Theodore A. Slotkin

Nicotine and the adolescent brain: Insights from an animal model

Tobacco use in adolescence represents one of the major challenges to the future of public health. Whereas numerous studies have explored the consequences of fetal or adult nicotine effects, little or no basic research has been conducted for nicotine during adolescence, the stage at which regular cigarette use typically begins. This review describes the recent development of a model of adolescent nicotine administration in rats that recapitulates the plasma levels of nicotine found in smokers. Adolescent nicotine evoked CNS nicotinic receptor up-regulation with a distinctly different regional pattern from that seen in the adult; increased receptor expression in male rats persisted for more than a month after discontinuing nicotine administration. We also identified evidence of cell damage and changes in cell size in the female hippocampus. These changes were accompanied by alterations in synaptic activity of cholinergic, noradrenergic, dopaminergic and serotonergic systems during nicotine administration and persisting for extended periods after the termination of exposure; behavioral alterations were commensurate with the neurochemical changes. In each case, the effects of adolescent nicotine differed not only from the adult, but also from the those seen after fetal exposure, indicating that adolescence represents a unique period of vulnerability for nicotine-induced misprogramming of brain cell development and synaptic function. Effects of nicotine on critical components of reward pathways and circuits involved in learning, memory and mood are likely to contribute to increased addictive properties and long-term behavioral problems seen in adolescent smokers.

Role of ornithine decarboxylase and the polyamines in nervous system development: a review

Development of nervous tissue is controlled, in part, by the ornithine decarboxylase (ODC)/polyamine system. Each brain region possesses a unique ontogenetic pattern for ODC, with highest levels of the enzyme associated with periods of most rapid growth. For this reason, perturbation of the ODC profile has proven useful in examinations of teratologic mechanisms and detection of adverse environmental effects during development. More recently, the replication of neuronal cells in developing brain has been shown to require the maintenance of polyamine levels and consequently, depletion of polyamines by alpha-difluoromethylornithine (DFMO, an ODC inhibitor) arrests brain cell maturation. DFMO also interferes with neuronal migration, axonogenesis and synaptogenesis, leading to disruption of the cytoarchitectural organization of brain structures: these results imply a similarly important role for polyamines in post-replicative events. Indeed, [3H]DFMO-autoradiographic localization of ODC in developing cerebellar lamina indicates high levels of activity associated with neuropil, areas of axonal outgrowth, and post-mitotic granule cells. Axonal outgrowth during regeneration after nerve damage in the mature nervous system may display some of the same characteristics as in developing neurons, suggesting that the two processes share common polyamine-dependent mechanisms.

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.

Brain anomalies in children exposed prenatally to a common organophosphate pesticide

Prenatal exposure to chlorpyrifos (CPF), an organophosphate insecticide, is associated with neurobehavioral deficits in humans and animal models. We investigated associations between CPF exposure and brain morphology using magnetic resonance imaging in 40 children, 5.9–11.2 y, selected from a nonclinical, representative community-based cohort. Twenty high-exposure children (upper tertile of CPF concentrations in umbilical cord blood) were compared with 20 low-exposure children on cortical surface features; all participants had minimal prenatal exposure to environmental tobacco smoke and polycyclic aromatic hydrocarbons. High CPF exposure was associated with enlargement of superior temporal, posterior middle temporal, and inferior postcentral gyri bilaterally, and enlarged superior frontal gyrus, gyrus rectus, cuneus, and precuneus along the mesial wall of the right hemisphere. Group differences were derived from exposure effects on underlying white matter. A significant exposure × IQ interaction was derived from CPF disruption of normal IQ associations with surface measures in low-exposure children. In preliminary analyses, high-exposure children did not show expected sex differences in the right inferior parietal lobule and superior marginal gyrus, and displayed reversal of sex differences in the right mesial superior frontal gyrus, consistent with disruption by CPF of normal behavioral sexual dimorphisms reported in animal models. High-exposure children also showed frontal and parietal cortical thinning, and an inverse dose–response relationship between CPF and cortical thickness. This study reports significant associations of prenatal exposure to a widely used environmental neurotoxicant, at standard use levels, with structural changes in the developing human brain.

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)