Introduction

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In spite of heightened education and prevention strategies, cigarette smoking remains a major health risk. Nicotine is believed to be the primary reason that people consume tobacco products. Indeed, substantial evidence now shows that nicotine is the addictive substance found in tobacco. In 1974, 31% of U.S. women and 43% of U.S. men were tobacco smokers. Current estimates indicate that the difference between men and women has narrowed considerably to a comparable rate (21-25%) (CDC, 1998). Furthermore, poorer outcome of women in smoking cessation trials, especially those involving nicotine replacement, has also been reported. In other words, nicotine replacement is less effective for smoking cessation than in men. There are several potential causes for this shift toward more women smokers. There are several possible explanations for this trend. Factors such as women's greater concern about weight gain, greater difficulty with negative mood (and higher prevalence of affective disorders), and greater need for social support to quit smoking could be of influence. Reduced availability of social support for cessation in women and greater impact of advertising on promoting smoking in women versus men may also have an influence. Broad cultural influences also have been advanced as an explanation for sex differences in smoking prevalence. Although cultural factors are clearly important in explaining the historical differences between the smoking patterns of American men and women earlier this century and in non-Western countries, they do not seem to be a likely explanation. Indeed, cultural restrictions in smoking behavior specific to women in Western societies are no longer in existence. One obvious possibility is the involvement of biological differences between the sexes might contribute to this gender difference.

Animal studies have found that male and female rodents have different sensitivities to the effects of nicotine. For example, female rats are less sensitive than male rats to the discriminative stimulus effects of nicotine (Schechter and Rosecrans, 1971), consistent with findings in humans (Perkins et al., 1999). Female mice were less sensitive to nicotine-induced suppression of Y-maze activity (Hatchell and Collins, 1980) and increase in active avoidance learning (Yilmaz et al., 1997). In addition, sex differences in receptor up-regulation after chronic exposure to nicotine was also reported with only male rats showing up-regulation of brain [³H]cytisine binding sites (Koylu et al., 1997). Conversely, female rats were more sensitive to nicotine's effects on prepulse inhibition (Faraday et al., 1999) and food intake (Grunberg et al., 1984). Female rats were also more sensitive to nicotine-induced antinociception compared with males in acute and chronic pain models (Craft and Milholland, 1998; Chiari et al., 1999; Lavand'homme and Eisenach, 1999). In addition, sensitization to nicotine-induced increase in locomotor activity was found to be greater in female rats after chronic i.v. administration of nicotine (Booze et al., 1999), but not after chronic s.c. injection (Kanyt et al., 1999). Such complexity and variability in the response of male and female rodents to nicotine may be due to differences in the dose and elimination profile of nicotine, age and specie of the test animal, route or duration of administration, time of evaluation, or the behavioral task used.

Another biological factor that could underlie sex differences is the influence of gonadal hormones. Levels of sex hormones change through the menstrual (human) or estrous (rodent) cycle. Human studies examining the effects of the phase of the menstrual cycle on cigarette smoking and on the psychoactive actions of nicotine are conflicting. However, most studies report that menstrual cycle phase may influence self-reported withdrawal symptoms in women (Benowitz and Hatsukami, 1998). Animal studies suggest that ovarian hormones could influence nicotine acute effects and reinforcement. Dluzen and Anderson (1997) reported that estrogen treatment to ovariectomized female rats increased the in vitro nicotine-induced release of dopamine from striatal slices, whereas the opposite effect was seen in castrated males.

Therefore, the aim of the present study was to conduct a comprehensive examination of the putative sex differences in the potency of nicotine between male and female mice using several pharmacological and behavioral tests. For that, the potency of nicotine in various pharmacological effects measures (antinociception, hypothermia, seizures, and motor activity, plus-maze activity) in animals was examined after systemic and intrathecal administration. A wide range of nicotinic effects is important to consider because it is believed that various nicotinic receptor subtypes mediate different pharmacological effects of nicotine. Because gonadal hormones may also mediate such sex differences, the effects of testosterone, 17-estradiol, and progesterone on nicotine's effects were examined. The in vivo effect of hormones was correlated with in vitro studies using the oocyte expression system. For that, the effect of sex hormones on the functional activity of the neuronal nAChRs ₄₂ (the major nicotinic receptor subtype in the brain) expressed receptors was studied.

	Materials and Methods

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Animals

Male and female ICR mice (20-25 g) matched for age were obtained from Harlan Laboratories (Indianapolis, IN). They were housed in groups of six and had free access to food and water. Animals were housed in an American Association of Laboratory Animal Care-approved facility, and the study was approved by the Institutional Animal Care and Use Committee of Virginia Commomwealth University.

Drugs

()-Nicotine was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI) and converted to the ditartrate salt as described by Aceto et al. (1979). ()-Epibatidine (hemi oxalate salt) was supplied by Dr. S. Fletcher (Merck Sharp and Dohme & Co., Essex, UK). Mecamylamine hydrochloride was supplied as a gift from Merck, Sharp and Dohme & Co. (West Point, PA). Testosterone, 17-estradiol, and progesterone water-soluble (cyclodextrin-encapsulated) and oil-soluble (testosterone decanoate) forms were purchased from Sigma Chemical Co. (St. Louis, MO); neostigmine was purchased from Research Biochemicals International (Natick, MA). Oil-soluble hormones were diluted in sesame oil (Sigma Chemical Co.). All other drugs were dissolved in physiological saline (0.9% sodium chloride) and given in a total volume of 1 ml/100 g of body weight for s.c. injections. All doses are expressed as the free base of the drug.

Intrathecal Injections

Intrathecal injections were performed free-hand between the L5 and L6 lumbar space in unanesthetized male mice according to the method of Hylden and Wilcox (1980). The injection was performed using a 30-gauge needle attached to a glass microsyringe. The injection volume in all cases was 5 µl. The accurate placement of the needle was evidenced by a quick "flick" of the mouse's tail.

Antinociceptive Tests

Tail-Flick Test. Antinociception was assessed by the tail-flick method of D'Amour and Smith (1941) as modified by Dewey et al. (1970). A control response (2-4 s) was determined for each mouse before treatment, and a test latency was determined after drug administration. To minimize tissue damage, a maximum latency of 10 s was imposed. Antinociceptive response was calculated as percentage maximum possible effect (%MPE), where %MPE = [(test  control)/(10  control)] × 100. Groups of 8 to 12 animals were used for each dose and for each treatment. The mice were tested 5 min after either s.c. or i.t. injections of nicotine.

Hot-Plate Test. The method is a modification of that described by Eddy and Leimbach (1953) and Atwell and Jacobson (1978). Mice were placed into a 10-cm-wide glass cylinder on a hot-plate (Thermojust Apparatus, Richmond, VA) maintained at 55.0°C. Two control latencies at least 10 min apart were determined for each mouse. The normal latency (reaction time) was 6 to 10 s. Antinociceptive response was calculated as %MPE, where %MPE = [(test  control)/(40  control) × 100]. The reaction time was scored when the animal jumped or licked its paws. Eight mice per dose were injected s.c. with nicotine and tested 5 min after injection.

Behavioral Testing

Locomotor Activity. Mice were placed into individual Omnitech photocell activity cages (28 × 16.5 cm) 5 min after s.c. administration of either 0.9% saline or nicotine. Interruptions of the photocell beams (two banks of eight cells each) were then recorded for the next 10 min. Data are expressed as number of photocell interruptions.

Body Temperature. Rectal temperature was measured by a thermistor probe (inserted 24 mm) and digital thermometer (Yellow Springs Instrument Co., Yellow Springs, OH). Readings were taken just before and at 30 min after the s.c. injection of either saline or nicotine. The difference in rectal temperature before and after treatment was calculated for each mouse. The ambient temperature of the laboratory was 21-24°C from day to day.

Seizure Activity. After a s.c. injection of nicotine at a dose of 9 mg/kg, each animal was placed in a 30- × 30-cm Plexiglas cage and observed for 5 min. Whether a clonic seizure occurred within a 5-min time period was noted for each animal after s.c. administration of different drugs. This amount of time was chosen because seizures occur very quickly after nicotine administration. Results are expressed as percentage seizure.

Elevated Plus-Maze. An elevated plus-maze, prepared with gray Plexiglas, consisted of two open arms (23 × 6.0 cm) and two enclosed arms (23 × 6 × 15 cm) that extended from a central platform. It was mounted on a base raised 60 cm above the floor. Fluorescent lights (350-lux intensity) located in the ceiling of the room provided the only source of light to the apparatus. The animals were placed in the center of the maze and the following variables were scored: 1) time spent in the open arms, 2) time spent in the closed arms, and 3) total number of crossings between arms. These variables are automatically recorded by a photocell beam system. The test lasted 5 min and the apparatus was thoroughly cleaned after removal of each animal. Readings were taken 15 min after the s.c. injection of either saline or nicotine. Anxiolytic response was calculated as the percentage of time spent in the open arm, where % time = [(test  300 s) × 100].

Oocyte Expression Studies

Oocyte Preparation. Oocytes preparation was performed according to the method of Mirshahi and Woodward (1995) with minor modifications. Briefly, oocytes were isolated from female adult oocyte-positive Xenopus laevis frogs. Frogs were anesthetized in a 0.2% 3-aminobenzoic acid ethyl ester solution (Sigma Chemical Co.) for 30 min and a fraction of the ovarian lobes was removed. The eggs were rinsed in Ca²⁺-free ND96 solution, treated with collagenase type IA (Sigma Chemical Co.) for 1 h to remove the follicle layer, and then rinsed again. Healthy stage V-VI oocytes were selected and maintained for up to 14 days after surgery in 0.5× L-15 media (Sigma Chemical Co.).

mRNA Preparation and Microinjection. ₄ and ₂ rat subunit cDNA contained within a pcDNAIneo vector were kindly supplied by Dr. James Patrick (Baylor College of Medicine, Houston, TX). The template was linearized downstream of coding sequence and mRNA was synthesized using an in vitro transcription kit from Ambion (Austin, TX). The quantity and quality of message were determined via optical density (spectrophotometer; Beckman Instruments Inc., Chaumburg, IL) and denaturing formaldehyde gel analysis. Oocytes were injected with either 51 ng (41 nl) of ₄ and ₂ mRNA mixed in a 1:1 ratio using a Variable Nanoject (Drummond Scientific Co., Broomall, PA). Oocytes were incubated in 0.5× L-15 media IA supplemented with penicillin, streptomycin, and gentimycin for 4 to 6 days at 19°C before recording.

Electrophysiological Recordings. Oocytes were placed within a Plexiglas chamber (total volume 0.2 ml) and continually perfused (10 ml/min) with buffer consisting of 115 mM NaCl, 1.8 mM CaCl₂, 2.5 mM KCl, 1.0 µM atropine, and 10.0 mM HEPES at pH 7.2. Oocytes were impaled with two microelectrodes containing 3 M KCl (0.3-3 M) and voltage-clamped at 70 mV using an Axon Geneclamp amplifier (Axon Instruments Inc., Foster City, CA). Oocytes were stimulated for 10 s with various concentrations of acetylcholine using a six-port injection valve. Except where noted, applications were separated by 5-min periods of washout. Currents were filtered at 10 Hz and collected by a Macintosh Centris 650 with a 16-bit analog digital interface board, and data were analyzed using Pulse Control voltage-clamp software running under the Igor Pro graphic platform (Wavemetrics, Lake Oswego, OR). Water-soluble forms of hormones were applied at different concentrations and concentration-response curves were normalized to the current induced by 1 µM ACh. The normalizing concentration of ACh was applied before and after drug application to each oocyte to check for desensitization. Data were rejected if responses to the normalizing dose fell below 75% of the original response. IC₅₀ values were determined using data from four to six oocytes.

Statistical Analysis

Data were analyzed statistically by an analysis of variance followed by the Fisher protected least-significant difference multiple comparison test. For time course studies, Dunnett's test was used. The null hypothesis was rejected at the 0.05 level. IC₅₀ (antagonist concentration 50%) and ED₅₀ (effective dose 50%) values with 95% confidence limits (CL) were calculated by unweighted least-squares linear regression as described by Tallarida and Murray (1987). Tests for parallelism were calculated according to the method of Tallarida and Murray (1987). If confidence limit values did not overlap, then the shift in the dose-response curve was considered significant.

	Results

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Antinociception Studies

Tail-Flick Test. Nicotine time course of action after an equipotent dose in male (2 mg/kg) and female (3.5 mg/kg) mice was evaluated in the tail-flick after s.c. injection. As seen in Fig. 1A, no significant difference was seen between male and female mice. Nicotine's effect disappeared completely within 30 min after s.c. administration in both sexes. Dose-response relationships were then established for nicotine in male and female mice by measuring antinociception at the time of maximal effect (5 min) (Fig. 1B). Nicotine was 3 times less potent in females compared with males. Nicotine produced a dose-responsive increase in the tail-flick latency in both male and female mice with ED₅₀ (±CL) values of 1.0 (0.6-1.3) and 2.9 (1.4-5.8) mg/kg, respectively. The basal latencies between male and female mice were not statistically different (2.5 ± 0.2 and 2.7 ± 0.2 s for male and female, respectively). Pretreatment with the centrally active noncompetitive nicotinic receptor antagonist mecamylamine (1 mg/kg s.c.) 10 min before nicotine blocked its antinociceptive activity in both male and female mice (Fig. 1C). Although mecamylamine seems to be more potent in blocking nicotine's effects in female mice [AD₅₀ (±CL) = 0.09 (0.01-0.4) compared with male AD₅₀ (±CL) = 0.2 (0.04-1.1)], the difference was not significant because confidence limits of the two curves overlapped. A similar sex difference was also observed with epibatidine, a very potent nicotinic agonist. Epibatidine produced a dose-responsive increase in the tail-flick latency (Fig. 2) in both male and female mice with ED₅₀ (±CL) values of 0.8 (0.5-1.1) and 1.7 (1.2-2.6) µg/kg, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Antinociceptive effects of nicotine in male () and female () female mice using the tail-flick test after s.c. administration. A, time course of antinociception induced by nicotine (2 and 3.5 mg/kg for male and female, respectively) after s.c. administration in mice. B, nicotine dose-response curves in both sexes 5 min after injection. C, comparison of mecamylamine blockade of antinociception induced by nicotine in male and female mice. Mecamylamine at different doses was injected s.c. 10 min before an equipotent dose of nicotine (ED84) was administered s.c. and mice were tested 5 min after the second injection. Each point represents the mean ± S.E. of 12 to 16 mice.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Antinociceptive effects of epibatidine in male () and female () mice using the tail-flick test after s.c. administration. Epibatidine dose-response curves were determined in both sexes 10 min after injection. Each point represents the mean ± S.E. of 12 to 16 mice.

Hot-Plate Test. Nicotine was then evaluated in both male and female mice in another acute pain test, the hot-plate assay. Dose-response relationships were established for nicotine in male and female mice by measuring antinociception at the time of maximal effect (5 min) (Fig. 3). Similar to that observed with the tail-flick test, female mice were less sensitive to the effect of nicotine compared with males. The difference of potency between the two sexes was less (1.8 times) than the one determined for the tail-flick test. Nicotine produced a dose-responsive increase in the hot-plate latency in both male and female mice with ED₅₀ (±CL) values of 0.5 (0.4-0.6) and 0.9 (0.8-1.2) mg/kg, respectively. The basal latencies between male and female mice were not statistically different (12.9 ± 0.8 and 11.5 ± 3.2 s for male and female, respectively).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Antinociceptive effects of nicotine in male () and female () mice using the hot-plate test after s.c. administration. Nicotine dose-response curves in both sexes were determined 5 min after injection. Each point represents the mean ± S.E. of 12 to 16 mice.

Antinociceptive Effects after i.t. Injection in the Tail-Flick Test. Nicotine potency was then evaluated in the tail-flick after i.t. injection in male and female mice. Similar to that observed with s.c. administration, female mice were less sensitive to the effect of nicotine compared with males (Fig. 4A). Nicotine produced a dose-responsive increase in the tail-flick latency in both male and female mice with ED₅₀ (±CL) values of 11.4 (9.5-13.7) and 23 (15.1-34.2) µg/animal, respectively. To determine whether the sex differences extended to cholinesterase inhibitors, neostigmine was evaluated by i.t. injection. The ED₅₀ values for neostigmine-induced antinociception in male and female mice were 0.004 and 0.003 µg/animal, respectively. Therefore, the effects of elevating endogenous ACh were not influenced by gender.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Antinociceptive effects of direct and indirect cholinergic agonists in male () and female () mice using the tail-flick test after i.t. administration. A, nicotine dose-response curves in both sexes 5 min after injection. B, neostigmine dose-response curves in both sexes 5 min after injection. Each point represents the mean ± S.E. of 12 to 16 mice.

Other Behavioral Effects of Nicotine

To further characterize sex differences, additional experiments were conducted to determine nicotine potency in other behavioral responses.

Effect on Plus-Maze Activity. Nicotine increased the time spent in the open arm of the maze when administered s.c. to male and female mice (Fig. 5A). Nicotine potency was higher in male mice compared with female mice with ED₅₀ (±CL) values of 0.40 (0.2-0.6) and 0.95 (0.8-1.2) mg/kg, respectively. No gender differences were seen for total crossings between the different arms in the plus-maze test (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Potency of nicotine in male () and female () female mice in different behavioral tests after s.c. injection. A, nicotine-induced increase in open arm time using the plus-maze test. Mice were tested 15 min after nicotine injection. B, nicotine-induced hypothermia. Mice were tested 30 min after nicotine injection. C, nicotine-induced hypomotility. Mice were tested 5 min after nicotine injection. D, nicotine-induced seizures. Mice were tested 5 min after nicotine injection. Each point represents the mean ± S.E. of 8 to 12 mice.

Effect on Body Temperature, Seizure Activity, and Locomotor Activity. Unlike the antinociceptive and anxiolytic effects, nicotine potency in male and female mice was similar in inducing hypothermia, hypomotility, and seizures production after s.c. administration (Fig. 5, B-D; Table 1). No sex differences were observed in baseline values in the spontaneous activity and body temperature (data not shown).

In Vivo Interaction of Sex Hormones with Nicotine with Expressed Neuronal Nicotinic Receptors

The interaction between nicotine and different sex hormones (testosterone, 17-estradiol, and progesterone) was studied in the tail-flick test after s.c. administration of nicotine. Time course studies were performed to determine the time of maximal blockade and then dose-response curves for each hormone in blocking nicotine-induced antinociception were carried out to determine the blockade potency.

Effect of Progesterone. Progesterone was evaluated for its ability to antagonize a 3.5-mg/kg dose of nicotine in female mice using the tail-flick procedure after i.p. injection. As shown in Fig. 6A, progesterone time dependently blocked nicotine-induced antinociception. The duration of action of progesterone in the tail-flick test was time-dependent with maximum blockade occurring between 3 and 4 h after injection of a dose of 20 mg/kg. Nicotine's effect started to recover within 6 h after pretreatment with progesterone. Additionally, as showed in Fig. 6B, progesterone dose dependently blocked nicotine-induced antinociception with an AD₅₀ (±CL) of 4.8 (2.8-8.4) mg/kg when given s.c. 4 h before nicotine.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Blockade of nicotine-induced antinociception in the tail-flick test by progesterone after i.p. injection in female mice. A, time course of progesterone effect on nicotine-induced antinociception (3 mg/kg s.c.) after i.p. administration of 20 mg/kg in mice. B, dose-response blockade of progesterone after i.p. administration in mice. Progesterone at different doses was administered i.p. 4 h before nicotine (3.0 mg/kg s.c.) and mice were tested 5 min later. Each point represents the mean ± S.E. of 8 to 12 mice. *p < 0.05 compared with correspondent zero time point.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Dose-response curve of progesterone blocking nicotine-induced antinociception in female mice chronically treated with progesterone () or vehicle () using the tail-flick test. Mice were injected with either vehicle (sesame oil i.p.) or progesterone (10 mg/kg i.p.) twice a day for 4 days. Twelve hours after the last injection, the potency of progesterone in blocking a challenge of nicotine (3.0 mg/kg s.c.) was assessed. Each point represents the mean ± S.E. of 8 to 12 mice.

Effect of 17-Estradiol and Testosterone. Similar to progesterone, 17-estradiol time and dose dependently blocked nicotine-induced antinociception in female mice. The duration of effect of 17-estradiol (20 µg/kg i.p.) was briefer than that of progesterone, with a maximum effect at 3 h after injection and a full recovery of nicotine's effects 6 h later (Fig. 8A). Additionally, as showed in Fig. 8B, 17-estradiol dose dependently blocked nicotine-induced antinociception with an AD₅₀ (±CL) of 5.5 (4.0-6.6) µg/kg when given s.c. 4 h before nicotine. In contrast to progesterone and 17-estradiol, testosterone (up to 500 mg/kg i.p.) did not significantly decrease nicotine-induced antinociception at the times (Fig. 9A) and doses tested (Fig. 9B).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Blockade of nicotine-induced antinociception in the tail-flick test by 17-estradiol after i.p. injection in female mice. A, time course of 17-estradiol's effect on nicotine-induced antinociception (3 mg/kg s.c.) after i.p. administration of 20 µg/kg in mice. B, dose-response blockade of 17-estradiol after i.p. administration in mice. 17-Estradiol at different doses was administered i.p. 3 h before nicotine (3.0 mg/kg s.c.) and mice were tested 5 min later. Each point represents the mean ± S.E. of 8 to 12 mice. *p < 0.05 compared with correspondent zero time point.

View larger version (45K): [in this window] [in a new window]   Fig. 9.   Lack of testosterone's effects on nicotine-induced antinociception in the tail-flick test after i.p. injection in male mice. A, effect of testosterone (250 mg/kg i.p.) at different times after injection on nicotine-induced antinociception (3 mg/kg s.c.) in mice. B, effect of testosterone at different doses on nicotine-induced antinociception (3 mg/kg s.c.) 4 h before nicotine. Mice were tested 5 min later. Each point represents the mean ± S.E. of 8 to 12 mice.

Interaction of Sex Hormones with Neuronal Nicotinic Receptors Expressed in Oocytes

Because progesterone and 17-estradiol blocked nicotine's behavioral effects, their potency as a blocker at expressed neuronal nicotinic receptors was investigated. Progesterone and 17-estradiol at 50 µM elicited little current when applied for 10 s to oocytes expressing the ₄₂ subunit combinations (5 ± 2 nA). Although they did not activate these expressed receptors, they antagonized the effects of nicotine in a concentration-related manner. The concentration that blocked 50% of the nicotinic current was determined to be 1.8 (1.1-2.9) and 25 (15-43) µM for progesterone and 17-estradiol, respectively (Fig. 10, A and B). At the lower concentration of 17-estradiol (0.01 µM), there was a small (12% increase) enhancement in the nicotinic current when the two drugs were coapplied (Fig. 10B). This enhancement disappeared at 0.1 µM 17-estradiol. The effect of progesterone and 17-estradiol on ₄₂ receptors was reversible after 5-min wash period (Fig. 10, A and B).

View larger version (36K): [in this window] [in a new window]   Fig. 10.   Concentration-dependence of progesterone (A) and 17-estradiol (B) inhibition of 42 nAChRs subtype expressed in oocytes. Responses of neuronal 42 receptor to 1 µM nicotine in the absence and presence of different concentrations of progesterone or 17-estradiol when they were preapplied at the tested concentration for 20 s and then followed by a coapplication of the hormone and nicotine. Each application is separated by 5-min washout. Oocytes were held at 70 mV. ACh "after" represents the current activated by an application of ACh after hormone washout.

	Discussion

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The primary findings of this study are that there are sex differences in nicotine's acute pharmacological effects, but these differences are response-dependent. This study also confirms that sex hormones are functional blockers of nicotinic receptors in in vivo and in vitro tests. Previous work suggested that gender was an important factor that influences certain behavioral effects of nicotine, such as those on feeding behavior (Grunberg et al., 1984), locomotor sensitization (Booze et al., 1999), and cognition (Kanyt et al., 1999). Among the responses to nicotine where significant sex differences were observed in our studies are the antinociceptive and the anxiolytic effects of nicotine. Female mice were found less sensitive to the acute effects of nicotine in these tests. Although gender differences in the plus-maze test have been not described before, the difference in nicotine-induced antinociception agrees well with a recent human study where women were less sensitive to nicotine than men (Jamner et al., 1998).

This sex difference is not limited to nicotine, but other nicotinic agonists such as epibatidine showed a similar difference. In addition, the fact that a similar sex difference was seen after spinal injection would also confirm the involvement of spinal mechanisms in mediating such a difference. However, no central administration of nicotine was performed in the present study. Furthermore, the lack of sex difference in the case of neostigmine suggests that sex interacted significantly with nicotinic and not muscarinic drugs because neostigmine-induced antinociception is mediated through muscarinic and not nicotinic receptors (Yaksh et al., 1985; Naguib and Yaksh, 1994).

Proposed Mechanisms Underlying Sex Differences in Nicotine Analgesia. One obvious possibility is that nicotine bioavailability might differ between the sexes. Although a sex difference in nicotine kinetics is probably not a major influence in humans, there is evidence of sex difference in nicotine metabolism in rats, where studies showed that male rats eliminate nicotine faster than females (Kyerematen et al., 1988; Nwoso and Crooks, 1988). Although plasma and brain concentrations of nicotine were not measured in our studies, our data argue against an exclusive role for pharmacokinetics in accounting for the differential analgesic response of male and female mice to nicotine. Indeed, the following arguments suggest that kinetic factors are probably playing a minor role: 1) the fact that sex differences were not seen in a uniform manner in all behavior responses measured; 2) sex differences were seen with more than one nicotinic agonist (epibatidine and nicotine); and 3) sex differences were seen in nicotine potency in the tail-flick test after i.t. administration.