Yael Abreu-Villaça

Prenatal nicotine exposure alters the response to nicotine administration in adolescence: effects on cholinergic systems during exposure and withdrawal.

Maternal smoking during pregnancy increases the likelihood that the offspring will become smokers in adolescence. In the current study, we evaluated effects of prenatal and adolescent nicotine exposure in rats to assess whether there is a biological basis for this relationship. Pregnant rats were given nicotine or vehicle throughout pregnancy and the offspring then again received nicotine or vehicle during adolescence (postnatal days PN30-47.5), using a regimen (6 mg/kg/day by subcutaneous infusion) that produces plasma nicotine levels similar to those in smokers. Evaluations were made in the cerebral cortex and midbrain during adolescent nicotine administration (PN45) and for up to 1 month after the end of treatment. We assessed the magnitude and persistence of nicotinic acetylcholine receptor (nAChR) upregulation; in addition, we evaluated cholinergic synaptic activity by comparing the effects on choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, with those on hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, which is regulated by nerve impulse activity. Prenatal nicotine exposure had only minor effects on nAChRs but produced persistent cholinergic hypoactivity (reduced HC-3 binding relative to ChAT) throughout adolescence and into adulthood (PN75). Adolescent nicotine exposure evoked robust nAChR upregulation and also suppressed cholinergic activity. Prenatal nicotine exposure reduced the upregulation of nAChRs evoked by adolescent nicotine but worsened the cholinergic hypoactivity during withdrawal. Our results indicate that prenatal nicotine exposure alters the subsequent response to nicotine in adolescence, effects that may contribute to the association between maternal smoking during pregnancy and subsequent adolescent smoking in the offspring.

Nicotine is a neurotoxin in the adolescent brain: critical periods, patterns of exposure, regional selectivity, and dose thresholds for macromolecular alterations.

In the fetus, nicotine is a neuroteratogen that elicits cell damage and loss and subsequent abnormalities of synaptic function. We explored whether these effects extend into adolescence, the period when most people begin smoking. Beginning on postnatal day 30, rats were given a 1 week regimen of nicotine infusions or twice-daily injections, at doses (0.6, 2 and 6 mg/kg/day) set to achieve plasma levels found in occasional to regular smokers. We assessed indices of cell packing density and cell number (DNA concentration and content), cell size (total protein/DNA ratio) and neuritic projections (membrane/total protein) in the midbrain, hippocampus and cerebral cortex, three regions known to be vulnerable to developmental effects of nicotine. With either route of administration, nicotine evoked shortfalls in DNA concentration and content, compensatory elevations of total protein/DNA, and reductions in the membrane/total protein ratio. Nearly all of the effects were apparent even at the lowest dose of nicotine and remained fully evident 1 month posttreatment. Although both males and females showed significant alterations, in general the effects were larger in females. Our results indicate that in adolescence, even a brief period of continuous or intermittent nicotine exposure, elicits lasting alterations in biomarkers associated with cellular and neuritic damage. As the effects are detected at exposures that produce plasma concentrations one-tenth of those in regular smokers, the exquisite sensitivity of the adolescent brain to nicotine neurotoxicity may contribute to lasting neurobehavioral damage even in occasional smokers.

Developmental aspects of the cholinergic system

Beyond its importance in sustaining or modulating different aspects of the activity of the central nervous system (CNS), the cholinergic system plays important roles during development. In the current review, we focus on the developmental aspects associated with major components of the cholinergic system: Acetylcholine, choline acetyltransferase, vesicular acetylcholine transporter, high-affinity choline transporter, acetylcholinesterase, nicotinic and muscarinic receptors. We describe when and where each one of these components is first identified in the CNS and the changes in their levels that occur during the course of prenatal and postnatal development. We also describe how these components are relevant to many events that occur during the development of the CNS, including progenitor cells proliferation and differentiation, neurogenesis, gliogenesis, neuronal maturation and plasticity, axonal pathfinding, regulation of gene expression and cell survival. It will be noticed that evidence regarding the developmental aspects of the cholinergic system comes mostly from studies that used agonists, such as nicotine, and antagonists, such as hemicholinium-3. Studies using immunohistochemistry and genetically altered mice also provided valuable information.

Does Prenatal Nicotine Exposure Sensitize the Brain to Nicotine-Induced Neurotoxicity in Adolescence?

Offspring of women who smoke during pregnancy are themselves more likely to take up smoking in adolescence. We evaluated neurotoxicant effects of prenatal and adolescent nicotine exposure in developing rats to evaluate whether these contribute to a biological basis for this relationship. Rats were given nicotine or vehicle throughout pregnancy and the offspring then again received nicotine or vehicle during adolescence (postnatal days PN30–47.5); this regimen reproduces the plasma nicotine levels found in smokers. Indices of neural cell number (DNA concentration and content), cell size (protein/DNA ratio), and cell membrane surface area (membrane/total protein) were then evaluated in brain regions during adolescent nicotine administration (PN45) and up to 1 month post-treatment. By itself, prenatal nicotine administration produced cellular alterations that persisted into adolescence, characterized by net cell losses in the midbrain and to a lesser extent, in the cerebral cortex, with corresponding elevations in the membrane/total protein ratio. The hippocampus showed a unique response, with increased DNA content and regional enlargement. Adolescent nicotine treatment alone had similar, albeit smaller effects, but also showed sex-dependence, with effects on protein biomarkers preferential to females. When animals exposed to nicotine prenatally were then given nicotine in adolescence, the net outcome was worsened, largely representing summation of the two individual effects. Our results indicate that prenatal nicotine exposure alters parameters of cell development lasting into adolescence, where the effects add to those elicited directly by adolescent nicotine; neurotoxicant actions may thus contribute to the association between maternal smoking and subsequent smoking in the offspring.

Individual differences in novelty-seeking behavior but not in anxiety response to a new environment can predict nicotine consumption in adolescent C57BL/6 mice

Considering that adolescence is associated with an increased motivation to seek out new stimuli and with low anxiety levels in exploring novel environments, and that both behavioral traits may be associated with substance abuse, we investigated whether the behavioral response to a novel environment can predict subsequent oral nicotine self-administration in adolescent C57BL/6 mice. On postnatal day 30 (PN30), the novelty-seeking behavior and anxiety levels were assessed in a hole board activity box. The total number of head-dips (DIP) was used to classify animals either into the high novelty (HN; DIP above median) or low novelty (LN; DIP below median) groups. The percentage of center squares crossed (CEN) was used to classify animals either into the high anxiety (HA; CEN below median) or low anxiety (LA; CEN above median) groups. From PN31 to PN41, all animals were given a free choice between tap water or a nicotine solution (10 microg/ml). LN mice did not change nicotine intake throughout the free choice procedure, however, HN mice presented a marked increase in consumption. There were no differences in consumption between HA and LA mice. Our results indicated that mice that presented a more intense novelty-seeking behavior increased their preference for nicotine during the free choice experiment but that anxiety levels did not predict nicotine consumption. These results suggest that higher motivation to seek out new experiences is a significant contributor to drug use in adolescents and that anxiety is probably not a major factor that determines differential nicotine consumption during adolescence.

Impact of adolescent nicotine exposure on adenylyl cyclase-mediated cell signaling: enzyme induction, neurotransmitter-specific effects, regional selectivities, and the role of withdrawal.

Recent animal studies indicate that the adolescent brain is especially vulnerable to nicotine-induced alterations in synaptic function, echoing the increased susceptibility to nicotine dependence and withdrawal noted for adolescent smokers. We administered nicotine to adolescent rats via continuous minipump infusions from PN30 to PN47.5, using 6 mg/kg/day, a dose rate that replicates the plasma nicotine levels found in smokers, and examined the effects on cell signaling mediated through adenylyl cyclase (AC) and its response to catecholamines. Studies were conducted during nicotine administration (PN45) and in the posttreatment, withdrawal period (PN50, 60, 75). Adolescent nicotine augmented AC activity as evidenced by increased responsiveness to the direct AC stimulants, forskolin and Mn(2+). The effects on AC were equally noted in brain regions enriched (striatum) or sparse (cerebellum) in cholinergic projections, implying that the effects are secondary to activation/repression of neural circuits, rather than representing direct effects on AC mediated by nicotinic cholinergic receptors. AC responses to dopaminergic and noradrenergic stimulants were also enhanced by nicotine exposure. However, in contrast to earlier work with serotonin-mediated responses, the effects on catecholaminergic stimulation were smaller and did not display the sex-dependence noted for serotonin. An alternate administration paradigm that maximizes episodic withdrawal (twice-daily nicotine injections) induced AC more rapidly at lower nicotine doses. Our results indicate that adolescent nicotine exposure elicits lasting alterations in synaptic signaling that intensify and persist during withdrawal. These findings support the concept that the adolescent brain is especially susceptible to persistent nicotine-induced alterations.

Combined Exposure to Nicotine and Ethanol in Adolescent Mice Differentially Affects Anxiety Levels during Exposure, Short-Term, and Long-Term Withdrawal

Smoking and consumption of alcoholic beverages are frequently associated during adolescence. This association could be explained by the cumulative behavioral effects of nicotine and ethanol, particularly those related to anxiety levels. However, despite epidemiological findings, there have been few animal studies of the basic neurobiology of the combined exposure in the adolescent brain. In the present work we assessed, through the use of the elevated plus maze, the short- and long-term anxiety effects of nicotine (NIC) and/or ethanol (ETOH) exposure during adolescence (from the 30th to the 45th postnatal day) in four groups of male and female C57BL/6 mice: (1) Concomitant NIC (nicotine free-base solution (50 μg/ml) in 2% saccharin to drink) and ETOH (ethanol solution (25%, 2 g/kg) i.p. injected every other day) exposure; (2) NIC exposure; (3) ETOH exposure; (4) Vehicle. C57BL/6 mice were selected, in spite of the fact that they present slower ethanol metabolism, because they readily consume nicotine in the concentration used in the present study. During exposure (45th postnatal day: PN45), our results indicated that ethanol was anxiolytic in adolescent mice and that nicotine reverted this effect. Short-term drug withdrawal (PN50) elicited sex-dependent effects: exposure to nicotine and/or ethanol was anxiogenic only for females. Although neither nicotine nor ethanol effects persisted up to 1 month postexposure (PN75), the coadministration elicited an anxiogenic response. In spite of the fact that generalizations based on the results from a single strain of mice are prone to shortcomings, our results suggest that the deficient response to the anxiolytic effects of ethanol in adolescents co-exposed to nicotine may drive higher ethanol consumption. Additionally, increased anxiety during long-term smoking and drinking withdrawal may facilitate relapse to drug use.

Increased apoptosis and reduced neuronal and glial densities in the hippocampus due to nicotine and ethanol exposure in adolescent mice.

It has been recently shown that nicotine and ethanol interact during adolescence affecting memory/learning and anxiety levels. Considering the role of the hippocampus in both anxiety and memory/learning, we investigated whether adolescent nicotine and/or ethanol administration elicit apoptotic cell death and whether this results in neuronal and/or glial density alterations in the following regions of the hippocampus: granular layer of the dentate gyrus (GrDG), molecular layer (Mol), CA1, CA2 and CA3. From the 30th to the 45th postnatal day, C57BL/6 male and female mice were exposed to nicotine free base (NIC) and/or ethanol (ETOH). Four groups were analyzed: (1) concomitant NIC (50 μg/ml in 2% saccharin to drink) and ETOH (25%, 2 g/kg i.p. injected every other day) exposure; (2) NIC exposure; (3) ETOH exposure; (4) vehicle. We evaluated cell degeneration (TUNEL assay), neuronal and glial densities (optical disector) and region thicknesses at the end of the period of exposure. Our results demonstrate that ETOH elicited an increase in TUNEL‐positive cells relative to the vehicle group in all hippocampal regions. NIC elicited less severe region‐dependent effects: the number of TUNEL‐positive cells was significantly increased in the Mol and CA1 when compared to the vehicle group. These results were paralleled by reductions in neuronal and glial cells densities, which indicate that both cell types are sensitive to the neurotoxic effects of these drugs. There were no effects on region thicknesses. On the other hand, concomitant NIC and ETOH reduced the adverse effects of the drugs when administered separately. This ability of nicotine and ethanol co‐exposure to lessen the adverse effects of nicotine and ethanol may contribute to adolescents co‐use and co‐abuse of tobacco and alcoholic beverages.

Anxiety-like behavior during nicotine withdrawal predict subsequent nicotine consumption in adolescent C57BL/6 mice

We have previously demonstrated that anxiety-like behavior assessed in the elevated plus maze does not predict subsequent nicotine consumption in naïve adolescent mice. However, an association between anxiety and relapse to drug use has been suggested. In the present study, we investigated whether anxiety levels during nicotine withdrawal predict subsequent nicotine consumption in adolescent mice. C57BL/6 mice were either exposed to (-)-nicotine-free base (nicotine, 50mug/ml) or tap water (water) from postnatal day 30 to 45 (PN30-PN45, priming period). By the end of PN48, all animals were submitted to the elevated plus maze and classified as either having high (HiAnx) or low (LoAnx) levels of anxiety. Immediately after finishing the test, all animals were returned to their home cages and were given a free choice (from PN49 to PN55, free-choice period) between two bottles, one containing a nicotine solution (10mug/ml) and the other tap water. Nicotine consumption during the free-choice period was affected by the priming treatment (nicotine or water) in a way that was dependent on the anxiety level (HiAnx or LoAnx): the nicotine HiAnx group had lower nicotine consumption than the other groups. No differences were observed between the nicotine LoAnx, water HiAnx and water LoAnx groups. The present study provides experimental evidence for the role of anxiety on the regulation of drug consumption. Specifically, our results suggest that the anxiety-like behavior during nicotine withdrawal is associated with subsequent nicotine self-administration.

Neonatal nicotine exposure causes insulin and leptin resistance and inhibits hypothalamic leptin signaling in adult rat offspring

Maternal nicotine (NIC) exposure during lactation leads to overweight, hyperleptinemia, and hypothyroidism in adult rat offspring. In this model, we analyzed adipocyte morphology, glucose homeostasis (serum insulin and adiponectin; liver and muscle glycogen), serum lipid, and the leptin signaling pathway. After birth, osmotic minipumps were implanted in lactating rats, which were divided into the groups NIC (6 mg/kg per day s.c. for 14 days) and control (C, saline). NIC and C offspring were killed at the age of 180 days. Adult NIC rats showed higher total body fat (+10%, P<0.05), visceral fat mass (+12%, P<0.05), and cross-sectional area of adipocytes (epididymal: +12% and inguinal: +43%, P<0.05). Serum lipid profile showed no alteration except for apolipoprotein AI, which was lower. We detected a lower adiponectin:fat mass ratio (-24%, P<0.05) and higher insulinemia (+56%, P<0.05), insulin resistance index (+43%, P<0.05), leptinemia (+113%, P<0.05), and leptin:adiponectin ratio (+98%, P<0.05) in the adult NIC group. These rats presented lower hypothalamic contents of the proteins of the leptin signaling pathway (leptin receptor (OB-R): -61%, janus tyrosine kinase 2: -41%, and p-signal transducer and activator of transcription 3: -56%, P<0.05), but higher suppressor of cytokine signaling 3 (+81%, P<0.05). Therefore, NIC exposure only during lactation programs rats for adipocyte hypertrophy in adult life, as well as for leptin and insulin resistance. Through the effects of NIC, perinatal maternal cigarette smoking may be responsible for the future development of some components of the metabolic syndrome in the offspring.