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Cocaine Directly Inhibits ?6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

ABSTRACT: Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits ?6N/?3C?2?3-nAChR (?6*-nAChRs) function. Human ?6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic ?6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure ?6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human ?6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 ?M. Interestingly, in the presence of 30 ?M cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both ?6N/?3C?2?3-nAChRs and ?6M211L/?3IC?2?3-nCAhRs similarly, suggesting that cocaine may not act on the ?3 transmembrane domain of chimeric ?6N/?3C?2?3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes ?6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed ?6*-nAChRs. These findings suggest that ?6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

SUBMITTER: Chen D

PROVIDER: S-EPMC6383119 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Chen Dejie D Gao Fenfei F Ma Xiaokuang X Eaton Jason Brek JB Huang Yuanbing Y Gao Ming M Chang Yongchang Y Ma Zegang Z Der-Ghazarian Taleen T Neisewander Janet J Whiteaker Paul P Wu Jie J Su Quanxi Q

Frontiers in pharmacology 20190214

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (V ...[more]

PMID: 30837868

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Project description:Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4β2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 μM) induced inward currents, co-application of l-THP (3 μM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 μM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4β2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4β2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42β23-nAChR) and cytisine-activated, low-sensitive one (α43β22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4β2-nAChR function, which may underlie its inhibition on nicotine addiction.

Project description:Midbrain dopamine (DA) neurons are considered a critical substrate for the reinforcing and sensitizing effects of nicotine and tobacco dependence. While the role of the α4 and β2 subunit containing nicotinic acetylcholine receptors (α4β2∗nAChRs) in mediating nicotine effects on DA release and DA neuron activity has been widely explored, less information is available on their role in the morphological adaptation of the DA system to nicotine, eventually leading to dysfunctional behaviors observed in nicotine dependence. In particular, no information is available on the role of α6∗nAChRs in nicotine-induced structural plasticity in rodents and no direct evidence exists regarding the occurrence of structural plasticity in human DA neurons exposed to nicotine. To approach this problem, we used two parallel in vitro systems, mouse primary DA neuron cultures from E12.5 embryos and human DA neurons differentiated from induced pluripotent stem cells (iPSCs) of healthy donors, identified using TH+ immunoreactivity. In both systems, nicotine 1-10 μM produced a dose-dependent increase of maximal dendrite length, number of primary dendrites, and soma size when measured after 3 days in culture. These effects were blocked by pretreatments with the α6∗nAChR antagonists α-conotoxin MII and α-conotoxin PIA, as well as by the α4β2nAChR antagonist dihydro-β-erythroidine (DHβE) in both mouse and human DA neurons. Nicotine was also ineffective when the primary DA neurons were obtained from null mutant mice for either the α6 subunit or both the α4 and α6 subunits of nAChR. When pregnant mice were exposed to nicotine from gestational day 15, structural plasticity was also observed in the midbrain DA neurons of postnatal day 1 offspring only in wild-type mice and not in both null mutant mice. This study confirmed the critical role of α4α6∗nAChRs in mediating nicotine-induced structural plasticity in both mouse and human DA neurons, supporting the translational relevance of neurons differentiated from human iPSCs for pharmacological studies.

Dataset Information

Cocaine Directly Inhibits ?6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Publications

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

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