To mimic nicotine exposure from light cigarette use during the initial experimentation stage, C57BL/6 N wild-type mice were treated with nicotine (0.5 mg/kg (-)-nicotine ditartrate in physiological saline, pH adjusted to 7.4) once daily for three days. On the following day, mice were treated with 0.3 mg/kg mecamylamine (MEC) to induce precipitated withdrawal (PW) from nicotine, while other mice were treated with saline to induce spontaneous withdrawal (SW). Mecamylamine or saline was administered 24 h after the last nicotine administration based on previous findings that the somatic signs of nicotine withdrawal intensify 24–48 h after cessation of nicotine administration [25, 26]. Behavioral tests were conducted 10 min after the last injection of MEC or saline. For all experiments, different mice were used and the experimenter was blinded to the experimental conditions during analysis. The overall injection scheme and experimental schedule are depicted in Fig. 1.

Drug injection and experimentation schedule. Mice were treated with saline or nicotine solution (0.175 mg/kg free-base) once daily for three days. On the following day, mice were treated with saline or mecamylamine solution (MEC; 0.3 mg/kg). All behavioral tests commenced 10 min after the last injection (saline or mecamylamine). Mice treated only with saline were designated as the control group (black). Mice treated with three days of saline followed by mecamylamine were designated the mecamylamine (MEC) group (gray). Mice treated with three days of nicotine followed by saline were designated the early spontaneous withdrawal (SW) group (blue). Mice treated with three days of nicotine followed by mecamylamine were designated the early precipitated withdrawal (PW) group (red)

The open field test was conducted to examine general locomotor function and anxiety-like behavior (Fig. 2A) (n = 10–11 mice/group). Precipitated withdrawal from nicotine caused a significant decrease in the distance moved compared to the control and spontaneous withdrawal groups (Fig. 2B) (Group effect, F(3,37) = 6.542, p = 0.0012; post-hoc analysis, **p = 0.0092 for Control vs. PW, **p = 0.0012 for SW vs. PW). In addition, precipitated nicotine withdrawal led to a significant increase in the time spent immobile compared to the control group (Fig. 2C) (Group effect, F(3,37) = 4.024, p = 0.0142; post-hoc analysis, *p = 0.0167 for Control vs. PW). Lastly, precipitated nicotine withdrawal significantly reduced the time spent in the center zone compared to the control and spontaneous withdrawal groups (Fig. 2D) (Group effect, F(3,37) = 4.600, p = 0.0078; post-hoc analysis, *p = 0.0265 for Control vs. PW, *p = 0.0110 for SW vs. PW). These findings show that early precipitated withdrawal from nicotine reduces locomotor activity and increases anxiety-like behavior in the open field, but not early spontaneous withdrawal.

Open field test. A Illustration of the open field test. B The distance moved was significantly reduced after early precipitated withdrawal (PW) from nicotine (asterisks). C The time spent immobile was significantly increased after PW from nicotine (asterisk). D The time spent in the center zone was significantly reduced after PW from nicotine (asterisks). Data represent the mean ± S.E.M. from 10–11 mice/group

Next, the elevated plus maze test was conducted to further examine anxiety-like behavior (Fig. 3A) (n = 7–12 mice/group). Unexpectedly, mecamylamine challenge and precipitated nicotine withdrawal caused a significant increase in the time spent in the closed arm (Fig. 3B) (Interaction effect, F(6,72) = 3.039, p = 0.015; post-hoc analysis, *p = 0.0245 for Control vs. MEC, *p = 0.0296 for Control vs. PW, *p = 0.0106 for MEC vs. SW, *p = 0.0120 for SW vs. PW). In addition, mecamylamine challenge and precipitated nicotine withdrawal caused a significant reduction in the number of entries into the closed arm (Fig. 3C) (Group effect, F(3,36) = 14.04, p < 0.0001; post-hoc analysis, **p = 0.0034 for Control vs. MEC, ****p < 0.0001 for Control vs. PW, **p = 0.0033 for MEC vs. SW, ****p < 0.0001 for SW vs. PW). On the other hand, only precipitated nicotine withdrawal caused a significant reduction in the number of entries into the open arm (Fig. 3C) (post-hoc analysis, **p = 0.0014 for Control vs. PW, **p = 0.0018 for SW vs. PW). These findings indicate that mecamylamine acutely increases anxiety-like behavior and reduces movement in the elevated plus maze.

Elevated plus maze test. A Illustration of the elevated plus maze test. B The time spent in the closed arm was significantly reduced after mecamylamine injection (MEC) or early precipitated withdrawal (PW) from nicotine (asterisks). C The number of entries into the open arm was significantly reduced after PW, and the number of entries into the closed arm was significantly reduced after MEC and PW (asterisks). Data represent the mean ± S.E.M. from 7–12 mice/group

Then, the somatic signs of early nicotine withdrawal were assessed to further examine the physical aspects. Previous studies have shown that the somatic signs of nicotine withdrawal in rodents include rearing, head shakes, forelimb shakes (paw tremor), body shakes, jumping, abdominal constrictions, teeth chattering/chewing, facial tremor, scratching, grooming, eye blinks, ptosis, genital licking, yawns, immobility, etc. [21, 25, 26]. Previous clinical studies have demonstrated that the reduction in hand steadiness or increased hand tremor is a prominent motor sign of nicotine withdrawal in humans [27], while macroscopic physical gestures such as head/body shakes and immobility can be readily translated into the clinic. However, most other somatic signs defined in rodents cannot be translated into the physical symptoms of nicotine withdrawal in humans, since those somatic signs are (1) not observed in the clinic, (2) largely rodent-specific, or (3) more appropriate when included in the category of natural rodent behavior. Moreover, preclinical data from pioneering studies have suggested that paw tremor is the single most replicable somatic sign of withdrawal in rodents observed after both low- and high-dose nicotine treatment [21, 25, 26]. Lastly, a seminal study has shown that episodes of locomotor immobility can be observed after precipitated nicotine withdrawal [21]. Therefore, three replicable and translatable signs of somatic nicotine withdrawal were selected for analysis: paw tremors, body shakes, and immobility.

In the analysis of the somatic signs of early nicotine withdrawal (Fig. 4A) (n = 10–11 mice/group), precipitated withdrawal from nicotine caused a significant increase specifically in the number of paw tremors compared to all other groups (Fig. 4B) (Group effect, F(3,39) = 4.540, p = 0.0080; Interaction effect, F(6,78) = 3.643, p = 0.0031; post-hoc comparison, ****p < 0.0001 for Control vs. PW, ****p < 0.0001 for MEC vs. PW, **p = 0.0042 for SW vs. PW). In addition, precipitated withdrawal from nicotine caused a significant increase in the overall number of somatic signs compared to the control and mecamylamine challenge groups (Fig. 4C) (Group effect, F(3,39) = 4.540; p = 0.0080; post-hoc comparison, *p = 0.0134 for Control vs. PW, *p = 0.0185 for MEC vs. PW). Additionally, both spontaneous and precipitated withdrawal from nicotine caused a significant increase in the overall number of somatic signs compared to a hypothetical value of 2 (the value was decided as the median of the control group, which was 2) (Fig. 4C) (SW, sum of signed ranks (W) = 49, ††p = 0.0098; PW, sum of signed ranks (W) = 55, ††p = 0.0020). Furthermore, precipitated nicotine withdrawal showed a significant distancing from other groups in the cumulative distribution plot of somatic signs (Additional file 1: Fig. S1A). Lastly, precipitated withdrawal from nicotine caused a largely consistent distribution of somatic events throughout time (Additional file 1: Fig. S1B). These findings show that early precipitated withdrawal from nicotine increases the number of somatic signs, mainly paw tremor.

Somatic withdrawal signs. A Illustration of the somatic withdrawal sign examination. B The number of paw tremors was significantly increased after early precipitated withdrawal (PW) from nicotine (asterisks). C The total number of somatic signs was significantly increased after PW and early spontaneous withdrawal (SW) from nicotine (asterisks). The total number of somatic signs was significantly different from the hypothetical value 2 after SW and PW (crosses). Data represent the mean ± S.E.M. from 10–11 mice/group

Next, the passive avoidance test was conducted to examine fear memory (Fig. 5A) (n = 9–12 mice/group). Early nicotine withdrawal did not alter the latency to enter the dark chamber (Fig. 5B), the time spent in the dark chamber (Fig. 5C), or the number of entries into the dark chamber (Fig. 5D) compared to the other groups. These findings suggest that early withdrawal from nicotine did not affect fear memory.

Passive avoidance test. A Illustration of the passive avoidance test. B The latency to enter the dark chamber did not significantly differ among groups. C The time spent in the dark chamber did not significantly differ among groups. D The number of entries into the dark chamber did not significantly differ among groups. Data represent the mean ± S.E.M. from 9–12 mice/group

Then, the spatial object recognition test was conducted to examine spatial recognition memory (Fig. 6A) (n = 6–10 mice/group). Early nicotine withdrawal did not affect the time spent sniffing all objects during either training or recall (Fig. 6B), the time spent sniffing displaced objects during recall (Fig. 6C), or the recognition index (Fig. 6D) compared to other groups. On the other hand, mice after early precipitated withdrawal from nicotine did not differ in the recognition index compared to the hypothetical value of 50% (Fig. 6D) (Control, Sum of signed ranks (W) = 28, †p = 0.0156; MEC, Sum of signed ranks (W) = 21, †p = 0.0313; SW, Sum of signed ranks (W) = 49, ††p = 0.0098). These findings suggest that early nicotine withdrawal did not grossly affect spatial recognition memory.

Spatial object recognition test. A Illustration of the spatial object recognition test. B The total time spent sniffing objects did not significantly differ among groups. C The time spent sniffing each object during recall did not significantly differ among groups. D The recognition index did not significantly differ among groups (NS: not significant). The recognition index was significantly different from the hypothetical value of 50% in the control group, after mecamylamine injection (MEC), and after early spontaneous withdrawal (SW) from nicotine (crosses). Data represent the mean ± S.E.M. from 6–10 mice/group

Finally, the social interaction test was conducted to examine social behavior (Fig. 7A) (n = 9–11 mice/group). Early nicotine withdrawal did not affect the time spent sniffing the empty or social object (Fig. 7B and C), or the social interaction ratio (Fig. 7D) compared to other groups. In addition, early nicotine withdrawal did not affect the social interaction ratio when compared to the hypothetical value of 1 (Fig. 7D) (Control, Sum of signed ranks (W) = 45, ††p = 0.0039; MEC, Sum of signed ranks (W) = 64, ††p = 0.0020; SW, Sum of signed ranks (W) = 55, ††p = 0.0020; PW, Sum of signed ranks (W) = 55, ††p = 0.0020). These findings suggest that early nicotine withdrawal did not affect social behavior.

Social interaction test. A Illustration of the social interaction test. B The time spent sniffing objects did not significantly differ among groups. C The total time spent sniffing each object did not significantly differ among groups. D The social interaction ratio did not significantly differ among groups (NS: not significant). The social interaction ratio was significantly different from the hypothetical value of 1 in all groups (crosses). Data represent the mean ± S.E.M. from 9–11 mice/group