Ronald J. Lukas

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Nicotinic acetylcholine receptors as targets for antidepressants

While the monoamine deficiency hypothesis of depression is still most commonly used to explain the actions of antidepressant drugs, a growing body of evidence has accumulated that is not adequately explained by the hypothesis. This article draws attention to contributions from another apparently common pharmacological property of antidepressant medications—the inhibition of nicotinic acetylcholine receptors (nAChR). Evidence is presented suggesting the hypercholinergic neurotransmission, which is associated with depressed mood states, may be mediated through excessive neuronal nicotinic receptor activation and that the therapeutic actions of many antidepressants may be, in part, mediated through inhibition of these receptors. In support of this hypothesis, preliminary evidence is presented suggesting that the potent, centrally acting nAChR antagonist, mecamylamine, which is devoid of monoamine reuptake inhibition, may reduce symptoms of depression and mood instability in patients with comorbid depression and bipolar disorder. If this hypothesis is supported by further preclinical and clinical research, nicotinic acetylcholine receptor antagonists may represent a novel class of therapeutic agents for treating mood disorders.

A Novel Nicotinic Acetylcholine Receptor Subtype in Basal Forebrain Cholinergic Neurons with High Sensitivity to Amyloid Peptides

Nicotinic acetylcholine receptors (nAChRs) containing α7 subunits are thought to assemble as homomers. α7-nAChR function has been implicated in learning and memory, and alterations of α7-nAChR have been found in patients with Alzheimers disease (AD). Here we report findings consistent with a novel, naturally occurring nAChR subtype in rodent, basal forebrain cholinergic neurons. In these cells, α7 subunits are coexpressed, colocalize, and coassemble with β2 subunit(s). Compared with homomeric α7-nAChRs from ventral tegmental area neurons, functional, presumably heteromeric α7β2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the β2 subunit-containing nAChR-selective antagonist, dihydro-β-erythroidine (DHβE). Interestingly, presumed, heteromeric α7β2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid β1–42 (Aβ1–42). Slow whole-cell current kinetics, sensitivity to DHβE, and specific antagonism by oligomeric Aβ1–42 also are characteristics of heteromeric α7β2-nAChRs, but not of homomeric α7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Aβ when elicited from MS/DB neurons derived from nAChR β2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric α7β2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Aβ1–42 suggests possible mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and might be targeted by disease therapies.

Alterations of Alzheimer's disease in the cholesterol-fed rabbit, including vascular inflammation. Preliminary observations.

Abstract: We determined the levels of endothelial inflammation using MECA‐32 antibody and a4 nicotinic receptor subunit densities employing [3H]epibatidine binding in the brains of Alzheimers disease (AD) patients, cholesterol‐fed rabbits, and appropriate controls. We also assessed rabbit brain for β‐amyloid levels and immunohistochemical localization, and for evidence of blood‐brain barrier breach using normally‐excluded Evans Blue dye. Dietary cholesterol induced a twofold increase in β‐amyloid concentration in rabbit hippocampal cortex, which may be related to the appearance of β‐amyloid immunoreactivity in the neuropil. Epibatidine binding was significantly decreased in AD superior frontal cortex, but unchanged in the superior frontal cortex of cholesterol‐fed rabbits. Increased vascular MECA‐32 immunoreactivity occurred in AD and cholesterol‐fed rabbit brain. Evans Blue dye could be found in the parenchyma of cholesterol‐fed rabbits only, and appeared as pockets of dye surrounding small blood vessels. The data suggest that vascular inflammation can lead to breach of the blood‐brain barrier, which may produce biochemical derangements in surrounding brain tissue that are conducive to production of β‐amyloid.

Antidepressants Noncompetitively Inhibit Nicotinic Acetylcholine Receptor Function

Abstract : Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter‐gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle‐type nAChR (α1βγδ) in TE671/RD cells, human autonomic nAChR (α3β4α5 ±β2) in SH‐SY5Y neuroblastoma cells, or chick V274T mutant α7‐nAChR heterologously expressed in native nAChR‐null SH‐EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle‐type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T‐α7‐nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.

Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand‐gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short‐term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle‐type (α1β1γδ) or ganglionic (α3β4) nAChR measured using 86Rb+ efflux assays in TE671/RD clonal or SH‐SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank‐order potencies for steroid‐mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as 125I‐labeled α‐bungarotoxin or [3H]acetylcholine to muscle‐type or ganglionic nAChR or to neuronal toxin‐binding nAChR that contain α7 subunits (α7‐nAChR). Steroid‐mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell‐impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle‐type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand‐binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid‐nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.

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 3 H-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 expression of nicotinic acetylcholine receptors containing rat α7 subunits in human SH‐SY5Y neuroblastoma cells

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned α7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these α7‐nAChR function as α‐bungarotoxin (Bgt)‐sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt‐sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional α7‐nAChR in cell lines have failed. Here we report the successful use of SH‐SY5Y human neuroblastoma cells, which naturally express low levels of endogenous α7 transcripts, to stably overexpress heterologous rat nAChR α7 transgenes. These transgenes are expressed as the appropriately‐sized α7 messages and protein, and stably transfected SH‐SY5Y cells have over 30‐times higher levels of specific Bgt binding sites than do wild‐type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of α7‐nAChR. We conclude that stable, functional expression of α7 transgenes in a mammalian cell line has been achieved for the first time.

β-Amyloid Directly Inhibits Human α4β2-Nicotinic Acetylcholine Receptors Heterologously Expressed in Human SH-EP1 Cells

Amyloid-β (Aβ) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimers 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 α4 and β2 subunits (hα4β2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Aβ on hα4β2-nAChR function have not been investigated in detail. We first confirmed expression of hα4 and hβ2 subunits as messenger RNA in transfected, human SHEP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed α4 and β2 subunit protein expression and co-assembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-hα4β2 cells of functional hα4β2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Aβ1-42 in a dose-dependent manner. Functional inhibition was selective for Aβ1-42 compared with the functionally inactive, control peptide Aβ40-1.Aβ1-42-mediated inhibition of hα4β2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5′-yl thiophosphate failed to prevent Aβ1-42-induced inhibition, suggesting that down-regulation of hα4β2-nAChR function by Aβ1-42 is not mediated by nAChR internalization. Sensitivity to Aβ1-42 antagonism at 1 nm was evident for hα4β2-nAChRs, but not for heterologously expressed human α7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nm Aβ1-42, with the magnitude of functional block being higher for 100 nm Aβ1-42 acting on hα7-nAChRs. These findings suggest that hα4β2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Aβ neurotoxicity in AD.

Similarity between rat brain nicotinic α-bungarotoxin receptors and stably expressed α-bungarotoxin binding sites

Abstract: The present results demonstrate stable expression of α‐bungarotoxin (α‐BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild‐type GH4C1 cells do not express α‐BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH4C1 cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH4C1 cells also express saturable, high‐affinity binding sites for 125I‐labeled α‐BGT, with a KD of 0.4 nM and Bmax of 3.2 fmol/106 intact cells. 125I‐α‐BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH4C1 cells. Furthermore, KD and Ki values for 125I‐α‐BGT binding sites on intact α7/GH4C1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α‐BGT binding sites expressed in α7/GH4C1 cells was similar to that of the native brain α‐BGT receptor. Chronic exposure of α7/GH4C1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α‐BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α‐BGT binding sites in transfected α7/GH4C1 cells resemble those for brain nicotinic α‐BGT receptors. If the heterologously expressed α‐BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α‐BGT receptor has a similar homooligomeric structure. Alternatively, if α‐BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α‐BGT receptor.