Stimulants such as methylphenidate (MPH) are the main pharmacological treatment for attention-deficit hyperactivity disorder (ADHD). These stimulants block the dopamine (DA) transporter, thereby increasing extracellular DA in the cortico-thalamic-striatal (CST) circuitry¹. While an increasing number of children are being prescribed MPH, the effects of MPH on human brain development are not well-characterized. As studies in juvenile animals exposed to MPH suggested long-lasting changes in DA function², characterizing those effects on human brain development is important. Therefore, the aim of this randomized controlled trial was to assess the effect of MPH treatment on the DA system in children and adult patients with ADHD using arterial spin labeling (ASL) pharmacological MRI (phMRI). PhMRI is a non-invasive imaging technique that can visualize alterations in the DA system in humans³ and has been shown to correlate with well with DA release and DA transporter availability⁴. In line with preclinical studies, we hypothesized that MPH treatment, when given to young children would, in contrast to adults, increase the phMRI response to a MPH challenge in CST areas. 50 boys (mean age=11.3y) and 49 men (mean age= 28.5y) were included in a double-blind randomized controlled trial and underwent an MRI session before and 17 weeks (including 1-week washout) after randomization to MPH treatment or placebo (1:1). Each MRI session, they underwent two ASL-phMRI scans to measure cerebral blood flow (CBF), one before and one 90 minutes after an oral MPH challenge (0.5 mg/kg) (Figure 1). MRI studies were performed on a 3T Philips scanner using an 8-channel receive-only head coil. PhMRI data were acquired with a pCASL sequence (TR/TE: 4000/14ms, resolution: 3x3x7 mm, 17 slices, labeling duration: 1650ms, delay: 1525ms, GE-EPI read-out, 75 control-label pairs, no background suppression). Data were analyzed with the Iris Pipeline⁵ (Figure 2). First, timeseries were motion-corrected with a group-wise method that uses a similarity metric based on principal component analysis⁶. Subsequently, control-label pairs were subtracted (Mdiff). Outlier rejection was performed for the Mdiff images, because in our ADHD dataset large motion influenced the ASL signal quality. Motion correction was performed on the remaining timepoints, and the resulting Mdiff images were averaged to obtain a perfusion-weighted image (ΔM). For each subject, probabilistic GM segmentations were registered to the ΔM images and CBF was quantified using a single-compartment model⁷. For further analysis, CBF maps were transformed to T1w space⁸. Then, regions of interest (ROIs) were defined using a multi-atlas approach using a majority voting algorithm to obtain a final ROI labeling^9,10. For the striatum, thalamus and anterior cingulate cortex (ACC), mean CBF values within GM were computed. Changes in CBF response to the MPH challenge from baseline to post-treatment (ΔiCBF) were statistically compared within groups using a paired t-test. Subsequently, treatment groups were compared using an independent t-test on ΔiCBF and an age-by-treatment interaction was tested using a two-way analysis of variance. Missing CBF values were replaced using nearest neighbor interpolation within age and treatment group.Paired t-tests demonstrated a significant increase (p=0.04) in ΔiCBF in the thalamus of children treated with MPH and a trend towards significant increases in the striatum and ACC (p=0.07 and p=0.06 respectively), suggesting increased DA reactivity. No effect was found in the placebo groups or adult group with MPH treatment (Figure 3). Furthermore, striatal ΔiCBF was significantly higher (p=0.03) in children treated with MPH when compared to the placebo condition, whereas no treatment effect was observed in adults. Finally, a two-way ANOVA showed a trend towards age-by-treatment interaction in the striatum (p=0.07).Our results suggest enduring effects of MPH treatment on the DA system after wash-out in children, but not adults with ADHD. This is in line with preclinical literature using phMRI showing long-lasting increases in regional CBV in juvenile-treated rats in several DA-rich brain regions². Epidemiological studies and clinical trials have associated chronic MPH treatment with lower substance abuse¹¹, but increased levels of anxiety and depression¹². Therefore, follow-up studies are needed to examine the association between these dopaminergic changes and behavioral consequences of long-term MPH exposure.