688 Cellular, molecular and developmental neuroscience
Enriched environment attenuates nicotine self-administration and induces changes in DFosB expression in the rat prefrontal cortex and nucleus accumbens Arturo Venebra-Mun˜oza, Aleph Corona-Moralesb, Juan Santiago-Garcı´ac, Montserrat Melgarejo-Gutie´rreza, Mario Cabad and Fabio Garcı´a-Garcı´aa Environment enrichment conditions have important consequences on subsequent vulnerability to drugs of abuse. The present work examined whether exposure to an enriched environment (EE) decreases oral selfconsumption of nicotine. Wistar rats were housed either in a standard environment (SE, four rats per standard cage) or in an EE during 60 days after weaning. EE consisted of eight animals housed in larger cages containing a variety of objects such as boxes, toys, and burrowing material that were changed three times a week. After this period, animals were exposed to nicotine for 3 weeks, where animals chose freely between water and a nicotine solution (0.006% in water). Fluid consumption was evaluated on a daily basis. DFosB immunohistochemistry in the prefrontal cortex and nucleus accumbens was also performed. Rats of the EE group consumed less nicotine solution (0.25±0.04 mg/kg/day) than SE rats (0.54±0.05 mg/kg/day). EE increased the number of DFos-immunoreactive (DFos-ir) cells in the nucleus accumbens core and shell and in the prefrontal cortex, compared with animals in the standard condition.
Introduction Compared with standard housing, an enriched environment (EE), for rats, provides a combination of increased physical exercise, social interactions, and continual exposure to learning tasks, as well as attenuates stress responses, which collectively induce morphological, physiological, and behavioral changes in the rodent brain [1,2]. As a result, improvements in learning and memory are observed [3]. Furthermore, a large body of evidence shows some degree of protection from brain damage or against the deleterious effects of aging in animals subjected to EE [4]. Importantly, housing animals in EE reduces the rewarding effects of psychostimulants, such as amphetamine, cocaine, and morphine [5–7], possibly by reducing the hedonic threshold of a drug of addiction. An EE also decreases the hyperlocomotor activity stimulated by subcutaneous injections of nicotine [8,9]. Nicotine, a psychoactive component of tobacco with strong addictive properties, is known to produce a variety of motivational effects. Around half of the individuals who try tobacco develop nicotine dependence [10]; therefore, understanding the conditions that regulate sensitivity to the rewarding properties of nicotine is an issue of substantial importance. Preclinical research has identified risk factors as strong and translatable c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
However, rats exposed to nicotine in the SE showed higher DFos-ir cells in the nucleus accumbens core and shell than nonexposed rats. Nicotine consumption did not modify DFos-ir cells in these brain areas in EE animals. These results support the idea of a possible protective effect of the EE on reward sensitivity and the development of an c addictive behavior to nicotine. NeuroReport 25:688–692 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:688–692 Keywords: addiction, dopaminergic system, neuroplasticity, nicotine a
Biomedicine Department, Health Sciences Institute, bPhysiology Laboratory, School of Nutrition, cBiological Sciences Institute and dBiomedical Research Center, Veracruzana University, Veracruz, Me´xico Correspondence to Fabio Garcı´a-Garcı´a, PhD, Health Sciences Institute, Veracruzana University, Av. Luis Castelazo s/n, Industrial Animas, Xalapa, Veracruz 91190, Me´xico Tel: + 52 228 8418925; fax: + 52 228 8418926; e-mail: [email protected] Present address: Arturo Venebra-Mun˜oz, Sciences School, Me´xico State University, Toluca 50000, Edo., Me´xico Received 28 January 2014 accepted 28 February 2014
predictors of nicotine abuse and initiation to drugs. Among these, social factors, adolescence, and environmental conditions are involved [11]. It has been shown that after administration of many drugs of abuse, proteins of the Fos family (transcription factors), which includes c-Fos, FosB, Fra1, Fra2, and DFosB, encoded by the fosB gene, are rapidly and transiently induced in specific brain regions related to addiction [12]. Particularly, DFosB expression is increased in the nucleus accumbens (NA) and dorsal striatum after the administration of numerous addictive drugs, including nicotine [13]. It has been suggested that the induction of DFosB in the NA facilitates the development of the addictive state [14]. As addiction is a result of genotype–environment interactions, we analyzed whether a different living environment EE versus a standard environment (SE) has an effect on nicotine self-administration and on DFosB expression in the prefrontal cortex (PFC) and NA in Wistar rats.
Materials and methods We used male Wistar rats that were 21 days old, weighing 60.5±2.4 g at the beginning of the experiments, obtained DOI: 10.1097/WNR.0000000000000157
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Environment modifies nicotine drinking Venebra-Mun˜oz et al. 689
from our animal facility. Subjects were housed in a temperature-controlled room under a 12/12 h dark/light cycle with ad-libitum access to food and water. All experimental procedures were approved and conducted according to the Institutional Ethical Committee, in agreement with national (NOM-062-ZOO-2003) and international guidelines (Society for Neuroscience) for the production, care, and use of laboratory animals. After weaning (21 days old, experimental day – ED1), animals from different litters were randomly distributed and housed in two different environmental conditions for 81 days (ED81). Rats under SE conditions were housed four per cage (47 33 19 cm) with no other stimuli, whereas animals under EE conditions were housed eight per cage (75 60 60 cm). Two different enrichment cages were used for the experiments, which contained two levels with ramps and freely manageable objects such as plastic tubes, polypropylene balls, burrowing material, and toys of different forms, sizes, and textures. EE animals were transferred every third day from one enrichment cage to the other, which had similar materials but a different spatial configuration. Brief bedding change and body weight recordings were made every third day, avoiding any other type of stimulation. After 2 months under these environmental conditions (SE or EE), nicotine consumption was initiated using the two-bottle free-choice test, where animals could choose freely between water or nicotine solution (0.006%, N5260; Sigma, St Louis, Missouri, USA) as described previously [15]. Daily liquid consumption and body weight were measured every morning at 8:00, during 3 weeks, from ED61 to ED81. The position of the two bottles in the cage was frequently exchanged to discard a place preference. The experiments were conducted twice with a total of 16 rats under the SE condition and 16 under the EE condition. After the nicotine test, in ED81, five animals per group were killed and their brains were processed for DFosB immunodetection. To distinguish changes in DFosB expression exclusively because of our environment conditions from those because of nicotine consumption, five animals from each group (SE and EE) without the nicotine test were killed at ED81 and their brains were also processed for DFosB immunohistochemistry.
reward and addiction [17]. Briefly, tissue sections were incubated with 0.5% hydrogen peroxide for 10 min, followed by a FosB antibody, 1 : 500 in phosphate buffer with 0.3% Triton X-100 (sc-48; Santa Cruz Biotechnology, Santa Cruz, California, USA) for 96 h at 41C, and with a biotinylated goat anti-rabbit antibody (81-6140; Invitrogen, Carlsbad, California, USA), 1 : 250 for 2 h at room temperature. The FosB antibody was raised against an internal region of FosB that recognizes both FosB and DFosB proteins [18]. Reaction was visualized with a solution of 0.06% diaminobenzidine, 1% nickel sulfate, and 1% cobalt chloride. In all cases, tissue sections from each of the different experiments were processed together. Control sections were processed as above, but without the primary antibody. Quantification of DFosB-ir cells in brain sections
DFosB-immunoreactivity was identified as a black-purple precipitate from the DAB-nickel/cobalt reaction in the cell nucleus. All slides were coded and DFosB-ir nuclei were counted in both hemispheres by two observers blinded to the experimental condition of subjects with a rectangular grid (6440 mm2) using a Nikon microscope (Eclipse E200; Nikon Instruments Inc., Melville, New York, USA) with a 40 objective. Four tissue sections per structure were analyzed [17] using an imaging analysis freeware (ImageJ 1.34; NIH, Bethesda, Maryland, USA). Statistical analysis
Results are expressed as mean±SEM. Data from nicotine consumption were compared between SE and EE groups using Student’s t-test. To determine significant differences in cell numbers of DFosB-ir nuclei under each condition (environment and nicotine consumption), a generalized linear model was fitted, using the number of cells (NC) as the dependent variable, on the basis of the two-way factorial analysis of variance model: NC = Nicotine + Environment + Nicotine Environment. The data were tested previously for homogeneity of variances and normality and rank-transformed when necessary. The analyses were carried out using JMP (Statistica 7; StatSoft Inc., Tulsa, Oklahoma, USA, 1984–2004), and post-hoc tests and Fisher test were also performed to compare the means. Values were considered statistically significant at a P value of up to 0.05.
Results DFosB immunohistochemistry
Nicotine consumption
Rats were deeply anesthetized with sodium pentobarbital intraperitoneally and transcardially perfused with saline solution (0.9%), followed by paraformaldehyde (4%) in 0.1 M phosphate buffer pH 7.4. Brains were removed, postfixed overnight, and then equilibrated to a gradient of sucrose solutions (10, 20, and 30%). Coronal sections of 40 mm of the PFC (Bregma 3.20 mm) and NA core and shell (Bregma 1.60 mm) [16] were obtained. These brain regions were selected because they are implicated in drug
Measurements taken throughout the 81 days of the experiments showed no difference in body weight gain between the SE and the EE groups (2.9±0.3 and 2.8±0.3 g/day/animal, respectively, t = 0.16, P > 0.05). The total fluid intake (tap water + nicotine solution) was also similar between groups during the 21 days of the nicotine test (SE, 37.9±1.0 vs. EE, 37.8±0.7 ml/day/ animal; t = 0.01, P > 0.05). In contrast, we observed a clear difference in the dose of nicotine ingested; SE rats
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690 NeuroReport 2014, Vol 25 No 9
consumed 0.54±0.05 mg/kg/day, whereas EE animals consumed 0.25±0.04 mg/kg/day (t = – 4.69, P < 0.01, Fig. 1). DFosB-ir cells in different brain regions
The mean number of DFos-ir cells in the NA core was higher in EE than SE animals under basal conditions (EE 33.55±2.7; SE 15.09±1.82; Fig. 2). SE animals exposed to nicotine showed a large increase in DFos-ir cells in the NA core; however, EE animals exposed to nicotine did not show a significant effect on DFos-ir cells (SE 37.63±2.59; EE 41.82±2.74; Fig. 2). Statistical analysis indicated a significant effect of the environment (SE vs. EE) (F1, 33 = 7.4; P < 0.001); of the treatment (basal vs. nicotine) (F1, 33 = 12.7; P < 0.001); the number of DFosir cells was lower under basal conditions. However, the interaction environment treatment did not show a difference (F1, 33 = 2.6; P = 0.1). Similar to the NA core, the number of DFos-ir cells in the NA shell was higher in EE than SE animals under basal conditions (EE 25.12±1.93; SE 12.59±1.48; Fig. 2), and SE animals exposed to nicotine showed an increase in the number of DFos-ir cells, whereas EE animals did not (SE 36.05±1.88; EE 28.61±1.86; Fig. 2). The statistical analysis indicated a significant difference between basal and nicotine groups (F1, 34 = 42; P < 0.001) and the interaction environment treatment (F1, 34 = 20.3; P < 0.001); however, no difference was found in the number of DFos-ir cells between environments (SE vs. EE) (F1, 34 = 0.4; P < 0.5). Fig. 1
Nicotine consumption per rat (mg/kg/day)
0.7
0.6
0.5
0.4 ∗∗
0.3
0.2
0.1
0.0
SE
EE
Nicotine consumption. Rats reared under a standard environment (SE, n = 16) consumed twice as much nicotine than those under an enriched environment (EE, n = 16) conditions. Nicotine consumption was recorded daily during 21 days of the two-bottle free-choice test. Data are expressed as mean±SEM (t = – 4.69, **P < 0.01).
In terms of the PFC, the number of DFos-ir cells was lower in SE than EE animals, both under basal and under nicotine conditions (basal SE 8.66±0.58, EE 30.42±2.17; nicotine SE 8.67±1.12, EE 19.22±1.37; Fig. 2). The statistical analysis showed significant differences between environments (F1, 35 = 60.9; P < 0.001) and the interaction environment treatment between groups (F1, 35 = 5; P = 0.03); however, no difference was found in response to the treatment (nicotine) (F1, 35 = 2.8; P = 0.1).
Discussion Our results show that animals reared under EE consumed less nicotine than those under standard conditions, suggesting a protective role of the EE in the development of addictive behaviors to this substance. Similar results have been reported for other addictive drugs [6,19,20]. In addition, several studies have reported that palatability does not play a role in voluntary selection of nicotine [15]. The behavioral effects of nicotine are mediated in part by dopamine transmission in the mesolimbic pathway. This pathway originates in the ventral tegmental area (VTA) and terminates in several forebrain structures including the NA, which has been shown to play an important role in mediating drug addiction [21]. However, it has been suggested that EE reduces the hedonic threshold of a drug of addiction and that animals reared under enriched conditions show greater sensitivity to the effects of psychostimulant drugs [22]. Our data strengthen this hypothesis and also reinforce previous studies showing that environmental conditions are sufficient to modify the sensitivity of the reward system [20,22,23]. Consistent with this, we found an increased number of DFosB-positive cells in the PFC of EE animals. DFosB is a transcription factor and also a secondary indicator of neuronal activity and neuronal plasticity implicated in the transcription of genes encoding neurotrophic factors or structural proteins in neurons [13]. In the current study, the number of DFosB-ir cells in the PFC was increased by the EE, but not by nicotine consumption, suggesting a higher level of neuronal activity. It has been suggested that increased neuronal activity in this region involves structural changes or induction of transcription factors promoted by EE [6,24]. Several studies have reported that the PFC is a region highly susceptible to the development of neural plasticity induced by environmental enrichment [24,25]. Therefore, the increase in DFosB-ir observed in the PFC may be because of the constant sensory stimulation provided by environmental enrichment, as in other brain regions such as the striatum [6]. PFC is a brain region involved in integrating information from different structures including the NA, hippocampus, and amygdala. PFC also sends projections to the striatum and VTA. Interestingly, the number of DFosB-ir cells in
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Environment modifies nicotine drinking Venebra-Mun˜oz et al.
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Fig. 2
Basal-SE
Basal-EE
Nicotine-SE
Nicotine-EE
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
PFC
PiL
IL
NA-shell
LV Aca
(i)
(j)
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B
50
SE Number of ΔFosB-ir cells means ± SEM
EE C
40
C B 30
B B
20
A A
0
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Nicotine
Basal
NA-core
Nicotine NA-shell
Basal
Nicotine PFC
DFosB-ir cells in different brain areas of SE or EE animals without (basal) or with nicotine consumption. The upper left side of the figure shows drawings indicating the brain regions where DFosB-ir cells were counted, in the prefrontal cortex (PFC) and nucleus accumbens shell (gray squares). Microphotographs of DFosB-ir cells in PFC (a–d) and nucleus accumbens shell (NA shell) (e–h) of rats reared under standard (SE) or enriched environment (EE) conditions. (i) DFosB-ir cells in the nucleus accumbens (NA) core. The number of positive cells was increased by both nicotine and enriched environment. (j) DFosB-ir cells in the NA shell. The number of positive cells was higher in EE than SE animals under basal conditions; however, nicotine consumption increased the number of DFosB-ir cells only in SE animals. (k) DFosB-positive cells in the PFC. The number of positive cells were increased by the enriched environment, but not by nicotine. Results are means±SEM, P < 0.001. Bars with different letters (A, B, C) are statistically different between them. Aca, anterior commissure; IL, infralimbic cortex; LV, lateral ventricle; PrL, prelimbic cortex. Magnification bar = 30 mm.
the PFC is lower in EE animals after nicotine consumption compared with EE animals not exposed to nicotine. Similar results have been found for cocaine administration in the striatum of EE rats [6]. The number of DFosB-positive cells in the NA shell increased as a result of the EE, suggesting that the
environment has an effect on the neuronal activity in this brain region. The NA shell is the purely limbic region of the accumbens, closely related to the amygdala and hippocampus, with a role in information processing of stimuli associated with the reinforcing effect of consumption of drugs [21]. Several studies have shown that nicotine increases dopamine release in the NA shell by fibers from the VTA [21]. In addition, the
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NA shell is the region most susceptible to the motivational mechanisms in the consumption of drugs. Nicotine exerts its effect in the NA shell through the activation of VTA dopaminergic neurons, thereby increasing dopamine levels and consequent activation of the neurons in the NA shell. In accordance with this, we observed that animals under standard conditions exposed to nicotine had higher levels of DFosB-ir than those not exposed. Notably, this effect was not observed under EE conditions, where nicotine exposure had no effect on DFosB-ir in the NA shell. This phenomenon was also observed in the NA core.
Conclusion Our results support the idea of a possible protective effect of the EE on reward sensitivity and on the development of an addictive behavior to nicotine, possibly by preventing the increase in neuronal activity promoted by nicotine, beyond the stimulation of EE.
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Acknowledgements The authors thank Oscar Prospero Garcı´a, MD, PhD, for reviewing the manuscript, Armando Martı´nez Chaco´n, PhD, for his help with the statistical analysis, Diana Milla´n-Aldaco for her technical support, and Mercedes Acosta for providing the animals.
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This work was supported by grants PROMEP/103.5/12/ 3510, 61344 to A.C.M. from CONACYT and 133178 to F.G.G. from CONACYT. Conflicts of interest
There are no conflicts of interest.
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References 1
2
3
4
Faherty CJ, Kerley D, Smeyne RJA. Golgi-Cox morphological analysis of neuronal changes induced by environmental enrichment. Brain Res Dev Brain Res 2003; 141:55–61. Nittianantharajah J, Hannan J. Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nat Rev Neurosci 2006; 7:697–709. Leggio MG, Mandolesi L, Federico F, Spirito F, Ricci B, Gelfo F. Environmental enrichment promotes improved spatial abilities and enhanced dendritic growth in the rat. Behav Brain Res 2005; 163:78–90. Andrade de Sousa A, Reis R, Bento-Torres J, Tre´via N, Lins N, Passos A, et al. Influence of enriched environment on viral encephalitis outcomes: behavioral and neurophatological changes in albino swiss mice. PLoS One 2011; 6:15597.
22
23
24
25
Bardo MT, Klebaur J, Valone J, Deaton C. Environmental enrichment decreases intravenous self-administration of amphetamine in female and male rats. Psychopharmacology (Berl) 2001; 155:278–284. Solinas M, Thiriet N, El Rawas R, Lardeux V, Jaber M. Environmental enrichment during early stages of life reduces the behavioral, neurochemical, and molecular effects of cocaine. Neuropsychopharmacology 2009; 34:1102–1111. Xu Z, Hou B, Gao Y, He F, Zhang C. Effects of enriched environment on morphine-induced reward in mice. Exp Neurol 2007; 204:714–719. Green T, Cain M, Thompson M, Bardo MT. Environmental enrichment decreases nicotine-induced hyperactivity in rats. Psychopharmacology (Berl) 2003; 170:235–241. Coolon RA, Cain ME. Individual differences in response to novelty and the conditioned locomotor effects of nicotine. Behav Pharmacol 2009; 20: 322–329. Pomerleau CS. Is mental health in childhood a major predictor of smoking in childhood? Addiction 1998; 93:1878. Redolat R, Pe´rez-Martı´nez M, Carrasco C, Meza P. Individual difference in novelty-seeking and behavioral response to nicotine: a review of animal studies. Curr Drug Abuse Rev 2009; 2:230–242. Kelz MB, Nestler EJ. DeltaFosB: a molecular switch underlying long-term neural plasticity. Curr Opin Neurol 2000; 13:715–720. Nestler EJ. Transcriptional mechanisms of addiction: role of deltaFosB. Philos Trans R Soc Lond B Biol Sci 2008; 363:3245–3255. Nestler EJ. Molecular mechanisms of drug addiction. Neuropharmacology 2004; 47:24–32. Dadmarz M, Vogel WH. Individual self-administration of nicotine by rats. Pharmacol Biochem Behav 2003; 76:425–432. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego, CA: Elsevier Academic Press; 2005. Perrotti LI, Weaver RR, Robison B, Renthal W, Maze I, Yazdani S, et al. Distinct patterns of DeltaFosB induction in brain by drugs of abuse. Synapse 2008; 62:358–369. Harris GC, Hummel M, Wimmer M, Mague SD, Aston-Jones G. Elevations of FosB in the nucleus accumbens during forced cocaine abstinence correlate with divergent changes in reward function. Neuroscience 2007; 147: 583–591. Laviola G, Hannan AJ, Macri S, Solinas M, Jaber M. Effects of enriched environment on animals models of neurodegenerative diseases and psychiatric disorders. Neurobiol Dis 2008; 31:159–168. Stairs DJ, Bardo MT. Neurobehavioral effects of environmental enrichment and drug abuse vulnerability. Pharmacol Biochem Behav 2009; 92: 377–382. Di Chiara G. Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 2002; 137:75–114. Rahman S, Bardo MT. Environmental enrichment increases amphetamineinduced glutamate neurotransmission in the nucleus accumbens: a neurochemical study. Brain Res 2008; 1197:40–46. Bardo MT, Robinet PM, Hammer JR. Effect of differential rearing environments on morphine-induced behaviors, opioid receptors and dopamine synthesis. Neuropharmacology 1997; 36:251–259. Kolb B, Gorny G, So¨derpalm AHV, Robinson TE. Environmental complexity has different effects on the structure of neurons in the prefrontal cortex versus the parietal cortex or nucleus accumbens. Synapse 2003; 48: 149–153. Zhu J, Apparsundaram S, Bardo MT, Dwoskin LP. Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex. J Neurochem 2005; 93:1434–1443.
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Enriched environment attenuates nicotine self-administration and induces changes in DFosB expression in the rat prefrontal cortex and nucleus accumbens Arturo Venebra-Mun˜oza, Aleph Corona-Moralesb, Juan Santiago-Garcı´ac, Montserrat Melgarejo-Gutie´rreza, Mario Cabad and Fabio Garcı´a-Garcı´aa Environment enrichment conditions have important consequences on subsequent vulnerability to drugs of abuse. The present work examined whether exposure to an enriched environment (EE) decreases oral selfconsumption of nicotine. Wistar rats were housed either in a standard environment (SE, four rats per standard cage) or in an EE during 60 days after weaning. EE consisted of eight animals housed in larger cages containing a variety of objects such as boxes, toys, and burrowing material that were changed three times a week. After this period, animals were exposed to nicotine for 3 weeks, where animals chose freely between water and a nicotine solution (0.006% in water). Fluid consumption was evaluated on a daily basis. DFosB immunohistochemistry in the prefrontal cortex and nucleus accumbens was also performed. Rats of the EE group consumed less nicotine solution (0.25±0.04 mg/kg/day) than SE rats (0.54±0.05 mg/kg/day). EE increased the number of DFos-immunoreactive (DFos-ir) cells in the nucleus accumbens core and shell and in the prefrontal cortex, compared with animals in the standard condition.
Introduction Compared with standard housing, an enriched environment (EE), for rats, provides a combination of increased physical exercise, social interactions, and continual exposure to learning tasks, as well as attenuates stress responses, which collectively induce morphological, physiological, and behavioral changes in the rodent brain [1,2]. As a result, improvements in learning and memory are observed [3]. Furthermore, a large body of evidence shows some degree of protection from brain damage or against the deleterious effects of aging in animals subjected to EE [4]. Importantly, housing animals in EE reduces the rewarding effects of psychostimulants, such as amphetamine, cocaine, and morphine [5–7], possibly by reducing the hedonic threshold of a drug of addiction. An EE also decreases the hyperlocomotor activity stimulated by subcutaneous injections of nicotine [8,9]. Nicotine, a psychoactive component of tobacco with strong addictive properties, is known to produce a variety of motivational effects. Around half of the individuals who try tobacco develop nicotine dependence [10]; therefore, understanding the conditions that regulate sensitivity to the rewarding properties of nicotine is an issue of substantial importance. Preclinical research has identified risk factors as strong and translatable c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
However, rats exposed to nicotine in the SE showed higher DFos-ir cells in the nucleus accumbens core and shell than nonexposed rats. Nicotine consumption did not modify DFos-ir cells in these brain areas in EE animals. These results support the idea of a possible protective effect of the EE on reward sensitivity and the development of an c addictive behavior to nicotine. NeuroReport 25:688–692 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:688–692 Keywords: addiction, dopaminergic system, neuroplasticity, nicotine a
Biomedicine Department, Health Sciences Institute, bPhysiology Laboratory, School of Nutrition, cBiological Sciences Institute and dBiomedical Research Center, Veracruzana University, Veracruz, Me´xico Correspondence to Fabio Garcı´a-Garcı´a, PhD, Health Sciences Institute, Veracruzana University, Av. Luis Castelazo s/n, Industrial Animas, Xalapa, Veracruz 91190, Me´xico Tel: + 52 228 8418925; fax: + 52 228 8418926; e-mail: [email protected] Present address: Arturo Venebra-Mun˜oz, Sciences School, Me´xico State University, Toluca 50000, Edo., Me´xico Received 28 January 2014 accepted 28 February 2014
predictors of nicotine abuse and initiation to drugs. Among these, social factors, adolescence, and environmental conditions are involved [11]. It has been shown that after administration of many drugs of abuse, proteins of the Fos family (transcription factors), which includes c-Fos, FosB, Fra1, Fra2, and DFosB, encoded by the fosB gene, are rapidly and transiently induced in specific brain regions related to addiction [12]. Particularly, DFosB expression is increased in the nucleus accumbens (NA) and dorsal striatum after the administration of numerous addictive drugs, including nicotine [13]. It has been suggested that the induction of DFosB in the NA facilitates the development of the addictive state [14]. As addiction is a result of genotype–environment interactions, we analyzed whether a different living environment EE versus a standard environment (SE) has an effect on nicotine self-administration and on DFosB expression in the prefrontal cortex (PFC) and NA in Wistar rats.
Materials and methods We used male Wistar rats that were 21 days old, weighing 60.5±2.4 g at the beginning of the experiments, obtained DOI: 10.1097/WNR.0000000000000157
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Environment modifies nicotine drinking Venebra-Mun˜oz et al. 689
from our animal facility. Subjects were housed in a temperature-controlled room under a 12/12 h dark/light cycle with ad-libitum access to food and water. All experimental procedures were approved and conducted according to the Institutional Ethical Committee, in agreement with national (NOM-062-ZOO-2003) and international guidelines (Society for Neuroscience) for the production, care, and use of laboratory animals. After weaning (21 days old, experimental day – ED1), animals from different litters were randomly distributed and housed in two different environmental conditions for 81 days (ED81). Rats under SE conditions were housed four per cage (47 33 19 cm) with no other stimuli, whereas animals under EE conditions were housed eight per cage (75 60 60 cm). Two different enrichment cages were used for the experiments, which contained two levels with ramps and freely manageable objects such as plastic tubes, polypropylene balls, burrowing material, and toys of different forms, sizes, and textures. EE animals were transferred every third day from one enrichment cage to the other, which had similar materials but a different spatial configuration. Brief bedding change and body weight recordings were made every third day, avoiding any other type of stimulation. After 2 months under these environmental conditions (SE or EE), nicotine consumption was initiated using the two-bottle free-choice test, where animals could choose freely between water or nicotine solution (0.006%, N5260; Sigma, St Louis, Missouri, USA) as described previously [15]. Daily liquid consumption and body weight were measured every morning at 8:00, during 3 weeks, from ED61 to ED81. The position of the two bottles in the cage was frequently exchanged to discard a place preference. The experiments were conducted twice with a total of 16 rats under the SE condition and 16 under the EE condition. After the nicotine test, in ED81, five animals per group were killed and their brains were processed for DFosB immunodetection. To distinguish changes in DFosB expression exclusively because of our environment conditions from those because of nicotine consumption, five animals from each group (SE and EE) without the nicotine test were killed at ED81 and their brains were also processed for DFosB immunohistochemistry.
reward and addiction [17]. Briefly, tissue sections were incubated with 0.5% hydrogen peroxide for 10 min, followed by a FosB antibody, 1 : 500 in phosphate buffer with 0.3% Triton X-100 (sc-48; Santa Cruz Biotechnology, Santa Cruz, California, USA) for 96 h at 41C, and with a biotinylated goat anti-rabbit antibody (81-6140; Invitrogen, Carlsbad, California, USA), 1 : 250 for 2 h at room temperature. The FosB antibody was raised against an internal region of FosB that recognizes both FosB and DFosB proteins [18]. Reaction was visualized with a solution of 0.06% diaminobenzidine, 1% nickel sulfate, and 1% cobalt chloride. In all cases, tissue sections from each of the different experiments were processed together. Control sections were processed as above, but without the primary antibody. Quantification of DFosB-ir cells in brain sections
DFosB-immunoreactivity was identified as a black-purple precipitate from the DAB-nickel/cobalt reaction in the cell nucleus. All slides were coded and DFosB-ir nuclei were counted in both hemispheres by two observers blinded to the experimental condition of subjects with a rectangular grid (6440 mm2) using a Nikon microscope (Eclipse E200; Nikon Instruments Inc., Melville, New York, USA) with a 40 objective. Four tissue sections per structure were analyzed [17] using an imaging analysis freeware (ImageJ 1.34; NIH, Bethesda, Maryland, USA). Statistical analysis
Results are expressed as mean±SEM. Data from nicotine consumption were compared between SE and EE groups using Student’s t-test. To determine significant differences in cell numbers of DFosB-ir nuclei under each condition (environment and nicotine consumption), a generalized linear model was fitted, using the number of cells (NC) as the dependent variable, on the basis of the two-way factorial analysis of variance model: NC = Nicotine + Environment + Nicotine Environment. The data were tested previously for homogeneity of variances and normality and rank-transformed when necessary. The analyses were carried out using JMP (Statistica 7; StatSoft Inc., Tulsa, Oklahoma, USA, 1984–2004), and post-hoc tests and Fisher test were also performed to compare the means. Values were considered statistically significant at a P value of up to 0.05.
Results DFosB immunohistochemistry
Nicotine consumption
Rats were deeply anesthetized with sodium pentobarbital intraperitoneally and transcardially perfused with saline solution (0.9%), followed by paraformaldehyde (4%) in 0.1 M phosphate buffer pH 7.4. Brains were removed, postfixed overnight, and then equilibrated to a gradient of sucrose solutions (10, 20, and 30%). Coronal sections of 40 mm of the PFC (Bregma 3.20 mm) and NA core and shell (Bregma 1.60 mm) [16] were obtained. These brain regions were selected because they are implicated in drug
Measurements taken throughout the 81 days of the experiments showed no difference in body weight gain between the SE and the EE groups (2.9±0.3 and 2.8±0.3 g/day/animal, respectively, t = 0.16, P > 0.05). The total fluid intake (tap water + nicotine solution) was also similar between groups during the 21 days of the nicotine test (SE, 37.9±1.0 vs. EE, 37.8±0.7 ml/day/ animal; t = 0.01, P > 0.05). In contrast, we observed a clear difference in the dose of nicotine ingested; SE rats
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690 NeuroReport 2014, Vol 25 No 9
consumed 0.54±0.05 mg/kg/day, whereas EE animals consumed 0.25±0.04 mg/kg/day (t = – 4.69, P < 0.01, Fig. 1). DFosB-ir cells in different brain regions
The mean number of DFos-ir cells in the NA core was higher in EE than SE animals under basal conditions (EE 33.55±2.7; SE 15.09±1.82; Fig. 2). SE animals exposed to nicotine showed a large increase in DFos-ir cells in the NA core; however, EE animals exposed to nicotine did not show a significant effect on DFos-ir cells (SE 37.63±2.59; EE 41.82±2.74; Fig. 2). Statistical analysis indicated a significant effect of the environment (SE vs. EE) (F1, 33 = 7.4; P < 0.001); of the treatment (basal vs. nicotine) (F1, 33 = 12.7; P < 0.001); the number of DFosir cells was lower under basal conditions. However, the interaction environment treatment did not show a difference (F1, 33 = 2.6; P = 0.1). Similar to the NA core, the number of DFos-ir cells in the NA shell was higher in EE than SE animals under basal conditions (EE 25.12±1.93; SE 12.59±1.48; Fig. 2), and SE animals exposed to nicotine showed an increase in the number of DFos-ir cells, whereas EE animals did not (SE 36.05±1.88; EE 28.61±1.86; Fig. 2). The statistical analysis indicated a significant difference between basal and nicotine groups (F1, 34 = 42; P < 0.001) and the interaction environment treatment (F1, 34 = 20.3; P < 0.001); however, no difference was found in the number of DFos-ir cells between environments (SE vs. EE) (F1, 34 = 0.4; P < 0.5). Fig. 1
Nicotine consumption per rat (mg/kg/day)
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Nicotine consumption. Rats reared under a standard environment (SE, n = 16) consumed twice as much nicotine than those under an enriched environment (EE, n = 16) conditions. Nicotine consumption was recorded daily during 21 days of the two-bottle free-choice test. Data are expressed as mean±SEM (t = – 4.69, **P < 0.01).
In terms of the PFC, the number of DFos-ir cells was lower in SE than EE animals, both under basal and under nicotine conditions (basal SE 8.66±0.58, EE 30.42±2.17; nicotine SE 8.67±1.12, EE 19.22±1.37; Fig. 2). The statistical analysis showed significant differences between environments (F1, 35 = 60.9; P < 0.001) and the interaction environment treatment between groups (F1, 35 = 5; P = 0.03); however, no difference was found in response to the treatment (nicotine) (F1, 35 = 2.8; P = 0.1).
Discussion Our results show that animals reared under EE consumed less nicotine than those under standard conditions, suggesting a protective role of the EE in the development of addictive behaviors to this substance. Similar results have been reported for other addictive drugs [6,19,20]. In addition, several studies have reported that palatability does not play a role in voluntary selection of nicotine [15]. The behavioral effects of nicotine are mediated in part by dopamine transmission in the mesolimbic pathway. This pathway originates in the ventral tegmental area (VTA) and terminates in several forebrain structures including the NA, which has been shown to play an important role in mediating drug addiction [21]. However, it has been suggested that EE reduces the hedonic threshold of a drug of addiction and that animals reared under enriched conditions show greater sensitivity to the effects of psychostimulant drugs [22]. Our data strengthen this hypothesis and also reinforce previous studies showing that environmental conditions are sufficient to modify the sensitivity of the reward system [20,22,23]. Consistent with this, we found an increased number of DFosB-positive cells in the PFC of EE animals. DFosB is a transcription factor and also a secondary indicator of neuronal activity and neuronal plasticity implicated in the transcription of genes encoding neurotrophic factors or structural proteins in neurons [13]. In the current study, the number of DFosB-ir cells in the PFC was increased by the EE, but not by nicotine consumption, suggesting a higher level of neuronal activity. It has been suggested that increased neuronal activity in this region involves structural changes or induction of transcription factors promoted by EE [6,24]. Several studies have reported that the PFC is a region highly susceptible to the development of neural plasticity induced by environmental enrichment [24,25]. Therefore, the increase in DFosB-ir observed in the PFC may be because of the constant sensory stimulation provided by environmental enrichment, as in other brain regions such as the striatum [6]. PFC is a brain region involved in integrating information from different structures including the NA, hippocampus, and amygdala. PFC also sends projections to the striatum and VTA. Interestingly, the number of DFosB-ir cells in
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Environment modifies nicotine drinking Venebra-Mun˜oz et al.
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Fig. 2
Basal-SE
Basal-EE
Nicotine-SE
Nicotine-EE
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SE Number of ΔFosB-ir cells means ± SEM
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DFosB-ir cells in different brain areas of SE or EE animals without (basal) or with nicotine consumption. The upper left side of the figure shows drawings indicating the brain regions where DFosB-ir cells were counted, in the prefrontal cortex (PFC) and nucleus accumbens shell (gray squares). Microphotographs of DFosB-ir cells in PFC (a–d) and nucleus accumbens shell (NA shell) (e–h) of rats reared under standard (SE) or enriched environment (EE) conditions. (i) DFosB-ir cells in the nucleus accumbens (NA) core. The number of positive cells was increased by both nicotine and enriched environment. (j) DFosB-ir cells in the NA shell. The number of positive cells was higher in EE than SE animals under basal conditions; however, nicotine consumption increased the number of DFosB-ir cells only in SE animals. (k) DFosB-positive cells in the PFC. The number of positive cells were increased by the enriched environment, but not by nicotine. Results are means±SEM, P < 0.001. Bars with different letters (A, B, C) are statistically different between them. Aca, anterior commissure; IL, infralimbic cortex; LV, lateral ventricle; PrL, prelimbic cortex. Magnification bar = 30 mm.
the PFC is lower in EE animals after nicotine consumption compared with EE animals not exposed to nicotine. Similar results have been found for cocaine administration in the striatum of EE rats [6]. The number of DFosB-positive cells in the NA shell increased as a result of the EE, suggesting that the
environment has an effect on the neuronal activity in this brain region. The NA shell is the purely limbic region of the accumbens, closely related to the amygdala and hippocampus, with a role in information processing of stimuli associated with the reinforcing effect of consumption of drugs [21]. Several studies have shown that nicotine increases dopamine release in the NA shell by fibers from the VTA [21]. In addition, the
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NA shell is the region most susceptible to the motivational mechanisms in the consumption of drugs. Nicotine exerts its effect in the NA shell through the activation of VTA dopaminergic neurons, thereby increasing dopamine levels and consequent activation of the neurons in the NA shell. In accordance with this, we observed that animals under standard conditions exposed to nicotine had higher levels of DFosB-ir than those not exposed. Notably, this effect was not observed under EE conditions, where nicotine exposure had no effect on DFosB-ir in the NA shell. This phenomenon was also observed in the NA core.
Conclusion Our results support the idea of a possible protective effect of the EE on reward sensitivity and on the development of an addictive behavior to nicotine, possibly by preventing the increase in neuronal activity promoted by nicotine, beyond the stimulation of EE.
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Acknowledgements The authors thank Oscar Prospero Garcı´a, MD, PhD, for reviewing the manuscript, Armando Martı´nez Chaco´n, PhD, for his help with the statistical analysis, Diana Milla´n-Aldaco for her technical support, and Mercedes Acosta for providing the animals.
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This work was supported by grants PROMEP/103.5/12/ 3510, 61344 to A.C.M. from CONACYT and 133178 to F.G.G. from CONACYT. Conflicts of interest
There are no conflicts of interest.
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References 1
2
3
4
Faherty CJ, Kerley D, Smeyne RJA. Golgi-Cox morphological analysis of neuronal changes induced by environmental enrichment. Brain Res Dev Brain Res 2003; 141:55–61. Nittianantharajah J, Hannan J. Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nat Rev Neurosci 2006; 7:697–709. Leggio MG, Mandolesi L, Federico F, Spirito F, Ricci B, Gelfo F. Environmental enrichment promotes improved spatial abilities and enhanced dendritic growth in the rat. Behav Brain Res 2005; 163:78–90. Andrade de Sousa A, Reis R, Bento-Torres J, Tre´via N, Lins N, Passos A, et al. Influence of enriched environment on viral encephalitis outcomes: behavioral and neurophatological changes in albino swiss mice. PLoS One 2011; 6:15597.
22
23
24
25
Bardo MT, Klebaur J, Valone J, Deaton C. Environmental enrichment decreases intravenous self-administration of amphetamine in female and male rats. Psychopharmacology (Berl) 2001; 155:278–284. Solinas M, Thiriet N, El Rawas R, Lardeux V, Jaber M. Environmental enrichment during early stages of life reduces the behavioral, neurochemical, and molecular effects of cocaine. Neuropsychopharmacology 2009; 34:1102–1111. Xu Z, Hou B, Gao Y, He F, Zhang C. Effects of enriched environment on morphine-induced reward in mice. Exp Neurol 2007; 204:714–719. Green T, Cain M, Thompson M, Bardo MT. Environmental enrichment decreases nicotine-induced hyperactivity in rats. Psychopharmacology (Berl) 2003; 170:235–241. Coolon RA, Cain ME. Individual differences in response to novelty and the conditioned locomotor effects of nicotine. Behav Pharmacol 2009; 20: 322–329. Pomerleau CS. Is mental health in childhood a major predictor of smoking in childhood? Addiction 1998; 93:1878. Redolat R, Pe´rez-Martı´nez M, Carrasco C, Meza P. Individual difference in novelty-seeking and behavioral response to nicotine: a review of animal studies. Curr Drug Abuse Rev 2009; 2:230–242. Kelz MB, Nestler EJ. DeltaFosB: a molecular switch underlying long-term neural plasticity. Curr Opin Neurol 2000; 13:715–720. Nestler EJ. Transcriptional mechanisms of addiction: role of deltaFosB. Philos Trans R Soc Lond B Biol Sci 2008; 363:3245–3255. Nestler EJ. Molecular mechanisms of drug addiction. Neuropharmacology 2004; 47:24–32. Dadmarz M, Vogel WH. Individual self-administration of nicotine by rats. Pharmacol Biochem Behav 2003; 76:425–432. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego, CA: Elsevier Academic Press; 2005. Perrotti LI, Weaver RR, Robison B, Renthal W, Maze I, Yazdani S, et al. Distinct patterns of DeltaFosB induction in brain by drugs of abuse. Synapse 2008; 62:358–369. Harris GC, Hummel M, Wimmer M, Mague SD, Aston-Jones G. Elevations of FosB in the nucleus accumbens during forced cocaine abstinence correlate with divergent changes in reward function. Neuroscience 2007; 147: 583–591. Laviola G, Hannan AJ, Macri S, Solinas M, Jaber M. Effects of enriched environment on animals models of neurodegenerative diseases and psychiatric disorders. Neurobiol Dis 2008; 31:159–168. Stairs DJ, Bardo MT. Neurobehavioral effects of environmental enrichment and drug abuse vulnerability. Pharmacol Biochem Behav 2009; 92: 377–382. Di Chiara G. Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 2002; 137:75–114. Rahman S, Bardo MT. Environmental enrichment increases amphetamineinduced glutamate neurotransmission in the nucleus accumbens: a neurochemical study. Brain Res 2008; 1197:40–46. Bardo MT, Robinet PM, Hammer JR. Effect of differential rearing environments on morphine-induced behaviors, opioid receptors and dopamine synthesis. Neuropharmacology 1997; 36:251–259. Kolb B, Gorny G, So¨derpalm AHV, Robinson TE. Environmental complexity has different effects on the structure of neurons in the prefrontal cortex versus the parietal cortex or nucleus accumbens. Synapse 2003; 48: 149–153. Zhu J, Apparsundaram S, Bardo MT, Dwoskin LP. Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex. J Neurochem 2005; 93:1434–1443.
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