Prenatal ethanol preferentially enhances reactivity of the dopamine D1 but not D2 or D3 receptors in offspring

Neurotoxicology and Teratology 27 (2005) 73 – 93 www.elsevier.com/locate/neutera

Prenatal ethanol preferentially enhances reactivity of the dopamine D1 but not D2 or D3 receptors in offspring Sonya K. Sobriana,*, Barbara L. Jonesa, Hutchinson Jamesa, Feremusu N. Kamaraa, R. Robert Holsonb a

Department of Pharmacology, Howard University College of Medicine, 520 W Street, NW, Washington, DC 20059, USA b Department of Psychology, New Mexico Tech, Socorro, NM 87801, USA Received 17 August 2004; received in revised form 3 September 2004; accepted 7 September 2004 Available online 13 October 2004

Abstract Reports of prenatal ethanol (ETOH) effects on the dopamine system are inconsistent. In an attempt to clarify this issue, dams were given 35% ethanol-derived calories as the sole nutrient source in a liquid diet from the 10th through the 20th day of gestation (ETOH). Controls were pair-fed (PF) an isocaloric liquid diet or given ad libitum access to laboratory chow (LC). Prenatal exposure to both liquid diets reduced body weight of offspring relative to LC controls, more so for ETOH than for PF exposure. Prenatal ETOH also decreased litter size and viability, relative to both LC and PF control groups. On postnatal days 21–23, male and female offspring were given an injection of saline vehicle or one of eight specific dopamine receptor agonists or antagonists. Immediately after injection subjects were placed in individual observation cages, and over the following 30 min, eight behaviors (square entries, grooming, rearing, circling, sniffing, yawning, head and oral movements) were observed and quantified. No prenatal treatment effects on drug-induced behaviors were observed for dopamine D2 (Apomorphine, DPAT or Quinpirole) or D3 (PD 152255, Nafadotride, Apo or Quin effects on yawning) receptor agonists or antagonists, or for the vehicle control. In contrast, prenatal treatment effects were seen with drugs affecting the dopamine D1 receptor. Both D1 agonists (SKF 38393) and antagonists (SCH 23390 and high doses of spiperone) altered behaviors, especially oral and sniffing behaviors, in a manner which suggested enhanced dopamine D1 drug sensitivity in both ETOH and PF offspring relative to LC controls. These results suggest that at this age, both sexes experience a prenatal undernutrition-linked increase in the behavioral response to dopamine D1 agonists and antagonists, which can be intensified by gestational exposure to alcohol. D 2004 Elsevier Inc. All rights reserved. Keywords: Prenatal ethanol exposure; Dopamine receptor subtypes; Drug challenge; Dopamine agonists; Dopamine antagonists; Sex factors; Fetal growth retardation; Undernutrition

1. Introduction Maternal consumption of alcohol during pregnancy can have profound teratological effects on the developing fetus. The spectrum of enduring manifestations are extensive and lie along a continuum, from fetal alcohol syndrome (FAS), which is defined by pre- and/or postnatal growth retardation, characteristic facial dysmorphology, and abnormal function

* Corresponding author. Tel.: +1 202 806 7901; fax: +1 202 806 4453. E-mail address: [email protected] (S.K. Sobrian). 0892-0362/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2004.09.002

of the central nervous system (CNS), to other alcoholrelated birth deficits (ARBD), which range from severe behavioral and cognitive dysfunction to minimal deficit [1,2,44]. The most devastating and far-reaching consequences of prenatal exposure to alcohol is its effect on the brain and the ensuing behavioral alterations that occur [60,90]. CNS dysfunction, one of the more consistent and persistent outcomes, is expressed as mental retardation, developmental delay, attention deficits, hyperactive behavior, impulsivity, hyper-responsiveness to stress, poor motor coordination, and learning disabilities [53]. While neuropathology is most

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often the result of heavy prenatal alcohol exposure, altered CNS function can occur even after relatively modest amount of alcohol exposure and can persist into adolescence and adulthood [79]. Although there is a considerable amount of data regarding the morphological and behavioral effects associated with prenatal alcohol exposure, the mechanisms underlying developmental defects caused by maternal ETOH consumption remain unclear [55]. Animal models, which mirror many of the physical anomalies and behavioral abnormalities observed clinically, have been used to define the neurochemical abnormalities that result from in utero ethanol exposure. These investigations have found ethanol-induced impairments in the development of most CNS neurotransmitter systems, including serotonin, norepinephrine, glutamate, g-aminobutyric acid, acetylcholine, histamine and opiate peptides [5,6,41,53,60, 78,90,91,108]. However, neurochemical, behavioral and pharmacological studies suggest that developing dopaminergic systems may be particularly targeted [41,95]. Prenatal ethanol exposure impairs the synthesis and/or secretion of dopaminergic trophic factors, which are essential for normal CNS development [75,111], and the ability of such factors to mediate differentiation and neurite elongation, thus adversely affecting subsequent neural development in dopaminergic and other systems [39]. Animal studies reveal that both chronic and episodic in utero exposure to ETOH results in structural abnormalities in the caudate nucleus and the cerebellum [7,42]. At the cellular level, Shetty et al. [106] found dendritic dysmorphology in the substantia nigra pars compacta of prenatally ETOH exposed rats. A very wide range of effects/no effects are reported for whole brain and/or regional content of DA and its metabolites. Gestational exposure to ethanol decreases basal concentrations of DA and its metabolite DOPAC in brain regions that are targets of dopaminergic projections, as well as those that contain cell bodies of dopaminergic neurons, increases tyrosine hydroxylase activity, alters DA receptor binding, adversely impacts the development of dopamine reuptake sites, and reduces the spontaneous activity of ventral tegmental area dopamine neurons [19,29,34, 35,38,49,53,63,74,89,95,108,113,127]. Therefore, the reduction of DA in target areas combined with a decreased concentration of reuptake sites suggests that exposure of the fetus to ethanol impairs the development of DA projections [37]. Morphological changes in DA neurons, including smaller cell bodies and retarded dendritic growth and branching, [106], as well as decreased activity in substantial nigra/VTA neurons [105,127] provide support for this hypothesis. However, despite the evidence suggesting that prenatal ethanol produces a hypofunctioning in DA systems, there is also a body of literature indicating no changes in DA, DOPAC or HVA in the whole brain or striatum and nucleus accumbens [18,19,27,28,78,83,96,115] or striatal DA reup-

take sites [53]. Moreover, there is some indication that prenatal ETOH exposure increases activity in central DA systems [20,49,76]. These inconsistencies emphasize the need for further research in this area. The selectivity of prenatal alcohol on dopamine systems is further suggested by the impact of this treatment on the number of functional dopamine receptors subtypes in the rodent brain. However, changes are age, sex, receptor subtype and species specific. Prenatal ETOH both decreases [38] and increases [53] D1 binding sites in the striatum and / or frontal cortex. In mouse, striatal D1 receptors are increased [19]. In both mice and rats, the effects on the D1 receptor subtype occur early in development and are transient [19,38]. Both no change [19,38] or a significant reduction in D2 binding sites have been reported in the striatum following prenatal ETOH [76,77,89,95]. These divergent findings prohibit the formulation of a definitive hypothesis regarding ethanol’s effect on DA receptor subtypes. Behavioral and pharmacological studies also indicate that prenatal ethanol exposure affects dopaminergic systems in the CNS. Rats and mice prenatally exposed to alcohol show alterations in behaviors supported by normal dopaminergic function, such as motor activity, catalepsy and stereotypy, motor coordination and reward-seeking behaviors (see Ref. [19] for review). Moreover, studies with acute and chronic drug challenges suggest that prenatal alcohol profoundly alters the behavioral response to dopaminergic drugs. Altered sensitivity of the DA receptor is supported by reports that rats exposed to ethanol prenatally show enhanced responsiveness to the indirect acting CNS stimulants, amphetamine and methylphenidate, with increased peak exploratory behavior, stereotypies and unilateral rotational behavior [17,48,81,119]. In contrast, sensitization of locomotor activity to repeated intermittent methylphenidate in adult rat offspring is unchanged [95], and amphetamine challenge reduced peak activity [76]. In addition to motor activity, lever pressing for a food reward is more disrupted by amphetamine in adult and middle-aged prenatal ethanol exposed male mice than controls [51,52]. Similarly, prenatal ethanol exposure affects the response to dopamine receptor agonists and antagonists. The D1/D2 mixed agonist, apomorphine, increased stereotypy in adult male rat prenatally exposed to ETOH [48]. It also induced greater locomotor activity at low doses but lower activity at high doses in prenatal ETOH exposed males, while female offspring showed less of an increase in this behavior than controls following high doses of the drug [62]. Although the direction of the response to apomorphine is opposite, the does–response effect seen in ethanol-exposed mice is similar to that seen in rat offspring. At higher doses, mice are less sensitive to the locomotor suppressant effect of apomorphine on baseline and ethanol-stimulated locomotor activity, but exhibited greater sensitivity to the suppressant effects of low doses of the drug [12]. Moreover, the D2 antagonist, haloperidol, produced a greater decrease in

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motor activity in prenatally ethanol exposed male rats than in controls [76]. Although the oral activity dose–response curve for the D1 agonist, SKF 38393, is unaltered from controls in male rats following in utero ethanol exposure, quinpiroleinduced yawning, linked to D3 stimulation, was markedly increased, suggesting that prenatal ethanol may predominantly diminish reactivity of the D3, rather than the D1 receptor subtype [22]. However, both reduced magnitude and delayed maturation of the cataleptic response to the D1 antagonist, SCH23390, have also been seen following prenatal exposure to ethanol [61]. The purpose of this experiment was to determine the effects of prenatal ethanol exposure on the development of dopamine receptor subtypes in an effort to address the inconsistencies in the current literature. Discrepancies in the existing literature may reflect (1) differences in the period of alcohol exposure, as well as the route of administration, (2) acute vs. chronic drug challenge, (3) the age and/or sex of the offspring at testing, and (4) the relationship between different sites of drug action in the CNS and the behavioral variable sampled. Although the majority of studies that have looked at the behavioral effects of offspring challenge with dopaminergic drugs have exposed dams to ethanol from gestations days 6–7 to 18–20 [11,12,17,49,61,62, 88,95,119,129], other schedules have employed total pregnancy [2] in combination with premating [22] and/or postnatal [76] exposure, as well as a single injection of ETOH on gestation day (GD) 8 [48]. The oral route of administration, either by liquid diet, in drinking water, or by intragastric intubation, was used in all but one study [41]. While acute drug challenge is the paradigm most often tested, repeated challenge with methylphenidate [81,95,119] has been used. Varied findings may also reflect the fact that many of the reported changes in receptor function and binding are sex and age specific. Blanchard et al. [17] have shown that sex contributes to the differences in susceptibility to or recovery from prenatal alcohol exposure, with males showing greater sensitivity than females after acute drug challenge. Female offspring appear to be consistently less affected by prenatal alcohol or to recover sooner from the alcohol-related effects of postnatal acute stimulant pharmacology [15]. Therefore, both male and female offspring were tested. Moreover, challenge studies have primarily evaluated only locomotor activity. However, the necessity of evaluating several behaviors simultaneously is suggested by the involvement of different brain regions in various behaviors. Apomorphine-induced hypoactivity is exerted via DA mechanisms in the mesolimbic system, specifically presynaptic DA receptors localized in the nucleus accumbens and not in the nigrostriatal system [93,121]. In contrast, apomorphine hypersensitivity is thought to be mediated either striatally [56], or by postsynaptic mesolimbic dopamine receptors [62]. While oral stereotypy is believed to result from the direct stimulating effects of apomorphine

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on striatal postsynaptic sites [69], and catalepsy is thought to involve D1 nigrostriatal function [61,62], stereotypic sniffing is mediated by mesolimbic release [30]. To assess the functional status of DA D1, D2 and D3 receptors following prenatal ethanol exposure, a behavioral assay that involved an acute drug challenge paradigm was used. We developed a DA receptor subtype behavioral profile, a strategy that involves the measurement of a variety of drug-appropriate behaviors. These included grooming, sniffing and oral movements, increases in which are the hallmark of D1 receptor activation [14,21,45,54], and sniffing, oral and head movements, rearing, grooming, and locomotion, which are elicited in a dose-dependent manner by stimulation of the D2 receptor with apomorphine or quinpirole [8,14,43,84,85]. Three doses of each DA receptor agonists and antagonists were used, in order to clarify the dose-dependent behavioral effects. This approach necessitated testing at one age. Postnatal day (PND) 21 was chosen for several reasons: (1) most of the neurochemical studies on the effects of prenatal ETOH were conducted on young rats; and (2) changes in receptor function are transient and occur early, suggesting that the response to DA drugs would be more likely altered in developing rather than adult offspring.

2. Methods 2.1. Subjects One hundred and sixty-eight nulliparous time-pregnant Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were delivered to our laboratory on gestation day (GD) 8. The morning that vaginal plugs were found was designated as GD 0, and births were expected on GD 21–22. Females were housed individually in polyethylene maternity cages (44
25
20 cm), lined with wood-chip bedding, under environmentally controlled conditions (0700 h lights on, 1900 h lights off, ambient temperature 20–23 8C) with ad libitum access to Purina Rat Chow and tap water. 2.2. Ethanol exposure Females were matched on the basis of GD 9 body weight and assigned by body weight to one of three treatment groups: ethanol (ETOH), pair-fed (PF), or lab chow (LC). On GD 9, both ETOH and PF groups were given ad libitum access to liquid diets. On GD 10, ETOH females were given access to liquid diet ( LD’82, Shake and Pour-Ethonal: BioServ, Frenchtown, NJ), containing 17% ethanol-derived calories. From GD 11–20, they had ad libitum access to a liquid diet containing 35% ethanol-derived calories. The PF dams were fed a control liquid diet (LD’82, Shake and Pour: Bio-Serv) but with dextrin-maltose substituted isocalorically for ethanol. The amount was restricted to the previous day’s intake of the ETOH dams, as calculated by the formula: [(Intake (ml) of 35% ETOH dam/Body Weight of 35%

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ETOH dam)
Body Weight of pair-fed dam)+4 ml] (Edward Riley, personal communication). No pair-fed dam was given less than 50 ml of the liquid diet per day. LC dams were given ad libitum access to laboratory rat chow and water. The inclusion of a pair-fed group in addition to the ad lib lab chow control allowed an assessment of the potential contribution of restricted caloric intake to any noted effects. The ethanol liquid diet and the control liquid diet provided approximately 1.01 and 0.99 kcal/ml, respectively. Both are nutritionally complete for rats, and served as the sole source of nutrition during the period of administration. Consumption of the diets was measured daily to the nearest 0.1 g; both diets were made fresh each day and presented between 1400 and 1500 h and were available for a 24-h period. Maternal blood alcohol levels (BAL) were determined on GD 15 from blood collected from the tail vein (approximately 75 Al), 2–3 h after lights off (1900–2000 h), a time at which peak blood concentrations can be obtained [122], and analyzed for blood alcohol content within 24 h by a micromodification of the alcohol dehydrogenase method [66] using the Sigma Alcohol Kit (Sigma Diagnostic, St. Louis, MO). With a liquid diet procedure the amount of ethanol intake is determined by how much the rat drinks; however, the average absolute alcohol intake typically varies between 10 and 14 g/kg/day using a 35% EDC diet [62]. From the morning of GD 21 until birth, cages of females were monitored discretely three times daily between 0800 and 1600 h. Females were allowed to deliver naturally and nurse their own young. At birth, postnatal day (PND) 0, pups were weighed, measured, sexed and examined for external malformations. Litters were culled to a maximum of 10 pups, balancing for gender when possible. Only body weight and crown-rump length data from litters with six or more pups were used for analyses. 2.3. Maternal behavior Within 4 h after births were recorded and litters had nursed at least once, dams were monitored for maternal behavior. Dams giving birth after 1600 h were not tested because of these restrictions and the timing of the onset of the dark cycle. A culled litter of 10 pups was dispersed throughout a maternity cage containing clean bedding and the dam was placed at one end. The latency to begin and complete both nest building and pup retrieval was measured to the nearest second with a stopwatch. The number of dams not completing pup retrieval, as well as the number of pups remaining out of the nest at the end of the 5-min test period was recorded. Thirty minutes after the initial test, dams were observed for presence in the nest and pup-directed behavior, as well as eating, drinking and self-grooming. The number of unretrieved pups was noted and they were placed in the nest at this time.

2.4. Drug challenge Pharmacological stimulation or blockade of the various receptor subtypes induces distinct behavioral effects that vary according to dose and to the preferential affinity of these drugs for different DA receptor subtypes [47]. A preliminary study was conducted in naive 21-day-old male and females pups to determine the appropriate doses, routes of injection and behaviors to be scored (see Ref. [110] for details). Briefly, five doses were tested for each drug except nafadotride; limited quantity of this compound necessitated determining doses from the literature on adult animals. Fifteen behaviors, most often reported in the literature following drug challenge, were originally monitored. Behaviors not observed in at least 10% of the pups tested were eliminated. Low, medium and high doses for each drug were selected on the bases of differential behavioral profiles, and the three doses for each drug and the eight behaviors scored are listed in Tables 1 and 2, respectively. We have recently reviewed the literature [110] regarding the relationship of these drug-induced behaviors to dopamine receptor subtype activity in both adult and developing rats. Testing was conducted at PND 21–23 because it is the time at which the complete adult-typical response pattern that results from D1 and D2 receptor synergism is first evident. Moreover, it appears to be the age around which the Table 1 Challenge drugs and doses Drugs Agonists SKF 38393 (s.c.) D1 Apomorphine (i.p.) D1 high dose D2 low dose Quinpirole (s.c.) D2 high dose D3 low dose 7-OH-DPAT (s.c.) D2 high dose D3 low dose Antagonists SCH 23390 (s.c.) D1 Spiperone (i.p.) D2 low dose D1 high dose Nafadotride (i.p.) D3 low dose D2 high dose PD 152255 (i.p.) D3 Control Saline (0.9%)a a

Drug doses (mg/kg) Low

Med

High

0.01

0.1

10.0

0.1

1.0

3.0

0.05

1.0

3.0

0.01

1.0

5.0

0.01

0.1

0.3

0.0625

0.5

1.0

0.1

1.0

10.0

1.0

3.0

10.0

0.1 ml/100 g body weight

Saline (0.9%) was the vehicle used for all drugs.

S.K. Sobrian et al. / Neurotoxicology and Teratology 27 (2005) 73–93 Table 2 Behavioral variables Behavior

Description

Activity Circling Grooming Head Movements Oral movements Rearing Sniffing Yawning

Total squares entered 3608 Turns Discrete bouts of 2–3 s duration Vertical and lateral when stationary Licking, chewing, biting, facial clonus Vertical extension on hind legs Discrete bouts of 2–3 s duration Involuntary wide opening of mouth

controversies surrounding the functional maturation of the D2 autoreceptor and the D3 receptor pivot (see Ref. [110] for details). In order to account for the small differences in ages, pups were randomly assigned to each of the 25 drug-dose cells. Pups were not weaned before testing. On PND 21–23, 6 male and 6 female offspring from each of the prenatal treatment groups were challenged with one of three doses of one of eight drugs or saline (see Table 1). Immediately after injection of the challenge drug/dose, each pup was placed into one of six observation chambers with clear Plexiglas fronts for 30 min. The floors of these chambers were divided into a 3
3 array of squares (15
15 cm) used to measure activity. The six boxes were arranged so that one observer, blind with respect to the prenatal treatment of the subject and the challenge drug, could score the behavior of six rats simultaneously. Prior to drug-challenge testing, the three observers were trained in the behavioral technique used until between rater reliability was at least 90%. Behaviors were scored using a 15-s time-sampling technique. Each rat was observed once every 3 min for a total of 10 observation periods. Activity was measured as total squares entered during each 15-s observation period. The behaviors assessed were activity, circling, grooming, head movements, oral movements, rearing, sniffing, and, yawning (see Table 2). Each pup was tested in only one drug/dose condition, and each litter was represented only once in each cell of the design. Nafadotride was a generous gift from Dr. Francois Sautel of the Centre Paul Broca de l’Institut National de la Sante et de la Recherche Medical, Paris France. PD 152255 was generously supplied by Parke-Davis Pharmaceutical Research, Ann Arbor, MI. The remaining drugs were purchased commercially: apomorphine hydrochloride (Sigma); SKF 38393 hydrochloride, SCH 23390 hydrochloride and spiperone hydrochloride (RBI, Natick, MA); 7OH-DPAT hydrobromide and quinpirole hydrochloride (Tocris, Ballwin, MO). It should be noted that most neuroactive drugs bind to a variety of receptors of different neurotransmitters. Data from whole-cell patch clamp studies indicate that SKF 38393, but not SCH 23390, is a NMDA channel ligand [25]. However, the compounds chosen bind with the highest affinity for the DA subtypes investigated in this paper (see Ref. [110] for review), and the behavioral profiles observed were for the

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most part similar to those seen with adults that have been attributed to activity at the D1 and/or D2 DA receptors. Although both apomorphine (0.05–3.0 mg/kg sc) and quinpirole (0.5 mg/kg sc) have been shown to increase 5HT output in the dorsal raphe [80], and there is evidence of a relationship between D1 and 5-HT2C receptor systems in the neostriatum [4], none of the behavioral effects observed resembled the serotoninergic syndrome. 2.5. Statistical analyses 2.5.1. Maternal and litter variables Only data from pregnant females were used for analyses of gestational weight gain and diet intake. Completely randomized (CR) one-factor (prenatal treatment) analyses of variance (ANOVAs) were used to analyze total maternal gestational weight gain and total liquid diet intake from GD 10–20. Daily weight gain, calculated by subtracting the current day’s reading from the previous day’s body weight, and diet intake were analyzed using a two-factor split-plot design ANOVAs, with days as the repeated measure. Birth statistics (percent of dams pregnant, period of gestation, litter size and mortality) and maternal behavior (latencies to nest building and pup retrieval) were subjected to a one-factor ANOVA for Prenatal Treatment effects, and Newman–Keuls was used for post hoc analyses of significant main effects. Behaviors observed 30 min later were subjected to chi-square. Two-factor ANOVAs (CRF) (Sex and Prenatal Treatment) were employed to analyze pups’ birth weights, crown-rump lengths, total litter size, number of viable pups, and the number of male and female pups (male/female ratio). Tukey’s post hoc analyses were used to compare significant main effects [70]. 2.5.2. Behavioral variables The use of eight drugs, each with three doses, plus a saline control, and seven dependent behavioral variables clearly presents substantial issues regarding analysis of treatment effects. Foremost among these issues is alpha inflation. Bonferonni’s correction was used for multiple comparisons throughout as the primary means of adjusting for alpha inflation. We began by analyzing each drug, plus the saline control group, individually for treatment effects. Since this required nine independent analyses (eight drugs plus the saline injection control), alpha was initially set at 0.0057. Thus, only if the parametric analyses or chi-square analyses, described below, for each of nine drug challenges gave Treatment effects, or Treatment
Sex, or Treatment
Dose effects significant at this level or below did we proceed with further analyses of that drug (for saline controls, there were no Treatment
Behavior effects). 2.5.2.1. The problem of zero scores. A second problem was presented by the two drugs (SCH 23390 and Spiperone) which had potent inhibitory effects on most behaviors. Use of these drugs, especially at higher doses, frequently

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produced only zero scores for many animals. This bbasementQ effect is a serious violation of parametric analysis assumptions, since it reduces within-cell variability to, or near, zero. Hence, for these two drugs, any behavior, which was reduced to zero in more than 40% of cases, was analyzed for treatment effects using a nonparametric chi-square approach. Scores for such behaviors were first replaced by a bivariate bsome behavior observed vs. no behavior observedQ categorization, then data were analyzed in a 3 (treatments) by 2, (behavior vs. not) chisquare matrix. For both SCH 23390 and Spiperone, the same 5 of 7 behavioral variables (all behaviors except oral and head movements) had to be so analyzed. For an initial analysis of these five behavioral variables collapsed across behaviors (the equivalent of a main effect of Treatment in a parametric analysis), behaviors were categorized slightly differently because of the preponderance of zero scores. For this initial analysis, behaviors were categorized into two slightly different categories: either two or less total observations of all of the five behaviors summed over the 10 observational periods, or more than two such observations. The remaining two variables, head and oral movements, were analyzed using the same parametric profile analysis described below. 2.5.2.2. Parametric profile analyses. The experimental design was mixed, involving repeated measures within subjects across the seven behavioral measures (see Table 2; yawning was eventually dropped because of the infrequency of this behavior), and between-subject measures on the primary design factors, including prenatal treatment, drug dose and gender of the subject. This design may be symbolized as subjects (Treatment
Dose
Sex)
Behaviors, with seven behaviors, two sexes, three drug doses and three prenatal treatments [ethanol (ETOH), lab chow (LC), and pair-fed controls (PF)]. Profile analysis was the multivariate technique used to evaluate the seven behavioral repeated measures [120]. This technique treats all repeated measures as a single k-dimensional multivariate vector; in this study, k=7, the number of individual behavioral measures used in this design. This multivariate approach has several advantages over the traditional mixed-model ANOVA repeated measures design, including fewer assumptions (symmetry is not required) and greater power. Typically, of greatest interest are the interaction terms between the repeated measures behavioral variable and the other fixed terms. For example, the Behavior
Treatment interaction determines whether the bprofileQ of the seven levels of the repeated behavioral variable is the same or different across the various levels of the treatment factor. However, the profile analysis multivariate technique requires the same general scale of measurement for all k repeated-measures variables. This was accomplished by converting all raw behavioral measures to z-scores. Each step of this multistage process is described in greater detail below.

2.5.2.3. Conversion of raw data to z-scores. A repeated measures ANOVA was conducted on just the saline challenge group, using the seven behavioral measures as seven levels of a single repeated-measures factor, and Treatment (LC or PF) and Sex as the between-subjects factors. Since in this analysis the LC and PF groups did not differ for any of the seven behaviors at pN0.20, means and standard deviations were calculated for each of the seven behaviors from the pooled LC and PF groups, and these means and standard deviations were then used to transform all raw behavioral scores for all subjects. This procedure was used rather than the more conventional approach of calculating the standard deviation across all groups, because in some cases, drug treatments altered not only group means but group variances as well. 2.5.2.4. Post hoc tests. The effect of each drug was then analyzed separately using profile analysis with Sex, Treatment and Drug Dose as between-subject measures and Behavior as the multivariate repeated measure, again with seven levels representing the seven behaviors. If the Behavior
Treatment, Behavior
Treatment
Dose, or Behavior
Treatment
Sex interaction was significant ( p=0.0057), individual behaviors were then analyzed for treatment effects using conventional univariate ANOVA tests, followed in the case of significance by a Duncan’s test for post hoc differences between the three treatment levels. Since there were seven behaviors potentially analyzed for such post hoc tests, Bonferonni’s correction set the alpha level at 0.0073 for each of seven individual behavioral comparisons. While Bonferroni’s correction is often excessively conservative, the observed drug effects on receptor subtypes were so large that virtually no effects were excluded by the use of this correction.

3. Results 3.1. Maternal variables 3.1.1. Treatment weight gain Only data from pregnant females were used to calculate treatment weight gain and liquid diet intake. Total and daily maternal weight gains during ETOH exposure are presented in Fig. 1A and B, respectively. Maternal body weights prior to the start of ETOH exposure (GD 9) were not significantly different [ F(2/123)=0.069, p=0.938]. Total weight gains, determined by calculating the change in weight from GD 10 (the morning of treatment onset) to GD 20, were significantly reduced in the both the ETOH and PF dams [ F(2/123)=78.35, pb0.001], which did not differ from each other. Analysis of daily weight gains indicated that significant differences occurred during three of the first 4 days after the liquid diet was introduced, although ETOH and PF dams showed reduced weight gains with respect to LC dams throughout the treatment

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Fig. 1. Prenatal treatment effects on maternal weight gain. (A) Total maternal body weight gain (gFS.E.M.) from gestational day 10 through gestational day 20, by prenatal treatment condition. (B) Daily weight change (gFS.E.M.), gestational days 10–20, by prenatal treatment conditions. For both panels, asterisks indicate statistically significant differences at the pV0.05 level.

period [Prenatal Treatment
Days: F(20/1230)=5.35, pb0.0001; Newman–Keuls, pb0.05]. 3.1.2. Liquid diet intake Total liquid diet intake did not differ between the ETOH and PF dams (Fig. 2A). However, small daily differences occurred (Fig. 2B). On GD 12 PF females consumed significantly less liquid diet than ETOH dams; this trend was reversed towards the end of the treatment period [Prenatal Treatment
Day: F(10/850)=3.52, pb0.001; Newman–Keuls, pb0.05]. 3.1.3. Pregnancy outcomes Gestational and birth statistics are listed in Table 3. Consumption of the liquid diet containing 35% ethanolderived calories resulted in blood levels of 0.14%. The percentage of non-pregnant females was small in each group

and did not differ as a function of prenatal treatment [X 2 (2)=0.1464]. In addition, neither length of the gestational period, nor the male/female ratios were significantly altered by prenatal treatments [ F(2/123)=2.25, pN0.10 and F(2/ 123)=2.11, pN0.01, respectively]. However, total litter size [ F(2/123)=4.15, pb0.02], and the number of viable pups in the ETOH litters [ F(2/123)=9.95, pb0.0001] was significantly reduced in comparison to those in PF and LC litters. Birth weight and crown-rump length were recorded only for litters with six or more pups. Both ETOH and PF offspring had significantly lower birth weights than LC offspring; ETOH pups were also significantly smaller than PF offspring at birth [ F(2/230)=56.59, pb0.001;]. Crownrump length was also reduced in the ETOH pups [ F(2/ 230)=13.14, pb0.001]. Physical abnormalities, determined by gross inspection of skull, limbs and snout, were not observed in offspring at birth and did not develop

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maternal behaviors, observed 30 min after initial 5-min testing, only pup grooming showed a significant difference. The percentage of females engaged in this behavior, i.e., ETOH: 23%; PF: 5%; and LC: 14%, was largest in the ETOH group [X 2(2)=11.57, pb0.01]. 3.2. Offspring body weight At PND 21–23, body weights of the LC male and female offspring were significantly larger than both the same gender ETOH and PF offspring, who were also significantly different from each other [Table 4: F(2/888)=159.7, pb0.0001]. While females in all groups were significantly lighter than males, the Treatment by Gender interaction was not significant [Table 4: F(1/888)=33.6, pb0.0001 and F(2/ 888)=0.93, p=0.396, for Sex effects and the Sex
Treatment interaction, respectively]. 3.3. Drug challenge

Fig. 2. Prenatal treatment effects on maternal liquid diet intake. Both panels show liquid diet intake for the two groups given these diets, the ethanol (ETOH) and pair-fed groups. (A) Mean daily liquid diet intake (mlFS.E.M.) averaged over 11 days, gestational days (GD) 10–20. (B) Mean daily liquid diet intake (mlFS.E.M.) for ETOH and PF dams, from GD 9, the day prior to the first ethanol exposure, through GD 20. Again asterisks indicate statistically significant differences at the pV0.05 level.

subsequently. However, pup mortality at birth and 3 weeks of age was increased by prenatal exposure to ETOH [ F(2/ 123)=4.57, pb0.012; F(2/123)=14.24, pb0.0001, respectively]. At PND 21–23 (Table 3), body weights of male and female ETOH pups were still lower than their PF and LC counterparts. However, while PF female pups did not differ from LC females, PF male offspring were still lighter than LC males [ F(2/893)=159.70, pb0.0001; Tukey’s, pb0.01]. 3.1.4. Maternal behavior When females were tested within 4 h of recording births, there were no significant differences among ETOH, PF and LC dams with respect to latencies to begin and complete nest building [ F(2/62)=0.051, p=0.951; F(2/62)=0.039, p=0.962, respectively], and retrieve pups [ F(2/64)=0.534, p=0.589; F(2/62)=1.367, p=0.262]. Of the pup-directed

3.3.1. Gender effects At the early age at which subjects were tested there were no significant effects of gender on behavioral responses to any of the seven drug challenges for which full parametric profile analyses were possible (Apomorphine, DPAT, Nafadotride, PD 152255, Quinpirole, SKF 38393 or saline controls). Furthermore, for these drug challenges there were no significant Behavior
Gender
Dose interactions, and only one statistically significant Behavior
Gender
Treatment interaction (PD 152255) and one statistically significant Behavior
Gender
Treatment
Dose interaction (Apomorphine). Similarly, chi-square analysis of behavioral effects pooled over five behaviors (square entries, rearing, grooming, circling and sniffing) were not significant for gender for the non-parametric SCH 23390 and Spiperone drug challenges. Nevertheless, for subsequent analyses gender was retained in the full parametric profile analysis model to avoid inflation of the error term. 3.3.2. Zero scores and yawning Drug- or saline-induced yawning was extremely rare at the age tested in this study (PND 21–23). Consequently, yawning was not included in the behavioral categories analyzed by drug. Indeed, so rare was this behavior (seen in only 4% of saline challenge offspring), that even analysis by chi-square was not possible for individual drugs, since typically half of all cells had expected counts of less than five cases. 3.3.3. Drug effects For five of the challenge drugs and saline, there were no significant Prenatal Treatment effects, or Treatment
Dose interactions (Fig. 3). Thus, the behavior by Treatment effect, and the Behavior
Dose
Treatment effect in the profile analyses did not attain significance for apomorphine [ F(12,176)=1.06, p=0.40 and F(24,308)=0.78, p=0.76], DPAT [ F(12, 170)=1.17, p=0.31 and F(24,298)=0.77,

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Table 3 Gestational and birth statistics for females given ethanol, pair-fed or lab chow diets on GD 10–20 (meanFS.E.M) Females (N)

Percent dams pregnant

Period of gestation (days)

Total litter size

Viable litter size

Serum ETOH (mg/dl)

ETOH [57] Pair-fed [56] Lab chow [55]

89.47% 91.07% 94.54%

22.27F0.08 22.00F0.09 22.16F0.13

10.27F0.42* 10.71F0.73 12.00F0.36

8.44F0.47*y 10.21F0.61 11.52F0.40

139.50F19.78

Litters (N)

Male/female ratio

Birth weight (g) Male

ETOH [47]a Pair-fed [39]a Lab chow [38]a

1:1.37F0.33 1:1.03F0.37 1:1.41F0.36

4.76F0.20*y 5.39F0.25* 6.33F0.22

a * y

Crown-rump length (cm)

Mortality

Female

Male

Female

PND 0

PND 21

4.67F0.21*y 5.11F0.25* 6.42F0.22

4.46F0.11* 4.65F0.13 4.85F0.12

4.37F0.21* 4.60F0.13 4.73F0.14

1.81F0.30*y 0.76F0.31 0.68F0.29

3.43F0.46*y 1.10F0.21 1.17F0.29

Number of litters with six or more pups. Significantly different from lab chow, pb0.001. Significantly different from pair-fed, pb0.001.

p=0.78], nafadotride [ F(12,168)=1.15, p=0.32 and F (24,294)=0.92, p=0.57], PD 152255 [ F(12,168)=1.15 and F(24, 294)=1.16, p=0.28], quinpirole [ F(12,168)=0.71, p=0.74 and F(24,294)=0.88, p=0.63], or saline [ F (12,50)=1.14, p=0.35). Three drugs, one agonist (SKF), and two antagonists (SPIP and SCH), did show significant Treatment effects, which are presented in detail below. 3.3.3.1. D1 agonists: SKF 38393. For this drug, the profile analyses indicated significant overall Treatment [ F (12,188)=2.89, pb0.0001] and Treatment
Dose effects [ F(24,329)=2.51, pb0.0001]. Fig. 4A shows data for each behavior pooled across doses. For the individual behaviors, the two-way treatment by dose ANOVAs gave significant effects for two behaviors, sniffing and oral movements. Sniffing was increased in both the ETOH and PF groups relative to LC controls; ETOH and PF groups did not differ from one another [Treatment effect: F(2,99)=6.52, p=0.002]. There was also a significant Treatment
Dose interaction for sniffing [ F(4,99)=6.93, pb0.0001], with the high dose being the most effective in producing this effect (Table 5). There were also significant Treatment [ F (2,99)=9.44, p =0.0002], and Treatment
Dose [ F(4,99)=3.86, p=0.006] effects for oral movements, with the LC and ethanol groups not differing from each other, but both showing significantly lower oral movements then the PF group. The interaction was complex. At the low dose, PF subjects showed a greater enhancement of drug effects on

Table 4 Prenatal treatment effects on offspring body weights by gender at 21–23 days of age Prenatal treatment

Mean body weight (g)FS.E.M. Female

Lab chow Pair-fed Ethanol

Male A

55.0F0.58 47.0F0.62B 43.2F0.67C

Sexes combined A

57.4F0.53 49.6F0.69B 47.2F0.70C

56.2F0.40A 48.3F0.47B 45.2F0.50C

A,B,C Values with different letters are significantly different from each other, pb0.0001.

oral movements than either other treatment group (Fig. 4B). In contrast, for the middle dose, oral movements were increased more for both the PF and ethanol groups, relative to the LC group (Fig. 4C). Fig. 4B–D (see also Table 5) presents the SKF data by dose. For the low SKF dose (Fig. 4B), only oral movements showed Treatment effects [ F(2,33)=6.32, pb0.005], with the PF offspring producing substantially higher oral movements than LC or ETOH, which did not differ. Oral movements were also increased by the medium dose of SKF 38393 (Fig. 4C), but now in both the ETOH and PF groups [ F(2,33)=6.07, p=0.006] relative to the LC group. In addition, both ETOH and PF showed a reduced number of square entries relative to LC animals at the medium dose [ F(2,33)=7.44, pb0.002]. In the high SKF 38393 dose group (Fig. 4D), sniffing was affected, with both ETOH and PF pups showing an increase in these behaviors relative to the LC controls [ F(2,33)=15.8, pb0.0001]. 3.3.3.2. D1 and D2 antagonists. As expected (see Ref. [89]), SPIP and SCH 23390 reduced 5 of 7 behaviors (square entries, rearing, grooming, sniffing and circling) to near-zero levels. The preponderance of zero scores for these behaviors limits the variance, and hence the size of the error term, violating basic assumptions of the ANOVA. These variables were, therefore, analyzed after conversion to a classificatory variable (behavior seen during observational session vs. not seen), as detailed in the Methods section. Only head movements and oral movements occurred at levels sufficient to justify a parametric profile analysis. SCH 23390. When results were pooled across dose and analyzed either by chi-square or by profile analysis, there were clear main effects of prenatal treatment. Starting with the profile analysis of the two parametric variables (oral and head movements), the main effect of Treatment [ F(2,99)=6.97, p=0.002], but not the Treatment
Dose interaction [ F(4,99)=0.98, p=0.714] were significant. For the two behaviors analyzed individually, there was a highly significant main effect of Treatments for oral movements

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Fig. 3. Behavioral effects of saline vehicle, dopamine D2 agonists or dopamine D3 antagonists by prenatal treatment. All six graphs show a profile of z-scores for seven different behaviors on the x-axis, and the seven behaviors on the y-axis, averaged across all three doses. Apomorphine (APO); quinpirole (QUIN); PD 152255, a novel D3 antagonist (PD); nafadotride, a second D3 antagonist (NAF); saline vehicle (SAL). Doses for each of the above compounds are shown in Table 2. Prenatal treatment groups did not differ with respect to any of the seven behaviors.

[ F(2,99)=9.45, pb0.0002]; ETOH offspring exhibited an increase in this behavior with respect to the PF and LC offspring, which did not differ (Fig. 5A). The Treatment
Dose interaction for oral movements was not significant [ F(4,99)=1.27, p=0.289]. There were no significant main effects or interactions for head movements

[ F(2,99)=3.64, p=0.03 for Treatment and F(4,99)=2.15, p=0.081 for the interaction; Fig. 5A]. For the five non-parametric behaviors (square entries, circling, grooming, rearing and sniffing), when all five were collapsed into a single chi-square analysis, there was a highly significant Treatment effect [v 2(2)=29.6, pb0.001],

Fig. 4. Behavioral effects of SKF38393, by dose and prenatal treatment. Behavioral profiles as in Fig. 3, by dose or pooled across doses. (A) Prenatal treatment effects pooled across dose. (B) Low dose of 0.01 mg/kg SKF 38393. (C) Medium dose of 0.10 mg/kg SKF 38393. (D) High dose of 10.0 mg/kg SKF 38393. Prenatal treatment groups for which the three prenatal treatment symbols have different-colored fills on any behavior differ significantly for that behavior.

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Table 5 Dose–response effects of prenatal ETOH and PF treatments Challenge drug Agonists SKF 38393 (all doses) SKF 38393 (low dose) SKF 38393 (medium dose) SKF 38393 (high dose)

Antagonists SCH 23390 (all doses) SCH 23390 (low dose) SCH 23390 (medium dose) SCH 23390 (high dose) Spiperone (all doses) Spiperone (low dose) Spiperone (medium dose) Spiperone (high dose)

Prenatal treatment

Squares

Circling

Grooming

Head movements

Oral movements

Rearing

Sniffing

Yawning

Receptor subtype

ETOH PF ETOH PF ETOH PF ETOH PF

D1

ETOH PF ETOH PF ETOH PF ETOH PF ETOH PF ETOH PF ETOH PF ETOH PF

D1

with 36% of LC controls and none of the ETOH or PF groups having more than two observations of the five pooled behaviors. When these data were collapsed across dose and analyzed individually by behavior, four of the five binary behavioral variables were significant (Fig. 5A), and

D1 D1 D1

D1 D1 D1 D1/D2 D2

D1

in all four of these cases, the LC control group differed from the PF and ETOH groups, which did not differ among themselves: Square entries [v 2(2)=39.2, pb0.001], with no PF subjects, one of 36 ETOH subjects, and 18 of 36 LC subjects entering at least one square; Circling [v 2(2)=20.4,

Fig. 5. Behavioral effects of SCH 23390, by dose and prenatal treatment. Behavioral profiles as in Fig. 3, by dose or pooled across doses. (A) Prenatal treatment effects pooled across dose. (B) Low dose of 0.01 mg/kg SCH 23390. (C) Medium dose of 0.10 mg/kg SCH 23390. (D) High dose of 3.0 mg/kg SCH 23390.

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pb0.00], with 0 and 2 subjects in the PF and ETOH groups, respectively, showing any circling, while 12 of 36 LC animals displayed circling behavior; Grooming [v 2(2)=13.1, pb0.001], with 4 and 3 of 36 PF and ETOH subjects, respectively, and 14 of 36 LC subjects showing some grooming; Sniffing [v 2(2)=11.4, pb0.003], with 7, 8 and 19 of 36 subjects showing some sniffing in the PF, ETOH and LC groups, respectively. There were no Treatment effects on rearing [v 2(2)=4.60, pb0.10]. With respect to individual doses (Fig. 5B–D; see also Table 5), the lowest dose of SCH 23390 (Fig. 5B) produced the least behavioral suppression and in consequence the greatest differentiation between treatments. Three of five non-parametric behaviors (square entries, circling and grooming) and both oral and head movements showed differences across prenatal treatments. For all non-parametric behaviors, v 2(2)=17.4, pb0.001, with 0, 0 and 7 out of 12 subjects in the ETOH, PF and LC groups, respectively, showing more than two incidents of the five behaviors: Square entries [v 2(2)=17.4, pb0.001], again with 0, 0 and 7 out of 12 subjects in the ETOH, PF and LC groups, respectively, showing square entries at least once; Circling [v 2(2)=17.4, pb0.001], with 1, 0 and 7 of 12 ETOH, PF and LC subjects, respectively, circling at least once; Grooming [v 2(2)=13.6, pb0.001], with 1, 1 and 8 of 12 ETOH, PF and LC subjects, respectively, grooming at least once. Neither sniffing [v 2(2)=9.0, p=0.01] nor rearing [v 2(2)=0.47, p=0.79] reached the requisite significance level of pb0.007. Parametric analyses of oral movements and head movements behaviors both produced highly significant Treatment effects [ F(2,33)=6.83, p=0.003 and F(2,33)=5.71, p=0.007, respectively]. For both behaviors, ETOH subjects displayed

significantly more behavior than either of the other two treatment groups, which did not differ among themselves. The medium SCH 23390 dose (Fig. 5C and Table 5) also differentiated between prenatal treatments. For three of the five non-parametric behaviors, v 2(2)=14.4, pb0.001 overall, with 0, 0 and 6 of 12 ETOH, PF and LC subjects, respectively, producing more than two incidences of any of the five behaviors: Squares [v 2(2)=20.6, pb0.001], with 0, 0 and 8 of 12 PF, ETOH and LC subjects, respectively, producing one or more square entries; Circling [v 2(2)=11.6, pb0.001], with 0, 0 and 5 of 12 ETOH, PF and LC subjects, respectively, circling at least once; Sniffing [v 2(2)=14.3, pb0.001], with 1, 2 and 9 of 12 PF, ETOH and LC subjects, respectively, sniffing at least once during the observation session. Significant Treatment effects were not obtained for either rearing [v 2(2)=6.55, pb0.04] or grooming [v 2(2)=5.3, pb0.07]. Parametric analyses of head movements [ F (2,33)=0.23, p=0.80], and oral movements [ F(2,33)=0.52, p=0.61] did not reached requisite levels of significance for treatment effects. The high dose of SCH 23390 (Fig. 5D) produced such severe behavioral inhibition that no Treatment effects were significant at requisite alpha levels. No animal in any treatment group produced more than two incidences of the five non-parametric behaviors. In addition, neither head movements [ F(2,33)=2.38, p=0.11], nor oral movements [ F(2,33)=3.24, p=0.052] differed among the prenatal treatment groups. Spiperone. Fig. 6A shows prenatal treatment effects collapsed across dose. With respect to the five nonparametric behavioral parameters collapsed across behaviors and dose (square entries, grooming, circling, rearing and

Fig. 6. Behavioral effects of spiperone, by dose and prenatal treatment. Behavioral profiles as in Fig. 3, by dose or pooled across doses. (A) Prenatal treatment effects pooled across dose. (B) Low dose of 0.0625 mg/kg Spiperone. (C) Medium dose of 0.5 mg/kg Spiperone. (D) High dose of 1.0 mg/kg Spiperone. Prenatal treatment groups for which the three prenatal treatment symbols have different-colored fills on any behavior differ significantly for that behavior.

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sniffing), or the profile analysis of the two parametric behaviors (head and oral movements), this drug clearly discriminated between prenatal treatment groups. For the parametric profile analysis, there was a highly significant main effect of Treatment [ F(2,93)=18.92, pb0.0001], but not a Treatment
Dose interaction [ F(4,93)=3.14, p=0.018]. Of the two parametric behaviors, only oral movements produced significant Treatment effects [ F(2,93)=13.91, pb0.0001] collapsed across Dose. Oral movements for each prenatal treatment group were significantly different from the other two, with ETOH pups showing the most and LC offspring the least (Fig. 6A). The Treatment
Dose interaction for this behavior was not significant [ F(4,93)=1.75, p=0.145]. Head movements did not distinguish between prenatal treatment groups [Treatment: F(2,93)=0.76, p=0.471; Treatment
Dose: F(4,93)=1.90, p=0.118]. For the five non-parametric behavioral comparisons pooled across behaviors and dose, there was a highly significant effect of Treatment [v 2(2)=30.9, pb0.00], with 7 of 36 PF animals, 7 of 30 ETOH subjects, and 28 of 36 LC controls showing more than two incidences of behaviors across all five behavioral categories. For individual nonparametric behaviors pooled across dose, all five behaviors provided significant Treatment effects at the conservative aV0.0073 level, and in all of these cases the ETOH and PF groups, which did not differ among themselves, showed less behavior that the LC group: Square entries [v 2(2)=31.4, pb0.001], with 14% of PF controls, 20% of ETOH animals and 72% of LC controls having at least one square entry; Circling [v 2(2)=22.2, pb0.001], with 19%, 20% and 67% of PF, ETOH and LC subjects, respectively, circling at least once; Grooming [v 2(2)=29.3, pb0.001], with 42%, 37% and 94% of PF, ETOH and LC subjects, respectively, grooming at least once; Rearing [v 2(2)=25.9, pb0.001] with 8%, 20% and 61% of PF, ETOH and LC subjects, respectively, rearing at least once; Sniffing [v 2(2)=12.4, pb0.002], with 44%, 53% and 83% of PF, ETOH and LC subjects, respectively, sniffing at least once. Turning to individual drug doses (Fig. 6B–D), and starting with the low dose (Fig. 6B and Table 5), the overall analysis of the Treatment effects for the five non-parametric behaviors was not significant at the required alpha level [v 2(2)=6.0, p=0.05], nor were any of these behaviors significant individually. In contrast, oral movements once again differed by treatment group, with both ETOH and PF subjects showing more drug-induced oral movements than the LC group [ F(2,31)=10.29, p=0.0004]. The medium dose of Spiperone produced substantive treatment effects (Fig. 6C and Table 5). Overall, the five non-parametric behaviors were diminished in the ETOH and PF groups in comparison to LC controls [v 2(2)=16.1, pb0.001], with 1 of 10 ETOH, 2 of 12 PF, and 10 of 12 LC controls showing more than two behavioral incidents. For individual non-parametric behaviors, all but circling showed the same general pattern: Square entries [v 2(2)=12.4, pb0.002], with 8%, 10% and 67% of PF,

85

ETOH and LC subjects, respectively, entering at least one square during the observation session; Circling [v 2(2)=8.02, pb0.018] did not attain the requisite alpha level; Grooming [v 2(2)=18.2, pb0.001], with 10%, 50% and 100% of ETOH, PF and LC subjects, respectively, grooming at least once; Rearing [v 2(2)=15.4, pb0.001], with 8%, 10% and 75% of PF, ETOH and LC subjects, respectively, rearing at least once; Sniffing [v 2(2)=16.9, pb0.001], with 20%, 33% and 100% of 33% ETOH, PF and LC subjects, respectively, sniffing at least once. This dose did not produce significant treatment effects on either head [ F(2,31)=0.86, p=0.435] or oral [ F(2,31)=3.84, p=0.032] movements. For the high Spiperone dose, there were significant Treatment effects over all pooled non-parametric behaviors, and for three of the five individual behaviors (Fig. 6D and Table 5). As with the medium dose, in all cases the ETOH and PF behavioral levels were lower than those in the LC group, and PF and ETOH groups did not differ. Overall for the five non-parametric behaviors, v 2(2)=12.91, pb0.002, with 1 of 10, 2 of 12, and 9 of 12 subjects producing at least two behaviors in the ETOH, PF and LC groups, respectively. Three of five individual non-parametric behaviors showed statistically significant Prenatal Treatment effects: Square entries [v 2(2)=12.9, pb0.001], with 1, 2 and 9 subjects in the ETOH, PF and LC groups, respectively, entering at least one square; Grooming [v 2(2)=18.6, pb0.001], with 20%, 25% and 100% of ETOH, PF and LC groups, respectively, grooming at least once; Rearing [v 2(2)=10.0, pb0.007], with 0%, 8% and 50% of ETOH, PF and LC subjects, respectively rearing at least once. Significant Treatment effects were not seen for either sniffing [v 2(2)=1.55, p=0.46] and circling [v 2(2)=8.5, pb0.014], or head movements [ F(2,31)=4.33, p=0.022] and oral movements [ F(2,31)=3.20, p=0.055].

4. Discussion The major finding of this study is that prenatal alcohol exposure selectively affects DA D1, but not the D2 or D3 receptor subtypes, resulting in an enhanced responsivity to D1 agonists and antagonists. While some behavioral responses to D1 antagonists were differentially impacted by prenatal ethanol exposure, increased sensitivity was evident in both liquid diet groups, with and without alcohol, and in both sexes, implicating the role of caloric restriction in these effects. 4.1. Maternal and litter data Maternal toxicity was seen in both ETOH and PF dams, as evidence by lower gestational weight gain in both groups. In agreement with the current finding, several previous studies report that dams in the ETOH and PF groups did not differ from each other with respect to this variable [17,6,27,62,119], although Becker et al. [10,12] found a

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further decrease in body weight in ethanol-exposed dams compared to pair-fed controls. However, reduced maternal weight gain was not sufficient to account for all ETOH effects. Prenatal ETOH alone increased pup mortality, reduced litter size, and exaggerated pup birth-weight disparity. While few report normal weights in both ETOH and PF offspring [19,83], reduced birth weights are more frequently seen in ETOH exposed and/or PF pups in comparison to LC offspring [61,119], and, as reported here, ETOH pups are also lighter than their pair-fed counterparts [10,17,62,102]. Although ETOH offspring continue to show growth deficits throughout the preweaning and periadolescent period, this deficiency does not usually persist in PF pups [17,19,29,83]. However, Becker et al. [10] reported that at PND 22 ETOH and PF offspring still had reduced body weights compared to LC offspring, a finding which coincides with data obtained here. Maternal blood levels, measured at the midpoint of gestational ETOH exposure, were 139 mg/dl. This concentration is comparable to levels in the literature using a variety of exposure methods. Typically, a liquid diet providing 35% ethanol-derived calories produces peak blood alcohol concentrations of 130–180 mg/dl [61], although BALs of 99.4 mg/dl and 108.9 mg/dl have been reported early in gestation, i.e., GD 5 and 10, respectively [81]. Chronic liquid diets of 6.6% (v/v) ethanol resulted in BAL concentrations of 75–120 mg/dl [53], while10% alcohol in drinking water produced BAL of 143 mg% in pregnant rats [22]. These values are comparable to BALs in humans that are associated with cognitive and sensory impairment, and have been linked to neurobehavioral effects seen after gestational alcohol exposure [36]. While acute and/or gavage methods produce a range of BALs, peak concentrations are generally higher (~260–340 mg/dl) than those reported with constant availability [2,27,75,105,127]. However, both chronic and acute exposure results in nearperfect correlations between dam and fetal blood alcohol concentrations [78]. 4.2. Sex differences The present study did not find any sex differences in the behavioral responses to receptor subtype-specific DA agonists and antagonists of offspring following gestational ethanol exposure. While the effects of prenatal exposure to ethanol have been predominantly studied in male offspring, when males and females have been used, finding of sex differences are inconsistent. However, the absence of sex differences reported here does parallel previous findings in young mice and rats, and adolescent rats. In mice, no gender differences were found with respect to striatal DA concentration and turnover, as well as number of DA D1 receptors and DA D1 and D2 receptor binding at PND 21 and 28 [19]. In 10-day-old ethanol-exposed rats, both sexes show a cocaine-induced decrease in DA content in the nucleus accumbens [27]. Moreover, in 9- to 10-week-old rats, sex of

the offspring did not influence the effect of prenatal ETOH on tyrosine hydroxylase, adenylate cyclase, or D1 or D2 binding in olfactory tubercles [57]. However, there is a report that gestational exposure to ethanol elevated DA levels in nucleus accumbens and striatum of males, but diminished DA release in the striatum of females at PND 90–120 in response to an ethanol challenge [18], suggesting that sexually dimorphic changes in neurochemical parameters may occur only in fully mature rodents. In contrast, there are literature reports of sex differences in prenatal ETOH effects on responses to several dopaminergic drugs in rats, and to ethanol challenge in both mice and rats. In rat, gestational ethanol has been reported to (1) enhance sensitivity to the locomotor stimulating effects of methlyphenidiate and amphetamine in 4-weekold males, while decreasing behavioral responsiveness to the latter drug in females [17]; (2) differentially alter the duration of the cataleptic response to the D1 agonist, SCH 23390, in males and females at PND 21, and delay the emergence of a mature response in males but not females [61]; and (3) shift the dose–response curve for APOinduced motor activity to the left in males but not females tested at PND 28 [61]. In mice, sex differences in locomotor activity following challenge doses of ETOH are both dose and age dependent. Male offspring prenatally exposed to ETOH exhibited reduced baseline activity and a blunted stimulant response to all challenge doses of ETOH at 30 but not 70 days of age [10]. In contrast, females exhibited similar responses to males following challenge with only the lowest dose of ethanol at PND 30, but the effects persisted into adulthood. Moreover, young and adult females in all treatment groups exhibited a similar stimulant response to higher doses of ethanol [8]. It should be noted that alcohol-induced sexually dimorphic changes in reaction to drug-challenge generally occurred in animals at least 4 weeks of age, and the one exception to the current findings may reflect strain differences. It would, therefore, appear, that in addition to its impact on sex differences, age at testing per se may account for the reported differences among studies. Because males are in general more affected than females on some measures of fetal alcohol effects, sex differences have been ascribed to alcohol-induced reductions in testosterone levels during neural development and decreased testicular steroidogenesis [15,61]. Male/female differences have also been attributed to differential susceptibility to, or recovery from, fetal alcohol effects, with females being either less affected or recovering earlier than males [17,67]. Both normal gender differences in the biobehavioral maturation of central DA functioning, with systems developing more slowly in males compared to females, as well as alcohol-induced shifts in this ontogenetic pattern have also been proposed as possible mechanisms [17,19]. However, none of these theories can account for all of the observed effects, and a more parsimonious explanation must await a reconciliation of the discrepancy between the neurochemical

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and behavioral findings with respect to sex differences in prenatal alcohol effects. 4.3. Dopamine receptor subtypes Ligand binding studies indicate that D1 and D2 receptors are present early in development. D1 and D2 receptor gene expression can be detected as early as gestational days 17 and 14, respectively [58,59], and are coupled to guanine nucleotides early in development [104]. Furthermore, D1 and D2 receptor subtypes are functional during the late prenatal period. D1 receptor activation induces an increase in activity, as well as forelimb, rear limb, and head movements in the 21-day-old rat fetus, while fetal rats exhibit a general suppression in movements following administration of a D2 agonist. Administration of a D1 antagonist results in substantial increases in mouthing movements, whereas a D2 antagonist has little or no observable effect on fetal behavior [86]. Given that DA receptor subtypes are present and functional during the prenatal period, and that functional activity in neural systems is an important determinant of their maturation, fetal exposure to interventions which alter the balance of receptor concentrations may induce abnormal changes in the synaptic environment that impact the functional response to postnatal challenges [60,90].

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cumulative and oral stereotypies to a 1.0 mg/kg dose of APO, indicative of enhanced sensitivity at PND 90. Despite a lack of consensus, all three studies show that DA receptor sensitivity to APO is altered following chronic or acute prenatal ETOH exposure. While opposite results may reflect species, drug dose, and gender differences of the offspring, these factors are unlikely to account for differences between the current report and Hannigan [61]. One possible explanation for these differences may be agedependent ETOH effects on D2 receptor density. There is some suggestion that ETOH-induced changes in D 2 receptors occurs later in postnatal life, with decreases in density reported at PND 63 and 6 months of age [76,95], although there are reports to the contrary [19]. In contrast, no changes in density have been reported at PND 1, 4 or 10 [74], or at 21 [16] and 35 days of age [38,40]. Reports of changes in D2 receptor number are also inconsistent. While ethanol-exposed mice show no changes in D2 receptor number at PND 28, rats exhibit a reduction in D2 striatal receptors at PND 30 but not earlier [89]. While this age effect might account for the lack of ETOH induced effect on the D2 receptor sensitivity reported here, it should be noted that this one change in D2 receptor binding does not satisfactorily explain the bidirectional alterations in sensitivity outlined in the literature above. 4.5. D3 receptor subtype

4.4. D2 receptor subtype No prenatal treatment effect was observed for any of the three dopamine D2 receptor agonists, apomorphine, quinpirole and 7-OH DPAT (see Fig. 3), indicative of a strong internal consistency. These findings are in contrast to several other reports, which themselves are contradictory. Two laboratories report dose-dependent, albeit opposite, changes in receptor sensitivity to apomorophine following chronic prenatal exposure to ETOH. Becker et al. [11,12] reported that daily prenatal exposure of mice to ETOH on GD 6–18 produced a dose–response alteration in drug sensitivity. While APO dose dependently decreased activity and blocked the stimulant effects of ethanol at all doses tested in control offspring at PND 90, male ETOH offspring showed less reduction in baseline and ethanol-stimulated activity at a high dose of APO, but enhanced sensitivity to the locomotor suppressant effects of low doses of the drug. Hannigan et al. [62] challenged male and female rats exposed to ETOH on GD 6–18 with apomorphine at PND 28. Increased sensitivity to APO was again specific to the ETOH males, as low doses of the drug induced greater locomotor activity than in controls. However, at high doses both sexes of ETOH-treated offspring showed a reduction in drug-stimulated activity in automated monitors in comparison to controls, but were more sensitive to the induction of stereotypies. Acute exposure to prenatal ETOH also alters receptor sensitivity. Male rats, exposed prenatally to two injections of ETOH on GD 8 [48], exhibited increased

There was no prenatal ETOH effect on the behavioral response to either D3 agonists, i.e., low dose quinpirole and 7-OH-DPAT, or antagonists (see Fig. 3). Research in this area has been minimal, in that only one other paper has examined the behavioral effects of D3 agonists following preweaning/prenatal ETOH exposure [22]. Yawning, a D3mediated behavior [33] was markedly impaired in 3-monthold ETOH-exposed male offspring challenged with low doses of quinpirole. Differences between these and the current study may reflect age of testing. D3 agonist-induced yawning is infrequently seen in rats at 3 weeks of age, a deficit which has been attributed to the immaturity of the presynaptic D3 receptor [110]. 4.6. D1 receptor subtype Stimulation of the D1 receptor characteristically results in enhanced sniffing and grooming [54,110], along with a slight increase in locomotor activity [54,82]. Prenatal treatments significantly altered one of these characteristic behavioral responses to the D1 agonist, SKF 38393 (see Fig. 4). Sniffing was enhanced in both liquid diet groups by high dose SKF 38393 (10 mg/kg) and overall (i.e., averaged all three doses), suggestive of augmented receptor sensitivity. This augmentation was also seen with oral movements, in that pair-fed offspring alone showed greater overall agonistinduced increase in this behavior. This finding is in agreement with Brus et al. [22], who also reported no

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change in SKF 38393-induced oral movements in ETOH treated male rats relative to lab chow controls. In contrast, in the current study no prenatal treatment effects on grooming were observed following challenge with this drug. The classic D1 antagonist, SCH 23390, induces catelepsy and a broad inhibition of behaviors [9], with the exception of oral movements [110]. Substantial prenatal treatment effects were seen for both ETOH and PF offspring, in that inhibition of activity, circling, grooming and sniffing was enhanced overall, and by low and medium but not high drug doses, in these two groups in comparison to LC controls. Additionally, oral movements, the sole behavior not suppressed by this drug, were increased only in prenatal ETOH offspring. This profile of effects again suggests that ETOH offspring, and in most cases PF pups as well, were more drug sensitive than controls. A dose- and gender-specific effect of prenatal treatment on SCH 23390-induced catalepsy was reported for PND 21 ETOH offspring [61]. In agreement with present results, ETOH males, challenged with a 1.0 mg/kg dose, exhibited increased catalepsy in comparison to PF and LC controls. However, at a lower dose (0.33 mg/kg, virtually identical to our high dose of 0.3 mg/kg), the cataleptic behavior was decreased in this group. In contrast, ETOH females exhibited an increased catalepsy at the high dose with respect to both control groups, while the lower dose was ineffective in altering this behavior. Where the two studies are most comparable (i.e., same two control groups, same age at drug challenge, and same SCH23390 dose, in both sexes), results show the greatest differences. While we find no sex differences in drug response, an increased responsivity to drug challenge at 0.01 and 0.1 mg/kg but not at 0.3 mg/kg, and a difference between the two control groups, Hannigan [61] reports sex differences, a decreased responsivity to the 0.33 mg/kg SCH 23390 dose in males but not in females, and no differences between LC and PF controls. We are unable to account for these differences, beyond noting the extreme complexity of the latter results. Prenatal treatment effects seen with SPIP, a mixed D1/D2 receptor antagonist, are similar to those of SCH 23390, in that the behaviors which are either increased or decreased following challenge with SCH were further altered in both liquid diet groups by SPIP. Drug challenge resulted in an increased inhibition of activity, circling, grooming, sniffing, as well as rearing, both overall and for medium and high but not low drug doses in ETOH and PF pups in comparison to LC controls. Again, oral movements were the only measured behavior which showed a drug-induced increase. Averaging over doses, this increase was higher in the ETOH group than in the PF group, and higher in the PF group than in LC controls. These effects were generally more pronounced at the two higher doses, which are thought to preferentially affect the D1 rather than the D2 receptor subtype. It is also likely that the enhanced oral activity shown especially at the two lower doses of SPIP by ETOH and PF pups is a D1-

mediated behavior. The literature suggests that ventral striatal control of oral movements involves a D1/D2 antagonism, such that D2 antagonists release D1-mediated oral movements [24,71–73,87,98,99,100] via an unusual phosphatidylinositol/non-cAMP linked D 1 receptor [3,32,64,98,101,118]. Although SPIP is a potent D2 antagonist, it can uncover a D1 mediated effect. Thus, the SPIP effects reported here seem more likely to be mediated via treatment-induced alterations in the dopamine D1 system. Our findings of enhanced behavioral drug sensitivity to D1 agonist and antagonists are supported by findings of increased binding activity of D1 receptors. These ETOHinduced changes in the D1 receptors occur early in postnatal life and may be transient in nature. A moderate increase in D1 receptor binding in the striatum of rats has been reported at PND 19 following in utero ETOH exposure [53], while similar increases in binding coefficients of striatal D1 receptors were found at PND 21 and 28 but not PND 63 in mice [19], or PND 90 in rats [95]. In contrast, the number of D1 receptors were reported to be decreased in the striatum of ETOH pups at PND 19 and 35 [38], while DA-stimulated adenylate cyclase in this structure was normal at PND 19 [40]. However, these findings may not be contradictory, in that binding may be altered independently of changes in receptor number, or increases in this parameter may represent a compensatory mechanism [53,127]. The present results indicate that the DA D1 receptor is preferentially targeted by our experimental manipulations. This receptor appears to have a special role in both prenatal and postnatal development. During the embryonic period, the D1 receptor is thought to regulate the morphogenic or neurotrophic effects of dopamine on neuronal development and differentiation [38], and appears to be also responsible for the interdependence of the serotonin and dopamine systems during development [126]. Stimulation of this receptor subtype has been shown to inhibit neuronal growth cone motility in retinal cells [75], and to alter the development of 5-HT fibers innervating the hippocampus and superior colliculus [103]. Postnatally, the density of D1 receptor sites in rat brain increases 14-fold from birth to a maximum at 35 day of age [54]. However, synthesis-modulating D1 receptors appear to arise and function transiently in some regions of the CNS between postnatal days 15 and 22, but cease to function by PND 60, suggesting that the early postnatal surge in D1 receptor density might include some D1 autoreceptor, followed by selective loss of this class of autoreceptor with further maturation [116]. The failure of these receptor types to arise and recede at the proper time may account for the neurobehavioral alterations and/or developmental delays reported following gestational exposure to ethanol. 4.7. Pair feeding Altered receptor sensitivity following drug challenge was seen in the present study in both liquid diet groups, with and

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without alcohol. Similarities between offspring of prenatal ethanol and pair-fed dams have been previously reported. ETOH and PF offspring exhibit similar baseline activity [10], as well as comparable locomotor responses following challenge with apomorphine, either alone [62] or in combination with ethanol [11,12], methylphenidate [120], amphetamine [17], or ethanol [10]. Neurochemically, ETOH and PF pups exhibit changes in DA turnover [29], as well as in levels of DA [34], tyrosine [35] and DOPAC [78] that differ significantly from LC offspring but not from each other. However, other studies indicate that PF offspring do not differ from LC controls with respect to locomotor activity following methylphenidate challenge [81], or nicotine-induced rearing [88]. As primary and secondary (i.e., intestinal malabsorption) malnutrition has been associated with ethanol intake in rats [92], pair-fed groups are often included in the research design as a control for caloric restriction. However, caloric restriction, per se, may decrease the availability of essential nutrients, trophic factors and antioxidants to the fetal tissue, which in turn alter brain development, resulting in behavioral dysfunction [60]. This raises the possibility that the dopamine D1 drug supersensitivity seen in the two growth-stunted groups, i.e., ETHO and PF, in this study is directly related to gestational nutritional deprivation or subsequent catch-up growth. There is suggestive evidence that this might be the case. The greatest treatment effects produced by the D1 agonists (SKF 38393) and antagonists (SCH 23390 and spiperone) were on oral movements, behaviors possibly linked to neural control of appetite. Furthermore, growth stunting was more severe for the ETOH than the PF group, and following challenge with SCH 23390 and spiperone, the more deprived ETOH group displayed significantly higher drug-induced oral movements than did the PF group. Consonant with the possibility that these abnormalities in drug-induced oral behaviors are linked to concurrent catchup growth (and probable concomitant alterations in appetite) is the finding that in mice D1 receptor density was reduced at PNDs 21 and 28 but not PND 56 [19]; at this later age (PND 56) catch-up growth velocity will have slowed considerably, and this slowing would presumably be reflected in a reduction in dopamine alterations. Finally, a link between fetal growth stunting and appetite is suggested by the finding that fetal growth restriction in humans is known to predispose offspring to the metabolic syndrome later in life, a syndrome that sometimes includes enhanced obesity [23,31,65]. Whether or not the ETOH and PF abnormalities found here are a consequence of gestational nutritional deprivation, it is important to ask why gestational growth stunting should impact the developing dopamine system. Such an effect is not implausible if it reflects alterations in appetite, since the dopamine system is well known to mediate appetite. Either neurochemical lesion of the dopamine system or dopamine knockout in mice produces pronounced

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hypophagia [50,112], while food-deprived rats and humans release dopamine on refeeding or even on the sight of palatable foodstuffs [109,125,128,123,124]. Drug studies of specific receptor mechanisms in the dopamine mediation of appetite are difficult to interpret, given the profound suppressive effects of dopamine antagonists on all behaviors. However, some studies have suggested an important role for either the dopamine D1 [117] or D3 receptor complexes [13]. That all of the above dopamine mechanisms may be appetite-specific is also supported by recent reports of insulin and leptin receptors in the mesencephalic sources of dopamine innervation of the forebrain [46]. If the dopamine system is responsive to gestational undernutrition, it would seem likely that such effects would have been reported in studies of gestational malnutrition and the brain. Unfortunately, this is not the case. The scientific literature on the effects of gestational undernutrition on the dopamine system is extremely scanty, and the few published reports are decades old [107]. Further complicating matters, these few studies have typically looked at only one form of malnutrition, specifically protein malnutrition. These studies thus may not be strictly comparable to the undernutrition produced by prenatal ethanol or pair feeding. In this latter case the diet is carefully balanced, so that any deprivation will involve not just proteins but all caloric sources. Furthermore, some of these studies combined prenatal and postnatal malnutrition, and it is unclear whether PF or ETOH dams in this study were producing inadequate quantities of milk postnatally, given that they were placed on ad lib diets from GD 21. Several reports have looked at some form of pre- and/or perinatal protein deprivation and its effects on the dopamine system. Combined prenatal and postnatal, or only postnatal protein deprivation reduced whole-brain dopamine content around PND 21 [94,107]. However, a more careful regional analysis of dopamine content following only prenatal protein deprivation resulted in contradictory findings. While Chen et al. [26] found no effect on dopamine content of brainstem, hippocampus, cortex, or caudate nucleus at any age, from PND 1 onward, Kehoe et al. [68] reported that prenatal protein malnutrition reduced dopamine content in hippocampus but not hypothalamus on PND 9. However, Chen et al. [26] also looked at dopamine release from hippocampal slices from rats from PND 15 through PND 220, and found dopamine release enhanced by prenatal protein malnutrition. Finally, we could find no reports which assessed the impact of prenatal undernutrition on dopamine receptors, and only two which looked at drug challenges similar to those used here. In these papers, rats were undernourished from birth through PND 21, at which age they were challenged with either SCH 23390, chlorpromazine or haloperidol. Drug effects on catalepsy and activity were assessed for some hours, but no nutritional effects were seen over the time period used in this study [97,114]. In conclusion, then, it is plausible but by no means certain that the relatively mild prenatal undernutrition and conse-

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quent growth stunting induced by prenatal ETOH or pair feeding could be accompanied by the sort of early postnatal changes in D1 receptor drug sensitivity reported here. In summary, prenatal ethanol exposure enhanced the functional drug sensitivity of the dopamine D1 receptor to both agonists and antagonists in male and female offspring at 21 days of age. Restricting the caloric intake of pregnant rats, without ethanol exposure, also resulted in a selective increase in D1 drug sensitivity in offspring of both sexes. These findings suggest that prenatal ethanol exposure and/or undernutrition can preferentially alter the development of dopamine receptor subtypes. This does not preclude the possibility that the D2 and D3 receptors, which may not be fully functional at 3 weeks of age [110], could show altered sensitivity following drug challenge in older animals.

Acknowledgments This work was supported by NIAAA/NIH grant number U24AA11898 (to S.K.S.).

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