Dopaminergic (DAergic) neuronal activity in the ventral tegmental area (VTA) is thought to contribute generally to pleasure, reward, and drug reinforcement and has been implicated in nicotine dependence. nAChRs expressed in the VTA exhibit diverse subunit compositions, but the functional and pharmacological properties are largely unknown. Here, using patch-clamp recordings in single DAergic neurons freshly dissociated from rat VTA, we clarified three functional subtypes of nAChRs (termed ID, IID and IIID receptors) based on whole-cell current kinetics and pharmacology. Kinetic analysis demonstrated that comparing to ID, IID receptor-mediated current had faster activation and decay constant and IIID receptor-mediated current had larger current density. Pharmacologically, ID receptor-mediated current was sensitive to the α4β2-nAChR agonist RJR-2403 and antagonist dihydro-β-erythroidine (DHβE); IID receptor-mediated current was sensitive to the selective α7-nAChR agonist choline and antagonist methyllycaconitine (MLA); while IIID receptor-mediated current was sensitive to the β4-containing nAChR agonist cytisine and antagonist mecamylamine (MEC). The agonist concentration-response relationships demonstrated that IID receptor-mediated current exhibited the highest EC 50 value compared to ID and IIID receptors, suggesting a relatively low agonist affinity of type IID receptors. These results suggest that the type ID, IID and IIID nAChR-mediated currents are predominately mediated by activation of α4β2-nAChR, α7-nAChR and a novel nAChR subtype(s), respectively. Collectively, these findings indicate that the VTA DAergic neurons express diversity and multiplicity of functional nAChR subtypes. Interestingly, each DAergic neuron predominantly expresses only one particularly functional nAChR subtype, which may have distinct but important roles in regulation of VTA DA neuronal function, DA transmission and nicotine dependence.

Amyloid-␤ (A␤) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human ␣4 and ␤2 subunits (h␣4␤2-nAChR). However, the relationships between these phenomena are uncertain, and effects of A␤ on h␣4␤2-nAChR function have not been investigated in detail. We first confirmed expression of h␣4 and h␤2 subunits as messenger RNA in transfected, human SH-EP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed ␣4 and ␤2 subunit protein expression and coassembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-h␣4␤2 cells of functional h␣4␤2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by A␤ 1-42 in a dosedependent manner. Functional inhibition was selective for A␤ 1-42 compared with the functionally inactive, control peptide A␤ 40 -1 . A␤ 1-42 -mediated inhibition of h␣4␤2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5-yl thiophosphate failed to prevent A␤ 1-42 -induced inhibition, suggesting that down-regulation of h␣4␤2-nAChR function by A␤ 1-42 is not mediated by nAChR internalization. Sensitivity to A␤ 1-42 antagonism at 1 nM was evident for h␣4␤2-nAChRs, but not for heterologously expressed human ␣7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nM A␤ 1-42 , with the magnitude of functional block being higher for 100 nM A␤ 1-42 acting on h␣7-nAChRs. These findings suggest that h␣4␤2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for A␤ neurotoxicity in AD.Alzheimer's disease (AD) 1 is a progressive, neurodegenerative disorder manifest as a severe impairment of learning and memory. Pathophysiological hallmarks of AD include extracellular deposits of ␤-amyloid peptide (A␤) in senile plaques, formation of intraneuronal neurofibrillary tangles, and cholinergic neuron death (1). Although the precise mechanisms of AD pathogenesis are only partially understood, it is now widely accepted that the accumulation and aggregation of A␤ 1-42 plays a key role in the disease (2). Evidence has indicated an interaction between A␤ and the cholinergic system (3). For example, very low concentrations (pico to nanomolar) of A␤ can directly induce cholinergic hypofunction (4 -6). It has been reported that solubilized A␤ inhibits several steps of acetylcholine synthesis and release (4, 7), inhibits cholinergic enzyme activity (6), impairs cholinergic metabolism and neurotransmission (8 -10), and depresses hippocampal synaptic function (11).Recent evidence suggests possible roles for nicotinic acetylcholine receptors (nAChRs) as central targets for A␤-induced neurotoxicity manifest as...

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BackgroundAlthough evidence suggests that the prevalence of Parkinson’s disease (PD) is lower in smokers than in non-smokers, the mechanisms of nicotine-induced neuroprotection remain unclear. Stimulation of the α7 nicotinic acetylcholine receptor (α7-nAChR) seems to be a crucial mechanism underlying the anti-inflammatory potential of cholinergic agonists in immune cells, including astrocytes, and inhibition of astrocyte activation has been proposed as a novel strategy for the treatment of neurodegenerative disorders such as PD. The objective of the present study was to determine whether nicotine-induced neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model occurs via α7-nAChR-mediated inhibition of astrocytes.MethodsBoth in vivo (MPTP) and in vitro (1-methyl-4-phenylpyridinium ion (MPP+) and lipopolysaccharide (LPS)) models of PD were used to investigate the role(s) of and possible mechanism(s) by which α7-nAChRs protect against dopaminergic neuron loss. Multiple experimental approaches, including behavioral tests, immunochemistry, and stereology experiments, astrocyte cell cultures, reverse transcriptase PCR, laser scanning confocal microscopy, tumor necrosis factor (TNF)-α assays, and western blotting, were used to elucidate the mechanisms of the α7-nAChR-mediated neuroprotection.ResultsSystemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, and protected against dopaminergic neuron loss and the activation of astrocytes and microglia in the substantia nigra. The protective effects of nicotine were abolished by administration of the α7-nAChR-selective antagonist methyllycaconitine (MLA). In primary cultured mouse astrocytes, pretreatment with nicotine suppressed MPP+-induced or LPS-induced astrocyte activation, as evidenced by both decreased production of TNF-α and inhibition of extracellular regulated kinase1/2 (Erk1/2) and p38 activation in astrocytes, and these effects were also reversed by MLA.ConclusionTaken together, our results suggest that α7-nAChR-mediated inhibition of astrocyte activation is an important mechanism underlying the protective effects of nicotine.