Prenatal phenytoin exposure and spatial navigation in offspring: effects on reference and working memory and on discrimination learning.

Previous research has shown that rats exposed to phenytoin (PHT) in utero demonstrate abnormal circling, decreased learning, hyperactivity, and delayed air righting reflex development. The effects of prenatal PHT on offspring learning have been found on multiple-T mazes (Biel and Cincinnati types) and on spatial navigation (Morris maze). However, the specificity of the latter effects is unknown. Herein, we tested the effects of prenatal PHT in a Morris maze using six different procedures: cued versus spatial reference memory-based learning, cued versus spatial working memory-based learning, and cued versus spatial discrimination learning. PHT-exposed offspring showed increased preweaning mortality, growth reduction, and abnormal circling as noted in previous studies. PHT offspring were separated into those exhibiting circling and those not. PHT noncircling offspring demonstrated impaired reference memory-based spatial learning (acquisition and reversal), but no other effects. By contrast, PHT circling offspring demonstrated not only impaired reference memory-based spatial learning, but also impaired cued platform learning, impaired spatial discrimination, and impaired working memory-based learning. These data confirm that prenatal PHT induces a specific reference memory-based spatial learning deficit even in asymptomatic (noncircling) offspring that is distinct from the impairment induced in littermates exhibiting the circling impairment.

Genetic differences in spatial learning between Dark Agouti and Sprague-Dawley strains: possible correlation with the CYP2D2 polymorphism in rats treated neonatally with methamphetamine.

Following neonatal exposure to d-methamphetamine, adult rats have previously been shown to exhibit augmented acoustic startle and spatial learning deficits. d-Methamphetamine is structurally similar to several phenylethylamines that are metabolized by CYP2D6. In humans, allelic differences in the CYP2D6 confer the extensive or poor metabolizer phenotype for the more than three dozen drugs that are members of the CYP2D6-mediated 'debrisoquine/sparteine panel.' An analogous genotype exists with the CYP2D2 gene in rats. Female Dark Agouti rats show the poor metabolizer phenotype, whereas Sprague-Dawley rats show the extensive metabolizer phenotype; male Dark Agouti rats are intermediate. We sought to test the possibility that these strains might exhibit altered d-methamphetamine-induced developmental neurotoxicity. Dark Agouti and Sprague-Dawley litters (11-20 days of age) were given d-methamphetamine or vehicle alone subcutaneously twice daily (15 mg/kg). Offspring were assessed as adults (beginning at 50 days of age) on acoustic startle, straight-channel swimming, and spatial learning and memory in a Morris hidden platform maze. Increases in d-methamphetamine-induced acoustic startle were found in both male and female Dark Agouti rats, but not Sprague-Dawley rats. In the Morris maze, d-methamphetamine-induced spatial navigation deficits were found in both strains among males, suggesting some mechanism other than the CYP2D2 polymorphism. In contrast, among females only the d-methamphetamine-treated Dark Agouti rats showed deficits in spatial navigation. The maze deficits in Dark Agouti females, and enhanced acoustic startle in Dark Agouti females and males, support the hypothesis that the CYP2D2 poor metabolizer phenotype confers increased vulnerability to d-methamphetamine-induced developmental neurotoxicity, indicating that the parent drug rather than a CYP2D2-mediated metabolite is responsible for this behavioural defect--which occurs in adults who had been exposed to d-methamphetamine during the neonatal period.

CYP2D1 polymorphism in methamphetamine-treated rats: genetic differences in neonatal mortality and effects on spatial learning and acoustic startle.

d-Methamphetamine (MA) is one of more than two dozen drugs included in the cytochrome P450-mediated "debrisoquine oxidation polymorphism" panel. The human gene (CYP2D6) is responsible for the "poor metabolizer" (PM) and "extensive metabolizer" (EM) phenotypes for drugs such as MA; a similar polymorphism (the CYP2D1 gene) exists in rats. Female Black or Dark Agouti rats exhibit the PM phenotype, whereas Sprague-Dawley (SD) rats show the EM trait. We sought to test the possibility that these strains of rats might exhibit altered MA-induced developmental neurotoxicity. Neonatal exposure to MA on days 11-20 has previously been shown to induce spatial learning deficits in Sprague-Dawley rats when tested as adults. Therefore, in the present experiment, on postpartum days 11 through 20, ACI (Black Agouti) and SD progeny were administered 30 mg/kg MA twice daily. MA treatment caused larger increases in mortality in ACI than in SD rats, suggesting that decreased MA metabolism leads to enhanced toxicity and lethality. Female offspring were assessed behaviorally as adults. No differences were observed in acoustic startle or straight swimming channel performance. In the Morris maze, both MA-treated rat strains showed longer latencies to find the hidden platform during acquisition, reinstatement, and shift trials, and spent less time in the target quadrant on probe trials; no strain differences in learning were found. Although these data do not support our hypothesis that MA-induced developmental neurotoxicity might be enhanced in the ACI rat, this interpretation is tempered by the high mortality rate (65%) of MA-treated ACI neonates, suggesting a possible "survivor effect" in this strain.

Behavioral effects of prenatal exposure to pulsed-wave ultrasound in unanesthetized rats.

The present experiment examined the developmental neurotoxicity of pulsed-wave (pw) ultrasound in rats, using an exposure system designed to eliminate restraint or anesthesia from the exposure conditions. Pregnant Sprague-Dawley CD rats trained to remain immobile in a water-filled ultrasound exposure tank were scanned with 3-MHz pw ultrasound at spatial peak temporal average intensities (ISPTA) of 0, 2, 20, or 30 W/cm2 on embryonic days 4-20 for approximately 10 min/day. The data showed that such insonation produced no adverse effects on maternal weight gain or reproductive outcome, nor on the postnatal growth or survival of the offspring. No exposure-related alterations in behavioral development were observed in the offspring of rats scanned with pw ultrasound during gestation. In addition, there was no consistent evidence of an ultrasound-associated change in the adult offspring behaviors tested; i.e., no treatment effects were found on measures of locomotor activity, water maze learning, and acoustic startle reactivity. An effect on tactile startle was observed on some trials in the low exposure group male offspring, but this effect was neither dose dependent nor consistent with any other finding. Overall, these results indicate that the neurobehavioral development of rats was not altered by prenatal exposure to pw ultrasound at ISPTA levels of up to 30 W/cm2.

Stage-specific effects of prenatal d-methamphetamine exposure on behavioral and eye development in rats.

Pregnant Sprague-Dawley rats were treated with 0, 5, 10, 15, or 20 mg/kg d-methamphetamine (MA), expressed as the free base, by SC injection (b.i.d., 8 h apart) on days 7-12 or 13-18 of gestation. Plasma concentration of MA and amphetamine were determined after the last dose. MA reduced gestation weight gain. The late exposure resulted in an increase in maternal and offspring mortality and reduced offspring growth. Offspring treated early in gestation with MA showed delayed development of early locomotion. In addition, memory impairment, evidenced by decreased target quadrant times and platform crossings on test trials and increased latency on reversal trials in the Morris spatial navigation maze, reduced spontaneous alternation, and lengthened passive avoidance retention latency was seen in the early treated high-dose groups. A reduction of serotonin was found in the nucleus accumbens following late exposure to MA at 20 mg/kg. Animals in both exposure groups had eye defects; however, the type of defect was dependent on the developmental stage at the time of dosing. Anophthalmia occurred only after early MA exposure, whereas folded retina was drug related only after late MA exposure. The behavioral effects did not show graded dose dependency; however, the effects were sensitive to exposure period. The early exposed animals had more alterations in behavior whereas the late exposed group showed higher mortality, reduced body weights, and neurochemical alterations.

Prenatal exposure to sodium phenytoin in rats induces complex maze learning deficits comparable to those induced by exposure to phenytoin acid at half the dose.

Gravid Sprague-Dawley CD (VAF) rats were administered sodium phenytoin suspended in corn oil by gavage once per day on embryonic days 7-18 at a dose of 100 mg/kg. Controls were administered corn oil alone by gavage on E7-18. Litters were randomly culled to 10. Offspring were regularly weighted, mortality noted, and males checked for preputial separation. At approximately 50 days of age offspring were evaluated in a straight water-filled channel for swimming proficiency and motivation to escape. Following this, rats were tested in the Cincinnati multiple T-water maze and scored for errors, latency to find the goal, and presence of phenytoin-induced abnormal circling behavior while swimming. Sodium phenytoin-exposed dams gained weight normally and delivered normally. Offspring mortality in the sodium phenytoin group was not increased above controls. No treatment effects on preputial separation or offspring growth were observed. No differences between groups in swimming proficiency in straight channel performance were obtained. In the Cincinnati maze, phenytoin offspring committed significantly more errors and had longer latencies to find the goal than controls. Among the phenytoin offspring, those exhibiting abnormal circling committed more errors than noncircling animals. When compared to previous data using the same maze and test protocol, it was found that 100 mg/kg of sodium phenytoin induced performance deficits similar to those induced by a dose of 200 mg/kg of phenytoin acid. Accordingly, the present data help explain why other investigators have reported sodium phenytoin to be more developmentally neurotoxic than phenytoin acid. Because the prenatal neurotoxic effects seen with the salt of phenytoin occur at lower doses, it suggests that phenytoin is more developmentally neurotoxic than previously believed.

Long-term learning deficits and changes in unlearned behaviors following in utero exposure to multiple daily doses of cocaine during different exposure periods and maternal plasma cocaine concentrations.

Although the possible behavioral neurotoxic effects of in utero exposure to cocaine have been the subject of numerous experiments, only a limited number of different types of animal models of cocaine exposure, critical periods, or long-term effects of such exposures have been investigated. In the present experiment, the effects of multiple daily SC exposures to cocaine (20 mg/kg/dose x 5 doses per day) were investigated when administered to gravid Sprague-Dawley CD rats on embryonic days E7-12 or E13-18 compared to weight-matched, vehicle injected, pair-fed controls. Effects of exposure were assessed on general development, olfactory orientation behavior, early locomotion, startle reactivity, spontaneous motor activity, and learning on two different tasks (Morris and Cincinnati water mazes). The multiple cocaine dosing regimen produced maternal peak serum concentrations of cocaine 3 times higher than that of a single dose (approximately 1550 vs. approximately 550 ng/mL). Early-exposed cocaine offspring had lower olfactory orientation scores and reduced postweaning rearing and hole-poke motor activity, whereas late-exposed cocaine offspring had increased postweaning locomotor, rearing, and hole-poke activity. On the Morris hidden platform maze, the cocaine early-exposed females had longer latencies on acquisition than controls. On the Cincinnati multiple-T water maze, the early-exposed cocaine females and the late-exposed cocaine males had increased errors, whereas the early-exposed cocaine males had reduced errors. The effects on measures of learning, when taken together, and in light of their being in the early-exposed group, suggest that embryonic cocaine exposure may have subtle effects on cognition in the offspring as adults. Such effects represent a form of neurotoxicity not previously associated with prenatal cocaine exposure.

Behavioral teratologic effects of prenatal exposure to continuous-wave ultrasound in unanesthetized rats.

While there are no known risks associated with diagnostic ultrasound, uncertainty about the safety of prenatal ultrasound exposure remains. The purpose of the present experiment was to evaluate the behavioral teratogenic potential of continuous-wave (cw) ultrasound in rats, in the absence of maternal anesthesia or restraint. Pregnant CD rats, trained to remain immobile in a water-filled ultrasound exposure tank, were scanned with 3 MHz cw ultrasound at levels of 0, 2, 10, 20, or 30 W/cm2 ISPTA (spatial peak, temporal average intensity) on gestational days 4-20 for approximately 10 min/day. Offspring were examined postnatally for survival, growth, physical landmarks of development, behavioral development, and the adult functions of locomotor activity, learning and memory, and startle reactivity. No effects of prenatal ultrasound were found on maternal characteristics, offspring survival or growth, physical or behavioral landmarks of development, or adult tests of passive avoidance or startle. Effects at the highest intensity were obtained on corner and side locomotor activity and in a multiple-T water maze on measures of errors of commission and time spent finding the goal. The results showed that prenatal cw ultrasound in rats can induce effects on some postnatal neurobehavioral functions at high exposure intensities (30 W/cm2), but at lower intensities (2-20 W/cm2) no consistent evidence of neurobehavioral effects was observed.

A developmental neurotoxicity evaluation of the effects of prenatal exposure to fluoxetine in rats.

Fluoxetine is a widely used serotonin reuptake inhibitor effective in the treatment of depression. This experiment assessed the potential developmental neurotoxicity of fluoxetine. Sprague-Dawley CD rats were treated once per day on Days 7-20 of gestation with 0, 1, 5, or 12 mg/kg of fluoxetine (free base) dissolved in distilled water. One control group received water by gavage; animals in this group were provided food and water ad libitum. The second control group (PF) also received water by gavage; animals in this group had their food and water restricted by pair-feeding and watering them to the 12 mg/kg fluoxetine group. Litters were culled to 12 after birth and offspring (male/female pairs) were tested neurobehaviorally at three developmental stages (preweaning, juvenile, and adult). At each stage, two pairs per litter received tests of locomotor activity, acoustic startle, and startle after administration of one of two pharmacological challenges (one pair each receiving fluoxetine or apomorphine). Two pairs were also tested for spontaneous alternation, passive avoidance, and complex learning in a water maze. At the highest dose, fluoxetine caused maternal weight loss during pregnancy, reduced litter sizes at birth, and increased neonatal mortality. No effects on long-term growth or survival were seen. Prenatal fluoxetine exposure produced no significant effects on locomotor activity, spontaneous alternation, passive avoidance, or water maze performance. A few scattered interactions involving treatment group were obtained on startle, but no pattern of treatment-related changes was evident. Regional wet and dry brain weights taken at each stage were not affected by prenatal fluoxetine exposure. The data suggest that fluoxetine is not developmentally neurotoxic in the rat.

Trans-2-ene-valproic acid is less behaviorally teratogenic than an equivalent dose of valproic acid in rats.

Although animal experiments have shown the trans-2-ene metabolite (t-2-ene-VPA) of valproic acid (VPA) to be pharmacologically equivalent to the parent compound in terms of anticonvulsant activity, it is considerably less teratogenic in studies which have examined prenatally exposed fetuses for morphological defects. This has made t-2-ene-VPA an attractive potential antiepileptic agent. However, while neurobehavioral alterations have also been observed in rats prenatally exposed to VPA, even at doses below those which produce malformations, the developmental neurotoxicity of t-2-ene-VPA had not previously been examined. The current study evaluated the long-term behavioral effects of prenatal exposure to t-2-ene-VPA. Pregnant CD rats were treated with 300 or 400 mg/kg t-2-ene-VPA by gavage on days 7-18 of gestation, doses previously shown to produce no teratogenicity. A VPA group was administered 300 mg/kg for comparison. The pharmacokinetic profiles of the two compounds were similar. Behavioral findings in offspring prenatally exposed to VPA were consistent with previous findings, i.e., VPA offspring exhibited decreased locomotor activity, increased swimming maze errors, and reduced tactile startle responding compared to controls. In the 400 mg/kg t-2-ene-VPA group, Cincinnati maze errors and auditory startle reactivity were increased, while no significant behavioral effects were detected in the 300 mg/kg t-2-ene group. These results indicate that the developmental neurotoxicity of t-2-ene-VPA is lower than that of VPA but is still significant.

Methamphetamine exposure during early postnatal development in rats: I. Acoustic startle augmentation and spatial learning deficits.

Methamphetamine (MA) induces neurotransmitter reductions and neurotoxicity at high doses in adult animals, but its effects on early brain development and behavior have received less attention. In this experiment the effects of MA exposure during a period equivalent to the human third trimester were examined. Rats (Sprague-Dawley CD) were injected subcutaneously with d-MA (30 mg/kg b.i.d.) early in postnatal development (days 1-10), later (postnatal days 11-20), or with water during both of these periods. Both early and later MA-exposed offspring exhibited augmented acoustic startle and impaired performance in a complex multiple-T water maze. Only the early MA exposure group showed a persistent deficit in weight while only the later MA exposure group showed impaired learning in the Morris hidden platform maze. Effects on locomoter activity are reported in the accompanying article. It was concluded that the effects of MA are both long lasting and stage dependent and involve cognitive as well as arousal functions.

Methamphetamine exposure during early postnatal development in rats: II. Hypoactivity and altered responses to pharmacological challenge.

Methamphetamine induces neurotransmitter reductions and neurotoxicity at high doses in adult animals, but its effects on early brain development and behavior have received little attention. In this experiment the effects of methamphetamine exposure during a period equivalent to the human third trimester were examined. Rats (Sprague-Dawley CD) were injected subcutaneously with d-methamphetamine (d-MA) (30 mg/kg b.i.d.) early in postnatal development (days 1-10), later (postnatal days 11-20), or with water during both of these periods. Both early and later MA-exposed offspring exhibited reduced locomotor activity. The effect was most evident at 30 days of age and was smaller at 45 and 60 days and only present at these latter ages in males. Only the early MA exposure group showed prolonged suppression of activity in response to a challenge dose of fluoxetine and a persistent deficit in weight while only the later MA exposure group showed attenuated suppression of activity in response to a challenge dose of fluoxetine. Based both on the present data and those in the preceding article, it was concluded that the effects of MA are both long lasting and stage dependent and involve arousal as well as cognitive functions.

Teratologic evaluation of rats prenatally exposed to pulsed-wave ultrasound.

While there are no known risks associated with diagnostic ultrasound, uncertainty about the safety of prenatal ultrasound exposure remains. The purpose of the present experiment was to evaluate the teratogenic potential of pulsed-wave (pw) ultrasound in rats, in the absence of maternal anesthesia or restraint. Pregnant CD rats, trained to remain immobile in a water-filled ultrasound exposure tank, were scanned with 3-MHz pw ultrasound at levels of 0, 2, 20 or 30 W/cm2 ISPTA (spatial peak, temporal average intensity) on gestational days 4-19 for approximately 10 min/day. Examination of fetuses on E20 revealed no increase in skeletal or visceral malformations in groups exposed to pw ultrasound in utero. The number of implantations/dam was significantly increased in all pw ultrasound exposure groups compared to sham-exposed animals and, in a possibly related finding, resorptions were increased in the two highest exposure groups. Although significantly increased compared to controls, resorption frequencies in these groups were low (< 10%). No exposure-related changes in fetal weights were observed in offspring of rats scanned with pw ultrasound during gestation. The results indicate that, under the conditions described, no overt embryotoxicity is associated with gestational exposure to pw ultrasound intensities of up to 30 W/cm2.