Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul;130(2):185-98.
doi: 10.1111/jnc.12721. Epub 2014 Apr 19.

α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration

Michael J Marks  1 , Sharon R Grady, Outi Salminen, Miranda A Paley, Charles R Wageman, J Michael McIntosh, Paul Whiteaker
Affiliations

α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration

Michael J Marks et al. J Neurochem. 2014 Jul.

Abstract

Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are down-regulated following chronic nicotine exposure (unlike other subtypes that have been investigated - most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR down-regulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than up-regulation of α4β2*-nAChR. In contrast, nAChR-mediated [(3) H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [(3) H]-DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function. This study examined dose-response relationships for murine α6β2*-nicotinic acetylcholine receptor (nAChR) down-regulation by chronic nicotine treatment. The ID50 value for α6β2* down-regulation (35 nM) is ≈ 3x lower than the ED50 value for α4β2* nAChR up-regulation (95 nM), both well within the range reached by human smokers. Chronic nicotine treatment altered α6β2*- and α4β2*-nAChR-mediated [(3) H]-dopamine release from striatal and olfactory tubercle synaptosomes, but dopaminergic parameters were largely unaffected. We conclude that functional changes are primarily driven by altered nAChR activity.

Keywords: dopamine receptor; dopamine transporter; dopaminergic terminal regions; nicotinic receptor; α4β2 subtype.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest associated with the work described in this manuscript.

Figures

Figure 1
Figure 1. Autoradiograms of [125I]α-conotoxinMII ([125I]α-CtxMII) and [125I]epibatidine binding
Representative examples are shown of radioligand binding patterns, recorded on X-ray film, using autoradiography approaches as described in the Methods section. Left column: [125I]α-CtxMII (500 pM) labeling. Right column: [125I]epibatidine (200 pM). Regions of interest are labeled as follows: DLG, dorsolateral geniculate nucleus; mHab, medial habunula; nAcc, nucleus accumbens; opt, optic tract; OPTN, olivary pretectal nucleus; OT, olfactory tubercle; SC, superior colliculus (superficial layers); Str, striatum; Th, thalamus; VLGN, ventrolateral geniculate nucleus.
Figure 2
Figure 2. Concentration dependence of decreased [125I]α-CtxMII binding site (α6β2* nAChR) expression following chronic nicotine administration
Mice were treated for 10 days with continuous infusion of sterile saline (control) or a range of nicotine doses (0.125 – 4 mg/kg/h). Expression of α6β2* nAChR was assessed using [125I]α-CtxMII autoradiography in sections (14 µm) collected from each brain. Specific labeling was determined as the difference in labeling by quantitation of total binding (500 pM [125I]α-CtxMII alone) and non-specific labeling (500 pM [125I]α-CtxMII in the presence of 100 µM nicotine) between adjacent sections or equivalently by binding to sections of β2 null mutant mice, by reference to known radioactive standards. Please see Methods section for experimental details. n = 9 for each point; points represent mean ± S.E.M.; * denotes a significant difference (p < 0.05) from no-nicotine control binding detected by one-way ANOVA within each region, followed by the Duncan post-hoc test. All other statistical analyses and calculated parameters are given in Table 1.
Figure 3
Figure 3. Concentration dependence of increased cytisine-sensitive [125I]epibatidine binding site (α4β2* nAChR) expression following chronic nicotine administration
As in Figure 2, mice were treated for 10 days with continuous infusion of sterile saline (control) or a range of nicotine doses (0.125 – 4 mg/kg/h). Expression of α4β2* nAChR was assessed using [125I]epibatidine autoradiography in sections (14 µm) collected from each brain. Specific labeling of α4β2* nAChR was determined as the difference in labeling by quantitation of total binding (200 pM [125I]epibatidine alone) and cytisine-sensitive labeling (200 pM [125I]epibatidine in the presence of 50 nM cytisine) between adjacent sections, by reference to known radioactive standards. Nonspecific binding measured by including 10 µM nicotine in the incubation did not differ from film background. Please see Methods section for experimental details. n = 5–6 for each point; points represent mean ± S.E.M; * denotes a significant difference (p < 0.05) from no-nicotine control binding detected by one-way ANOVA within each region, followed by the Duncan post-hoc test. All other statistical analyses and calculated parameters are given in Table 1.
Figure 4
Figure 4. Concentration-response curves for nicotine-evoked [3H]dopamine release mediated by α6β2* nAChR following chronic nicotine administration
Crude synaptosomes were produced from striatum and olfactory tubercles of animals treated for 10 d with saline (control) or three different nicotine doses as indicated. Synaptosomes were loaded with [3H]-DA, then superfused for 5 min in the presence or absence of 50 nM α-CtxMII just prior to stimulation with a range of nicotine concentrations (10 nM – 3 µM). α-CtxMII-sensitive release (mediated by α6β2* nAChR) of [3H]-DA was calculated by subtracting the resistant fraction from the total (n = 6–11 for each point; points represent mean values ± S.E.M), and is expressed as a multiple of baseline release just before stimulation. The curves for stimulation by nicotine represent the best fits of the data as one saturable component, for each chronic treatment regime, in each region, as described in the Methods. Calculated parameters and statistical analyses are given in Table 2.
Figure 5
Figure 5. Concentration-response curves for nicotine-stimulated [3H]dopamine release mediated by α4β2* nAChR following chronic nicotine administration
Crude synaptosomes were produced from striatum and olfactory tubercles of animals treated for 10 d with saline (control) or with the indicated different nicotine doses. Synaptosomes were loaded with [3H]-DA, then superfused for 5 min in the presence of 50 nM α-CtxMII just prior to stimulation with a range of nicotine concentrations (10 nM – 3 µM). The residual α-CtxMII-resistant release of [3H]-DA is mediated by α4β2* nAChR (n = 6–11 for each point; points represent mean values ± S.E.M), and is expressed as a multiple of baseline release just before stimulation. The curves for stimulation by nicotine represent the best fits of the data as one saturable component, for each chronic treatment regime, in each region, as described in the Methods. Calculated parameters and statistical analyses are given in Table 2.
Figure 6
Figure 6. Chronic nicotine administration does not affect expression of dopamine system markers
Expression of dopaminergic markers was assessed in striatum and olfactory tubercle membrane preparations of animals treated for 10 d with saline (control) or with the three indicated nicotine doses. Values obtained in striatal membranes are represented as black circles, while those measured in olfactory tubercles are shown as gray triangles (n = 6–7 per point; points represent mean ± S.E.M.). Expression of D1/D5 receptors was measured using [3H]-SCH23390 (1.7 nM; top panel), while that of D2/D4 receptors was measured using [3H]-raclopride (17 nM; middle panel). Dopamine transporter (DAT) expression was assessed by binding of [3H]-mazindol (200 nM; lower panel). One-way ANOVA was performed for each marker in each region to determine if chronic nicotine treatment significantly affected expression levels. No effect of nicotine dose was seen in any combination of region × marker.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baker LK, Mao D, Chi H, et al. Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug. European Journal of Neuroscience. 2013;37:1004–1011. - PMC - PubMed
    1. Barr JE, Holmes DB, Ryan LJ, Sharpless SK. Techniques for the chronic cannulation of the jugular vein in mice. Pharmacol Biochem Behav. 1979;11:115–118. - PubMed
    1. Bordia T, McIntosh JM, Quik M. The nicotine-mediated decline in l-dopa-induced dyskinesias is associated with a decrease in striatal dopamine release. J Neurochem. 2013;125:291–302. - PMC - PubMed
    1. Brunzell DH. Preclinical Evidence That Activation of Mesolimbic Alpha 6 Subunit Containing Nicotinic Acetylcholine Receptors Supports Nicotine Addiction Phenotype. Nicotine & Tobacco Research. 2012;14:1258–1269. - PMC - PubMed
    1. Cartier GE, Yoshikami D, Gray WR, Luo S, Olivera BM, McIntosh JM. A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J Biol Chem. 1996;271:7522–7528. - PubMed

Publication types

MeSH terms

LinkOut - more resources