Regulation Of Nicotinic Acetylcholine Receptors Research Articles

Swift regulation of nicotinic acetylcholine receptors (nAChRs) and glutathione S-transferase (GST) enables the rapid detoxification of thiacloprid in pine sawyer beetles

Thiacloprid, a neonicotinoid insecticide, has become one of the major control agents for the pine sawyer beetle, Monochamus alternatus Hope, however, the mechanism of detoxification is unknown. We demonstrate that glutathione S-transferases (GSTs) and nicotinic acetylcholine receptors (nAChRs) are involved in the rapid detoxification of thiacloprid in M. alternatus larvae. The activity of detoxification enzyme GSTs was significantly higher, while the activity of acetylcholinesterase (AChE) was inhibited under thiacloprid exposure. The inhibition of AChE activity led to lethal over-stimulation of the cholinergic synapse, which was then released by the rapid downregulation of nAChRs. Meanwhile, GSTs were overexpressed to detoxify thiacloprid accordingly. A total of 3 nAChR and 12 GST genes were identified from M. alternatus, among which ManAChRα2 and MaGSTs1 were predicted to confer thiacloprid tolerance. RNA interference (RNAi) was subsequently conducted to confirm the function of ManAChRα2 and MaGSTs1 genes in thiacloprid detoxification. The successful knock-down of the ManAChRα2 gene led to lower mortality of M. alternatus under LC30 thiacloprid treatment, and the suppression of the MaGSTs1 gene increased the mortality rate of M. alternatus. However, the mortality rate has no significant difference with controls when thiacloprid was fed together with both dsMaGSTs1 and dsManAChRα2. Molecular docking modeled the molecular basis for interaction between MaGSTs1/ManAChR and thiacloprid. This study highlights the important roles that ManAChRα2 and MaGSTs1 genes play in thiacloprid detoxification through transcriptional regulation and enzymatic metabolization, and proposes a new avenue for integrated pest management that combines pesticides and RNAi technology as an efficient strategy for M. alternatus control.

Effects of nicotine + morphine on reward‐related behavior and nicotinic acetylcholine receptor regulation in mouse midbrain.

In the United States about 480,000 deaths/year are caused by cigarette smoking and about 50,000 due to opioid abuse. Co‐use of more than one drug is common, therefore, it is vital to study the effects of drug combinations to understand how co‐use alters the brain. Especially given that in America, > 90% of opioid dependents are heavy smokers. The objective for this study is to determine how combined nicotine + morphine alters reward‐related behavior and nicotinic acetylcholine receptor (nAChR) expression in the midbrain. Our hypothesis is that co‐use of nicotine + morphine leads to enhanced GABA neuron disinhibition of VTA dopamine neurons. Female and male (3–5 months old) C57BL/6J background strain mice with or without α4‐mCherryα6‐GFP nAChRs were used in conditioned place (CPP) preference assays to examine reward‐related behavior with nicotine + morphine. We used confocal microscopy to analyze the density of α4‐containing (α4*), α6*, and α4α6* nAChRs in ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). In our CPP assays, a Two‐way ANOVA (factors: treatment and sex) showed a significant effect of sex (F (1, 42) = 5.75; p = 0.02) on reward‐related behavior, suggesting a sex‐dependent dose effect with morphine. In our microscopy assays, nicotine + morphine treatment downregulated α4* nAChRs on SNr GABA cells. Our CPP data supports that sex is an important variable in the effects of drugs on reward‐related behavior. Our receptor regulation data suggests that downregulation of α4* nAChRs on SNr GABA cells is important to the combined effects of nicotine + morphine.Support or Funding InformationSupport: Funding was provided through start‐up funds through the Marshall University Research Corporation.

Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors

Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine–mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine.

Drug-dependent behaviors and nicotinic acetylcholine receptor expressions in Caenorhabditis elegans following chronic nicotine exposure

Nicotine, the major psychoactive compound in tobacco, targets nicotinic acetylcholine receptors (nAChRs) and results in drug dependence. The nematode Caenorhabditis elegans’ (C. elegans) genome encodes conserved and extensive nicotinic receptor subunits, representing a useful system to investigate nicotine-induced nAChR expressions in the context of drug dependence. However, the in vivo expression pattern of nAChR genes under chronic nicotine exposure has not been fully investigated. To define the role of nAChR genes involved in nicotine-induced locomotion changes and the development of tolerance to these effects, we characterized the locomotion behavior combining the use of two systems: the Worm Tracker hardware and the WormLab software. Our results indicate that the combined system is an advantageous alternative to define drug-dependent locomotion behavior in C. elegans. Chronic (24-h dosing) nicotine exposure at 6.17 and 61.7μM induced nicotine-dependent behaviors, including drug stimulation, tolerance/adaption, and withdrawal responses. Specifically, the movement speed of naïve worms on nicotine-containing environments was significantly higher than on nicotine-free environments, suggesting locomotion stimulation by nicotine. In contrast, the 24-h 6.17μM nicotine-treated worms exhibited significantly higher speeds on nicotine-free plates than on nicotine-containing plates. Furthermore significantly increased locomotion behavior during nicotine cessation was observed in worms treated with a higher nicotine concentration of 61.7μM. The relatively low locomotion speed of nicotine-treated worms on nicotine-containing environments also indicates adaption/tolerance of worms to nicotine following chronic nicotine exposure. In addition, this study provides useful information regarding the comprehensive in vivo expression profile of the 28 “core” nAChRs following different dosages of chronic nicotine treatments. Eleven genes (lev-1, acr-6, acr-7, acr-11, lev-8, acr-14, acr-16, acr-20, acr-21, ric-3, and unc-29) were significantly up-regulated following 61.7μM nicotine treatment, in which worms showed significantly increased locomotion behavior. This study provides insights into the linkage between nicotine-induced locomotion behavior and the regulation of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor regulation of striatal dopamine transmission: The different requirement for diverse subtypes in ventral versus dorsal striatum

Nicotinic acetylcholine receptor regulation of striatal dopamine transmission: The different requirement for diverse subtypes in ventral versus dorsal striatum

Abstract 1803: Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung cancer.

Abstract Nicotine, the addictive component of cigarette smoke, has been shown to promote cell proliferation, migration, invasion, and angiogenesis in multiple cancer types. Earlier studies from our lab had shown that nicotine can promote the growth and metastasis of non-small cell lung carcinoma (NSCLC) in mouse models. While a broad range of nAChRs have been found to be expressed on NSCLC cell lines, nicotine-mediated proliferation, invasion, and migration are facilitated predominantly through the α7 subunit. Consistent with this, α7 nAChR levels are elevated in NSCLC in mice that were administered nicotine. Stimulation of α7 nAChR with nicotine has been reported to activate Src, resulting in inactivation of the retinoblastoma (Rb) tumor suppressor protein and enhancing E2F-mediated transcription. The Rb-E2F transcriptional regulation pathway is known to induce genes involved in cell cycle progression, angiogenesis, and metastasis implicating it in tumor survival and progression. Given this background, attempts were made to elucidate whether nAChR genes are regulated by the Rb-E2F pathway. Analysis of a 2000bp promoter region of the human α7 gene revealed the presence of multiple E2F binding sites. Transient transfection experiments showed α7 to be responsive to multiple E2Fs. E2F1 was found to associate with the α7 promoter via chromatin immunoprecipitation assays. Depletion of E2F genes via small interfering RNA demonstrated a differential regulation of α7 by E2F family members 1-5. These results raise the possibility that exposure to nicotine stimulates the α7 signaling cascade resulting in elevated E2F1-mediated activation of various proliferative promoters, including that of α7 itself in a positive feedback mechanism. In addition to the α7 nAChR, recent genome wide association studies (GWAS) have identified a susceptibility locus for human lung cancer at 15q25.1 which encodes for nAChR subunits α3 and α5. Promoter analysis has revealed that both α3 and α5 have multiple potential E2F binding sites, as well. Preliminary knock down of E2F family members via small interfering RNA has suggested these subunits may also be differentially regulated by the E2F family of transcription factors. Further studies are under way to elucidate the role of E2F in the regulation of nAChR α3 and α5, and how this impacts nicotine signaling as well as growth and progression of NSCLC. Citation Format: Courtney M. Schaal, Smitha Pillai, Jackie L. Johnson, Srikumar Chellappan. Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2013-1803

Bacterial expression of the water-soluble domain of lynx1, an endogenous neuromodulator of human nicotinic receptors

Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.

Parallel Anxiolytic-Like Effects and Upregulation of Neuronal Nicotinic Acetylcholine Receptors Following Chronic Nicotine and Varenicline

Clinical and preclinical studies suggest that regulation of nicotinic acetylcholine receptors (nAChR) maybe involved in the etiology of withdrawal symptoms. We evaluated heteromeric nAChR regulation via [³H]epibatidine binding following cessation of chronic nicotine or varenicline treatment. Animals were concurrently tested in the marble-burying test to evaluate treatment-related effects. We found that both nicotine (18 mg/kg/day, free base) and varenicline (1.8 mg/kg/day) chronically administered for 14 days upregulated nAChRs significantly in the cortex, hippocampus, striatum, and thalamus. The duration of upregulation (up to 72 hr) was both drug and region specific. In addition to nAChR upregulation, chronic administration of both nicotine and varenicline had anxiolytic-like effects in the marble-burying test. This effect was maintained for 48 hr following cessation of varenicline but was absent 24 hr following cessation from nicotine. Additionally, marble-burying behavior positively correlated to the regulation of cortical nAChRs following cessation of either treatment. Varenicline has been shown to be an efficacious smoking cessation aid, with a proposed mechanism of action that includes modulation of dopamine release in reward areas of the brain. Our studies show that varenicline elicits both anxiolytic effects in the marble-burying test as well as region- and time-specific receptor upregulation. These findings suggest receptor upregulation as a mechanism for its efficacy as a smoking cessation therapy.

Abnormal response of dopaminergic neurons to nicotine without perturbation of nicotinic receptors in αCGRP knock-out mice

Alpha-calcitonin gene-related peptide (αCGRP) is a neuropeptide with multiple biological properties, including the regulation of nicotinic acetylcholine receptors (nAChRs). We have previously reported a reduction of somatic withdrawal symptoms in αCGRP knock-out mice exposed to chronic nicotine, leading us to investigate the contribution of αCGRP to the regulations of ventral tegmental area (VTA) neurons and their response to nicotine. The electrophysiological activity of VTA dopaminergic (DA) neurons was recorded in vivo, under anesthesia. These neurons displayed identical spontaneous electrophysiogical activities in wild-type and αCGRP−/− mice. However, we found that intravenous administration of nicotine (30 μg/kg) had no significant effect on the activity of DA neurons in αCGRP−/− mice, whereas it induced a doubling of the firing rate in wild-type animals. A higher dose (90 μg/kg) produced a significant excitation in both strains, but this effect remained smaller in the mutants. To investigate this difference, we have studied the functional state of nAChRs in wild-type and αCGRP−/− mice. Both strains exhibited identical expression of α 7 and α 4β 2 nAChRs as revealed by autoradiographical studies in the VTA. In addition, focal application of acetylcholine on DA neurons recorded by patch-clamp revealed identical currents mediated by nAChRs in mutant animals, as compared to wild-type mice. These data outline the possibility of a contribution of αCGRP to the effects of nicotine on DA neurons, by a physiological pathway independent of VTA nicotinic receptors.

Muscle-like nicotinic receptor accessory molecules in sensory hair cells of the inner ear

Nothing is known about the regulation of nicotinic acetylcholine receptors (nAChRs) in hair cells of the inner ear. MuSK, rapsyn and RIC-3 are accessory molecules associated with muscle and brain nAChR function. We demonstrate that these accessory molecules are expressed in the inner ear raising the possibility of a muscle-like mechanism for clustering and assembly of nAChRs in hair cells. We focused our investigations on rapsyn and RIC-3. Rapsyn interacts with the cytoplasmic loop of nAChR α9 subunits but not nAChR α10 subunits. Although rapsyn and RIC-3 increase nAChR α9 expression, rapsyn plays a greater role in receptor clustering while RIC-3 is important for acetylcholine-induced calcium responses. Our data suggest that RIC-3 facilitates receptor function, while rapsyn enhances receptor clustering at the cell surface.

Long-term effects of chronic nicotine exposure on brain nicotinic receptors

Chronic nicotine exposure results in long-term homeostatic regulation of nicotinic acetylcholine receptors (nAChRs) that play a key role in the adaptative cellular processes leading to addiction. However, the relative contribution of the different nAChR subunits in this process is unclear. Using genetically modified mice and pharmacological manipulations, we provide behavioral, electrophysiological, and pharmacological evidence for a long-term mechanism by which chronic nicotine triggers opposing processes differentially mediated by beta2*- vs. alpha7*nAChRs. These data offer previously undescribed insights into the understanding of nicotine addiction and the treatment of several human pathologies by nicotine-like agents chronically acting on beta2*- or alpha7*nAChRs.

Novel Families of Toxin-like Peptides in Insects and Mammals: A Computational Approach

Most animal toxins are short proteins that appear in venom and vary in sequence, structure and function. A common characteristic of many such toxins is their apparent structural stability. Sporadic instances of endogenous toxin-like proteins that function in non-venom context have been reported. We have utilized machine learning methodology, based on sequence-derived features and guided by the notion of structural stability, in order to conduct a large-scale search for toxin and toxin-like proteins. Application of the method to insect and mammalian sequences revealed novel families of toxin-like proteins. One of these proteins shows significant similarity to ion channel inhibitors that are expressed in cone snail and assassin bug venom, and is surprisingly expressed in the bee brain. A toxicity assay in which the protein was injected to fish induced a strong yet reversible paralytic effect. We suggest that the protein may function as an endogenous modulator of voltage-gated Ca 2+ channels. Additionally, we have identified a novel mammalian cluster of toxin-like proteins that are expressed in the testis. We suggest that these proteins might be involved in regulation of nicotinic acetylcholine receptors that affect the acrosome reaction and sperm motility. Finally, we highlight a possible evolutionary link between venom toxins and antibacterial proteins. We expect our methodology to enhance the discovery of additional novel protein families.

Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons

We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons of Sprague–Dawley rats. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that α3 and α4 subunits declined and α6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of α4 mRNA in SNc than VTA at gestational day (G) 15, and of α5, α6 and β3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [ 3H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation.

Regulation of nicotinic acetylcholine receptors by tyrosine kinases in the peripheral and central nervous system: same players, different roles.

Nicotinic acetylcholine receptors (nAChRs) exist in many subtypes and are found in the peripheral and central nervous system where they mediate or modulate synaptic transmission. We review how tyrosine phosphorylation and kinases regulate muscle and neuronal nAChRs. Interestingly, although some of the same kinase players interact with the various receptor subtypes, the functional consequences are different. While concerted action of MuSK, Abl- and Src-family kinases (SFKs) regulates the synaptic distribution of nAChRs at the neuromuscular junction, SFKs activate heteromeric neuronal nAChRs in adrenal chromaffin cells, thereby enhancing catecholamine secretion. In contrast, the activity of homomeric neuronal nAChRs, as found in the hippocampus, is negatively regulated by tyrosine phosphorylation and SFKs. It appears that tyrosine kinases provide the means to regulate all nAChRs; but the functional consequences, even those caused by the same kinase family, are specific for each receptor subtype and location.

Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor.

Neuronal nicotinic receptors in PC12 cells were measured by binding with [3H]epibatidine and in functional studies with agonist-stimulated 86Rb+ efflux and [3H]norepinephrine release assays. Two subtypes of receptors labeled by [3H]epibatidine were found: one that was increased about 4-fold in cells grown for 2 to 4 days in the presence of nicotine and one that was increased 5-fold in cells grown for 2 to 4 days in the presence of nerve growth factor (NGF). The actions of the two treatments were superadditive, resulting in approximately a 13-fold increase in binding sites in cells grown in the combination of the two treatments. The pharmacology of the binding sites in the nicotine- and NGF-treated cells was compared with the pharmacology of defined alpha3beta2 and alpha3beta4 nicotinic acetylcholine receptor (nAChR) subtypes heterologously expressed in human embryonic kidney 293 cells. Nicotine treatment predominantly increased a receptor with characteristics of an alpha3beta2 subtype, whereas the NGF treatment exclusively increased a receptor with characteristics of an alpha3beta4 subtype. Nicotinic receptor-mediated function measured with the 86Rb+ efflux assay was evident only in the NGF-treated cells, and it had a pharmacological profile that was, again, nearly identical to that of the heterologously expressed alpha3beta4 receptor subtype. Receptor function measured with the [3H]norepinephrine release assay was measurable in both nicotine-treated and NGF-treated cells; however, cytisine-stimulated [3H]norepinephrine release indicated that nicotine treatment increased an nAChR containing beta2 subunits, whereas NGF increased a receptor containing beta4 subunits. NGF treatment increased mRNA only for beta4 subunits in these cells, whereas nicotine treatment did not affect mRNA for any of the subunits measured. After withdrawal of the treatments, the receptors increased by nicotine were much less stable than those increased by NGF.

Regulation of nicotinic acetylcholine receptors on human neuroblastoma cells during differentiation

Neuronal nicotinic acetylcholine receptors are expressed on a variety of cells in the nervous system where they play key roles in synaptic transmission and information transfer. Little is known, however, about the molecular mechanisms that control their expression, distribution, and function during nervous system development. We have investigated the control of expression during differentiation of one class of acetylcholine receptors that bind α-bungarotoxin of human neuroblastoma cells. We report that induction of differentiation of SH-SY5Y, SK-n-SH or IMR-32 cells by the phorbol ester 12- O-tetradecanoyl phorbol 13-myristate (10 nM, TPA) or by retinoic acid resulted in as much as a 70% decline in α-bungarotoxin receptors on the cells. The response to the phorbol ester was blocked by the protein kinase C inhibitors staurosporine and bisindolylmaleimide. The decrease in receptors induced by 10 μM retinoic acid was not affected by either agent. However, responses to lower (10 nM) concentrations of retinoic acid were blocked by staurosporine but not bisindolylmaleimide, suggesting a dual mechanism of action for retinoic acid in regulating acetylcholine receptors. It appears that acetylcholine receptors on neuroblastoma cells are regulated during differentiation by both protein kinase C-dependent and -independent mechanisms.

Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA

Previous studies had shown that MyoD promoted nicotinic acetylcholine subunit gene expression; the present experiments were done to determine whether this subsequently led to the development of functional nicotinic acetylcholine receptors. Transfection of C3H 10T1/2 cells with MyoD cDNA resulted in the appearance of [ 125I]α-bungarotoxin binding sites; radiolabelled α-toxin binding was not observed in cells transfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seven and 11 days after exposure to fusion medium. [ 125I]α-bungarotoxin binding was of high affinity ( K d = 1 nM ), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with ic 50s of 1–3 nM for α-bungarotoxin, 1–3 μM for d-tubocurarine and 3–10 μM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functional response. Carbachol resulted in uptake of 22Na into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response was effectively blocked by nicotinic receptor ligands, including α-bungarotoxin ( ic 50 = 2 to 6 nM ) and d-tubocurarine ( ic 50 = 0.1 to 0.4 μM ); muscarinic receptor ligands had no effect. A time course study showed that α-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the α-bungarotoxin recognition site. To conclude, the present studies show that transfection with MyoD results in the appearance of a functional cell surface muscle type nicotinic acetylcholine receptor. They further support the contention that MyoD plays a pivotal role in nicotinic acetylcholine receptor regulation in muscle.

Heterogeneity and Regulation of Nicotinic Acetylcholine Receptors

Publisher Summary This chapter focuses on nicotinic acetylcholine receptors (nAChRs). This component emphasizes studies done using mammalian tissues or their derivatives, and using vertebrate nonmammalian systems. The chapter foscuses on the bases, manifestations, and functional significance of nAChR diversity. There are at least two populations of putative nAChRs in brain defined based on their ability to interact with 3H-labeled agonists or radiolabeled α-bungarotoxin. These binding sites are differentially regulated during development and exhibit differential anatomical distributions. Studies done on nAChRs in the periphery at the neuromuscular junction or on neural crest-derived cells are largely consistent with this dogma, in that chronic agonist treatment produces a decrease in receptor numbers and usually a comparable or quantitatively larger decrease in functional responsiveness. Chronic administration of two pyrethroid xenobiotics, bioallethrin and deltamethrin, produces an increase in levels of nAChRs. It is also becoming clear that diversity in mechanisms involved in the regulation of nAChR expression and function is built on the diversity of the nAChR family.

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.

Changes in the number, function, and regulation of nicotinic acetylcholine receptors during neuronal development

Acetylcholine receptor (AChR) development was examined in neurons freshly isolated from chick ciliary ganglia. Between Embryonic Day 8 (E8) and 16, both the ACh response per unit membrane and the density of surface AChRs increased, while the apparent affinity for ACh decreased. AChRs had single-channel conductances of 25 and 40 pS. The distribution of single-channel events shifted during development; at E8 events of both conductances were equally rare, while by E14 there were many events and most were 40 pS. The open durations of 25 and 40 pS events had two mean values. The open lifetimes of the 25 pS events did not change between E8 and E14, while the lifetimes of the 40 pS events increased, and by E14 most were long. The ACh response of the neurons also became sensitive to regulation by a cAMP-dependent mechanism at about E10. The observed changes may reflect developmental control over processing required for receptor regulation and differential expression of AChR subtypes.