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Effect of cold acclimation and repeated swimming on opioid and nonopioid swim stress-induced analgesia in selectively bred mice
Physiology & Behavior 78 (2003) 345 – 350
Effect of cold acclimation and repeated swimming on opioid and nonopioid swim stress-induced analgesia in selectively bred mice Iwona B. Lapoa,*, Marek Konarzewskib, Bogdan Sadowskia,c a
Institute for Genetics and Animal Breeding, Polish Academy of Sciences, 05-552 Wo´lka Kosowska, Poland b Institute of Biology, University of Bialystok, 15-950 Bialystok, Poland c Department of Experimental Pathology, Medical Academy of Warsaw, 00-325 Warsaw, Poland Received 23 April 2002; received in revised form 17 September 2002; accepted 23 October 2002
Abstract Swiss – Webster mice selectively bred for high swim stress-induced analgesia (SSIA) were exposed to continuous ambient cold (5 °C) for 6 weeks or to daily 3-min swims for 14 consecutive days either in 20 or 32 °C water. Thereafter, mice subjected to the particular procedure were injected intraperitoneally with 10 mg/kg of naltrexone HCl and were tested for modification of the opioid and nonopioid component of SSIA. SSIA was reduced following swims at either water temperature and was antagonized by naltrexone to greater extent than in nonswimming mice. Thus, the nonopioid (i.e. naltrexone-resistant) portion of the overall SSIA was significantly reduced, whereas the opioid (naltrexone-sensitive) portion became relatively augmented. In contrast, SSIA differed neither in magnitude nor in sensitivity to naltrexone between cold-acclimated and unacclimated mice. Swim hypothermia as well as the nonopioid component of SSIA were decreased after repeated swimming at 32 and 20 °C, but remained unchanged after cold acclimation. This argues for the essential role of an extrathermal, probably emotional in nature, factor not only in the elicitation of nonopioid SSIA, but also in the modulation of thermoregulatory processes during swimming. We suggest that the emergency component of swim stress, together with initial moderate hypothermic challenge, first produces the opioid form of SSIA, and subsequently, as the swim continues, also affects the thermoregulatory processes maintaining thermal homeostasis. This causes further increase in swim hypothermia and raises its stressing property to induce the nonopioid form of SSIA. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cold acclimation; Repeated swimming; Naltrexone; Nonopioid analgesia; Opioid-mediated analgesia; Swim stress-induced analgesia
1. Introduction Laboratory rodents after being exposed to swimming manifest a decrease in pain sensitivity, termed swim stressinduced analgesia (SSIA). Two forms of SSIA are commonly distinguished: an opioid-mediated and a nonopioid one. The opioid form of SSIA is reversed by naloxone or naltrexone, whereas the nonopioid analgesia is insensitive to these opioid receptor antagonists. In addition to this distinction, a mixed opioid – nonopioid SSIA is also recognized. Activation of a particular form depends, to much extent, on the severity of swim parameters. Thus, swim-
* Corresponding author. Tel.: +48-22-756-1711; fax: +48-22-756-1699. E-mail address: [email protected] (I.B. Lapo).
ming in warm water and/or for short duration usually elicits an opioid SSIA, whereas swimming at more severe conditions produces a nonopioid SSIA [1– 3]. The idea that phenotypic differences in SSIA within outbred populations of laboratory rodents may reflect a genotypic differentiation of pain inhibitory mechanisms [4] prompted us to breed mice for divergent magnitudes of analgesia induced by 3-min swimming in 20 °C water. Using this strategy, we have developed a high- (HA) and a low-analgesia (LA) line, differing not only in the magnitude of SSIA [5], but also in its neurochemical nature [6]. Thus, a high SSIA in the HA line is partially reversed by naloxone or naltrexone, and therefore, is qualified as mixed opioid– nonopioid. The opioid involvement in SSIA of the HA line depends on the severity of swim parameters; that is, mild water temperatures and/or short swim duration produce a mixed opioid –nonopioid SSIA, whereas swim-
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(02)01004-1
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ming in cold water and/or for long duration causes nonopioid SSIA. In contrast, the LA line manifests only low SSIA, which is insensitive to the opioid receptor antagonists under all swim parameters, and thus, is nonopioid in nature [7,8]. Apart from the differences in the pattern of SSIA, HA and LA mice also differ in thermoregulatory responses to swimming—traits not intended in the selection protocol. Thus, high SSIA of HA mice, elicited by swimming in 20 °C water, is accompanied by greater postswim hypothermia and lower maximum oxygen consumption, as compared to low SSIA of LA mice [9]. One could then conceive that a decrease in the animal’s core temperature during swimming is essential to produce SSIA, and the magnitude of hypothermia might determine the neurochemical nature of SSIA. This possibility seems to be supported by the finding that 15-min exposure to ambient cold in helium – oxygen (Helox) atmosphere—a procedure causing an abrupt removal of heat from the animal’s body—elicits in HA mice a high analgesia that, depending on Helox temperature, is or is not antagonized by naltrexone [10]. In general, exposure to a relatively low Helox temperature causes a pronounced hypothermia together with purely nonopioid analgesia, whereas a higher temperature, causing little hypothermia, induces opioid – nonopioid analgesia. In a concurrent study, we found the Helox-induced analgesia, but not SSIA, to be attenuated in HA mice acclimated to ambient cold. On the other hand, repeated swims have significantly decreased SSIA, but exerted no effect on analgesia caused by exposure to Helox. We suggested that while Helox-induced analgesia can be regarded only as a consequence of a rapid removal of heat from the animal’s body, an extrathermal, perhaps emotional in nature, component of swim stress, interacting with swim hypothermia, plays an important role in the development of SSIA [11]. In the present study, we aimed to assess how a hypothermic and an extrathermal swim stress-related factor might interact in eliciting the opioid and the nonopioid component of SSIA. HA mice provide a unique model for this research because, together with high mixed opioid – nonopioid SSIA and low swim metabolism, they also manifest enhanced emotionality under appropriate behavioral tests [12]. In one experiment, we acclimated HA mice to ambient low temperature in order to find out whether a mere improvement of animals’ thermogenic capacity will alter the neurochemical background of SSIA. In another experiment, we exposed mice to a fortnight of daily swims in order to habituate them to the emergency component of the swim stress. Some mice swam in 20 °C water, thus being also exposed to repeated swim hypothermia, and others swam in 32 °C water, causing only little change in animal’s core temperature. Naltrexone, a prototypic opioid receptor antagonist, was administered to differentiate between the effects of the procedures on the opioid and on the nonopioid form of SSIA.
2. Material and methods 2.1. Animals Swiss –Webster mice were obtained from our colony, selectively bred for over 40 generations toward high SSIA produced by 3-min swimming in 20 °C water, as described elsewhere [5]. The mice were maintained on 0600 h lights on/1800 h lights off photoperiod and had unlimited access to murine chow and tap water. Before each experimental session, mice were weighted to the nearest 0.1 g. All trials were conducted between 0800 and 1800 h. Each experimental group counted equal number of males and females. 2.2. Acclimation to cold Upon weaning at the age of 3 weeks, 72 HA mice of the 44th generation were randomly assigned to two groups and individually maintained in separate cages (24 18 13 cm). We raised the thermogenic capacity of experimental mice by allowing them 6-week acclimation to ambient cold at 5 °C. The unacclimated (control) group was maintained at 21 – 22 °C. 2.3. Repeated swimming One hundred and forty-four HA mice of the 47th generation were housed four to five, same-sex, and same-family individuals per cage (29 21 10 cm) at ambient temperature of 20 – 21 °C. At the age of 7 weeks, they were randomly assigned to three groups. The first group was exposed for 14 consecutive days to 3-min swimming in 20 °C water, a condition used under the selection protocol and consistently causing a pronounced SSIA and hypothermia in the HA line. The second group was given the same daily swims, but at 32 °C, earlier found to produce a far lower SSIA and only little hypothermia in this line [8]. The animals swam individually in a plastic container filled with tap water up to 30 cm above the floor. The third (control) group did not swim, but was kept in the same room in order to experience comparable amount of environmental disturbance. 2.4. Swim test At the end of the continuous maintenance in ambient cold or of the repeated swimming, all mice were exposed to a 3-min swim test in 20 °C water to assess SSIA and postswim hypothermia. Swimming occurred in the same container, as described in the preceding section. Thirty minutes before swimming, half of each group intraperitoneally received 10 mg/kg of naltrexone HCl (Research Biochemicals International, Natick, MA) dissolved in 0.9% physiological saline, and the other half were injected with an equal volume of saline. This dose of naltrexone was
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earlier found effective to partially antagonize SSIA in HA mice [8,10].
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inary testing did not detect sex-related differences. Planned contrasts or Duncan test were used for detailed comparisons, where appropriate [13].
2.5. Algesiometry and temperature measurements 2.7. Bioethics Pain sensitivity was measured with the hot plate test. Each mouse was placed in a 15-cm-wide transparent box on a metallic plate heated to 56 °C and was carefully observed for the latency of a characteristic hind paw flick or lick response, which terminated the trial. If no such response occurred within 60 s, the animal was removed from the plate to avoid possible skin damage. Colonic temperature was taken to the nearest 0.1 °C with a digital thermometer (model BAT-12, Physitemp Instruments, Clifton, NJ) equipped with a RET-3 thermocouple probe. The measurements of pain sensitivity and core temperature were made before swimming (baseline) and at the end of 2-min drying after completion of the swim [8]. The magnitude of SSIA was quantified by converting the hot plate latencies to percent of maximum possible effect (%MPE) according to the formula:
The protocol of the experiments was approved by the Ethics Commission of the Institute for Genetics and Animal Breeding, Polish Academy of Sciences. The rules of humane treatment and animals’ welfare were strictly observed.
3. Results 3.1. Effects of acclimation to cold
2.6. Statistics
SSIA magnitude was not modified in mice acclimated to low ambient temperature, F(1,68) = 0.29, P=.59, but was significantly attenuated by naltrexone, F(1,68) = 19.12, P < .0001, two-way ANOVA, in unacclimated as in coldacclimated mice. This is indicated by nonsignificant cold acclimation naltrexone interaction, F(1,68) = 1.52, P=.22, and further supported by detailed comparisons within unacclimated, P < .001, and cold-acclimated mice, P < .05, planned contrasts (Fig. 1A). Cold acclimation did not affect the magnitude of swim hypothermia, F(1,67) = 1.42, P=.24, two-way ANCOVA (Fig. 1B). A slightly greater hypothermia seen in naltrex-
Since the animals used in the particular experiments belonged to two different generations, the effect of each procedure was compared to the control group within the same generation. This is because the magnitudes of swim hypothermia—indirectly selected trait, and to much lesser extent SSIA—the directly selected trait, vary across generations. Differences in SSIA were analyzed by means of an analysis of variance (ANOVA), using arcsine transformation of the computed %MPE values to normalize variances. In addition, to compare the relative effectiveness of naltrexone on SSIA in mice given repeated swimming at two water temperatures and in controls, naltrexone antagonism was expressed as the percentage of a mean computed for the matching saline-injected subgroup. Since the magnitude of swim hypothermia to some extent depends on animals’ body size, hypothermic data were analyzed by a model of analysis of covariance (ANCOVA), with animals’ body mass as a covariate. The subjects in particular experiments were mice belonging to different families, therefore, we first analyzed the data with the appropriate models of ANCOVA and ANCOVA with family as a nested random factor. Since between-family variations of SSIA and swim hypothermia were found nonsignificant, we excluded the family factor from further analyses. Likewise, the data obtained from either sex were combined in all analyses, because prelim-
Fig. 1. Analgesia (A) and hypothermia (B) induced in cold-acclimated (5 °C) and unacclimated (control) HA mice by 3-min swimming in 20 °C water. In this figure and in Fig. 2, the analgesic and the hypothermic data, respectively, represent means from ANOVA and least square means from ANCOVA corrected for animal’s body mass ± S.E. * P < .05, * * P < .001 with respect to saline (planned contrasts).
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ðpostswim latency baseline latencyÞ : ð60 baseline latencyÞ
Swim hypothermia was quantified as a decrease in core temperature from baseline.
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group it did not differ, P=.06 between the mice given repeated swims in 20 and 32 °C water, planned contrasts. Mice exposed to repeated swimming displayed reduced swim hypothermia as compared to nonswimming controls, F(2,137) = 46.75, P < .0001, two-way ANCOVA. No difference was found between the two water temperatures, P=.65, planned contrasts (Fig. 2B). The effect of naltrexone was not significant, F(1,137) = 0.36, P=.53, two-way ANCOVA. Body mass (males: 33.1 ± 0.3 g, females: 27.4 ± 0.3 g) was significant as covariate, F(1,137) = 10.65, P < .01.
4. Discussion
Fig. 2. Effects of repeated swimming in 32 and 20 °C water on analgesia (A) and hypothermia (B) induced by 3-min swimming in 20 °C water. The naltrexone antagonism is also shown as percentage of the decrease in swim analgesia in naltrexone-injected mice, with respect to saline means taken as 100% (A—solid line). * Different from saline, P < .001 (planned contrasts); y different from controls, P < .001 (Duncan test).
one-treated mice was significant in two-way ANCOVA, F(1,67) = 5.25, P < .05, but not in separate, one-way ANCOVAs within unacclimated, P=.17, and cold-acclimated mice, P=.07. Body mass of males (35.8 ± 0.5 g) and females (30.2 ± 0.2 g) was nonsignificant as covariate, F(1,67) = 0.32, P=.58, two-way ANCOVA. 3.2. Effects of repeated swimming Both repeated swimming and naltrexone significantly attenuated SSIA, F(2,138) = 47.87 and F(1,138) = 92.27, respectively, P < .0001, two-way ANOVA. Planned contrasts comparisons confirmed the effectiveness of naltrexone in controls as well as in experimental groups, P < .0001 (Fig. 2A). Percent attenuation of SSIA by naltrexone was greater after repeated swimming than in nonswimming mice, F(2, 69) = 12.29, P < .0001, one-way ANOVA, and P < .001, Duncan test. No significant difference was found between the two water temperatures of repeated swimming, P=.22, Duncan test (Fig. 2A). Separate one-way ANOVAs revealed that repeated swimming significantly attenuated SSIA both in mice injected with saline, F(2,69) = 26.03, and with naltrexone, F(2,69) = 24.11, P < .0001. The magnitude of SSIA in mice exposed to repeated swims was significantly lower than in controls (saline-injected mice swimming at 20 °C: P < .0001; salineinjected mice swimming at 32 °C: P < .025; P < .0001 for both swim temperatures in naltrexone-injected mice, planned contrasts). Within the saline-injected group, the magnitude of SSIA differed, P < .001, and within the naltrexone-injected
The main finding of this study is that the repeated swimming not only decreased overall SSIA magnitude in mice expressing genetically determined high opioid– nonopioid stress analgesia, but also modified the sensitivity of SSIA to naltrexone. Namely, the nonopioid (i.e. naltrexoneresistant) portion of the overall SSIA was significantly reduced, whereas the opioid (naltrexone-sensitive) portion became relatively augmented. In accordance with the concept of collateral inhibition between the opioid and the nonopioid systems [14 – 20], we assume that the repeated swimming primarily caused a suppression of the nonopioid analgesic system, which in turn led to an alleviation of the inhibitory action tonically exerted by the latter on the opioid one. In contrast to repeated swimming, SSIA magnitude and its neurochemical nature remained unchanged following continuous 6-week exposure to ambient 5 °C. This comparison is important since the acclimation of HA mice to ambient cold substantially improves their thermogenic capacity. When exposed to cold Helox atmosphere, they manifest far-reduced hypothermia and about 50% higher peak metabolic rates than unacclimated controls do [21]. Overall, these results are consistent with the hypothesis that the observed reversal in proportion between opioid and
Fig. 3. Hypothetical interaction between the emergency and the hypothermic component of swim stress in the development of the opioid and the nonopioid form of SSIA in HA mice.
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nonopioid components of SSIA resulting from repeated swimming was associated with habituation to an extrathermal component of swim stress that may be emotional in nature, not with an increased rate of thermogenesis. Moreover, the proposed habituation, and not the repeated exposure to low swim temperature, might account for the attenuation of swim hypothermia as well. This claim is justified by the finding that neither the attenuation of nonopioid SSIA nor the decrease in swim hypothermia depended on water temperature of the repeated swims, and also that the hypothermia, as SSIA, was not affected by cold acclimation. Modulation of animals’ thermogenic capacity by emotional factors has been described by several authors. For example, the attenuation of hypothermia in rats given repeated immersions in cold water was disrupted by changing the environmental context [22,23] and was facilitated by electric stimulation in brain reward sites [24,25]. Particularly interesting is the rise of nonshivering thermogenesis in rats after repeated immobilization stress [26], which means that not only the exposure to cold, but also a habituation to nonthermic stressors can improve animals’ thermogenic capacity. In accordance with these observations, our results not only argue for a major role of a stress – emotional component of swimming in the elicitation of opioid and nonopioid SSIA, but also point to the effect of an extrathermal factor on thermoregulatory processes. A net result would be swim hypothermia consistently encountered in HA mice, sensitive to emotional stimuli, than in the less fearful LA mice [12]. Based on the results of our earlier work, we propose that for HA mice swimming in 20 °C water, SSIA develops faster than hypothermia and for 3 min is more susceptible to naloxone than after 5 min of swimming (cf. Fig. 4C in Ref. [7]). It is then likely that the emergency component of swim stress, in connection with moderate hypothermia, initially produces the opioid form of SSIA. Subsequently, as swimming continues, this primary analgesic mechanism affects the thermoregulatory processes maintaining thermal homeostasis and further increases the stressing property of swim hypothermia. This ultimately induces the nonopioid form of SSIA. Thus, a nonopioid SSIA is particularly likely to develop in HA mice swimming in cold water or for a long duration (that is, when pronounced hypothermia develops). This is well supported by the results of the present as well as our previous studies showing that a profound swim hypothermia is not sensitive to naltrexone or naloxone [7,8]. As the hypothermia increases, the nonopioid component of SSIA begins to prevail and damps the opioid one. A graphical illustration of our hypothesis is presented in Fig. 3. Summing up, we conclude that the complex nature of opioid – nonopioid SSIA in the mouse stems from an integrated response of the animal to an extrathermal (i.e. emergency) and a thermal (i.e. hypothermic) component of swim stress.
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Acknowledgements The authors thank two anonymous reviewers for their effort involved in the improvement of the manuscript. We also thank I. Gasiorkiewicz and B. Sobolewska for technical assistance. This study was supported by Polish Committee for Scientific Research (KBN) Grants No. 6PO4C01616 and No. 6PO4C01818 to B. Sadowski.
References [1] O’Connor P, Chipkin RE. Comparisons between warm and cold water swim stress in mice. Life Sci 1984;35:631 – 9. [2] Terman GW, Morgan MJ, Liebeskind JC. Opioid and non-opioid stress analgesia from cold water swim: importance of stress severity. Brain Res 1986;372:167 – 71. [3] Marek P, Mogil JS, Sternberg WF, Panocka I, Liebeskind JC. Nmethyl-D-aspartic acid (NMDA) receptor antagonist MK-801 blocks non-opioid stress-induced analgesia: II. Comparison across three swim stress paradigms in selectively bred mice. Brain Res 1992; 578:197 – 203. [4] Bodnar RJ, Sikorszky V. Naloxone and cold-water swim analgesia: parametric considerations and individual differences. Learn Motiv 1983;14:223 – 37. [5] Panocka I, Marek P, Sadowski B. Inheritance of stress-induced analgesia in mice. Selective breeding study. Brain Res 1986;397:152 – 5. [6] Panocka I, Marek P, Sadowski B. Differentiation of neurochemical basis of stress-induced analgesia in mice by selective breeding. Brain Res 1986;397:156 – 60. [7] Mogil JS, Sternberg WF, Balian H, Liebeskind JC, Sadowski B. Opioid and non-opioid swim stress-induced analgesia: a parametric analysis in mice. Physiol Behav 1996;59:123 – 32. [8] Sadowski B, Sas R, S´widerski T, Trzasko M. Differentiation of opioid and non-opioid pain inhibitory mechanisms, and of thermoregulation in mice selectively bred for high and low swim stress-induced analgesia. Analgesia 1996;2:211 – 8. [9] Konarzewski M, Sadowski B, Jo´z´wik I. Metabolic correlates of selection for swim stress-induced analgesia in laboratory mice. Am J Physiol 1997;273(42):R337 – 43. [10] Sadowski B, Konarzewski M. Analgesia in selectively bred mice exposed to cold in helium/oxygen atmosphere. Physiol Behav 1999;66: 145 – 51. [11] Lapo I, Konarzewski M, Sadowski B. Analgesia induced by swim stress in the mouse: a relationship between analgesic and thermoregulatory mechanisms. Pflu¨gers Arch 2003 [in press]. [12] Blaszczyk J, Tajchert K, Lapo I, Sadowski B. Acoustic startle and open-field behavior in mice bred for magnitude of swim analgesia. Physiol Behav 2000;70:471 – 6. [13] SAS Institute. SAS/STAT users guide (version 6). 4th ed. Cary (NC): SAS Institute; 1990. [14] Greeley JD, Le AD, Poulos CX, Cappell H. ‘‘Paradoxical’’ analgesia induced by naloxone and naltrexone. Psychopharmacology 1988;96: 36 – 9. [15] Grisel JE, Fleshner M, Watkins LR, Mayer SF. Opioid and nonopioid interactions in two forms of stress-induced analgesia. Pharmacol Biochem Behav 1993;45:161 – 72. [16] Krichgessner AL, Bodnar RJ, Pasternak GW. Naloxazone and paininhibitory systems: evidence for a collateral inhibition model. Pharmacol Biochem Behav 1982;17:1175 – 9. [17] Rochford J, Stewart J. Activation and expression of endogenous pain control mechanisms in rats given repeated nociceptive tests under the influence of naloxone. Behav Neurosci 1987;101:87 – 103. [18] Steinman JL, Faris PL, Mann PE, Olney JW, Komisaruk BR, Wills WD, et al. Antagonism of morphine analgesia by nonopioid cold-
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I.B. Lapo et al. / Physiology & Behavior 78 (2003) 345–350 water swim analgesia: direct evidence for collateral inhibition. Neurosci Biobehav Rev 1990;14:1 – 7. Westbrook RF, Greeley JD. Some effects of the opioid antagonist, naloxone, upon the rat’s reactions to a heat stressor. Q J Exp Psychol 1990;42:1 – 40. Yoburn BC, Truesdell LS, Kest B, Inturrisi CE, Bodnar RJ. Chronic opioid antagonist treatment facilitates nonopioid stress-induced analgesia. Pharmacol Biochem Behav 1987;27:525 – 7. Lapo I, Konarzewski M, Sadowski B. Differential metabolic capacity of mice selected for magnitude of swim stress-induced analgesia. J Appl Physiol 2003 [in press]. Harrod SB, Metzger MM, Stempowski NK, Riccio DC. Cold tolerance behavioral differences following single or multiple cold exposures. Physiol Behav 2002;76:27 – 39.
[23] Riccio DC, MacArdy EA, Kissinger SC. Associative process in adaptation to repeated cold exposures in rats. Behav Neurosci 1991;105: 599 – 602. [24] Talan MI, Engel BT. Habituation and dishabituation to repeated mild cold exposures in C57BL/6J mice. Physiol Behav 1988;44:753 – 7. [25] Talan MI, Engel BT. Effect of electrical stimulation of ‘‘rewarding’’ areas of the hypothalamus on habituation and dishabituation to repeated mild cold exposures in C57BL/6J mice. Physiol Behav 1989; 45:603 – 6. [26] Kuroshima A, Habara Y, Uehara A, Murazumi K, Yahata T, Ohno T. Cross adaptation between cold and stress in rats. Eur J Physiol 1984; 402:402 – 8.
Effect of cold acclimation and repeated swimming on opioid and nonopioid swim stress-induced analgesia in selectively bred mice Iwona B. Lapoa,*, Marek Konarzewskib, Bogdan Sadowskia,c a
Institute for Genetics and Animal Breeding, Polish Academy of Sciences, 05-552 Wo´lka Kosowska, Poland b Institute of Biology, University of Bialystok, 15-950 Bialystok, Poland c Department of Experimental Pathology, Medical Academy of Warsaw, 00-325 Warsaw, Poland Received 23 April 2002; received in revised form 17 September 2002; accepted 23 October 2002
Abstract Swiss – Webster mice selectively bred for high swim stress-induced analgesia (SSIA) were exposed to continuous ambient cold (5 °C) for 6 weeks or to daily 3-min swims for 14 consecutive days either in 20 or 32 °C water. Thereafter, mice subjected to the particular procedure were injected intraperitoneally with 10 mg/kg of naltrexone HCl and were tested for modification of the opioid and nonopioid component of SSIA. SSIA was reduced following swims at either water temperature and was antagonized by naltrexone to greater extent than in nonswimming mice. Thus, the nonopioid (i.e. naltrexone-resistant) portion of the overall SSIA was significantly reduced, whereas the opioid (naltrexone-sensitive) portion became relatively augmented. In contrast, SSIA differed neither in magnitude nor in sensitivity to naltrexone between cold-acclimated and unacclimated mice. Swim hypothermia as well as the nonopioid component of SSIA were decreased after repeated swimming at 32 and 20 °C, but remained unchanged after cold acclimation. This argues for the essential role of an extrathermal, probably emotional in nature, factor not only in the elicitation of nonopioid SSIA, but also in the modulation of thermoregulatory processes during swimming. We suggest that the emergency component of swim stress, together with initial moderate hypothermic challenge, first produces the opioid form of SSIA, and subsequently, as the swim continues, also affects the thermoregulatory processes maintaining thermal homeostasis. This causes further increase in swim hypothermia and raises its stressing property to induce the nonopioid form of SSIA. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cold acclimation; Repeated swimming; Naltrexone; Nonopioid analgesia; Opioid-mediated analgesia; Swim stress-induced analgesia
1. Introduction Laboratory rodents after being exposed to swimming manifest a decrease in pain sensitivity, termed swim stressinduced analgesia (SSIA). Two forms of SSIA are commonly distinguished: an opioid-mediated and a nonopioid one. The opioid form of SSIA is reversed by naloxone or naltrexone, whereas the nonopioid analgesia is insensitive to these opioid receptor antagonists. In addition to this distinction, a mixed opioid – nonopioid SSIA is also recognized. Activation of a particular form depends, to much extent, on the severity of swim parameters. Thus, swim-
* Corresponding author. Tel.: +48-22-756-1711; fax: +48-22-756-1699. E-mail address: [email protected] (I.B. Lapo).
ming in warm water and/or for short duration usually elicits an opioid SSIA, whereas swimming at more severe conditions produces a nonopioid SSIA [1– 3]. The idea that phenotypic differences in SSIA within outbred populations of laboratory rodents may reflect a genotypic differentiation of pain inhibitory mechanisms [4] prompted us to breed mice for divergent magnitudes of analgesia induced by 3-min swimming in 20 °C water. Using this strategy, we have developed a high- (HA) and a low-analgesia (LA) line, differing not only in the magnitude of SSIA [5], but also in its neurochemical nature [6]. Thus, a high SSIA in the HA line is partially reversed by naloxone or naltrexone, and therefore, is qualified as mixed opioid– nonopioid. The opioid involvement in SSIA of the HA line depends on the severity of swim parameters; that is, mild water temperatures and/or short swim duration produce a mixed opioid –nonopioid SSIA, whereas swim-
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(02)01004-1
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ming in cold water and/or for long duration causes nonopioid SSIA. In contrast, the LA line manifests only low SSIA, which is insensitive to the opioid receptor antagonists under all swim parameters, and thus, is nonopioid in nature [7,8]. Apart from the differences in the pattern of SSIA, HA and LA mice also differ in thermoregulatory responses to swimming—traits not intended in the selection protocol. Thus, high SSIA of HA mice, elicited by swimming in 20 °C water, is accompanied by greater postswim hypothermia and lower maximum oxygen consumption, as compared to low SSIA of LA mice [9]. One could then conceive that a decrease in the animal’s core temperature during swimming is essential to produce SSIA, and the magnitude of hypothermia might determine the neurochemical nature of SSIA. This possibility seems to be supported by the finding that 15-min exposure to ambient cold in helium – oxygen (Helox) atmosphere—a procedure causing an abrupt removal of heat from the animal’s body—elicits in HA mice a high analgesia that, depending on Helox temperature, is or is not antagonized by naltrexone [10]. In general, exposure to a relatively low Helox temperature causes a pronounced hypothermia together with purely nonopioid analgesia, whereas a higher temperature, causing little hypothermia, induces opioid – nonopioid analgesia. In a concurrent study, we found the Helox-induced analgesia, but not SSIA, to be attenuated in HA mice acclimated to ambient cold. On the other hand, repeated swims have significantly decreased SSIA, but exerted no effect on analgesia caused by exposure to Helox. We suggested that while Helox-induced analgesia can be regarded only as a consequence of a rapid removal of heat from the animal’s body, an extrathermal, perhaps emotional in nature, component of swim stress, interacting with swim hypothermia, plays an important role in the development of SSIA [11]. In the present study, we aimed to assess how a hypothermic and an extrathermal swim stress-related factor might interact in eliciting the opioid and the nonopioid component of SSIA. HA mice provide a unique model for this research because, together with high mixed opioid – nonopioid SSIA and low swim metabolism, they also manifest enhanced emotionality under appropriate behavioral tests [12]. In one experiment, we acclimated HA mice to ambient low temperature in order to find out whether a mere improvement of animals’ thermogenic capacity will alter the neurochemical background of SSIA. In another experiment, we exposed mice to a fortnight of daily swims in order to habituate them to the emergency component of the swim stress. Some mice swam in 20 °C water, thus being also exposed to repeated swim hypothermia, and others swam in 32 °C water, causing only little change in animal’s core temperature. Naltrexone, a prototypic opioid receptor antagonist, was administered to differentiate between the effects of the procedures on the opioid and on the nonopioid form of SSIA.
2. Material and methods 2.1. Animals Swiss –Webster mice were obtained from our colony, selectively bred for over 40 generations toward high SSIA produced by 3-min swimming in 20 °C water, as described elsewhere [5]. The mice were maintained on 0600 h lights on/1800 h lights off photoperiod and had unlimited access to murine chow and tap water. Before each experimental session, mice were weighted to the nearest 0.1 g. All trials were conducted between 0800 and 1800 h. Each experimental group counted equal number of males and females. 2.2. Acclimation to cold Upon weaning at the age of 3 weeks, 72 HA mice of the 44th generation were randomly assigned to two groups and individually maintained in separate cages (24 18 13 cm). We raised the thermogenic capacity of experimental mice by allowing them 6-week acclimation to ambient cold at 5 °C. The unacclimated (control) group was maintained at 21 – 22 °C. 2.3. Repeated swimming One hundred and forty-four HA mice of the 47th generation were housed four to five, same-sex, and same-family individuals per cage (29 21 10 cm) at ambient temperature of 20 – 21 °C. At the age of 7 weeks, they were randomly assigned to three groups. The first group was exposed for 14 consecutive days to 3-min swimming in 20 °C water, a condition used under the selection protocol and consistently causing a pronounced SSIA and hypothermia in the HA line. The second group was given the same daily swims, but at 32 °C, earlier found to produce a far lower SSIA and only little hypothermia in this line [8]. The animals swam individually in a plastic container filled with tap water up to 30 cm above the floor. The third (control) group did not swim, but was kept in the same room in order to experience comparable amount of environmental disturbance. 2.4. Swim test At the end of the continuous maintenance in ambient cold or of the repeated swimming, all mice were exposed to a 3-min swim test in 20 °C water to assess SSIA and postswim hypothermia. Swimming occurred in the same container, as described in the preceding section. Thirty minutes before swimming, half of each group intraperitoneally received 10 mg/kg of naltrexone HCl (Research Biochemicals International, Natick, MA) dissolved in 0.9% physiological saline, and the other half were injected with an equal volume of saline. This dose of naltrexone was
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earlier found effective to partially antagonize SSIA in HA mice [8,10].
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inary testing did not detect sex-related differences. Planned contrasts or Duncan test were used for detailed comparisons, where appropriate [13].
2.5. Algesiometry and temperature measurements 2.7. Bioethics Pain sensitivity was measured with the hot plate test. Each mouse was placed in a 15-cm-wide transparent box on a metallic plate heated to 56 °C and was carefully observed for the latency of a characteristic hind paw flick or lick response, which terminated the trial. If no such response occurred within 60 s, the animal was removed from the plate to avoid possible skin damage. Colonic temperature was taken to the nearest 0.1 °C with a digital thermometer (model BAT-12, Physitemp Instruments, Clifton, NJ) equipped with a RET-3 thermocouple probe. The measurements of pain sensitivity and core temperature were made before swimming (baseline) and at the end of 2-min drying after completion of the swim [8]. The magnitude of SSIA was quantified by converting the hot plate latencies to percent of maximum possible effect (%MPE) according to the formula:
The protocol of the experiments was approved by the Ethics Commission of the Institute for Genetics and Animal Breeding, Polish Academy of Sciences. The rules of humane treatment and animals’ welfare were strictly observed.
3. Results 3.1. Effects of acclimation to cold
2.6. Statistics
SSIA magnitude was not modified in mice acclimated to low ambient temperature, F(1,68) = 0.29, P=.59, but was significantly attenuated by naltrexone, F(1,68) = 19.12, P < .0001, two-way ANOVA, in unacclimated as in coldacclimated mice. This is indicated by nonsignificant cold acclimation naltrexone interaction, F(1,68) = 1.52, P=.22, and further supported by detailed comparisons within unacclimated, P < .001, and cold-acclimated mice, P < .05, planned contrasts (Fig. 1A). Cold acclimation did not affect the magnitude of swim hypothermia, F(1,67) = 1.42, P=.24, two-way ANCOVA (Fig. 1B). A slightly greater hypothermia seen in naltrex-
Since the animals used in the particular experiments belonged to two different generations, the effect of each procedure was compared to the control group within the same generation. This is because the magnitudes of swim hypothermia—indirectly selected trait, and to much lesser extent SSIA—the directly selected trait, vary across generations. Differences in SSIA were analyzed by means of an analysis of variance (ANOVA), using arcsine transformation of the computed %MPE values to normalize variances. In addition, to compare the relative effectiveness of naltrexone on SSIA in mice given repeated swimming at two water temperatures and in controls, naltrexone antagonism was expressed as the percentage of a mean computed for the matching saline-injected subgroup. Since the magnitude of swim hypothermia to some extent depends on animals’ body size, hypothermic data were analyzed by a model of analysis of covariance (ANCOVA), with animals’ body mass as a covariate. The subjects in particular experiments were mice belonging to different families, therefore, we first analyzed the data with the appropriate models of ANCOVA and ANCOVA with family as a nested random factor. Since between-family variations of SSIA and swim hypothermia were found nonsignificant, we excluded the family factor from further analyses. Likewise, the data obtained from either sex were combined in all analyses, because prelim-
Fig. 1. Analgesia (A) and hypothermia (B) induced in cold-acclimated (5 °C) and unacclimated (control) HA mice by 3-min swimming in 20 °C water. In this figure and in Fig. 2, the analgesic and the hypothermic data, respectively, represent means from ANOVA and least square means from ANCOVA corrected for animal’s body mass ± S.E. * P < .05, * * P < .001 with respect to saline (planned contrasts).
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ðpostswim latency baseline latencyÞ : ð60 baseline latencyÞ
Swim hypothermia was quantified as a decrease in core temperature from baseline.
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group it did not differ, P=.06 between the mice given repeated swims in 20 and 32 °C water, planned contrasts. Mice exposed to repeated swimming displayed reduced swim hypothermia as compared to nonswimming controls, F(2,137) = 46.75, P < .0001, two-way ANCOVA. No difference was found between the two water temperatures, P=.65, planned contrasts (Fig. 2B). The effect of naltrexone was not significant, F(1,137) = 0.36, P=.53, two-way ANCOVA. Body mass (males: 33.1 ± 0.3 g, females: 27.4 ± 0.3 g) was significant as covariate, F(1,137) = 10.65, P < .01.
4. Discussion
Fig. 2. Effects of repeated swimming in 32 and 20 °C water on analgesia (A) and hypothermia (B) induced by 3-min swimming in 20 °C water. The naltrexone antagonism is also shown as percentage of the decrease in swim analgesia in naltrexone-injected mice, with respect to saline means taken as 100% (A—solid line). * Different from saline, P < .001 (planned contrasts); y different from controls, P < .001 (Duncan test).
one-treated mice was significant in two-way ANCOVA, F(1,67) = 5.25, P < .05, but not in separate, one-way ANCOVAs within unacclimated, P=.17, and cold-acclimated mice, P=.07. Body mass of males (35.8 ± 0.5 g) and females (30.2 ± 0.2 g) was nonsignificant as covariate, F(1,67) = 0.32, P=.58, two-way ANCOVA. 3.2. Effects of repeated swimming Both repeated swimming and naltrexone significantly attenuated SSIA, F(2,138) = 47.87 and F(1,138) = 92.27, respectively, P < .0001, two-way ANOVA. Planned contrasts comparisons confirmed the effectiveness of naltrexone in controls as well as in experimental groups, P < .0001 (Fig. 2A). Percent attenuation of SSIA by naltrexone was greater after repeated swimming than in nonswimming mice, F(2, 69) = 12.29, P < .0001, one-way ANOVA, and P < .001, Duncan test. No significant difference was found between the two water temperatures of repeated swimming, P=.22, Duncan test (Fig. 2A). Separate one-way ANOVAs revealed that repeated swimming significantly attenuated SSIA both in mice injected with saline, F(2,69) = 26.03, and with naltrexone, F(2,69) = 24.11, P < .0001. The magnitude of SSIA in mice exposed to repeated swims was significantly lower than in controls (saline-injected mice swimming at 20 °C: P < .0001; salineinjected mice swimming at 32 °C: P < .025; P < .0001 for both swim temperatures in naltrexone-injected mice, planned contrasts). Within the saline-injected group, the magnitude of SSIA differed, P < .001, and within the naltrexone-injected
The main finding of this study is that the repeated swimming not only decreased overall SSIA magnitude in mice expressing genetically determined high opioid– nonopioid stress analgesia, but also modified the sensitivity of SSIA to naltrexone. Namely, the nonopioid (i.e. naltrexoneresistant) portion of the overall SSIA was significantly reduced, whereas the opioid (naltrexone-sensitive) portion became relatively augmented. In accordance with the concept of collateral inhibition between the opioid and the nonopioid systems [14 – 20], we assume that the repeated swimming primarily caused a suppression of the nonopioid analgesic system, which in turn led to an alleviation of the inhibitory action tonically exerted by the latter on the opioid one. In contrast to repeated swimming, SSIA magnitude and its neurochemical nature remained unchanged following continuous 6-week exposure to ambient 5 °C. This comparison is important since the acclimation of HA mice to ambient cold substantially improves their thermogenic capacity. When exposed to cold Helox atmosphere, they manifest far-reduced hypothermia and about 50% higher peak metabolic rates than unacclimated controls do [21]. Overall, these results are consistent with the hypothesis that the observed reversal in proportion between opioid and
Fig. 3. Hypothetical interaction between the emergency and the hypothermic component of swim stress in the development of the opioid and the nonopioid form of SSIA in HA mice.
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nonopioid components of SSIA resulting from repeated swimming was associated with habituation to an extrathermal component of swim stress that may be emotional in nature, not with an increased rate of thermogenesis. Moreover, the proposed habituation, and not the repeated exposure to low swim temperature, might account for the attenuation of swim hypothermia as well. This claim is justified by the finding that neither the attenuation of nonopioid SSIA nor the decrease in swim hypothermia depended on water temperature of the repeated swims, and also that the hypothermia, as SSIA, was not affected by cold acclimation. Modulation of animals’ thermogenic capacity by emotional factors has been described by several authors. For example, the attenuation of hypothermia in rats given repeated immersions in cold water was disrupted by changing the environmental context [22,23] and was facilitated by electric stimulation in brain reward sites [24,25]. Particularly interesting is the rise of nonshivering thermogenesis in rats after repeated immobilization stress [26], which means that not only the exposure to cold, but also a habituation to nonthermic stressors can improve animals’ thermogenic capacity. In accordance with these observations, our results not only argue for a major role of a stress – emotional component of swimming in the elicitation of opioid and nonopioid SSIA, but also point to the effect of an extrathermal factor on thermoregulatory processes. A net result would be swim hypothermia consistently encountered in HA mice, sensitive to emotional stimuli, than in the less fearful LA mice [12]. Based on the results of our earlier work, we propose that for HA mice swimming in 20 °C water, SSIA develops faster than hypothermia and for 3 min is more susceptible to naloxone than after 5 min of swimming (cf. Fig. 4C in Ref. [7]). It is then likely that the emergency component of swim stress, in connection with moderate hypothermia, initially produces the opioid form of SSIA. Subsequently, as swimming continues, this primary analgesic mechanism affects the thermoregulatory processes maintaining thermal homeostasis and further increases the stressing property of swim hypothermia. This ultimately induces the nonopioid form of SSIA. Thus, a nonopioid SSIA is particularly likely to develop in HA mice swimming in cold water or for a long duration (that is, when pronounced hypothermia develops). This is well supported by the results of the present as well as our previous studies showing that a profound swim hypothermia is not sensitive to naltrexone or naloxone [7,8]. As the hypothermia increases, the nonopioid component of SSIA begins to prevail and damps the opioid one. A graphical illustration of our hypothesis is presented in Fig. 3. Summing up, we conclude that the complex nature of opioid – nonopioid SSIA in the mouse stems from an integrated response of the animal to an extrathermal (i.e. emergency) and a thermal (i.e. hypothermic) component of swim stress.
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Acknowledgements The authors thank two anonymous reviewers for their effort involved in the improvement of the manuscript. We also thank I. Gasiorkiewicz and B. Sobolewska for technical assistance. This study was supported by Polish Committee for Scientific Research (KBN) Grants No. 6PO4C01616 and No. 6PO4C01818 to B. Sadowski.
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