Brain Research, 572 (1992) 57-63 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
57
BRES 17424
Prenatal cocaine exposure disrupts the development of the serotonergic system H.M. Akbari a, H.K. Kramer 1, P.M. Whitaker-Azmitia 2, L.P. Spear 3 and E.C. Azmitia 1 1Department of Biology, New York University, New York, NY 10003 (U.S.A.), 2Department of Psychiatry, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) and 3Department of Psychology, State University of New York at Binghamton, Binghamton, NY 13901 (U.S.A.) (Accepted 1 October 1991) Key words: Cocaine; Serotonin; Neuronal development; Paroxetine; Immunocytochemistry; Cortex; Hippocampus
Prenatal cocaine exposure has been found to result in a number of neurobehavioral abnormalities in both clinical and laboratory studies. We have previously shown that cocaine inhibits the growth of developing serotonin neurons in culture. This study examines the effects of cocaine on the developing serotonin system in vivo. Pregnant rats were injected with cocaine (40 mg/kg s.c.) from gestational day 13 to parturition. One group of rats was additionally injected on postnatal days 1-5 with cocaine (10 mg/kg s.c.). [3H]Paroxetine, a selective ligand for the serotonin uptake carder, was used to quantify serotonin terminal fiber density at one day, one week, and four weeks postnatal. Cocaine exposure was found to significantly decrease [aH]paroxetine-labelled sites and thus the density of serotonin fibers in the cortex and hippocampus at one day and one week postnatal. By four weeks postnatal, no significant effect was observed, indicating that a recovery had occurred. Serotonin immunocytochemistry performed at one month revealed normal fiber distribution in the cortex but a loss of fibers in the CA1 and CA2 hippocampal fields. Postnatal treatment alleviated the effects of prenatal cocaine exposure, resulting in [3H]paroxetine binding levels at one week which were comparable to and, in the cortex, even higher than those of saline controls. We conclude that cocaine delays the maturation of the serotonin system when administered prenatally but may accelerate maturation when administered both pre- and postnatally. INTRODUCTION Previous animal studies have shown that prenatal exposure to cocaine causes a n u m b e r of teratogenic and neurobehavioral abnormalities. Increases in fetal hemorrhage and abruptio placentae have been reported following gestational cocaine administration to rats ~'~6. Teratogenic effects that have been shown include soft tissue, cardiovascular, and skeletal defectsa3; however, a lack of teratogenicity has been reported at doses as high as 60 mg/kg 16. Neurobehavioral effects of prenatal cocaine administration include increases in locomotor activity and attenuations in shock-precipitated wall climbing at postnatal day 1243. Such exposure has also been found to delay the acquisition of the righting reflex 25. However, using a different treatment regimen, Sobrian et al. have shown accelerated development of the surface righting reflex and startle reflex 42. In addition, impaired performance in an odor-association task and aversive conditioning task has been shown 44. Although much research has centered on the behavioral consequences of in utero cocaine exposure, little work has been done on the un-
derlying neurochemical changes. Increased brain glucose utilization 14'15 and decreased responsiveness to a catecholaminergic challenge 42 have been reported; however, no studies have focused on the neurochemical bases of these effects. Cocaine affects the developing central nervous system both directly and indirectly. Indirectly, cocaine may result in fetal hypoxia by vasoconstriction of the umbilical artery 51, thus adversely affecting the developing brain. Cocaine's anesthetic properties may also affect the developing nervous system, since prenatal exposure to lidocaine has been previously shown to result in behavioral deficits 41. Directly, cocaine interacts with a number of neurochemical systems. Cocaine exerts its psychotropic effects primarily by inhibition of the reuptake of dopamine ( D A ) , norepinephrine (NE), and serotonin and stimulation of dopamine release 5'21'24'28'36'a7. Other neurochemical effects of cocaine include stimulation of tyrosine hydroxylase activity 45, inhibition of tryptophan uptake a7, and reduction of serotonin synthesis a9. Cocaine would presumably have similar neurochemical effects on the developing nervous system. Cocaine has also
Correspondence: H.M. Akbari, Dept. of Biology, New York University, 1009 Main Bldg., 100 Washington Sq. East, New York, NY 10003, U.S.A.
58 been found to be a competitive antagonist at the 5-HT 3 receptor TM which has been described in the rat central nervous system 27. Serotonin (5-HT), one of the neurotransmitter systems affected by cocaine, has been shown to act as a developmental regulatory substance both on its own develo p m e n t and on the proliferation and m a t u r a t i o n of other n e u r o n a l populations 3'1°'29'3°. Specifically, 5-HT has been found to have an autoregulatory inhibitory effect on the growth of fetal cultured 5-HT n e u r o n s as assessed by [3H]5-HT uptake and 5-HT immunocytochemistry49"5°. Similarly, it has been concluded that 5-HT autoregulates the biochemical differentiation of serotonergic n e u r o n s in culture 26, inhibits growth cone activity, and causes neurite retraction in cultured 5-HT Helisoma neurons 22' 23,32. Thus the extracellular accumulation of 5-HT caused by cocaine may inhibit the outgrowth of serotonergic neutites. In previous in vitro experiments, we found cocaine to inhibit the m a t u r a t i o n of cultured 5-HT n e u r o n s as measured by [3H]5-HT uptake 2. In this study, the effects of cocaine on the development of the serotonergic system were investigated in vivo. Cocaine at a dose of 40 rag/ kg/3 ml or saline was administered from gestational day 13 to parturition. Gestational day 13 has been shown to be the earliest date that 5-HT immunoreactivity could be detected in the developing rat brain 46. The dose of cocaine used has been previously characterized as producing high fetal levels and behavioral teratogenicity without producing gross abnormalities44. 5-HT fiber density was assessed indirectly on postnatal days 1, 7, and 28 with [3H]paroxetine, a ligand for the 5-HT uptake cartier, and directly by 5-HT immunocytochemistry on postnatal day 28.
MATERIALS AND METHODS
Animal treatments Timed pregnant Sprague-Dawley rats (Taconic Farms, PA) were obtained on gestational day (GD) 10 and housed individually in plastic breeding cages with free access to food and water. On GD13, pregnant dams were randomly assigned to two treatment groups. Animals received subcutaneous injections of either 40 mg/ kg/3 ml cocaine HC1 (NIDA, Washington, DC) or an equivalent volume of saline vehicle from GD13 to parturition. Gestational exposure to this dose of cocaine has been previously shown to cause neurobehavioral and neuroehemical alterations in offspring43. On the day of birth, litters in both treatment groups were culled to 8 pups/litter to ensure standard nutrition. Litters from cocaine-treated dams were fostered to untreated dams to control for the residual effects of the drug on maternal behavior. In addition, one group of the cocaine-treated pups was given subcutaneous injections of 10 mg/kg cocaine HCl from postnatal day (PND) i to PND5 inclusive. [3H]Paroxetine binding On PND1, PNDV, and PND28, 4 pups from each treatment group were randomly selected and killed by decapitation for [3H]-
paroxetine binding to determine the density of 5-HT uptake sites. This method of quantification of 5-HT uptake sites with [3H]paroxetine as a measure of 5-HT fiber density has been previously shown to be feasible in the assessment of drug toxicity6'7. At least 3 different litters were represented for each group. Brains were rapidly removed and dissected on ice. Appropriate brain regions were weighed, placed in 40x volume by weight (v/w) 50 mM Tris buffer with 120 mM NaCI, 10 mM KCI, and 0.1% ascorbate (pH 7.4), rapidly frozen on dry ice, and kept frozen at -70°C until assay was performed. On day of assay, samples were thawed to 4°C and homogenized with a glass-Teflon homogenizer. Samples were centrifuged at 15,000 rpm for 10 min and the ensuing pellet was resuspended to 40x v/w in Tris buffer. One hundred and sixty td aliquots of the tissue homogenates were plated in 96-well plates (Nunc, Denmark) and allowed to equilibrate for 10 min with either 40/al Tris buffer for total binding or 40 ~1 10-5 (final concentration) fluoxetine (gift from Eli Lilly Co., Indianapolis, IN) to determine non-specific binding. Following this preincubation, the tissue was incubated for one hour with 0.125 nM [3H]paroxetene (final concentration, spec. act. 26.5 Ci/mmol; NEN-Dupont, Boston, MA). Samples were harvested in a Titertek harvester with ice-cold Tris buffer. The filters were collected and placed in 3 mt of Liquiscent (National Diagnostics, Manville, NJ) and counted for 1 min in a Beckman liquid scintillation counter (Model 1801; counting efficiency 38%). Protein content of samples was determined using the Lowry method 31 and CPM data was converted to fmol [3H]paroxetene bound/rag protein.
lmmunocytochemistry In addition to homogenate binding studies, immunocytochemistry was performed at PND28 to compare the innervation pattern of 5-HT fibers in the cocaine and saline groups. Four pups from each treatment group were randomly selected and perfused transcardially on PND28 with ice cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) over 15 min. Brains were removed and postfixed for 4 h. Sixty-#m sections were cut on a Series 1000 vibratome (Kramer) and collected in 0.1 M PBS. Following two washes in PBS, 3 washes in 0.1 M Tris-buffered saline (TBS) and a one hour incubation in blocking serum, sections were incubated overnight in primary antibody raised in goat directed against 5-HT diluted 1:3000 (Incstar Corp., StiUwater, MN). Antibodies were diluted in TBS containing 1% blocking serum and 0.3% Triton (Sigma). Sections were incubated for one hour each in biotinylated secondary rabbit anti-goat antibody and the avidin-biotin complex (Vector Labs., Burlingame, CA), then reacted with 0.05% diaminobenzidene with 0.003% hydrogen peroxide for 15 min. Sections were mounted, dehydrated and coverslipped with DPX mountant (DBH). Slides were viewed on a Leitz microscope and photographs taken with Ilford Pan F film (ASA 50). Statistics The statistical significance of the results from the [3H]paroxetine binding experiments was evaluated with the Student's t-test. Significance was assumed at the level of P < 0.05. All binding data are given as means + S.E.M.
RESULTS The density of 5-HT uptake sites in 3 different brain regions was assessed using [3H]paroxetine, a ligand at the 5-HT uptake carrier, at 3 postnatal ages (one day, one week, and 4 weeks postnatal) following prenatal cocaine exposure. O n PND1, there were significant decreases in the density of 5-HT uptake sites in the cerebral cortex in the cocaine-treated animals as shown by decreased [3H]paroxetine binding levels (66.76% of sa-
59
1 DAY POSTNATAL 140
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CORTEX SPINAL CORD prenatal saline prenatal cocaine
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Fig. 1. Effects of prenatal cocaine on the specific binding of [3H]paroxetine in the cerebral cortex, brainstem, and spinal cord at 1 da~, postnatal. Each bar represents the mean from 4 animals from at least 3 litters. Error bars represent the S.E.M. * Significant with regpect to saline control by Student's t-test, P < 0.05.
line control, P < 0.05) when compared to saline-treated animals (Fig. 1). However, no decreases were evident in either the spinal cord or the brainstem of these animals. At one week postnatal, a decrease in the n u m b e r of [3H]paroxetine-labelled sites in the cortex was again observed in prenatally treated animals when compared to saline controls (64.76%, P < 0.05). In addition, significant decreases in labelled uptake sites were found in the hippocampus in cocaine-treated animals (48.21% of saline control, P < 0.05). These results indicate a decrease in the density of 5-HT fibers in these two regions. In contrast to forebrain regions, no change could be de-
tected in the spinal cord (Fig. 2). As shown in Fig. 3, no significant differences in [3H]paroxetine binding were observed between the animals exposed to cocaine prenatally and the saline controls at 4 weeks postnatal in either the cortex or hippocampus. There was a trend towards higher binding levels in some of the prenatal cocaine animals; however, this did not demonstrate significance. These results indicate that a recovery of the serotonergic innervation of the cortex and hippocampus occurs between one week and four weeks postnatal. To determine whether the distribution of serotonergic fibers following recovery in the cocaine-treated animals was similar to the normal pattern, immunocytochemistry was performed on PND28 on an additional group of animals. The distribution of 5-HT immunoreactive fibers in all cortical regions examined (parietal, frontal, cingulate and entorhinal cortices) in prenatal cocaine-treated animals was similar to saline controls. However, the 5-HT immunoreactive fiber distribution in the hippocampus following gestational cocaine exposure was altered when compared to controls. Decreases were observed in the density of fibers in two regions as shown in Fig. 4. A loss of 5-HT fibers in the stratum lacunosum-moleculate of the CA1 hippocampal field and the stratum radiatum of the C A 2 field was seen 4 weeks postnatal following prenatal cocaine treatment. Thus, unlike the cortical 5-HT system, prenatal cocaine results in longterm alterations in the serotonergic innervation of the hippocampus. In contrast to the results obtained from animals prenatally treated with cocaine, no decreases in [3H]paroxetine binding were observed in animals treated perina-
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prenatal saline prenatal cocaine pre- & postnatal cocaine
Fig. 2. Effects of prenatal cocaine (solid bars) and pre- and postnatal cocaine (hatched bars) on the specific binding of [3H]paroxetine in the cerebral cortex, spinal cord, and hippocampus at 1 week postnatal. Each bar represents the mean from 4 animals. Error bars represent the S.E.M. * Significant with respect to saline control by Student's t-test, P < 0.05. ** Significant with respect to prenatal cocaine, P < 0.05. *** Significant with respect to prenatal cocaine, P < 0.01.
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CORTEX STRIATUM r ~ l prenatal saline prenatal cocaine pre- & postnatal cocaine
HIPPOCAMPUS
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Fig. 3. Specific binding of [3H]paroxetine in the cerebral cortex, striatum, and hippocampus at 4 weeks postnatal following prenatal cocaine (solid bars) or pre- and postnatal cocaine (hatched bars) administration. Each bar represents the mean from 4 animals. Error bars represent the S.E.M. * Significant with respect to prenatal cocaine by Student's t-test, P < 0.05.
60
Fig. 4. Darkfield photomontage of 60 ~m coronal section of the hippocampus immunostained for 5-HT. Serotonergic fibers are decreased along the stratum lacunosum-molecutare of the CA1 field (bold arrow) and stratum radiatum of the CA2 field (curved arrow) in section from prenatally cocaine-treated animal (B) when compared to control animal (A). Bar = 60 ~m.
tally with tical mals
(i.e. both pre- and postnatally; G D 1 3 - 2 2 ; P N D 1 - 5 ) cocaine. As can be seen in Fig. 2, on PND7, cor[3H]paroxetine binding in pefinatally treated aniwas significantly increased when compared to either
prenatal saline or prenatal cocaine-treated animals (123% and 201%; P < 0,05 and P < 0.01, respectively). In the hippocampus, [3H]paroxetine binding was significantly higher in animals treated perinatally with cocaine when
61 compared to prenatal cocaine-treated animals (209%, P < 0.01). Additionally, the levels of [3H]paroxetine binding in the spinal cord preparations from perinatally treated animals were significantly higher (206%, P < 0.05) than those of animals treated only prenatally. These levels were also higher than animals treated with saline, although this did not show significance (Fig. 2). By 4 weeks postnatal, no significant differences in [3H]paroxetine binding could be seen between the 3 groups. DISCUSSION Prenatal exposure to 40 mg/kg cocaine from GD13 to parturition results in significant changes in the serotonergic system during the early postnatal period. Our data indicate that cocaine inhibits the normal growth of 5-HT fibers to the cerebral cortex and hippocampus but not to the spinal cord. The lack of a noticeable effect on the [3H]paroxetine binding in spinal cord at one day and one week indicates that the decreases seen elsewhere are not a residual effect of the drug in the neonates. At one week, the cerebral cortex and hippocampus showed decreases in [3H]paroxetine binding in the cocaine-treated animals. The inhibition of fiber outgrowth by cocaine may be due to any one of a number of neurochemical changes which cocaine induces. The primary action of cocaine on serotonergic neurons is an inhibition of 5-HT reuptake 37 resulting in increased extraceUular concentrations of 5-HT. This extracellular accumulation of 5-HT would result in autoinhibition of serotonergic neurite outgrowth 5°. Thus cocaine's inhibition of neuronal reuptake of 5-HT may be directly responsible for the delayed innervation of forebrain regions following prenatal cocaine exposure. By 4 weeks postnatal, the [3H]paroxetine binding levels in prenatally cocaine-treated animals were not significantly lower than saline controls, indicating a recovery of the 5-HT fibers. Whether this is due to a regrowth from damaged axons or represents a delayed maturation is not clear, although the latter seems more likely due to the rapid recovery period. If fibers had initiated a normal innervation and then degenerated, a recovery period of 3 weeks would not be sufficient for complete recovery since in previous studies on 5-HT fiber regrowth only 50% of control values had been reached at one month following 5,7-dihydroxytryptamine lesions ~9. However, that study was performed in an adult animal and may not be an accurate measure of 5-HT fiber regrowth in the immature brain. Immunocytochemistry revealed a normal pattern of cortical 5-HT fibers at 4 weeks postnatal, indicating that the reinnervation was not merely quantitative, but qualitative. The 5-HT fiber distribution was similar in pre-
natal cocaine-treated animals and saline controls in all cortical regions examined. In the hippocampus, decreases in the density of 5-HT immunoreactive fibers were seen in the stratum radiatum of CA2 and stratum lacunosummoleculare of CA1. Both of these hippocampal regions receive fibers originating primarily from the median raphe nucleus 4 which raises the possibility that fibers from the median raphe nucleus may be more susceptible to the long-term effects of prenatal cocaine. The effects on [3H]paroxetine binding caused by continued treatment through the first 5 postnatal days were quite different from prenatal cocaine treatment alone. Paradoxically, these animals did not exhibit the decreases in [3H]paroxetine binding seen in the prenatal cocaine group, but instead appeared to exhibit an increased rate of development. At one week postnatal, animals treated with cocaine perinatally had increased binding both with respect to the prenatal cocaine group and, in the cortex, the prenatal saline group. With respect to neurite extension and maturation, 5-HT has been found to have a stimulatory effect in organotypic cultures 1°, indicating that neurite outgrowth is facilitated by 5-HT once target cells are reached. 5-HT has also been reported to stimulate a reinitiation of growth in embryonic neurons which have ceased to elongate 2°. Perinatal treatment with cocaine would maintain high extracellular concentrations of 5-HT, which could then stimulate neurites arrested in their growth by prenatal exposure to cocaine, reinitiating their outgrowth. In addition, glial maturation and proliferation continues at a high rate during the early postnatal period and may contribute to the stimulatory effects of cocaine. The release of growth factors by glial cells in response to 5-HT stimulation has been reported previously47'48. Thus the stimulation of serotonergic neurite growth by cocaine during the early postnatal period may reflect the activation of glial receptors by 5-HT rather than neuronal receptors, while prenatally primarily neuronal receptors would be activated. The inhibition of 5-HT reuptake by cocaine prenatally results in autoinhibition of neurite outgrowth, but postnatally results in the release of growth factors which stimulate neurite outgrowth. The results reported here show that gestational exposure to cocaine causes a neurochemical imbalance, at least in the early postnatal period. Cocaine delays the maturation of the serotonergic system and may result in inappropriate synaptic connectivities following recovery. The developmental effects of cocaine are not limited to the serotonergic system: we have found increased tyrosine hydroxylase immunoreactivity in the cerebral cortex following prenatal exposure 1. While no significant changes in [3H]paroxetine binding are detectable at 4 weeks postnatal, such early changes in the terminal out-
62 growth of 5-HT fibers may result in long-term behavioral
sleep patterns, increased tremulousness and reactivity to
deficits. In a recent study, Shemer and associates found that 5-methoxytryptamine administration prenatally at
sensorineural stimuli, a b n o r m a l hypoxic arousal responses, poor visual processing and an increased incidence of Sudden Infant Death Syndrome 9'12'13'34'35'38. This study
low doses resulted in alterations in 5-HT terminal fiber densities detectable
only on
PND1
but
behavioral
changes at PND30. The authors concluded that 5-HT not only autoregulates its own development but may also play a role in neurochemical imprinting4°.
shows that prenatal cocaine exposure affects the develo p m e n t of the serotonergic system which may help explain some of the behavioral deficits observed in h u m a n infants.
O u r findings are significant in light of the recent increase in the n u m b e r of infants exposed to cocaine in utero 8'1s. Clinically, infants exposed to cocaine in utero have been reported to suffer from a n u m b e r of behavioral and neurological deficits which include abnormal
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Acknowledgements. Research supported by NIDA contract 27190-7403 to E.C.A. The authors wish to thank Suzanne Akbari for help in preparation of the manuscript and Patrick Gannon for photographic assistance.
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Hollenbeck, A.R., Behavioral effects of midpregnancy administration of lidocaine and mepivacaine in the rat, Neurobehav. Toxicol. Teratol., 8 (1985) 691-695. Sobrian, S.K., Burton, L.E., Robinson, N.L., Ashe, W.K., James, H., Stokes, D.L. and Turner, L.M., Neurobehavioral and immunological effects of prenatal cocaine exposure in rat, Pharmacol. Biochem. Behav., 35 (1990) 571-575. Spear, L.P., Kirstein, C.L., Bell, J., Yoottanasumpun, V., Greenbaum, R., O'Shea, J., Hoffman, H. and Spear, N.E., Effects of prenatal cocaine exposure on behavior during the early postnatal period, Neurotoxicol. Teratol., 11 (1989) 57-63. Spear, L.P., Kirstein, C.L. and Frambes, N.A., Cocaine effects on the developing central nervous system: behavioral, psychopharmacological, and neurochemical studies. In D.E. Hutchings (Ed.), Prenatal Abuse of Licit and Illicit Drugs, Ann. N.Y.. Acad. Sci., Vol. 562, N.Y. Acad. Sci., New York, 1989, pp. 290-307. Taylor, D. and Ho, B.T., Effect of short- and long-term treatment with cocaine on rat brain tryptophan hydroxylase, Res. Commun. Chem. Pathol. Pharmacol., 15 (1976) 805-808. Wallace, J.A. and Lander, J.M., Development of the serotonergic system in the rat embryo: an immunocytochemical study, Brain Res. Bull., 10 (1983) 459-479. Whitaker-Azmitia, P.M., Murphy, R. and Azmitia, E.C., Stimulation of astroglial 5-HTla receptors releases the serotonergic growth factor, protein S-100, and alters astrogiial morphology, Brain Research, 528 (1990) 155-158. Whitaker-Azmitia, P.M. and Azmitia, E.C., Stimulation of astroglial serotonin receptors produces culture media which regulates growth of serotonergic neurons, Brain Research, 369 (1989) 80-85. Whitaker-Azmitia, P.M., Lauder, J.M., Shemer, A. and Azmitia, E.C., Postnatal changes in serotoninl receptors following prenatal alterations in serotonin levels: further evidence for functional fetal serotonin1 receptors, Dev. Brain Res., 33 (1987) 285-289. Whitaker-Azmitia, P.M. and Azmitia, E.C., Autoregulation of fetal serotonergic neuronal development: role of high affinity serotonin receptors, Neurosci. Lett., 67 (1986) 307-312. Woods, J.R., Plessinger, M.A. and Clark, K.E., Effect of cocaine on uterine blood flow and fetal oxygenation, J. Am. Med. Assoc., 257 (1987) 957-961.
57
BRES 17424
Prenatal cocaine exposure disrupts the development of the serotonergic system H.M. Akbari a, H.K. Kramer 1, P.M. Whitaker-Azmitia 2, L.P. Spear 3 and E.C. Azmitia 1 1Department of Biology, New York University, New York, NY 10003 (U.S.A.), 2Department of Psychiatry, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) and 3Department of Psychology, State University of New York at Binghamton, Binghamton, NY 13901 (U.S.A.) (Accepted 1 October 1991) Key words: Cocaine; Serotonin; Neuronal development; Paroxetine; Immunocytochemistry; Cortex; Hippocampus
Prenatal cocaine exposure has been found to result in a number of neurobehavioral abnormalities in both clinical and laboratory studies. We have previously shown that cocaine inhibits the growth of developing serotonin neurons in culture. This study examines the effects of cocaine on the developing serotonin system in vivo. Pregnant rats were injected with cocaine (40 mg/kg s.c.) from gestational day 13 to parturition. One group of rats was additionally injected on postnatal days 1-5 with cocaine (10 mg/kg s.c.). [3H]Paroxetine, a selective ligand for the serotonin uptake carder, was used to quantify serotonin terminal fiber density at one day, one week, and four weeks postnatal. Cocaine exposure was found to significantly decrease [aH]paroxetine-labelled sites and thus the density of serotonin fibers in the cortex and hippocampus at one day and one week postnatal. By four weeks postnatal, no significant effect was observed, indicating that a recovery had occurred. Serotonin immunocytochemistry performed at one month revealed normal fiber distribution in the cortex but a loss of fibers in the CA1 and CA2 hippocampal fields. Postnatal treatment alleviated the effects of prenatal cocaine exposure, resulting in [3H]paroxetine binding levels at one week which were comparable to and, in the cortex, even higher than those of saline controls. We conclude that cocaine delays the maturation of the serotonin system when administered prenatally but may accelerate maturation when administered both pre- and postnatally. INTRODUCTION Previous animal studies have shown that prenatal exposure to cocaine causes a n u m b e r of teratogenic and neurobehavioral abnormalities. Increases in fetal hemorrhage and abruptio placentae have been reported following gestational cocaine administration to rats ~'~6. Teratogenic effects that have been shown include soft tissue, cardiovascular, and skeletal defectsa3; however, a lack of teratogenicity has been reported at doses as high as 60 mg/kg 16. Neurobehavioral effects of prenatal cocaine administration include increases in locomotor activity and attenuations in shock-precipitated wall climbing at postnatal day 1243. Such exposure has also been found to delay the acquisition of the righting reflex 25. However, using a different treatment regimen, Sobrian et al. have shown accelerated development of the surface righting reflex and startle reflex 42. In addition, impaired performance in an odor-association task and aversive conditioning task has been shown 44. Although much research has centered on the behavioral consequences of in utero cocaine exposure, little work has been done on the un-
derlying neurochemical changes. Increased brain glucose utilization 14'15 and decreased responsiveness to a catecholaminergic challenge 42 have been reported; however, no studies have focused on the neurochemical bases of these effects. Cocaine affects the developing central nervous system both directly and indirectly. Indirectly, cocaine may result in fetal hypoxia by vasoconstriction of the umbilical artery 51, thus adversely affecting the developing brain. Cocaine's anesthetic properties may also affect the developing nervous system, since prenatal exposure to lidocaine has been previously shown to result in behavioral deficits 41. Directly, cocaine interacts with a number of neurochemical systems. Cocaine exerts its psychotropic effects primarily by inhibition of the reuptake of dopamine ( D A ) , norepinephrine (NE), and serotonin and stimulation of dopamine release 5'21'24'28'36'a7. Other neurochemical effects of cocaine include stimulation of tyrosine hydroxylase activity 45, inhibition of tryptophan uptake a7, and reduction of serotonin synthesis a9. Cocaine would presumably have similar neurochemical effects on the developing nervous system. Cocaine has also
Correspondence: H.M. Akbari, Dept. of Biology, New York University, 1009 Main Bldg., 100 Washington Sq. East, New York, NY 10003, U.S.A.
58 been found to be a competitive antagonist at the 5-HT 3 receptor TM which has been described in the rat central nervous system 27. Serotonin (5-HT), one of the neurotransmitter systems affected by cocaine, has been shown to act as a developmental regulatory substance both on its own develo p m e n t and on the proliferation and m a t u r a t i o n of other n e u r o n a l populations 3'1°'29'3°. Specifically, 5-HT has been found to have an autoregulatory inhibitory effect on the growth of fetal cultured 5-HT n e u r o n s as assessed by [3H]5-HT uptake and 5-HT immunocytochemistry49"5°. Similarly, it has been concluded that 5-HT autoregulates the biochemical differentiation of serotonergic n e u r o n s in culture 26, inhibits growth cone activity, and causes neurite retraction in cultured 5-HT Helisoma neurons 22' 23,32. Thus the extracellular accumulation of 5-HT caused by cocaine may inhibit the outgrowth of serotonergic neutites. In previous in vitro experiments, we found cocaine to inhibit the m a t u r a t i o n of cultured 5-HT n e u r o n s as measured by [3H]5-HT uptake 2. In this study, the effects of cocaine on the development of the serotonergic system were investigated in vivo. Cocaine at a dose of 40 rag/ kg/3 ml or saline was administered from gestational day 13 to parturition. Gestational day 13 has been shown to be the earliest date that 5-HT immunoreactivity could be detected in the developing rat brain 46. The dose of cocaine used has been previously characterized as producing high fetal levels and behavioral teratogenicity without producing gross abnormalities44. 5-HT fiber density was assessed indirectly on postnatal days 1, 7, and 28 with [3H]paroxetine, a ligand for the 5-HT uptake cartier, and directly by 5-HT immunocytochemistry on postnatal day 28.
MATERIALS AND METHODS
Animal treatments Timed pregnant Sprague-Dawley rats (Taconic Farms, PA) were obtained on gestational day (GD) 10 and housed individually in plastic breeding cages with free access to food and water. On GD13, pregnant dams were randomly assigned to two treatment groups. Animals received subcutaneous injections of either 40 mg/ kg/3 ml cocaine HC1 (NIDA, Washington, DC) or an equivalent volume of saline vehicle from GD13 to parturition. Gestational exposure to this dose of cocaine has been previously shown to cause neurobehavioral and neuroehemical alterations in offspring43. On the day of birth, litters in both treatment groups were culled to 8 pups/litter to ensure standard nutrition. Litters from cocaine-treated dams were fostered to untreated dams to control for the residual effects of the drug on maternal behavior. In addition, one group of the cocaine-treated pups was given subcutaneous injections of 10 mg/kg cocaine HCl from postnatal day (PND) i to PND5 inclusive. [3H]Paroxetine binding On PND1, PNDV, and PND28, 4 pups from each treatment group were randomly selected and killed by decapitation for [3H]-
paroxetine binding to determine the density of 5-HT uptake sites. This method of quantification of 5-HT uptake sites with [3H]paroxetine as a measure of 5-HT fiber density has been previously shown to be feasible in the assessment of drug toxicity6'7. At least 3 different litters were represented for each group. Brains were rapidly removed and dissected on ice. Appropriate brain regions were weighed, placed in 40x volume by weight (v/w) 50 mM Tris buffer with 120 mM NaCI, 10 mM KCI, and 0.1% ascorbate (pH 7.4), rapidly frozen on dry ice, and kept frozen at -70°C until assay was performed. On day of assay, samples were thawed to 4°C and homogenized with a glass-Teflon homogenizer. Samples were centrifuged at 15,000 rpm for 10 min and the ensuing pellet was resuspended to 40x v/w in Tris buffer. One hundred and sixty td aliquots of the tissue homogenates were plated in 96-well plates (Nunc, Denmark) and allowed to equilibrate for 10 min with either 40/al Tris buffer for total binding or 40 ~1 10-5 (final concentration) fluoxetine (gift from Eli Lilly Co., Indianapolis, IN) to determine non-specific binding. Following this preincubation, the tissue was incubated for one hour with 0.125 nM [3H]paroxetene (final concentration, spec. act. 26.5 Ci/mmol; NEN-Dupont, Boston, MA). Samples were harvested in a Titertek harvester with ice-cold Tris buffer. The filters were collected and placed in 3 mt of Liquiscent (National Diagnostics, Manville, NJ) and counted for 1 min in a Beckman liquid scintillation counter (Model 1801; counting efficiency 38%). Protein content of samples was determined using the Lowry method 31 and CPM data was converted to fmol [3H]paroxetene bound/rag protein.
lmmunocytochemistry In addition to homogenate binding studies, immunocytochemistry was performed at PND28 to compare the innervation pattern of 5-HT fibers in the cocaine and saline groups. Four pups from each treatment group were randomly selected and perfused transcardially on PND28 with ice cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) over 15 min. Brains were removed and postfixed for 4 h. Sixty-#m sections were cut on a Series 1000 vibratome (Kramer) and collected in 0.1 M PBS. Following two washes in PBS, 3 washes in 0.1 M Tris-buffered saline (TBS) and a one hour incubation in blocking serum, sections were incubated overnight in primary antibody raised in goat directed against 5-HT diluted 1:3000 (Incstar Corp., StiUwater, MN). Antibodies were diluted in TBS containing 1% blocking serum and 0.3% Triton (Sigma). Sections were incubated for one hour each in biotinylated secondary rabbit anti-goat antibody and the avidin-biotin complex (Vector Labs., Burlingame, CA), then reacted with 0.05% diaminobenzidene with 0.003% hydrogen peroxide for 15 min. Sections were mounted, dehydrated and coverslipped with DPX mountant (DBH). Slides were viewed on a Leitz microscope and photographs taken with Ilford Pan F film (ASA 50). Statistics The statistical significance of the results from the [3H]paroxetine binding experiments was evaluated with the Student's t-test. Significance was assumed at the level of P < 0.05. All binding data are given as means + S.E.M.
RESULTS The density of 5-HT uptake sites in 3 different brain regions was assessed using [3H]paroxetine, a ligand at the 5-HT uptake carrier, at 3 postnatal ages (one day, one week, and 4 weeks postnatal) following prenatal cocaine exposure. O n PND1, there were significant decreases in the density of 5-HT uptake sites in the cerebral cortex in the cocaine-treated animals as shown by decreased [3H]paroxetine binding levels (66.76% of sa-
59
1 DAY POSTNATAL 140
+
120. 100.
~-~ I -'r
,o 20
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CORTEX SPINAL CORD prenatal saline prenatal cocaine
BRAIN STEM
Fig. 1. Effects of prenatal cocaine on the specific binding of [3H]paroxetine in the cerebral cortex, brainstem, and spinal cord at 1 da~, postnatal. Each bar represents the mean from 4 animals from at least 3 litters. Error bars represent the S.E.M. * Significant with regpect to saline control by Student's t-test, P < 0.05.
line control, P < 0.05) when compared to saline-treated animals (Fig. 1). However, no decreases were evident in either the spinal cord or the brainstem of these animals. At one week postnatal, a decrease in the n u m b e r of [3H]paroxetine-labelled sites in the cortex was again observed in prenatally treated animals when compared to saline controls (64.76%, P < 0.05). In addition, significant decreases in labelled uptake sites were found in the hippocampus in cocaine-treated animals (48.21% of saline control, P < 0.05). These results indicate a decrease in the density of 5-HT fibers in these two regions. In contrast to forebrain regions, no change could be de-
tected in the spinal cord (Fig. 2). As shown in Fig. 3, no significant differences in [3H]paroxetine binding were observed between the animals exposed to cocaine prenatally and the saline controls at 4 weeks postnatal in either the cortex or hippocampus. There was a trend towards higher binding levels in some of the prenatal cocaine animals; however, this did not demonstrate significance. These results indicate that a recovery of the serotonergic innervation of the cortex and hippocampus occurs between one week and four weeks postnatal. To determine whether the distribution of serotonergic fibers following recovery in the cocaine-treated animals was similar to the normal pattern, immunocytochemistry was performed on PND28 on an additional group of animals. The distribution of 5-HT immunoreactive fibers in all cortical regions examined (parietal, frontal, cingulate and entorhinal cortices) in prenatal cocaine-treated animals was similar to saline controls. However, the 5-HT immunoreactive fiber distribution in the hippocampus following gestational cocaine exposure was altered when compared to controls. Decreases were observed in the density of fibers in two regions as shown in Fig. 4. A loss of 5-HT fibers in the stratum lacunosum-moleculate of the CA1 hippocampal field and the stratum radiatum of the C A 2 field was seen 4 weeks postnatal following prenatal cocaine treatment. Thus, unlike the cortical 5-HT system, prenatal cocaine results in longterm alterations in the serotonergic innervation of the hippocampus. In contrast to the results obtained from animals prenatally treated with cocaine, no decreases in [3H]paroxetine binding were observed in animals treated perina-
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prenatal saline prenatal cocaine pre- & postnatal cocaine
Fig. 2. Effects of prenatal cocaine (solid bars) and pre- and postnatal cocaine (hatched bars) on the specific binding of [3H]paroxetine in the cerebral cortex, spinal cord, and hippocampus at 1 week postnatal. Each bar represents the mean from 4 animals. Error bars represent the S.E.M. * Significant with respect to saline control by Student's t-test, P < 0.05. ** Significant with respect to prenatal cocaine, P < 0.05. *** Significant with respect to prenatal cocaine, P < 0.01.
0
CORTEX STRIATUM r ~ l prenatal saline prenatal cocaine pre- & postnatal cocaine
HIPPOCAMPUS
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Fig. 3. Specific binding of [3H]paroxetine in the cerebral cortex, striatum, and hippocampus at 4 weeks postnatal following prenatal cocaine (solid bars) or pre- and postnatal cocaine (hatched bars) administration. Each bar represents the mean from 4 animals. Error bars represent the S.E.M. * Significant with respect to prenatal cocaine by Student's t-test, P < 0.05.
60
Fig. 4. Darkfield photomontage of 60 ~m coronal section of the hippocampus immunostained for 5-HT. Serotonergic fibers are decreased along the stratum lacunosum-molecutare of the CA1 field (bold arrow) and stratum radiatum of the CA2 field (curved arrow) in section from prenatally cocaine-treated animal (B) when compared to control animal (A). Bar = 60 ~m.
tally with tical mals
(i.e. both pre- and postnatally; G D 1 3 - 2 2 ; P N D 1 - 5 ) cocaine. As can be seen in Fig. 2, on PND7, cor[3H]paroxetine binding in pefinatally treated aniwas significantly increased when compared to either
prenatal saline or prenatal cocaine-treated animals (123% and 201%; P < 0,05 and P < 0.01, respectively). In the hippocampus, [3H]paroxetine binding was significantly higher in animals treated perinatally with cocaine when
61 compared to prenatal cocaine-treated animals (209%, P < 0.01). Additionally, the levels of [3H]paroxetine binding in the spinal cord preparations from perinatally treated animals were significantly higher (206%, P < 0.05) than those of animals treated only prenatally. These levels were also higher than animals treated with saline, although this did not show significance (Fig. 2). By 4 weeks postnatal, no significant differences in [3H]paroxetine binding could be seen between the 3 groups. DISCUSSION Prenatal exposure to 40 mg/kg cocaine from GD13 to parturition results in significant changes in the serotonergic system during the early postnatal period. Our data indicate that cocaine inhibits the normal growth of 5-HT fibers to the cerebral cortex and hippocampus but not to the spinal cord. The lack of a noticeable effect on the [3H]paroxetine binding in spinal cord at one day and one week indicates that the decreases seen elsewhere are not a residual effect of the drug in the neonates. At one week, the cerebral cortex and hippocampus showed decreases in [3H]paroxetine binding in the cocaine-treated animals. The inhibition of fiber outgrowth by cocaine may be due to any one of a number of neurochemical changes which cocaine induces. The primary action of cocaine on serotonergic neurons is an inhibition of 5-HT reuptake 37 resulting in increased extraceUular concentrations of 5-HT. This extracellular accumulation of 5-HT would result in autoinhibition of serotonergic neurite outgrowth 5°. Thus cocaine's inhibition of neuronal reuptake of 5-HT may be directly responsible for the delayed innervation of forebrain regions following prenatal cocaine exposure. By 4 weeks postnatal, the [3H]paroxetine binding levels in prenatally cocaine-treated animals were not significantly lower than saline controls, indicating a recovery of the 5-HT fibers. Whether this is due to a regrowth from damaged axons or represents a delayed maturation is not clear, although the latter seems more likely due to the rapid recovery period. If fibers had initiated a normal innervation and then degenerated, a recovery period of 3 weeks would not be sufficient for complete recovery since in previous studies on 5-HT fiber regrowth only 50% of control values had been reached at one month following 5,7-dihydroxytryptamine lesions ~9. However, that study was performed in an adult animal and may not be an accurate measure of 5-HT fiber regrowth in the immature brain. Immunocytochemistry revealed a normal pattern of cortical 5-HT fibers at 4 weeks postnatal, indicating that the reinnervation was not merely quantitative, but qualitative. The 5-HT fiber distribution was similar in pre-
natal cocaine-treated animals and saline controls in all cortical regions examined. In the hippocampus, decreases in the density of 5-HT immunoreactive fibers were seen in the stratum radiatum of CA2 and stratum lacunosummoleculare of CA1. Both of these hippocampal regions receive fibers originating primarily from the median raphe nucleus 4 which raises the possibility that fibers from the median raphe nucleus may be more susceptible to the long-term effects of prenatal cocaine. The effects on [3H]paroxetine binding caused by continued treatment through the first 5 postnatal days were quite different from prenatal cocaine treatment alone. Paradoxically, these animals did not exhibit the decreases in [3H]paroxetine binding seen in the prenatal cocaine group, but instead appeared to exhibit an increased rate of development. At one week postnatal, animals treated with cocaine perinatally had increased binding both with respect to the prenatal cocaine group and, in the cortex, the prenatal saline group. With respect to neurite extension and maturation, 5-HT has been found to have a stimulatory effect in organotypic cultures 1°, indicating that neurite outgrowth is facilitated by 5-HT once target cells are reached. 5-HT has also been reported to stimulate a reinitiation of growth in embryonic neurons which have ceased to elongate 2°. Perinatal treatment with cocaine would maintain high extracellular concentrations of 5-HT, which could then stimulate neurites arrested in their growth by prenatal exposure to cocaine, reinitiating their outgrowth. In addition, glial maturation and proliferation continues at a high rate during the early postnatal period and may contribute to the stimulatory effects of cocaine. The release of growth factors by glial cells in response to 5-HT stimulation has been reported previously47'48. Thus the stimulation of serotonergic neurite growth by cocaine during the early postnatal period may reflect the activation of glial receptors by 5-HT rather than neuronal receptors, while prenatally primarily neuronal receptors would be activated. The inhibition of 5-HT reuptake by cocaine prenatally results in autoinhibition of neurite outgrowth, but postnatally results in the release of growth factors which stimulate neurite outgrowth. The results reported here show that gestational exposure to cocaine causes a neurochemical imbalance, at least in the early postnatal period. Cocaine delays the maturation of the serotonergic system and may result in inappropriate synaptic connectivities following recovery. The developmental effects of cocaine are not limited to the serotonergic system: we have found increased tyrosine hydroxylase immunoreactivity in the cerebral cortex following prenatal exposure 1. While no significant changes in [3H]paroxetine binding are detectable at 4 weeks postnatal, such early changes in the terminal out-
62 growth of 5-HT fibers may result in long-term behavioral
sleep patterns, increased tremulousness and reactivity to
deficits. In a recent study, Shemer and associates found that 5-methoxytryptamine administration prenatally at
sensorineural stimuli, a b n o r m a l hypoxic arousal responses, poor visual processing and an increased incidence of Sudden Infant Death Syndrome 9'12'13'34'35'38. This study
low doses resulted in alterations in 5-HT terminal fiber densities detectable
only on
PND1
but
behavioral
changes at PND30. The authors concluded that 5-HT not only autoregulates its own development but may also play a role in neurochemical imprinting4°.
shows that prenatal cocaine exposure affects the develo p m e n t of the serotonergic system which may help explain some of the behavioral deficits observed in h u m a n infants.
O u r findings are significant in light of the recent increase in the n u m b e r of infants exposed to cocaine in utero 8'1s. Clinically, infants exposed to cocaine in utero have been reported to suffer from a n u m b e r of behavioral and neurological deficits which include abnormal
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Acknowledgements. Research supported by NIDA contract 27190-7403 to E.C.A. The authors wish to thank Suzanne Akbari for help in preparation of the manuscript and Patrick Gannon for photographic assistance.
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