Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat

Life Sciences, Vol . 24, pp . 1223-1230 Printed in the U .S .A .

Pergamon Press

PERINATAL METHADONE ADDICTION AFFECTS BRAIN SYNAPTIC DEVELOPMENT OF BIOGENIC AMINE SYSTEMS IN THE RAT Theodore A. Slotkin, William L . Whitmore, Maria Salvaggio, and Frederic J . Seidler Department of Pharmacology Duke University Medical Center Durham, North Carolina 27710 USA (Received in final form February 16, 1979)

SUMMARY Administration of methadone to pregnant and nursing rats or direct treatment of developing pups with methadone resulted in deficits of development of body weight, brain weight and synaptosomal uptake of 5hydroxytryptamine, dopamine and norepinephrine . Defective synaptic development was moat apparent immediately after birth in pups whose mothers received methadone ; while some recovery occurred by 3 weeks of age, there was a subsequent deficit in synaptosomal uptake post-weaning . A similar pattern also was seen in development of synaptic vesicle amine uptake . Direct treatment of neonates with methadone also caused reductions in development of synaptosomàl and vesicular uptake mechanisms, but the patterns of alteration were different from those in the maternal treatment group . These studies show that the adverse effect of opiates on general brain growth are accompanied by a slowing of synaptic development of biogenic amine systems . Opiate exposure during gestation results in general retardation of growth and development . A large proportion of infants born to opiate-addicted mothers are underweight and display increased risk of mortality (1-3) . Accompanying the effects on birthweight and growth are disturbances in the development and function of the central nervous system (4-6), ultimately including alterations in behavioral ontogeny and motor activity (7, 8) . While a number of reports have demonstrated opiate-induced alterations of biochemical development of nucleic acids, polyamines and proteins, in the brain (6, 9-11), it is at present unclear whether these metabolic abnormalities can result in changes of synaptic development which in turn may participate in the production of behavioral disturbances . The current study demonstrates that biogenic amine synaptic development in the rat is adversely affected by perinatal opiate exposure . Methods Methadone doss e re imens and tissue re aration . Timed pregnant Sprague-Dawley rats (Ziv c-M ller) were given daily 0024-3205/79/131223-07502 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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injections of methadone hydrochloride : 2 .5 mg/kg s .c . on the 10th day of gestation, 3 .5 mg/kg on the 11th day and 5 mg/kg thereafter, continued through weaning . This dose zegimen was chosen because it was the maximal dose utilizable which largely did not affect litter size or neonatal mortality ; maternal mortality was less than 108 . For studies in developing rats utilizing direct methadone administration, pups were given daily subcutaneous injections beginning at one day of age . The dosages were 2 .5 mg/kg for the first administration, 3 .5 mg/kg for the second and 5 mg/kg thereafter . In both cases, control animals (dams or pups) received daily injections of saline equal in volume to the methadone injections (1 ml/kg) . Pups were weighed and killed at intervals of several days and brain excised, weighed and homogenized in 4 volumes of 0 .3 M sucrose buffered at pH 7 .4 with 2 .5 mM Tris and containing 10 uM iproniazid, using 5 up-down strokes in a glass homogenizer fitted with a Teflon pestle . na tosomal u take . The homogenate was diluted with an equal vo ume o sucrose-tris and centrifuged at 1000 x g for 10 min . Aliquots of the supernatant representing 10 mg original wet weight of tissue were added to Krebs-Henseleit medium containing final concentrations of 1 .25 uM iproniazid, 2 uM ascorbic acid and 0 .05 uM 9 H-norepinephrine, 9 H-dopamine or 3 H-5-hydroxytryptamine in a final incubation volume of 1 ml . Samples were incubated for 5 min at 37° while duplicate tubes were kept on ice to serve as blanks . Uptake was stopped by placing the tubes on ice and adding 3 ml of ice-cold Rrebs-Henseleit medium . The labeled synaptosomes were trapped by rapid vacuum filtration on Gelman cellulose acetate filters (0 .2 um pore size), washed twice with 3 ml of medium and counted by liquid scintillation spectrometry . This synaptosomal preparation has been characterized previously in preparations from both adult and neonatal rats (12, 13) . Vesicular u take . The original tissue suspension was rehomogen ze in an a -glass apparatus and the subcellular fraction containing synaptic vesicles prepared by the method of Seidler et al . (14) . The homogenate was then centrifuged at 1000 x g for 15 min and the supernatant recentrifuged at 20,000 x g for 30 min . The supernatant of the latter centrifugation was sedimented at 100,000 x g for 30 min in a Beckman No . 40 rotor and the supernatant solution was discarded . The crude, vesicle-containing pellet was resuspended gently in a volume of 130 mM potassium phosphate (pH 7 .4) equal to that of the glass-to-glass homogenate using 2 up-down strokes in a Teflon-to-glass homogenizer, and this suspension was used for subsequent incubations . Although this microsomal fraction contains many particles and organelles, uptake of amines in vitro appears to occur primarily into the synaptic vesicles preséntin the preparation (14-17) . This preparation also has been characterized fully in both adult (14-17) and developing (18, 19) rats as well as in brain tissue from other species (20, 21) . For determinations of vesicular uptake, standard incubations contained 0 .67 ml of the vesicle preparation (corresponding to 133 mg of original brain tissue), and final concentrations of 1 mM ATP-Mg 2+ , 2 uM ascorbic acid, 1 .25 uM iproniazid, 0 .05 uM ~Hnorepinephrine and phosphate buffer to make a final incubation

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volume of 1 .7 ml . Samples were incubated for 4 min at 30° while duplicate tubes were kept on ice to serve as blanks . Uptake was stopped by the addition of 1 .7 ml of ice-cold phosphate buffer and the labeled vesicles trapped and washed on cellulose acetate filter paper as described above and counted for radioactivity . Data are presented as means and standard errors of 6-12 rats in each group, with significance calculated by two-tailed paired and unpaired t-tests (22) . In the former, group means of two developmental curves were paired by age over the entire time course of development ; degrees of freedom were calculated as the number of paired means minus one . This mode of pairing enables comparisons of differences between the two curves taken as a whole, as opposed to comparison only of individual age points in the unpaired t-test . Materials . ß-Norepinephrine-7-~H (2 .2 Ci/mmol), dopamine- . 2-~H 1 . C mmol) and s-hydroxytryptamine-l,2-~H (25 .6 Ci/mmol) were obtained from New England Nuclear Corp, iproniazid phosphate from Sigma Chemicals and methadone HC1 from Merck Sharp & Dohme . Results and Discussion Exposure of developing rats to methadone, either through prenatal and postnatal treatment of the mother or through direct postnatal injections of the pups, resulted in extensive lags in development of body weight (Fig . 1) . In both cases, a deficit of 20-30$ was apparent by the age of weaning ; the low body weight persisted after weaning in the maternal treatment group despite the fact that drug exposure was terminated by 23 days o~f age . While either maternal or direct methadone administration also reduced brain weight during development, there was a difference in the developmental pattern between the two treatment groups . In the pups treated directly with methadone, the brain weight deficit generally parallelled that of body weight, whereas a definite recovery occurred by weaning in the maternal treatment group . The latter may reflect the lower postnatal dose in animals receiving drug via the milk compared to that from placental transfer or to the dose given directly to the pups . This observation supports earlier studies which concluded that at least some of the effects of maternal methadone on organ growth may be dose-related drug effects as opposed to secondary nutritional actions (10, 23) . In the maternal treatment group, a further post-weaning deficit in brain weight appeared after a period of apparent normality, and was associated with neurochemical changes (see below) . A deficit in brain weight need not in itself denote defective synaptogeneais or synaptic function . In order to evaluate whether synaptic development was indeed altered by perinatal methadone exposure, synaptosomal uptake of biogenic amines was measured . Previous studies have shown that this procedure provides an estimate of the numbers of synaptic terminals for each neurotransmitter (13, 19, 24) . in general, pups exposed to methadone via maternal administration displayed defects in synaptosomal uptake of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) neurons, demonstrating that perinatal methadone exposure does affect biogenic amine systems (Fig . 1) . While NE synaptosomal uptake was

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transmitter effects could be accounted for by brain growth deficits, as the developmental lag was significantly greater for synaptosomal uptake than for brain weight (by paired t-test, p < 0 .001 for 5-HT uptake deficit vs brain weight deficit, p < 0 .001 for DA, p < 0 .005 for NE) . Thus, maternal methadone may exert a more specific effect on development of synaptosomal uptake over and above influencing brain growth . The general downward trend of brain weight and synaptosomal uptakes in the postweaning period indicates the potential for delayed alterations to occur . A previous study suggests that this effect may be related to or result from long-term interference with brain RNA synthesis produced by maternal methadone administration (10), and the delayed neurotransmitter alterations ultimately may then cause brain dysfunction or behavioral abnormalities (10) . A role of withdrawal occurring at weaning in producing these late changes also cannot be ruled out . in the neonates receiving direct methadone injections, nearly all of the developmental lags in synaptosomal uptake could be accounted for by brain growth deficits, in contrast to the maternal methadone group . Indeed, in the "direct" group, there was little or no lag in development of synaptosomal uptake of NE . Thus, when administered directly to pups postnatally, the actions of methadone on synaptic development appear to be secondary to effects on general cellular development . Administration of opiates to mature animals does not itself alter synaptosomal uptake (26), and thus a direct effect on biogenic amine transport mechanisms seems unlikely to explain the effects of perinatal methadone on development of synaptosomal uptake . A partial confirmation that the number of neurons or terminals was affected by perinatal methadone, was provided by the experiments in which synaptic vesicle uptake . was measured . Poisoning of the synaptosomal uptake system by perinatal methadone should not alter the number or function of synaptic vesicles, whereas a reduction in the number of terminals should be accompanied by the presence of fewer vesicles . Both maternal and direct methadone exposures resulted in a reduction in NE vesicular uptake ; these results, coupled with the earlier finding that activities of catecholamine biosynthetic enzymes are reduced by perinatal methadone, suggest strongly that the number of synaptic terminals has been affected . The lack of a one-to-one relationship between deficits of NE vesicular uptake and synaptosomal uptake may result from the fact that NE uptake in vesicles harvested from whole brain involves both a mixed population of DA and NE vesicles from terminals and probably from axons and cell bodies as well (15, 17) . In conclusion, these studies demonstrate that perinatal methadone exposure retards synaptic development of 5-HT, DA and NE neurons in the brain . While general actions on brain growth and development play a role in this effect, more direct drug actions on synaptic development may contribute to the deficits seen in rats whose mothers received methadone . Acknowledgement Supported by USPHS DA-00465 and DA-00006 .

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 .

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