Frederic J. Seidler

Adolescent nicotine exposure causes persistent upregulation of nicotinic cholinergic receptors in rat brain regions.

Whereas numerous studies have explored the consequences of fetal or adult nicotine exposure, little or no basic research has been conducted for nicotine exposure during adolescence, the developmental period in which regular cigarette use typically begins. We administered nicotine to adolescent rats on postnatal days 30-47 via continuous infusion with implanted osmotic minipumps, using a dose rate (3-6 mg kg-1 day-1) set to achieve plasma nicotine levels found in smokers; results were compared to exposure of adult rats. During and after exposure, we assessed nicotinic cholinergic receptor binding in the midbrain, cerebral cortex, and hippocampus, using [3H]cytisine. Robust receptor upregulation was observed with both adolescent and adult nicotine exposure but there were major differences in the regional specificity and persistence of effect. In adolescents, upregulation was uniform across all regions during the infusion period, whereas in adults, there was a distinct regional hierarchy: midbrain < cerebral cortex < hippocampus; accordingly, receptors in the adolescent midbrain were upregulated far more than with adult exposure. In addition, adolescent nicotine treatment produced long-lasting effects on the receptors, with significant increases still apparent in male rats 1 month after the termination of drug exposure. We also obtained evidence for hippocampal cell damage in adolescent female rats exposed to nicotine, characterized by increases in total membrane protein concentration indicative of a decrease in overall cell size. Adolescent nicotine exposure thus elicits region- and gender-selective effects that differ substantially from those in adults, effects that may contribute to increased addictive properties and lasting deficits in behavioral performance.

Persistent behavioral consequences of neonatal chlorpyrifos exposure in rats

Chlorpyrifos (CPF) is a widely used insecticides which has been shown to alter brain cell development. The current project was conducted to determine whether there are persistent behavioral effects of early [1 mg/kg/day postnatal days (PNDs) 1-4] or late (5 mg/kg/day PNDs 11-14) postnatal CPF exposure in rats. We tested spontaneous alternation in a T-maze, locomotor activity in the Figure-8 apparatus and learning in the 16-arm radial maze, throughout adolescence and into adulthood. Exposure during either neonatal period elicited significant long-term effects on cognitive behavior. In the radial-arm maze, as has been seen previously, control male performed more accurately than control females. Early postnatal CPF exposure reversed this effect. With exposure on PNDs 1-4, females in the CPF group showed a reduction in working and reference memory errors in the radial maze, reducing their error rate to that seen in control males; in contrast, CPF-exposed males exhibited an increase in errors during the initial stages of training. When animals were exposed on PNDs 11-14 and then tested in adolescence and adulthood, males showed a significant slowing of response latency in the T-maze and the rate of habituation in the Figure-8 apparatus was slowed in both sexes. When females were challenged acutely with the muscarinic antagonist, scopolamine, they did not show reference memory impairment, whereas controls did; these results suggest that adaptations occur after CPF exposure that lead to loss of muscarinic cholinergic control of reference memory. No such changes were seen with a nicotinic cholinergic antagonist (mecamylamine). These results indicate that early neonatal exposure to CPF induces long-term changes in cognitive performance that, in keeping with the neurochemical changes seen previously, are distinctly gender-selective. Additional defects may be revealed by similar strategies that subject the animals to acute challenges, thus uncovering the adaptive mechanisms that maintain basal performance.

Prenatal chlorpyrifos exposure in rats causes persistent behavioral alterations.

Use of chlorpyrifos (CPF) has been curtailed due to its developmental neurotoxicity. In rats, postnatal CPF administration produces lasting changes in cognitive performance, but less information is available about the effects of prenatal exposure. We administered CPF to pregnant rats on gestational days (GD) 17-20, a peak period of neurogenesis, using doses (1 or 5 mg/kg/day) below the threshold for fetal growth impairment. We then evaluated performance in the T-maze, Figure-8 apparatus and 16-arm radial maze, beginning in adolescence and continuing into adulthood. CPF elicited initial locomotor hyperactivity in the T-maze. Females showed slower habituation in the Fig. 8 maze; no effects were seen in males. In the radial-arm maze, females showed impaired choice accuracy for both working and reference memory and again, males were unaffected. Despite the deficits, all animals eventually learned the maze with continued training. At that point, we challenged them with the muscarinic antagonist, scopolamine, to determine the dependence of behavioral performance on cholinergic function. Whereas control females showed impairment with scopolamine, CPF-exposed females did not, implying that the delayed acquisition of the task had been accomplished through alternative mechanisms. The differences were specific to muscarinic circuits, as control and CPF groups responded similarly to the nicotinic antagonist, mecamylamine. Surprisingly, adverse effects of CPF were greater in the group receiving 1 mg/kg as compared to 5 mg/kg. Promotional effects of acetylcholine (ACh) on cell differentiation may thus help to offset CPF-induced developmental damage that occurs through other noncholinergic mechanisms. Our results indicate that late prenatal exposure to CPF induces long-term changes in cognitive performance that are distinctly gender-selective. Additional defects may be revealed by similar strategies that subject the animals to acute challenges, thus, uncovering the adaptive mechanisms that maintain basal performance.

Developmental Exposure of Rats to Chlorpyrifos Leads to Behavioral Alterations in Adulthood, Involving Serotonergic Mechanisms and Resembling Animal Models of Depression

Developmental exposure to chlorpyrifos (CPF) causes persistent changes in serotonergic (5HT) systems. We administered 1 mg/kg/day CPF to rats on postnatal days 1–4, a regimen below the threshold for systemic toxicity. When tested in adulthood, CPF-exposed animals showed abnormalities in behavioral tests that involve 5HT mechanisms. In the elevated plus maze, males treated with CPF spent more time in the open arms, an effect seen with 5HT deficiencies in animal models of depression. Similarly, in an anhedonia test, the CPF-exposed group showed a decreased preference for chocolate milk versus water. Developmental CPF exposure also has lasting effects on cognitive function. We replicated our earlier finding that developmental CPF exposure ablates the normal sex differences in 16-arm radial maze learning and memory: during acquisition training, control male rats typically perform more accurately than do control females, but CPF treatment eliminated this normal sex difference. Females exposed to CPF showed a reduction in working and reference memory errors down to the rate of control males. Conversely, CPF-exposed males exhibited an increase in working and reference memory errors. After radial-arm acquisition training, we assessed the role of 5HT by challenging the animals with the 5HT2 receptor antagonist ketanserin. Ketanserin did not affect performance in controls but elicited dose-dependent increases in working and reference memory errors in the CPF group, indicating an abnormal dependence on 5HT systems. Our results indicate that neonatal CPF exposures, classically thought to be subtoxic, produce lasting changes in 5HT-related behaviors that resemble animal models of depression.

Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development of coordination skills and locomotor activity ☆

The widespread use of chlorpyrifos has raised concern about the potential consequences of fetal and childhood exposure. Previous studies have shown that apparently subtoxic doses of chlorpyrifos are nevertheless capable of affecting brain development by inhibiting mitosis, eliciting apoptosis, and altering neuronal activity and reactivity. To determine whether these biochemical changes elicit behavioral abnormalities, we evaluated coordination skills and open field behaviors in developing rats. Administration of 1 mg/kg s.c. of chlorpyrifos on postnatal (PN) days 1-4 elicited deficits in reflex righting on PN3-4 and in geotaxic responses on PN5-8, an effect that was specific to females. However, the ontogeny of more complex behaviors indicated a subsequent selectivity toward males. In the periweaning period, open-field locomotor activity and rearing were markedly reduced in male rats that had been exposed to chlorpyrifos on PN1-4, whereas no effect was detected in females. The gender-selective behavioral effects were associated with greater sensitivity of males to inhibition of cholinesterase in the first few hours after chlorpyrifos treatment. In contrast to the effects seen after administration on PN1-4, shifting the period of chlorpyrifos exposure to PN11-14 had a much less notable effect, even when higher doses were used: no decreases in locomotor activity and overall increases in rearing and grooming that were not significantly gender-selective. Administration on PN11-14 did not produce differential effects on cholinesterase in males and females. These studies indicate that chlorpyrifos given during a critical neonatal period, even at levels below the threshold for overt toxicity, can elicit both immediate and delayed gender-selective behavioral abnormalities. The ultimate evaluation of the developmental neurotoxicity of chlorpyrifos will thus require long-term assessments of neurobehavioral consequences of exposure during discrete developmental periods.

An animal model of adolescent nicotine exposure: effects on gene expression and macromolecular constituents in rat brain regions

Nearly all smokers begin tobacco use in adolescence, and approximately 25% of US teenagers are daily smokers. Prenatal nicotine exposure is known to produce brain damage, to alter synaptic function and to cause behavioral anomalies, but little or no work has been done to determine if the adolescent brain is also vulnerable. We examined the effect of adolescent nicotine exposure on indices of cell damage in male and female rats with an infusion paradigm designed to match the plasma levels found in human smokers or in users of the transdermal nicotine patch. Measurements were made of DNA and protein as well as expression of mRNAs encoding genes involved in differentiation and apoptosis (p53, c-fos) in cerebral cortex, midbrain and hippocampus. Following nicotine treatment from postnatal days 30-47.5, changes in macromolecular constituents indicative of cell loss (reduced DNA) and altered cell size (protein/DNA ratio) were seen across all three brain regions. In addition, expression of p53 showed region- and gender-selective alterations consistent with cell damage; c-fos, which is constitutively overexpressed after gestational nicotine exposure, was unaffected with the adolescent treatment paradigm. Although these measures indicate that the fetal brain is more vulnerable to nicotine than is the adolescent brain, the critical period for nicotine-induced developmental neurotoxicity clearly extends into adolescence. Effects on gene expression and cell number, along with resultant or direct effects on synaptic function, may contribute to increased addictive properties and long-term behavioral deficits.

Adrenomedullary function in the neonatal rat: responses to acute hypoxia.

The mechanism of release of catecholamines from the adrenal medulla of neonatal rats was examined, together with the role of these amines in the ability of the organism to withstand acute O2 deprivation. Splanchnic innervation of the rat adrenal is non‐functional until the end of the first postnatal week. Nevertheless, hypoxia caused depletion of adrenal catecholamines in 1‐day‐old rats as well as in 8‐day‐old animals. Pre‐treatment with cholinergic receptor blocking agents did not prevent the catecholamine response at 1 day but did in older animals; these results indicate that the depletion mechanism is not neurogenic in 1‐day‐old animals but is neurogenic in 8‐day‐old animals. The proportions of noradrenaline and adrenaline released by hypoxic stress also differed at the two ages, with preferential release of adrenaline by the neurogenic mechanism but not by the non‐neurogenic one. The ontogenetic replacement of non‐neurogenic adrenomedullary responses by the neurogenic mechanism was directly related to the onset of splanchnic nerve function. Treatments which accelerated the development of neuronal connexions (neonatal hyperthyroidism, maternal stress) caused premature loss of the non‐neurogenic response. Prior to the development of sympathetic nerve function, adrenal catecholamines plays a predominant role in enabling the neonate to survive hypoxia. Interference with the release of adrenal amines invariably increased mortality during hypoxia. In contrast, interference with sympathetic neural release of catecholamines did not affect the ability of 1‐day‐old rats to withstand hypoxia, indicating that survival during low PO2 conditions is not dependent on the sympathetic innervation at that stage of development. After functional development of the sympathetic nerves and disappearance of non‐neurogenic adrenomedullary responses, the neonatal rats became partially dependent upon catecholamines derived from sympathetic terminals; administration of bretylium at 8 days significantly compromised survival during hypoxia. Interference with adrenergic receptor function also interfered with the ability of neonatal rats to withstand low PO2. At 1 day of age, either phenoxybenzamine or ICI‐118551, but not atenolol, shortened the survival time during hypoxia. At 8 days, only phenoxybenzamine did so.(ABSTRACT TRUNCATED AT 400 WORDS)

Chlorpyrifos Interferes with Cell Development in Rat Brain Regions

Chlorpyrifos, one of the most widely used pesticides, exhibits greater toxicity during development than in adulthood. We administered chlorpyrifos to neonatal rats in doses spanning the threshold for systemic toxicity and examined developing brain regions (brainstem, forebrain, cerebellum) for signs of interference with cell development using markers for cell packing density and cell number (DNA concentration and content) and cell size (protein/DNA ratio). Neonatal rats given 5 mg/kg of chlorpyrifos on postnatal days 1-4 showed significant mortality and the survivors exhibited severe cell loss in the brainstem; brainstem growth was maintained by enlargement of the remaining cells. This effect was not seen at 1 mg/kg, a dose that did not compromise survival or growth, nor was there any adverse effect at either dose in the forebrain, despite the fact that both brainstem and forebrain possess comparable cholinergic projections. When chlorpyrifos was administered later, on days 11-14, the major target for cell loss shifted from the brainstem to the forebrain and in this case, effects were seen at doses that did not compromise survival or growth. The loss of forebrain cell number occurred between 15 and 20 days of age rather than during the chlorpyrifos treatment. The cerebellum differed from the other regions in that it showed short-term elevations of DNA after chlorpyrifos exposure in either early or late postnatal periods; nevertheless, values then regressed to subnormal in parallel with the loss of cells in other regions. Thus, chlorpyrifos likely causes delayed cell death. Although regions rich in cholinergic projections, such as brainstem and forebrain, may be more affected than noncholinergic regions (cerebellum), the maturational timetable of each region (brainstem earliest, forebrain intermediate, cerebellum last) appears to be more important in setting the window of vulnerability. These results indicate that, even when growth or survival are unaffected, chlorpyrifos produces cellular deficits in the developing brain that could contribute to behavioral abnormalities.

Loss of neonatal hypoxia tolerance after prenatal nicotine exposure: Implications for sudden infant death syndrome

Maternal cigarette smoking has a high correlation with sudden Infant Death Syndrome, a condition in which cardiorespiratory failure occurs during an hypoxic episode, as in sleep apnea. Pregnant rats were given nicotine infusions of 2 or 6 mg/kg/day throughout gestation, regimens that produce plasma nicotine levels spanning the range in smokers. The day after birth, animals in the high dose group displayed excessive mortality during hypoxic challenge. These animals were found to be deficient in an essential response component, namely adrenomedullary catecholamine release that is required to maintain neonatal cardiac rhythm during hypoxia; the defect was in adrenal cell function rather than in altered innervation or nicotinic receptor desensitization. We also examined brainstem and forebrain noradrenergic mechanisms that are involved in neonatal respiratory control. The nicotine group showed suppressed spontaneous neuronal activity, but were hyperresponsive to hypoxia. As these projections are inhibitory for respiration, the nicotine-induced sensitization would be expected to contribute to respiratory arrest during hypoxia. Prenatal nicotine exposure may thus provide a useful animal model with which to study the physiological mechanisms that underlie Sudden Infant Death Syndrome, while at the same time providing a biological explanation for the association of the syndrome with smoking.

Comparative Developmental Neurotoxicity of Organophosphates In Vivo: Transcriptional Responses of Pathways for Brain Cell Development, Cell Signaling, Cytotoxicity and Neurotransmitter Systems

Organophosphates affect mammalian brain development through a variety of mechanisms beyond their shared property of cholinesterase inhibition. We used microarrays to characterize similarities and differences in transcriptional responses to chlorpyrifos and diazinon, assessing defined gene groupings for the pathways known to be associated with the mechanisms and/or outcomes of chlorpyrifos-induced developmental neurotoxicity. We exposed neonatal rats to daily doses of chlorpyrifos (1mg/kg) or diazinon (1 or 2mg/kg) on postnatal days 1-4 and evaluated gene expression profiles in brainstem and forebrain on day 5; these doses produce little or no cholinesterase inhibition. We evaluated pathways for general neural cell development, cell signaling, cytotoxicity and neurotransmitter systems, and identified significant differences for >60% of 252 genes. Chlorpyrifos elicited major transcriptional changes in genes involved in neural cell growth, development of glia and myelin, transcriptional factors involved in neural cell differentiation, cAMP-related cell signaling, apoptosis, oxidative stress, excitotoxicity, and development of neurotransmitter synthesis, storage and receptors for acetylcholine, serotonin, norepinephrine and dopamine. Diazinon had similar effects on many of the same processes but also showed major differences from chlorpyrifos. Our results buttress the idea that different organophosphates target multiple pathways involved in neural cell development but also that they deviate in key aspects that may contribute to disparate neurodevelopmental outcomes. Equally important, these pathways are compromised at exposures that are unrelated to biologically significant cholinesterase inhibition and its associated signs of systemic toxicity. The approach used here demonstrates how planned comparisons with microarrays can be used to screen for developmental neurotoxicity.