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A test battery for measuring nicotine effects in mice
Abstract
A test battery consisting of measurement of respiration, startle response, Y-maze activity, heart rate, and body temperature has been developed to assess the effects of nicotine on the mouse. Results obtained using the test battery were compared to those obtained with each test individually in four inbred strains of mouse (BALB, C57BL, DBA and C3H). No significant differences between the results obtained using the test battery and those obtained with individual tests were found. The results did demonstrate, however, that the genotype of the mouse strongly influenced the responses in several of the tests.
... The acute effects of nicotine on locomotor activity, body temperature, and HPA activity were examined using a within-subjects design as part of a modified test battery (Marks et al., 1985;Marks et al., 1989;Kamens et al., 2015). Specifically, every animal in each experiment received intraperitoneal injections of both saline and nicotine (Experiment 1 − 0.5 mg/kg; Experiment 2 − 0.5 or 1 mg/kg; doses presented as freebase nicotine). ...
... Immediately following injection, each mouse was placed into a Y-maze for 10 min. Locomotor activity was analyzed by measuring total distance traveled in the first 5 min of the test because near-maximal effects on Y-maze locomotion are observed 5 min after nicotine injection (Marks et al., 1985). Following locomotor activity testing, mice were returned to holding cages until rectal body temperature was measured 15 min after the injection. ...
... All samples were collected within 3 min of initial cage disruption between 14:00-16:00 h. Nicotine doses and testing times were based on published methods (Marks et al., 1985;Marks et al., 1989;Kamens et al., 2015). Two primary dependent variables were used to assess acute nicotine responses: nicotine-induced change in locomotor activity (cm) and change in body temperature (°C). ...
Anxiety disorders and nicotine use are significant contributors to global morbidity and mortality as independent and comorbid diseases. Early-life stress, potentially via stress-induced hypothalamic-pituitary-adrenal axis (HPA) dysregulation, can exacerbate both. However, little is known about the factors that predispose individuals to the development of both anxiety disorders and nicotine use. Here, we examined the relationship between anxiety-like behaviors and nicotine responses following adolescent stress. Adolescent male and female BALB/cJ mice were exposed to either chronic variable social stress (CVSS) or control conditions. CVSS consisted of repeated cycles of social isolation and social reorganization. In adulthood, anxiety-like behavior and social avoidance were measured using the elevated plus-maze (EPM) and social approach-avoidance test, respectively. Nicotine responses were assessed with acute effects on body temperature, corticosterone production, locomotor activity, and voluntary oral nicotine consumption. Adolescent stress had sex-dependent effects on nicotine responses and exploratory behavior, but did not affect anxiety-like behavior or social avoidance in males or females. Adult CVSS males exhibited less exploratory behavior, as indicated by reduced exploratory locomotion in the EPM and social approach-avoidance test, compared to controls. Adolescent stress did not affect nicotine-induced hypothermia in either sex, but CVSS males exhibited augmented nicotine-induced locomotion during late adolescence and voluntarily consumed less nicotine during adulthood. Stress effects on male nicotine-induced locomotion were associated with individual differences in exploratory locomotion in the EPM and social approach-avoidance test. Relative to controls, adult CVSS males and females also exhibited reduced corticosterone levels at baseline and adult male CVSS mice exhibited increased corticosterone levels following an acute nicotine injection. Results suggest that the altered nicotine responses observed in CVSS males may be associated with HPA dysregulation. Taken together, adolescent social stress influences later-life nicotine responses and exploratory behavior. However, there is little evidence of an association between nicotine responses and prototypical anxiety-like behavior or social avoidance in BALB/cJ mice.
... were examined using a test battery developed at the Institute for Behavioral Genetics for measuring the behavioral effects of nicotine (Marks et al, 1985;Marks et al, 1989;Mexal et al, 2012). Briefly, locomotor activity was examined in a symmetrical Y-maze, consisting of three red Plexiglas arms (26 L X 6.1 W X 10.2 H; cm). ...
... Following activity testing, the mice were returned to the holding cages before being tested for rectal body temperature 13 minutes after the nicotine injection. Nicotine doses and testing times were based on published methods (Marks et al, 1985;Marks et al, 1989;Mexal et al, 2012). Two primary dependent variables were evaluated: nicotine-induced locomotor activity (beam crosses) and change in body temperature (°C). ...
Genetic factors explain approximately half of the variance in smoking behaviors, but the molecular mechanism by which genetic variation influences behavior is poorly understood. SNPs in the putative promoter region of CHRNB3, the gene that encodes the β3 subunit of the nicotinic acetylcholine receptor (nAChR), have been repeatedly associated with tobacco behaviors. In this work we sought to identify putative function of three SNPs in the promoter region of CHRNB3 on in vitro gene expression. Additionally, we used β3 null mutant mice as a model of reduced gene expression to assess the effects on nicotine behaviors. The effect of rs13277254, rs6474413, and rs4950 on reporter gene expression was examined using a luciferase reporter assay. A major and minor parent haplotype served as the background on which alleles at the three SNPs were flipped onto different backgrounds (e.g. minor allele on major haplotype background). Constructs were tested in three human cell lines: BE(2)-C, SH-SY5Y and HEK 293T. In all cell types the major haplotype led to greater reporter gene expression compared to the minor haplotype, and results indicate that this effect is driven by rs6474413. Moreover, mice lacking the β3 subunit showed reduced voluntary nicotine consumption compared that of wildtype animals. These data provide evidence that the protective genetic variant at rs6474413 identified in human genetic studies reduces gene expression and that decreased β3 gene expression in mice reduces nicotine intake. This work contributes to our understanding of the molecular mechanisms that contribute to the human genetic associations and tobacco behaviors.
Copyright © 2015. Published by Elsevier Ltd.
... All nicotine doses are expressed as free base. Behavioral test measures were selected based on previous studies (Marks et al., 1985;Tritto et al., 2004). Mice were habituated to the experimental room for one h prior to testing. ...
... Acute injection of nicotine elicits a dose-dependent decrease in locomotor activity and an induction of hypothermia in WT mice (Marks et al., 1985). β2 knock-out mice demonstrate reduced sensitivity to the acute injections of nicotine on these measures (Tritto et al., 2004). ...
Several mutations in α4 or β2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the β2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (β2VL). Previous studies indicated that the β2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of β2VL mice. No differences in β2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the β2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.
... More recently, epidemiological studies have reported an inverse correlation between the incidence of Alzheimer's disease and smoking (24,56), suggesting a neuroprotective effect of nicotine. A nicotinic hypothesis for the etiology and treatment of Alzheimer's disease is further supported by the observations that (i) there is a significant depletion of high affinity nicotinic receptors in Alzheimer's patients' brains (30,42), (ii) chronic nicotine administration has been demonstrated to upregulate nAChRs whose density is decreased in the brain of Alzheimer patients (1 1, 36,49) and (iii) nicotine administered via subcutaneous injection (46) or transdermal patch (59) to humans improves attention and learning. Additional mechanistic support for such an hypothesis can be gleaned from the distribution of nAChRs in the brain and the reported effects of nicotine on neurotransmitter release in vitro and in vivo, both of which are potentially relevant to acute therapeutic effects. ...
... Nicotine and RJR-2403 were compared using a physiological test battery in C57B1/6 mice, according to the methods in ref. 36. Mice were injected subcutaneously with saline, (S)-(-)-nicotine (2.5, 10, or 15 pmol/kg), or RJR-2403 (75, 100, or 125 pmol/kg). ...
The therapeutic benefit of nicotinic ligands in a variety of neurodegenerative pathologies involving the CNS has energized the search for nAChR subtype-selective ligands (4,6,22,27,51). Numerous studies have demonstrated that nicotinic pharmacology is beneficial in improvement of cognitive function both in animals and in humans (18,29,31,41), but the demonstration of potential beneficial effects of nicotinic pharmacology is hampered by the lack of CNS-selective ligands. As a result, there has been a concerted effort to develop nicotinic compounds with selectivity for CNS nAChRs (e.g., ABT-418 and RJR-2403) as potential pharmaceutical tools in the management of numerous disorders (4,10,18,33). RJR-2403 exhibits the desired subtype selectivity and the physiological and behavioral profiles to be of potential clinical use in the management of CNS pathologies. The nicotinic agonist RJR-2403 provides a wide therapeutic window. It convincingly discriminates between the major nAChR subtypes expressed in the periphery (heart and skeletal muscle) and the neuronal subtypes expressed in discrete brain regions. Although no specific subtypes have yet been assigned to any behavioral end-points, the enhancement of performance in cognitive tests elicited by RJR-2403, coupled with the lack of side effects observed at cardiovascular sites, demonstrate that different subtype(s) subserve the psychotherapeutic benefits and the side-effect liabilities of nicotinic ligands.
... However, although nicotine produces both locomotor activation and inhibition, it often produces locomotor inhibition in mice (e.g. [143,144]). Tolerance develops to these effects with repeated treatment [145], which can subsequently reveal locomotor stimulant and sensitized responses to nicotine [146,147]. ...
... In further exploration of the genetic basis of such effects it will be important to separately address the difference between nicotine effects on premorbid obesity, from those effects produced by chronic nicotine administration and withdrawal. In such models, it will be advantageous to monitor a number of physiological and behavioral parameters that might lead to nicotine self-treatment, for which tolerance develops at different rates [143]. Nicotine administration can regulate both food intake and energy expenditure. ...
Humans differ in their ability to quit using addictive substances, including nicotine, the major psychoactive ingredient in tobacco. For tobacco smoking, a substantial body of evidence, largely derived from twin studies, indicates that approximately half of these individual differences in ability to quit are heritable genetic influences that likely overlap with those for other addictive substances. Both twin and molecular genetic studies support overlapping influences on nicotine addiction vulnerability and smoking cessation success, although there is little formal analysis of the twin data that support this important point. None of the current datasets provides clarity concerning which heritable factors might provide robust dimensions around which individuals differ in ability to quit smoking. One approach to this problem is to test mice with genetic variations in genes that contain human variants that alter quit success. This review considers which features of quit success should be included in a comprehensive approach to elucidate the genetics of quit success, and how those features may be modeled in mice.
... Indeed, the use of inbred mouse strains and genetically modified mice has proven very useful in examining genetic contributions to nicotine physiology and behavior (Hatchell and Collins, 1977; Picciotto et al., 2000). Numerous behavioral tests assessing acute responses to nicotine or nicotine consumption have been conducted on panels of inbred mouse strains to determine differences in nicotine responses across strains (Marks et al., 1985). These tests revealed that while variations in nAChR expression and sensitivity contribute to nicotine responses, there are clearly additional genetic factors that contribute to the behavioral effects of nicotine (Marks et al., 1983). ...
... In summary, we have provided a thorough evaluation of the pharmacological and behavioral differences to nicotine as measured in several behavioral tests of aspects that contribute to nicotine addiction. While several previous studies have compared nicotine behavioral responses across B6, D2, and other inbred strains (Marks et al., 1983Marks et al., , 1985Marks et al., , 1989Marks et al., , 1991 Collins et al., 1988), the current study, in addition to nicotine's acute and chronic effects, provides characterization of withdrawal and reward, two important aspects of nicotine dependence, in inbred strains; thus, the current study is the most comprehensive simultaneous comparison of acute and chronic responses to nicotine in these two widely studied inbred strains. The results of the current study suggest that the B6 and D2 strains may be useful progenitors for future genetic studies on nicotine behaviors across batteries of mouse lines such as the recombinant inbred BXD panel. ...
Approximately 50-70% of the risk for developing nicotine dependence is attributed to genetics; therefore, it is of great significance to characterize the genetic mechanisms involved in nicotine reinforcement and dependence in hopes of generating better smoking cessation therapies. The overall goal of these studies was to characterize behavioral and pharmacological responses to nicotine in C57Bl/6 (B6) and DBA/2 (D2) mice, two inbred strains commonly used for genetic studies on behavioral traits. B6 and D2 mice where subjected to a battery of behavioral tests to measure nicotine's acute effects, calcium-mediated antinociceptive responses, tolerance to chronic treatment with osmotic mini pumps, and following three days of nicotine withdrawal. In general, D2 mice were less sensitive than B6 mice to the acute effects of nicotine, but were more sensitive to blockade of nicotine-induced antinociceptive responses by a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. B6, but not D2 mice, developed tolerance to nicotine and nicotine conditioned place preference (CPP). While B6 and D2 mice both expressed some physical withdrawal signs, affective withdrawal signs were not evident in D2 mice. These results provide a thorough, simultaneous evaluation of the pharmacological and behavioral differences to experimenter-administered nicotine as measured in several behavioral tests of aspects that contribute to smoking behavior. The B6 and D2 strains show wide phenotypic differences in their responses to acute or chronic nicotine. These results suggest that these strains may be useful progenitors for future genetic studies on nicotine behaviors across batteries of mouse lines such as the BXD recombinant inbred panel.
... Locomotor activity was detected using a photo beam frame (30 Â 24 Â 8 cm) with sensors arranged in an eight-beam array strip around the cage and recorded by an activity monitoring system (MED Associates, St. Albans, VT). The primary dependent variable of interest was locomotor activity (beam breaks) over 15 min because this time frame encompasses the near maximal effects of NIC injection on locomotor activity (Marks, Romm, Bealer, & Collins, 1985). ...
Chronic administration of nicotine or exposure to stress can produce long-lasting behavioral and physiological changes in humans and animals alike. Further, the impact of nicotine and stress exposure can be inherited by offspring to produce persistent changes in physiology and behavior. To determine if nicotine and stress interact across generations to influence offspring behavior we exposed F0 male mice to nicotine and F1 male and female mice to chronic unpredictable stress during adolescence. We then measured locomotor sensitization to repeated nicotine injections in the subsequent F2 and F3 generations. Stress exposure alone (F1) did not influence locomotor sensitization in any lineage. However, in the F1 male lineage, F0 nicotine exposure abrogated locomotor sensitization in F2 male and transiently enhanced locomotor sensitization in F2 female offspring. These effects were not passed down to the F3 generations or observed in the F1 female lineage. F1 stress exposure modulated the effects of prior F0 nicotine exposure in a sex-dependent manner. Specifically, stress blunted the nicotine-induced enhancement in locomotor sensitization observed in F2 female offspring of F1 males. The effect of F0 nicotine and F1 stress exposure in females appears to have skipped a generation and enhanced nicotine sensitization only in the F3 generation, and only in females. This novel multigenerational exposure paradigm examining the inheritance of two different environmental exposures demonstrates that nicotine responses can be modified by nicotine and stress exposure from previous generations, and these effects are strongly influenced by sex.
... Each animal received an i.p. injection of saline on days 1 and 2 and nicotine (0.5 or 1 mg/kg) or saline on day 3 immediately before placement in the Y-maze. Nicotine doses were based on prior literature [18][19][20]. Locomotor activity was monitored for 10 min (in 5 min epochs). The dependent variable was change in locomotor activity (cm; Day 3 nicotine response -Day 2 habituated baseline). ...
... Tolerance testing-Behavioral test measures were selected based on previous studies that have demonstrated dose-dependent responses to nicotine that can be altered by altering nAChR expression (Marks et al., 1985;O'Neill et al., 2013;Tritto et al., 2004). Chronic nicotine treatment had been discontinued for at least two hr before testing to allow clearance of nicotine (Petersen et al., 1984). ...
The role of neuronal nicotinic acetylcholine receptors (nAChR) containing the β4 subunit in tolerance development and nicotinic binding site levels following chronic nicotine treatment was investigated. Mice differing in expression of the β4-nAChR subunit [wild-type (β4++), heterozygote (β4+−) and null mutant (β4−−)] were chronically treated for 10 days with nicotine (0, 0.5, 1.0, 2.0 or 4.0 mg/kg/h) by constant intravenous infusion. Chronic nicotine treatment elicited dose-dependent tolerance development. β4−− mice developed significantly more tolerance than either β4++ or β4+− mice which was most evident following treatment with 4.0 mg/kg/h nicotine. Subsets of [125I]-epibatidine binding were measured in several brain regions. Deletion of the β4 subunit had little effect on initial levels of cytisine-sensitive [125I]-epibatidine binding (primarily α4β2-nAChR sites) or their response (generally increased binding) to chronic nicotine treatment. In contrast, β4 gene-dose-dependent decreases in expression 5IA-85380 resistant [125I]-epibatidine binding sites (primarily β4*-nAChR) were observed. While these β4*-nAChR sites were generally resistant to regulation by chronic nicotine treatment, significant increases in binding were noted for habenula and hindbrain. Comparison of previously published tolerance development in β2−− mice (less tolerance) to that of β4−− mice (more tolerance) supports a differential role for these receptor subtypes in regulating tolerance following chronic nicotine treatment.
... Conditioned tolerance occurs to some behavioral and endocrine effects of nicotine (Marks, et al., 1983;. Nicotine administration induced a dose dependent tolerance to nicotine's effects on heart rate, respiration rate, locomotor activity, acoustic startle responses, body temperature and seizure threshold (Marks, et al., 1983;Marks, et al., 1985;Hakan and Ksir, 1991). Pauly, et al., (1988) ran behavioral tests including the Y-maze and startle response, and monitored heart rate and body temperature of normal and adrenalectomized rats. ...
... When mice sensitized to nicotine were vaccinated with HexonAM1 and subsequently challenged with nicotine, there was a dramatic alleviation of nicotine-induced hypoactivity behavior in the mouse locomotor activity in multiple nicotine challenges. While it has been shown that large doses of nicotine are required for alterations in mouse behavior and physiology, it is not known what the threshold of nicotinic receptor-binding is required to effect these nicotine-induced changes (Marks et al., 1985;Collins et al., 1988;Collins and Marks, 1989). In this study, we compared both acute effects of nicotine binding in vaccinated mouse brains and chronic effects of daily challenges with boluses of nicotine solutions on vaccinated mouse behavior. ...
Despite anti-smoking campaigns, cigarette smoking remains a pervasive addiction with significant societal impact, accounting for 1 of every 5 deaths. Smoking cessation therapies to help smokers quit are ineffective with a high recidivism rate. With the knowledge that nicotine is the principal addictive compound of cigarettes, we have developed an anti-smoking vaccine based on the highly immunogenic properties of the hexon protein purified from the serotype 5 adenovirus (Ad) capsid. We hypothesized that an effective anti-nicotine vaccine could be based on coupling the nicotine hapten AM1 to purified Ad hexon protein. To assess this, AM1 was conjugated to hexon purified from serotype 5 Ad to produce the "HexonAM1" vaccine. C57Bl/6 mice were sensitized by 10 daily nicotine administrations (0.5 mg/kg, subcutaneous) to render the mice addicted to nicotine. Control groups were sensitized to PBS. The mice were then im-munized with HexonAM1 (4 µg, intramuscular) at 0, 3, and 6 wk. By 6 wk, the HexonAM1-vaccinated mice had serum anti-nicotine antibody titers of 1.1 x 106 ± 7.6 x 104. To demonstrate that these high anti-nicotine titers were sufficient to suppress the effects of nicotine, HexonAM1-vaccinated mice were evaluated for nicotine-induced hypoactive behavior with nicotine chal-lenges (0.5 mg/kg wt) over 5 wk. In all challenges, the HexonAM1-vaccinated mice behaved similarly to that of PBS-challenged naive mice. These data demonstrate that a vaccine comprised of a nicotine analog coupled to Ad hexon can evoke high level anti-nicotine antibodies sufficient to inhibit nicotine-induced behavior. The HexonAM1 vaccine represents a platform paradigm for vaccines against small molecules.
... The mouse was then placed in a holding cage for another 7 minutes, followed by a body temperature measurement. These post-injection times have previously shown to correspond with a maximal response to nicotine (Marks et al. 1985). The testing of mice during the dark phase of the light cycle was done under dim red light (< 5 Lux). ...
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.
... Nicotine is a major addictive component of tobacco smoke and a strong psychostimulant. From a behavioral point of view, nicotine has been involved in multiple cognitive, emotional, and psychomotor processes, either after acute or chronic exposure, in human (Heishman et al. 1994; Newhouse et al. 2004; Xu et al. 2007), primate (Valette et al. 2007), or rodent models (e.g., Stolerman et al. 1973; Marks et al. 1985; Gäddnäs et al. 2000; Besson et al. 2007; for review, see Matta et al. 2007). It activates the release of several neurotransmitters (Li and Eisenach 2002; Rossi et al. 2005; Villégier et al. 2006; Fallon et al. 2007), stimulates dopaminergic neuron firing (Mameli-Engvall et al. 2006) and dopamine striatal release (Di Chiara 2000; Pietila and Ahtee 2000), and enhances the transcription of multiple immediate early genes in various brain regions (Hiremagalur and Sabban 1995). ...
The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity beta2-containing nicotinic receptors (beta2*nAChRs) are located.
We intend to see which brain circuits are activated when nicotine is given in animals naïve for nicotine and whether the beta2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas.
We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and beta2 knockout (KO) mice.
Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, beta2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via alpha7 nicotinic receptors.
Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on beta2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice.
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, 1.0 and 4.0 mg/kg/h). Deletion or partial deletion of the α4, β2 or both nAChR subunits reduced the sensitivity of mice to acute nicotine administration. This reduced sensitivity was gene dose-dependent. Modification of α4 subunit expression elicited a greater reduction in sensitivity than the modification of β2 subunit expression. No measurable tolerance was observed for mice of any genotype following chronic treatment with 0.25 mg/kg/h nicotine. Modest tolerance was noted following treatment with 1.0 mg/kg/h. Greater tolerance was observed following treatment with 4.0 mg/kg/h. The extent of tolerance differed among the mice depending on genotype: wild-type (α4 and β2) developed measurable tolerance for all four tests. Heterozygotes (α4, β2 and α4/β2) developed tolerance for only Y-maze crossings and body temperature. Null mutants (α4 and β2) did not become tolerant. However, following chronic treatment with 4.0 mg/kg/h nicotine, wild type, α4 and α4 mice displayed increased Y-maze crossings following acute administration of 0.5 mg/kg nicotine that may reflect the activity of α6β2*-nAChR. These results confirm the importance of the α4 and β2 nAChR subunits in mediating acute and chronic effects of nicotine on locomotion and body temperature in the mouse.
The data suggest genetic factors play an important role in regulating vulnerability to becoming a smoker, but only limited progress has been made in humans in terms of identifying genes critical in regulating nicotine dependence. However, some progress has been made in understanding the regulation of responses to nicotine in the mouse. This chapter will outline strategies that have been, or could be, used to identify genes that regulate behaviors. Specific examples of how these strategies have been used to gain an understanding of genetic influences on nicotine-related behaviors will be provided.
Although psychoactive substances are readily available in today’s society, not everyone uses or abuses these agents. Some individuals may choose not to use a drug because of environmental influences or societal attitudes, but even when drugs are readily available and the societal attitudes permissive or supportive not everyone will use a drug and not everyone who experiments with the drug becomes dependent. Whenever individual differences of this sort are observed, potential genetic influences on the trait in question must be considered. This chapter will briefly review the literature that suggests that genetic factors contribute to the use and abuse of alcohol and will attempt to provide a comprehensive review of the literature that indicates that genetic factors may influence tobacco use. Similarly, a cursory review of the studies done in animals that have attempted to explain and identify the genetic underpinnings of human alcohol use will be presented along with a thorough review of the animal studies that have dealt with the genetics of nicotine-induced behavioral and physiological effects. Some emphasis will be placed on potential genetic regulation of the interactions between alcohol and nicotine.
In recent years molecular cloning studies have resulted in the discovery of multiple genes encoding a variety of structural and functional subunits for nicotinic cholinergic receptors (nAChR) (Galzi and Changeux, 1995), 11 having neuronal localization (α2–α9, β2–β4) and 5 expressed in skeletal muscle (α1, β1,γ, δ, ε). This molecular diversity opens up the possibility of complex receptor subtype expression both in the autonomic (PNS) and central (CNS) nervous systems, possibly explaining the complex pharmacological effects of nicotine and other classic nicotinic cholinergic agonists that do not discriminate among the various receptor subtypes expressed in the CNS and PNS. The importance of understanding this complexity is emphasized by the growing body of evidence suggesting that compromised CNS nicotinic cholinergic neurotransmission may play a key role in a variety of CNS and PNS pathologies. In this regard, there is growing interest in the use of nicotinic agonists for the treatment of Alzheimer’s disease (AD) (Williams et al., 1994). One consistent feature of this disease is a decline in the function of cholinergic systems. The loss of neurons that release acetylcholine, a key neurotransmitter in learning and memory mechanisms, initially motivated an intense but disappointing search for replacement therapies targeting muscarinic receptors. On the other hand, epidemiological studies have reported an inverse correlation between the incidence of AD and smoking (van Duijn and Hofman, 1991), supporting the notion that nicotinic pharmacology underlies an apparent protective effect.
Nicotine administration affects many behavioral and physiological systems, and for many responses both stimulant and depressant effects may be observed. Presumably these actions are initiated by the binding of nicotine to appropriate receptor systems. Results presented in this paper suggest that the responses to nicotine are regulated by two distinct systems that may be related to two different nicotinic receptor systems.
Recent advances in the understanding of human behaviors indicate that genetic factors often play a critical role in regulating variation in behavior. Special attention has been paid to abnormal behaviors ranging from psychopathology to drug, usually alcohol, dependence. This chapter summarizes the data that suggest that genetic factors regulate the nicotinic and muscarinic cholinergic systems. Studies that indicate that the use of tobacco products by humans is genetically regulated are discussed in detail, as are animal studies that indicate that genetically determined differences in several aspects of nicotine pharmacology exist. Special emphasis is placed on those animal studies that have identified biochemical processes that may play critical roles in regulating genetically influenced variation in nicotine’s actions. A more cursory discussion of the genetics of human affective disorders is followed by an equally cursory summation of the limited animal literature, which argues that genetically determined variation in muscarinic cholinergic systems exists.
Nicotinic cholinergic receptors in the central nervous system have been characterized extensively from a biochemical viewpoint and at least two classes of nicotinic receptors exist. The class of nicotinic receptors that binds the snake venom alpha-bungarotoxin (BTX) has been characterized extensively but its function remains largely unknown. Indeed, several electrophysiological investigations have suggested that the BTX binding site is not functional. It has been reported that alpha-BTX fails to inhibit neurotransmission in the autonomic ganglion of the cat (Chou and Lee, 1969), chick (Carbonetto et al, 1978) and rat (Ko et al, 1976) and in the spinal cord of the frog (Miledi and Szczepaniak, 1975) and cat (Duggan et al, 1976). Some of this failure to detect effects of BTX on nicotinic, cholinergic transmission may relate to methodological difficulties. For example, Marshall (1981) blocked nicotinic neurotransmission in frog sympathetic ganglia by treating the tissue with 1–5 uM BTX for 60–90 min. Marshall (1981) indicated that collagenase treatment was necessary to improve the access of the toxin to frog neurons. Since none of the studies that failed to observe an effect of BTX on neurotransmission used this procedure it must be concluded that the role of the BTX binding site in the CNS remains largely unknown.
Nicotine was infused via the jugular vein into DBA mice, and the effects of such treatment on brain nicotinic receptors and various behavioral and physiological responses to nicotine were determined. Nicotinic receptors were measured using [³H]nicotine or [125I]α-bungarotoxin (α-BTX) as the ligands. Infusion with nicotine resulted in tolerance to several of nicotine’s effects. This change in drug sensitivity was accompanied by an increase in Bmax, for both brain nicotinic receptors. A dose-response analysis indicated that [³Hlnicotine binding increased at lower infusion doses than did [125I]α-BTX binding. The maximal increase in nicotine binding was seen at a 4-mg/kg/hr infusion dose, while α-BTX binding continued to increase with nicotine dose up to a dose of 8 mg/kg/hr. Tolerance to nicotine’s effects correlated best with the changes in [³H]nicotine binding. Time course studies for the onset and offset of tolerance and changes in nicotinic receptors were also carried out. Alpha-BTX binding increased rapidly at the nicotine dose used and reached its new equilibrium value within 2 days. Nicotine binding reached its new equilibrium value in 4 days. Approximately 8 days were required, following termination of nicotine treatment, for nicotine binding to return to control levels, while α-BTX binding was at control levels at the earliest post-treatment test time (4 days). The rates of acquisition and loss of tolerance to nicotine paralleled the changes in brain nicotine binding. These results suggest that chronic tobacco use may lead to changes in brain nicotinic receptors. Such effects could explain not only tolerance to nicotine, but also effects of tobacco on such processes as learning and memory.
A growing body of evidence suggests that disruption of nicotinic cholinergic systems may be an important factor in the etiology of a number of different diseases, ranging from neurodegenerative diseases, such as Alzheimer's and Parkinson's, to ulcerative colitis. The mechanistic basis for such diverse nicotinic effects is likely to lie in the ever growing number of potential receptor subtypes. Therefore, the development of receptor subtype-selective probes is essential to understand the emerging complexity of nicotinic cholinergic systems and the mechanisms underlying diseases that may involve these systems. Toward this end, we have evaluated the nicotinic agonist metanicotine, (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine, using the following in vitro and in vivo methods: 1) receptor binding and up-regulation, 2) neurotransmitter release and ion flux in synaptosomes/cells, 3) in vivo microdialysis in rats, 4) reversal of scopolamine-induced amnesia in a step-through passive-avoidance paradigm, 5) water maze performance in mice, 6) radial-arm maze performance in brain-lesioned rats, 7) changes in heart rate and blood pressure, and 8) physiological depression of body temperature, locomotor activity, acoustic startle, and respiration rate. Our in vitro results indicate that metanicotine binds with high affinity to the major receptor subtype in brain (α4β2), evokes dopamine release from striatal synaptosomes and Rb+ efflux from thalamic synaptosomes, but does not activate ganglionic, muscle, or other peripheral type nicotinic receptors. These results suggest that metanicotine is selective for α4-containing central nervous system (CNS) nicotinic receptors and has reduced selectivity for peripheral nervous system (PNS) receptor subtypes. These conclusions are further supported by in vivo studies with metanicotine showing enhanced cognitive effects and significantly lower peripheral effects. Our in vivo results indicate that metanicotine increases the release of acetylcholine, norepinephrine, dopamine, and serotonin in cortex and is equal to or better than nicotine on measures of cognitive enhancement. By comparison, metanicotine is significantly less potent than nicotine in increasing heart rate and blood pressure and in causing physiological depression. These results are consistent with in vitro data indicating metanicotine's CNS receptor selectivity, and they suggest that this ligand may be a suitable tool for probing the relationships that underlie the complex central and peripheral pharmacology of nicotinic cholinergic systems. Furthermore, metanicotine may be a good lead candidate for developing nicotinic agonists as CNS therapeutics with reduced peripheral side effects. Drug Dev. Res. 38:169–176 © 1996 Wiley-Liss, Inc.
Several studies have suggested that ethanol interacts with muscarinic cholinergic systems in the brain. In order to assess whether muscarinic systems regulate sensitivity to ethanol, the effects of oxotremorine pretreatment on sensitivity to ethanol were determined in the long-sleep (LS) and short-sleep (SS) mice, which were selectively bred for differential sensitivity to ethanol. In addition, the relative sensitivity of these two lines to intraperitoneally (ip) injected oxotremorine and total muscarinic receptors, as measured by quinuclidinyl benzilate (QNB) binding, M1 receptor subtypes, as measured by pizenzepine (PZ) binding, and ratios of high and low agonist affinity were measured in seven brain regions. SS mice were more sensitive to oxotremorine-induced increases in sensitivity to ethanol but the LS mice were more sensitive to the effects elicited by ip oxotremorine injection. Because the effects of oxotremorine were blocked by scopolamine but not by methylscopolamine, it is likely that the effects of oxotremorine that were measured are centrally mediated. QNB binding did not differ between the LS and SS mice except for cortex where the SS mice exhibited slightly larger numbers. The mouse lines did not differ in the number of M1 receptors or in ratio of high to low affinity agonist sites. Therefore, it does not seem likely that differences in receptor numbers are important in regulating the differential sensitivities of the LS and SS mice to oxotremorine or ethanol. Differences in receptor coupling processes may be critically involved.
It has been shown before that unconditioned footshocks can augment the acoustic startle response in rats. In the present study, male mice of two strains, C57Bl/6N and BALB/c, were compared with regard to footshock-induced sensitization of the acoustic startle response. Presentation of footshocks did not affect the acoustic startle response in C57Bl/6N mice, while in contrast, footshock-induced sensitization was apparent in the BALB/c strain. Shocked C57Bl/6N mice, but not BALB/c mice, displayed robust conditioning to the startle context when re-tested the next day. These findings indicate that mice may exhibit footshock-induced sensitization of the acoustic startle response, but that the effects of footshocks on the acoustic startle are strain- and time-dependent.
The smoking cessation aid, varenicline, has higher affinity for the alpha4beta2-subtype of the nicotinic acetylcholine receptor (α4β2*-nAChR) than for other subtypes of nAChRs by in vitro assays. The mechanism of action of acute varenicline was studied in vivo to determine (a) subtype activation associated with physiological effects and (b) dose relationship as an antagonist of nicotine.
Acute doses of saline, nicotine, and varenicline were given to mice, and locomotor depression and hypothermia were measured. Subunit null mutant mice as well as selective antagonists were used to study mode of action of varenicline as an agonist. Varenicline as an antagonist of nicotine was also investigated.
Varenicline evokes locomotor depression and hypothermia at higher doses than necessary for nicotine. Null mutation of the α7- or β2-nAChR subunit did not decrease the effectiveness of varenicline; however, null mutation of the β4 subunit significantly decreased the magnitude of the varenicline effect. Effects of the highest dose studied were blocked by mecamylamine (general nAChR antagonist) and partially antagonized by hexamethonium (largely peripheral nAChR antagonist). No significant block was seen with ondansetron antagonist of 5-hydroxytryptamine 3 receptor. Using a dose of nicotine selective for β2*-nAChR subtype effects with these tests, dose-dependent antagonism by varenicline was seen. Effective inhibitory doses were determined and appear to be in a range consistent with binding affinity or desensitization of β2*-nAChRs.
Varenicline acts as a functional antagonist of β2*-nAChRs, blocking certain effects of nicotine. At higher doses, varenicline is an agonist of β4*-nAChRs producing physiological changes in mice.
The performance of three widely used rat lines (Sprague-Dawley, Wistar, and Long Evans hooded) were evaluated in behavioral test systems that are sensitive to benzodiazepines. The in vivo effects of flunitrazepam and the brain [3H]Ro 15-1788 binding were determined and compared in these rat lines. The behavioral end points evaluated in this study were anxiolysis, measured using the automated elevated plus-maze; sedation by modification of locomotor activity; hyperphagia following food deprivation; protection for pentylenetetrazol-induced convulsions; and hypothermia. There were comparable results in the hypnotic, hypothermic, anticonvulsant, and feeding tests in these lines following flunitrazepam administration. However, the behavior of the Long Evans hooded rat was most amenable to the detection of drug-induced changes in the anxiety test. There was no difference in the maximum number of binding sites (Bmax) or the affinity (Ki) of the Ro 15-1788 or flunitrazepam binding in either the cerebellum or whole brain (minus cerebellum) in the three rat lines as determined by the competitive binding against [3H]Ro 15-1788. Thus, while these rat lines exhibited similar behavioral profiles in most tests the modest differences in the baseline responses and the ability to detect anxiolysis at lower doses of flunitrazepam observed with Long Evans hooded rats makes them particularly suited for these types of studies.
A recent study from our laboratory has demonstrated that C57BL/6 male mice that are chronically injected with nicotine develop a profound tolerance to nicotine that is not associated with changes in brain nicotinic receptors. We have proposed that alterations in the secretion of corticosterone (CCS) may regulate tolerance development in chronically injected animals. In the present study we have directly tested this hypothesis. Female DBA/2 mice were injected three times each day for 12 days with saline or 2 mg/kg nicotine. Blood samples were collected at various time points during the course of treatment and plasma CCS levels were determined. The animals were divided into two groups following the last injection on day 12. The first group of animals was tested for nicotine-induced release of corticosterone on day 13 of the experiment and then sacrificed. The brains of these animals were subsequently used to measure nicotinic receptor binding. The second group of animals was adrenalectomized (ADX) or sham-operated on day 13 of the experiment and tested for nicotine sensitivity on day 14 of the experiment. Plasma CCS levels were significantly elevated in animals that were chronically injected with nicotine (versus saline controls) by the fourth day of the experiment. Chronic nicotine-injected animals were tolerant to nicotine-induced CCS release. Animals that were chronically injected with nicotine and sham-operated were tolerant to acute nicotine challenge; however, tolerance to nicotine was not detected in ADX animals. These data support the hypothesis that the capacity to release CCS may underscore the expression of tolerance to nicotine in chronically injected animals.
DBA and C3H mice were injected chronically with 2.0 mg/kg diisopropylfluorophosphate (DFP) every other day for 2 or 4 weeks. Although acetylcholinesterase (AChE) activity and muscarinic receptor numbers ([3H] quinuclidinyl benzilate (QNB) binding) were decreased in DFP-treated DBA and C3H mice, the number of nicotinic receptors (L-[3H]nicotine and alpha-[125I]bungarotoxin (BTX) binding) was unchanged by chronic DFP treatment. Sprague-Dawley rats injected chronically with lower doses of DFP than were used in mice exhibited a greater reduction in AChE activity, as well as accompanying decreases in [3H]QNB and [3H]nicotine binding. Neither species exhibited changes in alpha-[125I]BTX following chronic DFP injection. The effects of chronic DFP treatment on sensitivity to DFP and to nicotine were also assessed in the two mouse strains using a battery of behavioral and physiological tests that included rotarod performance, Y-maze crossing and rearing activity, heart rate, and body temperature. No tolerance to DFP was observed in either mouse strain after 2 weeks of treatment. Following 4 weeks of treatment, DFP-treated DBA mice exhibited modest tolerance to the effect of DFP on body temperature. C3H mice did not survive the 4-week treatment. Some evidence for reduced sensitivity to nicotine's effects was detected in the DFP-treated DBA mice, but cross-tolerance to nicotine was not observed in the DFP-injected C3H mice. Because chronic DFP treatment did not evoke a change in the number of brain nicotinic receptors, the reduced sensitivity to some of nicotine's effects seen in DBA mice must be due to some factor other than receptor downregulation.
Pharmacogenetics, the study of genetic factors underlying individual differences in response to drugs, has proven useful for demonstrating that there are large genetic differences in response to a number of abused drugs. Pharmacogenetics also provides a number of useful tools for studying mechanisms underlying the effects of drugs. This review discusses pharmacogenetic techniques with potential utility for drug abuse research and provides examples of their use in studies of the effects of acute and chronic nicotine, cocaine and opiate administration. The importance of using genetically standardized animal models in behavioral and pharmacological research is also discussed.
The possibility that common genetic factors regulate initial sensitivities to ethanol and nicotine as well as the development of cross-tolerance between these agents was explored using the long-sleep (LS) and short-sleep (SS) mice. The LS mice proved to be more sensitive to an acute challenge with nicotine than were the SS mice. Segregation analysis (F1, F2, backcross) indicated that ethanol sensitivity and nicotine sensitivity segregate together. Acute pretreatment with nicotine did not significantly affect sensitivity to ethanol, but ethanol pretreatment altered nicotine responsiveness. The LS mice develop more tolerance to nicotine and ethanol than do the SS and they also develop more cross-tolerance. These genetically determined differences in initial sensitivities, and tolerance and cross-tolerance development are not readily explained by differences in brain nicotinic receptor numbers.
Nicotine is the most potent psychoactive agent in tobacco, and many studies indicate that people adjust their tobacco use in an attempt to carefully titrate their plasma nicotine levels. Furthermore, pretreatment with the nicotinic receptor antagonist, mecamylamine, results in an increase in tobacco use. In addition, several studies have demonstrated that people differ in sensitivity to nicotine. The chapter discusses the role of genetic factors in regulating first dose sensitivity to nicotine and the development of tolerance to nicotine. The young males from families with a positive history for alcoholism are less affected by alcohol, both behaviorally and physiologically (prolactin and cortisol release), than are young males from families that do not have a history of alcoholism. Alternatively, it may be that those individuals who develop tolerance to the actions of nicotine are more likely to persist in using tobacco; i.e. smokers are those individuals who develop tolerance to the noxious actions of nicotine thereby uncovering the reinforcing actions. Receptor changes are more related to the development of dependence on nicotine. Analysis of this potential role will require the development of adequate methods of quantifying a nicotine withdrawal syndrome, if it exists, in the mouse.
Male mice from 19 inbred strains were tested for the effects of nicotine on six responses: respiratory rate, acoustic startle response, Y-maze crosses, Y-maze rears, heart rate and body temperature. Dose-response curves were constructed for each strain on each test in a multitest battery. Results indicated that the responses were strongly influenced by the genotype of the animal. Comparison of the results from the six tests measured in this study and the results previously reported for nicotine-induced seizures in these same strains indicated that the responses could be grouped into two major classes: a set characterized by Y-maze crosses, Y-maze rears and body temperature and a set characterized by seizure sensitivity and seizure latency. Responses observed for respiratory rate and startle response shared characteristics with both of these sets, while nicotine effect on heart rate was fairly unique. The results have identified strains of mice which are differentially sensitive to the effects of nicotine.
The cocaine sensitivity of male and female long-sleep (LS) and short-sleep (SS) mice, which have been selectively bred for differential ethanol-induced "sleep-time," was examined in a battery of behavioral and physiological tests. Differences between these two mouse lines were subtle and were seen primarily at high doses. At high doses, SS mice were more sensitive than LS mice, particularly to cocaine-induced hypothermia; however, significant hypothermia was not seen except at doses which were very near to the seizure threshold. During a 60-min test of locomotor activity, LS mice showed greater stimulation of Y-maze activity by 20 mg/kg cocaine than SS mice. Consistent with the finding of subtle differences in sensitivity to low doses of cocaine. LS and SS mice did not differ in sensitivity to cocaine inhibition of synaptosomal uptake of [3H]-dopamine, [3H]-norepinephrine or [3H]-5-hydroxytryptamine. However, consistent with the finding of differential sensitivity to high doses of cocaine, SS mice were more sensitive to the seizure-producing effects of the cocaine and lidocaine, a local anesthetic. It is hypothesized that the differential sensitivity of these mouse lines to high doses of cocaine is due to differential sensitivity to cocaine's actions on systems that regulate local anesthetic effects. Selective breeding for differential duration of alcohol-induced "sleep-time" may have resulted in differential ion channel structure or function in these mice.
In order to assess the anticonvulsant potency of ethanol, male and female long-sleep (LS) and short-sleep (SS) mice were pretreated with ethanol 7.5 min prior to challenge with an ED80 dose of nicotine (LS: 4.25 mg/kg; SS: 6.25 mg/kg). LS mice were more sensitive to the anticonvulsant effects of ethanol than were SS mice. In order to assess the effect of ethanol on the nicotine-induced behavioral desensitization to nicotine observed previously in these mice, animals were pretreated with saline, nonanticonvulsant doses of ethanol (0.25 g/kg, 0.75 g/kg or 1.5 g/kg), a subseizure-producing dose of nicotine (2.0 mg/kg) or a combination of these two drugs 15 or 30 min prior to nicotine challenge. Ethanol enhanced the nicotine-induced behavioral desensitization in both mouse lines; however, this effect was seen at lower ethanol doses and was more pronounced in LS mice. Ethanol pretreatment did not affect brain nicotine concentrations; therefore, the ethanol effect probably involves changes in brain sensitivity to nicotine.
Nicotine was administered intravenously to DBA mice through cannulae implanted in the jugular veins. Five groups of animals were treated: a control group which received saline and four nicotine treatment groups. All of the nicotine treatment groups received a dose of 4.0 mg/kg/hr. The first group received continuous infusion, the second group received 1 mg/kg pulses four times an hour, the third group received 2 mg/kg pulses twice an hour, and the fourth group received 4 mg/kg pulses once an hour. After a 10-day treatment period, the animals were tested for tolerance to an acute intraperitoneal administration of nicotine. Tolerance was measured using a test battery composed of the following tests: respiratory rate, acoustic startle response, Y-maze crosses and rears, heart rate, and body temperature. Mice from each of the four nicotine treatment groups were tolerant to the acute effect of nicotine, but the extent of tolerance varied among the groups as follows: continuous infusion less than 1 mg/kg pulses four times/hr less than 2 mg/kg pulses twice/hr less than 4 mg/kg pulse once/hr. Chronic nicotine infusion resulted in significant increases in the binding of L-[3H]nicotine in all six brain regions assayed and in significant increases in the binding of alpha-[125I]bungarotoxin binding in cerebral cortex and hippocampus. All increases in binding resulted from increases in Bmax for these ligands. In contrast to the effects observed for tolerance development, the increases in [3H]nicotine binding were not significantly affected by the kinetics of nicotine infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Female DBA/2Ibg mice were treated chronically (21 days) with ethanol- or dextrin-containing liquid diets or infused chronically with nicotine (8 mg/kg/h) or saline for 10 days. The responses of these animals to challenge doses of ethanol (2.5 g/kg) or nicotine (1 or 2 mg/kg) were measured using a test battery consisting of respiration rate, acoustic startle response, Y-maze crosses and rears, heart rate and body temperature. Chronic ethanol-treated animals were tolerant to the effects elicited by a challenge dose of ethanol on four of the six measures and were cross-tolerant to nicotine's effects on the acoustic startle test. Chronic nicotine-treated animals were tolerant to nicotine's effects on five of the six measures and cross-tolerant to ethanol's effects on heart rate and body temperature. Thus, partial cross-tolerance between ethanol and nicotine exists. Chronic nicotine treatment resulted in significant increases in L-[3H]-nicotine binding in six of seven brain regions and in alpha-[125I]-bungarotoxin binding in three of seven brain regions. Chronic ethanol treatment failed to alter the binding of either ligand. Therefore, the cross-tolerance that develops between ethanol and nicotine is not totally dependent on alterations in the number of brain nicotinic receptors.
Nineteen inbred mouse strains were tested for their relative sensitivity to nicotine's effects on respiratory rate, acoustic startle response, heart rate, Y-maze activity (crosses and rears) and body temperature. Separate animals were tested for their sensitivity to nicotine-induced seizures following IP injection or IV infusion. Dose-response curves were constructed for each measure. Large strain differences were obtained for all of these measures. Nicotine's effects on heart rate, Y-maze activity and body temperature segregated together into the various mouse strains whereas seizure sensitivity segregated independently which suggests that these responses are under separate genetic control. Evidence was obtained which suggests that nicotine-induced seizures are regulated, in part, by the number of hippocampal nicotinic receptors measured with alpha-bungarotoxin (BTX). Strain differences in the development of tolerance to nicotine were also observed. Four mouse strains were tested and one of these strains (C3H) did not exhibit tolerance to nicotine. The binding of (3H)nicotine and (125I)BTX increased in the brains of all four mouse strains. These changes may relate to tolerance in some mouse strains, but since C3H mice did not exhibit tolerance even though brain nicotinic receptors changed following chronic treatment, other explanations for the role of receptor changes in tolerance to nicotine must be sought.
Changes in plasma corticosterone (CCS) levels following intraperitoneal injections of nicotine were measured in four inbred mouse strains: DBA/2Ibg, C57BL/6Ibg, C3H/2Ibg, and A/J. In all four strains, nicotine produced a dose-dependent (0.5-2.0 mg/kg nicotine) increase in plasma CCS levels which peaked 10-30 min after injection. Saline increased plasma CCS levels in C57BL, A, and C3H, but not in DBA mice. After correcting for plasma CCS levels produced by saline injection, the nicotine-induced rise in plasma CCS was significantly lower for the C57BL strain than for the other three strains tested. These mouse strains also varied in their responses to saline injection with the rank order: C57BL greater than A = C3H greater than DBA. However, the two most divergent strains (C57BL and DBA) did not differ in the effects of a cold water stress. The response to nicotine was completely inhibited by mecamylamine in two strains tested (C3H and C57BL) whereas the response to saline injection was unaffected, suggesting that only the response to nicotine was mediated by nicotinic receptors. It is clear that elevations in plasma CCS induced either by saline injection or by nicotine are influenced by genetic factors.
The effects of cocaine on Y-maze activity and heart rate have been examined in four inbred strains of mouse (BALB, C57BL, C3H and DBA). In addition, brain [3H]-cocaine concentrations were measured at the time of maximal response to cocaine. Cocaine produced a dose-related increase in Y-maze cross activity in C3H, DBA and C57BL, with C3H mice being considerably more sensitive than DBA or C57BL. Cocaine was without effect on Y-maze cross activity in BALB mice. Cocaine produced a biphasic effect on rearing activity in C3H mice, a dose related depression in BALB mice, and was without effect on C57BL and DBA mice. At the highest dose studied (15 mg/kg), cocaine produced a small decrease in heart rate in C3H mice. Strain differences in behavior were maximal 15 minutes after a dose of 5 mg/kg, IP. At this dose and time interval, brain [3H]-cocaine concentrations were not significantly different among the four strains of mice. The results suggest a genetically-determined difference in CNS sensitivity to cocaine.
Male and female long-sleep (LS) and short-sleep (SS) mice were pretreated with a subseizure-producing dose of nicotine (2.0 mg/kg) 7.5, 15 and 30 minutes prior to challenge with seizure-producing doses of this drug. Nicotine pretreated animals were less susceptible to nicotine-induced seizures than were saline pretreated animals. The latency to seizure following nicotine challenge was greater in nicotine pretreated animals than in saline controls. Nicotine pretreated LS mice show a greater decrease in nicotine-induced seizure susceptibility than do nicotine pretreated SS mice. This decrease in seizure susceptibility is consistent with induction of nicotinic receptor desensitization via nicotine pretreatment. It is hypothesized that LS and SS mice might differ in sensitivity to nicotine in part because they differ in baseline levels of desensitized versus functional nicotinic receptors.
Nicotine response and nicotinic receptor binding were characterized in long-sleep (LS) and short-sleep (SS) mice which have been selectively bred for differential "sleep-time" following ethanol administration. LS mice are more sensitive than SS mice to nicotine as measured by a battery of behavioral and physiological tests and as measured by sensitivity to nicotine-induced seizures. The greater sensitivity of the LS mice is not due to differences in binding of [3H]nicotine. Unlike inbred mouse strains which differ in sensitivity to nicotine-induced seizures, these selected mouse lines do not differ in levels of binding of [125I]alpha-bungarotoxin (BTX) in the hippocampus. Significant differences in BTX binding were found in the cerebellum and striatum. Although these two mouse lines do not differ in blood levels of nicotine following nicotine administration, they differ slightly in brain levels of nicotine indicating differential distribution of the drug. Since this distribution difference is much smaller than the observed behavioral differences, these mice probably differ in CNS sensitivity to nicotine; however, follow-up studies are necessary to test whether the differential response of these mice is due to subtle differences in distribution of nicotine to the brain.
In order to explore the relationship between response to muscarinic agonists and brain muscarinic receptors, two mouse strains that differ in acute sensitivity (DBA and C3H) were injected chronically with DFP or infused with oxotremorine. Chronic DFP-treated DBA mice were not tolerant to DFP's effects on any measure, but they were cross-tolerant to the effects of oxotremorine on heart rate and body temperature. DFP-treated C3H mice were not tolerant to DFP or cross-tolerant to oxotremorine on any measure. Oxotremorine infusion resulted in tolerance to oxotremorine in both mouse strains, and chronically infused DBA mice were cross-tolerant to DFP on five of the six measures. Oxotremorine-infused C3H mice were cross-tolerant to DFP on two of the measures. These results suggest that genetic factors influence the development of tolerance or cross-tolerance. These genetic factors do not seem to be related to changes in brain QNB binding. Both mouse strains showed comparable changes in QNB binding following chronic DFP and oxotremorine with DFP eliciting reductions in QNB binding in striatum and hippocampus and oxotremorine eliciting reductions in nearly every brain region. However, tolerance and cross-tolerance did not seem to correlate with changes in binding which suggests that the relationship between receptor changes and responses to muscarinic agonists must be examined further.
Mice of four inbred strains (BALB, C57BL, DBA and C3H) were administered either saline or oxotremorine, a muscarinic agonist, at a dose of 0.5 mg/kg/hr by constant infusion through cannulas implanted in the right jugular veins. Chronic treatment resulted in the development of tolerance to the effects of oxotremorine both on rotarod performance and on body temperature. For drug-treated BALB mice, the dose-response curves for both measures were parallel to those for saline-treated mice, while for DBA and C3H mice the slopes of the dose-response curves were significantly less for treated mice than they were for controls. The equi-effective doses for the drug-treated animals were at least 8-fold greater than those for saline-treated mice. Drug treatment resulted in a significant decrease in the total number of muscarinic receptors in cortex as measured by the binding of [3H]quinuclidinyl benzilate (QNB) without effect on the KD for this ligand. Similarly, drug treatment did not affect the affinity of carbamylcholine as an inhibitor of QNB binding, but did significantly decrease the levels of both the high- and low-affinity agonist binding sites in cortex. The number of M1 muscarinic receptors measured by high affinity [3H]pirenzepine (PZ) binding was also significantly decreased in cortex without effect on the KD. The experiments were extended to five other brain regions. Full saturation curves were not constructed, however. Oxotremorine treatment significantly reduced QNB binding in every brain region. While the binding to agonist affinity states measured by carbamylcholine inhibition of QNB binding and M1 receptor levels measured by high affinity PZ binding tended to decrease with oxotremorine treatment not all changes were statistically significant. The changes in muscarinic receptor subtype levels induced by oxotremorine infusion did not differ among the strains. The results demonstrate that chronic treatment with a muscarinic agonist results in substantial tolerance to the effects of the drug in all four mouse strains. Although some differences in tolerance development exist, these differences are not readily explained by differences in the number or affinity states of brain muscarinic receptors.
Several studies have demonstrated that chronic treatment with organophosphates, such as DFP, elicits a decreased number of brain muscarinic receptors (measured by the binding of QNB) which has been presented as an explanation for tolerance to the organophosphates. The purpose of the studies presented here was to assess whether graded changes in QNB binding could be attained following different methods of chronic DFP treatment, and whether tolerance to DFP paralleled these changes. Male DBA mice were injected with DFP every 4 days or 2 days for 30 days or daily for 14 days. The animals were subsequently challenged with DFP or the muscarinic agonist, oxotremorine, and respiratory rate, heart rate, body temperature, Y-maze activity and rearing were recorded. Chronic DFP-treated animals were supersensitive to the effects of DFP on respiratory rate, heart rate, and body temperature whereas a modest tolerance to the effects of oxotremorine on respiratory rate, heart rate, and body temperature was seen. Neither tolerance nor supersensitivity were observed for the effects of DFP and oxotremorine on the Y-maze measures. Chronic DFP treatment elicited reduced binding of QNB in striatum, cortex, and hippocampus with the group that had been treated every other day exhibiting the greatest changes. The changes in drug response did not parallel changes in QNB binding which raises questions as to the cause of the reduction in binding.
Four hundred seventeen heterogeneous stock mice were tested for their relative sensitivity to a low dose of nicotine (0.75 mg/kg) using activity in an automated Y-maze and body temperature as response measures. A wide spectrum of individual responsiveness to nicotine, ranging from complete suppression of activity to stimulation above baseline activity, was found. Replicate measures taken 1 week later on the same animals showed the responses to nicotine to be reliable and reproducible. Activity levels and body temperatures following nicotine administration were highly correlated (r = 0.60, df = 415). From analysis of between-litter proportions of variance, the heritability of nicotine-influenced activity was estimated to be 0.12, indicating that selective breeding for differential responsiveness to nicotine would be possible. The 10 most activated and 10 most depressed male and female mice were chosen as breeders for replicate nicotine activated (NA) and nicotine depressed (ND) lines, respectively. The selection criterion was nicotine-induced activity corrected for baseline activity using regression residuals. After six generations of selective breeding a good response to selection was obtained, although the response was better for the ND than for the NA lines. Realized heritability for responsiveness to nicotine calculated from the six selected generations was found to be 0.20, or slightly greater than that estimated from the foundation population. There were no significant differences in response to selection between the replicate NA or ND lines. Nicotine-induced body temperature was measured as a correlated response to selection, and was found to remain highly correlated with nicotine-induced locomotor activity. The response was more robust for the ND lines than it was for the NA lines. In contrast to the large differences between the ND and NA lines in locomotor activity and body temperatures following nicotine administration, mean baseline activities and body temperatures remained nearly identical throughout. This indicates that selection acted specifically on nicotine-induced responses, and not on baseline measurements, as predicted for response to a selection criterion based on regression residuals.
Previous studies have shown that chronic corticosterone (CCS) treatment via subcutaneous pellets elicits reduced sensitivity to many actions of nicotine in mice as well as decreased brain alpha-bungarotoxin (alpha-BTX) binding. We report here the time courses of altered sensitivity to nicotine, as measured by acoustic startle, Y-maze crossing and rearing activities, heart rate, and body temperature, and alpha-BTX binding during and after CCS treatment. CCS treatment resulted in rapid decreases in sensitivity to nicotine for four of the five responses that were measured, as well as rapid changes in alpha-BTX binding. Sensitivity to nicotine returned to control levels within 3 days following pellet removal, but alpha-BTX binding returned to control levels in most brain regions 9-11 days after pellet removal. Because the restoration of control sensitivity to nicotine occurred long before alpha-BTX binding returned to control levels, it seems likely that factors other than changes in alpha-BTX binding cause chronic CCS-induced changes in sensitivity to nicotine.
Inbred mouse strains differ in sensitivity to a first dose of nicotine and in the development of tolerance to nicotine. The experiments reported here used six inbred mouse strains (A, BUB, C3H, C57BL/6, DBA/2, ST/b) that differ in sensitivity to an acute challenge dose of nicotine to determine whether differences in oral self-selection of nicotine exist. Animals were presented with solutions containing nicotine or vehicle (water or 0.2% saccharin) and their daily intake of the two fluids was measured for 4 days starting with a 10 micrograms/ml nicotine solution. This was followed by sequential 4-day testing with 20, 35, 50, 65, 80, 100, 125, 160 and 200 micrograms/ml nicotine solutions. The strains differed dramatically in their self-selection of nicotine and in maximal daily dose (mg/kg); the rank order of the strains was C57BL/6 > DBA > BUB > A > or = C3H > or = ST/b for both the tap water and 0.2% saccharin choice experiments. Correlations between nicotine consumption and sensitivity to nicotine, as measured by a battery of behavioral and physiological responses, were also calculated. Strain differences in nicotine intake were highly correlated with sensitivity to nicotine-induced seizures. As sensitivity to nicotine-induced seizures increases, oral self-selection of nicotine decreases. This finding may suggest that this toxic action of nicotine serves to limit intake.
Genetic differences in nicotine-induced conditioned taste aversion were examined using inbred mice. Adult male C57BL/6J, DBA/2J, BALB/cJ and C3H/heJ mice were adapted to a 2-h per day water access regimen. Subsequently, mice received nicotine injections (0.5, 1.0 or 2.0 mg/kg) immediately after 1-h access to a NaCl flavored solution. DBA and C3H mice developed dose-dependent aversions to the nicotine-paired flavor. BALB mice showed only minor reductions in intake with no difference between the nicotine dose groups. C57BL mice did not show development of nicotine-induced conditioned taste aversion. These results demonstrate that nicotine's aversion motivational effect is strongly influenced by genotype. Further, genetic sensitivity (DBA mice) or insensitivity (C57BL mice) to nicotine-induced conditioned taste aversion was similar to reports of genetic sensitivity to ethanol's aversive effect measured in this design.
Hirschmann has shown that after a moderate dose of nicotin stimulation of the sympathetic nerve in the neck causes no dilation of the pupil. He concludes that nicotin paralyses the endings of the dilator fibres in the pupil. In the course of some observations on the physiological action of nicotin, we had occasion to repeat Hirschmann’s experiment; we found in the rabbit that 30 to 40 mgrms. of nicotin injected into a vein stopped the effect of stimulating the sym pathetic in the neck, not only on the pupil, but also on the vessels of the ear.
Separate groups of two different rat breeding lines, Roman High Avoidance (RHA/Verh.) and Roman Low Avoidance (RLA/Verh.), treated with either saline, nicotine (0.2 mg/kg), or amphetamine (0.4 mg/kg) were compared for exploratory efficiency and for exploratory locomotion by using two different mazes on alternate testing days. The RHA/Verh. rats generally showed more locomotion but less intermaze transfer of exploratory efficency than the RLA/Verh. rats. Nicotine did not alter exploratory efficiency but stimulated locomotor activity in the RHA/Verh. rats, while it did not significantly alter either category of behavior in the RLA/Verh. rats. Amphetamine stimulated locomotor activity in both rat lines but this stimulation was weaker in comparison with that of nicotine. In contrast to nicotine, amphetamine impaired exploratory efficiency in the RHA/Verh. rats. Like nicotine, amphetamine did not significantly affect exploratory efficiency in the RLA/Verh. rats. The results demonstrate that, when the two lines of rats are compared, there is a dissociation of the two categories of behavior and a further differentiation between nicotine and amphetamine effects.
Spontaneous motor activity in a Y-maze was measured in DBA/2Ibg and C57BL/6Ibg mice which had received nicotine or saline injections three times a day for two, four or seven days. Both genotype and sex influenced the development of tolerance to nocotine's effects on spontaneous motor activity, with DBA males requiring the longest exposure to nicotine and C57 males requiring the shortest drug exposure for tolerance development. DBA and C57 females developed behavioral tolerance equally after two days of pretreatment, but the C57 females showed a greater degree of tolerance after seven days of injections than did the DBA females. The development of behavioral tolerance in DBA males after four days of nicotine pretreatment was associated with the development of behavioral tolerance in DBA males after four days of nicotine pretreatment was associated with the development of drug dispositional tolerance, with minimal evidence for a change in nervous system sensitivity. Drug dispositional tolerance in DBA females, C57 males and C57 females, however, did not seem to affect spontaneous motor activity.
Utilizing an automated, Dashiell-type hexagonal maze, it was demonstrated that RHA rats: 1) were more active, 2) reversed direction more often, 3) entered radial (blind) alleys less often, and 4) displayed shorter latencies than did RLA rats. Direction reversals (U-turns) tended to increase from day to day with the RHA rats, whereas the opposite was true for the RLA rats. Nicotine injections (0.2 mg/kg) increased activity and the number of U-turns, shortened the latencies and lessened the likelihood of entering radial alleys for both strains. The RHA rats were more sensitive to nicotine than were the RLA rats in all of these measurements, which varied, depending upon alley length and structural complexity, among the maze configurations.
Spontaneous locomotor activity has been used to assess the development and the dissipation of tolerance to nicotine in rats. Nicotine administered i.p. to experimentally naive rats depressed activity in a Y-shaped runway in a dose-related manner. After a single i.p. dose of nicotine, acute tolerance to the depressant action of a second dose developed with a definite time-course, becoming maximal after 2 h and wearing off after about 8 h. Repeated i.p. doses of nicotine (3 times daily for 8 days) elicited chronic tolerance, which was found to persist for at least 90 days after the end of regular treatment with the drug. Tolerance was also produced when nicotine was administered in rats' drinking water and through reservoirs implanted subcutaneously. We conclude that tolerance to nicotine in rats can develop quickly, may be measured easily, and persists for prolonged periods after withdrawal. A nicotine abstinence syndrome was not detected. The doses (mg/kg i.p.) of nicotine necessary to induce chronic tolerance in rats were similar to those probably obtained by cigarette smokers, but the different routes and rates of administration make precise comparisons difficult. However, it is suggested that relapse to tobacco use in man may be associated with the persistence of tolerance.
In experimentally naive rats, nicotine reduced spontaneous locomotor activity in a dose-related manner. After a single pretreatment with nicotine, acute tolerance developed; this was shown by a shift of the dose-response curve, such that the dose of nicotine required to produce a given decrement in activity was multiplied by a factor of about 2.4. In a second experiment, a range of doses of nicotine was found to induce tolerance, but the dose inducing the maximum degree of tolerance was rather critical. The results demonstrated the importance of using a range of pretreatment and challenge doses when assessing tolerance to nicotine. However, frequently repeated doses were not necessary, since tolerance developed when nicotine was administered to rats only once in every 3 days. In terms of tolerance liability in rats, it seems that nicotine is not discriminable from other drugs upon which dependence can be established.
Male and female rats of Reactive and Non-Reactive strains were injected with either 0.8 mg/kg dose of nicotine bitartrate, 1.0 mg/kg dose of picrotoxin, 10.00 mg/kg dose of penterbarbital sodium or distilled water before each daily trial. Nicotine and picrotoxin facilitated rearing activity whereas pentobarbital sodium inhibited it. The Non-Reactive rats feared more than the Reactive animal. Male rats were found to have higher rearing scores than the females.
Male and female rats of Maudsley Reactive and Non-Reactive, Roman High and Low Avoidance strains were injected with either 0.8 mg/kg dose of nicotine bitartrate or distilled water. Nicotine facilitated rearing frequency. Strain differences were found. The Roman High Avoidance and Non-Reactive strains were more sensitive to nicotine than their counterparts. Female rats were found to be superior to the males in rearing score.
An investigation of central cholinoceptors in the mouse has been made by injecting cholinomimetic drugs into the cerebral ventricles and seeing how their effects were modified by prior administration of atropine‐like substances and other drugs.
Carbachol or oxotremorine injected in small doses intracerebroventricularly into conscious mice caused hypothermia, gross tremor and a variety of parasympathomimetic effects including lachrymation and salivation. Acetylcholine injected in this way was active only in much larger doses.
Methacholine and pilocarpine also caused a variety of parasympathomimetic effects after intracerebroventricular injection but virtually no hypothermia or tremor.
Nicotine injected intracerebroventricularly caused mild hypothermia, fine tremor but no parasympathomimetic effects.
Atropine‐like drugs, tricyclic antidepressants and amphetamine antagonized the hypothermia induced by intracerebroventricular carbachol or oxotremorine.
The sites of action of the atropine‐like drugs are in the brain; those of the tricyclic antidepressants and amphetamine are in the periphery probably on heat generating β‐adrenoceptor mechanisms.
It is concluded that the atropine sensitive cholinoceptors in the brain vary in their sensitivities to cholinomimetic drugs, other than acetylcholine, and may exist in isoreceptor forms.
Peripheral atropine sensitive cholinoceptors may also exist in isoreceptor forms.
The disposition of nicotine, cotinine, and nicotine N-oxide was investigated in male C57BL, DBA, and C3H mice following an ip injection of nicotine (1.0 mg/ml). The half-lives (t1/2) of nicotine in blood were 5.9 to 6.9 min. The rapid elimination of nicotine was accompanied by a rapid accumulation of metabolites; maximal concentrations of cotinine in blood (204 to 364 ng/ml) were achieved in 10 min and nicotine N-oxide (23 ng/ml in C3H mice) in 15 min. The t1/2 in blood was 20.1 to 39.8 min for cotinine and 18.4 min for nicotine N-oxide. The t1/2 values for nicotine in brain were similar to those in blood, but the values for liver were slightly larger (6.3 to 9.2 min) and interstrain differences were significant. A large strain-related difference in the t1/2 for cotinine was found; the metabolite was eliminated from the blood of DBA mice at only about one-half the rate determined for the other strains. The t1/2 for nicotine N-oxide in liver ranged from 12.7 to 27.3 min; the values were significantly different with C57BL greater than DBA greater than C3H. Strain-related differences were also observed in response to chronic exposure to cigarette smoke. The t1/2 of injected nicotine appeared to be slightly decreased in C57BL and DBA mice but was increased by 60% in livers of C3H mice compared to a control group.
C3H/2lbg mice are more sensitive to nicotine-induced seizures than are DBA/2lbg mice. There are also differences in alpha-bungarotoxin (alpha-BTX) binding in the hippocampus and midbrain of these two strains, with the C3H mice having greater binding. Because alpha-BTX and nicotine appear to bind to nicotinic receptors in the central nervous system, it is possible that there may be a relationship between seizure sensitivity after a nicotine dose and nicotinic receptor concentration. To examine this relationship, a classical cross producing F1, F2 and backcross (F1 X C3H and F1 X DBA) generations from these two strains was utilized. Dose-response curves for nicotine-induced seizures were constructed for both parental strains and all crosses derived from them. Nicotinic receptors were also measured in three brain regions: cortex, midbrain and hippocampus. Both DL-[3H]nicotine and alpha-[125I]BTX were used to measure nicotinic receptors. The pattern of results for the six generations for alpha-BTX binding in the hippocampus paralleled that for seizure sensitivity. These results suggest that strain differences for both seizure sensitivity and receptor concentration in the hippocampus may be due to allelic differences at a single autosomal locus, with dominance for low seizure susceptibility and fewer alpha-BTX receptors.
Tobacco smoke was administered to male and female mice of four inbred strains and two lines selectively bred for high activity (HA) or low activity (LA) in an open field. Administration occurred during 14 daily 10-min pretreatment sessions in a box filled with smoke from a nonfiltered cigarette (2 mg nicotine/ cigarette; average density, 750 ppm carbon monoxide). When open-field activity was subsequently measured in the absence or presence of smoke (average density, 150 ppm carbon monoxide), pretreated mice had significantly lower activity scores than controls. Comparisons of open-field activity scores under smoke-present and smoke-absent conditions revealed that effects of this acute exposure were dependent upon genotype: C3H/2Ibg activity was almost tripled in the presence of smoke; DBA/2Ibg activity was increased; but HA, LA, and C57BL/6Ibg activity scores were depressed. As measured by open-field activity, development of tolerance to the effects of acute exposure to tobacco smoke after chronic pretreatment was also genotype-dependent.
Significant genotypic differences were found for nicotine remaining in liver, brain, and blood samples when mice were sacrificed 2.5 or 5 min after a weight-specific injection of radiolabeled nicotine. Tissue nicotine levels were also related to sex, time after injection, and pretreatment interactions with genotype. Strong positive correlations were found between the measures of brain and liver levels at 5 min after injection and the behavioral measure of open-field activity under the smoke-present condition.
The influences of genotype and sex on spontaneous motor activity in a Y-maze after nicotine administration and on nicotine concentrations in liver and brain were assessed in three inbred mouse strains. The rank order of liver nicotine elimination rates in these strains was found to be C57 > C3H = DBA for females and DBA > C3H = C57 for males. Within the C57 and C3H strains, females eliminated nicotine significantly faster than males, while DBA females and males eliminated nicotine at similar rates. The rank order of motor depression at early time points after nicotine administration was found to be DBA = C57 > C3H for both males and females. Females of all three strains demonstrated less sensitivity to nicotine's depressant effects than males. There did not appear to be any consistent association between rate of liver nicotine elimination or brain nicotine level and motor depression as measured in the Y-maze. Although variability in liver nicotine elimination and in brain nicotine content may account for some of the observed behavioral effects, these data suggest that strain and sex differences in tissue sensitivity to nicotine are of primary importance.
The effects of nicotine on five behavioral and physiological measures were determined in four inbred mouse strains (BALB, C57BL, DBA and C3H). In addition, the binding characteristics of nicotine and alpha-bungarotoxin, two ligands which appear to label different nicotinic receptors, were measured in seven discrete brain regions, as well as in whole brain. A number of differences in response to nicotine were found among the four inbred strains. Whereas nicotine depressed open-field activity of BALB, C57BL and DBA mice in a dose-dependent manner, low doses of nicotine increased locomotor activity in C3H mice. The doses of nicotine tested reduced Rotarod performance in DBA and C57BL mice but not in C3H and BALB mice. All four strains displayed a dose-dependent decrease in body temperature after nicotine administration. The BALB mice were more sensitive to the drug than were the C3H, whereas the effects on C57BL and DBA mice were intermediate. All four strains showed a transient increase in respiration only after a high (2.0 mg/kg) nicotine dose. No dose of nicotine was found to have an effect on the startle response after auditory stimulation in three of the strains; only the C3H mice exhibited enhanced startle after nicotine was administered. Differences in DL-[ 3H ]nicotine binding among the seven brain regions were noted in each strain, but no differences among strains were observed. The IC50 values for inhibition of this binding by nicotine did not differ among brain regions within any strain or within any region among strains. Similarly, nicotine inhibited alpha-[125I]bungarotoxin binding with equal potency in all brain regions of each of the four strains; however, the binding of this ligand was significantly lower in the midbrain and hippocampus of DBA mice than it was in these regions in the other three strains. Thus, genetic factors influence response to nicotine, but variation in response is not easily explained by differences in brain nicotinic receptors.
The effects of chronic nicotine treatment both on tolerance development and on three ligand binding sites were examined. Nicotine was continuously infused into DBA female mice through a cannula implanted in the right jugular vein. Three final infusion rates (0.2, 1.0 and 5.0 mg/kg/hr) were used. Saline-treated animals served as controls. Mice were maintained at their final treatment dose for 8 to 10 days before they were removed from the infusion chamber. Two hours after infusion was stopped, base-line levels of rotarod performance, heart rate and body temperature were measured. These parameters were again assessed after i.p. injection of 2.0 mg/kg of nicotine. Although nicotine infusion had no effect on base-line rotarod performance or body temperature, the heart rates of mice treated with 1.0 or 5.0 mg/kg/hr of nicotine were significantly lower than those of controls. Significant tolerance to the acute effects of nicotine were noted for both body temperature and rotarod performance. DL-[3H]nicotine binding was measured in seven brain regions. Nicotine infusion resulted in significant increases in binding in cortex, midbrain, hindbrain, hippocampus and hypothalamus. An increase in α-[125I]bungarotoxin binding was seen in midbrain and hippocampus. No change in L-[3H]quinuclidinyl benzilate binding occurred. Changes resulted from increases in maximum binding site (B(max)) values, whereas K(D) values were unaffected. Correlations between nicotine response and binding were sought, and a highly significant correlation between hypothermia and hypothalamic DL-[3H]nicotine binding was obtained. These results indicate that chronic treatment with nicotine results in tolerance to the drug and is accompanied by an increase in brain nicotinic receptors.
Rats received nicotine (50 mg nicotine base/1) in their drinking fluid for 28 days. The daily consumption of nicotine was about 4 mg/kg per day. Controls received plain water. Body weight, food consumption and fluid intake were registered during the whole treatment period and up to 31 days after withdrawal of nicotine. A significant lower body weight was noted in rats, treated with nicotine, in comparison with control rats during the whole treatment period. After withdrawal of nicotine the body weight returned to normal. Only slight effects were seen on the food intake (at the beginning of the nicotine treatment). The fluid intake, on the other hand, was significantly lower in the nicotine group during the whole period of treatment and a reduced intake persisted 28 days after withdrawal of nicotine. Tolerance to nicotine was measured in rats 24 hours after withdrawal of nicotine, using two behavioural tests. No tolerance to nicotine was found after 31 days of withdrawal of nicotine. The number of nicotine-like binding sites was found to be reduced in the midbrain 24 hours after withdrawal of nicotine, while no significant change was found in the cortex and hippocampus.