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Mating produced analgesia in the Syrian hamster
Physiology& Behavior,Vol. 38, pp. 601--606.Copyright©PergamonJournals Ltd., 1986.Printed in the U.S.A.
0031-9384/86$3.00 + .00
Mating Produced Analgesia in the Syrian Hamster S A R A E. C R U Z - M O R A L E S * f A N D R. G. NOBLE:~ 1
*Autonomous National University o f Mexico (UNAM) fEscuela National de Estudios Professionales-Iztacala, Mexico 5;Department o f Psychology, Rensselaer Polytechnic Institute, Troy, N Y 12180 Received 14 April 1986 CRUZ-MORALES, S. E. AND R. G. NOBLE. Mating produced analgesia in the Syrian hamster. PHYSIOL BEHAV 38(5) 601-606, 1986.--A series of experiments examined the hypothesis that mating produced analgesia in the Syrian hamster can be blocked by opiate antagonists. The first experiment established a dose response relation to painful footshock. Females were more responsive to footshock than males (p<0.001). The second experiment demonstrated that ten minutes of mating reduced responsiveness of both males and females to a moderate intensity of footshock. The third experiment replicated the effect of mating on the responsiveness of males to footshock, but did not demonstrate any effect of the opiate antagonist naltrexone on mating induced analgesia. The fourth experiment demonstrated that limited mating stimulation (five intromissions) has little effect on the responsiveness of sexually inexperienced males to footshock. The fifth experiment replicated the fourth experiment using sexually experienced males and obtained comparable results. Apparently mating induced analgesia in male hamsters is not blocked by opiate antagonists and which raises questions about biological activity of reported changes in endorphin activity during mating. Mating
Analgesia
Opiateantagonists
SEVERAL lines of evidence suggest a relationship between the endogenous opioid system and reproductive behavior. Historically, opiate use has been associated with impaired sexual performance [7,12]. More recently, laboratory studies have demonstrated that the acute administration of opiates or endogenous opioids selectively inhibits male copulatory performance in rats and hamsters at doses which do not affect general activity [10--12, 20]. The administration of opiate antagonists has been reported to facilitate copulatory behavior in sexually active rats [5] and to affect the copulatory performance of sexually active male rats [10, 14, 15], although these effects are not consistently obtained [9, 23, 25]. Mating produced increases in systematic levels of beta endorphin like immunoreactive material in male hamsters have been reported [13], an observation consistent with the proposed relation between the endogenous opioid system and mating behavior. The relation between reported increases in systemic levels of immunoreactive beta-endorphin and mating behavior is not clear. It is not certain that beta endorphin in the peripheral circulation enters the central nervous system in biologically significant amounts [22] and it has been reported that stimulation produced changes in systemic endorphin levels are frequently lower than the levels produced by minimally effective doses of beta endorphin administered systemically [2]. Since beta endorphin is a potent, centrally acting analgesic, it is possible that mating produced increases in systemic endorphin levels [13] reaching the brain in physiologically
significant amounts would produce analgesia. Mating has been reported to reduce responsiveness to nociceptive stimulation in rats [24] and hamsters [4] but it is not known whether this mating produced reduction in responsiveness is blocked by opiate antagonists. The present series of experiments was designed to evaluate the hypothesis that systemic increases in beta endorphin could be responsible for mating induced analgesia by determining if analgesia was present at different times during mating when systemic levels are high [13] and by determining if mating induced analgesia is blocked by opiate antagonists. We adopted this approach to evaluating the physiological significance of mating produced increases in beta endorphin because analgesia produced by exogenously administered beta endorphin is reversed or blocked by opiate antagonists [1] and the physiological effects of very large doses of beta endorphin on hamsters are completely reversed by opiate antagonists [26]. EXPERIMENT ONE The first experiment evaluated the suitability of electric footshock as a source of nociceptive stimulation. It also provided the basis for selecting the intermediate level of shock intensity used in subsequent studies. The selection of intermediate shock intensity was intended to maximize the likelihood that both increases and decreases in responsiveness to footshock could be detected.
1Requests for reprints should be addressed to Ralph G. Noble.
601
C R U Z - M O R A L E S A N D NOBLE
6O2
FEMALES U4 7 I MALES 6 ~
c~--o o,-
=
=
<~
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FEMALE
(3
4~
MALE
)~J Z UJ F-
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2=,
< On
08
t
SHOCK
13
16
20
m
MATED
UNMATED
MATED
UNMATED
{MA) O' POST
FIG. 1. The relationship between the shock intensity and the incidence of paw shake.
METHOD
Subjects
MATING
30'
POST
MATING
FIG. 2. The effect of mating on latency to paw shake.
chamber for 20-30 sec while the latencies to paw shake and to escape were recorded, and a new trial was started immediately after the measures were recorded.
Thirty-eight adult (60-90 days old) Syrian hamsters
(Mesocricetus Auratus), purchased from Engle's Laboratory Animals Inc., were used in this experiment. Females had been ovariectomized under sodium pentobarbital anesthesia and implanted subcutaneously with a capsule containing 17 Beta-Estradiol [16] at least 30 days before the experiment and injected with progesterone (0.5 mg in 0.1 ml corn oil, SC) four hours before testing to produce behavioral recepting. The subjects were housed in solid bottom plastic cages (30×33× 17 cm), with four animals of the same sex per cage. The colony room was maintained at 22°C on a reversed light-dark cycle (L:D 14:10) with the onset of the dark phase of the cycle at 1400. Purina rat chow and water were continually available in the home cages.
Design Each subject was randomly assigned to one of five groups with at least four males and three females in each group. Each group received a different nonimal intensity of footshock: 0.08, 0.1, 0.13, 0.16 or 0.20 mA.
Apparatus
RESULTS Because paw shake responses were not consistently observed at all current intensities, the number of trials during which the paw shake response occurred was used as the dependent variable. Both males and females showed an increase in the number of trials during which the paw shake response was observed as a function of shock intensity, F(4,28)=28.69, p<0.001, and females were more responsive than males, F(1,28)=15.07, p<0.001, particularly to low intensities [sex by intensity interaction, F(4,28)=4.99, p<0.004, see Fig. 1]. The escape latency decreased with "increasing shock intensities, F(7,196)=19.63, p<0.001, and escape latencies were larger for males, F(7,28)=3.02, p=0.09, especially at lower intensities [intensity × sex interaction, F(7,196)=3.47, p<0.002].
E X P E R I M E N T TWO
The test chamber was a 51/2 gal glass aquarium. The inner walls of the chamber were covered with wire mesh (1.25×1.25 cm). The floor of the test chamber was constructed from an etched copperplate and connected to a Grason-Stadler Co. shock generator.
The second experiment replicated a previous experiment [4], by examining the effects of ten minutes of mating on the response of male and female hamsters to nociceptive stimulation provided by electric footshock.
Procedure
Subjects and Design
The subjects received two sessions on consecutive days, with four trials in each session. At the beginning of each trial the animal was placed in the center of the chamber and footshock was delivered for 180 seconds or until the subject escaped by climbing onto the hardware cloth. During shock administration, subjects observed a paw shake response [4] and the latency to the first paw shake response was recorded. The animal was scored as having escaped as soon as all four paws were in contact with the hardware cloth. After each trial the subjects were kept in a separate holding
The subjects were 39 sexually experienced hamsters, 21 intact females and 18 males, from 60 to 90 days of age. All females were tested on the day of estrus. Animals were assigned to four groups: The animals in group 1 (11 females) and group 2 (9 males) were permitted to mate with stimulus animals, while subjects of group 3 (10 females) and group 4 (9 males) were placed in the mating chamber alone. Sexually active males and ovariectomized females in hormone induced receptivity were used as stimulus animals in the mating tests.
METHOD
MATING AND ANALGESIA
603
Procedure Mating session. Each subject was placed in the mating chamber (a 51/2 gal all glass aquarium) for ten minutes before testing. Then a stimulus animal of the appropriate sex was introduced. The animals were allowed to mate without interruption for nine minutes after the first intromission. Mating was then terminated after the next ejaculation. Animals were removed from the mating chamber within ten seconds after the ejaculation and tested for analgesia. The animals in the control groups were placed in the test chamber alone for ten minutes and then tested for analgesia. The subject was placed in the center of the test chamber within five seconds after being removed from the mating chamber and a shock of 0.13 mA DC was delivered for 180 seconds or until it escaped. All animals were tested again 30 minutes later.
18= LU CO
m Z~m
SALINE
LU v "ICO
12
10,
O p-
8 6
Z W
4,
,-I
2 0 MATED
O'POST
RESULTS
NALTREXONE
16=
UNMATED MATING
MATED
UNMATED
30' POST MATING
FIG. 3. The effect of mating and naltrexone on latency to paw shake.
Latency to Paw Shake Mating increased the latency to paw shake in both males and females, F(1,35)=7.23, p<0.01. Paw shake latency decreased significantly with repeated testing, F(1,35)=9.5, p<0.004. As shown in Fig. 2, there was a large effect immediately post mating but there were no significant differences among treatment groups thirty minutes post mating.
Latency to Escape Mating increased latency to escape in both males and females (see Fig. 3), [main effect of mating, F(1,35)=8.92, p<0.005], and escape latency decreased with repeated testing, F(1,35)=6.01, p<0.001. Thirty minutes post mating, there were no significant differences among groups.
DISCUSSION
Procedure Each male was weighed, then placed in the mating chamber for ten minutes before the female was introduced. After five minutes in the mating chamber it was injected and put back in the chamber. The male was permitted to achieve five ejaculations. The number of mounts, number of intromissions and the time required to achieve five ejaculations were recorded. The males in the unmated groups were kept in the test chamber for an additional ten minutes. Analgesia test. Within five seconds after being removed from the mating chamber the male was put into the test chamber and a 0.13 mA DC footshock was delivered for 180 seconds or until the subject escaped. Thirty minutes later, the analgesia test was repeated.
The results extend and confirm the previous [4] demonstration of transient mating produced analgesia in males and females. The effect of mating was much more pronounced in the present study.
EXPERIMENT THREE This experiment was designed to determine whether mating produced analgesia be blocked by an opiate antagonist. Naltrexone was selected for its potency as an opiate antagonist and its long lasting effects in rats and in hamsters [19,26]. METHOD
Subjects and Design Forty sexually experienced male hamsters, 5-6 months old, were randomly assigned to four groups (n/group= 10). Males in two groups were mated and males in the other two groups were placed in the mating chamber alone. Animals in one of the mated groups and in one of the unmated groups were injected with naltrexone (10 mg/kg, in physiological saline SC) and animals in the other groups were injected with an equivalent volume of physiological saline (1 ml/kg, SC).
RESULTS One subject which did not achieve five ejaculations within 15 minutes was deleted from the experiment and its scores were not included in the data analyses. A two way ANOVA for repeated measures was used to evaluate the data,
Paw Shake Latency Mating increased paw shake latencies, F(1,35)=25.36, p <0.001, but naltrexone administration did not produce any significant effects and none of the interactions with naltrexone administration were significant. Thirty minutes post mating the mated group's average latency to paw shake had decreased and was not different from the mean paw shake latency of the unmated group which did not decrease. This produced both a significant trials effect, F(1,35)=28.37, p<0.001, and a significant trials x group interaction, F(1,35)=29.31, p<0.001 (see Fig. 3).
Latency to Escape The mated groups showed longer latencies to escape than animals in the unmated groups immediately after mating, but
604
CRUZ-MORALES AND NOBLE METHOD 18
t.u o3
16
LU v' < 2; O3
14
Sal~iects
NALTREXONE
Thirty-nine sexually inexperienced male hamsters, 60 days old, were assigned at random to four groups. Males in groups one and two were permitted five intromissions with a stimulus female five minutes after being injected with either naltrexone (10 mg/kg, SC, group 1) or physiological saline in equivalent volume (group 2). Males in groups three and four were placed in the empty mating chamber for two minutes, five minutes after being injected with either naltrexone (10 mg/kg, SC, group 3) or physiological saline (group 4). The procedures were otherwise the same as in experiment three.
SALINE
12
lO o_ o
8
t3 z
w
4
_J
o MATED O' P O S T M A T I N G
MATED 3 0 ' POST M A T I N G
FIG. 4. The effect of five intromissions and naltrexone on latency to paw shake.
there were no significant differences among groups thirty minutes post mating which produced a significant mating by trials interactions, F(1,35)=23.56, p<0.001. There was an overall decrease in the latency to escape with repeated testing, F(1,35) = 200, p <0.001, but there was no significant main effect of naltrexone administration and none of the interactions with naltrexone administration were significant.
Male Behavior
The group that received naltrexone displayed fewer mounts before achieving five intromissions (mean_+ S.E.M.=6.0_+l.25) than the group injected with saline [mean+ S.E.M. = 13.7-+3.1; t(18)=2.3, p<0.05]. Naltrexone reduced the number of intromissions required to achieve five ejaculations (mean-+S.E.M. = 16.7-+ 1.1) compared with the saline group [mean-+S.E.M. =23.5_+2.9; t(18)=2.3, p<0.05].
DISCUSSION
The effects of naltrexone administration on mating behavior in the present experiment were comparable to effects obtained previously [17]. The effects of mating on paw shake latency were much more pronounced in the present experiment than in experiment two, and the difference is quite specific to paw shake latency immediately postmating. The only systematic difference between the two experiments was that both males and females were tested in the shock apparatus in experiment two. It is possible that the presence of female odor in the test chamber in experiment two attenuated the effect of mating on paw shake latency.
EXPERIMENT FOUR This experiment was designed to find out if five intromissions produce analgesia which is antagonized by naltrexone. Apparently five intromissions produces maximal increases in systemic levels of beta endorphin [13].
RESULTS
Latency to Paw Shake
Five intromissions produced a small increase in paw shake latency immediately after mating but there were no differences in paw shake latency 30 min post mating which produced a significant mating by trials interaction, F(1,35)=6.65, p<0.01. There were no other significant main effects or significant interactions (see Fig. 4). Latency to Escape
There were no significant effects of mating on escape latency. Naltrexone administration increased the escape latencies of both mated and unmated males immediately after mating but not thirty minutes after mating. This produced both a significant main effect of naltrexone, F(1,35)=7.40, p<0.01, and a significant trials by naltrexone interaction, F(1,35)=6.18, p<0.01). In all groups the latency to escape decreased with repeated testing trials, F(1,35)=57.48, p <0.001. DISCUSSION
Since the initial increase in escape latency was not associated with an increase in paw shake latency it seems unlikely that naltrexone had agonist effects as has been sometimes hypothesized [26]. It seems more probable that the naltrexone administration produced a general hyperactivity to arousing stimuli which interfered with complex motor functioning [21]. It is possible that sexually inexperienced males having been handled less and having less exposure to the testing procedures were more reactive initially and the naltrexone exaggerated this reactivity. Because naltrexone had an unexpected effect on sexually inexperienced males, we examined the effects of five intromissions on the response of sexually experienced males to nociceptive stimulation. Ten sexually experienced males were allowed to achieve five intromissions, while ten males were placed in the mating chamber alone for two minutes. Five intromissions had no significant effects on responsiveness to nociceptive stimulation, which in general is consistent with the minimal effects of five intromissions on the responsiveness of sexually inexperienced males to nociceptive stimulation.
GENERAL DISCUSSION Our results support the conclusion that mating produced
MATING AND ANALGESIA analgesia is not mediated by activation o f the endorphin system, and that the mating produced increase in endorphin like immunoreactive material reported by Murphy [13] is insufficient to produce analgesia. We base this conclusion on the observation that mating produced analgesia is not reduced by an amount of naltrexone sufficient to antagonize the effects of large amounts of exogenously administered beta endorphin [25] on hamsters. Mating produced reductions in responsiveness to nociceptive stimulation in male hamsters strongly resembles the analgesia produced by mechanisms which are neural and non-opioid according to the classification of Watkins and Mayer [27]. The available evidence strongly suggests that forms of stimulus produced analgesia which involve activation of the endogenous opioid system are blocked, at least partially by opiate antagonists in the doses used [2,27]. The relatively short duration of mating produced analgesia is consistent with the duration produced of analgesia by activation of non-hormonal non-opioid systems [27]. Pretreatment with opiate antagonists ,did affect male copulatory behavior in the present study and in previous studies [17]. The alteration of mating behavior by doses of opiate antagonists that do not affect mating produced analgesia suggests male copulatory behavior is more sensitive to endogenous opioids than are reactions to nociceptive stimulation. Since higher doses of morphine are required to produce analgesia effects in hamsters than are required to affect copulatory behavior [17,18], it is reasonable to hypothesize that opioids are present during mating in hamsters in quantities sufficient to alter copulatory behavior but insufficient to produce anopioid related analgesia. The effects of opiate antagonists on male copulatory behavior in hamsters is probably specific to actions of the antagonists at the opiate receptor. The available evidence
605 suggests that opiate antagonists act quite selectively in hamsters. Opiate antagonists do not affect female copulatory behavior when administered in a wide range of doses under a variety of conditions [19]. Opiate antagonists do not affect spontaneous feeding in hamsters but do affect drinking behavior [8] and diazepam induced feeding in hamsters [1]. It may not be appropriate to classify mating produced reductions in responsiveness to nociceptive stimulation as analgesia. In addition to reducing responsiveness to nociceptive stimulation, mating reduces responsiveness to many classes of stimuli. Male rats on a terminal food deprivation regimen do not eat when given access to both food and receptive females [23]. F o o d deprived male rats provided access to both food and receptive females only respond to the presence of food after mating extensively and feeding is largely restricted to the postejaculatory interval [3]. Lactating female rats provided with access to males during postpartum estrus do not display maternal behavior during an ejaculatory series and only respond to disruption of the nest and scattering of the pups during the postejaculatory interval [6]. It may be that reduced responsiveness to nociceptive stimulation during and shortly after mating reflects a decreased responsiveness to all stimuli not directly related to the sexual interaction.
ACKNOWLEDGEMENTS This research was supported by NSF grant BNS78-17860. We thank Ines Vicente for excellent technical assistance. The opiate antagonists used in this study were donated by Endo Laboratories.
REFERENCES and R. G. Noble. Naloxone antagonism of diazepam 10. Mclntosh, T. K., M. L. Vallano and R. J. Barfield. Effects of feeling in the Syrian hamster. Life Sci 29:1125-1131, morphine, beta-endorphin and naloxone on catecholamine levels and sexual behavior in the male rat. Pharmacol Biochem R. J., D. D. Kelly, M. Beutus and M. Gusman. StressBehav 13: 435-441, 1980. analgesia: Neural and hormonal determinants. I 1. Meyerson, B. J. and L. Terenius. Beta-endorphin and male sexNeurosci Biobehav Rev 4: 87-100, 1980. ual behavior. Eur J Pharmacol 42: 191-192, 1977. 3. Brown, R. E. and D. J. McFarland. Interaction of hunger and 12. Murphy, M. R. Methadone reduces sexual performance and sexual motivation in the male rat: A time-sharing approach. sexual motivation in the male Syrian golden hamster. PharAnimal Behavior 27: 887-896, 1979. macol Biochem Behav 14" 561-567, 1981. 4. Cruz, S. E., N. L. Ostrowski and R. G. Noble. Mating and 13. Murphy, M. R., D. L. Bowie and C. B. Pert. Copulation eleresponsiveness to a nociceptive stimulus. Bull Psychonomi Soc vates plasma beta-endorphin in the male hamster. Soc Neurosci 16: 55-56, 1980. Abstr 5: 470, 1979. 5. Gessa, G. L., E. Paglietti and B. Pellegrine Quarantolli. Induc14. Myers, B. M. and M. J. Banm. Facilitation b y opiate tion of copulatory behavior in sexually inactive rats by antagonists of sexual performance in the male rat. Pharmacol naloxone. Science 204: 203--204, 1979. Biochem Behav 10: 615-618, 1979. 6. Gilbert, A. N., R. J. Pelchat and N. T. Adler. Postpartum 15. Myers, B. M. and M. J. Baum. Facilitation of copulatory percopulatory and maternal behavior in Norway rats under formance in male rats by naloxone: Effects of hypophysectomy, seminatural conditions. Anita Behav 28: 989-995, 1980. 17 alpha-estradiol, and luteinizing hormone releasing hormone. 7. Lieblich, I., M. J. Baum, P. Diamond, N. Goldblum, C. Iser and Pharmacol Biochem Behav 22: 365-370, 1980. C. G. Pick. Inhibition of mating by naloxone or morphine in 16. Noble, R. G. Mounting in female hamsters: Effects of different recently castrated, but not intact male rats. Pharmacol Biochem hormone regimens. Physiol Behav 19: 519-526, 1977. Behav 22: 361-364, 1985. 17. Noble, R. G. Hamster sexual functioning: Modulation by opiate 8. Lowy, M. T. and G. K. W. Yim. Drinking but not feeding is agonists, opiate antagonists and exercise. Presented at the Eastopiate sensitive in hamsters. Life Sci 30: 1639-1644, 1982. ern Conference on Reproductive Behavior at the Rockefeller 9. McConnell, S. K., M. J. Baum and T. M. Badger. Lack of University, New York, NY, June 1980. correlation between naloxone-induced charges in sexual behavior and serum LH in male rats. Horm Behav 15: 16-35, 1981. 1. Birk, J. induced 1981. 2. Bodnar, induced
606 18. Ostrowski, N. L., J. M. Stapleton, R. G. Noble and L. D. Reid. Morphine and naloxone's effects on sexual behavior of the female golden hamster. Pharmacol Biochem Behav 11: 673-681, 1979. 19. Ostrowski, N. L., R. G. Noble and L. D. Reid. Opiate antagonists and sexual behavior in female hamsters. Pharmacol Biochem Behav 14: 881-888, 1981. 20. Pellegrini Quarantotti, B., M. G. Corda, E. Paglietti, G. Biggio and G. L. Gessa. Inhibition of copulatory behavior in male rats by D-Ala2-Met-enkephalinamide. Life Sci 23: 673-678, 1978. 21. Pert, A., A. DeWald, H. Liao and C. Sivit. Effects of opiates and opioid peptides on motor behaviors: Sites and mechanisms of action. In: Endorphin in Mental Health Research, edited by E. Usdin, W. E. Bunney and N. S. Kline. New York: Oxford University Press, 1979, pp. 45-61. 22. Rappaport, S. I., W. A. Klee, K. D. Pettigrew and K. Ohno. Entry of opioid peptides into the central nervous system. Science 207: 84-86, 1979.
CRUZ-MORALES
AND NOBLE
23. Sachs, B. D. Sexual behavior of male rats after one to nine days without food. J Cornp Physiol Psychol 60: 144--146, 1965. 24. Szechtman, H., R. Simantov and M. Hershkowitz. Effects of naloxone on copulation in rats and the role of endogenous opiates in a spontaneous rewarding behavior. Soe Neurosci Abstr 5: 541, 1979. 25. Tseng, L. F., T. J. Orstwald, H. L. Loh and Ch. H. Li. Behavioral activities of opioid peptides and morphine sulfate in golden hamsters and rats. Psychopharmaeology (Berlin) 64: 215-218, 1979. 26. Verebeley, K. and S. J. Mule. Naltrexone pharmacology, pharmacokinetics, and metabolism: Current status. Am J Drug Ah'ohol Abuse 2: 351-363, 1975. 27. Watkins, L. R. and D. J. Mayer. Organization of endogenous opiate and nonopiate pain control systems. Science 216:11851192, 1982.
0031-9384/86$3.00 + .00
Mating Produced Analgesia in the Syrian Hamster S A R A E. C R U Z - M O R A L E S * f A N D R. G. NOBLE:~ 1
*Autonomous National University o f Mexico (UNAM) fEscuela National de Estudios Professionales-Iztacala, Mexico 5;Department o f Psychology, Rensselaer Polytechnic Institute, Troy, N Y 12180 Received 14 April 1986 CRUZ-MORALES, S. E. AND R. G. NOBLE. Mating produced analgesia in the Syrian hamster. PHYSIOL BEHAV 38(5) 601-606, 1986.--A series of experiments examined the hypothesis that mating produced analgesia in the Syrian hamster can be blocked by opiate antagonists. The first experiment established a dose response relation to painful footshock. Females were more responsive to footshock than males (p<0.001). The second experiment demonstrated that ten minutes of mating reduced responsiveness of both males and females to a moderate intensity of footshock. The third experiment replicated the effect of mating on the responsiveness of males to footshock, but did not demonstrate any effect of the opiate antagonist naltrexone on mating induced analgesia. The fourth experiment demonstrated that limited mating stimulation (five intromissions) has little effect on the responsiveness of sexually inexperienced males to footshock. The fifth experiment replicated the fourth experiment using sexually experienced males and obtained comparable results. Apparently mating induced analgesia in male hamsters is not blocked by opiate antagonists and which raises questions about biological activity of reported changes in endorphin activity during mating. Mating
Analgesia
Opiateantagonists
SEVERAL lines of evidence suggest a relationship between the endogenous opioid system and reproductive behavior. Historically, opiate use has been associated with impaired sexual performance [7,12]. More recently, laboratory studies have demonstrated that the acute administration of opiates or endogenous opioids selectively inhibits male copulatory performance in rats and hamsters at doses which do not affect general activity [10--12, 20]. The administration of opiate antagonists has been reported to facilitate copulatory behavior in sexually active rats [5] and to affect the copulatory performance of sexually active male rats [10, 14, 15], although these effects are not consistently obtained [9, 23, 25]. Mating produced increases in systematic levels of beta endorphin like immunoreactive material in male hamsters have been reported [13], an observation consistent with the proposed relation between the endogenous opioid system and mating behavior. The relation between reported increases in systemic levels of immunoreactive beta-endorphin and mating behavior is not clear. It is not certain that beta endorphin in the peripheral circulation enters the central nervous system in biologically significant amounts [22] and it has been reported that stimulation produced changes in systemic endorphin levels are frequently lower than the levels produced by minimally effective doses of beta endorphin administered systemically [2]. Since beta endorphin is a potent, centrally acting analgesic, it is possible that mating produced increases in systemic endorphin levels [13] reaching the brain in physiologically
significant amounts would produce analgesia. Mating has been reported to reduce responsiveness to nociceptive stimulation in rats [24] and hamsters [4] but it is not known whether this mating produced reduction in responsiveness is blocked by opiate antagonists. The present series of experiments was designed to evaluate the hypothesis that systemic increases in beta endorphin could be responsible for mating induced analgesia by determining if analgesia was present at different times during mating when systemic levels are high [13] and by determining if mating induced analgesia is blocked by opiate antagonists. We adopted this approach to evaluating the physiological significance of mating produced increases in beta endorphin because analgesia produced by exogenously administered beta endorphin is reversed or blocked by opiate antagonists [1] and the physiological effects of very large doses of beta endorphin on hamsters are completely reversed by opiate antagonists [26]. EXPERIMENT ONE The first experiment evaluated the suitability of electric footshock as a source of nociceptive stimulation. It also provided the basis for selecting the intermediate level of shock intensity used in subsequent studies. The selection of intermediate shock intensity was intended to maximize the likelihood that both increases and decreases in responsiveness to footshock could be detected.
1Requests for reprints should be addressed to Ralph G. Noble.
601
C R U Z - M O R A L E S A N D NOBLE
6O2
FEMALES U4 7 I MALES 6 ~
c~--o o,-
=
=
<~
5 ~
FEMALE
(3
4~
MALE
)~J Z UJ F-
] ~
2=,
< On
08
t
SHOCK
13
16
20
m
MATED
UNMATED
MATED
UNMATED
{MA) O' POST
FIG. 1. The relationship between the shock intensity and the incidence of paw shake.
METHOD
Subjects
MATING
30'
POST
MATING
FIG. 2. The effect of mating on latency to paw shake.
chamber for 20-30 sec while the latencies to paw shake and to escape were recorded, and a new trial was started immediately after the measures were recorded.
Thirty-eight adult (60-90 days old) Syrian hamsters
(Mesocricetus Auratus), purchased from Engle's Laboratory Animals Inc., were used in this experiment. Females had been ovariectomized under sodium pentobarbital anesthesia and implanted subcutaneously with a capsule containing 17 Beta-Estradiol [16] at least 30 days before the experiment and injected with progesterone (0.5 mg in 0.1 ml corn oil, SC) four hours before testing to produce behavioral recepting. The subjects were housed in solid bottom plastic cages (30×33× 17 cm), with four animals of the same sex per cage. The colony room was maintained at 22°C on a reversed light-dark cycle (L:D 14:10) with the onset of the dark phase of the cycle at 1400. Purina rat chow and water were continually available in the home cages.
Design Each subject was randomly assigned to one of five groups with at least four males and three females in each group. Each group received a different nonimal intensity of footshock: 0.08, 0.1, 0.13, 0.16 or 0.20 mA.
Apparatus
RESULTS Because paw shake responses were not consistently observed at all current intensities, the number of trials during which the paw shake response occurred was used as the dependent variable. Both males and females showed an increase in the number of trials during which the paw shake response was observed as a function of shock intensity, F(4,28)=28.69, p<0.001, and females were more responsive than males, F(1,28)=15.07, p<0.001, particularly to low intensities [sex by intensity interaction, F(4,28)=4.99, p<0.004, see Fig. 1]. The escape latency decreased with "increasing shock intensities, F(7,196)=19.63, p<0.001, and escape latencies were larger for males, F(7,28)=3.02, p=0.09, especially at lower intensities [intensity × sex interaction, F(7,196)=3.47, p<0.002].
E X P E R I M E N T TWO
The test chamber was a 51/2 gal glass aquarium. The inner walls of the chamber were covered with wire mesh (1.25×1.25 cm). The floor of the test chamber was constructed from an etched copperplate and connected to a Grason-Stadler Co. shock generator.
The second experiment replicated a previous experiment [4], by examining the effects of ten minutes of mating on the response of male and female hamsters to nociceptive stimulation provided by electric footshock.
Procedure
Subjects and Design
The subjects received two sessions on consecutive days, with four trials in each session. At the beginning of each trial the animal was placed in the center of the chamber and footshock was delivered for 180 seconds or until the subject escaped by climbing onto the hardware cloth. During shock administration, subjects observed a paw shake response [4] and the latency to the first paw shake response was recorded. The animal was scored as having escaped as soon as all four paws were in contact with the hardware cloth. After each trial the subjects were kept in a separate holding
The subjects were 39 sexually experienced hamsters, 21 intact females and 18 males, from 60 to 90 days of age. All females were tested on the day of estrus. Animals were assigned to four groups: The animals in group 1 (11 females) and group 2 (9 males) were permitted to mate with stimulus animals, while subjects of group 3 (10 females) and group 4 (9 males) were placed in the mating chamber alone. Sexually active males and ovariectomized females in hormone induced receptivity were used as stimulus animals in the mating tests.
METHOD
MATING AND ANALGESIA
603
Procedure Mating session. Each subject was placed in the mating chamber (a 51/2 gal all glass aquarium) for ten minutes before testing. Then a stimulus animal of the appropriate sex was introduced. The animals were allowed to mate without interruption for nine minutes after the first intromission. Mating was then terminated after the next ejaculation. Animals were removed from the mating chamber within ten seconds after the ejaculation and tested for analgesia. The animals in the control groups were placed in the test chamber alone for ten minutes and then tested for analgesia. The subject was placed in the center of the test chamber within five seconds after being removed from the mating chamber and a shock of 0.13 mA DC was delivered for 180 seconds or until it escaped. All animals were tested again 30 minutes later.
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RESULTS
NALTREXONE
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UNMATED
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FIG. 3. The effect of mating and naltrexone on latency to paw shake.
Latency to Paw Shake Mating increased the latency to paw shake in both males and females, F(1,35)=7.23, p<0.01. Paw shake latency decreased significantly with repeated testing, F(1,35)=9.5, p<0.004. As shown in Fig. 2, there was a large effect immediately post mating but there were no significant differences among treatment groups thirty minutes post mating.
Latency to Escape Mating increased latency to escape in both males and females (see Fig. 3), [main effect of mating, F(1,35)=8.92, p<0.005], and escape latency decreased with repeated testing, F(1,35)=6.01, p<0.001. Thirty minutes post mating, there were no significant differences among groups.
DISCUSSION
Procedure Each male was weighed, then placed in the mating chamber for ten minutes before the female was introduced. After five minutes in the mating chamber it was injected and put back in the chamber. The male was permitted to achieve five ejaculations. The number of mounts, number of intromissions and the time required to achieve five ejaculations were recorded. The males in the unmated groups were kept in the test chamber for an additional ten minutes. Analgesia test. Within five seconds after being removed from the mating chamber the male was put into the test chamber and a 0.13 mA DC footshock was delivered for 180 seconds or until the subject escaped. Thirty minutes later, the analgesia test was repeated.
The results extend and confirm the previous [4] demonstration of transient mating produced analgesia in males and females. The effect of mating was much more pronounced in the present study.
EXPERIMENT THREE This experiment was designed to determine whether mating produced analgesia be blocked by an opiate antagonist. Naltrexone was selected for its potency as an opiate antagonist and its long lasting effects in rats and in hamsters [19,26]. METHOD
Subjects and Design Forty sexually experienced male hamsters, 5-6 months old, were randomly assigned to four groups (n/group= 10). Males in two groups were mated and males in the other two groups were placed in the mating chamber alone. Animals in one of the mated groups and in one of the unmated groups were injected with naltrexone (10 mg/kg, in physiological saline SC) and animals in the other groups were injected with an equivalent volume of physiological saline (1 ml/kg, SC).
RESULTS One subject which did not achieve five ejaculations within 15 minutes was deleted from the experiment and its scores were not included in the data analyses. A two way ANOVA for repeated measures was used to evaluate the data,
Paw Shake Latency Mating increased paw shake latencies, F(1,35)=25.36, p <0.001, but naltrexone administration did not produce any significant effects and none of the interactions with naltrexone administration were significant. Thirty minutes post mating the mated group's average latency to paw shake had decreased and was not different from the mean paw shake latency of the unmated group which did not decrease. This produced both a significant trials effect, F(1,35)=28.37, p<0.001, and a significant trials x group interaction, F(1,35)=29.31, p<0.001 (see Fig. 3).
Latency to Escape The mated groups showed longer latencies to escape than animals in the unmated groups immediately after mating, but
604
CRUZ-MORALES AND NOBLE METHOD 18
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NALTREXONE
Thirty-nine sexually inexperienced male hamsters, 60 days old, were assigned at random to four groups. Males in groups one and two were permitted five intromissions with a stimulus female five minutes after being injected with either naltrexone (10 mg/kg, SC, group 1) or physiological saline in equivalent volume (group 2). Males in groups three and four were placed in the empty mating chamber for two minutes, five minutes after being injected with either naltrexone (10 mg/kg, SC, group 3) or physiological saline (group 4). The procedures were otherwise the same as in experiment three.
SALINE
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MATED 3 0 ' POST M A T I N G
FIG. 4. The effect of five intromissions and naltrexone on latency to paw shake.
there were no significant differences among groups thirty minutes post mating which produced a significant mating by trials interactions, F(1,35)=23.56, p<0.001. There was an overall decrease in the latency to escape with repeated testing, F(1,35) = 200, p <0.001, but there was no significant main effect of naltrexone administration and none of the interactions with naltrexone administration were significant.
Male Behavior
The group that received naltrexone displayed fewer mounts before achieving five intromissions (mean_+ S.E.M.=6.0_+l.25) than the group injected with saline [mean+ S.E.M. = 13.7-+3.1; t(18)=2.3, p<0.05]. Naltrexone reduced the number of intromissions required to achieve five ejaculations (mean-+S.E.M. = 16.7-+ 1.1) compared with the saline group [mean-+S.E.M. =23.5_+2.9; t(18)=2.3, p<0.05].
DISCUSSION
The effects of naltrexone administration on mating behavior in the present experiment were comparable to effects obtained previously [17]. The effects of mating on paw shake latency were much more pronounced in the present experiment than in experiment two, and the difference is quite specific to paw shake latency immediately postmating. The only systematic difference between the two experiments was that both males and females were tested in the shock apparatus in experiment two. It is possible that the presence of female odor in the test chamber in experiment two attenuated the effect of mating on paw shake latency.
EXPERIMENT FOUR This experiment was designed to find out if five intromissions produce analgesia which is antagonized by naltrexone. Apparently five intromissions produces maximal increases in systemic levels of beta endorphin [13].
RESULTS
Latency to Paw Shake
Five intromissions produced a small increase in paw shake latency immediately after mating but there were no differences in paw shake latency 30 min post mating which produced a significant mating by trials interaction, F(1,35)=6.65, p<0.01. There were no other significant main effects or significant interactions (see Fig. 4). Latency to Escape
There were no significant effects of mating on escape latency. Naltrexone administration increased the escape latencies of both mated and unmated males immediately after mating but not thirty minutes after mating. This produced both a significant main effect of naltrexone, F(1,35)=7.40, p<0.01, and a significant trials by naltrexone interaction, F(1,35)=6.18, p<0.01). In all groups the latency to escape decreased with repeated testing trials, F(1,35)=57.48, p <0.001. DISCUSSION
Since the initial increase in escape latency was not associated with an increase in paw shake latency it seems unlikely that naltrexone had agonist effects as has been sometimes hypothesized [26]. It seems more probable that the naltrexone administration produced a general hyperactivity to arousing stimuli which interfered with complex motor functioning [21]. It is possible that sexually inexperienced males having been handled less and having less exposure to the testing procedures were more reactive initially and the naltrexone exaggerated this reactivity. Because naltrexone had an unexpected effect on sexually inexperienced males, we examined the effects of five intromissions on the response of sexually experienced males to nociceptive stimulation. Ten sexually experienced males were allowed to achieve five intromissions, while ten males were placed in the mating chamber alone for two minutes. Five intromissions had no significant effects on responsiveness to nociceptive stimulation, which in general is consistent with the minimal effects of five intromissions on the responsiveness of sexually inexperienced males to nociceptive stimulation.
GENERAL DISCUSSION Our results support the conclusion that mating produced
MATING AND ANALGESIA analgesia is not mediated by activation o f the endorphin system, and that the mating produced increase in endorphin like immunoreactive material reported by Murphy [13] is insufficient to produce analgesia. We base this conclusion on the observation that mating produced analgesia is not reduced by an amount of naltrexone sufficient to antagonize the effects of large amounts of exogenously administered beta endorphin [25] on hamsters. Mating produced reductions in responsiveness to nociceptive stimulation in male hamsters strongly resembles the analgesia produced by mechanisms which are neural and non-opioid according to the classification of Watkins and Mayer [27]. The available evidence strongly suggests that forms of stimulus produced analgesia which involve activation of the endogenous opioid system are blocked, at least partially by opiate antagonists in the doses used [2,27]. The relatively short duration of mating produced analgesia is consistent with the duration produced of analgesia by activation of non-hormonal non-opioid systems [27]. Pretreatment with opiate antagonists ,did affect male copulatory behavior in the present study and in previous studies [17]. The alteration of mating behavior by doses of opiate antagonists that do not affect mating produced analgesia suggests male copulatory behavior is more sensitive to endogenous opioids than are reactions to nociceptive stimulation. Since higher doses of morphine are required to produce analgesia effects in hamsters than are required to affect copulatory behavior [17,18], it is reasonable to hypothesize that opioids are present during mating in hamsters in quantities sufficient to alter copulatory behavior but insufficient to produce anopioid related analgesia. The effects of opiate antagonists on male copulatory behavior in hamsters is probably specific to actions of the antagonists at the opiate receptor. The available evidence
605 suggests that opiate antagonists act quite selectively in hamsters. Opiate antagonists do not affect female copulatory behavior when administered in a wide range of doses under a variety of conditions [19]. Opiate antagonists do not affect spontaneous feeding in hamsters but do affect drinking behavior [8] and diazepam induced feeding in hamsters [1]. It may not be appropriate to classify mating produced reductions in responsiveness to nociceptive stimulation as analgesia. In addition to reducing responsiveness to nociceptive stimulation, mating reduces responsiveness to many classes of stimuli. Male rats on a terminal food deprivation regimen do not eat when given access to both food and receptive females [23]. F o o d deprived male rats provided access to both food and receptive females only respond to the presence of food after mating extensively and feeding is largely restricted to the postejaculatory interval [3]. Lactating female rats provided with access to males during postpartum estrus do not display maternal behavior during an ejaculatory series and only respond to disruption of the nest and scattering of the pups during the postejaculatory interval [6]. It may be that reduced responsiveness to nociceptive stimulation during and shortly after mating reflects a decreased responsiveness to all stimuli not directly related to the sexual interaction.
ACKNOWLEDGEMENTS This research was supported by NSF grant BNS78-17860. We thank Ines Vicente for excellent technical assistance. The opiate antagonists used in this study were donated by Endo Laboratories.
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