ABSTRACT
Despite the evidence that there is a daily rhythm in smoking behavior and that the effects of drugs of abuse exhibit diurnal variations, very few studies have explored the extent to which sensitivity to the effects of nicotine vary over the course of the day. In the studies described in this report, the melatonin proficient mouse strain C3H/Ibg and the melatonin deficient mouse strains C57BL/6J and DBA/2J were assessed for diurnal variations in sensitivity to the effects of nicotine. Results indicated that there is significant variation in sensitivity to both activity and body temperature depressant effects of nicotine in the melatonin proficient C3H/Ibg strain with maximal sensitivity occurring during the latter third of the light period of the light cycle and minimal sensitivity taking place during the last third of the dark phase of the light cycle. The melatonin deficient strains did not exhibit diurnal differences in sensitivity to the effects of nicotine suggesting a potential role for melatonin in modulating the effects of nicotine. Experiments with knockout mice lacking both the Mtnr1a and Mtnr1b melatonin receptors confirmed that the reduced sensitivity observed during the dark phase is melatonin dependent. Diurnal variation in nicotinic receptor expression also was measured in cortex, hippocampus, hypothalamus and striatum using [(125)I]-α-bungarotoxin and [(125)I]-epibatidine. [(125)I]-α-bungarotoxin binding in hypothalamus of C3H mice exhibited a diurnal pattern with maximal binding observed in the latter third of the light portion of the light cycle. No other significant differences in binding were detected.
Subject(s)
Circadian Rhythm/genetics , Circadian Rhythm/physiology , Melatonin/physiology , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Animals , Body Temperature/drug effects , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Bungarotoxins/metabolism , Dose-Response Relationship, Drug , Female , Male , Maze Learning/drug effects , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Motor Activity/drug effects , Pyridines/metabolism , Radioligand Assay , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/metabolism , Sex Characteristics , Signal Transduction/physiology , Species SpecificityABSTRACT
The reasons why people smoke are varied, but research has shown that genetic influences on various aspects of nicotine addiction are a major factor. There also is a strong genetic influence on measures of nicotine sensitivity in mice. Despite the established contribution of genetics to nicotine sensitivity in mice and humans, no naturally occurring genetic variation has been identified that demonstrably alters sensitivity to nicotine in either species. However, one genetic variant has been implicated in altering nicotine sensitivity in mice is a T529A polymorphism in Chrna4, the gene that encodes the nicotinic receptor (nAChR) alpha4 subunit. The Chrna4 T529A polymorphism leads to a threonine to alanine substitution at position 529 of the alpha4 subunit. To more definitively address whether the Chrna4 T529A polymorphism does, in fact, influence sensitivity to nicotine, knock-in mice were generated in which the threonine codon at position 529 was mutated to an alanine codon. Compared with Chrna4 T529 littermate controls, the Chrna4 A529 knock-in mice exhibited greater sensitivity to the hypothermic effects of nicotine, reduced oral nicotine consumption and did not develop conditioned place preference to nicotine. The Chrna4 A529 knock-in mice also differed from T529 littermates for two parameters of acetylcholine-stimulated Rb+ efflux in midbrain: maximal efflux and the percentage of alpha4beta2* receptors with high sensitivity to activation by agonists. Results indicate that the polymorphism affects the function of midbrain alpha4beta2* nAChRs and contributes to individual differences in several behavioral and physiological responses to nicotine thought to be modulated by midbrain alpha4beta2* nAChRs.
Subject(s)
Gene Knock-In Techniques , Nicotine/pharmacology , Receptors, Nicotinic/genetics , Acetylcholine/pharmacology , Administration, Oral , Animals , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Mice , Mutation/genetics , Nicotine/administration & dosage , Organ Specificity/drug effects , Polymorphism, Single Nucleotide/genetics , Pyridines/metabolism , Receptors, Nicotinic/metabolism , Reproducibility of ResultsABSTRACT
Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.
