Characterization of pharmacological and behavioral differences to nicotine in C57Bl/6 and DBA/2 mice

Abstract

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.

Figure 1. Acute pharmacological response to nicotine in D2 and B6 mice

Different groups of D2 (open circles) and B6 (closed circles) mice were treated with increasing doses of nicotine, and nicotine’s effects on tail flick, hot plate, body temperature, locomotor activity, and plus maze were measured. Each point represents mean ± S.E.M. of 8–10 mice per group. * denotes p< 0.05 vs. lowest nicotine dose tested for each strain. MPE, maximum possible effect

Figure 2. Involvement of spinal CaMKII in nicotine-induced antinociception in D2 nd B6 mice

Different groups of D2 (open circles) and B6 (open squares) mice were treated with varying doses of the CaMKII inhibitor KN62 (i.t.), 5 minutes prior to injection of 25 or 15 µg/animal of nicotine respectively. Tail flick assessment revealed significant dose-dependent blockade of nicotine-induced antinociception by KN62 in both mouse strains; however, the effect was more potent in D2 mice compared to B6 mice. Each point represents mean ± S.E.M. of 8–10 mice per group. * denotes p< 0.05 vs. lowest nicotine dose tested for each strain

Figure 3. Evaluation of nicotine tolerance in B6 mice after chronic administration

Different groups of B6 mice chronically treated with saline or nicotine for 14 days received challenge injections of various nicotine doses. A dose response curve shift to the right was noted in A. the tail flick, B. hot plate, and C. body temperature assessment for nicotine treated B6 mice, indicating the development of tolerance to nicotine. Each point represents the mean ± S.E.M. of 8–10 mice per group. * denotes p< 0.05 vs. lowest nicotine dose tested for each strain

Figure 4. Evaluation of nicotine tolerance in D2 mice after chronic administration

Different groups of D2 mice chronically treated with saline or nicotine for 14 days received challenge injections of various nicotine doses. There were no differences between saline and nicotine treated mice in the A. tail flick, B. hot plate, or C. body temperature assessment, indicating that D2 mice did not develop tolerance to nicotine. Each point represents the mean ± S.E.M. of 8–10 mice per group.

Figure 5. Nicotine CPP in B6 and D2 mice

The rewarding effects of nicotine were measured using the CPP model. A dose response curve for nicotine CPP was generated in B6 and D2 mice. The x-axis represents nicotine dose in mg/kg and the y-axis represents the preference score in s. Each point represents the mean ± S.E.M. of 8–10 mice per group. *denotes p< 0.05 vs. vehicle

Figure 6. Nicotine withdrawal assessment in B6 mice

Mini pumps (MP) were removed from mice treated with saline (circles) or nicotine (squares) for 14 days, and testing initiated 18–24 hours after removal. Responses were measured in A. somatic signs, B. plus maze, C. tail flick, D. plantar stimulation, and E. locomotor activity for three days following mini pump removal. Each point represents the mean ± S.E.M. of 8–10 mice per group. * denotes p< 0.05 vs. saline

Figure 7. Nicotine withdrawal assessment in D2 mice

Mini pumps (MP) were removed from mice treated with saline (circles) or nicotine (square) for 14 days, and testing initiated 18–24 hours after removal. Responses were measured in A. somatic signs, B. plus maze, C. tail flick, D. plantar stimulation, and E. locomotor activity for three days following mini pump removal. Each point represents the mean ± S.E.M. of 8–10 mice per group. * denotes p< 0.05 vs. saline

Figure 8. Nicotine CPA in B6 and D2 mice

CPA, an affective component of nicotine withdrawal, was measured in B6 and D2 mice. Mecamylamine (mec, 3.5 mg/kg) precipitated aversion in B6 nicotine-treated mice, but not in D2 mice. The dose of mecamylamine utilized had no effect on saline treated mice. Each point represents the mean ± S.E.M. of 10–12 mice per group. * denotes p< 0.05 vs. saline B6 group. MP= mini pump.