Elsevier

Drug and Alcohol Dependence

Volume 132, Issue 3, 1 October 2013, Pages 562-570
Drug and Alcohol Dependence

Intravenous prenatal nicotine exposure increases orexin expression in the lateral hypothalamus and orexin innervation of the ventral tegmental area in adult male rats

https://doi.org/10.1016/j.drugalcdep.2013.04.003Get rights and content

Abstract

Background

Approximately 18% of pregnant women continue to smoke tobacco cigarettes throughout pregnancy. Offspring exposed to tobacco smoke in utero exhibit a higher incidence of drug use in later stages of development relative to non-exposed children. Animal models indicate that prenatal nicotine (PN) exposure alone alters the development of the mesocorticolimbic dopamine (DA) system, which, in part, organizes motivated behavior and reward. The orexin/hypocretin neuropeptide system, which originates in the lateral hypothalamus (LH), projects to key areas of the mesocorticolimbic DA pathway. Previous research suggests that orexin exerts a major influence on motivation and reward.

Methods

The present experiments determined if intravenous (IV) PN exposure alters (1) the expression of orexin neurons and melanin-concentrating hormone (MCH; positive control) in the LH; and (2) orexin projections from the LH onto DA neurons in the ventral tegmental area (VTA). Dams were injected with IV nicotine (0.05 mg/kg/injection) or saline 3×/day during gestational days 8–21. Tissues from adult male offspring (∼130 days) were examined using immunohistochemistry.

Results

Relative to controls, offspring of IV PN exposure showed (1) increased numbers of orexin neurons in the LH, and no changes in the expression of MCH; and (2) increased orexin appositions on DA cells in the VTA.

Conclusion

The findings indicate that the influence of PN exposure is enduring, and suggests that the PN-induced modification of orexin expression on mesolimbic circuitry may contribute to the reported changes in motivated behaviors related to food and drug reward observed in offspring prenatally exposed to nicotine.

Introduction

Between 2002 and 2011, approximately 18% of women in the US reported smoking during pregnancy (∼17.6% in 2010–2011; Substance Abuse and Mental Health Services Administration, 2012). In utero tobacco smoke exposure decreases fetal growth and causes developmental abnormalities including low birth weight, decreased lung growth and pulmonary function, and increased incidence of sudden infant death syndrome (Castles et al., 1999, Cornelius and Day, 2009, DiFranza et al., 2004, Fleming and Blair, 2007). Neurocognitive deficits, such as language acquisition delays, auditory processing deficits, and attention deficit hyperactivity disorder are reported following prenatal exposure to maternal smoking (Button et al., 2007, DiFranza et al., 2004, Fergusson et al., 1998, Linnet et al., 2003). Prenatal tobacco smoke exposure also increases the likelihood of drug use during adolescence and adulthood (Buka et al., 2003, Kandel et al., 1994, Weissman et al., 1999). These findings suggest that maternal smoking has enduring effects on the neurodevelopment and motivated behavior of offspring (Buka et al., 2003, Cornelius and Day, 2009, Kandel et al., 1994).

Nicotine is the key compound in tobacco that maintains cigarette smoking behavior (Benowitz et al., 2009, Corrigall and Coen, 1989). Various animal models are therefore used to assess the effects of prenatal nicotine (PN) on brain development (Dwyer et al., 2009). In these models, nicotine alone is administered continuously by osmotic minipump (Dwyer et al., 2008, Slotkin et al., 1987a, Slotkin et al., 1987b) orally through the animal's drinking water (Pauly et al., 2004, Schneider et al., 2010, Zhu et al., 1996, Zhu et al., 2012), or intravenously (Lacy et al., 2012, LeSage et al., 2006). Research investigating PN exposure demonstrates that PN acts as a teratogen: exposure to continuous PN reduces brain cell number (Slotkin et al., 1987a, Slotkin et al., 1987b), and alters cell replication, cell survival, and synaptogenesis in utero, relative to control animals (Navarro et al., 1989, Slikker et al., 2005, Slotkin, 2004).

A major focus of research investigating the effects of PN exposure is on the development of neural systems that organize motivated behavior (Dwyer et al., 2009). One hypothesis being tested is that PN alone alters the development of the mesocorticolimbic dopamine (DA) system, which is primarily composed of the ventral tegmental area (VTA), the nucleus accumbens (NAcc), and the prefrontal cortex (Edwards and Koob, 2010, Everitt et al., 2008, Kalivas, 2009, Robinson and Berridge, 2003, Wise and Bozarth, 1987), and that these modifications mediate the changes in motivated behavior observed in human offspring of maternal tobacco smoking, e.g., increasing the vulnerability to drug dependence (Kandel et al., 1994, Weissman et al., 1999). PN exposure produced alterations in DA neurons in fetal and preweanling rats (Navarro et al., 1988, Ribary and Lichtensteiger, 1989) and resulted in decreased striatal DA concentrations and D2 receptors in weanlings (Richardson and Tizabi, 1994). Adolescent offspring, exposed to PN, exhibited increased c-fos expression in the infralimbic cortex and NAcc core (Park et al., 2006) and decreased nicotine-evoked DA release in the NAcc shell (Kane et al., 2004). PN altered MAPK and PI3K signaling pathways (Wei et al., 2011) and produced increased mRNA expression and protein levels of brain-derived neurotrophic factor throughout the mesocorticolimbic DA system (Harrod et al., 2011, Wei et al., 2011), relative to saline treated controls. Together these results demonstrate that PN alters the development of the motivational system and suggests that nicotine exposure via maternal smoking directly contributes to the increased drug abuse liability observed in human offspring of maternal smoke exposure (Buka et al., 2003, Cornelius and Day, 2009, Kandel et al., 1994, Weissman et al., 1999).

Other neurophysiological systems modulate the activity of the mesocorticolimbic DA system and therefore influence motivated behavior. One example is the orexin/hypocretin system (Aston-Jones et al., 2009, Aston-Jones et al., 2010, Cason et al., 2010, Espana et al., 2011, Kenny, 2011), which originates in the lateral hypothalamus and projects to the VTA, NAcc, and PFC (Alberto et al., 2006, Fadel and Deutch, 2002, Mondal et al., 1999, Sakurai et al., 1998), as well as numerous other structures throughout the brain (Nambu et al., 1999, Peyron et al., 1998). Orexin neurons release the neuropeptides orexin A (OxA) which binds both the orexin-1 (Ox1R) and orexin-2 (Ox2R) receptors, and orexin B (OxB) which is selective for Ox2R (Sakurai et al., 1998). Orexin neurons exhibit reciprocal connections with DA neurons within the VTA (Alberto et al., 2006, Bubser et al., 2005) and these neuropeptides modulate DA release to the PFC (Vittoz and Berridge, 2006, Vittoz et al., 2008). Orexin knockout mice and rats treated with an Ox1R antagonist show altered basal DA signaling and diminished DA responses to cocaine (Espana et al., 2010). VTA DA signaling is also critical for drug-dependent behavioral sensitization and synaptic plasticity (Bonci and Borgland, 2009, Borgland et al., 2006). Cocaine seeking (Smith et al., 2009, Smith et al., 2010, Zhou et al., 2008), morphine place preference (Sharf et al., 2010a) and chronic alcohol intake (Lawrence et al., 2006, Stettner et al., 2011, Voorhees and Cunningham, 2011) are associated with increased indices of orexin signaling in the LH.

It is unlikely that PN-induced changes within the mesocorticolimbic DA system alone account for the modifications in the motivated behavior of rodents described above, given that PN exposure has been shown to modify receptor systems and signaling pathways in multiple brain regions of offspring (Harrod et al., 2011, Lawrence et al., 2006, Navarro et al., 1989, Park et al., 2006, Richardson and Tizabi, 1994, Slotkin et al., 1987a, Slotkin et al., 1987b, Stettner et al., 2011, Voorhees and Cunningham, 2011, Wei et al., 2011). The present set of experiments focused on the effects of PN on the orexin/hypocretin system because it is documented to play an important role in motivation and reward (Aston-Jones et al., 2009, Aston-Jones et al., 2010, Harris et al., 2005, Sharf et al., 2010b, Smith et al., 2009), and abundant evidence from adult animals suggests that the orexin system may mediate certain effects of acute or chronic nicotine administration. For example, nicotine alters orexin expression and neuronal activation (Corrigall, 2009, Kane et al., 2001, Kane et al., 2000, Pasumarthi and Fadel, 2008, Pasumarthi et al., 2006). Also, nicotine self-administration increases orexin receptor mRNA in the arcuate nucleus (LeSage et al., 2010), and administering nicotine directly into the LH area containing orexin neurons stimulates local glutamate and acetylcholine efflux (Pasumarthi and Fadel, 2010). In adult animals, chronic exposure to nicotine via daily IP injection (2–4 mg/kg for 14 days) resulted in increased expression of mRNA for prepro-orexin, OxA and OxB, and both orexin receptors, OX1R and OX2R, in the hypothalamus (Kane et al., 2000). Orexin signaling is also required for the maintenance of nicotine self-administration in adult rats (Hollander et al., 2008, Kenny, 2011). Moreover Boychuk and Hayward (2011) reported that continuous PN, delivered via osmotic minipump, resulted in decreased expression of prepro-orexin mRNA in the LH of adolescent rats.

The present experiments utilized a low-dose IV PN exposure method that has been shown to alter the expression of brain-derived neurotrophic factor in offspring (Harrod et al., 2011), and to produce alterations in various behavioral assays including prepulse inhibition of the acoustic startle response (Lacy et al., 2011), sucrose-maintained responding (Lacy et al., 2012), and methamphetamine self-administration (Harrod et al., 2012). We determined if PN exposure altered the expression of orexin in the LH and in its projections to the VTA using immunohistochemical techniques in tissue from adult offspring (∼130 days) that were prenatally exposed to nicotine or saline. This is the first experiment to investigate the effects of PN exposure on orexin expression in the VTA. Based on previous findings, it was hypothesized that IV PN exposure would result in altered expression of orexin in the LH and in the VTA.

Section snippets

Animals

A total of 45 female and 15 male experimentally naïve Sprague-Dawley rats were acquired from Harlan Industries, Inc. (Indianapolis, IN). Animals were transported to the University of South Carolina, and were allowed to acclimate to a colony room located in the Department of Psychology for seven days prior to breeding. Rodent food (ProLab Rat/Mouse/Hamster Chow 3000) and water were provided ad libitum throughout the course of the experiments. All animal cages were provided with Nylabones

Litter parameters

There were no differences between the PN and PS groups for the measures of maternal or pup weight gain. The weight gain data for the saline and nicotine dams throughout gestation and for the PN and PS pups across PND 1–21 are shown in Table 1. There were also no significant effects between PN and PS rats with regard to the number of pups born or litter composition. Additionally, there were no significant effects of PN treatment on the developmental milestones of righting reflex, negative

Discussion

The present experiments investigated if IV PN exposure resulted in modification of orexin/hypocretin expression in the LH and the VTA of adult, male offspring. Our results indicate that the low-dose IV PN exposure resulted in increased orexin expression, compared to saline controls. PN-exposed rats had significantly more orexin-positive cells in the LH, as well as more orexin appositions onto DA neurons in the VTA, compared to PS animals. PN exposure did not increase expression of MCH-positive

Role of funding source

Funding for this study was provided by the National Institute on Drug Abuse, DA021287 (SBH), National Institute of Health, 5 T32GM091740, National Institute on Aging AG030646 (JRF) and the University of South Carolina Research Productivity Scholar grant, KA-21 (SBH). None of these funding sources had a further role in the preparation of the experimental procedures, writing, or the submission of the manuscript.

Contributors

Steven Harrod and Jim Fadel designed the study. Ryan Lacy and Steven Harrod conducted the prenatal treatments and Amanda Morgan and Ryan Lacy managed the surrogate fostering procedures and care of litters and the developing offspring. Jim Fadel and Emily Stanley wrote the protocol for tissue preparation and the immunohistochemical methods. Amanda Morgan conducted the statistical analyses and wrote the first version of the manuscript, and all authors edited subsequent versions. The final version

Conflict of interest

All authors declare they have no conflict of interest.

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