Printed in Great Britain. All rights reserved 0306-3623/94 $7.00 + 0.00
Gender and Test Dependence of a Type of Kappa Mediated Stress Induced Analgesia
in Mice L U I S M E N E N D E Z , I* F E R N A N D O A N D R I ~ S - T R E L L E S , 2
A G U S T I N H I D A L G O J a n d A N A B A A M O N D E 1
ILaboratorio de Farmacologia, Departamento de Medicina, Facultad de Medicina, Universidad de Oviedo, Julidn Claverla s/n, 33071 Oviedo, Asturias, Spain [TeL 34-8-5103551; Fax 34-8-5232255] and
2Departamento de Farmacologia, Facultad de Medicina, Universidad Complutense de Madrid, 28040 Madrid, Spain
(Received 28 December 1993)
Abstract--1. In male mice, 80 inescapable footshocks (S-80) induce analgesic responses measured by the tail flick test that are blocked by naloxone and the kappa opioid antagonist, nor-binaltorphimine. We now study the nociceptive responses, induced after this particular stress, measured by the writhing test, the tail immersion test and a high intensity tail immersion test both in male and female mice.
2. In stressed males, analgesic responses are seen in all the nociceptive tests. Naloxone (10 mg/kg) does not prevent them.
3. In stressed females, in contrast with males, no analgesia is produced in the tail flick test. The writhing test and the tail immersion test registered analgesic responses that were not prevented by naloxone ( 10 mg/kg).
4. We conclude that only the antinociceptive kappa opioid mediated component of the stress we study is strongly dependent on gender, in contrast to other types of analgesia triggered by the same stress.
I N T R O D U C T I O N
It is well known that the response to stress includes
a decrease in the sensitivity to painful stimuli (stress
induced analgesia, SIA) which can often be related
to endogenous opioid mobilization and, thus, can be
prevented by naloxone. However not all the analgesic
responses to stress seem to depend on opioids and
some types of SIA resistant to naloxone have been
described (Lewis et al., 1980). The nature of SIA has
been related mainly to the type of stress (Watkins and
Mayer, 1982), and it has been shown that closely
related types (such as several forms of footshock) can
differ in the opioid or non opioid nature of the
analgesia they produce (Drugan et al., 1982; Tierney
et al., 1991; Menendez et al., 1993a). The responses
are also dependent on the analgesic test used and on physiological variables such as age, sex and even
"emot iona l" factors (Girardot and Holloway, 1985;
*To whom all correspondence should be addressed.
Bodnar et al., 1988; Baamonde et al., 1989). How-
ever, although there is a considerable amount of
literature on the subject, it is difficult to outline a
clear pattern. This might be partially due to the fact
that in many cases the specific opioid receptors
involved in the analgesic responses to stressors are
analgesic responses when measured by the tail flick
test and in male mice, in which spinal kappa opioid
receptors are clearly implicated (Menendez et al.,
1993b), although, as then discussed, gender or the
nociceptive test used could be crucial in its pro-
duction. In order to assess how these responses,
well-defined in terms of opioid receptor type, vary
with the nociceptive stimulus used and the sex of the
animals, we now compare them and the analgesic
responses to such stress when measured by other, currently used, testing methods both in male and
female mice.
903
904 Luls MENENDEZ et al.
MATERIALS AND METHODS
Animals
Male and female Swiss mice (Interfauna Iberica, Spain), weighing 28-32 g were used. The animals were housed in groups of twenty, exposed to a light-dark cycle of 12hr and had water and food available ad libitum. Each animal was used only once.
Footshock stress
Eighty inescapable and intermittent shocks (1 sec, 1.5 mA, 0.2 Hz) (S-80) were applied through a metal wire mesh surrounded by transparent plastic walls (8 × 8 × 5.5 cm) for 6 min 40 sec. A Letica LI2706 shock unit was used. Control animals were placed in the same environment for the same length of time but no shocks were applied to these (sham-stress procedure).
Antinociceptive assays
Tail-flick test (TFT). Based on the previously described method (D'Amour and Smith, 1941), at time 0, the mean of three consecutive measures (at 1 min intervals) was calculated (basal latency). Initially, the intensity of the thermal stimulus was adjusted in order to obtain a latency of 4 _+ 1 sec, and the same intensity was used for male and female mice. 23 min later, stress (corresponding to 80 shocks) or sham stress was applied (from time 23 until time 29 rain 40 sec) and immediately after, a second TFT measure was performed (experimental latency). To avoid tissue damage, a cut-off time of 10 sec was set. Trials were automat- ically terminated if a response did not occur within 10 sec.
High intensity tail-flick test (HITFT). In this test the intensity of the thermal stimulus was increased in order to obtain a basal latency of about 2 sec. To avoid tissue damage, the cut-off time was set at 6 sec and only two measures were made in each trial. The rest of the experimental procedure was the same as described for the TFT. This was only performed in males.
Tail-immersion test (TIT) . The method was similar to that described by Grotto and Sulman (1967). The noxious stimulus used in this test was water thermo- stathized at 52°C. Mice were manually restrained, their tails completely dipped in a water bath and the latencies to obtain a rapid tail movement (both basal and experimental as before) were measured by using a stopwatch. The value of the basal latencies was about 2.5 sec and the cut-off value was 6.5 sec. Only one measure of the tail-flick latency was made in each case.
Writhing test (WT). In this test, the number of abdominal contractions were counted for 20min after acetic acid injection (Koster et al., 1959). Acetic acid 1% (10 ml/kg) was administered i.p. just after the stress or sham-stress. This test can be made only once in each mouse and, thus, there is no comparison between basal and experimental scores, only between those of different groups of mice either stressed or sham stressed.
Drugs
Drugs were dissolved in 0.2 ml saline. Naloxone hydrochioride (Abell6, Spain) was administered subcutaneously (10 mg/kg) 20 min before the exper- imental TFT, TIT, HITFT or the writhing test. Nor-Binaltorphimine (RBI) was administered i.p. (4mg/kg) 30min before the experimental TFT in males. Control animals received, instead, the same volume of saline.
Statistical analysis
The Student's t-test for paired data was used to compare basal and experimental latencies obtained by the TFT, HITFT and TIT. In the WT, a Student's t-test for unpaired data was used to compare sham-stressed and stressed groups treated with the same drug. The level of significance was set at P < 0.05.
RESULTS
Analgesic responses in male mice
1. Tailflick test. As Fig. I(A) shows, the stress we use (80 footshocks, S-80) increases the latency of the tail-flick when measured by the tail-flick test immedi- ately after the end of the footshocks in male mice. As we have previously described in males (Menendez et al., 1993b), the analgesia so induced is prevented by naloxone (10 mg/kg s.c. 20 rain before) and nor- Binaltorphimine (4 mg/kg i.p. 30 rain before).
2. Writhing test. A lower number of writhings was obtained in male mice subjected to S-80 as compared to those unstressed (Fig. IB). These anal- gesic responses were not prevented by naloxone (10 mg/kg).
3. Tail immersion test. Male mice tested by the TIT after S-80 (Fig. 1C), showed analgesic responses not prevented by naloxone (10 mg/kg). Because the TIT and the TFT mainly differ in their basal iatencies (a higher latency in the TFT), we have tested the effect of this stress by using a modified TFT, i.e. a high intensity TFT (HITFT). In this test, the intensity was adjusted to obtain basal latencies of about the same value (2 sec) as those obtained in the TIT (2.5 sec). Also in this ease, male mice showed anal-
Gender, testing and stress analgesia
A) TAIL-FLICK 10
i i l~ l BASAL * * I l l EXPERIMENTAl. 50"
4O
( ~ 3 0 .
nili '
2" 10.
B) WRITHING TEST
-t
1
i
+ + 0 0
STRESS _ _ + + STRESS
NALOXONE - 10 -- NALOXONE 10 - 10 10 4 N-BNI 4
C) TAIL-IMMERSION D) HIGH INTENSITY TAIL-FLICK
r=.,SAL ** ! BA ,L
0 - -
STRESS - + + NALOXONE I0
NALOXONE 10 - I0
Fig. 1. Pain reactions measured in male mice either stressed (80 inescapable footshocks) or unstressed by using different nociceptive tests. The mean of the latencies (in seconds) to the flick of the tail (in A, C and D) or of the number of writhings (in B) + their SEM is represented for n = 8--10 data. When indicated, naloxone (10 mg/kg s.c.) and nor-binaltorphimine (n-BNI) (4 mg/kg i.p.) were used either 20 min or 30 min before, respectively. In A, C and D, comparisons are made between basal (first measure) and experimental (second measure, post stress or sham-stress) procedures; in B, between stressed and sham-stressed mice.
The level of significance was set at P < 0.05.
905
gesic responses that were not modified by naloxone (10 mg/kg) (Fig. ID).
Analgesic responses in female mice
In contrast to males, female mice did not show significant analgesic responses in the TFT after the stress (Fig. 2A). However, they showed analgesic responses both in the WT and the TIT, that were, as in the case of males, naloxone-insensitive (Figs 2B and C).
DISCUSSION
We have previously shown that this 80-shock stress induces analgesia as measured by the tail flick test in male mice. This analgesia is prevented by high doses
of naloxone (10 mg/kg s.c.) and by the kappa antag- onist nor-binaltorphimine both systemically and intrathecally administered and, therefore, spinal kappa opioid receptors are clearly involved in its production (Menendez et al., 1993b).
We now describe in detail that in male mice analgesic responses are also obtained using the same stress paradigm when noxious reactions are provoked by a different kind of thermal stimulus (tail immer- sion) or by a chemical stimulus (writhing test). In contrast with the results obtained in the TFT, these antinociceptive responses do not seem mediated by opioids because they are not prevented by prior administration of high doses of naloxone (10 mg/kg, enough to block all mu, delta and kappa opioid receptors).
906 Luis MENENDEZ et al.
A) TAIL-FLICK 10
E:::3 BASAL
EXPERIMENTAL
" , - - / 6 ' T
C)
2.
0
STRESS - -
NALOXONE I0
1ill + +
10
5 0
4 0
3 0
20
1 0
0
S T R E S S
N A L O X O N E
B) WRITHING TEST
10 -- 10
C) TAIL-IMMERSION
g
BASAL
5 . I l l EXPERIMENTAL
4
3
1
l o
0
STRESS NALOXONE
11 4- +
- 1 0
Fig. 2. Pain reactions measured in female mice either stressed (80 inescapable footshocks) or unstressed by using different nociceptive tests. The mean of the latencies (in seconds) to the flick of the tail (in A and C) or of the number ofwrithings (in B) + their SEM is represented for n = 8-10 data. When indicated, naloxone was used at a dose of 10 mg/kg s.c. 20 min before. In A and C, comparisons are made between basal (first measure) and experimental (second measure, post stress or sham-stress) procedures; in B,
between stressed and sham-stressed mice. The level of significance was set at P < 0.05.
It is well known that a thermal noxious stimulus in the tail (as in the TFT) affects nociceptive pathways different from those affected by a chemical stimulus administered i.p. (as in the writhing test), as analgesic drugs either opioid or non opioid show different potencies in the various tests (review in Franklin and Abbott , 1989). Although in some cases the results obtained with the analgesia induced by stress using different nociceptive tests are uniform (Tierney et al. ,
1991), it is not unusual to find different results when responses to stress are studied in relation to the analgesic method used (Kelly et al. , 1982). For example, it has been described that the same subject under a unique stress can display either analgesic or hyperalgesic responses depending on the testing method (Kelly et al. , 1982).
It is more striking that two very similar thermal stimuli (tail flick vs tail immersion; the tail immersion test is considered a common variation of the radiant heat tail flick) might elicit analgesic responses affected or not by naloxone in each case (only the T F T is naloxone-sensitive). The apparently greater difference between these tests is that the basal iatencies are longer in the tail flick test (4 + 1 sec) than in the tail immersion test (2.5 sec), this probably being due to the different heat intensities involved. This difference can be relevant considering the fact that, as pre- viously described (Menendez et al. , 1993b), kappa receptors are the opioid receptors responsible for the analgesic responses induced by S-80 in the tail flick test as it has been shown that no (or lower) analgesic responses can be induced by kappa agonists when
Gender, testing and stress analgesia 907
short basal latencies (high thermal intensity) are used in the tail flick test (Millan, 1989). Therefore, we tested the stressed mice with a tail flick adjusted to obtain shorter basal latencies. With this method (HITFT), mice behave as in the tail immersion test, that is, they show analgesic responses that are not affected by naloxone. On the one hand, this result is in accordance with the lack of kappa-opioid mediated responses when short basal iatencies are used and supports the idea that the knowledge of the particular receptors involved in SIA allows us to understand responses to stress under some conditions. On the other hand, it is confusing that not only the opioid response disappears, but a non opioid mechanism comes into play and induces analgesic responses and so it has to be presumed that complex interactions among several pain modulating systems might take place.
We also studied the influence of sex. Female mice, similar to male mice, showed analgesic reactions measured by the TIT or the WT, thus no gender differences were seen in these non opioid responses. However, in the TFT, in which males showed anal- gesia sensitive to naloxone, females showed no anal- gesic responses at all. In consequence, the only differ- ence related to sex that we have detected corresponds to the kappa-opioid analgesia.
It has been reported that males can show different analgesic responses than females to morphine (Beatty and Beatty, 1970; Chatterjee et al., 1982; Kepler et al.,
1989) and/or to stress (Romero and Bodnar, 1986; Wong, 1987; Ryan and Maier, 1988; Bodnar et al.,
1988; Baamonde et al., 1989; Mogil et al., 1993). Although few data relate these variations to specific opioid receptors, some reports show that the stimu- lation of spinal kappa opioid receptors can be depen- dent on hormonal factors. Pregnant rats have higher nociceptive thresholds than non pregnant ones, and spinal kappa opioid receptors have been shown to be mainly involved in these nociceptive differences (Sander et al., 1988, 1989). Furthermore, Ryan and Maier (1988) have demonstrated the dependence on hormonal factors in a type of opioid stress-induced analgesia in rats (80-100 tailshocks) but not in a similar non opioid one (10-20 tailshocks). In their experiments, diestrus females exhibited the same pattern of analgesia as males, the non-opioid anal- gesia was slightly attenuated during estrus but the opioid analgesia was markedly reduced. Ovariectomy only slightly altered non opioid analgesia but elimi- nated opioid analgesia. More recently, Watkins et al.
(1992a), have demonstrated the involvement of spinal kappa opioid receptors in the hormone-modulated analgesia in male rats in the above study (Ryan and Maier, 1988). These latter results are in accordance
with ours in that they establish a relationship between the stimulation by stress of spinal kappa receptors and hormonal factors. However, in our case, the lack of responses of female mice in the TFT were un- affected by the estrus stage (estrus vs diestrus) and the different responses in males and females remained unaltered after gonadectomy (Menendez L., PhD Thesis, University of Oviedo, Spain 1992, unpub- lished results), and, thus, we can not identify gonadal hormones as the gender dependent factor modulating the impact of stressors on the endogenous kappa mediated pain inhibition we study.
A final general point is worth raising. As we have just discussed, the analgesic response to 80 footshock stress in which spinal kappa opioid receptors partici- pate, is seen when using some methods of nociceptive testing but not others, and shows remarkable gender differences. This is not the case with other analgesic responses induced by the same stress and detected by other methods of nociceptive testing. It is thus very likely that stressors can simultaneously trigger a variety of responses some of which are mediated by opioids (conceivably acting through different receptors) and some not. In consequence, these exper- iments, as well as others, such as the recent ones by Watkins et al. (1992b) who have reported that some stress-induced analgesias previously considered as non opioid appear in fact to cause a parallel acti- vation of multiple opioid systems, further stress that a classifying criterion of the analgesia inducing stresses as, simply opioid or non opioid, is inadequate and should not be used.
Acknowledgements--This work was supported by a grant from the Spanish Fondo de Investigaciones de la Seguridad Social (FIS 92/0896) and the Universidad de Oviedo (DF 92/65).
REFERENCES
Baamonde A. I., Hidalgo A. and Andres-Trelles F. (1989) Sex related differences in the effects of morphine and stress on visceral pain. Neuropharmacology 28, 967-970.
Beatty W. W. and Beatty P. A. (1970) Hormonal determi- nants of sex differences in avoidance behavior and reactiv- ity to electric shock in the rat. J. Comp. Physiol. Psychol. 73, 446-455.
Bodnar R. J., Romero M. T. and Kramer E. (1988) Organismic variables and pain inhibition: roles of gender and aging. Brain Res. Bull. 21, 947-953.
Chatterjee T. K., Das S., Banerjee P. and Ghosh J. J. (1982) Possible physiological role of adrenal and gonadal steroids in morphine analgesia. Eur. J. Pharmacol. 77, 119-121.
D'Amour F. E. and Smith D. L. (1941) A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74-79.
Drugan R. C., Moye T. B. and Maier S. F. (1982) Opioid and nonopioid forms of stress-induced analgesia: some environmental determinants and characteristics. Behav. Neural Biol. 35, 251-264.
908 Luls MENENDEZ et al.
Franklin K. B. J. and Abott F. V. (1989) Techniques for assessing the effects of drugs on nociceptive responses. In Psychopharmacology (Edited by Boulton A. A., Baker G. B. and Greenshaw A. J.), pp. 145-211. Humana Press, Clifton, New Jersey.
Girardot M. N. and Holloway F. A. (1985) Chronic stress, aging and morphine analgesia: chronic stress affects the reactivity to morphine in young mature but not old rats. J. PharrnacoL Exp. Ther, 233, 545-553.
Grotto M. and Sulman F. G. (1967) A modified receptacle method for animal analgesimetry. Arch. Int. Pharma- codyn. 165, 152-159.
Kelly D. D., Lemaire P. A. and Leitner D. J. (1982) Hyperalgesia and analgesia induced by the same stressor. Soc. Neurosci. Abstr. 8, 511.
Kepler K. L., Kest B., Kiefel J. M., Cooper M. L. and Bodnar R. J. (1989) Roles of gender, gonadectomy and estrous phase in the analgesic effects of intracerebro- ventricular morphine in rats. Pharmacol. Biochem. Behav. 34, 119-127.
Koster R., Anderson M. and DeBeer E. J. (1959) Acetic acid for analgesic screening. Fed. Proc. 18, 412.
Lewis J. W., Cannon J. T. and Liebeskind J. C. (1980) Opioid and non opioid mechanisms of stress analgesia. Science 208, 623~25.
Menendez L., Andres-Trelles F., Hidalgo A. and Baamonde A. (1993a) Opioid footshock-induced analgesia in mice acutely falls by stress prolongation, Physiol. Behav. 53, 1115-1119.
Menendez L., Andres-Trelles F., Hidalgo A. and Baamonde A. (1993b) Involvement of spinal kappa opioid receptors in a type of footshock induced analgesia in mice. Brain Res. 611, 264-271.
Millan M. J. (1989) Kappa-opioid receptor-mediated anti- nociception in the rat. I. Comparative actions of mu- and kappa-opioids against noxious thermal, pressure and electrical stimuli. J. Pharmacol. Exp. Ther. 251, 334-341.
Mogil J. S., Sternberg W. F., Kest B., Przemyslaw M. and Liebeskind J. C. (1993) Sex differences in the antagonism of swim stress-induced analgesia: effects of gonadectomy and estrogen replacement. Pain 53, 17-25.
Romero M. T. and Bodnar R. J. (1986) Gender differences in two forms of cold-water swim analgesia. Physiol. Behav. 37, 893-897.
Ryan S. and Maier S. F. (1988) The estrous cycle and estrogen modulate stress-induced analgesia. Behav. Neurosci. 102, 371-380.
Sander H. W., Portoghese P. S. and Gintzler A. R. (1988) Spinal x-opiate receptor involvement in the analgesia of pregnancy: effects of intrathecal nor-binaltorphimine, a ~c-selective antagonist. Brain Res. 474, 343-347.
Sander H. W., Kream R. M. and Gintzler A. R. (1989) Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera. Eur. J. Pharmacol. 159, 205-209.
Tierney G., Carmody J. and Jamieson D. (1991) Stress analgesia: the opioid analgesia of long swims suppresses the non-opioid analgesia induced by short swims in mice. Pain 46, 89-95.
Watkins L. R. and Mayer D. J. (1982) Organization of endogenous opiate and nonopiate pain control systems. Science 216, 1185-1192.
Watkins L. R., Wiertelak E. P. and Maier S. F. (1992a) Kappa opiate receptors mediate tail-shock induced anti- nociception at spinal levels. Brain Res. 582, 1-9.
Watkins L. R., Wiertelak E. P., Grisel J. E., Silbert L. H. and Maier S. F. (1992b) Parallel activation of multiple spinal opiate systems appears to mediate "non-opiate" stress-induced analgesias. Brain Res. 594, 99-108.
Wong C. L. (1987) The effect of gonadectomy on antinoci- ception induced in mice by swim stress. Eur. J. Pharmacol. 142, 159-161.
