Prenatal nicotine exposure mouse model showing hyperactivity, reduced cingulate cortex volume, reduced dopamine turnover, and responsiveness to oral methylphenidate treatment

Abstract

Cigarette smoking, nicotine replacement therapy, and smokeless tobacco use during pregnancy are associated with cognitive disabilities later in life in children exposed prenatally to nicotine. The disabilities include attention deficit hyperactivity disorder (ADHD) and conduct disorder. However, the structural and neurochemical bases of these cognitive deficits remain unclear. Using a mouse model we show that prenatal nicotine exposure produces hyperactivity, selective decreases in cingulate cortical volume, and radial thickness, as well as decreased dopamine turnover in the frontal cortex. The hyperactivity occurs in both male and female offspring and peaks during the "active" or dark phase of the light/dark cycle. These features of the mouse model closely parallel the human ADHD phenotype, whether or not the ADHD is associated with prenatal nicotine exposure. A single oral, but not intraperitoneal, administration of a therapeutic equivalent dose (0.75 mg/kg) of methylphenidate decreases the hyperactivity and increases the dopamine turnover in the frontal cortex of the prenatally nicotine exposed mice, once again paralleling the therapeutic effects of this compound in ADHD subjects. Collectively, our data suggest that the prenatal nicotine exposure mouse model has striking parallels to the ADHD phenotype not only in behavioral, neuroanatomical, and neurochemical features, but also with respect to responsiveness of the behavioral phenotype to methylphenidate treatment. The behavioral, neurochemical, and anatomical biomarkers in the mouse model could be valuable for evaluating new therapies for ADHD and mechanistic investigations into its etiology.

Figure 1.

The effects of two different doses (0.05 and 0.1 mg/ml drinking water) of prenatal nicotine treatment on spontaneous locomotor activity were compared to the effects of prenatal treatment with saccharin vehicle alone (SAC, 2% in drinking water) in male mice at P42 and P60. Locomotor activity was analyzed for a 4 h period beginning at 08:00 A.M. (n = 8–13 for each group). *p < 0.05.

Figure 2.

Locomotor activity was analyzed over a 21 h period between 3:00 and 12:00 P.M. in male and female mice aged P42–P56 from the PNE, SAC, and WATER groups. The lights-off period was from 7:00 P.M. to 7:00 A.M. A–F, Hourly (A, B) or cumulative (C–F) locomotor activity measurements are shown. Both male (A) and female (B) mice in the PNE group showed significantly higher locomotor activity compared to their counterparts in the SAC and WATER groups throughout the 21 h period of observation (A–C). Activity between the SAC and WATER groups was not significantly different. When the activity was analyzed separately for the two lights-on and one lights-off sessions (C–E), PNE group showed significant increases during the lights-off period (E) and during the lights-on period immediately following (F) but not preceding (D) the lights-off period. There were no significant differences between SAC and WATER groups nor between male and female mice within any given prenatal treatment group. ANOVA and multiple comparison tests were used to test statistical significance of the experimental effects (male, n = 10–15; female, n = 10–15).

Figure 3.

A, B, The effects of intraperitoneal (A) or oral (B) administration of saline or MPH on locomotor activity in 42-d-old male mice in the SAC and PNE groups. Locomotor activity was recorded for 4 h. Mice in the PNE group showed significantly increased activity compared to their counterparts in the SAC group when saline was administered to both the groups either intraperitoneally (A) or orally (B). Intraperitoneal MPH did not produce significant changes in the locomotor activity in PNE group at any of the five doses (A). However, it increased locomotor activity in the SAC group at 3.75 and 7.5 mg/kg doses (A). Oral MPH administration did not affect locomotor activity in the SAC group at either dose (B). However, 0.75 mg/kg MPH significantly decreased locomotor activity in the PNE group. ANOVA and multiple comparison tests were used to test statistical significance of the experimental effects (n = 6 for each group).

Figure 4.

A, B, The effects of a single oral dose (0.75 mg/kg) of MPH on locomotor activity in 42-d-old mice from the PNE and SAC exposure groups. The analysis was performed separately for male (A) and female (B) mice over a period of 12 h during the lights-off period. MPH or SAL was gavaged at 7:00 P.M. (open arrows) and locomotor activity was recorded for a 12 h period (7:00 P.M. to 7:00 A.M.) from the time of MPH or saline administration until the lights were turned on. When saline was administered, both male (A) and female (B) mice in the PNE group showed significantly higher activity compared to their counterparts in the SAC group. MPH produced significant decreases in locomotor activity in both male and female mice in the PNE group such that the activity in the MPH–PNE group was not significantly different than that in the SAL–SAC group. MPH did not produce significant changes in locomotor activity in the SAC group of male or female mice. ANOVA and multiple comparison tests were used to test statistical significance of the experimental effects (female, n = 12 for each group; male, n = 8–11).

Figure 5.

A–C, Coronal sections of the forebrain from 42-d-old mice stained with cresyl violet. The boxed area in A shows the location of the cingulate cortex (CG). We analyzed the volume of the cingulate cortex using stereological methods in mice from the PNE and SAC exposure groups. In addition, the length (radial thickness) and width (height) of the cingulate cortex were analyzed in coronal sections representing its entire rostrocaudal extent. Representative micrographs are shown to illustrate reduced radial thickness (length) of the cingulate cortex in a mouse from the PNE group (C) compared to its counterpart from the SAC group (B). D, The decrease in length was statistically significant, whereas no statistically significant difference was found in the width between the PNE and SAC groups. *p < 0.05.