Combined exposure to tobacco smoke and ethanol in ...

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Dec 10, 2012 - the effects of tobacco smoke and/or ethanol exposure during adolescence .... natal day 21 = PN21), animals were separated by sex in groups.
Nicotine & Tobacco Research, Volume 15, Number 7 (July 2013) 1211–1221

Original Investigation

Combined Exposure to Tobacco Smoke and Ethanol in Adolescent Mice Elicits Memory and Learning Deficits Both During Exposure and Withdrawal Yael Abreu-Villaça PhD1, Anna Caroline de Carvalho Graça MSc1, Anderson Ribeiro-Carvalho PhD2, Victor de Freitas Naiff BSc1, Alex C. Manhães PhD1, Cláudio C. Filgueiras PhD1

Corresponding Author: Yael Abreu-Villaça, Laboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. Prof. Manuel de Abreu 444, 5 andar, Vila Isabel, Rio de Janeiro, RJ, 20550-170, Brazil. Telephone: (5521) 2587 6295; Fax: (5521) 2587 6129; E-mail: [email protected] Received July 7, 2012; accepted October 13, 2012

Abstract Introduction: Adolescents often associate tobacco smoking and consumption of alcoholic beverages. In spite of that, little is known about the neurobehavioral consequences of the dual exposure in the adolescent brain. In the present work, we assessed the effects of tobacco smoke and/or ethanol exposure during adolescence on memory/learning. Methods: From postnatal day 30 to 45 (PN30-45), male and female Swiss mice were exposed to tobacco smoke (SMK— generated from research cigarettes type 3R4F, whole body exposure, 8 hr/day) and/or ethanol (ETOH—25% solution, 2 g/kg intraperitoneally injected every other day) as follows: (a) SMK+ETOH exposure; (b) SMK exposure; (c) ETOH exposure; (d) Control. Memory/learning was evaluated during exposure (PN44-45) and during short- (PN49-50) and longstanding withdrawal (PN74-75). At each timepoint, mice were trained and tested in a step-down passive avoidance task (0.3 mA, 3 s footshock). Two retention tests were carried out in each animal, one at 3 hr after training to measure short-term memory and another at 24 hr to measure long-term memory. Results: During exposure, the short-term memory was impaired in all groups and the long-term memory was impaired in SMK and SMK+ETOH. During the short-standing withdrawal, a significant impairment was observed only in long-term memory of the male SMK+ETOH mice. At long-standing withdrawal, there were no significant differences between groups. Conclusions: Tobacco smoke and ethanol exposures during adolescence of mice negatively affect learning/memory performance. Deficits that were still present during SMK+ETOH short-standing withdrawal suggest that the combined exposure elicits a worsened memory/learning outcome and that males are more susceptible.

Introduction Like most other recreational drugs, tobacco is frequently taken in combination with other substances (Degenhardt & Hall, 2001). In this sense, both cigarette smoking and alcoholic beverage consumption initiate during adolescence (Centers for Disease Control Prevention, 2010; Doubeni, Reed, & Difranza, 2010; Spear, 2000), and epidemiological studies show that there is a strong relationship between smoking and drinking, so that the cooccurrence of alcohol consumption and smoking is very common (Larsson & Engel, 2004). Moreover, there is an inverse correlation between age at onset of smoking and the incidence of alcoholism, so that the younger the adolescent

when he starts smoking, the greater the frequency of alcoholism (Difranza & Guerrera, 1990; Grant, 1998), characterizing adolescence as a period of vulnerability to both drugs. Despite that, the biological bases and the effects of tobacco and ethanol coconsumption have received little attention. Considering that nicotine has been described as an active component of cigarette smoke responsible for a wide variety of nervous system effects resulting from tobacco consumption (for review: Fowler, Arends, & Kenny, 2008; Slotkin, 2002), most studies in animal models have focused on behavioral and biochemical effects of this drug and, more recently, on the effects of nicotine + ethanol combined exposure. In adolescent mice, it was demonstrated that the coexposure elicits

doi:10.1093/ntr/nts250 Advance Access publication December 10, 2012 © The Author 2012. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: [email protected].

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1Laboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, 20550-170, Brazil; 2Departamento de Ciências, Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro, São Gonçalo, RJ, 24435-005, Brazil

Combined exposure to tobacco and ethanol

Methods Animals and Treatment All experiments were carried out with the approval of the Animal Care and Use Committee of the Universidade do Estado do Rio de Janeiro (CEA/014/2011), in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals. Swiss mice were bred and maintained in a vivarium next to our laboratory. The animals were kept in a temperature-controlled room on a 12-hr light/dark cycle (lights on at 1:00 a.m.). Access to food and water was ad libitum. We only used litters of 8 to 12 pups. At weaning (postnatal day 21 = PN21), animals were separated by sex in groups of 2 to 5 mice and allowed free access to food and water. From PN30 to PN45, male and female Swiss mice were exposed to tobacco smoke and/or ethanol. PN30 to PN45 is the approximate age range during which rodents of most breeding stock exhibit adolescent-typical behavioral, neurochemical, and endocrine characteristics (Spear, 2000). During this period, mice were exposed to tobacco smoke generated from reference research cigarettes (University of Kentucky) type 3R4F

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(nicotine  =  0.73 mg/cigarette). Whole body exposure to cigarette smoke was for 8 hr/day, from 8 a.m. to 4 p.m., 7 days/week in a chamber that received the smoke generated from an automatic cigarette smoking machine (Teague Enterprises) as a surrogate for active smoking (Abreu-Villaça et al., 2010; Slotkin et al., 2001). Detailed description of smoke exposure characteristics is described as Supplementary Material. Control mice were exposed to air in an identical chamber. As for ethanol exposure, 25% ethanol (2 g/kg) solution (vol/vol) in saline was injected (intraperitoneally) every other day in order to mimic cyclical patterns of “binge” drinking in humans (Maier & West, 2001; Nunes et al., 2011; Tokunaga, Silvers, & Matthews, 2006; White, Ghia, Levin, & Swartzwelder, 2000). Control mice were exposed to saline. Accordingly, male and female mice from each litter were distributed into four treatment groups: VEH (air + injected saline), SMK (tobacco smoke + injected saline), ETOH (air + injected ethanol), and those receiving the combined treatment: SMK+ETOH (tobacco smoke + injected ethanol). From the beginning of the experimental period onwards, mice from each treatment group were segregated into separate cages. Body mass was measured every day. Behavioral Testing Learning/memory performance was assessed in the stepdown passive avoidance test. The test apparatus contained one chamber, 25  × 25  × 25 cm (length × width × height). Mice were submitted to three testing sessions: Initially, in a training/ acquisition session (T0), subjects were placed in a circular platform (diameter = 6.5 cm) and allowed up to 3 min to descend from it, whereupon they received a mild foot shock (0.3 mA/3 s). Three (T3) and twenty-four (T24) hours later, the animals were retested and allowed up to 3 min to descend from the platform. (Shock was not administered.) The test at 3 hr after training assesses short-term memory, whereas the test at 24 hr after training assesses long-term memory (Izquierdo et  al., 1998). Animals were tested in alternating groups and sexes. The latency (L) to descend from the platform on the first (L0), second (L3), and third (L24) sessions was noted. The learning/ memory component of the passive avoidance task is expressed as an increase in the time the animal takes to descend from the platform in the T3 and T24 sessions. Therefore, in order to visualize more clearly differences between groups, the learning/memory component of the task was evaluated by calculating memory/learning indices as follows: (L3 − L0)/L0 and (L24 − L0)/L0. Separate groups of mice were tested by the end of the drug administration period (PN44-45), during a short-standing withdrawal (PN49-50), or during a long-standing withdrawal (PN74-75). Pups from 33 litters (119 females and 115 males) were evenly distributed among subgroups so that there were 8–12 mice per timepoint (age), treatment group, and sex. For mice tested by the end of drug exposure, on PN44, at 3–4 hr of smoke exposure, animals were removed from the exposure chamber and allowed to habituate for 20 min in the testing room before T0. After the test, the animals were returned to the exposure chamber until T3, when the same procedure was performed. After T3, the animals returned to the exposure chamber to complete the 8 hr of tobacco smoke exposure. The next day, again at 3–4 hr of smoke exposure, animals were removed from the exposure chamber and allowed to habituate for 20 min in the testing room before T24. As for ethanol exposure, in

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distinct behavioral and biochemical effects when compared with nicotine or ethanol exposures, which indicates that these drugs interact, affecting the functioning of the central nervous system during this period of development (Abreu-Villaça et al., 2007; Abreu-Villaça, Nunes, Queiroz-Gomes, Manhães, & Filgueiras, 2008; Ribeiro-Carvalho, Lima, Filgueiras, Manhães, & Abreu-Villaça, 2008; Ribeiro-Carvalho et  al., 2009, 2011; Trezza, Baarendse, & Vanderschuren, 2009). Both tobacco and ethanol are known to affect cognitive parameters. Regarding nicotine, in rodents, it was shown to diminish attention performance during adolescence (Counotte et  al., 2009) and to improve memory/learning during withdrawal (AbreuVillaça et al., 2007; Trauth, Seidler, & Slotkin, 2000a). As for ethanol, consumption by adolescent rodents impairs memory/ learning during exposure and withdrawal (Abreu-Villaça et al., 2007; Sircar & Sircar, 2005; White & Swartzwelder, 2004). Detrimental effects of nicotine and ethanol on memory/learning were shown to represent a worsened outcome from the dual exposure, while, in contrast, negative effects of the combined exposure failed to persist during withdrawal (Abreu-Villaça et al., 2007). Interestingly, in spite of the large number of animal studies on the basic neurobiology of nicotine exposure that is made available every year (for review: Fowler et al., 2008; Slotkin, 2002), there is a lack of experimental studies that investigate the effects of tobacco smoke. In addition, despite recent studies that describe the effects of the combined nicotine and ethanol effects (Abreu-Villaça et al., 2007; Abreu-Villaça et al., 2008; Ribeiro-Carvalho et al., 2008, 2009, 2011), there is a lack of experimental studies on the effects of tobacco smoke and ethanol coexposure during adolescence. This issue is particularly relevant given that teenagers who smoke and drink alcohol are exposed to several other substances present in tobacco smoke besides nicotine, which have the potential to interact with both nicotine and ethanol. Accordingly, the purpose of this study was to examine the effects of adolescent tobacco smoke and/ or ethanol exposures on memory/learning during drug administration and withdrawal.

Nicotine & Tobacco Research order to avoid acute drug effects such as motor impairment and to allow the investigation of the effects of coexposure, the last injection was administered on PN44 after T3. Cotinine and Ethanol Serum Levels

Data Analyses Data Analysis of Body Mass A repeated measures analysis of variance (rANOVAs) was carried out. The following factors were used as between-subjects factors: Treatment (VEH, SMK, ETOH, and SMK+ETOH) and Sex. Day was considered the within-subjects factor. Data Analysis of L0, L3, L24, and (L3 – L0)/L0 and (L24 – L0)/L0 Indices Data are compiled as means and standard errors. To reduce the likelihood of type 1 statistical errors that might result from repeated testing of the global L0, L3, and L24 dataset, results were evaluated first by a global rANOVA on all factors: Treatment, Age (PN44-45, PN49-50, PN74-75), and Sex. Session (for L0, L3, and L24 data and for (L3 − L0)/L0 and (L24  − L0)/L0 indices) was considered the within-subjects factor. Significant Treatment interactions were followed by lower order ANOVAs and by pairwise post-hoc analyses by using Fisher’s Protected Least Significant Difference (FPLSD) tests. Main treatment effects were followed by FPLSD. Figures were only segmented by sex when significant Treatment × Sex interactions were observed. Effects were considered significant when p