This study demonstrates sex differences in C57BL/6J mice in response to chronic nicotine in measures of anxiety and locomotor activity. Chronic nicotine was anxiogenic in females in the EPM, but showed no effect in males. Females were also less sensitive to the locomotor stimulatory effects of nicotine, with locomotor activation exhibited only at the highest dose in females (200µg/ml) and at the middle and high (100, 200µg/ml) doses in males.
Chronic nicotine was anxiogenic in female mice, with decreases in time spent in open arms of the EPM in nicotine-treated females. Anxiogenic effects of chronic nicotine could not be explained by general decreases in locomotor activity, as measured be entries into the closed arms of the EPM. In contrast, males showed no effect of chronic nicotine in the EPM. Because males and females treated with the high dose of nicotine (200µg/ml) showed equivalent nicotine intake and plasma cotinine, sex differences in dose or metabolism of nicotine did not account for sex differences in behavioral responses. Both β2* knockout and wildtype females showed similar reductions in the percent time in the open arms in the EPM, suggesting that these anxiogenic effects are not mediated by β2* nAChRs. Finally, no overall differences in anxiety-like behavior were noted between β2* knockout and wildtype mice, confirming that β2* receptors do not mediate baseline anxiety levels in mice [37
Previous studies have shown that nicotine can be anxiolytic [6
], anxiogenic [31
], inactive, or can antagonize the anxiogenic effect of other drugs [21
] in the EPM. Nicotine dose, length and timing of nicotine administration, strain, or baseline levels of anxiety have been well-characterized [35
], few experiments have examined how sex may impact nicotine’s effects on anxiety. In one study in adolescent rats, nicotine was anxiogenic in the EPM in females but anxiolytic in males [12
]. Sex differences were also reported in the social interaction test, with adolescent female rats showing greater sensitivity to the anxiolytic actions of nicotine [8
]. In adult rats, however, no sex differences were found in acute effects of nicotine in the EPM, and nicotine was anxiogenic in both males and females [12
]. In contrast, in adult mice, acute nicotine had anxiolytic effects in the EPM, with females showing less sensitivity than males [9
]. In the present experiment, mice treated with chronic nicotine showed a unique pattern of response, with females exhibiting an anxiogenic response, and males showing no effect. Future studies examining nicotine and anxiety should determine how sex interacts with the other variables described here.
The present study tested whether β2* nAChRs contribute to chronic nicotine’s effects on anxiety-like behavior. The magnitude of response to chronic nicotine was equivalent in female β2* receptor knockout and wildtype mice, suggesting that β2* receptors are not involved in anxiogenic-like effects of chronic nicotine in the EPM, although β2* receptors may be involved in other aspects of anxiety. Promising candidates for mediating anxiety like behavior in the EPM are β4* and β3* subunits. Knockout mice lacking β4* [42
] or β3* [3
] subunits showed less anxiety-like behavior than wildtype mice in the EPM, suggesting that endogenous ACh may produce anxiogenic effects under conditions of stress mediated by β3* and β4* nAChRs.
Sex differences were observed in the psychostimulant effects of nicotine in response to chronic exposure. This finding expands on our previous report [24
], showing that the high (200µg/ml) dose of chronic nicotine resulted in locomotor activation in both females and males. In the present study, only males showed activation at the middle dose (100µg/ml). It is unlikely that this difference in sensitivity can be explained by sex differences in nicotine intake or plasma levels of nicotine. At the 100µg/ml concentration, nicotine intake was actually higher in females than males, resulting in equivalent plasma levels of cotinine for both sexes. The current results differ from previous reports showing greater sensitivity in female rats to the locomotor stimulant effects of chronic nicotine [4
]. Future studies will determine if the nature of these differences are due to species (mouse vs. rat) or route of administration (drinking water in the present study vs. iv administration [4
] and minipump [14
Several potential mechanisms may explain sex differences in response to chronic nicotine. First, pharmacokinetic variations in plasma or brain levels of nicotine could explain sex differences. One study found that C57BL/6 females eliminated nicotine faster than males [18
]. Studies in humans have found that women metabolize nicotine more rapidly, an effect related to estrogen [1
] Second, chronic nicotine differentially regulates nicotinic receptors in males and females, with males showing more pronounced nAChR upregulation than females [26
]. Third, steroid hormones modulate responses to nicotine and both progesterone and estradiol blocked nicotine-induced analgesia in one study [9
]. Estrogen also enhances dopamine release from striatal slices in females, but decreases dopamine release in males [10
]. As nicotine-induced increased locomotor in the current model is dopamine-mediated [24
], estrogen-induced modulation of dopamine release could mediate sex differences observed in this test.
It should be emphasized that sex differences in the present study do not reflect general increases or decreases in sensitivity for a particular sex, but rather different sensitivities dependent upon the behavioral measure. This is consistent with other reports. Females were more sensitive to nicotine in oral self-administration [25
], intravenous self-administration [11
], operant response to visual cues [7
], and reductions in body weight and feeding [13
]. Females were less sensitive than males to the effect of nicotine in analgesia [9
] and cognition (active avoidance) [46
] tests, and were less sensitive to the anxiogenic effects of nicotine during ethanol withdrawal [19
The pattern of behavioral response in the current mouse model, with females showing greater anxiety-like behavior but less sensitivity to dopamine-mediated locomotor activation, may represent an appropriate model of female smokers. It has been proposed that women are less sensitive than men to dopamine-dependent positive reinforcing effects of nicotine (see [34
]). Furthermore, nicotine may contribute to negative mood states and anxiety in smokers (see [32
]), an effect that may be more pronounced in women, who have higher rates of anxiety and depressive disorders than men [22
]. Future investigation into the hormonal and neurochemical mechanisms underlying sex differences in behavioral responsiveness to nicotine will further aid in understanding the relationship between smoking and anxiety disorders.
In summary, female mice were less sensitive to the anxiogenic and locomotor activating effects of nicotine. These data suggest that non-nicotine therapeutics may be more useful for smoking cessation in women as compared to men. Future studies will determine whether similar sex differences are important for the lower cessation rates in women.