T’s t-test (SigmaPlot 11.0, Systat Software Inc, Chicago, USA). Significance was set at P,0.05.Transcranial ultrasoundThe maximum subjective rating of the bone window was calculated for each subject and the average was 1.660.8 (i.e. good to excellent; median = 1 excellent). The diameter of the 3rd ventricle was normal in all subjects (maximum diameter: 4.94 mm) and the average diameter (right, left) did not significantly differ between groups (control: 1.5160.08 mm, stimulant: 1.4460.07 mm; cannabis: 1.0460.03 mm). Figure 1A shows single subject images of the area of substantia nigra echogenicity in 1 control subject, 1 cannabis subject, and 1 stimulant subject. For a given side (right), the average area of substantia nigra echogenicity was 0.16360.044 cm2 for operator 1 and 0.16660.051 cm2 for operator 2. The area of substantia nigra echogenicity exhibited acceptable inter-rater reliability (Cronbach’s alpha = 0.720; Spearman rank order correlation: r = 0.591, P = 0.005) with moderate to strong reproducibility (intraclass correlation coefficient; single measures = 0.577; average measures = 0.732). There was no significant difference between measurements obtained on machine 1 and 2 in the control group. Single subject data suggested that the area of substantia nigra echogenicity was greater in stimulant subjects than in control and cannabis subjects. Figure 2 shows group data for the area of substantia nigra echogenicity. In the control group, the average area of substantia nigra echogenicity was 0.18160.055 cm2 on the right sideResults Subject characteristicsTwo subjects were excluded due to insufficient bone window for transcranial sonography (1 control and 1 stimulant user). The characteristics of the remaining 77 subjects are presented in Table 1. There was a significant difference between the groups regarding age (F2,74 = 8.007, P,0.001) but not weight or height. The average age of subjects in the stimulant group was ,6.5 yrs older than subjects in the control (P = 0.001) and cannabis groups (P = 0.009). There was also a significant main effect of group on years of education (F2,74 = 3.268, P = 0.044) and a trend for a main effect of group on symptoms of MedChemExpress 478-01-3 depression (i.e. BDI-II score; F2,73 = 2.743, P = 0.071). Subjects in the stimulant group had undertaken ,1 less year of education compared to the control group (P = 0.041) and subjects in the stimulant and cannabis groups tended to have more symptoms of depression. Seven subjects in the stimulant group and 3 subjects in the cannabis group had received a formal diagnosis of depression (4 wereStimulant Drugs and Substantia Nigra MorphologyTable 1. Subject characteristics for the control, stimulant, and cannabis groups.Control (n = 29) Age (yrs) 15900046 Gender Weight (kg) Height (cm) Handedness Education (yrs) BDI-II score Depression diagnosis Head injuries Drug overdose Lifetime alcohol (total drinks) Lifetime tobacco (total cigarettes) 2465 11 M, 18 F 69622 161637 23 right, 6 left 1662 666 0 0 0 4796620Stimulant (n = 36) 3169 *Cannabis (n = 12) 2567 6 M, 6 F 75621 170611 10 right, 2 left 1562 12610 2 1 0 2,24462,159 * 11,BTZ043 site 011623,314 *21 M, 15 F 74616 165641 32 right, 4 left 1563 * 1068 7 10 4 12,384615,661 *1 64,0966101,413 *Data are mean6standard deviation. *Significantly different from control group (P,0.05). 1 Significant difference between stimulant group and cannabis group (P,0.05). doi:10.1371/journal.pone.0056438.t(median: 0.175) and 0.17660.057 cm2 on the left side (median: 0.180).T’s t-test (SigmaPlot 11.0, Systat Software Inc, Chicago, USA). Significance was set at P,0.05.Transcranial ultrasoundThe maximum subjective rating of the bone window was calculated for each subject and the average was 1.660.8 (i.e. good to excellent; median = 1 excellent). The diameter of the 3rd ventricle was normal in all subjects (maximum diameter: 4.94 mm) and the average diameter (right, left) did not significantly differ between groups (control: 1.5160.08 mm, stimulant: 1.4460.07 mm; cannabis: 1.0460.03 mm). Figure 1A shows single subject images of the area of substantia nigra echogenicity in 1 control subject, 1 cannabis subject, and 1 stimulant subject. For a given side (right), the average area of substantia nigra echogenicity was 0.16360.044 cm2 for operator 1 and 0.16660.051 cm2 for operator 2. The area of substantia nigra echogenicity exhibited acceptable inter-rater reliability (Cronbach’s alpha = 0.720; Spearman rank order correlation: r = 0.591, P = 0.005) with moderate to strong reproducibility (intraclass correlation coefficient; single measures = 0.577; average measures = 0.732). There was no significant difference between measurements obtained on machine 1 and 2 in the control group. Single subject data suggested that the area of substantia nigra echogenicity was greater in stimulant subjects than in control and cannabis subjects. Figure 2 shows group data for the area of substantia nigra echogenicity. In the control group, the average area of substantia nigra echogenicity was 0.18160.055 cm2 on the right sideResults Subject characteristicsTwo subjects were excluded due to insufficient bone window for transcranial sonography (1 control and 1 stimulant user). The characteristics of the remaining 77 subjects are presented in Table 1. There was a significant difference between the groups regarding age (F2,74 = 8.007, P,0.001) but not weight or height. The average age of subjects in the stimulant group was ,6.5 yrs older than subjects in the control (P = 0.001) and cannabis groups (P = 0.009). There was also a significant main effect of group on years of education (F2,74 = 3.268, P = 0.044) and a trend for a main effect of group on symptoms of depression (i.e. BDI-II score; F2,73 = 2.743, P = 0.071). Subjects in the stimulant group had undertaken ,1 less year of education compared to the control group (P = 0.041) and subjects in the stimulant and cannabis groups tended to have more symptoms of depression. Seven subjects in the stimulant group and 3 subjects in the cannabis group had received a formal diagnosis of depression (4 wereStimulant Drugs and Substantia Nigra MorphologyTable 1. Subject characteristics for the control, stimulant, and cannabis groups.Control (n = 29) Age (yrs) 15900046 Gender Weight (kg) Height (cm) Handedness Education (yrs) BDI-II score Depression diagnosis Head injuries Drug overdose Lifetime alcohol (total drinks) Lifetime tobacco (total cigarettes) 2465 11 M, 18 F 69622 161637 23 right, 6 left 1662 666 0 0 0 4796620Stimulant (n = 36) 3169 *Cannabis (n = 12) 2567 6 M, 6 F 75621 170611 10 right, 2 left 1562 12610 2 1 0 2,24462,159 * 11,011623,314 *21 M, 15 F 74616 165641 32 right, 4 left 1563 * 1068 7 10 4 12,384615,661 *1 64,0966101,413 *Data are mean6standard deviation. *Significantly different from control group (P,0.05). 1 Significant difference between stimulant group and cannabis group (P,0.05). doi:10.1371/journal.pone.0056438.t(median: 0.175) and 0.17660.057 cm2 on the left side (median: 0.180).