/kg: F I , 1 4 = 38.62, P < 0.01; 10 mg/kg: El,14 = 60.14, P < 0.01). Particularly after 10 mg/kg of morphine, paw-lick latencies in most of footshocked animals reached cut-off time 60 min later and remained much longer than in control animals even 150 min after the injection.
Opioid receptor binding assay Table I shows means + S.E.M. of the dissociation constant (Kd) and maximum binding capacity (Bmax) from independent Scatchard analysis of each group. There was no significant difference in both K d and B m a x for the [ 3 H ] naloxone and [3H]DADL binding sites between the 3 groups. Slight increases in B m a x for high affinity [3H]naloxone and [3H]DADL were observed in footshocked animals compared with two control groups of animals, though the differences were not statistically significant among them.
TABLE I
Receptor numbers (Bmax)and dissociation constants (Ke,) in each group using [~H]naloxone or [JH]o-Ala2,D-LeuS-enkephalin (DADL) as the labelled ligand Each value is expressed as m e a n + S.E.M.
[ 3H] naloxone High affinity B . . . . (fmol/mg protein) Control 170 _+ 3 Sham-control 179 + 13 Footshock 185 + 15 K~ (nM) Control 1.69 + 0.12 Sham-control 1.45 + 0.06 Footshock 1.61 + 0.16
[ 3H]D A D L Low affinity
4 6 3 + 18 510 + 32 509 + 43
147__+8 152 + 11 153 + 14
8.05 + 0.26 7.79 + 0.28 7.96 __+0.27
7.29 __+0.38 6.96 __+0.27 7.33 __+0.55
DISCUSSION
Behavioral results obtained in this study demonstrated that chronic footshock treatment during the preweanling period in the rat decreased pain sensitivity and increased the efficacy of morphine for producing analgesia, when tested in adulthood by the hot-plate method. Footshocked animals exhibited 1.5- to 2-fold longer paw-lick latency on the hot-plate at 56 °C than shamcontrol and control animals. On the other hand, no significant difference was found in paw-lick latency between the two control groups. The hotplate temperature necessary to produce equal baseline paw-lick latency (8-10 s) was significantly higher in footshocked animals than that in the controls. Furthermore, morphine-induced analgesia was significantly enhanced in footshocked animals in comparison with the controis at all doses tested. These findings are not consistent with a previous study 3 reporting that in rat pups exposed to chronic footshock for 21 days after birth the efficacy of morphine in producing antinociceptive effect was reduced. This inconsistency may be due to the difference in the time of testing for morphine. The previous study examined morphine-induced analgesia immediately following the last footshock, whereas in the present study we examined it in neonatally footshocked rats when they reached 90-100 days of age. Chronic stress of saline injection during maturation enhanced morphine efficacy when tested at maturity in mice, while saline injection for 4 weeks in adult mice had no effect on the analgesic effect of morphine 16. These results are in line with our results. It is likely that the exposure to chronic stress specifically during maturation results in a decrease in the pain sensitivity and an increase in the sensitivity to the analgesic effect of morphine, when tested at full maturity. However, the effects of neonatal chronic stress on changes in pain responsiveness and morphine efficacy observed in adulthood could not be attributed to the changes in opioid receptor binding to brain homogenates. No significant difference was found in the Bma x and Ka of [3H]DADL between footshocked and control animals. In contrast, Torda 26 found an increase in [3H]naloxone bind-
110 ing in rats chronically exposed to hot-plate stress during early postnatal life. On the other hand, it has also been reported that chronic footshock stress in the preweanling period did not alter #-type opioid receptor ontogeny 3. These results may indicate that different stressful stimulation brings about different influences on the endogenous ligand-receptor system. The present results obtained by using [ 3 H ] D A D L indicate that the chronic footshock treatment during the preweanling period does not alter b-type opioid receptor activity as well. Thus, it may be suggested that the development of the endogenous opioid system at the postsynaptic level is not affected by a class of stressor such as the footshock. Rather, at the presynaptic level, functional changes in the endogenous opioid system may occur. These cause the changes in pain responsiveness and morphine efficacy as observed in the present study. In adults, chronic footshock stress has been reported to deplete the central opioid leveP 9'2°, probably because of the disturbance in turnover, that is, the synthesis of opioid peptides cannot keep up with the excessive release of the peptides. However, when a developing endogenous opioid system was forced to synthesize and release endogenous opioid substances frequently by chronic footshock, the ability of the system to synthesize endogenous opioids in response to nociceptive stimuli might be increased, since it is well known that in the preweanling period protein synthesis such as an increase in enzymatic activity develops rapidly. The present findings that neonatally footshocked animals displayed decreased pain sensitivity and were more responsive to the analgesic effect of morphine, might be interpreted as that more abundant endogenous opioid peptides are synthesized and released in response to nociceptive situation such as the hot-plate in footshocked rats compared with the controls, since the hot-plate itself is a stressor which is known to increase the blood level of fl-endorphin 15, so that enhanced release of endogenous opioid peptides caused the elevation of the threshold for pain stimuli, and consequently analgesia is much more enhanced by increasing the number of receptors to be occupied by morphine after the injection of it. How-
ever, such an interpretation might not be limited to the opioid system, since even a large dose of naloxone (2 mg/kg) could not completely antagonize the antinociceptive effect of morphine in footshocked animals (Fig. 2), which may indicate the involvement ofnon-opioid neuropeptides. For example, it has been reported that stress-induced analgesia was reduced by hypophysectomy 18. So, in addition to assaying the turnover rate of endogenous opioid peptides, further studies which investigate the changes in the non-opioid system are needed. REFERENCES 1 Akil, H., Madden, J., Patrick, R.L. and Barchas, J.D., Stress-induced increase in endogenous opioid peptide: concurrent analgesia and its partial reversal by naloxon. In H. Kosteritz (Ed.), Opiate and Endogenous Opiate Peptides. Elsevier, Amsterdam, 1976, pp. 63-70. 2 Amir, S., Brown, Z.W. and Amit, Z., The role of endorphins in stress: evidence and speculations, Neurosci. Biobehav. Rev., 4 (1980) 44-86. 3 Bardo, M.T., Bhatnager, R.K. and Gebhart, G.F., Opiate receptor ontogeny and morphine-induced effects: influence of chronic footshock stress in preweanling rats, Dev. Brain Res., 1 (1981) 487-495. 4 Bodnar, R.J., Kelly, D.D., Brutus, M. and Glusman, M., Stress-induced analgesia: neural and hormonal determinants, Neurosci. Biobehav. Rev. 4 (1980) 87-100. 5 Bodnar, R.J., Kelly, D.D., Spiaggia, A., Ehrenberg, C. and Glusman, M., Dose-dependent reduction by naloxone of analgesia induced by cold-water stress, Pharmacol. Biochem. Behav., 8 (1978) 667-672. 6 Chance, W.T. Autoanalgesia: opiate and non-opiate mechanism, Neurosci. Biobehav. Rev., 4 (1980) 55-67. 7 Chance, W.T., White, A.C., Krynock, G:M. and Rosecrans, J.A., Conditioned fear-induced antinociception and decreased binding of [3H]-N-Leu-enkephalin to rat brain, Brain Res., 141 (1978) 371-374. 8 Chesher, G.B. and Chan, B., Footshock-induced analgesia in mice. Its reversal by and cross-tolerance with morphine, Life Sci., 21 (1977) 1569-1574. 9 Christie, M.J., Chesher, G.B. and Bird, K.D., The correlation between swim-stress induced antinociception and [3H]Leu-enkephalin binding to brain homogenates in mice, Pharmacol. Biochem. Behav., 15 (t981) 853-857. 10 Clendeninn, N.J., Petrait, M. and Simon, E.J., Ontological development of opiate receptors in rodent brain, Brain Res., 118 (1976) 157-160. 11 Coyle, J.T. and Pert, C.B., Ontogenic development of [3H]naloxonebinding in rat brain, Neuropharmacology, 15 (1976) 555-560. 12 Denenberg, V.H., Early experience and emotional development, Sci. Am., 208 (1963) 138-146.
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