Participants

40 participants (18–25 years old, Mean=21.33, SD=1.99; men/women=15/25; 23 Asian, 7 Hispanic or Latino, 6 White (not Hispanic or Latino), 2 Black or African-American, 2 more than one race) underwent PET and MRI scanning. Power analyses determined that this sample size is sufficient to detect relationships of r=0.32 with achieved power of 0.80. Power analyses were conducted with G*Power 3.1.7 (Faul et al, 2007). The Institutional Review Boards at the University of California, Berkeley and Lawrence Berkeley National Laboratory approved the study. All participants provided written consent and received monetary compensation for participating in the study.

Participants were recruited as part of a larger ongoing study of dopaminergic mechanisms of cognitive control, which included three fMRI sessions, and self-report questionnaires. Analysis of fMRI results, and self-report measures is ongoing, and is not presented in the current report. Prior to enrollment, participants underwent medical screening and physical examination by a medical doctor or nurse practitioner. Participants did not have a history of neurological, psychological, or psychiatric disorder. Four participants reported having seen a psychiatrist or psychologist to treat school or family stress that was resolved at the time of enrollment, and did not require pharmacological treatment. Participants reported no symptoms of depression, anxiety, paranoia or hallucinations, homicidal thoughts or acts, violent or threatening behavior, suicidal thoughts or acts, or suicide attempts. Self-report measures were collected through paper and pencil questionnaires.

Exclusion criteria included consumption of more than 7 alcoholic drinks per week, and use of psychoactive drugs within 2 weeks of enrollment or 10 times in the past year. We assessed drug history using a written screening form. Participants indicated their drug use history for the following list of specific drugs as well as broader drug categories: cocaine, stimulants (other than caffeine), amphetamines, hallucinogens, ‘ecstasy’, opiates, sedatives, pain or sleeping pills, and marijuana. In addition, we tested drug and alcohol use via urine drug screening and alcohol breath test prior to enrollment. No participant tested positive for any psychoactive drug, and alcohol breath test confirmed alcohol concentration below 0.05%. Prior to the PET and MRI sessions, participants underwent additional screening for self-reported drug use including screening for methylphenidate, dexmethylphenidate, dextroamphetamine, lisdexamfetamine, amphetamine and methamphetamine. Reported medications were limited to birth control, antibiotics, asthma and allergy medication, and non-prescription pain relievers. Participants did not use nicotine with the exception of two participants who reported smoking 1–2 cigarettes per week. Exclusion of these two participants does not change the significance of our analyses (data not shown).

Structural MRI Scan

Images were acquired using a Siemens 3 T Trio Tim scanner with a 12-channel coil. Each participant was scanned 3 times using a high-resolution T1-weighted magnetization prepared rapid gradient echo (MPRAGE) whole brain scan (TR=2,300 ms; TE=2.98 ms; FA=9°; matrix=240 × 256; FOV=256; sagittal plane; voxel size=1 × 1 × 1 mm; 160 slices). MPRAGE scans were aligned, averaged and segmented using FreeSurfer version 5.1 (http://surfer.nmr.mgh.harvard.edu/) and were used for coregistration with the PET data. The 3 MPRAGE scans were averaged to minimize the effect of head motion on the quality of image segmentation.

[18F]FMT PET Data Acquisition

Participants underwent an [18F]FMT PET scan to measure dopamine synthesis capacity. [18F]FMT is similar to DOPA ligands as both tracers are substrates for aromatic amino acid decarboxylase, an enzyme in the dopamine synthesis pathway. Though not the rate-limiting step, its activity provides an estimate of dopamine synthesis capacity when provided with enough substrate (DeJesus, 2003). [18F]FMT does not undergo post-release processing as DOPA ligands do, but is instead trapped in the presynaptic terminal after its conversion to 6-[18F]fluorohydroxyphenylacetic acid (Jordan et al, 1997). Furthermore, it is not subject to methylation by catechol-O-methyltransferase as DOPA ligands are, with the consequence that radiolabeled metabolites do not enter the brain. Both of these factors result in improved signal to noise ratio in [18F]FMT images compared to DOPA ligands.

[18F]FMT was synthesized at Lawrence Berkeley National Laboratory using methods previously described (VanBrocklin et al, 2004). Participants ingested 2.5 mg/kg of carbidopa ~1 h before scanning to minimize the peripheral decarboxylation of [18F]FMT (Boyes et al, 1986; Firnau et al, 1988; Hoffman et al, 1992; Melega et al, 1990). All PET data were acquired using a Siemens Biograph Truepoint 6 PET/CT scanner (Siemens Medical Systems, Erlangen, Germany). After a short CT scan, participants were injected with approximately 2.5 mCi of [18F]FMT as a bolus in an antecubital vein (M±SD; specific activity=947.30±140.26 mCi/mmol; dose=2.43±0.06 mCi). Dynamic acquisition frames were obtained over 90 min in 3D mode (25 frames total: 5 × 1, 3 × 2, 3 × 3, 14 × 5 min). Data were reconstructed using an ordered subset expectation maximization algorithm with weighted attenuation, corrected for scatter, and smoothed with a 4 mm full width at half maximum (FWHM) kernel.

[11C]raclopride PET Data Acquisition

Participants received two [11C]raclopride PET scans an average of 21.65 days before or after the [18F]FMT scan (median=7 days) to measure D2/3 receptor occupancy and dopamine release. [11C]raclopride is a D2/3 receptor antagonist with relatively low affinity (Kd=1.2 nM) that competes with endogenous dopamine (Kohler et al, 1985; Seeman et al, 1989). [11C]raclopride was synthesized at Lawrence Berkeley National Laboratory using methods previously described (Volkow et al, 1993). To measure baseline D2/3 receptor occupancy, participants ingested a placebo pill approximately 1 h before [11C]raclopride scan 1. The placebo scan was always performed first. To measure dopamine release, participants ingested 30 mg (M±SD mg/kg: 0.46±0.08) of methylphenidate ~1 h before [11C]raclopride scan 2. Endogenous dopamine release was measured as the percent change in BP ND from [11C]raclopride scan 1 to [11C]raclopride scan 2 ((placebo [11C]raclopride−methylphenidate [11C]raclopride)/placebo [11C]raclopride). Scans were conducted on the same day, 2 h apart and participants were blind to whether placebo or methylphenidate was administered. The 30 mg pill provides a smaller dose than the 60 mg pill used in previous studies (Broft et al, 2012; Clatworthy et al, 2009; Martinez et al, 2011; Martinez et al, 2012; Volkow et al, 2001; Volkow et al, 2002). The fixed mg amount used here and by others has the disadvantage of not accounting for individual differences in body weight. Our pilot testing determined the 30 mg pill produced a percent reduction in [11C]raclopride signal within the range of signal reduction in [11C]raclopride BP ND associated with cognitive task performance: 5.3–10.2% (Jonasson et al, 2014; Monchi et al, 2006). For both [11C]raclopride scan 1 and [11C]raclopride scan 2, after a short CT scan, participants were injected with approximately 10 mCi of [11C]raclopride as a bolus in an antecubital vein. Mean specific activity and dose were not significantly different for [11C]raclopride scan 1 (M±SD; specific activity=5280.45±1359.41 Ci/mmol, dose=9.83±0.07 mCi) and [11C]raclopride scan 2 (specific activity=5092.98±1533.82 Ci/mmol, dose=9.83±0.09 mCi) as assessed by paired t-tests (specific activity t(39)=1.08, p=0.29, dz=0.17; dose t(39)=0.27, p=0.79, dz=0.00). Dynamic acquisition frames were obtained over 60 min in 3D mode (19 frames total: 5 × 1, 3 × 2, 3 × 3, 8 × 5). Reconstruction was performed as described above.

PET Data Analysis

PET data were preprocessed using SPM8 software (Friston et al, 2007). To correct for motion between frames, images were realigned to the middle frame. The first five images were summed prior to realignment to improve realignment accuracy, as these early images have relatively low signal contrast. Structural images were coregistered to PET images using the mean image of frames corresponding to the first 20 min of acquisition as a target. The mean image for the first 20 min was used rather than the mean image for the whole scan time because it provides a greater range in image contrast outside of striatum thus making it a better target for coregistration.

For [18F]FMT PET, graphical analysis for irreversible tracer binding was performed using Patlak plotting (Patlak and Blasberg, 1985; Sossi et al, 2003) implemented using in-house software and Matlab version 8.2 (The MathWorks, Natick, MA). Without measurement of the arterial input function, both [18F]FMT and [11C]raclopride PET analysis used reference region models. Such analyses rely on the existence of a tissue region with few specific binding sites (Blomqvist et al, 1989; Cunningham et al, 1991). Cerebellar gray matter was used as the reference region because this region shows very little tracer uptake, and has an extremely low density of dopamine receptors and metabolites relative to striatum (Camps et al, 1989; Farde et al, 1986; Hall et al, 1994; Levey et al, 1993). The most anterior ¼ of cerebellar gray was removed from the reference region to limit contamination of signal from the substantia nigra and ventral tegmental area. Exclusion of the anterior portion of the cerebellar gray has been reported previously (Aarts et al, 2014; Berry et al, 2016; Braskie et al, 2011; Braskie et al, 2008; Dang et al, 2017; Dang et al, 2012a; Dang et al, 2012b, 2013; Dang et al, 2016; Klostermann et al, 2012; Smith et al, 2016; Wallace et al, 2014), and was performed by manually removing the anterior ¼ of coronal slices from individual participants’ native space cerebellar gray FreeSurfer segmentation using Mango software (http://ric.uthscsa.edu/mango/). K i images were generated from PET frames corresponding to 25 to 90 min (Ito et al, 2006; Ito et al, 2007), which represent the amount of tracer accumulated in the brain relative to the reference region. K i can be expressed as K i =k 2 k 3 /(k 2 +k 3 ), where k 2 is the rate constant for the return of free [18F]FMT from brain back to plasma and k 3 is the rate constant for the trapping of brain [18F]FMT by aromatic amino acid decarboxylase. These images are comparable to K i images obtained using a blood input function but are scaled to the volume of tracer distribution in the reference region (Figure 1a).

Figure 1 Within-subject measures of dopamine synthesis capacity and D2/3 receptor binding. (a) [18F]FMT K i signal reflecting dopamine synthesis capacity was measured throughout striatum. The axial slice illustrates the extent of the striatal K i signal for a representative subject overlaid on the subject’s native space T1 MPRAGE. (b) [11C]raclopride BP ND displayed for placebo (baseline) scan as well as post-methylphenidate scan for the same representative subject. Methylphenidate administration reduced [11C]raclopride BP ND. (c) Striatal regions showing significantly reduced [11C]raclopride BP ND following methylphenidate administration across all participants. The t-map for the paired t-test comparing baseline and post-methylphenidate [11C]raclopride BP ND is displayed on the normalized mean T1 MPRAGE for all subjects. PowerPoint slide Full size image

For [11C]raclopride PET, reversible tracer binding was quantified using simplified reference tissue model analysis (SRTM; Lammertsma and Hume, 1996). Specifically, a basis function version of the SRTM was applied as previously described (Gunn et al, 1997) with posterior cerebellar gray matter used as the reference region. Using this method, the time-activity curve of the brain region of interest is described relative to the reference region. This analysis assumes the reference region has no specific binding and that both regions have the same level of nondisplaceable binding (Gunn et al, 1997; Lammertsma and Hume, 1996; Salinas et al, 2015). The SRTM analysis was performed using in-house software provided by Dr Roger Gunn and Matlab version 8.2. SRTM analysis was used to determine BP ND , which can be defined as:

BP ND = f ND × B avail /K D

where B avail is the concentration of D2/3 receptors, K D is the inverse of the affinity of the radiotracer for D2/3 receptors, and f ND is the free fraction of the ligand in the nondisplaceable tissue compartment (Innis et al, 2007; Slifstein and Laruelle, 2001). A BP ND voxel-wise map was generated for each participant (Figures 1b and c).

The use of BP ND relies on the assumption that nondispaceable binding is independent of treatment effects. Methylphenidate administration has been shown not to alter cerebellar [11C]raclopride signal following 60 mg oral administration (Volkow et al, 2001; Volkow et al, 2002). It is possible that intravenous methylphenidate administration reduces cerebellar distribution volume (Volkow et al, 2014), though these results are not consistent (Volkow et al, 1999). Without measurement of the arterial input function, we could not directly test the effect of 30 mg oral administration cerebellar BP. We did, however, confirm that the cerebellar region of interest (ROI) did not show significant changes in BP ND between [11C]raclopride scans 1 and 2 when using occipital cortex as the reference region (t(39)=0.70, p=0.49, dz=0.11). Occipital cortex also did not show significant changes in BP ND between [11C]raclopride scans 1 and 2 when posterior cerebellar gray was used as the reference region (t(39)=0.47, p=0.64, dz=0.07).

Regions of Interest

An ROI approach was used to test relationships between [18F]FMT K i , baseline [11C]raclopride BP ND , and percent change in [11C]raclopride BP ND (dopamine release). ROI analyses were conducted in two ways. First, a single striatal ROI mask (henceforth referred to as ‘whole striatum’) was generated from group level voxel-wise analyses of K i and BP ND maps. K i and BP ND maps were spatially normalized to the TPM.nii template in MNI space, and smoothed with a 4 mm FWHM kernel in SPM 12. Two one-sample t-tests were performed to define significant voxels for [18F]FMT K i and baseline [11C]raclopride BP ND . Paired t-test determined voxels for which methylphenidate significantly reduced BP ND . An initial cluster forming threshold of p<0.001 was applied. An additional minimum cluster extent threshold (k=55, p <0.05) was applied using 3dClustSim in AFNI (https://afni.nimh.nih.gov/). The whole striatum mask was comprised of the intersection of voxels (7097 mm3) surviving group level testing for [18F]FMT K i and baseline [11C]raclopride BP ND one-sample t-tests, and change in [11C]raclopride BP ND paired t-test ([18F]FMT K i ∩baseline [11C]raclopride BP ND ∩placebo [11C]raclopride BP ND >methyphenidate [11C]raclopride BP ND ).

Secondary, exploratory analyses examined the consistency of relationships between [18F]FMT K i and [11C]raclopride BP ND measures in striatal subregions. Striatal subregions were manually drawn for each participant. ROIs were drawn in native space on each participant’s averaged MPRAGE MRI scan using Mango software. The dorsal caudate, dorsal putamen, and ventral striatum were drawn as previously described (Mawlawi et al, 2001). This manual segmentation protocol was designed to create structurally defined ROIs that reflect the dorsal–ventral functional organization of the striatum. Specifically, ventral aspects of caudate and putamen are included in the ventral striatum ROI along with nucleus accumbens. These ventral portions of caudate and putamen partially surround nucleus accumbens, and share cortical and subcortical inputs from the limbic system (Haber et al, 1994; Poletti and Creswell, 1977; Russchen et al, 1985; Van Hoesen et al, 1981; Yeterian and Van Hoesen, 1978). Inter-rater reliability was high for manually drawn striatal subregions. For ROIs of five participants drawn by 3 raters, the Sorensen-Dice coefficient ranged from 0.80 to 0.89, and the intra-class correlation coefficient ranged from 0.87 to 0.99 for PET [18F]FMT K i signal extracted from ROIs. Mean±SD ROI volumes were 2042±377 mm3 for dorsal caudate, 3759±608 mm3 for dorsal putamen, and 1788±330 mm3 for ventral striatum.

Statistical Analyses

Statistical analyses were performed using SPSS, version 24. Analyses compared [18F]FMT and [11C]raclopride measures in striatum. Pearson correlations tested relationships between striatal [18F]FMT K i , [11C]raclopride BP ND , and dopamine release (% change: ([11C]raclopride BP ND placebo−[11C]raclopride BP ND methylphenidate)/[11C]raclopride BP ND placebo). Primary correlation analyses were performed for whole striatum defined in MNI space. Secondary analyses tested correlations within striatal subregions (dorsal caudate nucleus, dorsal putamen, ventral striatum) as described above.

Shapiro-Wilk tests confirmed distributions were normal for PET signal in all regions with the exception of percent change in [11C]raclopride BP ND for whole striatum; a Spearman correlation is reported for the analysis of its relationship with [18F]FMT K i . Correlations between [18F]FMT and [11C]raclopride % change are corrected for individual differences in body weight. We report r and p-values along with 95% confidence intervals for the r-values based on 1000 bootstrap samples (r, (confidence interval), p).

Complementary analyses demonstrated the limited impact of partial volume effects on our results. For analyses on manually drawn striatal subregions, we confirmed that all correlations described above remained significant after covarying ROI volume. Second, we confirmed that applying ROI-based partial volume correction (PVC; Rousset et al, 1998) to PET data did not affect our main conclusions. These analyses were performed in native space (non-normalized data) and correct for between-subject differences in the inclusion of white matter and CSF in the measured volumes. To apply the PVC in native space, we used FreeSurfer-generated ROIs for gray matter cortical and subcortical regions, white matter, and cerebral spinal fluid with manually drawn striatal ROIs substituting for the automated striatal segmentation. PVC results are reported in Supplementary information.