Prenatal cocaine exposure potentiates paroxetine ...

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Neuropharmacology 46 (2004) 942–953 www.elsevier.com/locate/neuropharm

Prenatal cocaine exposure potentiates paroxetine-induced desensitization of 5-HT2A receptor function in adult male rat offspring Zhuo Chen, Kate Waimey, Louis D. Van de Kar, Gonzalo A. Carrasco, Michelle Landry, George Battaglia  Department of Pharmacology and Center for Serotonin Disorders Research, Loyola University of Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA Received 15 July 2003; received in revised form 22 December 2003; accepted 19 January 2004

Abstract This study investigated the ability of prenatal exposure to cocaine to alter serotonin2A (5-HT2A) receptor function and paroxetine-induced desensitization of 5-HT2A receptor function in rat offspring. Following exposure to saline or ()cocaine (15 mg/kg, s.c., b.i.d.), during gestational days 13–20, adult male offspring were treated with either saline or paroxetine (10 mg/kg/day, i.p. 14 days). Eighteen hours post-treatment, changes in the stimulation of oxytocin, adrenocorticotropic hormone (ACTH) and corticosterone by ()4-iodo-2,5-dimethoxyphenylisopropylamine (DOI, 0.5 or 2.0 mg/kg, s.c.) and in 5-HT2A receptor densities were determined. Prenatal cocaine exposure did not alter 5-HT2A receptor-mediated neuroendocrine responses or 5-HT2A receptor densities. In contrast, paroxetine treatment reduced cortical 5-HT2A receptors (18–25%) and desensitized 5-HT2A receptor-mediated oxytocin responses in both offspring groups. Furthermore, in cocaine offspring, paroxetine produced an inhibition of 5-HT2A receptor-mediated increase in plasma ACTH levels and a greater attenuation of the oxytocin responses to ()DOI. Paroxetine-induced reductions in body weight gain (8.8%) were comparable in both offspring groups. These data, demonstrating that prenatal exposure to cocaine potentiates paroxetine-induced desensitization of 5-HT2A receptor function, may be clinically relevant with respect to treating mood disorders in adults exposed in utero to cocaine. # 2004 Elsevier Ltd. All rights reserved. Keywords: SSRIs; Serotonin; DOI; Development; Neuroendocrine

1. Introduction Cocaine abuse among pregnant women has received a great deal of scientific and media attention in recent years. Data from clinical and preclinical studies indicate that prenatal exposure to cocaine produces cognitive, behavioral and neurochemical impairments in offspring, and some of these effects may be long-lasting (Dow-Edwards et al., 1999). As cocaine can affect serotonergic function, potential prenatal cocaine-induced changes in serotonergic systems may be clinically relevant with respect to the etiology and/or treatment of mood disorders in cocaine-exposed offspring.  Corresponding author. Tel.: +1 708 216 5601; Fax: +1 708 216 6596. E-mail address: [email protected] (G. Battaglia).

0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.01.013

In adults, cocaine alters serotonergic neurotransmission primarily via blockade of 5-HT transporters and elevation of extracellular serotonin (5-HT) levels (Teneud et al., 1996). Since cocaine can cross the fetalplacental barrier (Meyer et al., 1996; Spear et al., 1989) when administered during pregnancy, cocaine is likely to block fetal 5-HT transporters and alter 5-HT levels in fetal brain. Prior to playing a role as a neurotransmitter, 5-HT influences the outgrowth of 5-HT fibers and the differentiation of 5-HT target tissues in fetal brain (Lauder, 1990). Therefore, 5-HT levels are likely to be critically regulated during fetal brain development in order to establish the appropriate synaptic connections of 5-HT pathways. Consequently, cocaine’s blockade of 5-HT transporters would be expected to alter the development of serotonergic pathways. Various reports have supported the contention that cocaine

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exposure during gestation can alter the development of 5-HT neurons (Akbari et al., 1992; Meyer et al., 1996). Previous studies in our laboratory demonstrated that prenatal cocaine exposure results in deficits in forebrain serotonergic nerve terminals, as assessed from reduced hormone responses to a 5-HT releaser (Cabrera et al., 1993), and in decreases of serotonin content in frontal cortex and hippocampus in adult offspring (Cabrera-Vera et al., 2000). These data suggest that prenatal cocaine exposure can produce long-term alterations in presynaptic components of serotonergic systems in offspring. In addition, prepubescent offspring exposed prenatally to cocaine exhibit increases in postsynaptic 5HT1A (Battaglia and Cabrera, 1994) and 5-HT2A receptor-mediated neuroendocrine responses (Battaglia et al., 2000). The increases in Emax for the 5-HT receptormediated stimulation of plasma hormones may represent initial compensatory responses to the presynaptic 5-HT deficits produced by prenatal cocaine. The consequences of prenatal cocaine on postsynaptic 5-HT2A receptor function in adult offspring is addressed in the present study. Serotonin2A (5-HT2A) receptors are located on postsynaptic membranes of 5-HT target cells and are widely distributed in many brain areas, including hypothalamus, amygdala and cortex (Pazos et al., 1985). These receptors are involved in the regulation of many behavioral (Koek et al., 1992), physiological and neuroendocrine effects (Levy et al., 1994). Dysfunction of serotonergic neurotransmission has been associated with several mood disorders such as depression, anxiety and premenstrual syndrome (Joffe and Cohen, 1998; Lucki, 1998). Various studies indicate that an increased function of the 5-HT2 receptor system may be associated with psychiatric disorders such as depression (D’haenen et al., 1992; Hrdina et al., 1993) while a reduction in 5-HT2 receptors has been associated with other neurological and psychiatric disorders such as Alzheimer disease (Blin et al., 1993). Thus, drugs that target 5-HT2A receptors are used clinically to treat psychiatric disorders, such as depression and schizophrenia (Blier and de Montigny, 1999; Aghajanian and Marek, 2000). Given the changes in 5-HT2A receptor function associated with various clinical disorders and our previous findings that 5-HT2A receptor function is increased in prepubescent cocaine offspring, the present study investigated the functional status of postsynaptic 5-HT2A receptor systems in adult male rats with respect to the effects of prenatal cocaine exposure and the effectiveness of paroxetine treatment to produce neuroadaptive changes in 5-HT2A receptor function. Paroxetine is a member of the class of drugs known as selective serotonin reuptake inhibitors (SSRIs), which are effective in treating a wide spectrum of mood disorders including depression, panic disorder and anxiety (Hyttel, 1994). The immediate effects of SSRIs

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are to selectively inhibit the reuptake of 5-HT into presynaptic nerve terminals thereby increasing synaptic concentrations of 5-HT (Gardier et al., 1996). However, the clinical efficacy of SSRIs is related to their ability to produce a gradual increase of 5-HT neurotransmission via desensitization of somatodendritic 5-HT1A and 5-HT1B autoreceptors (thereby reducing feedback inhibition on 5-HT neurons) following longterm treatment. Treatment of humans or male rats with SSRIs alters the sensitivity of postsynaptic 5-HT2A receptors (Li et al., 1993, 1997b; Tilakaratne et al., 1995), suggesting a role for neuroadaptive changes in postsynaptic 5-HT2A receptor as well. To date, there are no data available on the ability of SSRIs to effect changes in 5-HT2A receptor function in prenatal cocaine exposed offspring. Given that prenatal cocaine exposure can produce long-term reductions in serotonergic nerve terminal function and compensatory increases in postsynaptic serotonergic function in immature offspring (Battaglia et al., 1998), it is important to determine whether prenatal cocaine exposure will: (1) alter 5-HT2A receptor function in adult offspring and (2) alter the responsiveness of serotonergic systems to the neuroadaptive changes in 5-HT2A receptor function produced by SSRIs such as paroxetine. We report here that paroxetine produces a functional desensitization of postsynaptic 5-HT2A receptor-mediated neuroendocrine responses which are enhanced in male offspring exposed to cocaine prenatally. 2. Materials and methods 2.1. Animals Timed-pregnant Sprague–Dawley rats (280–320 g) were purchased from Taconic (Indianapolis, IN) to arrive in the laboratory at gestation day (GD) 5 (experimental animals; n ¼ 28) and GD 6 (untreated foster dams; n ¼ 28). The presence of a copulatory plug was defined as GD 0. The rats were housed individually in a light- (12 h light/dark, lights on from 7:00 AM to 7:00 PM), humidity- (50–55%) and temperav ture- (22–24 C) controlled room. Food and water were available ad libitum. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health, and approved by Loyola University Institutional Animal Care and Use Committee (IACUC). 2.2. Drugs ()Cocaine HCl was generously provided by the National Institute on Drug Abuse and was injected subcutaneously (s.c.) at a dose of 15 mg/kg twice daily.

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Paroxetine was obtained from Smith-Kline Beecham Pharmaceuticals (Philadelphia, PA) and was injected (10 mg/kg, i.p.) daily. Paroxetine was dissolved in 0.9% saline at 5 mg/ml and injected in a volume of 2 ml/kg. ()4-iodo-2,5-dimethoxyphenylisopropylamine HCl [()DOI] was purchased from Sigma Chemical Co. (St. Louis, MO). ()DOI was dissolved in 0.9% saline at two concentrations (0.5 and 2 mg/ml) in a volume of 1 ml/kg. All solutions were made fresh before administration. [3H]-ketanserin (specific activity, 88 Ci/mmol) and [125I]-DOI (specific activity, 1786.4 Ci/mmol) were obtained from Perkin-Elmer Life Sciences, Inc. (Boston, MA). 2.3. Experimental protocols 2.3.1. Pregnant dams Experimental animals (n ¼ 28) were assigned randomly to two groups (n ¼ 14 per group) to receive either saline or cocaine treatments. Starting on GD 8, and continuing throughout the injection period, all experimental animals were given ad libitum access to a nutritionally balanced liquid diet consisting of chocolate flavored Boost1 (Mead Johnson) supplemented with Vitamin Diet Fortification Mixture (ICN Nutritional Biochemicals), Salt Mixture XIV (ICN Nutritional Biochemicals) and water (Cabrera et al., 1993). Experimental dams were weighed daily and received injections of either saline (1 ml/kg, s.c.; n ¼ 14), or ()cocaine hydrochloride (15 mg/kg, s.c.; n ¼ 14) twice daily beginning on GD 13 and ending on GD 20. Foster dams had ad libitum access to rat chow, were weighed daily and received animal care identical to the experimental dams but were not subjected to any experimental procedures. 2.3.2. Offspring At birth (i.e. postnatal day (PD) 0), all offspring from each of the experimental groups were fostered to non-drug treated lactating dams in order to eliminate the possible influence of drug-induced differences in nurturing. At the time of fostering, the litters were culled to 10 pups per litter. On PD 20, prenatal saline and cocaine offspring were weaned and assigned to their respective experimental groups for subsequent adult treatment with saline or paroxetine and challenge drug injection. Each of the postnatal experimental groups contained pups from different litters. At the time of weaning, rats were housed in groups of two per cage, and had free access to food and water. On PD 62, adult male progeny from the prenatal saline and cocaine groups were treated with either saline (1 ml/kg) or paroxetine (10 mg/kg/day; i.p.) daily for 14 days through PD 75. Body weights were monitored throughout the paroxetine treatment period. On PD 76, rats were

subjected to a pharmacological challenge as described below. 2.3.3. Pharmacological challenge Rats were handled for four consecutive days before the experiment to minimize stress. Functional alterations in 5-HT2A receptor systems were determined in male progeny on PD 76 by measuring the magnitude of elevation in plasma hormones following a single s.c. injection of either saline or the selective 5-HT2A/2C receptor agonist ()DOI (0.5 and 2 mg/kg). The rats were sacrificed (between 10:00 and 13:00 h) by decapitation 30 minutes post-injection and the trunk blood was collected into centrifuge tubes containing 0.5 ml of a 0.3 M EDTA (pH 7.4) solution. After centrifugation, v the plasma was stored at 80 C for subsequent radioimmunoassays of plasma ACTH, corticosterone and oxytocin. The frontal cortex, described as the area anterior to the corpus collusum, was dissected immediately after decapitation and frozen in liquid nitrogen, v and then stored at 80 C for receptor binding assays. 2.4. Radioimmunoassays of hormones 2.4.1. Plasma oxytocin radioimmunoassay Radioimmunoassay of oxytocin is a double-antibody assay. It was performed by a method modified from Li et al. (1997a). Briefly, plasma (1 ml) was mixed with 2 ml ice-cold acetone and centrifuged at 2000 rpm for 30 v min at 4 C. The supernatant was then added to 5 ml cold petroleum ether and mixed immediately. After v centrifugation at 2000 rpm for 15 min at 4 C, the top layer was aspirated off and the extracted plasma samv ple was dried by blowing air at 4 C. The dried extract was dissolved in 1 ml cold assay buffer (0.05 M phosphate buffer, 0.125% bovine serum albumin, 0.01% sodium azide, 0.001 M EDTA, pH 7.4). The extracted plasma or oxytocin standard (Bachem Fine Chemicals, Torrance, CA) were incubated with 0.1 ml rabbit antioxytocin serum (donated by Dr Lanny Keil, Ames Research Center, Sunnyvale, CA) at a final dilution of v 1:250,000 for 24 h at 4 C. A total of 0.1 ml [125I]-oxytocin (3000 cpm, Du Pont NEN, Boston, MA) was v then added and incubated for 72 h at 4 C. A 0.1 ml of the second antibody (goat anti-rabbit c-globulin; CalBiochem, San Diego, CA) was added at a dilution of 1:20 and a 0.1 ml of normal rabbit serum (1:120 dilution) was also added. After incubation overnight at v 4 C, the samples were centrifuged at 3000 rpm for 40 v min at 4 C. The supernatant was decanted, and the radioactivity of the pellet was counted for 5 min by a Micromedic 4/200 plus c-counter and analyzed from the standard curve using RIA-AID computer program (Robert Maciel Associates, Arlington, MA). The concentrations of plasma oxytocin were calculated using a correction factor for the percent recovery of oxytocin

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following the extraction. Extraction recovery was calculated by adding various concentrations of oxytocin standard to 1 ml samples of normal plasma that were extracted with all our plasma samples. This extraction procedure gave 65.8% recovery of oxytocin. The sensitivity of this assay is 1 pg/tube and the typical intraand inter-assay variabilities are 8.1 and 8.6%, respectively. 2.4.2. Plasma ACTH radioimmunoassay Radioimmunoassay of ACTH was performed on plasma samples (10 and 50 ll) according to a previously published protocol (Li et al., 1993). Briefly, the ACTH antiserum was obtained from IgG Corp. (Nashville, TN). ACTH standard (1–39) was obtained from CalBiochem and [125I]-ACTH from Diasorin (Stillwater, MN). The sequence recognition of the antiserum is the 5–18 segment of ACTH. This antiserum does not significantly recognize a-MSH, b-MSH, b-endorphin, b-lipotropin, ACTH 11–24, or ACTH 1–16-amide. In this doubleantibody radioimmunoassay, samples or standards were incubated with the ACTH antibody (final dilution v 1:30,000, 32% total binding) for 24 h at 4 C in a 0.01 M phosphate buffer (pH 7.6) containing 1% bovine serum albumin (Sigma), 0.025 M EDTA, 0.5% normal rabbit serum (CalBiochem) and 25 KIU/ml aprotinin (Sigma Chemical Co). Then [125I]-ACTH (2000 cpm, DiaSorin, Stillwater, MN) was added and incubated for v 72 h at 4 C. The second antibody (goat anti-rabbit-cglobulin, CalBiochem) was added at a final dilution of v 1:20 and incubated overnight at 4 C. Cold phosphate buffer saline (PBS) was then added to the tubes and they v were centrifuged at 3000 rpm for 40 min at 4 C. The supernatant was decanted, and the radioactivity of the pellet was counted for 5 min by a Micromedic 4/200 plus c-counter and analyzed from the standard curve using RIA-AID computer program (Robert Maciel Associates). The sensitivity of the assay is 0.25 pg/tube and the typical intra- and inter-assay variabilities are 4.2 and 14.6%, respectively. 2.4.3. Plasma corticosterone radioimmunoassay Radioimmunoassay of corticosterone was performed on plasma samples in which binding proteins have been denatured by placing the samples in a hot water bath v of 80 C for 20 min as previously described (Li et al., 1993). The radioactive [3H]-corticosterone tracer was obtained from Du Pont NEN Research Products. Antiserum (final dilution 1:11,200, 46% total binding) from ICN Biochemicals (Irvine, CA). After incubation overv night at 4 C, a cold mixture of charcoal and DextranT70 was added and the tubes were centrifuged at 3000 v rpm for 20 min at 4 C. The radioactivity of the supernatant containing the antibody was counted for 2 min in a Beckman LS5000TD scintillation counter at an efficiency of 60%. The sensitivity of this assay is 0.02

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ng/tube and the typical intra- and inter-assay variabilities are 4.5 and 11.9%, respectively. 2.5. Radioligand binding assay for 5-HT2A receptors 2.5.1. Tissue preparation Rat cortex was weighed and homogenized (3 s) in 30 volumes of ice-cold 0.32 M sucrose using a Tekmar Tissumizer (Cincinnati, OH). The homogenate was cenv trifuged at 30; 000  g for 15 min at 4 C. After centifugation, the tissue pellet was washed by resuspending it in 30 volumes of ice-cold 50 mM Tris buffer (pH 7.4, v 25 C) containing 10 mM MgSO4 and 0.5 mM EDTA v (1:30 w/v), and incubated at 37 C for 15 min. The tissue was centrifuged at 30; 000  g for 10 min and finally resuspended in assay buffer (50 mM Tris, pH 7.4; 0.5 mM EDTA; 10 mM MgSO4; 0.1% ascorbate; 10 lM pargyline) to reach a concentration of 20 mg wet weight/ml. Only tissues from saline-injected rats were used for the receptor binding assays. Protein content was determined by using a BCA protein assay Kit (Pierce, Rockford, IL). 2.5.2. [3H]-Ketanserin-labeled 5-HT2A receptors Densities of antagonist-labeled 5-HT2A receptors were determined by incubating 0.8 nM [3H]-Ketanserin with 2 mg of frontal cortex in 1 ml of 50 mM Tris (pH 7.4), 0.5 mM EDTA, 10 mM MgSO4, 0.1% ascorbate and 10 lM pargyline during 60 min at room temperature. Prazosin (30 nM) was included in all assays to preclude binding to a1 receptors. Non-specific binding was defined in the presence of 107 M MDL 100,907, a 5-HT2A selective antagonist. The reactions were terminated by immediate vacuum filtration through Whatman GF/C glass fiber filters (pretreated with 1% polyethylenimine) and then the filters were washed with 25 ml of ice-cold 50 mM Tris buffer (pH 7.4). The radioactivity of the filters was counted in 5 ml scintillation liquid at 54% efficiency, using a Beckman LS 6500 scintillation counter. 2.5.3. [125I]-DOI-labeled 5-HT2A receptors Densities of 5-HT2A receptors in the agonist highaffinity state were performed by incubating 0.2 nM of [125I]-DOI with 2 mg wet weight/ml of tissue in a total volume of 0.5 ml of 50 mM Tris (pH 7.4), 0.5 mM EDTA, 10 mM MgSO4, 0.1% ascorbate and 10 lM pargyline. Non-specific binding was defined in the presence of 107 M MDL 100,907, a 5-HT2A selective antagonist. The tissues were incubated at room temperature for 90 min. The reaction was stopped by rapid vacuum filtration over Whatman GF/C glass fiber filters (pretreated with 1% polyethylenimine). The samples were then washed with 25 ml of 50 mM Tris buffer (pH 7.4). Filters were then added to 75  12 mm glass

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test tubes and counted for 5 min by a Micromedic 4/ 200 plus c-counter at an efficiency of 80%.

no effect of cocaine on the average number of pups per litter before culling (control, 10:3  0:4; cocaine, 10:4  0:3).

2.6. Statistical analysis All data are represented as group means and the standard errors of the mean (SEM). The hormone data were analyzed by two-way or three-way analysis of variance (ANOVA). The radioligand binding data were analyzed by two-way ANOVA. Individual group means were compared by the Newman–Keuls multiplerange test. Body weights during paroxetine treatment were analyzed by a repeated measures ANOVA. A statistics software program (GB-STAT; Dynamic Microsystems, Inc., Silver Spring, MD) was used for all of the statistical analyses.

3. Results 3.1. Effect of prenatal cocaine exposure on growth parameters The administration of cocaine to pregnant dams from GD 13–20 did not significantly alter maternal weight gain during the course of treatment (control, 75:0  5:6 g; cocaine, 72:8  4:2 g). Furthermore, the administration of liquid diet did not alter maternal weight gain during pregnancy, as there was no significant difference in weight gain between the above control dams receiving a liquid diet and chow-fed foster dams (74:2  3:3 g; n¼27). Consistent with the lack of effect on maternal weight gain, prenatal exposure to cocaine did not result in a significant difference in male progeny weights at birth (control, 6:3  0:1 g; cocaine, 6:2  0:1 g) or at PD 20 (control, 46:9  0:7 g; cocaine, 47:7  0:7 g) when the rats were assigned to experimental groups. There was

3.2. Neuroendocrine responses to the 5-HT2 receptor agonist ()DOI: lack of effect of prenatal cocaine exposure on 5-HT2A receptor-mediated neuroendocrine function As indicated in Table 1, basal levels (i.e. levels in saline-injected rats) of plasma oxytocin, ACTH and corticosterone were not significantly altered by prenatal exposure to cocaine in adult male progeny. ()DOI increased plasma levels of oxytocin in a dose-dependent manner. Plasma oxytocin levels at each of the doses (0.5 and 2.0 mg/kg) of ()DOI were elevated to a similar degree in saline- and cocaine-exposed male progeny. A two-way ANOVA for the oxytocin data indicated a significant main effect of ()DOI (F ð2;44Þ ¼ 67:19, p < 0:01), but no significant main effect of prenatal cocaine (F ð1;44Þ ¼ 0:58, p > 0:05) and no significant interaction between prenatal cocaine and ()DOI (F ð2;44Þ ¼ 0:77, p > 0:05). The Newman–Keuls test indicated that ()DOI significantly elevated (p < 0:01) the levels of plasma oxytocin in male progeny independent of the prenatal treatment. ()DOI increased plasma levels of ACTH in a dosedependent manner. Plasma ACTH levels at each of the doses (0.5 and 2.0 mg/kg) of ()DOI were elevated to a similar degree in saline- and cocaine-exposed male progeny. A two-way ANOVA for the ACTH data indicated a significant main effect of ()DOI (F ð2;45Þ ¼ 56:73, p < 0:01), but no significant main effect of cocaine (F ð1;45Þ ¼ 1:11, p > 0:05) and no significant interaction between cocaine and ()DOI (F ð2;45Þ ¼ 1:09, p > 0:05). The Newman–Keuls test indicated that ()DOI significantly elevated (p < 0:01) the levels of

Table 1 Prenatal exposure to cocaine does not alter 5-HT2A receptor-mediated hormone responses in adult male offspringa Prenatal Exposure Group

Saline

()DOI 0.5 mg/kg

()DOI 2.0 mg/kg

Oxytocin (pg/ml) Saline Cocaine

6:1  0:5 7:0  0:7

33:7  4:0 32:2  4:8

50:2  2:0++ 52:2  5:0++

ACTH (pg/ml) Saline Cocaine

56:8  2:6 55:1  3:6

936:4  93:3 899:7  96:2

1289:4  268:8+ 1401:0  140:0++

Corticosterone (lg/dl) Saline Cocaine

4:4  1:1 3:5  1:1

26:9  1:5 25:5  1:5

32:4  2:1+ 33:7  0:7+

a Data represent group means  SEM from 8 to 10 rats per challenge group, with each rat within a group being obtained from a different litter. For each of the hormones, data were analyzed by a two-way ANOVA followed by a Newman–Keuls test. The  indicates a significant difference (p < 0:01) from the corresponding saline-injected group. The + and ++ indicate a significant difference (at p < 0:05 and p < 0:01, respectively) from the corresponding lower dose of ()DOI.

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plasma ACTH in male progeny independent of the prenatal treatment. As observed for oxytocin and ACTH, ()DOI increased plasma levels of corticosterone in a dosedependent manner. Plasma corticosterone levels at each of the doses (0.5 and 2.0 mg/kg) of ()DOI were elevated to a similar degree in saline- and cocaine-exposed male progeny. A two-way ANOVA for the corticosterone data indicated a significant main effect of ()DOI (F ð2;47Þ ¼ 170:23, p< 0:01), but no significant main effect of cocaine (F ð1;47Þ ¼ 0:01, p> 0:05) and no significant interaction between cocaine and ()DOI (F ð2;47Þ ¼ 0:20, p > 0:05). The Newman–Keuls test indicated that ()DOI significantly elevated (p < 0:01) the levels of plasma corticosterone in male progeny independent of the prenatal treatment. 3.3. Effect of postnatal paroxetine treatment on weight gain Daily injections of paroxetine (10 mg/kg/day, i.p.) significantly inhibited weight gain during 14 days of treatment in prenatal saline (Fig. 1A) and in cocaine (Fig. 1B) offspring. Prenatal saline-exposed offspring (Fig. 1A) receiving daily injections of saline gained weight steadily from 343  6 g on treatment day 1 to 399  7 g on day 14. In contrast, rats that received daily injections of paroxetine gained less weight throughout the treatment period (from 346  5 g on day 1 to 364  5 g on day 14). Prenatal cocaineexposed offspring (Fig. 1B) receiving daily injections of saline exhibited weight gains comparable to that of their prenatal saline counterparts (from 344  6 g on day 1 to 397  7 g on day 14), whereas rats that received daily injections of paroxetine exhibited a significantly lower weight gain (from 344  4 g on day 1 to 363  4 g on day 14). A repeated measures ANOVA indicated a significant main effect for paroxetine (F ð1;1427Þ ¼ 11:98, p < 0:01) and a significant main effect for days of injection (F ð13;1427Þ ¼ 99:43, p < 0:01), but no significant main effect for cocaine (F ð1;1427Þ ¼ 0:14, p > 0:05). The interaction between paroxetine and days of injection was significant (F ð13;1427Þ ¼ 28:64, p < 0:01), but the interactions between paroxetine and cocaine (F ð1;1427Þ ¼ 0:01, p > 0:05) and between cocaine and days of injection (F ð13;1427Þ ¼ 0:93, p > 0:05) were not significant. There was no significant interaction among paroxetine, cocaine and days of injection (F ð13;1427Þ ¼ 1:10, p > 0:05). The post hoc Newman– Keuls multiple-range test indicated that a significant inhibition of body weight gain occurred in the paroxetine-treated rats following 5 days of injections in saline offspring and following 4 days of injections in cocaine exposed offspring. There was no significant difference in body weight between saline-exposed and cocaine-

Fig. 1. Effect of treatment with paroxetine (10 mg/kg/day, i.p. 14 days) on body weight gain in adult male offspring exposed in utero to either (A) saline or (B) cocaine. The data represent the means  SEM of 24–26 rats per group. The data were analyzed by a three-way ANOVA with repeated measures and a Newman–Keuls test. The  indicates a significant difference (p < 0:01) from the respective salinetreated controls.

exposed groups, indicating that prenatal cocaine exposure did not affect body weight gain, and it did not affect the inhibition of body weight gain induced by paroxetine treatment. 3.4. Paroxetine induced desensitization on 5-HT2A receptor function in saline and cocaine offspring 3.4.1. Oxytocin Fig. 2 illustrates basal and ()DOI-stimulated increases in plasma levels of oxytocin in saline (Fig. 2A) and cocaine (Fig. 2B) offspring, following 14 days of paroxetine treatment. In both saline and cocaine offspring, comparable 5-HT2A receptor-mediated oxytocin responses to ()DOI were observed in the chronic saline

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between cocaine and paroxetine (F ð1;79Þ ¼ 5:16, p < 0:05). There was no significant main effect for cocaine (F ð1;79Þ ¼ 0:51, p > 0:05) and no significant interaction between cocaine and ()DOI (F ð2;79Þ ¼ 0:19, p > 0:05). There was no significant interaction among cocaine, ()DOI and paroxetine (F ð2;79Þ ¼ 1:18, p > 0:05). The Newman–Keuls test indicated that chronic paroxetine treatment significantly attenuated the oxytocin responses to ()DOI in both groups of adult offspring, but significant differences in the magnitude of the desensitization were observed between groups. In prenatal saline offspring (Fig. 2A), both doses of ()DOI significantly elevated plasma oxytocin levels above basal, and chronic paroxetine significantly attenuated the oxytocin responses to 0.5 mg/kg (–39%) and 2 mg/kg doses of ()DOI (–48%). In prenatal cocaine offspring (Fig. 2B), paroxetine produced a significantly greater inhibition (–54 and –73%, respectively) of oxytocin responses to ()DOI at 0.5 and 2.0 mg/kg than was observed in saline offspring, and neither dose of ()DOI was able to elevate oxytocin significantly above basal.

Fig. 2. Effect of treatment with paroxetine (10 mg/kg/day, i.p. 14 days) on ()DOI-stimulated plasma oxytocin levels in adult male offspring exposed in utero to either (A) saline or (B) cocaine. The data represent the means  SEM of 8 to 10 rats per group, and were analyzed by a three-way ANOVA followed by a Newman–Keuls multiple range test. The  and  indicate a significant difference (at p < 0:05 and p < 0:01, respectively) from the corresponding salineinjected group. The + indicate a significant difference (p < 0:01) from the corresponding lower dose of ()DOI. The # and ## indicate a significant difference (at p < 0:05 and p < 0:01, respectively) from corresponding saline-treated / DOI-challenged group. The @ indicates a significant difference (p< 0:05) from the respective paroxetinetreated prenatal saline offspring group.

treated groups. Chronic paroxetine treatment did not alter basal levels of oxytocin in either prenatal saline or cocaine exposed offspring. However, treatment with paroxetine resulted in a diminished oxytocin response to ()DOI (Fig. 2). Three-way ANOVA of oxytocin data indicated significant main effects for paroxetine (F ð1;79Þ ¼ 53:34, p < 0:01), for ()DOI (F ð2;79Þ ¼ 55:30, p < 0:01) and a significant interaction between paroxetine and ()DOI (F ð2;79Þ ¼ 15:83, p < 0:01) and

3.4.2. ACTH Fig. 3 illustrates basal and ()DOI-stimulated increases in plasma levels of ACTH in saline (Fig. 3A) and cocaine (Fig. 3B) offspring following 14 days of paroxetine treatment. Consistent with the oxytocin data, no differences in 5-HT2A receptor-mediated ACTH responses to either dose of ()DOI were observed between saline and cocaine offspring. Chronic paroxetine treatment did not alter basal levels of ACTH in either prenatal saline or cocaine-exposed offspring. However, in cocaine offspring, treatment with paroxetine resulted in a diminished ACTH response to ()DOI (Fig. 3B). Three-way ANOVA of ACTH data indicated a significant main effect of paroxetine (F ð1;73Þ ¼ 5:82, p < 0:05) and a significant main effect of ()DOI (F ð2;73Þ ¼ 80:0, p < 0:01), but no significant main effect of cocaine (F ð1;73Þ ¼ 2:47, p > 0:05). There was no significant interaction between cocaine and paroxetine (F ð1;73Þ ¼ 3:64, p > 0:05), cocaine and ()DOI (F ð2;73Þ ¼ 0:74, p > 0:05) or paroxetine and ()DOI (F ð2;73Þ ¼ 2:60, p > 0:05). There was no significant interaction among cocaine, paroxetine and ()DOI (F ð2;73Þ ¼ 1:83, p > 0:05). A post hoc Newman–Keuls test indicated that ()DOI significantly elevated the plasma ACTH levels above basal in both saline and cocaine offspring. In prenatal saline offspring, paroxetine treatment did not alter the ACTH response to either dose of ()DOI. In contrast, chronic paroxetine treatment significantly (p < 0:05) attenuated (54%) the ACTH response to the 2 mg/kg dose of ()DOI in prenatal-cocaine offspring (Fig. 3B), and the magnitude of this response was significantly lower than that in the

Z. Chen et al. / Neuropharmacology 46 (2004) 942–953

Fig. 3. Effect of treatment with paroxetine (10 mg/kg/day, i.p. 14 days) on ()DOI-stimulated plasma ACTH levels in adult male offspring exposed in utero to either (A) saline or (B) cocaine. The data represent the means  SEM of 8 to 10 rats per group. Data were analyzed by a three-way ANOVA followed by a Newman–Keuls multiple range test. The  indicates a significant difference (p < 0:01) from the corresponding saline-injected group. The + indicates a significant difference (p < 0:05) from the corresponding lower dose of ()DOI. The # indicates a significant difference (p < 0:05) from corresponding saline-treated / DOI-challenged group. The @ indicates a significant difference (p< 0:05) from respective paroxetine-treated prenatal saline offspring group.

respective group of offspring not exposed to cocaine in utero. 3.4.3. Corticosterone Fig. 4 illustrates basal and ()DOI-stimulated increases in plasma levels of corticosterone in saline (Fig. 4A) and cocaine (Fig. 4B) offspring following 14 days of paroxetine treatment. Consistent with the oxytocin and ACTH data, no differences in 5-HT2A receptor-mediated corticosterone responses to either dose of ()DOI were observed between saline and cocaine off-

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Fig. 4. Effect of treatment with paroxetine (10 mg/kg/day, i.p. 14 days) on ()DOI-stimulated plasma corticosterone levels in adult male offspring exposed in utero to either (A) saline or (B) cocaine. The data represent the means  SEM of 8 to 10 rats per group, and were analyzed by a three-way ANOVA followed by a Newman– Keuls multiple range test. The  indicates a significant difference (p < 0:01) from the corresponding saline-injected group. The + indicates a significant difference (p < 0:05) from the corresponding lower dose of ()DOI.

spring. Three-way ANOVA of corticosterone data indicated a significant main effect for ()DOI (F ð2;83Þ ¼ 317:83, p < 0:01) but no significant main effect for paroxetine (F ð1;83Þ ¼ 0:01, p > 0:05) or cocaine (F ð1;83Þ ¼ 0:01, p > 0:05). There was no significant interaction between cocaine and ()DOI (F ð2;83Þ ¼ 0:02, p > 0:05) , cocaine and paroxetine (F ð1;83Þ ¼ 0:01, p > 0:05), paroxetine and ()DOI (F ð2;83Þ ¼ 1:04, p > 0:05) or an interaction among cocaine, paroxetine and ()DOI (F ð2;83Þ ¼ 0:23, p > 0:05). This indicated that paroxetine treatment did not significantly change the plasma levels of corticosterone stimulated by either dose of

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()DOI in prenatal saline-exposed offspring (Fig. 4A) or in prenatal cocaine-exposed offspring (Fig. 4B). 3.5. Paroxetine-induced decrease in 5-HT2A receptor binding in saline and cocaine offspring The effects of 14-day paroxetine treatment on the [3H]-ketanserin and [125I]-DOI labeled 5-HT2A receptors in the rat cortex are illustrated in Fig. 5. Prenatal cocaine exposure did not alter the densities of 5-HT2A receptors labeled by either [3H]-ketanserin (Fig. 5A) or [125I]-DOI (Fig. 5B). In contrast, the densities of cortical 5-HT2A receptors labeled by [3H]-ketanserin (Fig. 5A) were significantly (p < 0:01) reduced by par-

oxetine treatment in both saline (25%) and cocaine (18%) offspring. Likewise, chronic paroxetine treatment significantly (p < 0:01) reduced the densities of [3H]-DOI labeled 5-HT2A receptors (Fig. 5B) in offspring exposed to saline (23%) or cocaine (19%). A two-way ANOVA indicated a significant main effect of paroxetine on [3H]-ketanserin binding (F ð1;29Þ ¼ 36:61, p < 0:01) and [125I]-DOI binding (F ð1;29Þ ¼ 24:28, p < 0:01). There was no significant main effect for cocaine on [3H]-ketanserin binding (F ð1;29Þ ¼ 1:72, p > 0:05) or [125I]-DOI binding (F ð1;29Þ ¼ 0:27, p > 0:05), and there was no significant interaction between cocaine and paroxetine on [3H]-ketanserin or [125I]-DOI binding (F ð1;29Þ ¼ 1:57, p > 0:05; F ð1;29Þ ¼ 0:32, p> 0:05, respectively). The Newman–Keuls test indicated that paroxetine treatment significantly reduced the density of [3H]-ketanserin or [125I]-DOI-labeled 5-HT2A receptors to a comparable extent (18–25%) in both prenatal saline and cocaine offspring.

4. Discussion

Fig. 5. Effect of treatment with paroxetine (10 mg/kg/day, i.p. 14 days) on 0.8 nM [3H]-ketanserin or 0.2 nM [125I]-DOI labeled 5-HT2A receptors in cortical homogenates from adult male offspring exposed in utero to either (A) saline or (B) cocaine. The data represent the means  SEM of six to eight rats per group. For each radioligand, data were analyzed by a two-way ANOVA followed by a Newman–Keuls multiple range test. The  indicates a significant difference (p < 0:01) from the corresponding saline-treated control group.

We have previously reported that prenatal exposure to cocaine produces marked alterations in pre- and postsynaptic components of serotonergic systems in rat offspring which differ at different developmental ages (Battaglia et al., 1998). Given an increasing number of individuals that may have been exposed to cocaine in utero, it is important to determine whether the changes in serotonergic systems produced by prenatal cocaine exposure will alter the neuroadaptive responses in postsynaptic 5-HT receptor systems to clinically prescribed serotonergic drugs. To our knowledge, the present study provides the first and only information to date on the ability of the clinically prescribed SSRI, paroxetine (Paxil1), to produce neuroadaptive changes in postsynaptic 5-HT2A receptor function after prenatal exposure to cocaine. The main findings of this study are that: (1) chronic treatment with paroxetine produces a significant desensitization in postsynaptic 5HT2A receptor function in adult male rat offspring, evidenced by attenuated oxytocin responses to ()DOI; and (2) prenatal exposure to cocaine potentiates the paroxetine-induced desensitization of 5-HT2A receptor function. Although DOI exhibits similar affinities for 5-HT2A and 5-HT2C receptors (Hoyer, 1988), DOI-induced increases in plasma ACTH, corticosterone and oxytocin levels can be completely blocked by low doses of the selective 5-HT2A receptor antagonist MDL 100,907 (Hemrick-Luecke and Fuller, 1996; Van de Kar et al., 2001). Furthermore, a direct injection of MDL 100,907 into the hypothalamic paraventricular nucleus blocks the hormone responses to a peripheral injection of

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()DOI (Zhang et al., 2002). These data indicate that the neuroendocrine effects of DOI are mediated by hypothalamic 5-HT2A receptors. Thus, treatmentinduced changes in plasma hormone levels after a ()DOI challenge reflect changes in the functional status of postsynaptic 5-HT2A receptor systems. Furthermore, changes in hormone responses to ()DOI can be used as a peripheral index of changes in hypothalamic 5-HT2A receptor function, which may reflect comparable changes occurring in other brain regions. In the present study, the doses of ()DOI (0.5 and 2 mg/kg) were chosen based on data indicating that 2 mg/kg ()DOI mediates maximal increases in oxytocin, ACTH and corticosterone via activation of the 5-HT2A receptor subtype (Van de Kar et al., 2001). Prenatal cocaine exposure produces an increase in postsynaptic 5-HT2A receptor function in prepubescent male offspring (Battaglia et al., 2000). The increases in 5-HT2A receptor-mediated neuroendocrine responses were observed using a maximally effective dose (2 mg/ kg) of DOI, suggesting an increase in the Emax of 5HT2A receptor function. In contrast, our present data indicate that, when cocaine-exposed male offspring reach adulthood, the 5-HT2A receptor-mediated increase in plasma hormones is indistinguishable from that in control rats (Table 1). These findings indicate that the initial functional increase in the Emax of postsynaptic 5-HT2A receptor systems in prepubescent cocaine offspring does not persist into adulthood and there is no change in the sensitivity (i.e. ED50) of 5HT2A receptors to ()DOI at this age. These data suggest that the normal developmental changes associated with sexual maturation may interact with the initial effects of in utero cocaine exposure to affect postsynaptic 5-HT2A receptor function in offspring. Consequently, prenatal cocaine exposure can differentially alter postsynaptic 5-HT2A receptor function at different developmental ages. Developmental differences in the responsiveness of 5-HT2A receptor systems have also been observed following prenatal exposure to fluoxetine (Cabrera and Battaglia, 1994), a drug which more selectively blocks 5-HT transporters during fetal brain development. In prenatal fluoxetine-exposed offspring, no differences were observed in DOI-induced increases in ACTH or in the density of hypothalamic [125I]-DOIlabeled 5-HT2 receptors when the male offspring were prepubescent. However, littermates tested at an adult age exhibited a marked reduction (58%) in the ACTH response to DOI and a marked reduction (35%) in hypothalamic [125I]-DOI-labeled receptors. Again, these data suggest that hormonal changes associated with sexual maturation may represent one of a number of age-related events that can affect the expression of neuroadaptations in serotonin pathways in offspring exposed to serotonergic drugs during gestation.

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Paroxetine treatment of prenatal saline-exposed offspring produced a marked attenuation of the oxytocin response to both doses of ()DOI (Fig. 2A), but no change in the ACTH response to either dose of ()DOI (Fig. 3A). As 5-HT2A receptors have been shown to be present on oxytocin and CRH-containing cells (Gray et al., 2003), these data suggest that treatment with paroxetine produces a significant and differential marked desensitization of 5-HT2A receptors on oxytocin but not CRH-containing cells in the hypothalamus. These findings are consistent with other studies from our laboratory that indicate that treatment with fluoxetine, another SSRI, produced a selective desensitization of 5-HT2A receptor-mediated increase in oxytocin, but not ACTH responses, in prepubescent male rats (Shankaran et al., 2001). A previous study indicated that fluoxetine increased 5-HT2A receptor function in adult rats (Li et al., 1993), but this has not been supported by more recent data (Damjanoska et al., 2003). In offspring prenatally exposed to cocaine, paroxetine treatment was able to completely block the 5HT2A receptor-mediated stimulation of oxytocin, as no significant elevations above basal were elicited by either of the doses of ()DOI (Fig. 2B) and the oxytocin responses to both doses of ()DOI were significantly lower than in their prenatal saline counterparts. These data suggest that prenatal exposure to cocaine facilitates the ability of paroxetine to desensitize 5-HT2A receptor function on oxytocin-containing magnocellular cells and that the desensitization is primarily mediated via a reduction in Emax and/or a marked shift to the right in the ED50. Furthermore, in prenatal cocaine-exposed offspring, paroxetine treatment attenuated the ()DOI-induced increase in ACTH response to the maximal dose of ()DOI (Fig. 3B), suggesting a decrease in the Emax of 5-HT2A receptor function on CRH-containing parvocellular cells of the hypothalamus. This could be due to prenatal cocaine-induced reductions in receptor reserve for 5-HT2A receptor-mediated stimulation of oxytocin and ACTH in the paraventricular nucleus or reductions in levels of respective G proteins and/or the activity of respective second messenger enzymes. These possibilities are currently under investigation. In order to determine whether the attenuation of oxytocin and ACTH responses to a 5-HT2A receptor agonist was due to receptor changes, densities of 5HT2A receptors were measured in cortical homogenates using a single concentration (below the respective Kd) of the antagonist [3H]-ketanserin, or the agonist [125I]DOI. This approach renders radioligand binding sensitive to changes in either receptor affinity and/or density. Prenatal exposure to cocaine did not alter the binding of agonist- or antagonist-labeled 5-HT2A receptors. However, paroxetine treatment significantly

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reduced the density of both [125I]-DOI- and [3H]-ketanserin-labeled 5-HT2A receptors in both saline and cocaine offspring. As [125I]-DOI labels only those receptors that exist in the ‘‘high affinity state’’ (e.g. receptors coupled to a guanine nucleotide regulatory protein), selective changes in the density of [125I]-DOIlabeled receptors provide a useful index to access alterations in the agonist high affinity component of the receptor population. In contrast to the present data, chronic fluoxetine treatment has been shown to increase [125I]-DOI labeled 5-HT2 receptors without concomitant changes in [3H]-ketanserin binding or levels of Gq and G11 protein (Li et al., 1997b). The comparable magnitude of paroxetine-induced reductions in the binding of both radioligands (18 to 25%) indicates that the reductions in binding are most likely due to a reduction in receptor density rather than to changes in receptor affinity. Presumably, if there is little receptor reserve for 5-HT2A receptor-mediated neuroendocrine responses, then reductions in 5-HT2A receptors may be, in part, responsible for the paroxetine-induced reductions in oxytocin and ACTH to an Emax dose of the 5-HT2A agonist ()DOI. Previous studies have shown an absence of spare receptors for 5HT1A receptor-mediated inhibition of adenylyl cyclase activity (Yocca et al., 1992) but large differences in receptor reserve for 8-OH-DPAT stimulated prolactin, ACTH and corticosterone responses (Meller and Bohmaker, 1994). A recent literature review failed to identify any comparable data for 5-HT2A receptor-mediated neuroendocrine responses. Consequently, it is possible that the paroxetine-induced reductions in receptors and the corresponding reductions in the Emax of oxytocin and ACTH responses to ()DOI, are due to the absence or limited 5-HT2A receptor reserve for these hormone responses. Furthermore, a reduction in receptor reserve due to developmental exposure to cocaine would be consistent with the greater magnitude of paroxetine-induced attenuation in 5-HT2A receptor mediated oxytocin levels in the prenatal cocaine-exposed offspring (Fig. 2B). Additional studies are necessary to address this possibility as well as the ability of paroxetine to produce discrete changes in 5-HT2A receptors in hypothalamic and extrahypothalamic brain regions. In contrast to the effect of paroxetine in reducing the ACTH response to an Emax dose of ()DOI (Fig. 3B), corticosterone responses to ()DOI were unaltered by paroxetine treatment in prenatal saline or cocaine offspring. A simple explanation for the lack of changes in plasma corticosterone response following paroxetine treatment is that ACTH levels in all ()DOI-injected groups remained in excess of approximately 200–300 pg/ml, levels required to saturate adrenal ACTH receptors and produce maximal coricosterone responses (Engeland et al., 1981; Bagdy et al., 1989). As ACTH levels stimulated by ()DOI were greater than

600 pg/ml in all groups (Fig. 3), it would be expected that any of the paroxetine-induced reductions in ACTH responses to ()DOI would not be reflected by any corresponding reductions in plasma corticosterone. Paroxetine significantly attenuated weight gain in adult offspring, independent of prenatal-cocaine exposure. These effects on weight gain were apparent after 4–5 days of treatment and continued throughout the paroxetine treatment period (Fig. 1). These data are consistent with previously reported reductions in weight gain in adult rats after 5 days of injections with another SSRI fluoxetine (Zhang et al., 2000). While the mechanism underlying the reduction in body weight gain remains to be discerned, these data suggest that prenatal exposure to cocaine does not affect this phenomenon. In summary, the data presented here demonstrate that: (1) paroxetine treatment results in an attenuation of 5-HT2A receptor-mediated stimulation of oxytocin, but not ACTH or corticosterone, and (2) prenatalcocaine exposure potentiates paroxetine-induced desensitization of 5-HT2A receptor-mediated oxytocin responses and promotes the desensitization of 5-HT2A receptor-mediated ACTH in adult male rat offspring. These findings may be clinically relevant with respect to the therapeutic effectivenesss of antidepressants or antipsychotics that target 5-HT2 receptors in treating adults exposed in utero to cocaine.

Acknowledgements The authors express their gratitude to Dr. James Sincore for providing his expertise on the statistical analysis of data and Franciscia Garcia for her excellent technical assistance. This Study was supported by USPHS grants DA07741 (G.B.) and DA13669 (L.V.D.K.).

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