Effects on Offspring of Chronic Maternal Methamphetamine Exposure JOAN C. MARTIN Department of Psychiatry and Behavioral Sciences Utiiversity of Washington Seattle, Washington
Twenty-five Sprague-Dawley derived rats were administered 1.0, 3.0, 01 5.0 mg/kg of methamphetamine HCI or saline twice daily throughout gestation beginning on Day 1 of pregnancy. Rats were allowed t o deliver normally: offspring were culled to 8 and sexed on Day 7, and weaned on Day 21. All females had viable litters except at the 5.0 mg/kg dose where 4 of 7 failed to deliver. The rats given methamphetamine delivered earlier than did controls. Weight gain over gestation decreased as a function o f increasing drug dose. No gross anomalies were visible in the offspring. Litter size decreased as a function of increased dose and eye opening was delayed in the drug groups: the 5.0 mg/kg offspring made more conditioned avoidance responses than did the 3.0 mg/kg and saline offspring.
Amphetamine, a central nervous system (CNS) stimulant with sympathomimetic properties and a catecholamine-releasing action, owes its behaviorally facilitative action to norepinephrine release (Stein & Wise, 1970). The amphetamines are among the most widely abused of the prescription drugs, with the amount produced yearly variously estimated at 2- 10 billion dosage units (Amphetamine Legislation, 1972). d-Amphetamine, d,l-amphetamine, and methamphetamine are a series of chemically related compounds, which although they differ to some extent structurally, exert similar functional effects (Valzelli. 1973). The terms methamphetamine and amphetamine are therefore used interchangeably in this study. No developmental studies have been found which have contrasted the several compounds. The extent of amphetamine abuse in pregnant women was examined by Nora, Nora, Somerville, Hill, and McNamara (1967), who found that 27% of their sample of 240 gravid females had taken prescription appetite suppressants during at least 1 trimester of their gestational period. If this study is at all indicative of the extent of usage in gravidas, then the potential exposure to human offspring is very high. However, no studies have been located which have examined the physical, developmental, or behavioral consequences to infants and children of maternal ingestion of amphetamines during pregnancy. Middaugh, Blackwell, Sautos, and Zemp (1 974) administered methamphetamine to inice during the 3rd trimester of pregnancy. The offspring exhibited a transient increase in brain catecholamine levels and increased activity in the open field at 30 days of age. Kasirsky and Tansy (1971) administered methamphetamine during the 3rd Keceived for publication 4 June 1974 Revised for publication 7 October 1974 Uevelopnicrital Psychobiology, 8(5): 397-404 (1975) 0 1975 by John Wiley & Sons, Inc.
397
398
MARTIN
trimester of pregnancy to mice and rabbits and noted with higher doses an increase in anomalies (e.g., exencephaly, cleft palate, and eye deformities) in offspring. Because in the above studies the drug was administered only during the last trimester of pregnancy, the results may not be applicable to human prescription use and certainly are not typical of human abuse patterns. The following study was designed to investigate the effects on the offspring of 3 doses of methamphetamine administered over the entire gestational period.
Method Sti bjec ts Subjects were twenty-five 225 -250 g Sprague-Dawley derived, caesarian-borne, barrier-sustained, virgin female rats (Rattus norvegicus; supplied by Tyler Laboratories, Bellevue, Washington) which had been impregnated the previous night.
Procedure The pregnant rats were randomly assigned to 1 of 4 treatment groups: 6 rats in each of 3 groups with 7 in the group given the highest drug dose. Twice daily, at 0830 and 1430 hours, the subjects were given subcutaneous injections. These were begun on’ the morning following impregnation (Day 1) and were terminated on Day 21. All animals were housed individually, with rat chow and water available ad lib. Drug dosage. Methamphetamine HCl (Abbott Laboratories) was dissolved in a .9% isotonic saline solution with the volume held constant in doses of 1.0, 3.0, or 5.0 mg/kg of body weight. The control animals received a comparable volume of the vehicle alone. Because the amphetamines have a half-life of 14 hr in the human (Amphetamine Legislation, 1972), the twice-daily regimen was an attempt to maximize the period that the drug or its metabolites would remain in the organism. The doses are well within the range used in behavioral studies (Fog, 1969; Teitelbaum & Derks, 1958). The 2 higher doses typically resulted in stereotyped head and sniffing movements which last for 3 - 5 hr. Drug injections. Pregnant dams were injected subcutaneously over the lower dorsal surface with a 26%-gauge needle. The volume of fluid was .I25 ml for a 250-g rat, with control animals receiving comparable amounts. Volume was held constant over the 3 dose levels. The rats were weighed daily, but the daily drug dose was based upon the Day 1 weights because this more nearly approximates the pattern in human drug users who probably do not increase a drug dose as their weight increases over pregnancy. Animals were allowed to deliver normally. Offspring were not cross-fostered at birth but were raised by their natural mothers, again, as a model for human drug abuse. Therefore, this design did not allow a separation of fetal vs neonatal factors, but only a comparison of offspring whose dams received the drug with those which received saline injections during the same time period. Offspring were sexed and culled to 8 on Postnatal Day 7, with litters weighed every 7 days thereafter and
MATERNAL METHAMPHETAMINE EXPOSURE
399
weaned on Day 21. Two males were randomly chosen from each litter for behavioral studies on Day 28. Because only 2 litters survived at the highest dose level, 5 males were retained from each of these litters. Developmental measure. Eye opening was assessed on Postnatal Day 14 for each rat in a litter. (The eyes open in the rat between Days 14-17 [Farris & Griffith, 19491 .) Behavioral measure. The offspring were tested at 100-120 days of age in a Lehigh Valley Electronics shuttle-box (#146-04) for acquisition and extinction of the conditioned avoidance response (CAR). The shuttle-box consisted of two 22 x 19 x 19 cm Lucite compartments with a 4%-cm metal divider and a grid floor. A Mallory Sonalert audio oscillator/amplifier at 2.8 kHz and 70 dB was positioned above the divider. Stimulus lamps positioned on the end walls of the box were triggered with the onset of an auditory stimulus and provided an additional cue. A 15-sec interstimulus interval (ISI) was followed by a 3-sec tone-on period followed by .3 mA of continuous shock to the feet. An escape over the barrier terminated shock, tone, and light. An avoidance response reset the IS1 clock. Ninety minutes of acquisition were followed by a 10-min extinction period in which tone and light were operative but not shock. Dependent measures included: (1) avoidance during the pretone period, (2) avoidance during the conditioned stimulus-unconditioned stimulus (CS-UCS) interval, and (3) escape responses. The 1.0-mg/kg group was used to test the equipment and to determine timing parameters, but was then excluded from the statistical analysis. Two saline offspring died in a nonrelated laboratory accident, leaving an n = I0 in each of the 3.0- and 5.0-mg/kg groups and ad n = 8 in the saline control group.
Results
Maternal Measures Deliveries. Four of 7 of the 5.O-mglkg group failed to deliver. Of the 3 remaining in that group, 1 dam delivered only 1 offspring. This animal was not included in any of the studies. Because all other groups delivered viable litters and assignment to a group was random, nondelivery was probably due to either implantation failure or dose-dependent resorption. (Nidation sites were not examined in the rats which failed to deliver.) One dam in the 3.0-mg/kg group cannibalized her litter of 12 between Days 1-7. No other litters suffered neonatal loss. Maternal weight gain. The Kruskal-Wallis nonparametric rank test for differences among 4 groups resulted in an H = 10.7, with a p = .006. The 5.O-mg/kg females (n = 2) were omitted from the analysis, although the group is graphed for comparative purposes. (See Fig. 1.) Gestation length. All groups which received methamphetamine injections delivered on Day 22. One of the saline-injected rats delivered on Day 22, 1 on Day 21, and the remaining 4 on Day 23. Fisher’s Exact Test was performed on the delivery day data for the Saline, 1.0-, and 3.0-mglkg dams (n = 6). The null hypothesis was rejected with a p = .002.
400
MARTIN
Ofjipriiig i2leamrc~s Litter size. Fisher's Exact Test on the number of live-born young per litter in the 1.0-, 3.0-mg/kg, and Saline groups yielded a p = .007. The 5.0-mg/kg group was omitted in these analyses in that n = 2. (See Fig. 1 .) Neonatal weight gain. Saline offspring maintained a slight but non-significant weight advantage at birth and on eveiy postnatal weighing except on Day 7. Deilcloprnental 17leasiires. A x2 analysis was perfoirned on the number in each litter with both eyes open on Day 14. This resulted in a value of 11.4 ( p < .005). Seventy-one percent of the saline offspring had their eyes open, 38.8% of the 1 .O-mg/kg, 17.57 of the 3.0-nig/kg, and 25.0% of the two litters of 5.0-nig/kg young. (See Fig. 3.) Conditional czvoitlance response. A Kruskal-Wallis Test on the niean nuniber of avoidance responses yielded an H = 4.9 (p = .09). A similar test of the number of escape responses failed to reject the null hypothesis. (See Fig. 3.) So few extinction responses occurred that a test on frequency was not feasible. Instead, the number of animals which made at least 1 avoidance or escape response was compared with those which tnade n o responses. Sixty percent of the 5.O-mg/kg rats, 40% o f the 3.0-mg/kg rats, but only 12%% of the Saline offspring made at least 1 tesponse. The xz test failed to reject the null hypothesis. The result was not unexpected considering the small number and the insensitivity of this USP of the statistical test.
i70
7......
L ...... ...... ...... ...... ...... ...... ...... ......
2c n
I ,
10 3 0 5 0 Maternal
treatment
l,'ig. 1. I,et't side: h1e:in niatcrnal weight gain over gestation as a f u n c t i o n of twice daily treatment during gestation with 0.0 (Saline), I .O mg/kg. 3.0 nig/kp, 5.0 m:/kg of nicthamphetnrninc. Right side: Me;in nuinber o f live young born/litter as a function of the same treatments.
MATERNAL METHAMPHETAMlNE EXPOSURE
401
ao70-
;GOb I
r
$500
3
3 40-
1
P
$ 30-
h
s8
20-
10-
0
L 0.0
1.0
3.0
5.0
Maternal treatment
Pig. 2. Mcan percent o f offspring cyes open on Postnatal Day 14 as a consequence of maternal injcctions during gcstation 01' 0.0 (Saline), 1.0 mg/kg, 3.0 mg/kg, or ' 5.0 ma/kg of methamphetamine.
Previous investigations in our laboratory with nicotine, another CNS stimulant, showed that chronic administration in utero resulted in fewer live births and significantly underweight offspring which had a poorer chance for neonatal survival (Bccker & Martin, 1971). Such offspring also had a shorter life span than did control offspring (Martin & Becker, 1972). In our own study, the methamphetamine dams had smaller sized litters with the number of live offspring directly related t o dose. Three young were found stillborn in the groups which received the drug. Observations were made only twice daily; hcnce, only newly delivered stillborn rats were found. Over half of the rats at the highest dose failed t o deliver, which suggests that methamphetamine may havc an embryotoxic effect at this dose. Kasirsky and Tansy (1971) found no increase in anomalies in rnicc offspring whose dams were administered 5.0 mg/kg during the last third of gestation. They found a 13.6% malformation incidence with a dosc of 10.0 mg/kg. No gross anomalies were observcd in our surviving offspring. Because an examination of uterine contents prior to delivery was not attcmptcd, we cannot state that n o anomalies existed. Such offspring might have been sclectively cannibalized following birth. The only
402
MARTIN
275
50
250 u,
225
$
200
P .$
%
5
175
0
2
88 kj Q
$
150 125
100
?
g
75 50 0
Avoidance Escape Acquisition Period Fig. 3. Mean number of shuttle-box responses over acquisition by male offspring whose dams received either Saline, 3.0 mg/kg, or 5.0 mg/kg of methamphetamine during the entire gestational period.
developmental index taken, eye opening, demonstrated that these offspring lagged significantly behind Saline animals. A study by Krsiak (1973) found that a very small (.25 mg/kg) daily injection of 2icotine to gravid animals resulted in a significant retardation in offspring of eye opening, incisor eruption, and righting reflex. The earlier delivery by methamphetamine-treated females as compared with saline-injected rats is at variance with the results of nicotine studies of rats, which have found that treated animals have a longer gestational period (Becker, Little, & King, 1958; Becker & Martin, 1971). As is evident from Figure 3, the highest dose level was somewhat effective in increasing shuttle-box avoidance. These results are only tentative in that the numbers were small and the study not replicated. Middaugh et al. (1974) found that prenatal administration of amphetamine in mice caused an elevation of brain norepinephrine levels as measured from birth to 75 days of age. This was postulated as an explanation for the non-directed bursts of activity and stereotyped responses which were found in these offspring. Whether such changes persisted in adulthood was not determined. Possibly such animals became conditioned to a different activity level due to the norepinephrine change, which would then persist after the catecholamine levels had returned t o normal.
MATERNAL METHAMPHETAMINE EXPOSURE
403
A number of studies comparing behavioral change following administration of nicotine and one of the amphetamines to adult animals have been published recently. Newman (1972) found that both drugs resulted in facilitation of self-stimulation behavior in the rat, but that pretreatment with a cholinergic antagonist blocked nicotine facilitation but not amphetamine response facilitation. He hypothesized a 2-component cholingergic system. Fulginiti and Orsingher (1973) studied shuttle-box behavior and found that pretreatment by alpha-methyl-tyrosine markedly depressed response rate in both drug groups, which was counteracted when nialamide was administered simultaneously. They postulated a common adrenergic mechanism for the 2 drugs. Rats apparently do not confuse nicotine and amphetamine, in that nicotine used as a discriminative stimulus does not generalize to amphetamine or to a nicotine derivative (Schechter & Rosecrans, 1972). Pradhan and Dutta (1970) found similar facilitory effects in timing behaviors across several dose levels of both drugs. Thus, although the 2 cholinergic agonists result in similar changes in a variety of responses following administration in adult animals, biochemical mediation is only similar up to a point, and the animal can learn to discriminate between the 2 drugs. Whether the similarities between nicotine and amphetamine are due merely to widespread systemic effects which are found with other embryotoxic agents as well, or whether the effects are due to the catecholamine shifts in brain peculiar to CNS activators, remains to be seen.
Notes This research was supported in part by grant RR-05432, NIH/PHS, and by the Graduate School Research Fund of the University of Washington, Seattle, Washington. Reprints may be obtained from the author at the Department of Psychiatry and Behavioral Sciences, RP-10, University of Washington, Seattle, Washington 98195, U.S.A.
-
References Amphetamine Legislation. (1972). Hearings before the Subcommittee to Investigate Juvenile Delinquency of the Committee on the Judiciary of the United States Senate, 92nd Congress. July 15-16, 1971. Washington, D.C.: U.S. Government Printing Office. 70-190-0. Becker, R. F., Little, C. R. D., and King, J. E. (1968). Experimental studies on nicotine absorption in rats during pregnancy. Am. J. Obstet. GynecoL, 100: 957-968. Becker, R. F., and Martin, J . C. (1971). Vital effects of chronic nicotine absorption and chronic hypoxic stress during pregnancy and the nursing period. Am. J. Obstet. Gynecol., 110: 522-533. Farris, E. J., and Griffith, J . Q., Jr. (1949). The Rat in Laboratory Investigation. Philadelphia: Lippinco tt. Fog, R. (1969). Stereotyped and non-stereotyped behavior in rats induced by various stimulant drugs. Psychopharmacologia, 14: 299-304. Fulginiti, S., and Orsingher, 0. (1973). Further evidence in support of a common adrenergic mechanism for the facilitatory action on learning of amphetamine and nicotine in rats. J. Pharm. Pharrnacol., 25: 580-581. Kasirsky, G., and Tansy, M. F. (1971). Teratogenic effects of methamphetamine in mice and rabbits. Teratology, 4: 131-134. Krsiak, J. E. (1973). The effect of nicotine administration during pregnancy on the postnatal development of offspring. Act. Nerv. Super. (Praha), 15: 148.
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MARTIN
Martin, J. C., and Becker, R. F. (1972). The effects of chronic maternal absorption of nicotine or hypoxic episodes upon the life span of the offspring. Psyckonom. ScL, 29: 145-146. Middaugh, L. D., Blackwell, L. ,4., Santos, C. A., 111, and Zemp, J. W. (1974). Effects of d-amphetamine sulfate on activity and on catecholamines in the brains of offspring. Dev. Psychobiol, 7: 429438. Newman, L. M. (1972). Effects of cholinergic agonists and antagonists on self-stimulation behavior in the rat. J. Comp. Physiol. Psychol., 79: 394413. Nora, J. J., Nora, A. H., Sommerville, R. J., Hill, R. M., and McNamara, D. G. (1967). Maternal exposure to potential teratogens. JAMA, 202: 1065-1069. Pradhan, S . N., and Dutta, S. N. (1970). Comparative effects of nicotine and amphetamine on timing behavior in rats. Neuropharmacology, 2: 9-16. Schechter, M. D., and Rosecrans, J. A. (1972). Nicotine as a discriminative cue in rats: Inability of related drugs to produce a nicotine-like cueing effect. Psyckopharmacologth, 27: 379-387. Stein, L., and Wise, C. D. (1970). Mechanism of facilitation effects of amphetamine on behavior. In D. H. Efron (Ed.), Psychotomimetic Drugs. New York: Raven. Pp. 123-145. Teitelbaum, P., and Derks, P. (1958). The effect of amphetamine on forced drinking in the rat. J. Comp. Physiol. PsychoZ., 51 : 801-810. Valzelli, L. (1 973). Psychopharmacology: An Introduction to Experimental and Clinical Principles New York: Wiley.
Twenty-five Sprague-Dawley derived rats were administered 1.0, 3.0, 01 5.0 mg/kg of methamphetamine HCI or saline twice daily throughout gestation beginning on Day 1 of pregnancy. Rats were allowed t o deliver normally: offspring were culled to 8 and sexed on Day 7, and weaned on Day 21. All females had viable litters except at the 5.0 mg/kg dose where 4 of 7 failed to deliver. The rats given methamphetamine delivered earlier than did controls. Weight gain over gestation decreased as a function o f increasing drug dose. No gross anomalies were visible in the offspring. Litter size decreased as a function of increased dose and eye opening was delayed in the drug groups: the 5.0 mg/kg offspring made more conditioned avoidance responses than did the 3.0 mg/kg and saline offspring.
Amphetamine, a central nervous system (CNS) stimulant with sympathomimetic properties and a catecholamine-releasing action, owes its behaviorally facilitative action to norepinephrine release (Stein & Wise, 1970). The amphetamines are among the most widely abused of the prescription drugs, with the amount produced yearly variously estimated at 2- 10 billion dosage units (Amphetamine Legislation, 1972). d-Amphetamine, d,l-amphetamine, and methamphetamine are a series of chemically related compounds, which although they differ to some extent structurally, exert similar functional effects (Valzelli. 1973). The terms methamphetamine and amphetamine are therefore used interchangeably in this study. No developmental studies have been found which have contrasted the several compounds. The extent of amphetamine abuse in pregnant women was examined by Nora, Nora, Somerville, Hill, and McNamara (1967), who found that 27% of their sample of 240 gravid females had taken prescription appetite suppressants during at least 1 trimester of their gestational period. If this study is at all indicative of the extent of usage in gravidas, then the potential exposure to human offspring is very high. However, no studies have been located which have examined the physical, developmental, or behavioral consequences to infants and children of maternal ingestion of amphetamines during pregnancy. Middaugh, Blackwell, Sautos, and Zemp (1 974) administered methamphetamine to inice during the 3rd trimester of pregnancy. The offspring exhibited a transient increase in brain catecholamine levels and increased activity in the open field at 30 days of age. Kasirsky and Tansy (1971) administered methamphetamine during the 3rd Keceived for publication 4 June 1974 Revised for publication 7 October 1974 Uevelopnicrital Psychobiology, 8(5): 397-404 (1975) 0 1975 by John Wiley & Sons, Inc.
397
398
MARTIN
trimester of pregnancy to mice and rabbits and noted with higher doses an increase in anomalies (e.g., exencephaly, cleft palate, and eye deformities) in offspring. Because in the above studies the drug was administered only during the last trimester of pregnancy, the results may not be applicable to human prescription use and certainly are not typical of human abuse patterns. The following study was designed to investigate the effects on the offspring of 3 doses of methamphetamine administered over the entire gestational period.
Method Sti bjec ts Subjects were twenty-five 225 -250 g Sprague-Dawley derived, caesarian-borne, barrier-sustained, virgin female rats (Rattus norvegicus; supplied by Tyler Laboratories, Bellevue, Washington) which had been impregnated the previous night.
Procedure The pregnant rats were randomly assigned to 1 of 4 treatment groups: 6 rats in each of 3 groups with 7 in the group given the highest drug dose. Twice daily, at 0830 and 1430 hours, the subjects were given subcutaneous injections. These were begun on’ the morning following impregnation (Day 1) and were terminated on Day 21. All animals were housed individually, with rat chow and water available ad lib. Drug dosage. Methamphetamine HCl (Abbott Laboratories) was dissolved in a .9% isotonic saline solution with the volume held constant in doses of 1.0, 3.0, or 5.0 mg/kg of body weight. The control animals received a comparable volume of the vehicle alone. Because the amphetamines have a half-life of 14 hr in the human (Amphetamine Legislation, 1972), the twice-daily regimen was an attempt to maximize the period that the drug or its metabolites would remain in the organism. The doses are well within the range used in behavioral studies (Fog, 1969; Teitelbaum & Derks, 1958). The 2 higher doses typically resulted in stereotyped head and sniffing movements which last for 3 - 5 hr. Drug injections. Pregnant dams were injected subcutaneously over the lower dorsal surface with a 26%-gauge needle. The volume of fluid was .I25 ml for a 250-g rat, with control animals receiving comparable amounts. Volume was held constant over the 3 dose levels. The rats were weighed daily, but the daily drug dose was based upon the Day 1 weights because this more nearly approximates the pattern in human drug users who probably do not increase a drug dose as their weight increases over pregnancy. Animals were allowed to deliver normally. Offspring were not cross-fostered at birth but were raised by their natural mothers, again, as a model for human drug abuse. Therefore, this design did not allow a separation of fetal vs neonatal factors, but only a comparison of offspring whose dams received the drug with those which received saline injections during the same time period. Offspring were sexed and culled to 8 on Postnatal Day 7, with litters weighed every 7 days thereafter and
MATERNAL METHAMPHETAMINE EXPOSURE
399
weaned on Day 21. Two males were randomly chosen from each litter for behavioral studies on Day 28. Because only 2 litters survived at the highest dose level, 5 males were retained from each of these litters. Developmental measure. Eye opening was assessed on Postnatal Day 14 for each rat in a litter. (The eyes open in the rat between Days 14-17 [Farris & Griffith, 19491 .) Behavioral measure. The offspring were tested at 100-120 days of age in a Lehigh Valley Electronics shuttle-box (#146-04) for acquisition and extinction of the conditioned avoidance response (CAR). The shuttle-box consisted of two 22 x 19 x 19 cm Lucite compartments with a 4%-cm metal divider and a grid floor. A Mallory Sonalert audio oscillator/amplifier at 2.8 kHz and 70 dB was positioned above the divider. Stimulus lamps positioned on the end walls of the box were triggered with the onset of an auditory stimulus and provided an additional cue. A 15-sec interstimulus interval (ISI) was followed by a 3-sec tone-on period followed by .3 mA of continuous shock to the feet. An escape over the barrier terminated shock, tone, and light. An avoidance response reset the IS1 clock. Ninety minutes of acquisition were followed by a 10-min extinction period in which tone and light were operative but not shock. Dependent measures included: (1) avoidance during the pretone period, (2) avoidance during the conditioned stimulus-unconditioned stimulus (CS-UCS) interval, and (3) escape responses. The 1.0-mg/kg group was used to test the equipment and to determine timing parameters, but was then excluded from the statistical analysis. Two saline offspring died in a nonrelated laboratory accident, leaving an n = I0 in each of the 3.0- and 5.0-mg/kg groups and ad n = 8 in the saline control group.
Results
Maternal Measures Deliveries. Four of 7 of the 5.O-mglkg group failed to deliver. Of the 3 remaining in that group, 1 dam delivered only 1 offspring. This animal was not included in any of the studies. Because all other groups delivered viable litters and assignment to a group was random, nondelivery was probably due to either implantation failure or dose-dependent resorption. (Nidation sites were not examined in the rats which failed to deliver.) One dam in the 3.0-mg/kg group cannibalized her litter of 12 between Days 1-7. No other litters suffered neonatal loss. Maternal weight gain. The Kruskal-Wallis nonparametric rank test for differences among 4 groups resulted in an H = 10.7, with a p = .006. The 5.O-mg/kg females (n = 2) were omitted from the analysis, although the group is graphed for comparative purposes. (See Fig. 1.) Gestation length. All groups which received methamphetamine injections delivered on Day 22. One of the saline-injected rats delivered on Day 22, 1 on Day 21, and the remaining 4 on Day 23. Fisher’s Exact Test was performed on the delivery day data for the Saline, 1.0-, and 3.0-mglkg dams (n = 6). The null hypothesis was rejected with a p = .002.
400
MARTIN
Ofjipriiig i2leamrc~s Litter size. Fisher's Exact Test on the number of live-born young per litter in the 1.0-, 3.0-mg/kg, and Saline groups yielded a p = .007. The 5.0-mg/kg group was omitted in these analyses in that n = 2. (See Fig. 1 .) Neonatal weight gain. Saline offspring maintained a slight but non-significant weight advantage at birth and on eveiy postnatal weighing except on Day 7. Deilcloprnental 17leasiires. A x2 analysis was perfoirned on the number in each litter with both eyes open on Day 14. This resulted in a value of 11.4 ( p < .005). Seventy-one percent of the saline offspring had their eyes open, 38.8% of the 1 .O-mg/kg, 17.57 of the 3.0-nig/kg, and 25.0% of the two litters of 5.0-nig/kg young. (See Fig. 3.) Conditional czvoitlance response. A Kruskal-Wallis Test on the niean nuniber of avoidance responses yielded an H = 4.9 (p = .09). A similar test of the number of escape responses failed to reject the null hypothesis. (See Fig. 3.) So few extinction responses occurred that a test on frequency was not feasible. Instead, the number of animals which made at least 1 avoidance or escape response was compared with those which tnade n o responses. Sixty percent of the 5.O-mg/kg rats, 40% o f the 3.0-mg/kg rats, but only 12%% of the Saline offspring made at least 1 tesponse. The xz test failed to reject the null hypothesis. The result was not unexpected considering the small number and the insensitivity of this USP of the statistical test.
i70
7......
L ...... ...... ...... ...... ...... ...... ...... ......
2c n
I ,
10 3 0 5 0 Maternal
treatment
l,'ig. 1. I,et't side: h1e:in niatcrnal weight gain over gestation as a f u n c t i o n of twice daily treatment during gestation with 0.0 (Saline), I .O mg/kg. 3.0 nig/kp, 5.0 m:/kg of nicthamphetnrninc. Right side: Me;in nuinber o f live young born/litter as a function of the same treatments.
MATERNAL METHAMPHETAMlNE EXPOSURE
401
ao70-
;GOb I
r
$500
3
3 40-
1
P
$ 30-
h
s8
20-
10-
0
L 0.0
1.0
3.0
5.0
Maternal treatment
Pig. 2. Mcan percent o f offspring cyes open on Postnatal Day 14 as a consequence of maternal injcctions during gcstation 01' 0.0 (Saline), 1.0 mg/kg, 3.0 mg/kg, or ' 5.0 ma/kg of methamphetamine.
Previous investigations in our laboratory with nicotine, another CNS stimulant, showed that chronic administration in utero resulted in fewer live births and significantly underweight offspring which had a poorer chance for neonatal survival (Bccker & Martin, 1971). Such offspring also had a shorter life span than did control offspring (Martin & Becker, 1972). In our own study, the methamphetamine dams had smaller sized litters with the number of live offspring directly related t o dose. Three young were found stillborn in the groups which received the drug. Observations were made only twice daily; hcnce, only newly delivered stillborn rats were found. Over half of the rats at the highest dose failed t o deliver, which suggests that methamphetamine may havc an embryotoxic effect at this dose. Kasirsky and Tansy (1971) found no increase in anomalies in rnicc offspring whose dams were administered 5.0 mg/kg during the last third of gestation. They found a 13.6% malformation incidence with a dosc of 10.0 mg/kg. No gross anomalies were observcd in our surviving offspring. Because an examination of uterine contents prior to delivery was not attcmptcd, we cannot state that n o anomalies existed. Such offspring might have been sclectively cannibalized following birth. The only
402
MARTIN
275
50
250 u,
225
$
200
P .$
%
5
175
0
2
88 kj Q
$
150 125
100
?
g
75 50 0
Avoidance Escape Acquisition Period Fig. 3. Mean number of shuttle-box responses over acquisition by male offspring whose dams received either Saline, 3.0 mg/kg, or 5.0 mg/kg of methamphetamine during the entire gestational period.
developmental index taken, eye opening, demonstrated that these offspring lagged significantly behind Saline animals. A study by Krsiak (1973) found that a very small (.25 mg/kg) daily injection of 2icotine to gravid animals resulted in a significant retardation in offspring of eye opening, incisor eruption, and righting reflex. The earlier delivery by methamphetamine-treated females as compared with saline-injected rats is at variance with the results of nicotine studies of rats, which have found that treated animals have a longer gestational period (Becker, Little, & King, 1958; Becker & Martin, 1971). As is evident from Figure 3, the highest dose level was somewhat effective in increasing shuttle-box avoidance. These results are only tentative in that the numbers were small and the study not replicated. Middaugh et al. (1974) found that prenatal administration of amphetamine in mice caused an elevation of brain norepinephrine levels as measured from birth to 75 days of age. This was postulated as an explanation for the non-directed bursts of activity and stereotyped responses which were found in these offspring. Whether such changes persisted in adulthood was not determined. Possibly such animals became conditioned to a different activity level due to the norepinephrine change, which would then persist after the catecholamine levels had returned t o normal.
MATERNAL METHAMPHETAMINE EXPOSURE
403
A number of studies comparing behavioral change following administration of nicotine and one of the amphetamines to adult animals have been published recently. Newman (1972) found that both drugs resulted in facilitation of self-stimulation behavior in the rat, but that pretreatment with a cholinergic antagonist blocked nicotine facilitation but not amphetamine response facilitation. He hypothesized a 2-component cholingergic system. Fulginiti and Orsingher (1973) studied shuttle-box behavior and found that pretreatment by alpha-methyl-tyrosine markedly depressed response rate in both drug groups, which was counteracted when nialamide was administered simultaneously. They postulated a common adrenergic mechanism for the 2 drugs. Rats apparently do not confuse nicotine and amphetamine, in that nicotine used as a discriminative stimulus does not generalize to amphetamine or to a nicotine derivative (Schechter & Rosecrans, 1972). Pradhan and Dutta (1970) found similar facilitory effects in timing behaviors across several dose levels of both drugs. Thus, although the 2 cholinergic agonists result in similar changes in a variety of responses following administration in adult animals, biochemical mediation is only similar up to a point, and the animal can learn to discriminate between the 2 drugs. Whether the similarities between nicotine and amphetamine are due merely to widespread systemic effects which are found with other embryotoxic agents as well, or whether the effects are due to the catecholamine shifts in brain peculiar to CNS activators, remains to be seen.
Notes This research was supported in part by grant RR-05432, NIH/PHS, and by the Graduate School Research Fund of the University of Washington, Seattle, Washington. Reprints may be obtained from the author at the Department of Psychiatry and Behavioral Sciences, RP-10, University of Washington, Seattle, Washington 98195, U.S.A.
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