Subsets of acetylcholine-stimulated 86Rb+ efflux and [125I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment
Introduction
Chronic nicotine treatment increases the numbers of the predominant nicotinic acetylcholine receptor (nAChR) subtype, which is measured by high-affinity binding of agonists such as nicotine, cytisine or ACh in experimental animals (Marks et al., 1983, Schwartz and Kellar, 1983, Pauly et al., 1991, Flores et al., 1992, Sanderson et al., 1993), cell lines (Peng et al., 1994, Gopalakrishnan et al., 1996, Gopalakrishnan et al., 1997, Whiteaker et al., 1998), and, relevantly, the brains of human smokers (Benwell et al., 1988, Breese et al., 1997, Perry et al., 1999). These high-affinity binding sites reside primarily on α4β2-nAChR (Whiting and Lindstrom, 1988, Flores et al., 1992, Picciotto et al., 1995, Zoli et al., 1998, Marubio et al., 1999).
α4β2-nAChR have higher affinity for nicotine than other subtypes (Parker et al., 1998). Consequently, it is reasonable to expect that α4β2-nAChR would respond to treatment with lower nicotine doses. Indeed, the nicotine doses required to elicit increases in the density of α-bungarotoxin (αBTX) binding sites (α7-nAChRs) in mouse brain are higher than those required for agonist binding sites (α4β2-nAChR) (Marks et al., 1993, Pauly et al., 1991). However, affinity differences do not explain all of the variability in response to chronic treatment since upregulation of [125I]-αBTX differs markedly among brain regions and some mouse strains (including C57BL/6 used in the current study) are resistant to change (Marks et al., 1991).
Response of other nAChR subtypes expressed in the central nervous system (CNS) to chronic nicotine treatment is less well studied. Nicotinic binding sites in brain, adrenal glands, superior cervical ganglia and pineal gland, which are neither α4β2-nAChR nor α7-nAChR, are unaffected by chronic nicotine treatment (Flores et al., 1997, Davila-Garcia et al., 2003). Furthermore, the subset of [125I]-epibatidine binding sites in rat brain with properties of α4β2-nAChR are increased relatively selectively following chronic nicotine treatment (Nguyen et al., 2003). Perhaps nicotine concentrations in vivo are not sufficiently high to induce reliable, long-lasting changes in nAChR subtypes with lower affinity for nicotine.
While increases in high-affinity nicotinic binding sites appear to be generally diagnostic of chronic nicotine exposure, changes in receptor function will ultimately regulate behavioral adaptation to chronic nicotine. Several studies have investigated the effects of nicotine treatment on receptor function (see Gentry and Lukas, 2002 for a recent review). Decreased nAChR-mediated neurotransmitter release has been observed following chronic nicotinic agonist treatment in vivo (e.g. Lapchak et al., 1989, Marks et al., 1993, Jacobs et al., 2002), indicating that the response to chronic nicotine exposure is not only an increase in the number of binding sites, but a decrease in function. Nicotine-stimulated prolactin release in vivo is also reduced following chronic treatment (Hulihan-Giblin et al., 1990). Incubation of cells expressing nAChR with nicotine can lead to inactivation of functional responses (reviewed in Gentry and Lukas, 2002). Nicotine concentrations at which complete functional inactivation is observed in vitro are generally higher (as high as 1 mM) than the plasma concentrations reported in human smokers (Benowitz et al., 1982, Rose et al., 1999). However, functional inactivation has not been universally observed following treatment with nicotinic agonists. Indeed, increases in function have been described (Rowell and Wonnacott, 1990, Yu and Wecker, 1994, Gopalakrishnan et al., 1996, Gopalakrishnan et al., 1997, Buisson and Bertrand, 2001). The agonist studied, its concentration, and the expression system appear to affect the functional response observed after chronic exposure.
We have previously demonstrated that chronic nicotine treatment produces a dose-dependent decrease in agonist stimulated 86Rb+ efflux in mouse midbrain synaptosomes but had no significant effect in cerebral cortex (Marks et al., 1993). The current study extends the examination of the effects of nicotine treatment to 12 brain regions, measures the effects of chronic treatment on several binding sites, and provides measures of the concentration of nicotine in the blood plasma of mice to provide a more complete picture of the functional consequences of chronic nicotine exposure in vivo.
Section snippets
Materials
The radioisotopes 86RbCl (1–12 Ci/mg) and [125I]-epibatidine (2200 Ci/mmol) were purchased from New England Nuclear, Boston, MA. NaCl, KCl, CaCl2, MgSO4, glucose, tetrodotoxin, bovine serum albumin (fraction V), nicotine (free base), nicotine tartrate, acetylcholine iodide (ACh), diisopropylfluorophosphate (DFP), polyethylenimine, cytisine, and 3-(2-(S)-azetidinylmethoxy)pyridine dihydrochloride (A85380) were purchased from Sigma Chemical Company, St. Louis, MO. CsCl was obtained from Research
ACh-stimulated 86Rb+ efflux in cerebral cortex and thalamus
We have previously reported that chronic nicotine treatment resulted in a dose-dependent decrease in nicotine-stimulated 86Rb+ efflux from thalamic synaptosomes with no significant change in cortical synaptosomes (Marks et al., 1993). It has subsequently been demonstrated that agonist-stimulated 86Rb+ efflux is biphasic, with components differing in sensitivity to activation by ACh and other nicotinic agonists (Marks et al., 1999). In order to evaluate the effects of chronic treatment with 4.0
Discussion
The results reported here provide several novel observations. (1) Plasma nicotine and cotinine concentrations in mouse plasma increase linearly with infusion dose. (2) Mice must be treated with approximately 10-fold higher doses of nicotine than rats (Rowell and Li, 1997) to achieve comparable plasma nicotine concentrations. (3) Increases in cytisine-sensitive [125I]-epibatidine binding sites (likely to correspond to α4β2-nAChR) in mouse brain occur at nicotine concentrations in mouse plasma
Overall summary
Chronic nicotine treatment increased cytisine-sensitive [125I]-epibatidine binding sites in mouse brain at plasma nicotine concentrations comparable to those of human smokers. Changes in function measured with ACh-stimulated 86Rb+ efflux were relatively modest. However, the amount of 86Rb+ efflux per unit [125I]-epibatidine binding site decreased with increasing chronic dose. This observation suggests that a balance between receptor number and function per unit receptor occurs during chronic
Acknowledgements
This work was supported by a research grant from the National Institutes of Health (DA-03194). A.C. Collins is also supported by a Research Scientist Award from the National Institutes of Health (DA-00197). S.E. McCallum is a recipient of a Postdoctoral Fellowship from the National Institutes of Health (DA-14152).
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2019, NeuropharmacologyCitation Excerpt :Datasets for nAChR-mediated [3H]-DA release were analyzed by one-way ANOVA with group (F1 Vehn=18, F1 NICn=21, or F2 NICn=14) as the sole factor, and, where appropriate, Holm-Sidak's multiple comparisons post-hoc test was applied. The effect of DNE on ACh concentration ([Ach])–response relations measured by 86Rb+ efflux was quantitated via least squares non-linear regression using a biphasic [agonist]-response model:RACh = (RHS ∗ [ACh])/(ECHS + [ACh]) + (RLS ∗ [ACh])/(ECLS + [ACh])Where RACh is ACh-evoked 86Rb+ efflux at each ACh concentration tested, RHS and RLS are the estimated maximal rates of 86Rb+ efflux with higher and lower sensitivity to ACh stimulation, and ECHS and ECLS are the estimated ACh concentrations eliciting half-maximal 86Rb+ efflux with higher and lower sensitivity to ACh stimulation, respectively (Marks et al., 1999, 2004, 2007, 2010, 2012). Datasets for frontal cortical nAChR-mediated 86Rb+ efflux were analyzed by one-way ANOVA with group (F1 Vehn=12, F1 NICn=12, or F2 NICn=11) as the sole factor, and, where appropriate, Holm-Sidak's multiple comparisons post-hoc test was applied.
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- 1
Current address: School of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA.
- 2
Current address: Parkinson’s Institute, Sunnyvale, CA, USA.