Mean activity during the dark period

Overall mean activity of minute-by-minute cumulative data for the 12 h dark period (7:00–19:00) assessed across the three timepoints: the last day of administration (Day 10), the first day of drug discontinuation (acute WD), and after 10 days of discontinuation (prolonged WD) (Fig. 2A). A 3-way (treatment x age x sex) ANOVA of mean activity during the 12 h dark period on Day 10 (left panel), revealed a significant main effect of treatment [F (2, 97) = 4.004, P = 0.021], age [F (1, 97) = 5.462, P = 0.022], sex [F (1, 97) = 8.043, P = 0.006], treatment x age [F (2, 97) = 11.79, P < 0.001], and age x sex [F (1, 97) = 7.397, P = 0.008]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of treatment [F (2, 103) = 3.631, P = 0.030], age [F (1, 103) = 4.983, P = 0.028], and an interaction [F (2, 103) = 10.69, P < 0.001]. Post-hoc comparisons show that adolescent rats treated with MPH 1 mg/kg displayed significantly more activity during the dark period compared to controls (P < 0.001) and MPH 2 mg/kg (P = 0.021) treated adolescent rats. Additionally, adult rats treated with MPH 2 mg/kg displayed more activity during the dark period compared to controls (P = 0.026) and MPH 1 mg/kg treated adults (P = 0.002). An additional follow-up 2-way (sex x age) ANOVA revealed a significant main effect of sex [F (1, 105) = 6.806, P = 0.010], age [F (1, 105) = 4.186, P = 0.043], and an interaction [F (1, 105) = 5.713, P = 0.019]. Post-hoc comparisons show that adult female rats displayed significantly more activity during the dark period compared to adolescent females (P = 0.013) and adult male rats (P = 0.004). A 3-way (treatment x age x sex) ANOVA of activity during the 12 h dark period on acute WD (middle panel), revealed a significant main effect of sex [F (1, 87) = 4.925, P = 0.029] and treatment x age [F (2, 87) = 6.464, P = 0.002]. A follow-up 2-way (treatment x age) ANOVA revealed no main effects, but a significant interaction [F (2, 93) = 6.029, P = 0.004]. Tukey’s post-hoc comparisons show that adolescent rats treated with MPH 1 mg/kg displayed significantly more activity during the dark period compared to controls (P = 0.004) and MPH 2 mg/kg (P = 0.004) treated adolescent rats. A 3-way (treatment x age x sex) ANOVA of activity during the 12 h dark period on prolonged WD (right panel), revealed a significant main effect of sex [F (1, 95) = 6.967, P = 0.010] and age x sex [F (1, 95) = 18.120, P < 0.001]. A follow-up 2-way (age x sex) ANOVA revealed a main effect of age [F(1,103) = 7.250, P = 0.008] and an interaction [F (1,103) = 17.220, P < 0.001]. Tukey’s post-hoc comparisons show that adult male rats were significantly less active during the dark period compared to adult females (P < 0.001) and male adolescent rats (P = 0.005).

Fig. 2

Overall mean activity of minute-by-minute cumulative data for the (A) 12 h dark period and (B) 12 h light period assessed on the last day of administration (Day 10, left panel), the first day of drug discontinuation (acute WD, middle panel), and after 10 days of discontinuation (prolonged WD, right panel). Male (blue bars) and female (grey bars), adolescent (plain bars) and adult (hatch bars) rats were treated with MPH (1, 2 mg/kg) or control and assessed at these three timepoints. Drug administration took place at 9:00 and 17:00 each day. Overall mean activity of minute-by-minute cumulative data was also taken on the last day of treatment during the (C) 3 h block after the morning administration (9:00–12:00) and (D) 3 h block after the evening administration (17:00–20:00). *vs. control, # vs. MPH 1 mg/kg, a vs. age-matched controls, b vs. age-matched MPH 1 mg/kg, p < 0.05

Mean activity during the light period

Overall mean activity for the 12 h light period (19:00–7:00) was assessed across the same three timepoints (Fig. 2B). A 3-way (treatment x age x sex) ANOVA of activity during the 12 h light period on Day 10 (left panel), revealed only a significant main effect of treatment [F (2, 94) = 4.471, P = 0.014]. There were no other main effects or interactions between treatment, age, and sex. Tukey’s post-hoc comparisons show that irrespective of age or sex, rats treated with MPH 2 mg/kg displayed significantly more activity during the light period compared to controls (P = 0.001). A 3-way (treatment x age x sex) ANOVA of activity during the 12 h light period on acute WD (middle panel), revealed no significant differences. A 3-way (treatment x age x sex) ANOVA of activity during the 12 h light period on prolonged WD (right panel), revealed a significant main effect of treatment [F (2,93) = 8.609, P < 0.001] and age [F (1,93) = 5.332, P = 0.023]. There was no significant main effect of sex or any interactions between variables. Adolescent rats were overall more active during the light period compared to adult rats. Additionally, Tukey’s post-hoc comparisons showed that rats treated with MPH 1 mg/kg displayed significantly less activity during the light period compared to controls (P = 0.043) and MPH 2 mg/kg (P < 0.001) treated rats.

Mean activity during the 3 h post administration

To determine whether MPH treatment increases initial locomotor activity after administration, data from 3 h post-administration during the morning (9:00–12:00) and evenings (17:00–20:00) was assessed for mean activity on Day 10. A 3-way (treatment x age x sex) ANOVA of the first 3 h of activity after morning administration on Day 10 (Fig. 2C), revealed a significant main effect of treatment [F(2,97) = 10.35, P < 0.001], age [F(1,97) = 23.090, P < 0.001], and age x sex interaction [F(1,97) = 4.926, P = 0.029]. Tukey’s post-hoc comparisons show that irrespective of age or sex, rats treated with MPH 2 mg/kg displayed significantly more activity after the morning injection on Day 10 compared to controls (P < 0.001) and MPH 1 mg/kg (P = 0.038) treated rats. A follow-up 2-way (age x sex) ANOVAs revealed a significant main effect of age [F (1, 105) = 18.23, P < 0.001] and an interaction [F(1,105) = 4.082, P = 0.046]. Post-hoc analysis found that adult female rats were more active after the morning administration compared to adolescent female rats (P < 0.001). A 3-way (treatment x age x sex) ANOVA of the first 3 h of activity after evening administration on Day 10 (Fig. 2D), revealed a significant main effect of treatment [F (2, 94) = 16.180, P < 0.001] and age [F (1, 94) = 6.240, P = 0.014]. Adult rats were also more active after the evening injection compared to adolescent rats. Tukey’s post-hoc comparisons showed that rats treated with MPH 1 mg/kg (P = 0.003) and MPH 2 mg/kg (P < 0.001) displayed significantly more activity after the evening injection on Day 10 compared to control rats.

Active episodes during the dark period

As nocturnal animals, rats tend to be more active when the lights are off (dark period). As shown in Fig. 3, active episodes were investigated during the 12 h dark period (7:00–19:00) on three separate days: Day 10 of administration (left panels), first day of withdrawal from treatment (Acute WD, middle panels), and after 10 days of withdrawal from treatment (Prolonged WD, right panels). All activity data from the dark period is also included in Table 1.

Fig. 3

Active episodes were assessed during the 12 h dark period, when the animals spend more time awake. Male (blue bars) and female (grey bars), adolescent (plain bars) and adult (hatch bars) rats were treated with MPH (1, 2 mg/kg) or control were assessed on the last day of administration (Day 10, left panel), the first day of drug discontinuation (acute WD, middle panel), and after 10 days of discontinuation (prolonged WD, right panel). For the active episodes, the variables assessed across the three timepoints included the (A) mean number or count of active episodes, (B) maximum duration of active episodes, (C) mean active episode duration, and (D) the active fragmentation ratio. * vs. control, # vs. MPH 1 mg/kg, a vs. age-matched controls, b vs. age-matched MPH 1 mg/kg group. p < 0.05

Table 1 Summary of results from data collected during the dark and light periods Number of active episodes

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean number (or count) of active episodes during the dark period for each of the three timepoints (Fig. 3A). For the number of active episodes that took place during the dark period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of age [F (1, 97) = 4.995, P = 0.028], sex [F (1, 97) = 7.566, P = 0.007], and age x treatment interaction [F (2, 97) = 3.575, P = 0.032]. There was no significant main effect of treatment or other interactions for the number of active episodes. Male rats displayed more active episodes compared to female rats. A follow-up 2-way (age x treatment) ANOVAs revealed a significant main effect of age [F (1, 103) = 4.663, P = 0.033] but not treatment. There was a significant interaction [F (2, 103) = 3.411, P = 0.037], but no significant post-hoc analyses. For the number of active episodes that took place during the dark period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 93) = 5.825, P = 0.004] and age [F (1, 93) = 9.060, P = 0.003]. There was no significant main effect of sex or any interactions between the three variables. A Tukey’s post-hoc analysis revealed that irrespective of sex or age, MPH 1 mg/kg treated rats displayed more active episode counts than controls (P = 0.003). Additionally, adult rats overall displayed more active episode counts compared to adolescent rats. For the number of active episodes that took place during the dark period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 96) = 4.643, P = 0.012], sex [F (1, 96) = 16.71 P < 0.001], and age x sex interaction [F (1, 96) = 5.573, P = 0.020]. There was no significant main effect of age or any other interactions for the number of active episodes. A Tukey’s post-hoc analysis revealed that irrespective of sex or age, MPH 2 mg/kg treated rats displayed more active episode counts than MPH 1 mg/kg treated rats (P = 0.008). A follow-up 2-way (age x sex) ANOVA revealed a significant main effect of age [F (1, 104) = 4.338, P = 0.040], sex [F (1, 104) = 15.44, P = 0.0002], and an interaction [F (1, 104) = 6.055, P = 0.0155]. Tukey post-hoc analysis revealed that adult female rats display significantly more active episode counts compared to adolescent female rats (P = 0.011) and adult male rats (P < 0.001).

Maximum active episode duration

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean maximum active episode duration during the dark period for each of the three timepoints (Fig. 3B). For the maximum active episode duration that took place during the dark period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 97) = 11.32, P < 0.0001], age [F (1, 97) = 4.306, P = 0.0406], sex [F (1, 97) = 5.998, P = 0.0161], and an interaction between age x treatment [F (2, 97) = 5.519, P = 0.0054] and age x sex [F (1, 97) = 9.049, P = 0.0033]. A follow-up 2-way (age x treatment) ANOVAs revealed a significant main effect of treatment [F (2, 103) = 9.906, P = 0.0001], but not age, and a significant interaction [F (2, 103) = 4.874, P = 0.0095] with adult rats treated with MPH 2 mg/kg displaying longer maximum active episodes compared to controls (P < 0.001) and MPH 1 mg/kg (P < 0.001) treated adults. An additional follow-up 2-way (age x sex) ANOVAs revealed a significant main effect of sex [F (1, 105) = 4.721, P = 0.032] and an interaction [F (1, 105) = 6.871, P = 0.010]. There was no main effect of age. Tukey post-hoc analysis revealed that adult female rats display significantly longer maximum active episodes compared to adolescent female rats (P = 0.013) and adult male rats (P = 0.006). For the maximum active episode duration that took place during the dark period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 94) = 4.578, P = 0.013] but no other significant main effects or interactions between age, sex, and treatment. Post-hoc comparisons revealed MPH 2 mg/kg treated rats display significantly longer maximum active episode durations on acute withdrawal compared to MPH 1 mg/kg treated rats. For the maximum active episode duration that took place during the dark period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of sex [F (1, 95) = 13.28, P = 0.0004] and an interaction between age x sex [F (1, 95) = 6.842, P = 0.0104] but no significant main effect of treatment, age, or an interaction between the three variables. A follow-up 2-way (age x sex) ANOVAs revealed a significant main effect of sex [F (1, 103) = 13.32, P < 0.001], and an interaction [F (1, 103) = 7.499, P = 0.007]. Post-hoc analysis revealed that adult female rats display significantly longer maximum active episodes compared to adult male rats (P < 0.001).

Mean active episode duration

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean active episode duration during the dark period for each of the three timepoints (Fig. 3C). For the mean active episode duration that took place during the dark period on Day 10, a 3-way (treatment x age x sex) ANOVA revealed no significant main effect of treatment but a significant main effect of age [F (1, 97) = 5.548, P = 0.0205], sex [F (1, 97) = 9.098, P = 0.0033], treatment x age [F (2, 97) = 6.314, P = 0.0026], and age x sex [F (1, 97) = 4.760, P = 0.0315]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of sex [F (1, 103) = 4.976, P = 0.028], and an interaction [F (2, 103) = 5.800, P = 0.004]. There was no significant main effect of treatment. Tukey post-hoc analysis revealed that adult rats treated with MPH 2 mg/kg displayed significantly longer mean active episode durations compared to adult rats treated with MPH 1 mg/kg (P = 0.002). Another follow-up 2-way (age x sex) ANOVA revealed a significant main effect of age [F (1, 105) = 190.1, P < 0.001], sex [F (1, 105) = 129.2, P < 0.001], and an interaction [F (1, 105) = 118.3, P < 0.001]. Post-hoc analysis revealed that adult female rats display significantly longer mean active episodes compared to adolescent female rats (P < 0.001) and adult male rats (P < 0.001). For the mean active episode duration that took place during the dark period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 93) = 3.758, P = 0.0270], age [F (1, 93) = 11.12, P = 0.001], and sex [F (1, 93) = 10.35, P = 0.0018]. There were no significant interactions across treatment, age, and sex. Post-hoc comparisons revealed that irrespective of sex or age, MPH 1 mg/kg treated rats displayed shorter mean active episode durations compared to controls during acute withdrawal (P = 0.028). Additionally, female rats had longer active episode durations compared to males, and adolescent rats were active for longer episodes compared to adults. For the mean active episode duration that took place during the dark period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of sex [F (1, 97) = 30.78, P < 0.0001] and age x sex interaction [F (1, 97) = 6.437, P = 0.0128]. There was no significant main effect of treatment, age or any other interactions. A follow-up 2-way (age x sex) ANOVA revealed a significant main effect of sex [F (1, 105) = 29.96, P < 0.001] and an interaction [F (1, 105) = 6.923, P = 0.010]. There was no main effect of age. Tukey post-hoc analysis revealed that adult female rats display significantly longer mean active episodes compared to adolescent female rats (P = 0.021) and adult male rats (P < 0.001).

Active episode fragmentation ratio

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean active fragmentation ratio during the dark period for each of the three timepoints (Fig. 2D). For the mean active fragmentation that took place during the dark period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of sex [F (1, 97) = 5.042, P = 0.027], and treatment x age [F (2, 97) = 5.298, P = 0.007]. There was no significant main effect of treatment, age or any other interactions between the three variables. Males display more fragmented activity compared to females. A follow-up 2-way (treatment x age) ANOVA revealed no significant main effects (age or treatment) but there was a significant interaction between the two [F (2, 103) = 5.220, P = 0.0069]. Post-hoc comparisons show that adult rats treated with MPH 2 mg/kg displayed significantly less fragmented activity compared to MPH 1 mg/kg treated adults on Day 10 (P = 0.014). For the mean active fragmentation ratio that took place during the dark period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 93) = 4.213, P = 0.018] and age [F (1, 93) = 8.674, P = 0.004]. There was no significant main effect of sex or any interaction between the three variables. Follow-up comparisons demonstrate that irrespective of age or sex, rats treated with MPH 1 mg/kg displayed more fragmented activity during acute withdrawal compared to controls (P = 0.013). Additionally, male rats overall displayed significantly more active fragmentation compared to females. For the mean active fragmentation ratio that took place during the dark period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of sex [F (1, 96) = 17.61, P < 0.001] and age x sex [F (1, 96) = 5.739, P = 0.0185]. There was no significant main effect of treatment, age, or any interaction between the three variables. A follow-up 2-way (age x sex) ANOVA revealed a significant main effect of age [F (1, 105) = 10.85, P = 0.001], sex [F (1, 105) = 44.97, P < 0.001], and an interaction [F (1, 105) = 23.16, P < 0.001]. Post-hoc analysis revealed that adult female rats display significantly more fragmented active episodes compared to adolescent female rats (P < 0.001) and adult male rats (P < 0.001).

Rest episodes during the light period

As nocturnal animals, rats tend to be less active when the lights are on (12 h light period). Rest episodes are when the animal is moving minimally, as defined in the methods. As shown in Fig. 4, rest episodes were investigated during the 12 h light period (19:00–7:00) on three separate days; Day 10 of administration (left panels), first day of withdrawal from treatment (Acute WD, middle panels), and after 10 days of withdrawal from treatment (Prolonged WD, right panels). All activity data from the light period is also included in Table 1.

Fig. 4

Rest episodes were assessed during the 12 h light period, when the animals spend more time asleep. Male (blue bars) and female (grey bars), adolescent (plain bars) and adult (hatch bars) rats were treated with MPH (1, 2 mg/kg) or control were assessed on the last day of administration (Day 10, left panel), the first day of drug discontinuation (acute WD, middle panel), and after 10 days of discontinuation (prolonged WD, right panel). For the rest episodes, the variables assessed across the three timepoints included the (A) mean number or count of rest episodes, (B) maximum duration of rest episodes, (C) mean rest episode duration, and (D) the rest fragmentation ratio. *vs. control, a vs. age-matched controls, b vs. age-matched MPH 1 mg/kg, c vs. sex-matched controls, d vs. sex-matched MPH 1 mg/kg. p < 0.05

Number of rest episodes

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean number (or count) of active episodes during the light period for each of the three timepoints (Fig. 4A). For the number of rest episodes that took place during the light period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of age [F (1, 93) = 4.379, P = 0.039], treatment x age [F (2, 93) = 4.824, P = 0.010], and treatment x sex [F (2, 93) = 5.305, P = 0.007]. There was no significant main effect of sex, treatment or any other interactions for the number of rest episodes. A follow-up 2-way (age x treatment) ANOVAs revealed a significant interaction [F (2, 99) = 4.422, P = 0.015] with adolescent rats treated with MPH 1 mg/kg displaying increased rest episodes count compared to age-matched controls (P = 0.001). There was no main effect of treatment or sex. Another follow-up 2-way (sex x treatment) ANOVAs revealed a significant interaction [F (2, 99) = 4.566, P = 0.013] with female rats treated with MPH 1 mg/kg displaying increased rest episodes count compared to sex-matched controls (P = 0.001) and MPH 2 mg/kg (P = 0.029). There was again no main effect of treatment or sex. For the number of rest episodes that took place during the light period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 91) = 6.529, P = 0.002], age [F (1, 91) = 4.365, P = 0.0395], sex [F (1, 91) = 5.435, P = 0.0219], and treatment x age [F (2, 91) = 8.113, P = 0.001]. Overall, female rats display more rest episodes than males. A follow-up 2-way (treatment x age) ANOVAs revealed a significant main effect of treatment [F (2, 97) = 6.100, P = 0.0032], age [F (1, 97) = 4.441, P = 0.0377], and interaction [F (2, 97) = 7.997, P = 0.001] with adolescent rats treated with MPH 1 mg/kg displaying increased rest episodes count compared to age-matched controls (P < 0.001) and MPH 2 mg/kg treated rats (P = 0.001). For the number of rest episodes that took place during the light period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 95) = 10.74, P < 0.0001], age [F (1, 95) = 12.54, P = 0.0006], and sex [F (1, 95) = 7.889, P = 0.0060]. There were no significant interactions between treatment, age, and sex. Post-hoc comparisons show that irrespective of age and sex, MPH 1 mg/kg (P < 0.001) and MPH 2 mg/kg (P = 0.039) treated rats displayed significantly more rest episodes during the light period compared to controls. Additionally, female rats overall have more rest episodes than males, and adolescent rats overall have more episodes than adult rats.

Maximum rest episode duration

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean maximum rest episode duration during the light period for each of the three timepoints (Fig. 4B). For the maximum rest episode duration that took place during the light period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 93) = 4.578, P = 0.013] and treatment x age [F (2, 93) = 4.526, P = 0.013]. There was no significant main effect of age, sex or any other interaction between the three variables. A follow-up 2-way (age x treatment) ANOVAs revealed a significant main effect of treatment F (2, 99) = 4.553, P = 0.013], but no significant main effect of sex. There was a significant interaction [F (2, 99) = 3.989, P = 0.022] with adolescent rats treated with MPH 1 mg/kg (P = 0.004) and MPH 2 mg/kg (P = 0.020) displaying shorter maximum rest episodes compared to age-matched controls. For the maximum rest episode duration that took place during the light period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 88) = 3.755, P = 0.027], and age [F (1, 88) = 7.310, P = 0.008], and age x sex [F (1, 88) = 11.59, P = 0.0010]. Post-hoc comparisons show that irrespective of age or sex, MPH 2 mg/kg rats display significantly shorter rest episodes compared to controls (P = 0.021). A follow-up 2-way (age x sex) ANOVA revealed a significant main effect of age [F (1, 96) = 6.906, P = 0.010] and an interaction [F (1, 96) = 9.701, P = 0.002]. Post-hoc analysis revealed that adult male rats display significantly longer rest episodes compared to adolescent male rats (P = 0.001). For the maximum rest episode duration that took place during the light period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed no significant main effects or interactions.

Mean rest episode duration

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean rest episode duration during the light period for each of the three timepoints (Fig. 4C). For the mean rest episode duration that took place during the light period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed no significant main effect of treatment or sex. However, there was a significant main effect of age [F (1, 93) = 13.79, P = 0.0003] and treatment x sex [F (2, 93) = 5.580, P = 0.005]. Adolescent rats displayed shorter rest durations compared to adult rats. A follow-up 2-way (treatment x sex) ANOVA revealed a significant interaction [F (2, 99) = 4.087, P = 0.020] with female rats treated with MPH 1 mg/kg display shorter mean rest episode durations compared to sex-matched controls (P = 0.011). There was no significant main effect of sex or treatment. For the mean rest episode duration that took place during the light period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed again no significant main effect of treatment or sex. However, there was a significant main effect of age [F (1, 90) = 15.49, P < 0.001] and treatment x age [F (2, 90) = 3.361, P = 0.039]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of age [F (1, 96) = 16.32, P < 0.001] and an interaction [F (2, 96) = 3.506, P = 0.034]. Tukey’s post-hoc analysis revealed that adolescent MPH 1 mg/kg treated rats display significantly shorter rest episode durations compared to age-matched controls (P = 0.023). For the mean rest episode duration that took place during the light period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 95) = 7.268, P = 0.001], age [F (1, 95) = 17.64, P < 0.0001], and sex [F (1, 95) = 7.764, P = 0.006]. There were no significant interactions between the variables. Post-hoc comparisons show that irrespective of age and sex, MPH 1 mg/kg (P = 0.033) and MPH 2 mg/kg (P = 0.001) treated rats had shorter mean rest durations compared to controls. Additionally, adult rats overall had shorter rest durations compared to adolescents, and male rats overall had shorter rest durations in comparison to female rats.

Rest episode fragmentation ratio

Separate three-way (age x sex x treatment) ANOVAs were conducted on the mean rest fragmentation ratio during the light period for each of the three timepoints (Fig. 4D). For the mean rest fragmentation that took place during the light period on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 92) = 3.269, P = 0.043], age [F (1, 92) = 8.720, P = 0.004] and treatment x sex [F (2, 92) = 5.441, P = 0.006]. There was no significant main effect of sex or any other interactions. Irrespective of sex or treatment, adolescent rats displayed more fragmented rest compared to adult rats. A follow-up 2-way (treatment x sex) ANOVA revealed a significant main effect of treatment [F (2, 98) = 4.268, P = 0.017] and an interaction [F (2, 98) = 4.892, P = 0.009]. There was no significant main effect of sex. Tukey post-hoc comparisons show that female rats treated with MPH 1 mg/kg displayed significantly more fragmented rest compared to controls (P < 0.001) and MPH 2 mg/kg (P = 0.021) treated female rats. For the mean rest fragmentation that took place during the light period on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 90) = 5.756, P = 0.004], age [F (1, 90) = 12.92, P = 0.001], and treatment x age [F (2, 90) = 6.426, P = 0.003]. There is no significant main effect of sex or any other interactions. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of treatment [F (2, 96) = 5.800, P = 0.004], age [F (1, 96) = 12.57, P = 0.001], and an interaction [F (2, 96) = 6.005, P = 0.004]. Post-hoc comparisons show that adolescent rats treated with MPH 1 mg/kg displayed significantly more fragmented rest compared to controls (P < 0.001) and MPH 2 mg/kg (P = 0.004) treated rats. For the mean rest fragmentation that took place during the light period on prolonged WD (right panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 94) = 9.666, P < 0.001], age [F (1, 94) = 19.59, P < 0.001], and sex [F (1, 94) = 8.566, P = 0.004]. There were no significant interactions between these variables. Post-hoc comparisons show that irrespective of age and sex, MPH 1 mg/kg (P < 0.001) and MPH 2 mg/kg (P = 0.002) treated rats show significantly more fragmented rest compared to controls. Additionally, adult rats overall had lower rest fragmentation ratio compared to adolescents, and male rats overall had higher rest fragmentation in comparison to female rats.

Circadian Measures

As depicted in Fig. 5, rats were assessed for changes in circadian measures after 10 days of MPH treatment (Day 10), during the first day of discontinuation from treatment (acute WD) and then 10 days of discontinuation (prolonged WD).

Fig. 5

Circadian measures were assessed in male (blue bars) and female (grey bars), adolescent (plain bars) and adult (hatch bars) rats treated with MPH (1, 2 mg/kg) or control on the last day of administration (Day 10, left panel), during 24 h acute withdrawal (middle panel), and after 10 days of prolonged withdrawal (right panel). Circadian measures included (A) MESOR, (B) amplitude, and (C) acrophase. *vs. control, a vs. age-matched controls, b vs. age-matched MPH 1 mg/kg, c vs. sex-matched controls, p < 0.05

MESOR

Separate three-way (age x sex x treatment) ANOVAs were conducted on mesor (rhythm-adjusted mean) during 24 h period for each of the three timepoints (Fig. 5A). For the 24 h mesor taken on Day 10 (left panel), a 3-way (age x sex x treatment) ANOVA revealed a significant main effect of treatment [F (2, 91) = 6.039, P = 0.003], sex [F (1, 91) = 5.734, P = 0.019], treatment x age [F (2, 91) = 5.387, P = 0.006] and age x sex [F (1, 91) = 5.872, P = 0.017]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of treatment [F (2, 97) = 5.760, P = 0.004] and an interaction [F (2, 97) = 4.632, P = 0.012]. There was no significant main effect of age. Tukey’s post-hoc comparisons revealed adolescent MPH 1 mg/kg treated rats had significantly higher mesor compared to age-matched controls (P = 0.010) and adult MPH 2 mg/kg treated had significantly higher mesor compared to age-matched controls (P = 0.003) and MPH 1 mg/kg treated rats (P = 0.035). For 24 h mesor on acute WD (middle panel), a 3-way (age x sex x treatment) ANOVA revealed a significant main effect of sex [F (1, 92) = 7.660, P = 0.007] and treatment x age [F (2, 92) = 3.840, P = 0.025]. There was no significant main effect of treatment, age or any other interactions between variables. A follow-up 2-way (treatment x age) ANOVA revealed no main effect of treatment or age, but a significant interaction [F (2, 98) = 3.121, P = 0.049]. Tukey’s post-hoc comparison show that adolescent rats treated with MPH 1 mg/kg display higher mesor compared to age-matched controls (P = 0.020). For 24 h mesor on prolonged WD (right panel), a 3-way (age x sex x treatment) ANOVA revealed a significant main effect of age [F (1, 94) = 4.732, P = 0.032], sex [F (1, 94) = 8.522, P = 0.004], treatment x age [F (2, 94) = 4.409, P = 0.015], and age x sex [F (1, 94) = 8.896, P = 0.004]. There was no significant main effect of treatment or any other interactions between variables. A follow-up 2-way (treatment x age) ANOVA revealed no main effect of treatment or age. There was a significant interaction F (2, 100) = 3.388, P = 0.038], however, there were no significant post-hoc results. An additional follow-up 2-way (sex x age) ANOVA revealed a main effect of sex [F (1,102) = 7.449, P = 0.008] and an interaction [F (1,102) = 8.369, P = 0.005]. Tukey’s post-hoc analysis shows adult male rats had a significant lower mesor compared to adult female (P = 0.001) and adolescent male rats (P = 0.006).

Amplitude

Separate three-way (age x sex x treatment) ANOVAs were conducted on amplitude during 24 h period for each of the three timepoints (Fig. 4B). For the 24 h amplitude on Day 10 (left panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of treatment [F (2, 91) = 5.875, P = 0.004], age [F (1, 91) = 35.87, P < 0.001], sex [F (1, 91) = 4.944, P = 0.029], and treatment x age [F (2, 91) = 3.492, P = 0.035]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect of treatment [F (2, 97) = 5.736, P = 0.0044], age [F (1, 97) = 34.95, P < 0.001] and an interaction [F (2, 97) = 3.262, P = 0.043]. Tukey’s post-hoc shows adolescent MPH 1 mg/kg rats have higher amplitude compared to adolescent controls (P = 0.030), and MPH 2 mg/kg treated adults have higher amplitude in comparison to adult controls (P = 0.003). For the 24 h amplitude on acute WD (middle panel), a 3-way (treatment x age x sex) ANOVA revealed a significant main effect of age [F (1, 92) = 19.35, P < 0.001], sex F (1, 92) = 7.388, P = 0.008], and treatment x age [F (2, 92) = 4.931, P = 0.009]. A follow-up 2-way (treatment x age) ANOVA revealed a significant main effect age [F (1, 98) = 19.92, P < 0.001] and an interaction [F (2, 98) = 4.633, P = 0.012]. Tukey’s post-hoc shows adolescent MPH 1 mg/kg rats have higher amplitude compared to adolescent MPH 2 mg/kg treated rats (P = 0.016). For the 24 h amplitude on prolonged WD (right panel), a 3-way (age x sex x treatment) ANOVA revealed a significant main effect of treatment [F (2, 94) = 5.794, P = 0.004], age [F (1, 94) = 4.168, P = 0.044], sex F (1, 94) = 19.86 P < 0.001], treatment x age [F (2, 94) = 3.297, P = 0.041], age x sex [F (1, 94) = 8.179, P = 0.005], and treatment x age x sex [F (2, 94) = 4.365, P = 0.015]. Tukey’s post-hoc analysis for treatment x age x sex interaction shows that adult female control rats had higher amplitude compared to adolescent female controls (P < 0.001) and adult male controls (P < 0.001). Additionally, adolescent female rats treated with MPH 1 mg/kg had significantly higher amplitude compared to adolescent female control rats (P = 0.020).

Acrophase

Separate three-way (age x sex x treatment) ANOVAs were conducted on acrophase during 24 h period for each of the three timepoints (Fig. 5C). For the 24 h acrophase on Day 10 (left panel), a 3-way (age x sex x treatment) ANOVA revealed no main effect and only a significant interaction between treatment x sex [F (2, 91) = 3.320, P = 0.041]. A follow-up 2-way (treatment x sex) ANOVA revealed no main effect of treatment or sex, but there was a significant interaction between the two [F (2, 97) = 3.114, P = 0.049]. Post-hoc analysis revealed that male rats treated with MPH 1 mg/kg (P = 0.014) and MPH 2 mg/kg (P = 0.008) had significantly delayed acrophase compared to sex-matched controls. For acrophase on acute WD (middle panel), a 3-way (age x sex x treatment) ANOVA revealed a significant main effect of treatment [F (2, 92) = 3.286, P = 0.042] and sex [F (1, 92) = 7.047, P = 0.009]. There was no significant main effect of age or any interactions. Female rats had significantly longer acrophase compared to males. Post-hoc analysis also shows that irrespective of sex or age, MPH 2 mg/kg treated rats have a delayed acrophase compared to controls (P = 0.049) after acute withdrawal. For acrophase on prolonged WD (right panel), a 3-way (age x sex x treatment) ANOVA revealed no significant main effects, with only one interaction between age x sex [F (1, 94) = 5.156, P = 0.025]. A follow-up 2-way (sex x age) ANOVA revealed no main effect of treatment or age. There was a significant interaction F (1, 102) = 5.202, P = 0.025], however, there were no significant post-hoc results.