Suckling-induced changes in responsivity to the hypoalgesic effect of morphine

209

Fain, 59 (1994) 209-217

0 1994 Elsevier Science B.V. Ail rights reserved 0304-3959/94/$07.00

PAIN 2589

Suckling-induced changes in responsivity to the hypoalgesic effect of morphine Barbara

C. Woodside

a, Belinda

A. Lee a and Joseph

Rochford

b,*

a Center for

Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, Montreal, Quebec H4B lR6 (Canada) and b Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal, Quebec H4H lR3 (Canada)

(Received 12 November 1993, revision received 11 February 1994, accepted date 3 March 1994)

Summary The hypoalgesic effect of mo~hine in lactating rats was assessed using the hot-plate test. At midlactation (days 12 and 18 postpartum) females nursing litters of 8 pups were less responsive to the hypoalgesic effect of morphine than ovariectomised or cychng females and females on days 6 or 24 of lactation. Subsequent studies showed that the hypoalgesic response to morphine was inhibited in lactating rats at a number of time points after drug administration and across a variety of doses. This effect was not dependent on milk delivery but was dependent on the hormonal state of the female since separation of dams and their litters for 96 h was sufficient to reinstate the response to morphine if it resulted in a reappearance of vaginal estrus. Key words: Morphine; Lactation; Suckling; Antinociception;

There is accumulating evidence demonstrating that the hypoalgesic efficacy of both endogenous and exogenous opiates can be modulated by natural and experimentally induced variations in gonadal function. Gonadectomy has been shown to modulate the antinociceptive potency of both CL-and &opiate receptor-selective ligands (Chatterjee et al. 1982; Berglund et al. 1988; Kepler et al. 1989, 1991; Islam et al. 1993). Gonadectomy has also been shown to influence the magnitude of both opioid and non-opioid forms of stress-induced hypoalgesia (Romero et al. 1987, 1988; Ryan and Maier 1988). Further, most of these effects can be reversed by gonadal steroid replacement, which provides more direct evidence that they are mediated by changes in gonadal status. There is also evidence suggesting that modulation of opiate antinociception corresponds to more acute fluctuations in gonadal function. In the female rat, the

* Corresponniing author: Dr. 3. Rochford, Douglas Hospital Research Center, 6875 LaSalle Boulevard, Verdun, Quebec H4H lR3, Canada. SSDl 0304-3959(94)00065-M

(Rat)

hypoalgesic efficacy of morphine has been shown to vary across the estrous cycle; morphine exerts its most pronounced antinociceptive effect when administered on the afternoon of diestrous, but generally has Iittle or no effect when administered on the afternoon of proestrous (Banerjee et al. 1983; Berglund and Simpkins 1988; Kepler et al. 1989). Wieland and Wise (1990) have shown that this variation across the estrous cycle is correlated with changes in opiate receptor density. Changes in j3-endorphin content across the estrous cycle have also been observed in a number of hypothalamic nucleii (Barden et al. 19811, suggesting that synthesis and/or release of endogenous opioids can be regulated by gonada steroids. This variability in the function of opioid antinociceptive substrates is not restricted to the estrous cycle. Changes in opiate receptor density (Hammer and Bridges 1987; Hammer et al. 1992), opioid peptide levels (Wardlaw and Frantz 1983; Baron and Gintzler 1987; Bridges and Rosenheim 1987; Medina et al. 1993) and opioid-mediated hypoalgesia (Gintzler 1980; Baron and Gintzler 1984, 1987) have been demonstrated during pregnancy and parturition. Further, it has been demonstrated that changes in opiate receptor density in the medial preoptic area can be induced by treating ovariectomised animals with

hormones so as to mimic the pattern of estrogen and progesterone exposure seen during pregnancy (Hammer et al. 1992). Fluctuations in the levels of gonadal steroids are not restricted to events occurring during the estrous cycle or during pregnancy. During lactation, progesterone levels rise and reach a peak approximately 12 days postpartum (Greta and Eik-Nes 1967). Levels of circulating prolactin are also high during the first 2 weeks postpartum (Simpson et al. 19731. These changes are accompanied by a reduction in gonadotropin-releasing hormone release (Smith and Lee 1989) and low levels of estradiol and Iuteinizing hormone secretion (Ford and Melampy 1973; Smith and Neil1 1977, Fox and Smith 1984). In contrast to the wealth of information pertaining to gonadectomy-, estrous cycle- and pregnancy-related changes in opiate antinociceptive action, relatively little attention has been paid to the question of whether opiate function may be altered during lactation. A recent study reported by Janik et al. (1993) found that the hypoalgesic effect of morphine on the tail-flick test was reduced in animafs tested between days 6 and 10 of lactation. In the present series of experiments, we attempted to expand upon this limited data base by assessing the hypoalgesic effect of morphine with the use of a different test of pain sensitivity, the hot-plate test. In Exp. 1, we assessed the effect of morphine administration across the full span of the lactational period (6-24 days postpartum), and compared these effects to those observed in ovariectomized animals as well as animals that had returned to estrous cyclicity. In Exp. 2 we examined the time course of morphine’s hypoalgesic effect, whereas in Exp. 3 we assessed the dose-response curve in lactating and non-lactating animals. In each of these experiments, we observed that the hypoalgesic response to morphine was inhibited in lactating females. Consequently, Exps. 4 and 5 were conducted to determine what factors associated with lactation contribute to the effect. SuckIing stimulation from the pups both maintains the female in the hormonal profile typical of lactation and stimulates milk production (Ota and Yokayama 1967; Moltz et al. 1969). This latter effect places a heavy metabolic demand on the female and also produces changes in the morphology of the animal by increasing the mass of the mammary glands (Brady et al. 1938). Either of these factors could contribute to the change in responsiveness of lactating females to exogenous morphine administration. To determine whether milk production and its associated morphologic and metabolic changes or the hormonal status of the dam made the major contribution to the effects observed in Exps. 1-3, in Exp. 4 milk delivery was prevented in suckled, postparturient females by severing the galactophores (i.e., the

ducts that carry milk from the mammary glands to the nipples) prior to mating. Since pups continue to suckle at the nipples of these females, their hormonal status is similar to that of lactating dams (Desclin 1947). Finally, given the importance of suckling stimulation in maintaining the hormonal state of lactating females, Exp. 5 explored the effects of litter removal for varying periods of time prior to testing.

Method Subjects Ail subjects used in this experiment were virgin Wistar rats obtained from Charles River Breeding Farms (St. Constant, Quebec). Rats were maintained throughout the study on a 12-h light/dark cycle at a temperature of 21 f 2°C with ad lib access to food and water. All procedures were conducted within the first 5 h of the light portion of the cycle.

Surgery Surgeries were conducted under Metofane anaesthesia (Janssen Pha~aceuticals, Mississauga, Onta~o) and females were treated post-o~ratively with Ayercillin (Parke Davis). Qvariectomies (Rxp. 1) were performed 4 weeks prior to testing via bilateral dorsal incisions in the abdominal wall. Galactophore cuts (Exp. 4) were carried out 2 weeks prior to mating. A midline incision was made between both the upper 3 and the lower 3 pairs of nipples. The galactophores were exposed by reflected the skin, and each galactophore was severed. The midline incisions were then sutured. The success of the operations were verified by weighing litters before and after suckling and by postmortem examination of the mammary glands. Identical procedures were followed for sham-operated animals but the galactophores were left intact.

Apparatus

and drugs

The hot-plate apparatus consisted of a 20 x 38 X 20 clear Plexiglas chamber mounted on a O.&cm-thick piece of sheet metal. A hinged, wire-mesh top prevented the animals from escaping. Plate temperature was controlled by immersing the sheet metal in a water bath heated by a Haake E2 immersion/Open Bath Circulator (Berlin, Germany). In each experiment the water bath was heated to 52.Oi 0.2”C. The apparatus was located in a test room illuminated by 2 red light bulbs (25 W). During the interval between injection in the test room and analgesic testing, the animals were isolated in separate 30 x 20 x 15 cm wooden holding boxes. Morphine sulfate (Abbott Laboratories, Mississauga, Ontario) was dissolved in 0.9% saline and administered S.C.in the dorsal neck area, Doses were calculated based on the salt. The injection volume was 1 ml/kg.

Procedure Mating

Females were housed in group cages with a male rat. Eighteen days later those showing signs of pregnancy were removed to polypropylene maternity cages (33 x 45 x 17 cm) with beta chip bedding and remained in those cages throughout the remainder of

211

each experiment. Ovariectomised females were housed under similar conditions to females in the other groups.

mine whether dams were in lactational had experienced an estrous.

Hot-plate testing

Experiment 2 Assessment of the time course of morphine’s hypoalgesic effect. Females were assigned to either a lactating

In Exps. 1-4, all animals were preexposed to the testing procedure and the plate apparatus for 4 consecutive days prior to testing in order to eliminate the possibility that animals within the different groups may have been differentially sensitive to the hypoalgesic effects of novelty-stress (Rochford and Stewart 1987). During these exposures animals were transported to the test room, placed in the holding boxes for 30 min, and then exposed to the non-functional (i.e., room temperature) hot-plate apparatus for 60 sec. On the test day animals were transported to the test room and 5 min later administered a hot-plate test. Animals were placed on the surface of the plate and the latency to lick a hind paw (paw-lick latency: PLL) was measured. If no response was observed within 60 set the test was terminated and a PLL of 60 set was recorded. Animals were then administered morphine. In Exps. 1, 2, 4 and 5 the dose employed was 5 mg/kg. This dose lies in the midrange of the dose response curve (O’Callaghan and Holtzman 1975) and is commonly employed in studies assessing the hypoalgesic effect of morphine. Moreover, the hypoalgesic effect of this dose has been shown to be sensitive to variations in gonadal hormonal status (Banerjee et al. 1983; Berglund and Simpkins 1988; Islam et al. 1993; Janik et al. 1993). Exp. 3 (the dose-response study) assessed the effects of 5, 10 and 20 mg/kg. Save for Exp. 2, which assessed the time course of morphine’s hypoalgesic effect (see below), animals were administered a second hot-plate test 30 min after morphine administration. Each experiment was conducted blind in that the observer was unaware of the lactational state of the animal during hot-plate testing. Experiment 1 Assessment of morphine’s effect across the lactational phase. Six experimental groups were tested in this

study: an ovariectomised group (OVX) (n = ll), 4 groups of lactating females tested either on day 6 (n = lo), day 12 (n = 91, day 18 (n = 8) or day 24 (n = 10) postpartum, and a group of females who had given birth but whose litters had been removed on day 1 postpartum (LR; n = 9). In this and subsequent experiments, litters of dams assigned to the lactating groups were culled to 8. Females in the litter-removed group had returned to cyclicity and were run on the diestrous day closest to day 12 postpartum (range: days 11-13). The weight of the females and, where appropriate, litter weights were recorded daily for this and subsequent experiments. Daily vaginal smears were also taken in order to assess the stage of the cycle for the females in the litter-removed group and to deter-

diestrous

or

or litter-removed group (n = 8 per group) on day 1 postpartum. Dams in the lactating group were tested on day 12 postpartum, those in the litter-removed group on the diestrous day closest to day 12. Hot-plate assessments were conducted prior to, as well as 15, 30, 45 and 90 min following, administration of 5 mg/ kg morphine. Experiment 3 Assessment of the dose-response

curve. Animals were assigned to either a lactating or litter-removed group on day 1 postpartum. In this study these 2 groups of animals were further subdivided into 3 drug-dose groups: 5, 10, or 20 mg/kg of morphine sulphate (n = 7 per group). Testing was conducted on day 12 postpartum (or on the diestrous day closest to day 121, and hot-plate tests were conducted prior to and 30 min following morphine administration.

Experiment 4 The effect of galactophore cuts. In this study 3 groups

of animals were employed: galactophore-cut (n = 6), sham-operated/ lactating (n = 7) and sham-operated/ litter-removed (n = 5). To ensure that similar suckling stimulation was received by females in the lactating and galactophore-cut groups, and to prevent starvation of the litters of galactophore-cut dams, each of these females was paired with a sham-operated dam on the basis of day of parturition and litters rotated between galactophore-cut and sham-operated dams every 12 h (09:OOand 21:00 h) for the first 7 days postpartum and thereafter every 24 h. Habituation and testing procedures were as described for Exp. 1. Experiment 5 The effect of varying litter removal duration. On the

day after parturition the females were assigned to 1 of 5 groups: lactating dams &AC, n = 7) and litters were left undisturbed until the day of testing. In the litterremoved groups, litters were removed either on day 1 postpartum (LR, n = 71, 18 h prior to testing (LR-18h, n = 71, 36 h prior to testing (LR-36h, n = 6) or 96 h prior to testing (LR-96h). In this latter group, 8 females showed an estrous smear between pup removal and testing, whereas 7 animals continued to show a vaginal smear typical of lactational diestrous. Hence this group was subsequently divided into 2 groups: those that had shown an estrus smear (LR-96h-E) and those that did not (LR-96h-LD). All litters were culled to 8 pups in number on the day following parturition

Results

and vaginal smears were taken daily of all females. In this experiment animals were not pre-exposed to the apparatus prior to test. Since pre-exposure required 4 days, some females (i.e.. those in the LR-18h and LR-36h groups) would have experienced some pre-exposures with their litters present, and the remainder with their litters removed. Thus, to remove this potential confound, the habituation procedure was eliminated altogether. All females were tested on day 12 postpartum with the exception of those in the litter-removed group which were tested on the diestrous day closest to day 12 postpartum. The dose of morphine administered was 5 mg/ kg and hot-plate tests were conducted prior to and 30 min following morphine administration.

Experiment 1: morphine’s effect at dijjerent stages oj lactation

The mean PLLs for the 6 groups for both the preand post-morphine hot-plate tests are shown in Fig. I. There were no significant differences in PLLs among the 6 groups on the pre-morphine test. The groups did differ in the post-morphine test. Dams tested on days 12 and 18 postpartum did not display a significant elevation in PLLs relative to the pre-morphine test while all other groups did. Further, the effect of morphine was more pronounced in the OVX group in comparison to the days 6, 24 and litter-removed groups. On the test day the dams in the litter-removed group had a lower body weight (mean: 292.71 of:4.63 g) than all the other groups, which did not differ from each other (combined mean: 341.52 f. 2.40 g). None of the animals in lactating groups day 6, day 12 or day 18 postpartum had shown an estrus smear since the postpartum estrous. All the animals in the day 24 group had shown at least one estrus smear and had entered a second diestrous stage. All the animals in the litter-removed group had shown at least one estrous smear.

Statistical analyses

PLL scores from Exps. 1 and 2 were anaIyzed by individual spIit plot, Group X Test (pre- vs. postmorphine) analyses of variance (ANOVA). In all experiments, preliminary data anaIysis revealed that there were no differences among groups during the premorphine tests. Thus, for ease of data presentation, in Exps. 3-5, difference scores (post-morphine PLLs pre-morphine PLLs) were calculated and analyzed by between-subject ANOVAs. In Exp. 3, group and dose served as factors; for Exps. 4 and 5, groups served as the unique factor. Weight data from each experiment were analyzed by l-way ANOVAs. Analyses of significant interactions were conducted with F tests for simple main effects (Winer 1971). When required, pairwise post-hoc comparisons were computed using Tukey’s ‘honestly significant difference’ test. In each case the criterion for statistical significance was defined as P < 0.05.

& a.

cI

50

@ f

40

:: 3

30

L e s ti L! s iz

Experiment 2: time course

Fig. 2 displays the mean PLLs for the lactating and Iitter-removed groups for each of the hot-plate assessments of Exp. 2. As in Exp. 1, there were no differences in the PLLs during the pre-morphine test. These groups also did not differ on the 15 min post-morphine test. The latencies of the litter-removed group, however, continued to increase at the 30 and 45 min tests while those for the lactating group decreased slightly, and the differences in PLLs at these time periods were

**n 5

T

PRE-MORPHINE

n P0ST-UWRWNE

20 10

0

DAY 6

DAY12

DAY

16

DAY 24

ovx

Utter Removed

Group Fig. 1. Mean PLLs prior to and 30 min following administration of 5 mg/ kg morphine for dams tested on days 6, 12, 18, 24 of lactation, and for ovariectomized and litter-removed females. Error bars indicate 1 SEM. Asterisks indicate significant (P < 0.05) differences between pre- and ~st-mo~hine latencies within a group. Daggers reflect significant (P < 0.05) differences in the ~st-mo~hine iatencies between groups.

213

1 iM_tK L

f

Pre-morphine

30

- Time post-morphlne

Fig. 2. moved tration reflect

90

45

(min)

Dose

(mg/kg)

---+

Mean PLLs for lactat inr.g dams tested on day 12 and litter-refemales prior to and 15, 30 45 and 90 min following adminisof 5 mg/kg morphine. Error bars indicate 1 SEM. Asterisks significant (P < 0.05) differences between groups at each time point.

significant. There was no difference between the groups 90 min post-injection. As in the previous study, dams in the lactating group weighed significantly more on the test day (mean: 323.7 + 3.18 g> than females in the litter removed group (mean: 277.58 + 4.48 g). In addition, all females within the litter-removed group had shown at least one estrous smear prior to test, whereas those in the lactating group had not. Experiment 3: dose response

Mean difference scores (PLL post-morphine - PLL pre-morphine) for all groups are shown in Fig. 3. Dams on day 12 of lactation showed a smaller response to the hypoalgesic effect of morphine than females in the litter-removed group at both the 5 mg/ kg and 10 mg/kg doses. There was no difference between the groups at the 20 mg/kg dose. Analysis of weights revealed that females within the litter-removed groups (combined mean: 280.3 + 3.95 g) weighed less than those from the lactating groups (combined mean: 312.31 + 4.96 g>. Estrus smear analyses indicated that all females within the litter-removed groups had returned to cyclic&y by the test day, whereas animals in the lactating groups had not.

Fig. 3. Mean PLL difference scores (post-morphine PLL-premorphine PLL) for lactating (day 12) and litter-removed females administered 5, 10 and 20 mg/kg. Error bars indicate 1 SEM. Asterisks reflect significant (P < 0.05) differences between groups at each dose.

of pups gained on average 28.3 + 1.23 g/day while being suckled by sham-operated females and lost an average of 8.29 + 2.16 g/day while being suckled by galactophore-cut dams. This difference was significant. All females in both the sham-operated and galactophore-cut groups failed to show an estrus smear prior to test, whereas all females in the litter-removed groups had returned to cyclicity. Experiment 5: litter removal

The mean difference scores (PLL following morphine - PLL at baseline) for all groups are shown in Fig. 5. The mean PLLs were increased more in the litter-removed and LR-96h-E groups following morphine than in any of the other groups, which did not differ from each other. Analysis of body weights revealed that dams in the LR-96h-LD group weighed Ific:antly less than those in the litter-removed group.

iii

Experiment 4: galactophore cut

Fig. 4 shows the mean difference scores (PLL following morphine - PLL at baseline) for all groups. The mean difference score for the litter-removed group was significantly larger than those for the lactating and galactophore-cut groups. These latter 2 groups did not differ from one another. Analysis of the test-day body weights for females in all 3 groups showed that dams in both the sham-operated, lactating and galactophore-cut groups weighed significantly more than the females in the sham-operated/litter-removed group (mean: 326.5 k 7.00 g; 340.9 + 3.55 g and 292.3 k 2.52 g, respectively). Litters

Sham-Operated Lactating

Galactophore cut

_. snam-Operated Litter

Removed

Group

Fig. 4. Mean PLL difference scores (post-morphine PLL-premorphine PLL) for sham-operated/lactating (day 121, galactophorecut, and sham-operated/litter-removed females. Error bars indicate 1 SEM. Asterisks reflect the finding that the mean from the shamoperated/litter-removed group was significantly (P < 0.05) greater than the means for both other groups.

LAC

LR

LR-18h

LR-36h

LR.96h.LO

LR-96h-E

Group

Fig 5. Mean PLL difference scores (post-morphine PLL - pre-morphine PLL) for lactating (day 121, litter-removed females and females whose litters were removed either 18, 36, or 96 h prior to test. Females in the LR-96h-LD group were tested while in lactational diestrous, whereas those LR-%h-E group had returned to estrous cycficity prior to test. Error bars indicate 1 SEM. Asterisks reflect the finding that the means for the LR and LR-96h-E groups were significantly (P < 0.05) greater than the means for all other groups,

Body weights among the other groups did not differ significantly.

Discussion The results from the present series of experiments suggest that the hypoalgesic efficacy of morphine is reduced during mid-lactation. In Exp. 1, morphine administration failed to significantly augment PLLs in females tested on days 12 and 18 of lactation, whereas morphine did significantly increase latencies in animals tested on days 6 and 24 of lactation, as well as ovariectomized females and females that had their litters removed following parturition. These data confirm and extend those recently reported by Janik et al. (19931, who found that females tested between days 6 and 10 of lactation on the tail-flick test were hyporesponsive to the antinociceptive effect of morphine. The results from Exps. 2 and 3 demonstrated that the insensitivi~ to morphine in lactating females is also apparent when the time course and the dose-response relationship is examined. The time course of morphine-induced hypoalgesia was attenuated in lactating females; whereas both lactating and litter-removed dams displayed similar PLLs 15 min following morphine a~inistration, lactating females displayed lower PLLs 30 and 45 min following injection. Lactating females also displayed a less pronounced antinociceptive response to 5 and 10 mg/ kg morphine, although the response to the highest dose employed (20 mg/ kg) was similar in both Iactating and litter-removed animals. The results of Exps. l-3 suggest that insensitivity to morphine during midlactation is not simply a function of testing females in the postpartum period. The litter-removed females that were used as comparison

groups in these studies had also experienced pregnancy and were tested at a comparable time postpartum to the lactating females. Litter-removed females did, however, display elevated Iatencies in response to morphine administration. Another possibility that may be invoked to account for the blunted effect of morphine during midlactation is that lactating females may possess increased levels of adipose tissue relative to litterremoved females. Since mo~hine is a highly lipophilic ligand, the possibility exists that the drug was absorbed to a greater extent by the higher levels of adipose tissue in lactating females. As such, less ligand would be available to stimulate central nervous system opiate receptors involved in antinoci~ption. However, some of the results from the present experiments suggest that there is a lack of correspondence between changes in body weight and sensitivity to morphine. In Exp. 1, animals tested during the preliminary (day 61 and late (day 24) stages of lactation did display hypoalgesia in response to mo~h~e, despite the fact that the weights for these animals were comparable to the females tested on days 12 and 18. In Exp. 4, there was a pronounced difference in the response to morphine between females whose pups were removed for 96 h and had returned to cyclicity (group LR-96h-E) and those that had not returned to cyclicity (group LR-96hLD). However, the mean weights of these 2 groups did not differ. A lack of correspondence between body weight and morphine’s antinociceptive potency has also been observed in studies that have examined the effect of gonade~tomy on pain reactivity (for review, see Bodnar et al. 1988). Further, Callahan et al. (1988a) demonstrated that both systemic and intracerebroventricular morphine administration is ineffective in stimulating prolactin release in lactating females. These considerations suggest that the hyporesponsiveness to

215

morphine seen in midlactation is not attributable to changes in body weight or composition. It could also be suggested that the present results may be attributable to a change in morphine’s sedative and/or motor effects, rather than on nociception. According to this argument, morphine may have differentially influenced the ability of lactating and litter-removed females to perform the paw-lick response. We believe this argument is unlikely. As mentioned previously, Janik et al. (1993) have reported a blunted hypoalgesic response to morphine in lactating females as assessed by the tail-flick test. ~though the tail-flick reflex is modulated by descending, supraspinal fibers (e.g., Watkins et al. 19841, both the afferent and efferent neurocircuitry involved in initiating and executing the reflex is located exclusively within the spinal cord (Grossman et al. 1982). Consquently, this response is considered to be relatively impervious to alterations in general activity levels. As such, the finding that morphine is less able to modify the behavioral indices of thermal nociception in both the tail-flick and the hotplate tests suggests that the effects observed are not the result of changes in sedation and/or motor function. Given the wealth of evidence (see Introduction) indicating that morphine-induced antinociception is modulated by gonadal hormones, it is not unreasonable to advance the hypothesis that the hyporesponsiveness to morphine seen in midlactation is best attributed to the hormonal state correlated with lactation. This conclusion is supported by the results of Exp. 4, in which dams with galactophore cuts were equally hyporesponsive to morphine in comparison to lactating, sham-operated dams. This result suggests that the hypor~sponsiveness to morphine is not attributable to the increased metabolic demands that milk production places upon the dam. More importantly, since galactophorecut dams that continue to receive suckling stimulation from their pups are maintained in a similar hormonal status as lactating dams (Desclin 1947), this result is consistent with the hypothesis that the hormonal correlates of lactation are responsible for the effect. This possibility was further corroborated by the results of Exp. 5. The principal finding from this experiment was that removal of the suckling stimulus (by removing the litter) for 96 h was sufficient to reinstate responsivity to morphine, but only in those animals that had returned to cyclic&y. Dams that remained in lactational diestrous following litter removal did not respond to morphine administration. Thus, it appears that suckhng-induced maintenance of the hormonal state characteristic of lactation is responsible for the reduced sensitivity to morphine. The time course of recovery from the suppression of the response to morphine’s hypoalgesic effect is very similar to that reported for the suppression of the

response to the prolactin-stimulating effects of morphine reported by Callahan et al. (1988b). These investigators also found that 96 h of pup separation was sufficient to restore the prolactin-stimulating effect of morphine in about 50% of previously lactating females. Although vaginal smears were not taken in the latter study it is tempting to speculate that the difference between the 2 subsets of animals was that one group had experienced a vaginal estrous and the other had not. The occurrence of vaginal estrous is dependent upon increases in circulating estradiol levels (Feder 1981). This does not necessarily mean, however, that the rise in plasma estradiol levels is itself responsible for the difference in response to morphine administration between the LR-96h-E and LR-96h-LD groups of Exp. 5. Suckling stimulation from the pups maintains high circulating levels of prolactin, adrenocorticotrophic hormone and glucocorticoids (Voogt et al. 1969; Simpson et al. 1973) and low Iuteinizing hormone and estrogen levels in lactating female rats. Prolactin itself appears to maintain the corpora lutea of lactation and hence the increased circulating progesterone levels typical of lactating females (Tomogane et al. 1975). Prolactin levels have been reported to fall very rapidly following pup removal, which results in the involution of the corpora Iutea and a fall in progesterone levels. In parallel with these events there is an increase in pituitary gonadotropin hormone-releasing hormone receptor density (Smith and Lee 1989) and a consequent increase in luteinizing hormone and estrogen levels (Smith and Neil1 1977). The appearance of an estroustype vaginal smear, that is one in which the dominant cell type is cornified epithelial cells, is an indication of a decrease in prolactin and progesterone levels as well as an increase in estrogen levels. Which of these hormonal events restores the response to morphine is not presently known. In fact, it is possible that gonadal hormone modulation of opiate antinociception may be attributable to a relationship not previously considered. In previous studies the simple presence or absence of a particular hormone (most often estrogen or progesterone) has been manipulated, suggesting it is the presence of a given hormone that is responsible for modulation of opiate function, The data reported here suggest that the relationship between hormonal status and the response to morphine may be more complex. For example, in early lactation progesterone levels are high and estrogen levels are low. A similar pattern of response is seen on day 12 of lactation, yet the response to morphine differs in animals tested at these stages of lactation. Similarly, pregnancy is a period of high progesterone levels and it has been shown that endogenous opioid systems are potentiated at this time (Gintzler 1980; Baron and Gintzler 1984, 1987). It would seem, therefore, that inhibition of

opiate function may not always depend simply on the presence or absence of a given hormone, but on the overall pattern and duration of hormonal secretions to which the animal has been exposed. Experiments assessing the relative influence of individual hormones, and the combination of multiple hormones, on the efficacy of morphine-induced hypoalgesia are currently in progress. It is unclear at the present time how the hormonal state observed during lactation inhibits the hypoalgesic response to morphine. There are at least two possibilities. First, lactation may alter the metabolism and disposition of the drug. This possibility is suggested by the results from Exp. 2. In this study, lactating females displayed a hypoalgesic response similar to Iitter-removed females 15 min postadministration. but a blunted response at the later assessment intervals (30 and 45 min). This time course could be explained by the assumption that morphine is more rapidly metabolized and disposed of in lactating dams. The second possibility is that the hormonal state observed during lactation may alter opiate receptor density. As mentioned earlier, changes in receptor density have been correlated with changes in gonadal status (Hammer and Bridges 1987; Weiland and Wise 1990; Hammer et al. 1992). Moreover, there is evidence to suggest that both I*- and &receptor function is altered during lactation (Callahan et al. 1988b; Janik et al. 19931, and variations in gonadal status have also been shown to influence K-receptor-mediated effects (Sander et ai. 1988, 1989). Although morphine is most selective for the p-receptor, this ligand also exhibits affinity for both the 6- and the K-receptor subtypes (Pasternak and Wood 1986). Thus, further research will be required to address the issue of whether the reduction in sensitivity to morphine observed during lactation can be accounted for by a change in opiate receptor density, as well as the sub-type of opiate receptor that may be affected.

This work was supported by grants from the Medical Research Council of Canada (BCW) and the Natural Sciences and Engineering Council of Canada (JR). JR is a research scholar of the Fonds pour la Recherche en SantC du QuCbec. BAL is now at Nova University, Ffa. We thank Cheryl Renaud and Alfonso Abizaid for their expert technical assistance.

References Banejee, P., Chatterjee, T. and Ghosh, J.J., Ovarian steroids and modulation of mo~hine-induced anafgesia and catalepsy in female rats. Eur. J. Pharmacoi., 96 (1983) 291-294.

Harden. N., Merand. Y., Rouleau, D., Garon, M. and Dupont, A., Changes in @-endorphin content in discrete hy~thaiami~ nuclei during the estrous cycle of the rat, Brain Res., 204 (1981) 441-445. Baron, S.A. and Gintzier. A.R., Pregnant-induced analgesia: effects of adrenalectomy and glucocorticoid replacement, Brain Reh., 321 (1984) 341-346. Baron, S.A. and Gintzier, A.R.. Effects of hypophysectomy and dexamethasone treatment on plasma /3-endorphin and pain threshold during pregnancy. Brain Res., 418 (t987) 138-145. Berglund. L.A. and Simpkins, J.W., Alterations in brain opiate receptor mechanisms on proestrus afternoon, Neuroendocrinology, 48 (1988) 394-400. Berglund. L.A., Derendorf, H. and Simpkins, J.W., Desensitization of brain opiate receptor mechanisms by gonadal steroid treatments that stimulate luteinizing hormone secretion, Endocrinoiogy, 122 (198% 27f8-2726. Bodnar. R.J., Romero, M.T. and Kramer, E., Organismic variables and pain inhibition: roles of gender and aging. Brain Res. Bull.. 21 (1988) 947-953. Bridges, R.S. and Ronsheim, P.M., lmmunoreactive ,?I-endorphin concentrations in brain and plasma during pregnancy in rats: possible modulation by progesterone and estradioi. Neuroendocrinoiogy, 45 (1987) 381-388. Brody. S., Riggs, J., Kaufman, K. and Herring, V., Energy metabolism levels during gestation, lactation and post-lactation rest. Res. Bull. 281, University of Missouri Agriculutural Experimental Station, 1938. Callahan, P., Janik, J.. Grandison, L. and Rabii, J., Morphine does not stimulate proiactin release during lactation, Brain Res., 442 (1988a) 214-222. Callahan, P., Janik, J. and Rabii, J., Time course of t!le insensitivity of prolactin release to morphine administration in the Iactating female rat, Life Sci., 43 (1988b) 49-57. Chatterjee, T.K., Das, S., Banerjee, P. and Ghosh, J.J., Possible physiological role for adrenal and gonadal steroids in morphine analgesia, Eur. J. Pharmaco!., 77 (1982) 119-121. Desciin. A.. Concerning the mechanism of diestrum during lactation in the albino rat, Endocrinology, 40 (1947) 14-29. Feder, H.H., Estrous cyclic& in mammals. In: N.A. Adler (Ed.!, Neuroendocrinology of Reproduction: Physiology and Behavior. Plenum Press, New York, 1981, pp. 279-348. Ford, J.J. and Meiampy, R.M., Gonadotropin levels in lactating rats. Effect of ovariectomy, Endocrinology, 93 (1973) 540-547. Fox, S.R. and Smith, M.S., The suppression of puisatiie iuteinizin~ hormone secretion during lactation in the rat, Endocrinoio~, tlS (1984) 2045-2051. Gintzier. A.R., Endorphin-mediated increases in pain threshold during pregnancy, Science, 210 (1980) 193-195. Grossman. M.L., Basbaum, A.I. and Fields, H.L., Afferent and cfferent connections of the rat tail flick reflex (a mode! used to analyze pain control mechanisms), J. Comp. Neuroi., 206 (1982) O-16. Greta, L. and Eik-Nes, K., Plasma progesterone concentrations during pregnancy and lactation in the rat, J. Reprod. Fert., 13 (lY67) 83-91. Hammer, R.P. and Bridges, R.S., Preoptic area opioids and opiate receptors increase during pregnancy and decrease during iactation, Brain Res., 420 (1987) 48-56. Hammer, R.P., Mateo, A.R. and Bridges, R.S., Hormonal regulation of medial preoptic p-opiate receptor density before and after parturition, Neuroendocrinoiogy, 56 (1992) 38-45. Islam, A.K., Cooper, M.L. and Bodnar, R.J., Interactions among aging, gender, and gonadectomy effects upon morphine antinociception in rats, Physioi. Behav.. 54 (1993) 45-53. Janik, J., Callahan, P. and Rabii, J., Morphine induced analgesia is attenuated in post-partum lactating rats, Life Sci., 52 (1993) ‘71-279.

217 Kepler, K.L.. Kest, B., Kiefel, J.M., Cooper, M.L., Bodnar, R.J. Roles of gender, gonadectomy and estrous phase in the analgesic effects of intracerebroventricular morphine in rats, Pharmacol. Biochem. Behav., 34 (1989) 119-127. Kepler, K.L., Standifer, KM., Paul, D., Pasternak, G.W., Kest, B. and Bodnar, R.J., Differential gender effects upon central opioid analgesia, Pharmacol. Biochem. Behav., 34 (1991) 87-95. Medina, V.M., Wang, L. And Gintzler, A.R., Spinal cord dynorphin: positive region-specific modulation during pregnancy and parturition, Brain Res., 623 (1993) 41-46. Moltz, H., Levin, R. and Leon, M., Profactin in the postpa~um rat: synthesis and release in the absence of suckling stimulation, Science, 163 (1969) 1083-1084. O’Callaghan, J.P. and Holtzman, S.G., Quantification of the analgesic activity of narcotic antagonists by a modified hot-plate procedure, J. Pharmacol. Exp. Ther., 192 (1975) 497-505. Ota, K. and Yokayama, A., Body weight and food consumption of lactating rats nursing various sizes of litters, J. Endocrinol., 38 (1967) 263-268. Pasternak, G.W. and Wood, P.L., Multiple mu opiate receptors, Life Sci., 38 (1986) 1889-1898. Rochford, J. and Stewart, J., Activation and expression of endogenous pain control mechanisms in rats given repeated nociceptive tests under the influence of naloxone, Behav. Neurosci., 101 (1987) 87-103. Romero, M.T., Cooper, M.L., Komisaruk, B.R. and Bodnar, R.J. Gender-specific and gonadectomy-specific effects upon swim analgesia: role of steroid replacement therapy, Physiol. Behav., 44 (1988) 257-265. Romero, M.T., Kepler, K.L., Cooper, M.L., Komisaruk, B.R. and Bodnar, R.J., Modulation of gender-specific effects upon swimanalgesia in gonadectomized rats, Physiol. Behav., 40 (1987) 39-45. Ryan, S.M. and Maier, S.F., The estrous cycle and estrogen modulate stress-induced analgesia, Behav. Neurosci.. 102 (1988) 371380.

Sander, H.W., Kream, R.M. and Gintzler, A.R., Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera, Eur. J. Pharmacol., 159 (1989) 205-209. Sander, H.W., Portoghese, P.S. and Gintzler, A.R., Spinal K-opiate receptor involvement in the analgesia of pregnancy: effects of intrathecal nor-binaltophimine, a K-selective antagonist, Brain Res., 474 (1988) 343-347. Simpson, A.A., Simpson, M.H.W., Sinha, Y.N. and Schmidt, G.H. Changes in concentration of prolactin and adrenal corticosterosteroids in rat plasma during pregnancy and lactation, J. Endocrinol., 58 (1973) 675-676. Smith, MS. and Lee, L.R., Modulation of pituitary gonadotropin-releasing hormone receptors during lactation in the rat, Endocrinology, 124 (1989) 1456-1461. Smith, MS and Neill, J.D., Inhibition of gonadotropin secretion during lactation in the rat: relative contribution of suckling and ovarian steroids. Biol. Repro., 17 (1977) 255-261. Tomogane, H., Ota, K. and Yokoyama, A., Suppression of progesterone secretions in lactating rats by administration of egocornine and effect of prolactin replacement, J. Reprod. Fertil., 47 (1975) 347-349. Voogt, J.L., Sar, M. and Meites, J., Influence of cycling, pregnancy, labor and suckling on corticosterone-ACTH levels, Am. J. Physiol., 216 (1969) 655-658. Wardlaw, S.L. and Frantz, A.G., Brain ~-endorphin during pregnancy, parturition and the postpartum period, Endocrinology, 113 (1983) 1664-1668. Watkins, L.R., Drugan, R., Hyson, R.L., Moye, T.B., Ryan, SM., Mayer, D.J. and Maier, S.F., Opiate and nonopiate analgesia induced by inescapable footshock: effects of dorsolateral funiculus lesions and decerebration, Brain Res., 291 (1984) 325-336. Wieland, N.G. and Wise, P.M., Estrogen and progesterone regulate opiate receptor densities in multiple brain regions, Endocrinology, 126 (1990) 804-808. Winer, B.J., Statistical Principles in Experimental Design, 2nd edn, McGraw-Hill, New York, 1971, 438 pp.