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The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat
Neurotoxicologyand Teratology, Vol. 13, pp. 161-166. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
The Effect of Prenatal Methadone Exposure on Development and Nociception During the Early Postnatal Period of the Rat E. K A R L E N T E R S , H O N G Z H I G U O , U S H A P A N D E Y , D A I J I N K O A N D S U S A N E. R O B I N S O N 1
Department of Pharmacology and Toxicology, Medical College of Virginia Virginia Commonwealth University, Richmond, VA 23298-0613 R e c e i v e d 6 A u g u s t 1990
ENTERS, E. K., H.-Z. GUO, U. PANDEY, D. KO AND S. E. ROBINSON. The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat. NEUROTOXICOL TERATOL 13(2) 161-166, 1991.--The effects of prenatal exposure to methadone via Alzet osmotic minipump on early postnatal development and on nociceptive behavioral endpoints were assessed in Sprague-Dawley rat pups during the first three postnatal weeks. This treatment regimen appeared to produce no maternal toxicity, with dams developing and maintaining dependence upon methadone through parturition. Methadoneexposed dams exhibited a withdrawal syndrome consisting of wet-dog shakes, diarrhea, vocalizations and irritability when challenged with naloxone 24 h postpartum. Pups exhibited a similar withdrawal syndrome following naloxone challenge consisting of mouthing and licking, hyperactive response to touch and vocalizations 24 h postpartum. Although no significant difference in litter size was evident in methadone-treated litters, a 16% pup mortality rate was observed in these litters. Prenatal methadone-exposed pups exhibited a significant body weight reduction at birth that resolved by postnatal day 2 (P2) in males and P4 in females. Methadone-exposed pups exhibited significant developmental delay in the expression of the negative geotaxic response and ability to perform a vertical screen task. On P4 methadone-exposed pups exhibited enhanced analgesic response to a morphine challenge while, conversely, 21-day-old pups exhibited a significantly reduced analgesic response to this challenge. These studies indicate that this method of prenatal exposure to methadone can produce dependence in the dam and offspring without substantial mortality, induce developmental delay and alter analgesic responses to opiate challenge in exposed pups during the preweanling period. Methadone Analgesia Osmotic minipumps
Developmental toxicity
Behavioral teratology
METHADONE readily crosses the placenta and has been found to concentrate in the fetal brain, delaying postnatal brain growth in the rat (21, 30-32). It is well accepted that infants born to mothers maintained on methadone have low birth weights and undergo an abstinence syndrome (2, 4, 12, 18). These same findings are duplicated in animal studies: prenatal exposure to methadone produces behavioral changes suggestive of neonatal abstinence (10,24) followed by a more protracted disturbance in rest-activity cycles (11). Even more interesting and potentially of greater social impact are reports that adult rats exposed prenatally to opioids, including methadone, demonstrate a difference in sensitivity to centrally acting &rugs with abuse potential and, in one study, enhanced self-administration of morphine (8, 28, 33). Some problems are associated with the dosing regimens used in the above studies, as the plasma half-life of methadone is much shorter in the rat than in the human (19). Many of the above studies involve once daily opiate administration, which may in-
Morphine
Naloxone
crease maternal morbidity and possibly induce repeated fetal withdrawal. Attempts to circumvent this problem by increasing the size of the dose have increased maternal mortality, or by modifying the dosing regimen have resulted in a reduction in litter size, at best (1,9). However, the use of an osmotic minipump to deliver methadone continually to pregnant rats has recently been reported (29). Nociceptive challenges have been shown to be reliable behavioral indices of opiate activity during the early postnatal period (5,25). The present paper reports the effects of prenatal exposure to methadone via osmotic minipump on nociceptive behavioral endpoints in 4- and 21-day-old rat pups. METHOD Nulliparous 100-day-old female Sprague-Dawley rats (Dominion Labs, Dublin, VA) weighing 240-270 g were placed with male rats of the same strain for breeding. Mating was established
1Requests for reprints should be addressed to Susan E. Robinson, Department of Pharmacology and Toxicology, Box 613, MCV Station, Richmond, VA 23298-0613.
161
162
by the presence of a sperm plug, with this date denoted as day 0 of gestation. Animals were then randomly assigned to the treatment group or one of two control groups, housed individually in plastic breeding cages containing sterile wood chips, and given Purina Lab Chow and water ad lib. Food and water intake, as well as maternal weight, were measured dally. Food pellets were available to the animals in a standard wire hopper placed inside the breeding cage. Using small plastic trays placed under the hoppers, food intake (g/kg/day) was measured correcting for spillage. Water bottles were weighed daily, and intake (g/kg/day) was calculated by subtracting botde weight from the previous day's bottle weight. These data were transformed to ml/kg/day for statistical analysis. On day 8 of gestation, two groups of the female rats were briefly anesthetized with methoxyflurane and implanted subcutaneously with 14-day osmotic minipumps (Alza, Palo Alto, CA) filled with sterile water or methadone hydrochloride (initial dose rate, 9 mg/kg). This dosing regimen has previously been demonstrated to produce maternal dependence without significant maternal mortality (29). An additional group of untreated control dams was also included. Within 24 h of delivery, pups were weighed, sexed, the litter size reduced to 10, and the pups fostered to untreated foster dams. Attempts were made to maintain equivalent numbers of each sex, with the pups falling furthest from the litter mean being those excluded when litters were reduced to 10. Those litters with 8-10 total pups were fostered intact. To avoid potential postnatal nutritional and rearing confounds, litters with fewer than 8 pups were not used in these experiments. After fostering, water- and methadone-treated dams and any culled pups were assessed for dependence by naloxone challenge (2 mg/kg, SC). The water- or methadone-treated dams were euthanized with an overdose of Equithesin and implantation sites counted. Dams in the untreated control group were also used as foster dams; consequently, implantation site data could not be obtained for this control group. Pups were weighed daily, and on either postnatal day 4 or 21, their responses to opiate challenge (morphine: 0.4 mg/kg, SC on P4; 1.5 mg/kg, SC on P21), or blockade (naloxone, 1 mg/kg, SC) in the presence of a nociceptive stimulus were measured by use of the tail-flick test (3). The intensity of the tail-flick stimulus source beam was adjusted for each age of testing to maintain similar baselines allowing for characterizations of both potential increases and decreases in nociceptive responding. The challenge dose of morphine was adjusted to achieve similar levels of analgesia in both P4 and 21-day-old pups. An additional baseline group (saline, 20 ixl, SC) was included for statistical comparisons. Individual pups were used only one time, at one age and in only one drug condition, in the tall-flick test. Pups were kept at a temperature of 37°C at all times during which they were separated from their dams. Those pups not tested until 21 days postnatally were assessed for developmental delay. Beginning on postnatal day 2 (P2), pups were tested for acquisition of selected developmental markers which included: righting reflex, negative geotaxis, vertical screen, eye opening and ear opening (26). Once the animal presented a given marker, testing for that marker was discontinued, and no animal was assessed past P16. Litter data regarding prenatal measures of maternal food and water consumption, and weight were analyzed by repeated measure one-way analysis of variance (ANOVA). Additional postnatal litter data regarding litter size, implantation sites, pup sex, postnatal weights and developmental tasks were also analyzed by one-way ANOVAs. To account for any within-litter variability across either the prenatal treatment or postnatal drug conditions nociceptive data were analyzed using a mixed effects ANOVA model (20). The Tukey-Kramer method (15) was used for post
ENTERS ET AL.
hoc multiple comparisons of treatment means for each drug and sex group. Methadone hydrochloride and morphine sulfate were obtained from the National Institute for Drug Abuse (NIDA) and naloxone hydrochloride was the kind gift of Endo Laboratories (Garden City, NY). RESULTS The initial dose of methadone delivered by the minipump was 9 mg/kg/day; however, by the end of the pregnancy the dose had diminished to 5.6---0.2 mg/kg/day, due to the increase in the weight of the dams. This treatment regimen appeared to produce no maternal toxicity. Dams treated with water or methadone exhibited no significant differences in weight gain or food or water consumption from that of the untreated dams (Fig. 1, Table 1). Dams developed and retained dependence upon methadone through parturition. Dependence was determined in dams and randomly selected pups within 24 hours of delivery. When administered naloxone (2 mg/kg, SC), dams in the methadone group exhibited a withdrawal syndrome consisting of wet-dog shakes, diarrhea, vocalizations and irritability (data not shown). Pups born to the methadone-treated dams similarly underwent naloxone-induced withdrawal, as indicated by hyperactivity, vocalization, tonguing and mouthing behavior (data not shown). In pilot experiments, methadone-exposed pups fostered to nontreated dams exhibited no withdrawal symptoms when tested at 48 h after birth. Pups born in the other two treatment groups had no change in behavior when challenged with naloxone at 24 or 48 h post delivery. There were no differences in litter size or sex distribution among the 3 treatment groups (Table 1). Methadone exposure did not affect the number of implantation sites (Table 1). Although there were no dead pups in the litters born to the untreated and watertreated dams, a small, but significant, number of dead pups were observed in the litters born to the methadone-treated dams [F(2,23) = 6.949, p<0.005; Table 1]. The total mortality rate was 16% in the methadone-treated group, with all deaths occurring on or before day 1. Additionally, the weights of both female pups and male pups in the water and the methadone treatment groups were slightly, but significantly, reduced on the first postnatal day [female: F(2,23)=12.916, p<0.001; male: F(2,23)=8.844, p<0.005; Fig. 2]. By postnatal day 2, the weight of the wateror methadone-treated male pups no longer differed from those of the untreated controls. The weights of the female pups remained significantly reduced through the third postnatal day [day 2: F(2,23) =6.645, p<0.01; day 3: F(2,23)=4.315, p<0.05]. Methadone-exposed pups exhibited significant developmental delay in acquisition of the negative geotaxis reflex when compared to either the untreated or water-exposed pups [F(2,34)= 4.613, p<0.05; Table 2]. Similarly, methadone-exposed pups exhibited a significant delay in ability to perform the vertical screen task when compared to pups of untreated dams [F(2,34) = 5.378, p<0.01; Table 2]. No other differences in physiological or reflexive development were uncovered in our assessments. On postnatal day 4 or 21, the response of the pups to a nociceptive stimulus was tested using the tail-flick test. As expected, morphine administration induced significant analgesia in pups from all three treatment groups at both ages. However, methadone-exposed 4-day-old male pups exhibited a significantly enhanced analgesic response to the morphine challenge (0.4 mg/ kg), with female pups exhibiting a similar, but statistically insignificant, enhanced analgesic response to morphine (Fig. 3). In contrast, methadone-exposed 21-day-old male pups exhibited a significantly reduced analgesic response to the morphine challenge (1.5 mg/kg), with female pups exhibiting a similar, although
PRENATAL METHADONE AND NOCICEPTION
163
TABLE 1 EFFECT OF PRENATALEXPOSURETO METHADONEON DAMS AND PUPS
A.
25-~20-
Treatment (n)
v t-
'~ 15
Control (7)
Water (8)
Methadone (8)
(.-1 •~
Maternal Weight Day 0 % Increase
lO
Q
s Ne
3
6
9
12
15
18
21
BA'40
12.4 ___ 0.6 0
13.4 --- 0.9 0
5.6 _ 0.4 6.9 - 0.8
6.5 -+ 0.9 6.9 +-- 0.6
Implantation Sites Excess Implantation Sites
~35
g
12.2 --- 1.2 2.00 ___ 0.7* 4.7 5.5
--- 0.9 _+ 0.5
14.3 - 1.0
13.8 ± 1.3
0.9 +-- 0.5
1.6 ± 0.7
All data expressed in mean ± SEM. *p<0.05, as compared to control and water by ANOVA and Fisher's lsd test.
82o "O
o O
No. Pups Born Total Dead Live Male Female
253 --- 8.2 270 --- 8.7 258 +-- 4.1 63.7 --- 2.8 57.2 ± 3.6 61.1 ± 4.1
15
U.
10
i
I
i
i
i
i
3
6
9
12
15
18
i
21
controls. Additionally, a main effect of sex was observed at both ages, with males exhibiting shorter latencies at both P4 and P21, when data were pooled across all treatment and drug conditions [F(1,110)=7.39, p < 0 . 0 1 ; F(1,110)=4.02, p < 0 . 0 5 , respectively]. There was no statistical difference in tail-flick latencies following saline (20 I~1, SC) or naloxone (1 mg/kg) in female and male pups from the untreated, water, and methadone groups (Figs. 3 and 4).
C°
DISCUSSION
80" ~
70-
g, 403O 0
|
|
!
!
|
|
|
3
6
9
12
15
18
21
Day of Gestation FIG. 1. (A) Maternal gestational weight gain in grams/kilogram for control (untreated), water-treated and methadone-treated dams. (B) Maternal gestational food consumption in grams/kilogram for control, water- and methadone-treated dams. (C) Maternal gestational water consumption in milliliters/kilogram for all three groups. All data expressed as mean ---SEM. No significant differences were observed. nonsignificant, trend towards a reduced analgesic response to morphine (Fig. 4). Indeed, main effects of prenatal exposure were observed at both ages IF(2,110) = 3.617, p<0.05; F(2,110) = 6.00, p<0.005, respectively], with P4 methadone-exposed pups exhibiting an enhanced analgesic state as compared to pups from untreated litters. Again in contrast, P21 methadone-exposed pups exhibited a reduced analgesic response as compared to untreated
This study conf'Lrms that the osmotic minipump is an effective vehicle for delivery of methadone to pregnant female rats, producing a dependent state without maternal or excessive fetal mortality. The dosage of methadone in the present study was adjusted to the individual dams, so that no dam received more than 9 mg/ kg methadone on the first day of administration; however, by the last day of gestation, the average dose of methadone was 5.64 mg/kg. Using this dosing regimen there were no significant differences among the three treatment groups in the rate of weight gain, water consumption, or food consumption. The methadoneassociated mortality rate in the present study is much lower than that reported by researchers using other routes of administration of methadone (1, 7, 9), supporting the likelihood that the high rates of mortality in studies involving other routes of administration may result from repeated intrauterine withdrawal. Furthermore, in the present study, only a 16% mortality rate was observed in methadone-exposed litters, with no mortality observed after postnatal day 1. We infer that postnatal withdrawal may provide a significant contribution to the mortality rate, as the methadoneexposed pups appear to undergo spontaneous withdrawal during this time. Any acute withdrawal syndrome appears to resolve within 24 h after birth, and no deaths were observed from this time point on. A significant reduction in birth weight was observed in both the water- and methadone-exposed groups in the present study. However, the pup weights returned to control levels by postnatal day 4. The fact that both the water- and the methadone-exposed pups had significantly reduced weights on postnatal day 1 might suggest that at least part of this treatment effect on pup weight
ENTERS ET AL.
164 Female pup weight
4-day-old
Female
A. 8
A. 15 12
6 Q
v
9
[ ] Control [ ] Water [ ] Methadone
Z-
el
6
Q
[ ] saline [ ] naloxone • ~hlne
4
E F-2
3
0 1
2 3 Postnatal Day
control
4
Male pup weight
4-day-old
B. 15
B. 8
12
6
"" O)
"~ 3:
water
methadone
Treatment
Male
O
9
• Control [ ] Water [ ] Methadone
6
4
[] saline
• S I-
I~J naloxone • morphine
3
1
2 3 Postnatal Day
control
4
was related to maternal surgical stress. Methadone-exposed pups exhibited significant developmental delay in expression of negative geotaxis and ability to perform a vertical screen task. Given that only the methadone-exposed pups exhibited this developmen-
TABLE 2 EFFECT OF PRENATAL EXPOSURE TO METHADONE ON DEVELOPMENTAL TASKS
Righting reflex Negative geotaxis Vertical screen Eye opening Ear opening
Control (6) 2.4 8.4 8.3 13.7 13.7
± ± ± ±
0.1 0.3 0.2 0.2 0.2
methadone
Treatment
FIG. 2. Mean postnatal control (untreated), water- and methadone-exposed pup weights in grams (±SEM) from postnatal day 1 (P1) to P4, for (A) females and (B) males. Both female and male water- and methadone-exposed pups were lighter on P1, with these differences resolving by P2 in males, and by P4 in females. *p<0.05 significance as compared to untreated controls by ANOVA and Fisher's lsd test.
Treatment: (n)
water
Water (8) 2.5 8.7 9.0 13.5 13.8
± ± ± ---
0.2 0.4 0.3 0.2 0.2
Methadone (6) 2.7 9.6 9.3 13.4 13.9
- 0.2 ± 0.4* --+ 0.4* ± 0.2 ± 0.3
All data expressed in days until expression - SEM. * p < 0 . 0 5 , as c o m p a r e d to control b y A N O V A a n d Fisher's lsd test.
FIG. 3. Mean tail-flick latencies in seconds (_ SEM) for (A) female and (B) male, 4-day-old control (untreated), water- and methadone-exposed rat pups. All groups showed significant analgesia following morphine administration. Male methadone-exposed pups exhibited a significantly enhanced analgesia to morphine. *p<0.05 significance as compared to within-group saline administration by ANOVA and Tukey-Kramer multiple comparisons. **p<0.05 significance as compared to both within-group saline administration and control morphine responding by ANOVA and Tukey-Kramer multiple comparisons.
tal delay, these deficits appear unrelated to the reductions in body weight observed early in the postnatal period, as water-exposed pups failed to show similar developmental deficits. Indeed, these findings are consistent with other reports that perinatal methadone exposure produces developmental neurobehavioral deficits (23). Prenatal exposure to methadone enhanced the analgesic response to morphine in male pups at P4, with similar trends in female pups, as measured by the tall-flick test. It is possible that this response is related to some amount of methadone remaining in the pups from the maternal exposure. Levitt et al. (15,16) have reported a postnatal persistence of methadone following prenatal exposure during the first postnatal week. Additionally, it is possible that the more robust effects observed in male offspring in this study, and indeed even the main effects of sex observed as well, may be more related to the propensity of dams to give preferentially more care and attention to male pups (22). This increase in attention received from the dams in the first 24 hours postpartum may result in increased nursing behavior, with males taking in more methadone than their female littermates prior to
PRENATAL METHADONE AND NOCICEPTION
165
21-day-old Female A. 7 6 -fO -Q
5
~
4
•E
3
t-
2
[ ] saline [ ] naloxone • morphine
1 0 control
water
methadone
Treatment
21-day-old
Male
B.
~-t) @
5
~4
r'l saline [ ] naloxone • morphine
m )-
2
control
water
methadone
Treatment
FIG. 4. Mean tail-flick latencies in seconds (___SEM) for (A) female and (B) male 21-day-old control (untreated), water- and methadone-exposed rat pups. All groups showed significant analgesia following morphine administration. Male methadone-exposed pups exhibited a significantly reduced analgesic response to morphine. *p<0.05 significance as compared to saline administration by ANOVA and Tukey-Kramer multiple comparisons. **p<0.05 significance as compared to both within-group saline administration and control morphine responding by ANOVA and TukeyKramer multiple comparisons.
fostering. However, in the present study, these methadone-exposed pups did not exhibit an enhanced analgesic response to saline, which might be expected if they retained methadone from the prenatal exposure or early postpartum period, nor did they differ from control groups when challenged by naloxone. In contrast to the enhanced analgesic response to morphine
challenge observed at P4, methadone-exposed male pups exhibited a significantly reduced analgesic respose to this challenge at P21, with females reflecting a similar trend. These findings are in opposition to those of other researchers who have reported enhanced sensitivity to opiates in weanling rats exposed to methadone perinatally (32,33). Using a hot-plate measure of analgesia, Zagon and McLaughlin found that adult males which were exposed to methadone prenatally exhibited an enhanced analgesic response to morphine. Additionally, adult females which were exposed to methadone prenatally exhibited longer latencies in the hot-plate test with no drug treatment. These same researchers demonstrated that both male and female rats exposed to methadone prenatally exhibit protracted analgesia over the ages of 21 days to 120 days in the hot-plate test (31). It is difficult to appraise our 21-day-old results in light of these previous reports, given that these previous investigations used both different regimens of prenatal exposure, as well as a different nociceptive measure. Several tenable hypotheses might be considered. Given previous observations of alterations in spinal cord opiate receptors and spinal mediation of tail-flick response following prenatal morphine exposure (13,14), it is possible that the present data reflect exposure-related alterations in nociceptive responding at the spinal level. Conversely, as lesions of hindbrain raphe nuclei have been observed to alter opiate-mediated tail-flick nociception as early as the postnatal period (6), it is possible that the present data are reflecting exposure-related supraspinal alterations. Indeed, some recent reports of receptor alterations following prenatal opiate exposure support the possibility that responses to nociceptive stimuli might be altered in the direction of reduced analgesic response to opiate challenge. Wang et al. (29) have reported that prenatal exposure to methadone results in persistent reductions in the number of I• and ~ receptors in the hypothalamus and the cerebral cortex. Similar results have been reported for Ix receptors in pups exposed prenatally to morphine; however, these receptor alterations appeared to be more transient, resolving during the third postnatal week (27). However, these studies do not rule out the possibility that the opioids administered prenatally may remain in the brain and interfere with the binding of ligands to the receptors. Taken together, these receptor alterations along with the methodology issues noted above might support the contention that nascent neural systems are sensitive to subtle variations in either teratogen exposure or neurobehavioral/neuroanatomicai assessment or both. Nevertheless, the mechanism of action of the reduced analgesic response in the present study remains unclear. In summary, we have found that prenatal exposure to methadone administered to the dam via osmotic minipumps produces little effect on maternal health or litter size but is sufficient to produce dependence in both the dam and pups. Furthermore, methadone-exposed pups exhibited significant developmental delay, an enhanced analgesic response to morphine at P4, and a reduced analgesic response to morphine at 21 days postnatally. ACKNOWLEDGEMENT
These studies were supported in part by NIDA grant No. DA05274.
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0892-0362/91 $3.00 + .00
The Effect of Prenatal Methadone Exposure on Development and Nociception During the Early Postnatal Period of the Rat E. K A R L E N T E R S , H O N G Z H I G U O , U S H A P A N D E Y , D A I J I N K O A N D S U S A N E. R O B I N S O N 1
Department of Pharmacology and Toxicology, Medical College of Virginia Virginia Commonwealth University, Richmond, VA 23298-0613 R e c e i v e d 6 A u g u s t 1990
ENTERS, E. K., H.-Z. GUO, U. PANDEY, D. KO AND S. E. ROBINSON. The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat. NEUROTOXICOL TERATOL 13(2) 161-166, 1991.--The effects of prenatal exposure to methadone via Alzet osmotic minipump on early postnatal development and on nociceptive behavioral endpoints were assessed in Sprague-Dawley rat pups during the first three postnatal weeks. This treatment regimen appeared to produce no maternal toxicity, with dams developing and maintaining dependence upon methadone through parturition. Methadoneexposed dams exhibited a withdrawal syndrome consisting of wet-dog shakes, diarrhea, vocalizations and irritability when challenged with naloxone 24 h postpartum. Pups exhibited a similar withdrawal syndrome following naloxone challenge consisting of mouthing and licking, hyperactive response to touch and vocalizations 24 h postpartum. Although no significant difference in litter size was evident in methadone-treated litters, a 16% pup mortality rate was observed in these litters. Prenatal methadone-exposed pups exhibited a significant body weight reduction at birth that resolved by postnatal day 2 (P2) in males and P4 in females. Methadone-exposed pups exhibited significant developmental delay in the expression of the negative geotaxic response and ability to perform a vertical screen task. On P4 methadone-exposed pups exhibited enhanced analgesic response to a morphine challenge while, conversely, 21-day-old pups exhibited a significantly reduced analgesic response to this challenge. These studies indicate that this method of prenatal exposure to methadone can produce dependence in the dam and offspring without substantial mortality, induce developmental delay and alter analgesic responses to opiate challenge in exposed pups during the preweanling period. Methadone Analgesia Osmotic minipumps
Developmental toxicity
Behavioral teratology
METHADONE readily crosses the placenta and has been found to concentrate in the fetal brain, delaying postnatal brain growth in the rat (21, 30-32). It is well accepted that infants born to mothers maintained on methadone have low birth weights and undergo an abstinence syndrome (2, 4, 12, 18). These same findings are duplicated in animal studies: prenatal exposure to methadone produces behavioral changes suggestive of neonatal abstinence (10,24) followed by a more protracted disturbance in rest-activity cycles (11). Even more interesting and potentially of greater social impact are reports that adult rats exposed prenatally to opioids, including methadone, demonstrate a difference in sensitivity to centrally acting &rugs with abuse potential and, in one study, enhanced self-administration of morphine (8, 28, 33). Some problems are associated with the dosing regimens used in the above studies, as the plasma half-life of methadone is much shorter in the rat than in the human (19). Many of the above studies involve once daily opiate administration, which may in-
Morphine
Naloxone
crease maternal morbidity and possibly induce repeated fetal withdrawal. Attempts to circumvent this problem by increasing the size of the dose have increased maternal mortality, or by modifying the dosing regimen have resulted in a reduction in litter size, at best (1,9). However, the use of an osmotic minipump to deliver methadone continually to pregnant rats has recently been reported (29). Nociceptive challenges have been shown to be reliable behavioral indices of opiate activity during the early postnatal period (5,25). The present paper reports the effects of prenatal exposure to methadone via osmotic minipump on nociceptive behavioral endpoints in 4- and 21-day-old rat pups. METHOD Nulliparous 100-day-old female Sprague-Dawley rats (Dominion Labs, Dublin, VA) weighing 240-270 g were placed with male rats of the same strain for breeding. Mating was established
1Requests for reprints should be addressed to Susan E. Robinson, Department of Pharmacology and Toxicology, Box 613, MCV Station, Richmond, VA 23298-0613.
161
162
by the presence of a sperm plug, with this date denoted as day 0 of gestation. Animals were then randomly assigned to the treatment group or one of two control groups, housed individually in plastic breeding cages containing sterile wood chips, and given Purina Lab Chow and water ad lib. Food and water intake, as well as maternal weight, were measured dally. Food pellets were available to the animals in a standard wire hopper placed inside the breeding cage. Using small plastic trays placed under the hoppers, food intake (g/kg/day) was measured correcting for spillage. Water bottles were weighed daily, and intake (g/kg/day) was calculated by subtracting botde weight from the previous day's bottle weight. These data were transformed to ml/kg/day for statistical analysis. On day 8 of gestation, two groups of the female rats were briefly anesthetized with methoxyflurane and implanted subcutaneously with 14-day osmotic minipumps (Alza, Palo Alto, CA) filled with sterile water or methadone hydrochloride (initial dose rate, 9 mg/kg). This dosing regimen has previously been demonstrated to produce maternal dependence without significant maternal mortality (29). An additional group of untreated control dams was also included. Within 24 h of delivery, pups were weighed, sexed, the litter size reduced to 10, and the pups fostered to untreated foster dams. Attempts were made to maintain equivalent numbers of each sex, with the pups falling furthest from the litter mean being those excluded when litters were reduced to 10. Those litters with 8-10 total pups were fostered intact. To avoid potential postnatal nutritional and rearing confounds, litters with fewer than 8 pups were not used in these experiments. After fostering, water- and methadone-treated dams and any culled pups were assessed for dependence by naloxone challenge (2 mg/kg, SC). The water- or methadone-treated dams were euthanized with an overdose of Equithesin and implantation sites counted. Dams in the untreated control group were also used as foster dams; consequently, implantation site data could not be obtained for this control group. Pups were weighed daily, and on either postnatal day 4 or 21, their responses to opiate challenge (morphine: 0.4 mg/kg, SC on P4; 1.5 mg/kg, SC on P21), or blockade (naloxone, 1 mg/kg, SC) in the presence of a nociceptive stimulus were measured by use of the tail-flick test (3). The intensity of the tail-flick stimulus source beam was adjusted for each age of testing to maintain similar baselines allowing for characterizations of both potential increases and decreases in nociceptive responding. The challenge dose of morphine was adjusted to achieve similar levels of analgesia in both P4 and 21-day-old pups. An additional baseline group (saline, 20 ixl, SC) was included for statistical comparisons. Individual pups were used only one time, at one age and in only one drug condition, in the tall-flick test. Pups were kept at a temperature of 37°C at all times during which they were separated from their dams. Those pups not tested until 21 days postnatally were assessed for developmental delay. Beginning on postnatal day 2 (P2), pups were tested for acquisition of selected developmental markers which included: righting reflex, negative geotaxis, vertical screen, eye opening and ear opening (26). Once the animal presented a given marker, testing for that marker was discontinued, and no animal was assessed past P16. Litter data regarding prenatal measures of maternal food and water consumption, and weight were analyzed by repeated measure one-way analysis of variance (ANOVA). Additional postnatal litter data regarding litter size, implantation sites, pup sex, postnatal weights and developmental tasks were also analyzed by one-way ANOVAs. To account for any within-litter variability across either the prenatal treatment or postnatal drug conditions nociceptive data were analyzed using a mixed effects ANOVA model (20). The Tukey-Kramer method (15) was used for post
ENTERS ET AL.
hoc multiple comparisons of treatment means for each drug and sex group. Methadone hydrochloride and morphine sulfate were obtained from the National Institute for Drug Abuse (NIDA) and naloxone hydrochloride was the kind gift of Endo Laboratories (Garden City, NY). RESULTS The initial dose of methadone delivered by the minipump was 9 mg/kg/day; however, by the end of the pregnancy the dose had diminished to 5.6---0.2 mg/kg/day, due to the increase in the weight of the dams. This treatment regimen appeared to produce no maternal toxicity. Dams treated with water or methadone exhibited no significant differences in weight gain or food or water consumption from that of the untreated dams (Fig. 1, Table 1). Dams developed and retained dependence upon methadone through parturition. Dependence was determined in dams and randomly selected pups within 24 hours of delivery. When administered naloxone (2 mg/kg, SC), dams in the methadone group exhibited a withdrawal syndrome consisting of wet-dog shakes, diarrhea, vocalizations and irritability (data not shown). Pups born to the methadone-treated dams similarly underwent naloxone-induced withdrawal, as indicated by hyperactivity, vocalization, tonguing and mouthing behavior (data not shown). In pilot experiments, methadone-exposed pups fostered to nontreated dams exhibited no withdrawal symptoms when tested at 48 h after birth. Pups born in the other two treatment groups had no change in behavior when challenged with naloxone at 24 or 48 h post delivery. There were no differences in litter size or sex distribution among the 3 treatment groups (Table 1). Methadone exposure did not affect the number of implantation sites (Table 1). Although there were no dead pups in the litters born to the untreated and watertreated dams, a small, but significant, number of dead pups were observed in the litters born to the methadone-treated dams [F(2,23) = 6.949, p<0.005; Table 1]. The total mortality rate was 16% in the methadone-treated group, with all deaths occurring on or before day 1. Additionally, the weights of both female pups and male pups in the water and the methadone treatment groups were slightly, but significantly, reduced on the first postnatal day [female: F(2,23)=12.916, p<0.001; male: F(2,23)=8.844, p<0.005; Fig. 2]. By postnatal day 2, the weight of the wateror methadone-treated male pups no longer differed from those of the untreated controls. The weights of the female pups remained significantly reduced through the third postnatal day [day 2: F(2,23) =6.645, p<0.01; day 3: F(2,23)=4.315, p<0.05]. Methadone-exposed pups exhibited significant developmental delay in acquisition of the negative geotaxis reflex when compared to either the untreated or water-exposed pups [F(2,34)= 4.613, p<0.05; Table 2]. Similarly, methadone-exposed pups exhibited a significant delay in ability to perform the vertical screen task when compared to pups of untreated dams [F(2,34) = 5.378, p<0.01; Table 2]. No other differences in physiological or reflexive development were uncovered in our assessments. On postnatal day 4 or 21, the response of the pups to a nociceptive stimulus was tested using the tail-flick test. As expected, morphine administration induced significant analgesia in pups from all three treatment groups at both ages. However, methadone-exposed 4-day-old male pups exhibited a significantly enhanced analgesic response to the morphine challenge (0.4 mg/ kg), with female pups exhibiting a similar, but statistically insignificant, enhanced analgesic response to morphine (Fig. 3). In contrast, methadone-exposed 21-day-old male pups exhibited a significantly reduced analgesic response to the morphine challenge (1.5 mg/kg), with female pups exhibiting a similar, although
PRENATAL METHADONE AND NOCICEPTION
163
TABLE 1 EFFECT OF PRENATALEXPOSURETO METHADONEON DAMS AND PUPS
A.
25-~20-
Treatment (n)
v t-
'~ 15
Control (7)
Water (8)
Methadone (8)
(.-1 •~
Maternal Weight Day 0 % Increase
lO
Q
s Ne
3
6
9
12
15
18
21
BA'40
12.4 ___ 0.6 0
13.4 --- 0.9 0
5.6 _ 0.4 6.9 - 0.8
6.5 -+ 0.9 6.9 +-- 0.6
Implantation Sites Excess Implantation Sites
~35
g
12.2 --- 1.2 2.00 ___ 0.7* 4.7 5.5
--- 0.9 _+ 0.5
14.3 - 1.0
13.8 ± 1.3
0.9 +-- 0.5
1.6 ± 0.7
All data expressed in mean ± SEM. *p<0.05, as compared to control and water by ANOVA and Fisher's lsd test.
82o "O
o O
No. Pups Born Total Dead Live Male Female
253 --- 8.2 270 --- 8.7 258 +-- 4.1 63.7 --- 2.8 57.2 ± 3.6 61.1 ± 4.1
15
U.
10
i
I
i
i
i
i
3
6
9
12
15
18
i
21
controls. Additionally, a main effect of sex was observed at both ages, with males exhibiting shorter latencies at both P4 and P21, when data were pooled across all treatment and drug conditions [F(1,110)=7.39, p < 0 . 0 1 ; F(1,110)=4.02, p < 0 . 0 5 , respectively]. There was no statistical difference in tail-flick latencies following saline (20 I~1, SC) or naloxone (1 mg/kg) in female and male pups from the untreated, water, and methadone groups (Figs. 3 and 4).
C°
DISCUSSION
80" ~
70-
g, 403O 0
|
|
!
!
|
|
|
3
6
9
12
15
18
21
Day of Gestation FIG. 1. (A) Maternal gestational weight gain in grams/kilogram for control (untreated), water-treated and methadone-treated dams. (B) Maternal gestational food consumption in grams/kilogram for control, water- and methadone-treated dams. (C) Maternal gestational water consumption in milliliters/kilogram for all three groups. All data expressed as mean ---SEM. No significant differences were observed. nonsignificant, trend towards a reduced analgesic response to morphine (Fig. 4). Indeed, main effects of prenatal exposure were observed at both ages IF(2,110) = 3.617, p<0.05; F(2,110) = 6.00, p<0.005, respectively], with P4 methadone-exposed pups exhibiting an enhanced analgesic state as compared to pups from untreated litters. Again in contrast, P21 methadone-exposed pups exhibited a reduced analgesic response as compared to untreated
This study conf'Lrms that the osmotic minipump is an effective vehicle for delivery of methadone to pregnant female rats, producing a dependent state without maternal or excessive fetal mortality. The dosage of methadone in the present study was adjusted to the individual dams, so that no dam received more than 9 mg/ kg methadone on the first day of administration; however, by the last day of gestation, the average dose of methadone was 5.64 mg/kg. Using this dosing regimen there were no significant differences among the three treatment groups in the rate of weight gain, water consumption, or food consumption. The methadoneassociated mortality rate in the present study is much lower than that reported by researchers using other routes of administration of methadone (1, 7, 9), supporting the likelihood that the high rates of mortality in studies involving other routes of administration may result from repeated intrauterine withdrawal. Furthermore, in the present study, only a 16% mortality rate was observed in methadone-exposed litters, with no mortality observed after postnatal day 1. We infer that postnatal withdrawal may provide a significant contribution to the mortality rate, as the methadoneexposed pups appear to undergo spontaneous withdrawal during this time. Any acute withdrawal syndrome appears to resolve within 24 h after birth, and no deaths were observed from this time point on. A significant reduction in birth weight was observed in both the water- and methadone-exposed groups in the present study. However, the pup weights returned to control levels by postnatal day 4. The fact that both the water- and the methadone-exposed pups had significantly reduced weights on postnatal day 1 might suggest that at least part of this treatment effect on pup weight
ENTERS ET AL.
164 Female pup weight
4-day-old
Female
A. 8
A. 15 12
6 Q
v
9
[ ] Control [ ] Water [ ] Methadone
Z-
el
6
Q
[ ] saline [ ] naloxone • ~hlne
4
E F-2
3
0 1
2 3 Postnatal Day
control
4
Male pup weight
4-day-old
B. 15
B. 8
12
6
"" O)
"~ 3:
water
methadone
Treatment
Male
O
9
• Control [ ] Water [ ] Methadone
6
4
[] saline
• S I-
I~J naloxone • morphine
3
1
2 3 Postnatal Day
control
4
was related to maternal surgical stress. Methadone-exposed pups exhibited significant developmental delay in expression of negative geotaxis and ability to perform a vertical screen task. Given that only the methadone-exposed pups exhibited this developmen-
TABLE 2 EFFECT OF PRENATAL EXPOSURE TO METHADONE ON DEVELOPMENTAL TASKS
Righting reflex Negative geotaxis Vertical screen Eye opening Ear opening
Control (6) 2.4 8.4 8.3 13.7 13.7
± ± ± ±
0.1 0.3 0.2 0.2 0.2
methadone
Treatment
FIG. 2. Mean postnatal control (untreated), water- and methadone-exposed pup weights in grams (±SEM) from postnatal day 1 (P1) to P4, for (A) females and (B) males. Both female and male water- and methadone-exposed pups were lighter on P1, with these differences resolving by P2 in males, and by P4 in females. *p<0.05 significance as compared to untreated controls by ANOVA and Fisher's lsd test.
Treatment: (n)
water
Water (8) 2.5 8.7 9.0 13.5 13.8
± ± ± ---
0.2 0.4 0.3 0.2 0.2
Methadone (6) 2.7 9.6 9.3 13.4 13.9
- 0.2 ± 0.4* --+ 0.4* ± 0.2 ± 0.3
All data expressed in days until expression - SEM. * p < 0 . 0 5 , as c o m p a r e d to control b y A N O V A a n d Fisher's lsd test.
FIG. 3. Mean tail-flick latencies in seconds (_ SEM) for (A) female and (B) male, 4-day-old control (untreated), water- and methadone-exposed rat pups. All groups showed significant analgesia following morphine administration. Male methadone-exposed pups exhibited a significantly enhanced analgesia to morphine. *p<0.05 significance as compared to within-group saline administration by ANOVA and Tukey-Kramer multiple comparisons. **p<0.05 significance as compared to both within-group saline administration and control morphine responding by ANOVA and Tukey-Kramer multiple comparisons.
tal delay, these deficits appear unrelated to the reductions in body weight observed early in the postnatal period, as water-exposed pups failed to show similar developmental deficits. Indeed, these findings are consistent with other reports that perinatal methadone exposure produces developmental neurobehavioral deficits (23). Prenatal exposure to methadone enhanced the analgesic response to morphine in male pups at P4, with similar trends in female pups, as measured by the tall-flick test. It is possible that this response is related to some amount of methadone remaining in the pups from the maternal exposure. Levitt et al. (15,16) have reported a postnatal persistence of methadone following prenatal exposure during the first postnatal week. Additionally, it is possible that the more robust effects observed in male offspring in this study, and indeed even the main effects of sex observed as well, may be more related to the propensity of dams to give preferentially more care and attention to male pups (22). This increase in attention received from the dams in the first 24 hours postpartum may result in increased nursing behavior, with males taking in more methadone than their female littermates prior to
PRENATAL METHADONE AND NOCICEPTION
165
21-day-old Female A. 7 6 -fO -Q
5
~
4
•E
3
t-
2
[ ] saline [ ] naloxone • morphine
1 0 control
water
methadone
Treatment
21-day-old
Male
B.
~-t) @
5
~4
r'l saline [ ] naloxone • morphine
m )-
2
control
water
methadone
Treatment
FIG. 4. Mean tail-flick latencies in seconds (___SEM) for (A) female and (B) male 21-day-old control (untreated), water- and methadone-exposed rat pups. All groups showed significant analgesia following morphine administration. Male methadone-exposed pups exhibited a significantly reduced analgesic response to morphine. *p<0.05 significance as compared to saline administration by ANOVA and Tukey-Kramer multiple comparisons. **p<0.05 significance as compared to both within-group saline administration and control morphine responding by ANOVA and TukeyKramer multiple comparisons.
fostering. However, in the present study, these methadone-exposed pups did not exhibit an enhanced analgesic response to saline, which might be expected if they retained methadone from the prenatal exposure or early postpartum period, nor did they differ from control groups when challenged by naloxone. In contrast to the enhanced analgesic response to morphine
challenge observed at P4, methadone-exposed male pups exhibited a significantly reduced analgesic respose to this challenge at P21, with females reflecting a similar trend. These findings are in opposition to those of other researchers who have reported enhanced sensitivity to opiates in weanling rats exposed to methadone perinatally (32,33). Using a hot-plate measure of analgesia, Zagon and McLaughlin found that adult males which were exposed to methadone prenatally exhibited an enhanced analgesic response to morphine. Additionally, adult females which were exposed to methadone prenatally exhibited longer latencies in the hot-plate test with no drug treatment. These same researchers demonstrated that both male and female rats exposed to methadone prenatally exhibit protracted analgesia over the ages of 21 days to 120 days in the hot-plate test (31). It is difficult to appraise our 21-day-old results in light of these previous reports, given that these previous investigations used both different regimens of prenatal exposure, as well as a different nociceptive measure. Several tenable hypotheses might be considered. Given previous observations of alterations in spinal cord opiate receptors and spinal mediation of tail-flick response following prenatal morphine exposure (13,14), it is possible that the present data reflect exposure-related alterations in nociceptive responding at the spinal level. Conversely, as lesions of hindbrain raphe nuclei have been observed to alter opiate-mediated tail-flick nociception as early as the postnatal period (6), it is possible that the present data are reflecting exposure-related supraspinal alterations. Indeed, some recent reports of receptor alterations following prenatal opiate exposure support the possibility that responses to nociceptive stimuli might be altered in the direction of reduced analgesic response to opiate challenge. Wang et al. (29) have reported that prenatal exposure to methadone results in persistent reductions in the number of I• and ~ receptors in the hypothalamus and the cerebral cortex. Similar results have been reported for Ix receptors in pups exposed prenatally to morphine; however, these receptor alterations appeared to be more transient, resolving during the third postnatal week (27). However, these studies do not rule out the possibility that the opioids administered prenatally may remain in the brain and interfere with the binding of ligands to the receptors. Taken together, these receptor alterations along with the methodology issues noted above might support the contention that nascent neural systems are sensitive to subtle variations in either teratogen exposure or neurobehavioral/neuroanatomicai assessment or both. Nevertheless, the mechanism of action of the reduced analgesic response in the present study remains unclear. In summary, we have found that prenatal exposure to methadone administered to the dam via osmotic minipumps produces little effect on maternal health or litter size but is sufficient to produce dependence in both the dam and pups. Furthermore, methadone-exposed pups exhibited significant developmental delay, an enhanced analgesic response to morphine at P4, and a reduced analgesic response to morphine at 21 days postnatally. ACKNOWLEDGEMENT
These studies were supported in part by NIDA grant No. DA05274.
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