Elsevier

Brain Research

Volume 867, Issues 1–2, 9 June 2000, Pages 29-39
Brain Research

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

https://doi.org/10.1016/S0006-8993(00)02208-3Get rights and content

Abstract

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.

Introduction

The growing worldwide use of tobacco is spearheaded by the targeting of adolescents, the next generation of smokers. In the US alone, the recent rise in adolescent smoking runs counter to the trend of decreased smoking in the overall population, and currently involves one quarter of all adolescents, over 3 million teenagers [14], [26], [27]. The long-term impact of tobacco use in adolescence is clear: nearly all adult smokers tried cigarettes by age 20 and more than three quarters of them were daily smokers by that age [49], [53]. Initiating smoking during adolescence correlates with greater addiction liability, higher daily consumption and reduced probability of quitting [15].

In light of the epidemiology of tobacco use in adolescence, it is surprising that little or no basic research has been done to characterize the biochemical and behavioral effects of nicotine in the adolescent brain. It is increasingly evident that brain development, in the form of cell acquisition, apoptosis, synaptogenesis and synaptic programming, all continue into adolescence [2], [4], [5], [28], [45], [60] and studies with other psychoactive drugs show that the adolescent brain responds differently from that of the adult [44], [73]. Two recent studies indicate that adolescent nicotine treatment elicits long-term changes in the programming of reward pathways [33], persistent upregulation of nicotinic cholinergic receptor expression and membrane alterations suggestive of cell damage [77]. The actions of nicotine on the adolescent brain may thus represent an extension of the critical period in which this agent disrupts brain development. Nicotine is a neuroteratogen which mimics the actions of the endogenous transmitter acetylcholine, discoordinating the timing of trophic events linked to cholinergic nicotinic receptors that are present in the developing brain [37], [63], [64], [65]. Gestational nicotine exposure elicits inappropriate expression of genes involved in differentiation and apoptosis, leading to mitotic arrest, cell death, and eventual shortfalls in cell number [9], [37], [59], [63], [64], [65]. The current work addresses whether these effects are evoked when nicotine is administered to adolescent rats.

We chose to deliver nicotine by continuous infusion via implanted osmotic minipumps, a technique that avoids the episodic hypoxic–ischemic episodes that accompany nicotine injections, and that also sidesteps the inherent problems of repeated handling stress and episodic withdrawal between injections. To characterize this model, we measured plasma nicotine and cotinine levels both during the infusion period and after the end of the infusion so as to verify the cessation of exposure. We studied three brain regions that, based on studies of fetal nicotine exposure, represent likely targets for adverse effects of nicotine, and in which we have already demonstrated unique effects of adolescent nicotine exposure [77]: midbrain, cerebral cortex and hippocampus. We then evaluated the effects on the expression of mRNAs encoding two proteins that play critical roles in cell differentiation and apoptosis, c-fos and p53.

Although transient expression of c-fos is associated with short-term cell stimulation [17], [18], constitutive overexpression elicits cell death, even in otherwise healthy cells [18], [70], [80], and we have identified both constitutive c-fos overexpression and apoptosis in the gestational nicotine model [59], [78]. Similarly, the tumor-suppressor gene, p53 plays an important role both during differentiation and apoptosis [22], [46] and nicotine has been shown to elevate p53 expression in hippocampal precursor cells in association with apoptosis [9]. In the gestational nicotine exposure model, these adverse effects at the level of cell biochemistry culminate in cell loss, typified by a decrease in DNA content and a rise in protein/DNA ratio; accordingly, we have also determined whether these cell parameters are affected similarly in the adolescent nicotine exposure model. Finally, our earlier work indicated gender selectivity of some of the effects of adolescent nicotine treatment on nicotinic receptor expression and other cell membrane markers [77], so we have contrasted effects in males and females. In all cases, these studies were modeled specifically after previous work with gestational nicotine [37], [63], [64], [65] and, therefore, comparisons have been made throughout between the effects of adolescent treatment with those seen after fetal exposure.

Section snippets

Animal treatments and blood levels of nicotine and cotinine

All studies were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed pregnant Sprague–Dawley rats, dams with litters, or adult male rats (Zivic-Miller Laboratories, Allison Park, PA) were shipped by climate-controlled truck (total transit time less than 12 h). Shipping occurred on the second day of gestation (pregnant rats), or on about PN20 (litters) or

Results

During the course of nicotine administration, adolescent rats grew from initial weights (PN30) of 114±2 g for males and 98±2 g for females, to PN45 values of 238±4 and 173±2 g, respectively. Accordingly, although the initial dose rate was 6 mg/kg/day, by the end of the infusion period, the value fell to 3.0±0.1 mg/kg/day in males and 3.6±0.1 mg/kg/day in females. Despite the 20% dosage difference between genders, plasma levels of nicotine were virtually identical on PN45 (Fig. 1) and were

Discussion

The present results indicate that brain development remains vulnerable to nicotine into adolescence. Although effects on gene expression and on indices of cell number and size were smaller than those seen previously with models of gestational nicotine exposure [37], [63], [64], [65], the adolescent brain nevertheless exhibited changes in the same direction and over a similar post-treatment time course. In the current study, we found significant decreases in total cell number as assessed by DNA

Acknowledgements

Supported by a grant from the Smokeless Tobacco Research Council and by a STAR fellowship from the US Environmental Protection Agency.

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