- Email: [email protected]
Convergent control of serotonin transporter expression by glucocorticoids and cocaine in fetal and neonatal rat brain1
Developmental Brain Research 104 Ž1997. 209–213
Short communication
Convergent control of serotonin transporter expression by glucocorticoids and cocaine in fetal and neonatal rat brain 1 K.E. McGrath, F.J. Seidler, T.A. Slotkin
)
Department of Pharmacology, Duke UniÕersity Medical Center, Box 3813, Durham, NC 27710, USA Accepted 19 August 1997
Abstract Serotonin plays a trophic role in brain cell differentiation. In this study, expression of the serotonin presynaptic transporter protein, which regulates the extracellular serotonin concentration, was measured with w 3 Hxparoxetine in rats exposed to dexamethasone or cocaine prenatally. Within 24 h of a single dose of dexamethasone, significant increases were seen in fetal brain, and the effect persisted into the postnatal period. Chronic prenatal cocaine exposure elicited similar changes. These data indicate that exposures to apparently disparate drugs can elicit similar endpoints that may lead to behavioral teratogenesis. q 1997 Elsevier Science B.V. Keywords: Brain development; Cocaine; Dexamethasone; Glucocorticoid; Paroxetine; Serotonin; Transporter
In addition to their role in neurotransmission, biogenic amines are now known to provide important trophic inputs that influence differentiation of neural cells. This relationship has been best explored for serotonin, which controls the assembly of the nervous system from its origins in the neural crest and neural tube, all the way through the terminal events of differentiation including axonogenesis and synaptogenesis w12,15,16,19,47,50x. The presence of serotonin-synthesizing enzymes and the requisite neurotransmitter receptors in developing brain have been demonstrated clearly w16,50x. Although the ability to manufacture and release serotonin is an essential component of this neurotrophic process, major regulation of the extracellular serotonin concentration is provided by the high-affinity serotonin transporter that pumps the amine back into the cell, and the development of this protein has received recent attention as a target for developmental disrupters w19,31,35x. Most obviously, drugs like cocaine that bind directly to the transporter and inhibit its activity have been shown to affect the development and function of this protein w1,19,20,31x; in addition, factors like prenatal stress and glucocorticoids also have been found to elicit long-term alterations in transporter expression or in responses that are influenced by transporter function w13,27,35x.
) Corresponding author. Fax: q1 Ž919. 684-8197; E-mail: [email protected] 1 Supported by USPHS MH-40159.
0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 1 4 4 - 2
We recently found that late gestational exposure of rats to the synthetic glucocorticoid, dexamethasone, produced persistent elevations of serotonin transporter expression in adolescence and adulthood w35x, an effect which did not occur when dexamethasone was given to mature rats w38,39x. Because such changes influence serotonergic function, these findings are important for understanding the neurobehavioral teratogenesis associated with gestational glucocorticoid treatment w3x. They also raise the question of whether transporter overexpression occurs similarly in the fetal brain during the period in which serotonin influences cell differentiation and synaptogenesis, thus providing a mechanistic tie between effects on fetal serotonergic systems and adverse behavioral outcomes. In the current study, we have examined the immediate effects of prenatal dexamethasone treatment on the expression of transporter protein using the specific radioligand, w 3 Hxparoxetine w10,11x. We have contrasted the effects of single or repeated dexamethasone administration with those of cocaine. Timed pregnant Sprague–Dawley rats ŽZivic–Miller Laboratories, Allison Park, PA. were shipped by climatecontrolled truck Žtotal transit time: 12 h. and were housed individually in breeding cages with free access to food and water. On gestational day 17, dams were given 0.05, 0.2 or 0.8 mgrkg s.c. of dexamethasone phosphate ŽMerck Sharp and Dohme, Rahway, NJ., whereas controls received equivalent volumes of isotonic saline vehicle Ž1 mlrkg.. This dose range has been shown previously to span the
210
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
threshold for nervous system abnormalities and selective promotion of neuronal differentiation w3,23,24,36,37,41x. On gestational day 18 Ž24 h after dexamethasone treatment., the dams were decapitated, fetuses were removed and the brain dissected and frozen immediately on dry ice. Tissues were stored at y458C until assayed; preliminary work showed that w 3 Hxparoxetine binding was unaffected by freezing. An additional set of measurements was made immediately after birth. Animals received three treatments with the intermediate dose Ž0.2 mgrkg. of dexamethasone on gestational days 17, 18 and 19. The day after birth Žpostnatal day 1., animals were decapitated and the brain rapidly dissected by making blunt cuts through the cerebellar peduncles, removing the cerebellum, and making a cut rostral to the thalamus to separate the brainstem from the forebrain. This division follows the natural dissection planes of neonatal rat brain so that the region designated as ‘brainstem’ includes the colliculi, pons and medulla oblongata Žbut not cervical spinal cord., as well as the thalamus Žwhich is ordinarily not considered part of this region.. This region was selected because prior work has identified long-term changes in brainstem serotonin transporter expression after prenatal dexamethasone exposure w35x. In a third set of experiments, beginning on gestational day 8 Žjust after implantation of the fetus in the uterine wall. pregnant rats were given daily s.c. injections of 30 mgrkg of cocaine HCl ŽMallinckrodt, Inc., St. Louis, MO. divided into three doses injected into separate sites on the back so as to avoid tissue necrosis; controls received three injections of isotonic saline vehicle in the equivalent volume Ž3 mlrkg divided into three injection sites.. Cocaine was discontinued after the 20th day of gestation Ž2 days before birth. so as not to interfere with parturition. This dose and route have been shown to approximate plasma cocaine levels in human users w32,44x, to affect brain development w2,14x and to cause long-term neurobehavioral alterations w7,44,45x. Higher doses of cocaine cause convulsions, severe maternal weight loss and increased maternal mortality w30x, effects which were entirely absent at the dose used here Ždata not shown.. Cocaine attaches directly to the serotonin transporter within the developing brain w19x and hence, w 3 Hxparoxetine binding studies performed during gestation would be confounded by the presence of this competitor; accordingly, evaluations in the cocaine study were conducted the day after birth Žpostnatal day 1.. All studies using live animals were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Membrane fractions were prepared by techniques described previously w22x. In brief, tissues were thawed and homogenized ŽPolytron, Brinkmann Instruments, Westbury, NY. in 39 volumes of 120 mM NaCl, 5 mM KCl, 50
mM Tris-HCl ŽpH 7.4., sedimented at 40 000 = g for 15 min, and the supernatant solution was discarded. The pellet was washed twice by resuspension ŽPolytron. in the original volume of buffer, followed each time by resedimentation at 40 000 = g, after which the final pellet was resuspended Žsmooth glass homogenizer fitted with a Teflon pestle. to achieve a protein concentration of approximately 2.5 mgrml. This suspension was then used immediately for the w 3 Hxparoxetine binding assay w22x. Aliquots of membrane suspension corresponding to approximately 100 mg protein w17x were incubated in a total volume of 2 ml of 120 mM NaCl, 5 mM KCl, 50 mM Tris-HCl ŽpH 7.4. containing 100 pM paroxetinewphenyl-6X- 3 Hx Žspecific activity: 25.4 Cirmmol; New England Nuclear Corp., Boston, MA. with or without addition of 100 mM serotonin ŽSigma Chemical Co., St. Louis, MO. to displace specific binding. Incubations lasted 120 min at 208C, after which incubations were stopped by addition of 5 ml of ice-cold buffer, followed by vacuum filtration onto Whatman GFrC filters that were pre-soaked in 0.05% polyethyleneimine ŽSigma.; filters were washed 3 times with 5 ml of buffer, after which radioactivity was counted. Preliminary experiments were conducted to verify that the incubation conditions permitted equilibration of radioligand with the binding sites. Non-specific binding was 10–25% of the total depending upon age and brain region. All determinations were run in duplicate. In order to have sufficient tissue for analysis, brains and brain regions from several fetuses or pups within the same litter were combined to make a single sample. The large number of tissues involved in this study, six treatment groups, two age points, two brain regions, and up to 23 animals per treatment group, produced a total of over 100 membrane preparations to be examined. This fact, and the small amount of tissue available from each animal, precluded the practicability of running Scatchard analyses. Therefore, we examined binding using a w 3 Hxparoxetine concentration Ž100 pM. 3–4 times above the K d found in developing rat brain w38,39x, thus ensuring that measurements would reflect changes in maximal binding primarily, rather than in ligand affinity for the receptor. Because of the high binding affinity of w 3 Hxparoxetine Žin the pM range., we also verified that the free concentration of ligand at the end of the incubation period was not significantly changed from the initial value Žrange: 95–98 pM.. Previous studies with older animals indicate that the effects of dexamethasone on w 3 Hxparoxetine binding reflect alterations in the number of transporter sites w38,39x. For all experiments, a given dam or litter contributed to only a single sample, so that the number of determinations represents the numbers of dams or litters. Data are reported as means and standard errors and treatment effects were established using ANOVA, with data log-transformed whenever variance was heterogeneous. For the cocaine studies, there were two factors ŽTreatment, Region. and because a significant interaction of the factors was found,
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
211
Table 1 Effects of dexamethasone Žgestational day 18. Treatment Control Dexamethasone Dexamethasone Dexamethasone
0.05 mgrkg 0.2 mgrkg 0.8 mgrkg
Weight Žmg.
Membrane protein Žmgrg tissue.
w 3 HxParoxetine binding Žfmolrmg protein.
72 " 3 67 " 5 70 " 4 74 " 5
26.0 " 0.5 25.4 " 0.6 25.8 " 0.5 24.8 " 1.1
38.6 " 2.3 45.2 " 1.2 ) 46.2 " 1.4 ) 47.6 " 0.7 )
Data represent means and standard errors from 5–6 determinations in each treatment group. ANOVA across all treatments indicates no significant effects of dexamethasone on brain weight Ž P ) 0.7. or membrane protein Ž P ) 0.8., but a significant effect on w 3 Hxparoxetine binding Ž P - 0.003.. The effect on binding is significant for all three doses individually Ž ) P - 0.01..
separate analyses were conducted post-hoc ŽFisher’s Protected Least Significant Difference. to determine which regionŽs. showed significant effects. For all tests, significance was assumed at P - 0.05. Administration of dexamethasone did not cause significant inhibition of fetal brain growth within the first 24 h nor were there any adverse effects on membrane protein ŽTable 1.. Nevertheless, all three doses produced significant elevations of w 3 Hxparoxetine binding. Repeated administration of the intermediate dose Ž0.2 mgrkg. on gestational days 17, 18 and 19 led to significant elevations in w 3 Hxparoxetine binding on postnatal day 1 Žcontrol, 200 " 9 fmolrmg protein, n s 6; dexamethasone, 232 " 7 fmolrmg protein, n s 4, P - 0.04.; again, these were not accompanied by alterations in tissue weight or protein concentration Ždata not shown.. The magnitude of the effects on w 3 Hxparoxetine binding seen during gestation and just after birth Ž15–25% elevation. was comparable to that seen previously in adolescence and adulthood after repeated fetal dexamethasone administration w35x. Chronic prenatal cocaine administration also had no discernible effect on brain region weights or membrane protein concentrations measured on postnatal day 1; nevertheless, w 3 Hxparoxetine binding was significantly elevated, with the effect restricted to the brainstem as opposed to forebrain ŽTable 2.. Table 2 Effects of cocaine Žpostnatal day 1. Treatment and region Brainstem Control Cocaine Forebrain Control Cocaine
Weight
Membrane protein
w 3 HxParoxetine binding
Žmg.
Žmgrg tissue.
Žfmolrmg protein.
109"1 111"1
27.1"0.3 26.6"0.5
226"5 242"5 )
168"2 170"3
19.0"0.3 19.2"0.2
94"2 93"2
Data represent means and standard errors from 19–23 determinations for each region in each treatment group. Two-factor ANOVA ŽTreatment, Region. indicates no significant effects of cocaine on brain region weight Ž P ) 0.2. or membrane protein Ž P ) 0.8. but a significant, regionally dependent effect on w 3 Hxparoxetine binding ŽTreatment, Region, P 0.05.. The effect on binding is significant in the brainstem Ž ) P - 0.02. but not in the forebrain.
In our previous work, we showed that repeated prenatal dexamethasone treatment leads to up-regulation of serotonin transporter expression, assessed postnatally in adolescent and adult brainstem w35x. The current results indicate that the up-regulation actually begins within 24 h of a single dose of dexamethasone, so that transporter overexpression is also present during fetal brain development. This effect of exogenous glucocorticoid administration most probably occurs also with endogenous steroid release: prenatal administration of ACTH has been shown to increase the uptake of serotonin into brainstem nerve terminals w13x, the functional correlate of transporter overexpression. Thus, either exogenous or endogenously released glucocorticoids are likely to affect the availability of extracellular serotonin to interact with its receptor sites. The neurotrophic role of serotonin is now known to extend beyond the neural tube stage, to incorporate axonogenesis, synaptogenesis and gliogenesis, events which are all occurring during the late gestational period in which dexamethasone was given in the present study w50x. One specific role for serotonin is to accelerate synaptic maturation, so that synaptogenesis occurs at the expense of further axonal growth w50x. Similarly, dexamethasone treatment has been shown to evoke acceleration of the onset of synaptic activity but with eventual compromise of the total synaptic complement w3,34,36,37,40x. Accordingly, the impact on serotonergic neurotrophic control may represent one of the mechanisms underlying the neurobehavioral teratology of glucocorticoids. It should be noted that the ability of glucocorticoids to up-regulate serotonin transporter expression is unique to development as no such effect is seen with comparable treatment of adult rats w38,39x. Many abused substances are known to evoke glucocorticoid release, including nicotine w5,29,48x, opiates w4x and cocaine w21,26,28x. Indeed, the effects of developmental exposure to nicotine on high-affinity serotonin uptake into synaptosomes parallels that of adrenocortical stimulation w13x. In the current study, we examined the effects of chronic cocaine treatment and also observed effects similar to those of glucocorticoid administration; cocaine’s selectivity for transporter expression in the brainstem parallels previous findings with nicotine and ACTH w13x and matches the regional targeting of dexamethasone at the same age
212
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
w35x. Furthermore, cocaine has been shown to evoke corticosteroid release in the maternal–fetal unit w26x. It is therefore likely that some of the adverse effects of cocaine on brain development reflect indirect actions mediated by glucocorticoid release and its subsequent impact on serotonergic mechanisms. However, there are at least two other factors that need to be considered. First, cocaine interacts directly with the serotonin transporter Žas well as those for norepinephrine and dopamine. to inhibit its function. Given the short biologic half-life of cocaine, these direct effects can be expected to terminate rapidly after each dose, so that the blockade is only episodic. Prenatal exposure to amine uptake inhibitors other than cocaine affects brain development and subsequent behavioral performance w6,33,46x, but a study specifically comparing the behavioral effects of developmental exposure to cocaine with that of a serotonin uptake inhibitor found no overlap w8x. A second potential contributor to cocaine’s effects on serotonin transporter expression is hypoxiarischemia from the vasoconstrictor effects of the drug w18,25x. However, hypoxic brain damage elicits synaptic deficits which would produce a fall in the concentration of transporter molecules, not an increase as seen here. For example, repeated gestational administration of opiates in doses that produce respiratory depression causes the expected fall in serotonin transporter capacity w42x. In fact, when cocaine is given in higher doses and drug treatment is initiated later in gestation, transporter expression similarly is suppressed in conjunction with visible defects in serotonergic innervation w1x. Reducing the cocaine dose and beginning treatment earlier in gestation does not produce this synaptic loss w9,43,49x. The difference between commencing cocaine in late gestation as opposed to early gestation Žas used here. is that, with early treatment, vasoconstrictor effects most likely will be absent during later developmental phases of axonogenesis and synaptogenesis because of down-regulation and desensitization of peripheral adrenergic receptors; with later treatment, vasoconstriction remains a factor during these later developmental events. It is thus critical, not only for understanding effects in animal models of maternal cocaine abuse but also for extension of these findings to the potential impact on human brain development, that issues of dose and duration be considered. Accordingly, our results indicate that the effects of cocaine on brain development are likely to exhibit convergent mechanisms with those of the glucocorticoids that are released by cocaine administration, but that additional effects are superimposed on these actions. References w1x H.M. Akbari, H.K. Kramer, P.M. Whitaker-Azmitia, L.P. Spear, E.C. Azmitia, Prenatal cocaine exposure disrupts the development of the serotonergic system, Brain Res. 572 Ž1992. 57–63.
w2x T. Anderson-Brown, T.A. Slotkin, F.J. Seidler, Cocaine acutely inhibits DNA synthesis in developing rat brain regions: evidence for direct actions, Brain Res. 537 Ž1990. 197–202. w3x M.C. Bohn, Glucocorticoid induced teratologies of the nervous system, in: J. Yanai ŽEds.., Neurobehavioral Teratology, Elsevier, Amsterdam, 1984, pp. 365–387. w4x J. Borrell, I. Llorens, S. Borrell, Adrenal, plasma and urinary ´ corticosteroids during single or repeated administration of morphine in cats, Eur. J. Pharmacol. 31 Ž1975. 237–242. w5x G.R. Cam, J.R. Bassett, K.D. Cairncross, The action of nicotine on the pituitary-adrenal cortical axis, Arch. Int. Pharmacodyn. Ther. 237 Ž1979. 49–66. w6x V. Cuomo, Perinatal neurotoxicology of psychotropic drugs, Trends Pharmacol. Sci. 8 Ž1987. 346–350. w7x D.L. Dow-Edwards, Long-term neurochemical and neurobehavioral consequences of cocaine use during pregnancy, Ann. NY Acad. Sci. 562 Ž1989. 280–289. w8x D.L. Dow-Edwards, Modification of acoustic startle reactivity by cocaine administration during the postnatal period: comparison with a specific serotonin reuptake inhibitor, Neurotoxicol. Teratol. 18 Ž1996. 289–296. w9x E.M. Factor, R.P. Hart, G.M. Jonakait, Neurochemical development of the raphe after continuous prenatal cocaine exposure, Brain Res. Bull. 31 Ž1993. 49–56. w10x E.V. Gurevich, J.H. Joyce, Comparison of w 3 Hxparoxetine and w 3 Hxcyanoimipramine for quantitative measurement of serotonin transporter sites in human brain, Neuropsychopharmacology 14 Ž1996. 309–323. w11x E. Habert, D. Graham, L. Tahraoui, Y. Claustre, S.Z. Langer, Characterization of w 3 Hxparoxetine binding to rat cortical membranes, Eur. J. Pharmacol. 118 Ž1985. 107–114. w12x M. Hamon, S. Bourgoin, C. Chanez, F. De Vitry, Do serotonin and other neurotransmitters exert a trophic influence on the immature brain?, Dev. Neurobiol. 12 Ž1989. 171–183. w13x J.A. King, M. Davila-Garcia, E.C. Azmitia, F.L. Strand, Differential effects of prenatal and postnatal ACTH or nicotine exposure on 5-HT high affinity uptake in the neonatal rat brain, Int. J. Dev. Neurosci. 9 Ž1991. 281–286. w14x S.M. Koegler, F.J. Seidler, J.R. Spencer, T.A. Slotkin, Ischemia contributes to adverse effects of cocaine on brain development: suppression of ornithine decarboxylase activity in neonatal rat, Brain Res. Bull. 27 Ž1991. 829–834. w15x J.M. Lauder, Roles for neurotransmitters in development: possible interaction with drugs during the fetal and neonatal periods, in: M. Marois ŽEds.., Prevention of Physical and Mental Congenital Defects, Alan R. Liss, New York, 1985, pp. 375–380. w16x J.M. Lauder, H. Krebs, Serotonin as a differentiation signal in early neurogenesis, Dev. Neurosci. 1 Ž1978. 15–30. w17x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–270. w18x M.P. Mahalik, R.F. Gautieri, D.E. Mann Jr., Mechanisms of cocaine-induced teratogenesis, Res. Comm. Subst. Abuse 5 Ž1984. 279–302. w19x J.S. Meyer, L.P. Shearman, L.M. Collins, Monoamine transporters and the neurobehavioral teratology of cocaine, Pharmacol. Biochem. Behav. 55 Ž1996. 585–593. w20x J.S. Meyer, L.P. Shearman, L.M. Collins, R.L. Maguire, Cocaine binding sites in fetal rat brain: implications for prenatal cocaine action, Psychopharmacology 112 Ž1993. 445–451. w21x R.L. Moldow, A.J. Fischman, Cocaine induced secretion of ACTH, b-endorphin and corticosterone, Peptides 8 Ž1987. 819–822. w22x C. Moret, M. Briley, Platelet 3 H-paroxetine binding to the serotonin transporter is insensitive to changes in central serotonergic innervation in the rat, Psychiat. Res. 38 Ž1991. 97–104.
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213 w23x H.A. Navarro, E.M. Kudlacz, J.P. Eylers, T.A. Slotkin, Prenatal dexamethasone administration disrupts the pattern of cellular development in rat lung, Teratology 40 Ž1989. 433–438. w24x H.A. Navarro, J. Lachowicz, J. Bartolome, W.L. Whitmore, T.A. Slotkin, Effects of prenatal dexamethasone on development of ornithine decarboxylase activity in brain and peripheral tissues of rats, Pediatr. Res. 24 Ž1988. 465–469. w25x G.D. Olsen, Potential mechanisms of cocaine-induced developmental neurotoxicity: a minireview, Neurotoxicology 16 Ž1995. 159–167. w26x J.R. Owiny, M.T. Jones, D. Sadowsky, T. Myers, A. Massmann, P.W. Nathanielsz, Cocaine in pregnancy: the effect of maternal administration of cocaine on the maternal and fetal pituitary-adrenal axis, Am. J. Obstet. Gynecol. 164 Ž1991. 658–663. w27x R.E. Poland, P. Lutchman-Singh, D. Au, S. McGeoy, M. Que, S. Acosta, J.T. McCracken, Exposure to threshold doses of nicotine in utero: 3. Augmentation of the prolactin and ACTH response to 8-OH DPAT by desipramine treatment is compromised in adult male offspring, Neurotoxicology 17 Ž1996. 351–358. w28x F. Rougepont, M. Marinelli, M. LeMoal, H. Simon, P.V. Piazza, Stress-induced sensitization and glucocorticoids: 2. sensitization of the increase in extracellular dopamine induced by cocaine depends on stress-induced corticosterone secretion, J. Neurosci. 15 Ž1995. 7189–7195. w29x R.P. Rubin, W. Warner, Nicotine-induced stimulation of steroidogenesis in adrenocortical cells of the cat, Br. J. Pharmacol. 53 Ž1975. 357–362. w30x F.J. Seidler, T.A. Slotkin, Prenatal cocaine and cell development in rat brain regions: effects on ornithine decarboxylase and macromolecules, Brain Res. Bull. 30 Ž1993. 91–99. w31x L.P. Shearman, L.M. Collins, J.S. Meyer, Characterization and localization of wI-125xRTI-55-labeled cocaine binding sites in fetal and adult rat brain, J. Pharmacol. Exp. Ther. 277 Ž1996. 1770–1783. w32x R.K. Siegel, Cocaine smoking, J. Psychoactive Drugs 14 Ž1982. 271–359. w33x J.W. Simpkins, F.P. Field, G. Torosian, E.E. Soltis, Effects of prenatal exposure to tricyclic antidepressants on adrenergic responses in progeny, Dev. Pharmacol. Ther. 8 Ž1985. 17–33. w34x T.A. Slotkin, G. Barnes, C. Lau, F.J. Seidler, P. Trepanier, S.J. Weigel, W.L. Whitmore, Development of polyamine and biogenic amine systems in brains and hearts of neonatal rats given dexamethasone: role of biochemical alterations in cellular maturation for producing deficits in ontogeny of neurotransmitter levels, uptake, storage and turnover, J. Pharmacol. Exp. Ther. 221 Ž1982. 686–693. w35x T.A. Slotkin, G.A. Barnes, E.C. McCook, F.J. Seidler, Programming of brainstem serotonin transporter development by prenatal glucocorticoids, Dev. Brain Res. 93 Ž1996. 155–161. w36x T.A. Slotkin, S.E. Lappi, E.C. McCook, M.I. Tayyeb, J.P. Eylers, F.J. Seidler, Glucocorticoids and the development of neuronal function: effects of prenatal dexamethasone exposure on central noradrenergic activity, Biol. Neonate 61 Ž1992. 326–336.
213
w37x T.A. Slotkin, S.E. Lappi, M.I. Tayyeb, F.J. Seidler, Dose-dependent glucocorticoid effects on noradrenergic synaptogenesis in rat brain: ontogeny of w 3 Hxdesmethylimipramine binding sites after fetal exposure to dexamethasone, Res. Comm. Chem. Pathol. Pharmacol. 73 Ž1991. 3–19. w38x T.A. Slotkin, E.C. McCook, J.C. Ritchie, B.J. Carroll, F.J. Seidler, Serotonin transporter expression in rat brain regions and blood platelets: aging and glucocorticoid effects, Biol. Psychiatry 41 Ž1997. 172–183. w39x T.A. Slotkin, E.C. McCook, J.C. Ritchie, F.J. Seidler, Do glucocorticoids contribute to the abnormalities in serotonin transporter expression and function seen in depression? An animal model, Biol. Psychiatry 40 Ž1996. 576–684. w40x T.A. Slotkin, E.C. McCook, F.J. Seidler, Glucocorticoids regulate the development of intracellular signaling: enhanced forebrain adenylate cyclase catalytic subunit activity after fetal dexamethasone exposure, Brain Res. Bull. 32 Ž1993. 359–364. w41x T.A. Slotkin, F.J. Seidler, R.J. Kavlock, J.V. Bartolome, Fetal dexamethasone exposure impairs cellular development in neonatal rat heart and kidney: effects on DNA and protein in whole tissues, Teratology 43 Ž1991. 301–306. w42x T.A. Slotkin, W.L. Whitmore, M. Salvaggio, F.J. Seidler, Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat, Life Sci. 24 Ž1979. 1223–1230. w43x A.M. Snyder-Keller, R.W. Keller, Prenatal cocaine increases striatal serotonin innervation without altering the patchrmatrix organization of intrinsic cell types, Dev. Brain Res. 74 Ž1993. 261–267. w44x L.P. Spear, N.A. Frambes, C.L. Kirstein, Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats, Psychopharmacology 97 Ž1989. 427–431. w45x L.P. Spear, C.L. Kirstein, N.A. Frambes, Cocaine effects on the developing nervous system: behavioral, psychopharmacological and neurochemical studies, Ann. NY Acad. Sci. 562 Ž1989. 290–307. w46x S.R. Tonge, Catecholamine concentrations in discrete areas of the rat brain after the pre- and neonatal administration of phencyclidine and imipramine, J. Pharm. Pharmacol. 25 Ž1973. 164–166. w47x K. Turlejski, Evolutionary ancient roles of serotonin: long-lasting regulation of activity and development, Acta Neurobiol. Exp. 56 Ž1996. 619–636. w48x D.M. Turner, The role of adrenal catecholamines in the release of corticosterone and fatty acids by nicotine in the rat, Res. Comm. Chem. Pathol. Pharmacol. 12 Ž1975. 645–655. w49x X.H. Wang, P. Levitt, A.O. Jenkins, E.H. Murphy, Normal development of tyrosine hydroxylase and serotonin immunoreactive fibers innervating anterior cingulate cortex and visual cortex in rabbits exposed prenatally to cocaine, Brain Res. 715 Ž1996. 221–224. w50x P.M. Whitaker-Azmitia, Role of serotonin and other neurotransmitter receptors in brain development: basis for developmental pharmacology, Pharmacol. Rev. 43 Ž1991. 553–561.
Short communication
Convergent control of serotonin transporter expression by glucocorticoids and cocaine in fetal and neonatal rat brain 1 K.E. McGrath, F.J. Seidler, T.A. Slotkin
)
Department of Pharmacology, Duke UniÕersity Medical Center, Box 3813, Durham, NC 27710, USA Accepted 19 August 1997
Abstract Serotonin plays a trophic role in brain cell differentiation. In this study, expression of the serotonin presynaptic transporter protein, which regulates the extracellular serotonin concentration, was measured with w 3 Hxparoxetine in rats exposed to dexamethasone or cocaine prenatally. Within 24 h of a single dose of dexamethasone, significant increases were seen in fetal brain, and the effect persisted into the postnatal period. Chronic prenatal cocaine exposure elicited similar changes. These data indicate that exposures to apparently disparate drugs can elicit similar endpoints that may lead to behavioral teratogenesis. q 1997 Elsevier Science B.V. Keywords: Brain development; Cocaine; Dexamethasone; Glucocorticoid; Paroxetine; Serotonin; Transporter
In addition to their role in neurotransmission, biogenic amines are now known to provide important trophic inputs that influence differentiation of neural cells. This relationship has been best explored for serotonin, which controls the assembly of the nervous system from its origins in the neural crest and neural tube, all the way through the terminal events of differentiation including axonogenesis and synaptogenesis w12,15,16,19,47,50x. The presence of serotonin-synthesizing enzymes and the requisite neurotransmitter receptors in developing brain have been demonstrated clearly w16,50x. Although the ability to manufacture and release serotonin is an essential component of this neurotrophic process, major regulation of the extracellular serotonin concentration is provided by the high-affinity serotonin transporter that pumps the amine back into the cell, and the development of this protein has received recent attention as a target for developmental disrupters w19,31,35x. Most obviously, drugs like cocaine that bind directly to the transporter and inhibit its activity have been shown to affect the development and function of this protein w1,19,20,31x; in addition, factors like prenatal stress and glucocorticoids also have been found to elicit long-term alterations in transporter expression or in responses that are influenced by transporter function w13,27,35x.
) Corresponding author. Fax: q1 Ž919. 684-8197; E-mail: [email protected] 1 Supported by USPHS MH-40159.
0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 1 4 4 - 2
We recently found that late gestational exposure of rats to the synthetic glucocorticoid, dexamethasone, produced persistent elevations of serotonin transporter expression in adolescence and adulthood w35x, an effect which did not occur when dexamethasone was given to mature rats w38,39x. Because such changes influence serotonergic function, these findings are important for understanding the neurobehavioral teratogenesis associated with gestational glucocorticoid treatment w3x. They also raise the question of whether transporter overexpression occurs similarly in the fetal brain during the period in which serotonin influences cell differentiation and synaptogenesis, thus providing a mechanistic tie between effects on fetal serotonergic systems and adverse behavioral outcomes. In the current study, we have examined the immediate effects of prenatal dexamethasone treatment on the expression of transporter protein using the specific radioligand, w 3 Hxparoxetine w10,11x. We have contrasted the effects of single or repeated dexamethasone administration with those of cocaine. Timed pregnant Sprague–Dawley rats ŽZivic–Miller Laboratories, Allison Park, PA. were shipped by climatecontrolled truck Žtotal transit time: 12 h. and were housed individually in breeding cages with free access to food and water. On gestational day 17, dams were given 0.05, 0.2 or 0.8 mgrkg s.c. of dexamethasone phosphate ŽMerck Sharp and Dohme, Rahway, NJ., whereas controls received equivalent volumes of isotonic saline vehicle Ž1 mlrkg.. This dose range has been shown previously to span the
210
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
threshold for nervous system abnormalities and selective promotion of neuronal differentiation w3,23,24,36,37,41x. On gestational day 18 Ž24 h after dexamethasone treatment., the dams were decapitated, fetuses were removed and the brain dissected and frozen immediately on dry ice. Tissues were stored at y458C until assayed; preliminary work showed that w 3 Hxparoxetine binding was unaffected by freezing. An additional set of measurements was made immediately after birth. Animals received three treatments with the intermediate dose Ž0.2 mgrkg. of dexamethasone on gestational days 17, 18 and 19. The day after birth Žpostnatal day 1., animals were decapitated and the brain rapidly dissected by making blunt cuts through the cerebellar peduncles, removing the cerebellum, and making a cut rostral to the thalamus to separate the brainstem from the forebrain. This division follows the natural dissection planes of neonatal rat brain so that the region designated as ‘brainstem’ includes the colliculi, pons and medulla oblongata Žbut not cervical spinal cord., as well as the thalamus Žwhich is ordinarily not considered part of this region.. This region was selected because prior work has identified long-term changes in brainstem serotonin transporter expression after prenatal dexamethasone exposure w35x. In a third set of experiments, beginning on gestational day 8 Žjust after implantation of the fetus in the uterine wall. pregnant rats were given daily s.c. injections of 30 mgrkg of cocaine HCl ŽMallinckrodt, Inc., St. Louis, MO. divided into three doses injected into separate sites on the back so as to avoid tissue necrosis; controls received three injections of isotonic saline vehicle in the equivalent volume Ž3 mlrkg divided into three injection sites.. Cocaine was discontinued after the 20th day of gestation Ž2 days before birth. so as not to interfere with parturition. This dose and route have been shown to approximate plasma cocaine levels in human users w32,44x, to affect brain development w2,14x and to cause long-term neurobehavioral alterations w7,44,45x. Higher doses of cocaine cause convulsions, severe maternal weight loss and increased maternal mortality w30x, effects which were entirely absent at the dose used here Ždata not shown.. Cocaine attaches directly to the serotonin transporter within the developing brain w19x and hence, w 3 Hxparoxetine binding studies performed during gestation would be confounded by the presence of this competitor; accordingly, evaluations in the cocaine study were conducted the day after birth Žpostnatal day 1.. All studies using live animals were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Membrane fractions were prepared by techniques described previously w22x. In brief, tissues were thawed and homogenized ŽPolytron, Brinkmann Instruments, Westbury, NY. in 39 volumes of 120 mM NaCl, 5 mM KCl, 50
mM Tris-HCl ŽpH 7.4., sedimented at 40 000 = g for 15 min, and the supernatant solution was discarded. The pellet was washed twice by resuspension ŽPolytron. in the original volume of buffer, followed each time by resedimentation at 40 000 = g, after which the final pellet was resuspended Žsmooth glass homogenizer fitted with a Teflon pestle. to achieve a protein concentration of approximately 2.5 mgrml. This suspension was then used immediately for the w 3 Hxparoxetine binding assay w22x. Aliquots of membrane suspension corresponding to approximately 100 mg protein w17x were incubated in a total volume of 2 ml of 120 mM NaCl, 5 mM KCl, 50 mM Tris-HCl ŽpH 7.4. containing 100 pM paroxetinewphenyl-6X- 3 Hx Žspecific activity: 25.4 Cirmmol; New England Nuclear Corp., Boston, MA. with or without addition of 100 mM serotonin ŽSigma Chemical Co., St. Louis, MO. to displace specific binding. Incubations lasted 120 min at 208C, after which incubations were stopped by addition of 5 ml of ice-cold buffer, followed by vacuum filtration onto Whatman GFrC filters that were pre-soaked in 0.05% polyethyleneimine ŽSigma.; filters were washed 3 times with 5 ml of buffer, after which radioactivity was counted. Preliminary experiments were conducted to verify that the incubation conditions permitted equilibration of radioligand with the binding sites. Non-specific binding was 10–25% of the total depending upon age and brain region. All determinations were run in duplicate. In order to have sufficient tissue for analysis, brains and brain regions from several fetuses or pups within the same litter were combined to make a single sample. The large number of tissues involved in this study, six treatment groups, two age points, two brain regions, and up to 23 animals per treatment group, produced a total of over 100 membrane preparations to be examined. This fact, and the small amount of tissue available from each animal, precluded the practicability of running Scatchard analyses. Therefore, we examined binding using a w 3 Hxparoxetine concentration Ž100 pM. 3–4 times above the K d found in developing rat brain w38,39x, thus ensuring that measurements would reflect changes in maximal binding primarily, rather than in ligand affinity for the receptor. Because of the high binding affinity of w 3 Hxparoxetine Žin the pM range., we also verified that the free concentration of ligand at the end of the incubation period was not significantly changed from the initial value Žrange: 95–98 pM.. Previous studies with older animals indicate that the effects of dexamethasone on w 3 Hxparoxetine binding reflect alterations in the number of transporter sites w38,39x. For all experiments, a given dam or litter contributed to only a single sample, so that the number of determinations represents the numbers of dams or litters. Data are reported as means and standard errors and treatment effects were established using ANOVA, with data log-transformed whenever variance was heterogeneous. For the cocaine studies, there were two factors ŽTreatment, Region. and because a significant interaction of the factors was found,
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
211
Table 1 Effects of dexamethasone Žgestational day 18. Treatment Control Dexamethasone Dexamethasone Dexamethasone
0.05 mgrkg 0.2 mgrkg 0.8 mgrkg
Weight Žmg.
Membrane protein Žmgrg tissue.
w 3 HxParoxetine binding Žfmolrmg protein.
72 " 3 67 " 5 70 " 4 74 " 5
26.0 " 0.5 25.4 " 0.6 25.8 " 0.5 24.8 " 1.1
38.6 " 2.3 45.2 " 1.2 ) 46.2 " 1.4 ) 47.6 " 0.7 )
Data represent means and standard errors from 5–6 determinations in each treatment group. ANOVA across all treatments indicates no significant effects of dexamethasone on brain weight Ž P ) 0.7. or membrane protein Ž P ) 0.8., but a significant effect on w 3 Hxparoxetine binding Ž P - 0.003.. The effect on binding is significant for all three doses individually Ž ) P - 0.01..
separate analyses were conducted post-hoc ŽFisher’s Protected Least Significant Difference. to determine which regionŽs. showed significant effects. For all tests, significance was assumed at P - 0.05. Administration of dexamethasone did not cause significant inhibition of fetal brain growth within the first 24 h nor were there any adverse effects on membrane protein ŽTable 1.. Nevertheless, all three doses produced significant elevations of w 3 Hxparoxetine binding. Repeated administration of the intermediate dose Ž0.2 mgrkg. on gestational days 17, 18 and 19 led to significant elevations in w 3 Hxparoxetine binding on postnatal day 1 Žcontrol, 200 " 9 fmolrmg protein, n s 6; dexamethasone, 232 " 7 fmolrmg protein, n s 4, P - 0.04.; again, these were not accompanied by alterations in tissue weight or protein concentration Ždata not shown.. The magnitude of the effects on w 3 Hxparoxetine binding seen during gestation and just after birth Ž15–25% elevation. was comparable to that seen previously in adolescence and adulthood after repeated fetal dexamethasone administration w35x. Chronic prenatal cocaine administration also had no discernible effect on brain region weights or membrane protein concentrations measured on postnatal day 1; nevertheless, w 3 Hxparoxetine binding was significantly elevated, with the effect restricted to the brainstem as opposed to forebrain ŽTable 2.. Table 2 Effects of cocaine Žpostnatal day 1. Treatment and region Brainstem Control Cocaine Forebrain Control Cocaine
Weight
Membrane protein
w 3 HxParoxetine binding
Žmg.
Žmgrg tissue.
Žfmolrmg protein.
109"1 111"1
27.1"0.3 26.6"0.5
226"5 242"5 )
168"2 170"3
19.0"0.3 19.2"0.2
94"2 93"2
Data represent means and standard errors from 19–23 determinations for each region in each treatment group. Two-factor ANOVA ŽTreatment, Region. indicates no significant effects of cocaine on brain region weight Ž P ) 0.2. or membrane protein Ž P ) 0.8. but a significant, regionally dependent effect on w 3 Hxparoxetine binding ŽTreatment, Region, P 0.05.. The effect on binding is significant in the brainstem Ž ) P - 0.02. but not in the forebrain.
In our previous work, we showed that repeated prenatal dexamethasone treatment leads to up-regulation of serotonin transporter expression, assessed postnatally in adolescent and adult brainstem w35x. The current results indicate that the up-regulation actually begins within 24 h of a single dose of dexamethasone, so that transporter overexpression is also present during fetal brain development. This effect of exogenous glucocorticoid administration most probably occurs also with endogenous steroid release: prenatal administration of ACTH has been shown to increase the uptake of serotonin into brainstem nerve terminals w13x, the functional correlate of transporter overexpression. Thus, either exogenous or endogenously released glucocorticoids are likely to affect the availability of extracellular serotonin to interact with its receptor sites. The neurotrophic role of serotonin is now known to extend beyond the neural tube stage, to incorporate axonogenesis, synaptogenesis and gliogenesis, events which are all occurring during the late gestational period in which dexamethasone was given in the present study w50x. One specific role for serotonin is to accelerate synaptic maturation, so that synaptogenesis occurs at the expense of further axonal growth w50x. Similarly, dexamethasone treatment has been shown to evoke acceleration of the onset of synaptic activity but with eventual compromise of the total synaptic complement w3,34,36,37,40x. Accordingly, the impact on serotonergic neurotrophic control may represent one of the mechanisms underlying the neurobehavioral teratology of glucocorticoids. It should be noted that the ability of glucocorticoids to up-regulate serotonin transporter expression is unique to development as no such effect is seen with comparable treatment of adult rats w38,39x. Many abused substances are known to evoke glucocorticoid release, including nicotine w5,29,48x, opiates w4x and cocaine w21,26,28x. Indeed, the effects of developmental exposure to nicotine on high-affinity serotonin uptake into synaptosomes parallels that of adrenocortical stimulation w13x. In the current study, we examined the effects of chronic cocaine treatment and also observed effects similar to those of glucocorticoid administration; cocaine’s selectivity for transporter expression in the brainstem parallels previous findings with nicotine and ACTH w13x and matches the regional targeting of dexamethasone at the same age
212
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213
w35x. Furthermore, cocaine has been shown to evoke corticosteroid release in the maternal–fetal unit w26x. It is therefore likely that some of the adverse effects of cocaine on brain development reflect indirect actions mediated by glucocorticoid release and its subsequent impact on serotonergic mechanisms. However, there are at least two other factors that need to be considered. First, cocaine interacts directly with the serotonin transporter Žas well as those for norepinephrine and dopamine. to inhibit its function. Given the short biologic half-life of cocaine, these direct effects can be expected to terminate rapidly after each dose, so that the blockade is only episodic. Prenatal exposure to amine uptake inhibitors other than cocaine affects brain development and subsequent behavioral performance w6,33,46x, but a study specifically comparing the behavioral effects of developmental exposure to cocaine with that of a serotonin uptake inhibitor found no overlap w8x. A second potential contributor to cocaine’s effects on serotonin transporter expression is hypoxiarischemia from the vasoconstrictor effects of the drug w18,25x. However, hypoxic brain damage elicits synaptic deficits which would produce a fall in the concentration of transporter molecules, not an increase as seen here. For example, repeated gestational administration of opiates in doses that produce respiratory depression causes the expected fall in serotonin transporter capacity w42x. In fact, when cocaine is given in higher doses and drug treatment is initiated later in gestation, transporter expression similarly is suppressed in conjunction with visible defects in serotonergic innervation w1x. Reducing the cocaine dose and beginning treatment earlier in gestation does not produce this synaptic loss w9,43,49x. The difference between commencing cocaine in late gestation as opposed to early gestation Žas used here. is that, with early treatment, vasoconstrictor effects most likely will be absent during later developmental phases of axonogenesis and synaptogenesis because of down-regulation and desensitization of peripheral adrenergic receptors; with later treatment, vasoconstriction remains a factor during these later developmental events. It is thus critical, not only for understanding effects in animal models of maternal cocaine abuse but also for extension of these findings to the potential impact on human brain development, that issues of dose and duration be considered. Accordingly, our results indicate that the effects of cocaine on brain development are likely to exhibit convergent mechanisms with those of the glucocorticoids that are released by cocaine administration, but that additional effects are superimposed on these actions. References w1x H.M. Akbari, H.K. Kramer, P.M. Whitaker-Azmitia, L.P. Spear, E.C. Azmitia, Prenatal cocaine exposure disrupts the development of the serotonergic system, Brain Res. 572 Ž1992. 57–63.
w2x T. Anderson-Brown, T.A. Slotkin, F.J. Seidler, Cocaine acutely inhibits DNA synthesis in developing rat brain regions: evidence for direct actions, Brain Res. 537 Ž1990. 197–202. w3x M.C. Bohn, Glucocorticoid induced teratologies of the nervous system, in: J. Yanai ŽEds.., Neurobehavioral Teratology, Elsevier, Amsterdam, 1984, pp. 365–387. w4x J. Borrell, I. Llorens, S. Borrell, Adrenal, plasma and urinary ´ corticosteroids during single or repeated administration of morphine in cats, Eur. J. Pharmacol. 31 Ž1975. 237–242. w5x G.R. Cam, J.R. Bassett, K.D. Cairncross, The action of nicotine on the pituitary-adrenal cortical axis, Arch. Int. Pharmacodyn. Ther. 237 Ž1979. 49–66. w6x V. Cuomo, Perinatal neurotoxicology of psychotropic drugs, Trends Pharmacol. Sci. 8 Ž1987. 346–350. w7x D.L. Dow-Edwards, Long-term neurochemical and neurobehavioral consequences of cocaine use during pregnancy, Ann. NY Acad. Sci. 562 Ž1989. 280–289. w8x D.L. Dow-Edwards, Modification of acoustic startle reactivity by cocaine administration during the postnatal period: comparison with a specific serotonin reuptake inhibitor, Neurotoxicol. Teratol. 18 Ž1996. 289–296. w9x E.M. Factor, R.P. Hart, G.M. Jonakait, Neurochemical development of the raphe after continuous prenatal cocaine exposure, Brain Res. Bull. 31 Ž1993. 49–56. w10x E.V. Gurevich, J.H. Joyce, Comparison of w 3 Hxparoxetine and w 3 Hxcyanoimipramine for quantitative measurement of serotonin transporter sites in human brain, Neuropsychopharmacology 14 Ž1996. 309–323. w11x E. Habert, D. Graham, L. Tahraoui, Y. Claustre, S.Z. Langer, Characterization of w 3 Hxparoxetine binding to rat cortical membranes, Eur. J. Pharmacol. 118 Ž1985. 107–114. w12x M. Hamon, S. Bourgoin, C. Chanez, F. De Vitry, Do serotonin and other neurotransmitters exert a trophic influence on the immature brain?, Dev. Neurobiol. 12 Ž1989. 171–183. w13x J.A. King, M. Davila-Garcia, E.C. Azmitia, F.L. Strand, Differential effects of prenatal and postnatal ACTH or nicotine exposure on 5-HT high affinity uptake in the neonatal rat brain, Int. J. Dev. Neurosci. 9 Ž1991. 281–286. w14x S.M. Koegler, F.J. Seidler, J.R. Spencer, T.A. Slotkin, Ischemia contributes to adverse effects of cocaine on brain development: suppression of ornithine decarboxylase activity in neonatal rat, Brain Res. Bull. 27 Ž1991. 829–834. w15x J.M. Lauder, Roles for neurotransmitters in development: possible interaction with drugs during the fetal and neonatal periods, in: M. Marois ŽEds.., Prevention of Physical and Mental Congenital Defects, Alan R. Liss, New York, 1985, pp. 375–380. w16x J.M. Lauder, H. Krebs, Serotonin as a differentiation signal in early neurogenesis, Dev. Neurosci. 1 Ž1978. 15–30. w17x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–270. w18x M.P. Mahalik, R.F. Gautieri, D.E. Mann Jr., Mechanisms of cocaine-induced teratogenesis, Res. Comm. Subst. Abuse 5 Ž1984. 279–302. w19x J.S. Meyer, L.P. Shearman, L.M. Collins, Monoamine transporters and the neurobehavioral teratology of cocaine, Pharmacol. Biochem. Behav. 55 Ž1996. 585–593. w20x J.S. Meyer, L.P. Shearman, L.M. Collins, R.L. Maguire, Cocaine binding sites in fetal rat brain: implications for prenatal cocaine action, Psychopharmacology 112 Ž1993. 445–451. w21x R.L. Moldow, A.J. Fischman, Cocaine induced secretion of ACTH, b-endorphin and corticosterone, Peptides 8 Ž1987. 819–822. w22x C. Moret, M. Briley, Platelet 3 H-paroxetine binding to the serotonin transporter is insensitive to changes in central serotonergic innervation in the rat, Psychiat. Res. 38 Ž1991. 97–104.
K.E. McGrath et al.r DeÕelopmental Brain Research 104 (1997) 209–213 w23x H.A. Navarro, E.M. Kudlacz, J.P. Eylers, T.A. Slotkin, Prenatal dexamethasone administration disrupts the pattern of cellular development in rat lung, Teratology 40 Ž1989. 433–438. w24x H.A. Navarro, J. Lachowicz, J. Bartolome, W.L. Whitmore, T.A. Slotkin, Effects of prenatal dexamethasone on development of ornithine decarboxylase activity in brain and peripheral tissues of rats, Pediatr. Res. 24 Ž1988. 465–469. w25x G.D. Olsen, Potential mechanisms of cocaine-induced developmental neurotoxicity: a minireview, Neurotoxicology 16 Ž1995. 159–167. w26x J.R. Owiny, M.T. Jones, D. Sadowsky, T. Myers, A. Massmann, P.W. Nathanielsz, Cocaine in pregnancy: the effect of maternal administration of cocaine on the maternal and fetal pituitary-adrenal axis, Am. J. Obstet. Gynecol. 164 Ž1991. 658–663. w27x R.E. Poland, P. Lutchman-Singh, D. Au, S. McGeoy, M. Que, S. Acosta, J.T. McCracken, Exposure to threshold doses of nicotine in utero: 3. Augmentation of the prolactin and ACTH response to 8-OH DPAT by desipramine treatment is compromised in adult male offspring, Neurotoxicology 17 Ž1996. 351–358. w28x F. Rougepont, M. Marinelli, M. LeMoal, H. Simon, P.V. Piazza, Stress-induced sensitization and glucocorticoids: 2. sensitization of the increase in extracellular dopamine induced by cocaine depends on stress-induced corticosterone secretion, J. Neurosci. 15 Ž1995. 7189–7195. w29x R.P. Rubin, W. Warner, Nicotine-induced stimulation of steroidogenesis in adrenocortical cells of the cat, Br. J. Pharmacol. 53 Ž1975. 357–362. w30x F.J. Seidler, T.A. Slotkin, Prenatal cocaine and cell development in rat brain regions: effects on ornithine decarboxylase and macromolecules, Brain Res. Bull. 30 Ž1993. 91–99. w31x L.P. Shearman, L.M. Collins, J.S. Meyer, Characterization and localization of wI-125xRTI-55-labeled cocaine binding sites in fetal and adult rat brain, J. Pharmacol. Exp. Ther. 277 Ž1996. 1770–1783. w32x R.K. Siegel, Cocaine smoking, J. Psychoactive Drugs 14 Ž1982. 271–359. w33x J.W. Simpkins, F.P. Field, G. Torosian, E.E. Soltis, Effects of prenatal exposure to tricyclic antidepressants on adrenergic responses in progeny, Dev. Pharmacol. Ther. 8 Ž1985. 17–33. w34x T.A. Slotkin, G. Barnes, C. Lau, F.J. Seidler, P. Trepanier, S.J. Weigel, W.L. Whitmore, Development of polyamine and biogenic amine systems in brains and hearts of neonatal rats given dexamethasone: role of biochemical alterations in cellular maturation for producing deficits in ontogeny of neurotransmitter levels, uptake, storage and turnover, J. Pharmacol. Exp. Ther. 221 Ž1982. 686–693. w35x T.A. Slotkin, G.A. Barnes, E.C. McCook, F.J. Seidler, Programming of brainstem serotonin transporter development by prenatal glucocorticoids, Dev. Brain Res. 93 Ž1996. 155–161. w36x T.A. Slotkin, S.E. Lappi, E.C. McCook, M.I. Tayyeb, J.P. Eylers, F.J. Seidler, Glucocorticoids and the development of neuronal function: effects of prenatal dexamethasone exposure on central noradrenergic activity, Biol. Neonate 61 Ž1992. 326–336.
213
w37x T.A. Slotkin, S.E. Lappi, M.I. Tayyeb, F.J. Seidler, Dose-dependent glucocorticoid effects on noradrenergic synaptogenesis in rat brain: ontogeny of w 3 Hxdesmethylimipramine binding sites after fetal exposure to dexamethasone, Res. Comm. Chem. Pathol. Pharmacol. 73 Ž1991. 3–19. w38x T.A. Slotkin, E.C. McCook, J.C. Ritchie, B.J. Carroll, F.J. Seidler, Serotonin transporter expression in rat brain regions and blood platelets: aging and glucocorticoid effects, Biol. Psychiatry 41 Ž1997. 172–183. w39x T.A. Slotkin, E.C. McCook, J.C. Ritchie, F.J. Seidler, Do glucocorticoids contribute to the abnormalities in serotonin transporter expression and function seen in depression? An animal model, Biol. Psychiatry 40 Ž1996. 576–684. w40x T.A. Slotkin, E.C. McCook, F.J. Seidler, Glucocorticoids regulate the development of intracellular signaling: enhanced forebrain adenylate cyclase catalytic subunit activity after fetal dexamethasone exposure, Brain Res. Bull. 32 Ž1993. 359–364. w41x T.A. Slotkin, F.J. Seidler, R.J. Kavlock, J.V. Bartolome, Fetal dexamethasone exposure impairs cellular development in neonatal rat heart and kidney: effects on DNA and protein in whole tissues, Teratology 43 Ž1991. 301–306. w42x T.A. Slotkin, W.L. Whitmore, M. Salvaggio, F.J. Seidler, Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat, Life Sci. 24 Ž1979. 1223–1230. w43x A.M. Snyder-Keller, R.W. Keller, Prenatal cocaine increases striatal serotonin innervation without altering the patchrmatrix organization of intrinsic cell types, Dev. Brain Res. 74 Ž1993. 261–267. w44x L.P. Spear, N.A. Frambes, C.L. Kirstein, Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats, Psychopharmacology 97 Ž1989. 427–431. w45x L.P. Spear, C.L. Kirstein, N.A. Frambes, Cocaine effects on the developing nervous system: behavioral, psychopharmacological and neurochemical studies, Ann. NY Acad. Sci. 562 Ž1989. 290–307. w46x S.R. Tonge, Catecholamine concentrations in discrete areas of the rat brain after the pre- and neonatal administration of phencyclidine and imipramine, J. Pharm. Pharmacol. 25 Ž1973. 164–166. w47x K. Turlejski, Evolutionary ancient roles of serotonin: long-lasting regulation of activity and development, Acta Neurobiol. Exp. 56 Ž1996. 619–636. w48x D.M. Turner, The role of adrenal catecholamines in the release of corticosterone and fatty acids by nicotine in the rat, Res. Comm. Chem. Pathol. Pharmacol. 12 Ž1975. 645–655. w49x X.H. Wang, P. Levitt, A.O. Jenkins, E.H. Murphy, Normal development of tyrosine hydroxylase and serotonin immunoreactive fibers innervating anterior cingulate cortex and visual cortex in rabbits exposed prenatally to cocaine, Brain Res. 715 Ž1996. 221–224. w50x P.M. Whitaker-Azmitia, Role of serotonin and other neurotransmitter receptors in brain development: basis for developmental pharmacology, Pharmacol. Rev. 43 Ž1991. 553–561.