Partial and full deletion of nicotinic acetylcholine receptor α4 and β2 subunits reduces sensitivity to acute nicotine administration and development of tolerance following chronic nicotine administration.

The diversity of nicotinic cholinergic receptor (nAChR) subunits underlies the complex responses to nicotine. Mice differing in the expression of α4 and β2 subunits, which are most widely expressed in brain, were evaluated for the responses to acute nicotine administration on Y-maze crossings and rears, open-field locomotion and body temperature following chronic treatment with nicotine (0, 0.25, 1.

Presynaptic GABAB autoreceptor regulation of nicotinic acetylcholine receptor mediated [(3)H]-GABA release from mouse synaptosomes.

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [(3)H]-GABA and [(3)H]-dopamine ((3)H-DA) from mouse striatal synaptosomes.

Oral Nicotine Self-Administration in Rodents.

Nicotine addiction is a complex process that begins with self-administration. Consequently, this process has been studied extensively using animal models. A person is usually not called "smoker" if s/he has smoked for a week or a month in a lifetime; in general, a smoker has been smoking for many years.

Regulation of the distribution and function of [(125)I]epibatidine binding sites by chronic nicotine in mouse embryonic neuronal cultures.

Chronic nicotine produces up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) (* denotes that an additional subunit may be part of the receptor). However, the extent of up-regulation to persistent ligand exposure varies across brain regions. The aim of this work was to study the cellular distribution and function of nAChRs after chronic nicotine treatment in primary cultures of mouse brain neurons.

A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus.

Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs.

Individual differences in oral nicotine intake in rats.

To study individual differences in nicotine preference and intake, male and female rats were given free access to a choice of oral nicotine (10 or 20 mg/L) or water for 24 h/day for periods of at least six weeks, starting at adolescence or adulthood. A total of 341 rats, were used in four different experiments; weight, nicotine intake and total liquid consumption were recorded weekly. Results show that rats can discriminate nicotine from water, can regulate their intake, and that there are readily detected individual differences in nicotine preference.

Increased nicotinic acetylcholine receptor protein underlies chronic nicotine-induced up-regulation of nicotinic agonist binding sites in mouse brain.

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of α4β2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion.

86Rb+ efflux mediated by alpha4beta2*-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization.

The nicotinic acetylcholine receptors (nAChR) assembled from alpha4 and beta2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of alpha4beta2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry.

Mouse striatal dopamine nerve terminals express alpha4alpha5beta2 and two stoichiometric forms of alpha4beta2*-nicotinic acetylcholine receptors.

Wild-type and alpha5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate alpha-conotoxin MII (alpha-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration-effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the alpha5 null mutant. Deleting the alpha5 gene also resulted in decreased maximal ACh-stimulated alpha-CtxMII-resistant DA release.

John Daly's compound, epibatidine, facilitates identification of nicotinic receptor subtypes.

The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the alpha7, beta2, or beta4 subunit. In agreement with general belief, the alpha4beta2*-nAChR and alpha7-nAChR subtypes are major components of the epibatidine binding sites.

Pharmacological and immunochemical characterization of alpha2* nicotinic acetylcholine receptors (nAChRs) in mouse brain.

Aim: alpha2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha2* nAChRs in these mouse brain regions.

Methods: alpha2 nAChR subunit-null mutant mice were engineered.

The road to discovery of neuronal nicotinic cholinergic receptor subtypes.

The discovery that mammalian brain expresses the mRNAs for nine different nicotinic cholinergic receptor subunits (alpha2-alpha7, beta2-beta4) that form functional receptors when expressed in Xenopus laevis oocytes suggests that many different types of nicotinic cholinergic receptors (nAChRs) might be expressed in the mammalian brain., Using an historical approach, this chapter reviews some of the progress made in identifying the nAChR subtypes that seem to play a vital role in modulating dopaminergic function. nAChR subtypes that are expressed in dopamine neurons, as well as neurons that interact with dopamine neurons (glutamatergic, GABAergic), serve as the focus of this review.

Acetylcholine-stimulated [3H]GABA release from mouse brain synaptosomes is modulated by alpha4beta2 and alpha4alpha5beta2 nicotinic receptor subtypes.

Nicotinic acetylcholine receptor (nAChR) agonists stimulate the release of GABA from GABAergic nerve terminals, but the nAChR subtypes that mediate this effect have not been elucidated. The studies reported here used synaptosomes derived from the cortex, hippocampus, striatum, and thalamus of wild-type and alpha4-, alpha5-, alpha7-, beta2-, and beta4-null mutant mice to identify nAChR subtypes involved in acetylcholine (ACh)-evoked GABA release. Null mutation of genes encoding the alpha4 or beta2 subunits resulted in complete loss of ACh-stimulated [(3)H]GABA release in all four brain regions.

Partial deletion of the nicotinic cholinergic receptor alpha 4 or beta 2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of alpha 4 and beta 2 subunits.

Alpha4 and beta2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated (86)Rb(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha4beta2(*)-nAChR, because deletion of either the alpha4 or beta2 subunit reduces response at least 90%.

Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L;V16A], a selective alpha7 nicotinic acetylcholine receptor antagonist.

The alpha7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-alpha-bungarotoxin is commonly used to identify alpha7 nAChRs specifically. However, alpha-bungarotoxin also interacts potently with alpha1* and alpha9alpha10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the alpha7 subtype.

The CHRNA5/A3/B4 gene cluster variability as an important determinant of early alcohol and tobacco initiation in young adults.

Background: One potential site of convergence of the nicotine and alcohol actions is the family of the neuronal nicotinic acetylcholine receptors. Our study examines the genetic association between variations in the genomic region containing the CHRNA5, A3, and B4 gene cluster (A5A3B4) and several phenotypes of alcohol and tobacco use in an ethnically diverse young adult sample. Significant results were then replicated in a separate adult population-representative sample.

The neuronal nicotinic receptor subunit genes (CHRNA6 and CHRNB3) are associated with subjective responses to tobacco.

Neuronal nicotinic acetylcholine receptors have been implicated in various measures of nicotine dependence. In this paper, we present findings from an exploratory study of single nucleotide polymorphisms (SNPs) in the CHRNB3 and CHRNA6 genes with tobacco and alcohol phenotypes, including frequency of use and three subjective response factors occurring shortly after initiation of use. Subjects were 1056 ethnically diverse adolescents ascertained from clinical and community settings.

The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum.

This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes.

Chronic nicotine cell specifically upregulates functional alpha 4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally.

Gene targeting demonstrates that alpha4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine.

[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD approximately 0.02 nM) and lower affinity (KD approximately 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha7, beta2 or beta4 subunits.

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers.

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression.

Decreased anxiety-like behavior in beta3 nicotinic receptor subunit knockout mice.

Nicotine, via a family of nicotinic acetylcholine receptors, elicits many physiological responses, including alterations in anxiety. Studies suggest that the effects of nicotine on anxiety may support smoking behaviors. We reported previously that mice lacking the beta3 nicotinic receptor subunit demonstrate increased activity in the open field arena.

Pharmacology of alpha-conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice.

Subtypes of nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds alpha-conotoxin MII (alpha-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the alpha6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of alpha6beta2* nAChRs expressed in vivo for assessing pharmacology of alpha6beta2* nAChRs.

Association of the neuronal nicotinic receptor beta2 subunit gene (CHRNB2) with subjective responses to alcohol and nicotine.

Nicotine addiction and alcohol dependence are highly comorbid disorders that are likely to share overlapping genetic components. We have examined two neuronal nicotinic receptor subunit genes (CHRNA4 and CHRNB2) for possible associations with nicotine and alcohol phenotypes, including measures of frequency of use and measures of initial subjective response in the period shortly after first using the drugs. The subjects were 1,068 ethnically diverse young adults participating in ongoing longitudinal studies of adolescent drug behaviors at the University of Colorado, representing both clinical and community samples.