Expression Of Nicotinic Acetylcholine Receptor Subunits Research Articles

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and β1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

Nicotinic acetylcholine receptor subunit expression in the gastrocnemius and in the orbicularis oris before and after facial nerve injury in rats

ABSTRACTObjectives: To observe the expression of nicotinic acetylcholine receptor (AChR) subunits in normal orbicularis oris and gastrocnemius muscles and to explore the relationships between the expression of AChR subunits and the severity of facial nerve injury.Methods: Gene and protein expression of AChR subunits in the orbicularis oris and gastrocnemius muscles of male Sprague–Dawley rats was measured by reverse transcription polymerase chain reaction and western blotting, respectively, 1–90 days after graded facial nerve injury.Results: Expression of ε-AChR in the normal orbicularis oris was significantly higher than that in the gastrocnemius, whereas no γ subunit expression was observed. Expression of α, β, δ, ε, and γ subunits was upregulated in the orbicularis oris and was positively correlated with the degree of facial nerve injury.Discussion: We demonstrated the higher expression of the AChR subunits in the orbicularis oris, compared to gastrocnemius muscles. The differences in expression of these subunits between muscles innervated by the facial nerve and somatic nerves and the correlation of AChR subunit expression with the degree of facial nerve injury yield insights into the sensitivity to muscle relaxants during intraoperative facial nerve monitoring.

Memory-Relevant Mushroom Body Output Synapses Are Cholinergic

SummaryMemories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. Here we demonstrate a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses.

Identification of nicotinic acetylcholine receptor subunit expression in early avian embryos

Most previous studies on nicotine used relatively high concentrations of nicotine, administered later in development. Using chick embryos as our model system, we have found a single low dose of nicotine (100 nM) administered during early development significantly retarded embryo growth, reduced heart rate, and increased the incidence of defects four days later. It is often assumed that nicotine's teratogenic effects are mediated by nicotinic acetylcholine receptors (nAChRs), whose expression at very early stages of embryonic development has not been thoroughly investigated. The objective was to identify which nAChR subunits are expressed during early avian embryogenesis. We performed RT‐PCR expression analysis for eleven nAChR subunits, including alpha1‐alpha7, alpha9, alpha10, beta3, and beta4, at 24‐, 48‐, 72‐, and 96‐hrs of development. Surprisingly, we find nAChR expression is widespread during early development, which suggests the teratogenic effects of nicotine were mediated by the early activation of nAChRs.Nebraska NIH‐INBRE Program; Nebraska Health and Human Services (LB595); Ferlic Undergraduate Summer Research Program, Creighton University College of Arts and SciencesGrant Funding Source: none current

Retinoic acid and nerve growth factor induce differential regulation of nicotinic acetylcholine receptor subunit expression in SN56 cells

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [(3)H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [(3)H]epibatidine binding sites and the mRNA and protein level of the alpha3 and beta2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the alpha4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the alpha7 nAChR subunit. NGF treatment increased the mRNA and protein level of the alpha3 and beta2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the alpha4 and alpha7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells.

Nicotine-induced Ca 2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line

We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M 3 mAChRs on lymphocytes increases the intracellular free Ca 2+ concentration ([Ca 2+] i) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca 2+] i in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca 2+-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca 2+, nicotine (30 μM) evoked rapid, transient increases in [Ca 2+] i. This effect was concentration-dependently inhibited by the α7 nAChR subunit antagonists, α-bungarotoxin (0.01-10 μM) and methyllycaconitine (0.01-10 mM), suggesting that the α7 nAChR subunit mediates Ca 2+ signaling in T-lymphocytes. Nicotine (0.01–10 μM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the α6 and α7 subunits was down-regulated within 1 week, while expression of the α3 and α5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine — and therefore likely smoking — affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca 2+ signaling in lymphocytes.

Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of α and β subunits with specific structural, functional and pharmacological properties. In this study the distribution of α3, α4, α7, β2 and β4 nAChR subunits in the human hippocampus was investigated using immunohistochemistry. Most pyramidal neurons, pre-alpha cells of the entorhinal cortex and dentate granule cells were immunoreactive for all subunits. Small islands of α7 immunoreactive cells were present in the outer presubiculum. α4 and β2, and α3, α4 and β2 immunoreactive fibre tracts were present in the stratum radiatum and subiculum, respectively, suggesting nAChRs may play a role in modulating inputs to the hippocampus via Schaffer collaterals and along the perforant pathway. Some astrocytes were immunoreactive for α3, α7 and β4 subunits. Immunoreactivity to all subunits was noted in association with blood vessels. These results indicate the involvement of multiple nAChR subtypes in the modulation of both neuronal and non-neuronal functions in the human hippocampus.