Research ReportChronic neonatal nicotine upregulates heteromeric nicotinic acetylcholine receptor binding without change in subunit mRNA expression
Introduction
Despite warnings, it is estimated that 20% of pregnant woman use tobacco products regularly, resulting in low birth weight and cognitive and behavioral deficits in the offspring (Adams et al., 2002, Bauman et al., 1991, Weissman et al., 1999, Weitzman et al., 1992). Nicotine, the major psychoactive ingredient in tobacco, is believed to be responsible for most of the long- and short-term effects in children exposed to nicotine in utero. Rodent models developed to assess the effects of developmental nicotine exposure have shown that pre- and postnatal chronic nicotine can induce behavioral and cognitive changes (Levin et al., 1993, Narayanan et al., 2002, Peters and Ngan, 1982, Sorenson et al., 1991, Yanai et al., 1992), but the underlying mechanisms remain unknown.
Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs) located in the central and peripheral nervous system. nAChRs are ligand-gated cation channels composed of α and β subunits. To date, in the mammalian brain five ligand binding α (α2, α3, α4, α6, α7), two structural β (β2, β4) and two supportive subunits (α5, β3) have been identified, which form pharmacologically and physiologically distinct nAChR subtypes depending on their subunit combination (Le Novere et al., 2002, Sargent, 1993). Radioligand binding studies have previously defined two major classes of nAChRs in brain; homomeric and heteromeric subtypes. Homomeric nAChRs are characterized by their high affinity for the nAChR antagonist α-bungarotoxin (Clarke et al., 1985) and consist of five α7 subunits (Couturier et al., 1990). The vast majority of heteromeric neuronal nAChRs are characterized by their high affinity for nicotine and is composed of the widely expressed α4 and β2 subunits (Conroy and Berg, 1998, Flores et al., 1992, Whiting et al., 1991, Zoli et al., 1998).
Chronic nicotine exposure upregulates high-affinity nicotine binding sites in the brain (Marks et al., 1983, Marks et al., 1992, Mugnaini et al., 2002, Schwartz and Kellar, 1983, Tizabi and Perry, 2000). Although, there have been reports that different brain nAChR subtypes are subject to receptor upregulation, at nicotine concentrations equivalent to those found in smokers, heteromeric α4/β2 nAChRs are the most affected subtype (Nguyen et al., 2004), which is supported by the finding that deletion of the β2 subunit abolishes upregulation in response to chronic nicotine (Mugnaini et al., 2002, Tizabi and Perry, 2000). In contrast to the increase in receptor numbers, most studies agree that nAChR subunit mRNA expression is not changed in response to chronic nicotine exposure (Marks et al., 1998, Miao et al., 1998, Pauly et al., 1996) but is the result of posttranscriptional events that could involve receptor trafficking, turnover and stabilization (Corringer et al., 2006, Peng et al., 1994, Sallette et al., 2005, Vallejo et al., 2005, Wang et al., 1998).
While it has been shown that prenatal chronic nicotine increases nAChR binding sites (Eriksson et al., 2000, Miao et al., 1998, Sershen et al., 1982, Slotkin et al., 1987a, Van de Kamp and Collins, 1994), the effect of postnatal chronic nicotine exposure on nAChR binding sites is largely unknown. Only one study has shown that nicotine administered via maternal milk results in a dose-dependent increase in high-affinity nicotine binding in cortical membranes (Narayanan et al., 2002). We have developed a postnatal gastric intubation model (Huang et al., 2006), with dosing intervals closely mimicking the peaks and troughs seen in smoking mothers to study the effects of nicotine during a developmental period that corresponds to the third trimester in humans and is characterized by rapid brain growth, also called the “brain growth spurt” period (Dobbing and Sands, 1979, Huang et al., 2006). During this postnatal time several nAChR subunit mRNAs are transiently upregulated, including α7, α5, α3 and β4 (Adams et al., 2002, Broide et al., 1995, Winzer-Serhan and Leslie, 1997, Winzer-Serhan and Leslie, 2005, Zhang et al., 1998) as well as nAChR binding sites (Adams, 2003, Adams et al., 2002, Bina et al., 1995, Fuchs, 1989, Naeff et al., 1992, Zhang et al., 1998), suggesting a role for heteromeric and homomeric receptors in postnatal brain development and rendering this period particularly sensitive to the effects of nicotine. Chronic nicotine could interfere with the developmental function of nAChRs and increase nAChR binding sites, although, it is not clear if chronic nicotine results in an increase in nAChR numbers and an increase in functional receptors. However, recent studies indicate that contrary to initial indications (Dani and Heinemann, 1996), upregulated high-affinity nAChRs respond to nicotine (Buisson and Bertrand, 2002, Nguyen et al., 2004, Sallette et al., 2005). Thus, developmental chronic nicotine exposure could not only increased nAChR expression but chronically activate receptors, rendering the brain even more vulnerable to nicotine during a critical period of development.
Using our postnatal rat model (Huang et al., 2006), we determined whether an interval treatment regiment could result in upregulation of heteromeric and/or homomeric nAChR binding sites identified by 125I-epibatidine (125I-Epi) and 125I-α-bungarotoxin (125I-αBTX), respectively, and whether mRNA expression pattern of the three major neuronal nicotinic receptor subunits, α4, β2 and α7 are altered in hippocampus and cortex, two brain areas associated with cognitive functions and in thalamus, a relay station for incoming sensory information and strong expression of heteromeric nAChRs. In addition, the pattern of 125I-Epi binding in response to dihydro-beta-erythroidine (DHβE) was examined. DHβE, a selective antagonist for α4β2 heteromeric nAChRs, blocks the effects of nicotine on body weight (Huang et al., 2006) and could also reverse nicotine's effects on receptor upregulation.
Section snippets
Expression of nAChR binding sites and nAChR mRNA in adult brain sections
Total and nonspecific binding of 125I-Epi and 125I-α-BTX to heteromeric and homomeric nAChRs, respectively, was determined in lightly fixed adult rat brain sections (Figs. 1A–D). The distribution of total 125I-Epi binding (Fig. 1A) exhibited the highest number of binding sites in the medial habenula (MHb), followed by strong, moderate and low levels in thalamus, cortex and hippocampus, respectively. Excess nicotine completely displaced 125I-Epi binding in all brain regions except for a
Discussion
Although, numerous studies have established that chronic nicotine results in the upregulation of heteromeric nAChRs, this is the first anatomical study to examine the effects of chronic nicotine on nAChR regulation during early postnatal development. We have used 125I-Epi and 125I-αBTX binding to study the expression of heteromeric and homomeric receptors, respectively, in response to chronic neonatal nicotine. 125I-αBTX is an analogue from the snake toxin alpha-bungarotoxin and in rat brain
Animal and drug administration
Timed-pregnant Sprague-Dawley rat dams purchased from Harlan (Houston) arrived on gestation days 14–15 and were housed under standard condition at the College of Medicine's Animal Care Facility according to the rules of Texas A&M University Laboratory Animal Care Committee. The day rat pups were born was considered as postnatal day 0 (P0). On P1, 8 healthy pups from each litter were randomly assigned to different treatment groups with four animals per treatment, thus, two different treatment
Acknowledgment
This study was supported by NIH grant #DA016487.
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