Tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats

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Research Report

Tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats Adrie W. Bruijnzeela,⁎, Rayna M. Bauzoa , Vikram Munikotib , Gene B. Rodricka , Hidetaka Yamadaa , Casimir A. Fornalc , Brandi K. Ormerodb , Barry L. Jacobsc a

Department of Psychiatry, College of Medicine, McKnight Brain Institute, University of Florida, 100 S. Newell Dr., Gainesville FL 32610, USA J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32610, USA c Princeton Neuroscience Institute, Princeton University, Princeton, NJ 08544, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

Brain disorders and environmental factors can affect neurogenesis and gliogenesis in the

Accepted 19 July 2011

hippocampus. These studies investigated the effects of chronic exposure to tobacco smoke

Available online 24 July 2011

on progenitor cell proliferation and the survival and phenotype of new cells in the dentate gyrus of adolescent rats. The rats were exposed to tobacco smoke for 4 h/day for 14 days. To

Keywords:

investigate cell proliferation, the exogenous marker 5-bromo-2′-deoxyuridine (BrdU,

Tobacco smoke

200 mg/kg, ip) was administered 2 h into the 4-h smoke exposure session on day 14. The

BrdU

rats were sacrificed 2–4 h after the administration of BrdU. To investigate cell survival, the

Cell proliferation

same dose of BrdU was administered 24 h before the start of the 14-day smoke exposure

Cell survival

period. These rats were sacrificed 24 h after the last smoke exposure session. Tobacco

Adolescent

smoke exposure decreased both the number of dividing progenitor cells (−19%) and the

Rats

number of surviving new cells (−20%), labeled with BrdU in the dentate gyrus. The decrease in cell proliferation was not associated with an increase in apoptotic cell death, as shown by TUNEL analysis. Colocalization studies indicated that exposure to tobacco smoke decreased the number of new immature neurons (BrdU/DCX-positive) and transition neurons (BrdU/DCX/NeuN-positive) and increased the number of new glial cells (BrdU/GFAPpositive). These findings demonstrate that exposure to tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats. These effects may play a role in the increased risk for depression and cognitive impairment in adolescent smokers. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Tobacco addiction is a chronic disorder that is characterized by a loss of control over smoking, withdrawal symptoms, and relapse after periods of abstinence (American Psychiatric Association, 2000). Smoking has detrimental effects on the

health of smokers and the health of people who are exposed to second hand tobacco smoke. Smoking leads to the premature death of approximately 435,000 people per year in the United States and 3–5 million people worldwide (Ezzati and Lopez, 2003; Mokdad et al., 2004). The positive reinforcing effects of smoking have been suggested to play an important role in the initiation of

⁎ Corresponding author at: University of Florida, Department of Psychiatry, 100 S. Newell Dr. PO Box 100256, Gainesville, FL 32610, USA. Fax: +1 352 392 8217. E-mail address: [email protected] (A.W. Bruijnzeel). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.07.041

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smoking. Abrupt cessation of smoking in humans is characterized by negative affective symptoms including depressed mood and anxiety as well as somatic symptoms such as bradycardia and gastrointestinal discomfort (Hughes et al., 1991). The negative emotional state associated with tobacco withdrawal provides a powerful motivation for the continuation of smoking (Bruijnzeel and Gold, 2005; Koob and Le Moal, 2005). The negative mood state associated with nicotine withdrawal may be mediated by a hyperactivity of brain stress systems (Bruijnzeel et al., 2009, 2010). Smoking has extensive effects on brain function. It has been shown to affect attention, learning and memory, and mood states (Goodman and Capitman, 2000; Jacobsen et al., 2005, 2007). Smoking may alter brain function through its effects on postnatal hippocampal neurogenesis and/or gliogenesis. Neurogenesis or the birth of new neurons, and gliogenesis were first described in adult rats in the 1960s (Altman, 1962, 1963, 1966). Neurogenesis has been detected in many species including birds, fish, and mammals (Altman, 1962; Goldman and Nottebohm, 1983; Raymond and Easter, 1983). There are two areas in the brain where, under normal conditions, neural progenitor cells proliferate and differentiate into significant numbers of new neurons and glial cells. Neural progenitor cells located in the subventricular zone give rise to cells that migrate through the rostral migratory pathway to the olfactory bulb. Neural progenitor cells located in the subgranular zone (SGZ) of the dentate gyrus give rise to neuroblasts that migrate deeper into the granule cell layer (GCL) as they mature primarily into granule neurons and some glial cells (Suh et al., 2009). The studies described in this manuscript focused on investigating whether smoking influences hippocampal neurogenesis as there is evidence for a relationship between mood states, cognitive function, and neurogenesis in the hippocampus (Jacobs et al., 2000; Lu and Chang, 2004). There is also extensive evidence that the rate of neurogenesis can be altered by environmental, chemical, and genetic factors (Zhao et al., 2008). Overall, experimental studies suggest that behaviors or chronic drug treatments that lead to improved mood states promote neurogenesis and vice versa (David et al., 2010). For example, antidepressant drugs and electroconvulsive therapy (ECT) increase progenitor cell proliferation in the dentate gyrus of adult rats (Encinas et al., 2006; Madsen et al., 2000; Nakagawa et al., 2002). In contrast, exposure to stressors, which has been associated with the development of negative mood states, leads to a decrease in cell proliferation in the dentate gyrus of rats (Gould et al., 1997; Malberg and Duman, 2003). Drugs of abuse also have a negative effect on neurogenesis. Chronic administration of alcohol and the self-administration of morphine and heroin have been shown to inhibit cell proliferation and cell survival in rats (Eisch et al., 2000; Scerri et al., 2006). Furthermore, chronic subcutaneous nicotine administration has been shown to inhibit cell proliferation in the dentate gyrus (Scerri et al., 2006). The effect of nicotine on cell survival was not reported in the aforementioned study. Another study reported that chronic nicotine administration decreases the number of mature NeuNpositive neurons in the granule cell layer (Shingo and Kito, 2005). In contrast to the chronic administration of high doses of nicotine, endogenous nicotinic acetylcholine receptor (nAChR) activation may contribute to the maintenance of cell proliferation among progenitor cells and the survival of their progeny.

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This is supported by the observation that cell proliferation is decreased in mice that lack the β2-subunit of the nAChR and cell survival is decreased in mice that lack the α7-subunit of the nAChR (Campbell et al., 2010; Harrist et al., 2004). In order to model smoking in humans more closely, we have developed an animal model in which rats are passively exposed to tobacco smoke (Small et al., 2010; Yamada et al., 2010). Passive exposure to tobacco smoke leads to the development of nicotine dependence as indicated by mecamylamine-induced affective and somatic withdrawal signs (Small et al., 2010; Yamada et al., 2010). Passive exposure to tobacco smoke also leads to the upregulation of central nAChRs, which is a hallmark feature of nicotine dependence (Dani and Heinemann, 1996; Small et al., 2010). The aim of the present studies was to investigate the effects of passive exposure to tobacco smoke on neurogenesis (cell proliferation and survival) and gliogenesis in the dentate gyrus of the hippocampus in adolescent rats. In the cell survival experiment, the phenotype of the new cells was assessed with fluorescent immunostaining for BrdU (new cells), doublecortin (DCX, marker for immature neurons), neuronal nuclei (NeuN, marker for mature neurons), glial fibrillary acidic protein (GFAP, marker for astrocytes), and NG2 (marker for oligodendrocyte precursors). In the cell proliferation experiment, a deoxynucleotidyl transferase-mediated dUTP-nick-end labeling (TUNEL) analysis was done to investigate if exposure to tobacco smoke increased apoptotic cell death in new cells. The rats were exposed to tobacco smoke from postnatal days (PN) 29–42. It has been suggested that PN28–42 is the prototypic age range for adolescence in rats (Spear, 2000). These studies investigated the effects of tobacco smoke on progenitor cell proliferation and cell survival during adolescence because a great majority of the smokers start smoking during this developmental period (Gilman et al., 2003; Schulze and Mons, 2005). Recent studies have started to investigate the effects of nicotine on adolescent rats (O'Dell et al., 2006; Vastola et al., 2002). However, still very little is known about the effects of drugs of abuse on the brain of adolescents. It has been suggested that environmental factors that disrupt normal adolescent brain development may have delayed negative effects later in life (Crews et al., 2007). Therefore, studies into the effects of drugs of abuse on adolescent brain development are of great clinical relevance.

2.

Results

2.1.

Tobacco smoke and cell proliferation and cell death

There were no differences in body weights between the aircontrol group and the tobacco group prior to the onset of the tobacco smoke exposure sessions (Table 1; t(13) = 1.04, n.s.). Exposure to tobacco smoke decreased body weight gain during the exposure period (Table 1; Time: F13,182 = 551.05, P < 0.0001; Time × Treatment: F13,182 = 15.19, P < 0.0001). Proliferating cells in the dentate gyrus were identified by immunostaining for the DNA synthesis marker BrdU (Fig. 1, top panel). As shown in Fig. 2, chronic exposure to tobacco smoke for 14 days significantly decreased the number of dividing (BrdU-positive)

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Table 1 – Effect of tobacco smoke exposure on body weights. Experiment

Pre

Post

Control Tobacco Control Expt. 1 (proliferation) 83.1 ± 3.0 Expt. 2 (survival) 84.4 ± 2.3

88.1 ± 3.9 84.3 ± 2.4

Tobacco

179.0 ± 7.9 161.0 ± 5.5** 180.4 ± 4.8 149.6 ± 5.1**

Data are expressed as means (± S.E.M.). Pre refers to body weights obtained one day prior to the onset of smoke exposure. Post refers to body weights obtained on day 13 of smoke exposure. Asterisks (**P < 0.01) indicate decreased body weight gain over 13 days of tobacco smoke exposure.

cells observed in the dentate gyrus of adolescent rats (−19.0%; t(13) = 2.32; P < 0.05). This suppressant effect was observed in both the dorsal and ventral portions of the dentate gyrus (−20.2% and − 18.4%, respectively; data not shown). When proliferation was examined specifically in the SGZ (the primary site of neuron-producing neural progenitor cells), there was no statistically significant difference between the smoke-exposed and control animals (−10.7%; t(13) = 1.03; n.s.). When the hilus was examined separately, there was a significant decrease in cell proliferation (−30.7%; t(13) = 2.26; P < 0.05), suggesting a more robust effect of tobacco smoke exposure on the production of new cells in this hippocampal

Fig. 2 – Exposure to tobacco smoke decreases cell proliferation in the dentate gyrus of adolescent rats. Cell proliferation was measured in the dentate gyrus (DG) and this total is subdivided into counts in the subgranular zone (SGZ) and hilus. Animals were sacrificed 2–4 h after BrdU injection. Values are means ±S. E.M.; n = 8 for air-control; n = 7 for tobacco smoke exposure. *P < 0.05 vs. air-control. Exposure to tobacco smoke reduced cell proliferation relative to control.

region. In a separate analysis, we examined the proportion of new cells (BrdU-positive) in the SGZ that co-labeled with the apoptotic cell death marker TUNEL (Fig. 3). This analysis revealed no significant difference between the control group and the tobacco group (t(12) = 1.89; n.s.). This suggests that exposure to tobacco smoke did not increase apoptotic cell death in the newborn cells.

2.2.

Fig. 1 – Photomicrographs (1000× magnification) showing BrdU labeling in the dentate gyrus 2–4 h (top, cell proliferation) or 15 days (bottom, cell survival) after BrdU administration (200 mg/kg, ip) to adolescent rats. Immunoreactive cells appear brown in cresyl violet-stained sections. The top panel shows a cluster of newly generated BrdU-positive cells (total of 6) at the border of the granule cell layer (GCL) and the hilus, in the subgranular zone. The bottom panel shows several mature BrdU-positive cells migrating into the GCL. Scale bar = 20 μm.

Tobacco smoke and cell survival

There were no differences in body weights between the rats in the air-control group and rats in the tobacco group prior to the onset of tobacco smoke exposure (Table 1; t(14) = 0.048, n.s.). Exposure to tobacco smoke decreased body weight gain during the exposure period (Table 1; Time: F13,169 = 329.05, P < 0.0001; Time × Treatment: F13,169 = 8.42, P < 0.0001). To determine whether chronic exposure to tobacco smoke affects the survival of new hippocampal cells, we quantified the number of BrdU-labeled cells in the dentate gyrus 15 days after BrdU administration (Fig. 1, bottom panel). As shown in Fig. 4, exposure to tobacco smoke significantly reduced the number of surviving BrdU-positive cells in the dentate gyrus (− 19.9%; t(14) = 2.48; p < 0.05). This effect was observed in both the dorsal and ventral dentate gyrus (− 24.3% and − 17.2%, respectively; data not shown). Similarly, there was a significant decrease in BrdU-positive cell counts in the SGZ/GCL (− 25.1%; t(14) = 2.93; P < 0.05), whereas no significant change in BrdU labeling was seen in the hilus (− 7.8%; t(14) = 0.96; n.s.). The percentage of 15-day old BrdU-positive cells that coexpressed markers of immature neurons (DCX), transitioning neurons (DCX/NeuN), mature neurons (NeuN), astrocytes (GFAP), or oligodendrocyte precursors (NG2) was also quantified (Fig. 5). Fluorescence immunohistochemistry revealed new neurons and glial cells (Figs. 5A, B). As shown in Fig. 5C, exposure to tobacco smoke decreased the proportion of BrdU-positive cells that co-expressed markers for immature (DCX, t(14) = 3.18;

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Fig. 4 – Exposure to tobacco smoke decreases cell survival in the dentate gyrus of adolescent rats. Cell survival was measured in the dentate gyrus (DG) and this total is subdivided into counts in the subgranular zone/granule cell layer (SGZ/GCL) and hilus. Animals were sacrificed 15 days after BrdU injection. Values are means ± S.E.M.; n = 9 for air-control; n = 7 for tobacco smoke exposure. Data were normalized to percentages of control values. Control cell counts were: DG = 14731 ± 863; SGZ/GCL = 10281 ± 698; Hilus = 4449 ± 208. *P < 0.05 vs. air-control.

3.

Fig. 3 – Exposure to tobacco smoke does not affect cell death among dividing neural progenitor cells in the dentate gyrus of adolescent rats. (A) Example of a new (BrdU-positive) cell (red) in the SGZ that was co-labeled with TUNEL (green). The inset shows the cell that is depicted by the arrow and confirms the co-localization of BrdU (red; top panel) and TUNEL (green; middle panel) in a single DAPI-positive nucleus (blue; bottom panel). (B) Percentage of dividing (BrdU-positive) cells in the SGZ that was co-labeled with TUNEL. Values are means ± S.E.M.; n = 8 for air-control; n = 7 for tobacco smoke exposure.

P < 0.007) and transitioning neurons (DCX/NeuN, t(14) = 4.46; P < 0.001) in the SGZ/GCL. Tobacco smoke increased the proportion of new cells that co-expressed the astrocyte marker GFAP (t(14) = 7.19; P < 0.0001) and did not affect the proportion of cells that co-expressed the marker for mature neurons (t(14) = 1.83; n. s.) or the marker for oligodendrocyte precursors (Fig. 5C; t(14) = 0.88; n.s.). In addition, exposure to tobacco smoke decreased the total number of new immature neurons (t(14) = 4.57; P < 0.0001) and the total number of transitioning neurons (t(14) = 3.41; P < 0.005) and increased the total number of astrocytes (t(14) = 3.41; P < 0.005) in the SGZ/GCL (Fig. 5D). Tobacco smoke did not affect the total number of new mature neurons (t(14) = 0.77; n.s.) or oligodendrocyte precursors (t(14) = 0.88; n.s.) in the SGZ/GCL.

Discussion

These studies investigated the effects of exposure to tobacco smoke on progenitor cell proliferation and the survival of new cells in the dentate gyrus of adolescent rats. Exposure to tobacco smoke decreased the total number of dividing progenitor cells in the dentate gyrus (hilus but not SGZ). Exposure to tobacco smoke also decreased the total number of surviving cells in the dentate gyrus (SGZ/GCL but not hilus). Colocalization studies showed that exposure to tobacco smoke decreased the number of young neurons and increased the number of new astrocytes in the SGZ/GCL. Overall these data demonstrate that chronic exposure to tobacco smoke reduces neurogenesis by increasing the vulnerability of immature and transitioning neurons to death and/or by decreasing the probability that neuronal progenitor cells adopt a neuronal fate. In the present study, exposure to tobacco smoke decreased the number of immature (BrdU/DCX-positive) and transitioning neurons (BrdU/DCX/NeuN-positive) but did not affect the number of mature (BrdU/NeuN-positive) neurons. This pattern of results is most likely due to the time course of the expression of the neuronal markers DCX and NeuN in newborn neurons. DCX expression in newborn neurons is high from day 1 to day 14 and then gradually decreases. NeuN expression is very low during the first 14 days and then the expression levels increase (Esposito et al., 2005; Ge et al., 2006; Palmer et al., 2000). Very high levels of NeuN have been detected around day 28 (Esposito et al., 2005; Ge et al., 2006; Palmer et al., 2000). The percentage of newborn neurons that co-express NeuN and DCX increases from day 7 to day 14 and after this time period there is a rapid decrease in the number of cells that co-express NeuN and DCX (Esposito et al., 2005). In our survival study, brains were removed 15 days after the administration of BrdU. This was an optimal time point to detect an effect of tobacco smoke on the number of immature (DCX-positive) and transition neurons (NeuN/DCX-

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Fig. 5 – Exposure to tobacco smoke decreases the number of young neurons in the dentate gyrus of adolescent rats. (A) Confocal image of a dentate gyrus section stained with anti-BrdU (new cells; red), anti-DCX (immature neurons; blue), anti-NeuN (mature neurons; green) and DAPI (nuclei; white) 15 days after BrdU administration. Insets show a new transitioning neuron (BrdU/DCX/NeuN/DAPI-positive; yellow arrow) and a new mature neuron (BrdU/NeuN/DAPI-positive; white arrow). (B) Confocal image of a dentate gyrus section stained with anti-BrdU (new cells; red), anti-GFAP (astrocytes; blue), anti-NG2 (oligodendrocyte precursors; green), and DAPI (nuclei; white) 15 days after BrdU administration. Inset shows a new astrocyte (BrdU/GFAP/DAPI-positive; white arrow). (C) Percentage of new (BrdU-positive, 15 days) cells that acquired neuronal or glial fates in the SGZ/GCL of tobacco smoke and air-control animals. (D) Cumulative effects of tobacco smoke on the proliferation of neuronal progenitor cells and the fate choices and survival of their progeny. The number of cells was calculated by multiplying the total number of new, 15-day old, cells counted in the SGZ/GCL by the proportion of new cells that co-expressed neuronal and glial markers. Values are means ± S.E.M.; n = 9 for air-control; n = 7 for tobacco smoke exposure. *P < 0.05, **P < 0.01 vs. air-control.

positive) but it was too early to detect an effect on mature (NeuN-positive) neurons. The present finding that passive exposure to tobacco smoke decreases cell proliferation in the dentate gyrus is consistent with previous studies reporting that many drugs of abuse have a negative effect on hippocampal cell proliferation. For instance, alcohol has been shown to inhibit cell proliferation in the dentate gyrus of adolescent and adult rats (Morris et al., 2010; Nixon and Crews, 2002). Other drugs of abuse that inhibit cell proliferation include the psychostimulants nicotine and cocaine and the opioids morphine and heroin (Eisch et al., 2000; Scerri et al., 2006; Yamaguchi et al., 2005). In the present study, exposure to tobacco smoke reduced cell proliferation in the SGZ (−10.7%) and hilus (−30.7), but this effect was only significant in the hilus. A similar pattern of results has been observed after the administration of the psychostimulants caffeine and modafinil to rats. Both caffeine and modafinil inhibit cell proliferation in

the hilus but not in the SGZ (Kochman et al., 2009). Furthermore, sleep deprivation induces a greater decrease in cell proliferation in the hilus (−60%) than in the SGZ (−29%) (Tung et al., 2005). These findings suggest that stimulants and sleep deprivation affect cell proliferation in the hilus to a greater extent than cell proliferation in the SGZ. At this point, the functional consequences of this are not known. Whether progenitor cells in the SGZ and hilus represent distinct populations guided by independent mechanisms or a single population that responds differentially in two distinct niches (SGZ versus hilus) is currently unknown. This question would be interesting to address in future work. There are several possible mechanisms by which exposure to tobacco smoke may affect the proliferation of progenitor cells and the fate and survival of their neuronal progeny in the dentate gyrus. First, tobacco smoke may affect cell proliferation by stimulating the release of glutamate in the hippocampus.

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Evidence indicates that nicotine, one of the main psychoactive components of tobacco smoke, promotes the release of glutamate and other excitatory amino acids in the hippocampus (Fedele et al., 1998; McGehee et al., 1995). It has been reported that the N-methyl-D-aspartate (NMDA) receptor agonist NMDA inhibits cell proliferation in the dentate gyrus (Cameron et al., 1995). In contrast, the noncompetitive NMDA receptor antagonist MK-801 and the competitive NMDA antagonist CGP 37849 stimulate cell proliferation in the dentate gyrus (Cameron et al., 1995; Okuyama et al., 2004; Ormerod et al., 2003). Lesioning of the entorhinal cortex, which provides the main excitatory input to the hippocampus, also stimulates cell proliferation (Cameron et al., 1995). Second, tobacco smoke may affect cell proliferation by increasing the release of glucocorticoids. Extensive evidence indicates that smoking increases cortisol levels in humans (Mendelson et al., 2005; Steptoe and Ussher, 2006; Winternitz and Quillen, 1977). Tobacco smoke exposure and nicotine administration have also been shown to increase corticosterone levels in rodents (Andersson et al., 1985; Balfour et al., 1975; Cam et al., 1979). Several studies have shown that glucocorticoids exert a powerful inhibitory effect on cell proliferation. Conversely, adrenalectomy, which prevents the production of corticosterone in rodents, increases cell proliferation and this effect is reversed by corticosterone replacement (Gould et al., 1992). Furthermore, the administration of corticosterone to intact non-adrenalectomized rats inhibits cell proliferation (Cameron and Gould, 1994). It is interesting to note that the NMDA receptor antagonist MK-801 inhibits the corticosteroneinduced decrease in cell proliferation (Cameron et al., 1998). Therefore, it might be possible that corticosterone inhibits cell proliferation by stimulating glutamate release in the hippocampus. At present, very little research has been conducted to investigate the role of glucocorticoids in cell survival. It has been shown that adrenalectomy has a negative effect on the survival of granule cells in the dentate gyrus and that this can be prevented by corticosterone replacement (Gould et al., 1990). Future studies that examine differences in the expression of receptors on regionally distinct populations of dividing progenitor cells and their daughter cells (SGZ versus hilus, for example) may provide important insight into the pathways that influence cell proliferation and the survival of new cells. Third, it was considered a possibility that tobacco smoke exposure decreased cell proliferation by increasing apoptotic cell death. However, there was no difference in the number of BrdU/TUNEL-positive cells in the SGZ of the animals in the tobacco group and the aircontrol group. This suggests that exposure to tobacco smoke does not promote cell death among dividing cells. In future studies, it may be of interest to investigate TUNEL expression in new neurons at various time points following BrdU administration (e.g., 4 h–4 weeks). This time frame is based on the observation that about 80% of the new cells die within one month and very few cells die after this time period (Kempermann et al., 2003). The present results may have clinical implications for adolescent smokers and adolescents exposed to high levels of second hand tobacco smoke. Experimental studies suggest that there is a positive correlation between cell proliferation/ survival and cognitive performance (Zhao et al., 2008). Strains of mice that have a high baseline level of hippocampal neurogenesis outperform strains with lower baseline levels of

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neurogenesis on Morris water maze training trials (Kempermann and Gage, 2002). In addition, factors that increase hippocampal neurogenesis generally improve performance on hippocampusdependent tasks, while those that decrease neurogenesis are associated with performance decrements. For example, exercise increases neurogenesis by promoting progenitor cell proliferation and mice exposed to a running wheel for several weeks exhibit improved Morris water maze performance (Fordyce and Wehner, 1993; Van Praag et al., 1999). Environmental enrichment increases the survival of new cells in the dentate gyrus and also improves performance in the Morris water maze task (Nilsson et al., 1999; Young et al., 1999). Finally, chronic nicotine administration decreases cell proliferation in the dentate gyrus and impairs spatial learning and retention in the Morris water maze task (Scerri et al., 2006). The aforementioned studies suggest that genetic and environmental factors that promote neurogenesis also improve cognition. Clinical studies have shown that adolescent smokers have impairments in working memory performance compared to adolescents who do not smoke (Jacobsen et al., 2005, 2007). The tobacco smoke-induced decrease in cell proliferation and the survival of new neurons could play a role in the cognitive deficits observed in adolescent smokers. In addition to affecting cognition, there is also evidence that cell proliferation and the survival of young neurons play a role in the regulation of mood states. The antidepressant and selective serotonin reuptake inhibitor fluoxetine has been shown to increase progenitor cell proliferation and the survival of new cells in the dentate gyrus (David et al., 2009; Encinas et al., 2006). Further evidence for a role of neurogenesis in the regulation of mood state is provided by the observation that hippocampal x-irradiation inhibits cell proliferation in the dentate gyrus and blocks the antidepressant-like effects of fluoxetine in a variety of behavioral tests (Santarelli et al., 2003). In addition, chronic exposure to stressors has been shown to lead to negative mood states in humans and in animal models (Holsboer, 2000; McEwen, 2003). Chronic stress also leads to a suppression of cell proliferation in the dentate gyrus (Gould et al., 1997; Malberg and Duman, 2003). In contrast, behavioral and environmental manipulations that improve mood states such as running have been shown to increase neurogenesis (Van Praag et al., 1999). Several studies have reported that smoking during adolescence increases the risk for developing clinical depression (Goodman and Capitman, 2000; Wu and Anthony, 1999). Tobacco smoke-induced suppression of cell proliferation and cell survival may contribute to the development of negative mood states in adolescent smokers. It should be noted, however, that the relationship between neurogenesis and mood states is extremely complex. This is illustrated by the fact that a recent study showed that the antidepressant-like effects of fluoxetine are mediated by both neurogenesis dependent and independent mechanisms (David et al., 2009). At present, very little is known about the behavioral implication of the tobacco smoke-induced decrease in neurogenesis and increase in gliogenesis. Additional studies are warranted to investigate the role of neurogenesis and gliogenesis in the behavioral effects of tobacco smoke exposure. In addition, it is not known whether nicotine free cigarettes will have the same effects on neurogenesis and gliogenesis as regular cigarettes.

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4.

Experimental procedures

4.1.

Animals

Male Wistar rats (Charles River, Raleigh, NC, USA) weighing 55– 65 g (21 days of age; n = 31) at the beginning of each experiment were used. Animals were pair-housed in a temperature and humidity-controlled vivarium and maintained on a 12-h reversed light–dark cycle (lights off at 8 AM). All testing occurred at the beginning of the dark cycle. Food and water were available ad libitum in the home cages. All subjects were treated in accordance with the National Institutes of Health guidelines regarding the principles of animal care. Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the University of Florida Institutional Animal Care and Use Committee.

4.2.

Drugs

BrdU was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and dissolved in sterile saline (0.9% sodium chloride). Research cigarettes (3R4F) were purchased from the University of Kentucky (College of Agriculture, Reference Cigarette Program, Lexington, KY, USA). 4.3.

Tobacco smoke exposure

The rats were exposed to tobacco smoke in standard polycarbonate rodent cages (38 × 28 × 20 cm; L × W × H) with corncob bedding and a wire top as previously described by our research group (Small et al., 2010; Yamada et al., 2010). The rats were unrestrained (whole body exposure) during the tobacco smoke exposure sessions and water was freely available. The rats were moved to the exposure cages immediately prior to the tobacco smoke exposure session and returned to their home cages after the exposure session. Tobacco smoke was generated using a microprocessor-controlled cigarette smoking-machine (model TE-10, Teague Enterprises, Davis, CA, USA) (Teague et al., 1994). Tobacco smoke was generated by burning filtered 3R4F reference cigarettes using a standardized smoking procedure (35 cm3 puff volume, 1 puff/min, 2 s/puff). Mainstream and sidestream smoke was transported to a mixing and diluting chamber. The smoking machine produced a mixture of approximately 10% mainstream smoke and 90% sidestream smoke; based on total suspended particle matter. The smoke was aged for 2–4 min and diluted with air to a concentration of 30–100 mg of total suspended particles per m3 before being introduced into the exposure chambers. The concentration of the smoke was dependent on the stage of the experiment. Exposure conditions were monitored for carbon monoxide (CO) and total suspended particulate matter. CO levels were assessed using a continuous CO analyzer that accurately measures levels between 0 and 2000 ppm (Monoxor II, Bacharach, New Kensington, PA, USA). Total suspended particle matter in the exposure chambers was determined by measurement of samples collected from the chamber onto pre-weighed filters. During the experiment, the tobacco smoke concentration and exposure duration were gradually increased according to the following sequence: Day 1,

1 cigarette at a time for 2 h (5 cigarettes/h); Day 2, 1 cigarette at a time for 4 hrs (5 cigarettes/h); Day 3, 2 cigarettes at a time for 4 h (10 cigarettes/h); Day 4 and onward, 3 cigarettes at a time for 4 h (15 cigarettes/h). From days 4 to 14, the average total suspended particulate matter was about 100 mg/m3 and the CO level about 350 ppm. A previous study by our laboratory demonstrated that these tobacco smoke exposure conditions led to nicotine dependence in rats and plasma nicotine levels of approximately 45 ng/ml and cotinine levels of 250 ng/ml (Small et al., 2010). 4.4.

BrdU Administration and Animal Perfusion

The thymidine analog BrdU was dissolved in sterile 0.9% saline (containing 0.007 N NaOH) at a concentration of 20 mg/ml. The BrdU was dissolved immediately before administration. To study cell proliferation, a single BrdU injection (200 mg/kg, ip) was administered during the middle of the last smoke exposure session, as described above. This dose is based on a previous study that investigated the effects of the dose of BrdU on the number of BrdU labeled cells in the dentate gyrus of adult rats (Cameron and McKay, 2001). A relatively high dose of BrdU (200 mg/kg) labels a larger proportion of dividing progenitor cells than lower doses of BrdU and does not label cells that are not in the S-phase (Cameron and McKay, 2001). The 200 mg/kg dose of BrdU was used in previous studies by our group and others that showed that sleep deprivation, maternal deprivation, and psychostimulants decrease cell proliferation in the dentate gyrus of rats (Kochman et al., 2009; Mirescu et al., 2004; Tung et al., 2005). Subjects were perfused 2–4 h after BrdU, at the end of the 4-hr smoke exposure period. To study cell survival, 24-hr prior to the initiation of chronic smoke exposure, a single BrdU injection (200 mg/kg, ip.) was administered, to label dividing progenitor cells in the hippocampus. The effect of tobacco smoke on the subsequent survival of these cells was then evaluated at the end of the study. Subjects were sacrificed 15 days after BrdU, 24 h after the last smoke exposure session. At the end of each experiment, rats were anesthetized deeply with sodium pentobarbital (100 mg/kg, ip) and then perfused transcardially with cold physiological saline, followed by freshly prepared ice cold 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed, postfixed in perfusate overnight, and then stored in 30% sucrose, until sectioned on a microtome. 4.5.

BrdU immunohistochemistry

The rat brains were shipped cold in 30% sucrose via courier from the University of Florida to Princeton University for immunohistochemical analysis. Frozen coronal sections (40-μm thick) were cut throughout the entire hippocampus and a 1-in-12 series of tissue was then processed for BrdU, using a slide-mounted peroxidase technique (Fornal et al., 2007). Briefly, sections were heated in citric acid for antigen retrieval, permeabilized with trypsin, denatured with hydrochloric acid, and then incubated with a mouse monoclonal antibody raised against BrdU (Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) for 48 h at 4 °C. Following the primary antibody incubation, sections were incubated at room temperature with a biotinylated horse anti-mouse IgG and with avidin–biotin complex (Vector Laboratories, Burlingame, CA), and then reacted with 3,3′-diaminobenzidine (DAB) to

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visualize labeled cells. Sections were then counterstained with cresyl violet, dehydrated and coverslipped with DPX mountant. Slides were then coded prior to analysis. 4.6.

Immunofluorescence staining

The brains were sectioned at Princeton University and then the sections were returned to the University of Florida by courier. The sections were shipped on ice in 30% sucrose. Sections from the cell proliferation and the cell survival experiment were used. Three to five sections from the proliferation experiment were used to identify dying (TUNEL-positive) new (BrdU-positive) cells. The same number of sections was used from the survival experiment to identify the neuronal (DCX-positive, DCX/NeuNpositive or NeuN-positive) or glial (GFAP-positive or NG2positive) phenotypes of new cells. Apoptotic new (BrdU-positive) cells were detected by using a TUNEL Apoptosis Detection Kit according to kit instructions (Genscript, Piscataway, NJ, USA). Briefly, free floating sections were rinsed several times in tris-buffered saline (TBS) and permeabilized in a solution of 0.1% Triton-X and 0.1% sodium citrate in water for 2 min on ice before being wet-mounted on microscope slides (2–3 sections per slide). The sections were then incubated in a TUNEL Reaction Mixture (45 μl of reaction buffer, 1 μl of FITC-12-dUTP and 4 μl of TdT/slide) for 1 h at 37 °C in a light-resistant container. The sections were then removed from the microscope slides and rinsed repeatedly in 0.9% NaCl before being incubated in 2 MHCl at 37 °C for 20 min and then in rat anti-BrdU (1:500; Accurate, Westbury, New Jersey) overnight at 4 °C. The following day, the sections were incubated in minimally cross-reactive secondary IgG conjugated fluorophores (1:500; Jackson Immunoresearch, West Grove, PA, USA) for 4 h at room temperature before being DAPI-stained (1:20,000 in TBS for 20 min; Calbiochem, San Diego, CA, USA) and coverslipped under PVA-DABCO. The phenotype of the new cells was revealed by using immunohistochemical procedures that have been described previously (Palmer et al., 2000; Seifert et al., 2010). Briefly, free-floating sections were washed in TBS (pH 7.4) several times before staining and between each step. The sections were blocked in a solution of 3% normal donkey serum and 0.1% Triton-X in TBS before being incubated overnight at 4 °C in a cocktail of goat anti-doublecortin (1:500; Santa Cruz, CA, USA) and mouse anti-NeuN (1:500; Chemicon, Temecula, CA, USA) or chicken anti-GFAP (1:500; a gift from Dr. Gerry Shaw at the University of Florida) and rabbit anti-NG2 (1:500; Chemicon, Temecula, CA, USA) in a solution of 1% normal donkey serum and 0.1% Triton-X in TBS. The following day, the sections were incubated in minimally crossreactive secondary IgG conjugated fluorophores (1:500; Jackson Immunoresearch, West Grove, PA, USA) for 4 h at room temperature and then fixed in 4% paraformaldehyde for 10 min. Following several 0.9% NaCl rinses, the sections were incubated in 2 M HCl at 37 °C for 20 min and then in rat antiBrdU (1:500; Accurate, Westbury, NJ, USA) overnight at 4 °C. The following day, the sections were incubated in minimally crossreactive secondary IgG conjugated fluorophores (1:500; Jackson Immunoresearch, West Grove, PA, USA) for 4 h at room temperature before being DAPI-stained (1:20,000 in TBS for 20 min; Calbiochem, San Diego, CA, USA) and coverslipped under PVA-DABCO.

4.7.

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Stereological estimates of total new cell number

Slides were analyzed blind with respect to treatment, using an Olympus BX-60 light microscope (Olympus America Inc., Melville, NY, USA). In every 12th section, BrdU-positive cells (stained brown) were counted bilaterally in the dentate gyrus at high magnification (400× or 600×). The dentate gyrus included the granule cell layer (GCL), subgranular zone (SGZ), and the hilus. The cell counts for each animal were summed across all sections and then multiplied by 12, to obtain an estimate of the total labeled cell number in the dentate gyrus. In addition, the dentate gyrus was divided into anterior (dorsal) and posterior (ventral) portions as described previously (Guzman-Marin et al., 2008). For the cell proliferation study, labeled cells were also counted separately in the SGZ and in the hilus. The GCL was not investigated separately because there were very few proliferating cells in this area. For the cell survival study, labeled cells were counted separately in the SGZ/GCL and in the hilus. Cells located within two cell-body widths (~20 μm) from the border of the GCL were considered to be in the SGZ; cells located more distally were considered to be in the hilus. 4.8.

Analysis of BrdU-positive Cell Survival and Phenotypes

The number of BrdU-positive cells that were positive for TUNEL and the phenotypes of BrdU-positive cells was assessed with a Zeiss confocal microscope (model LSM 710 fully spectral meta system with 405, 440, 488, 532, 635 laser lines, Carl Zeiss MicroImaging, Jena, Germany). Brain sections from the proliferation study were used to determine the number of cells that were positive for BrdU and TUNEL. Brain sections from the survival study were used to determine the phenotype of the BrdU-positive cells. BrdU-labeled cells were scored as co-labeled when a full “zdimension” scan revealed that its nucleus contained TUNEL staining or was unambiguously associated with a lineage specific marker. BrdU-positive cells in the hilus did not co-label with any of the neuronal or glial markers used in this study. Therefore, we only report the phenotypes of BrdU-positive cells in the SGZ/GCL. At least 80 BrdU-positive cells were scanned on more than 2 dentate gyri per animal. We report the percentage of BrdUpositive cells that are co-labeled with each marker (TUNEL or phenotypic) and the total number (stereological estimate×proportion co-labeled) of new immature neurons (BrdU/DCX-positive), transitioning neurons (BrdU/DCX/NeuN-positive), mature neurons (BrdU/NeuN-positive), astrocytes (BrdU/GFAP-positive) and oligodendrocyte precursors (BrdU/NG2-positive). 4.9.

Experimental design

4.9.1. Tobacco smoke and cell proliferation The rats were handled for 3 days prior to the onset of the experimental treatments. The rats in the tobacco group (n = 7) were exposed to tobacco smoke from PN29–42 (14 days). These rats were exposed to tobacco smoke for 4 h/day and the smoke exposure sessions were conducted between 9:00 AM and 1:00 PM. The control rats (n = 8) were placed in an animal testing room adjacent to the smoking room during the tobacco smoke exposure sessions. The control rats were never placed in the room with the smoking machine in order to prevent exposure to tobacco fumes. On day 14 of tobacco smoke

40

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exposure (PN42), the rats in the tobacco group were exposed to tobacco smoke for 2 h and were then injected ip with 200 mg/ kg of BrdU. After the injections the rats were exposed to tobacco smoke for another 2 h and then they were deeply anesthetized with pentobarbital and perfused. The rats were perfused within 2 h after the end of the tobacco smoke exposure session (2–4 h after BrdU injection). The control animals were also perfused 2–4 h after the BrdU injections. 4.9.2. Tobacco smoke and cell survival The experimental procedures in the second experiment were the same as in the first experiment with the exception that the rats received the BrdU injections at a different time point. All the rats received an ip injection of BrdU on PN28. The rats in the tobacco group (n = 7) were exposed to tobacco smoke from PN29–42 (14 days). The rats in the control group (n = 9) were placed in the animals testing room adjacent to the smoke room during the tobacco smoke exposure sessions. The rats were deeply anesthetized with pentobarbital and perfused 24 h after the last tobacco smoke exposure session (PN43). 4.10.

Statistical analysis

The body weights over the course of the tobacco smoke exposure period were analyzed by two-way repeated measures analysis of variance (ANOVA) with time (days of tobacco smoke exposure) as the within subjects factor and treatment (control or tobacco smoke) as the between subjects factor. The body weights of the rats on the last day of tobacco smoke exposure (day 14) were not included in the overall analyses because not all the body weights were recorded at this time point. The baseline body weights of the control group and the tobacco group were compared by twotailed unpaired t-tests. BrdU cell counts and the percentage of proliferating (BrdU-positive) cells co-labeled with TUNEL were compared between groups using two-tailed unpaired t-tests. The cell survival data were normalized by expressing cell counts as a percentage of control values for two separate trials. This was done to control for the variability in absolute cell counts observed across the experimental trials. Group differences in the proportion of new cells expressing neuronal or glial phenotypes and the total number of new neurons and glia were revealed using twotailed unpaired t-tests. A probability value (P) < 0.05 was taken as statistically significant. The statistical analyses were performed using GraphPad Prism version 5.0c for Mac OS and PASW Statistics 18 or Statistica for Windows.

Acknowledgments We thank Sarah Hoffman and Claire Gutierrez for their excellent technical assistance. This research was funded by a Flight Attendant Medical Research Institute Young Clinical Scientist Award (Grant nr. 52312) to A. Bruijnzeel.

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