Study design

20 mg d-amphetamine sulfate (AMP), 20 mg methamphetamine hydrochloride (MA; Desoxyn®), and placebo (PBO) were investigated in a counterbalanced, double blind, within-subjects crossover design.

Recruitment

Eligible participants received a physical exam and electrocardiogram. Exclusion and inclusion criteria were similar to previous studies [40, 41] and excluded those younger than 18 or older than 35; those with BMI below 18 or over 30; left-handedness; less than a high-school education/GED; lack of fluency in English; chronic medical conditions; abnormal EKG; current or past Axis I psychiatric disorder; smoking more than five cigarettes per week; and in females, current or intended pregnancy or lactation. The study was approved by the Institution Review Board for research with human subjects at Brown University and the Memorial Hospital of Rhode Island in accordance with the Code of Federal Regulations (Title 45, Part 46) “Protection of Human Subjects” adopted by the National Institutes of Health and the Office for Protection from Research Risks. The study was conducted ethically in accordance with the Helsinki Declaration of 1964 (revised 2013) and the National Advisory Council on Drug Abuse Recommended Guidelines for the Administration of Drugs to Human Subjects. Written informed consent was obtained from all participants.

Participants

Twenty-six healthy right-handed individuals participated. Participants were 18–28 years of age (mean = 22.3 years, SD = 3.27). The sample was rigorously balanced by gender (13 female, 13 male). Participants were of normal body weight (mean BMI = 22.3, SD = 1.86; mean body weight = 145.1, SD = 17.4 lb), and weight did not differ in male and female participants (male mean (SD) = 150.9 (16.6); female mean (SD) = 139.4 (16.9); t(24) = 1.74, p = .10, n.s.). Racial composition was 73% White, 19% Asian, 4% African American, and 4% Multiracial/other. Ethnicity was 77% non-Hispanic and 23% Latino/Hispanic. The sample was well educated, with 8% reporting a high-school diploma, the majority (61%) reporting some college education and 31% reporting a Bachelor’s degree or more.

Procedures

The study consisted of seven sessions on separate days: psychological screening, medical screening, orientation, three MRI test sessions, and an exit session. Experimental design appears in Fig. 1. Each test session (AMP, MA, and PBO) was 5.5 h in duration, conducted at a fixed time of day for each participant to control for circadian effects. Sessions were approximately a week apart (mean interval 9(±8) days), with AMP and MA at least 3 days apart to avoid sensitization. For blinding purposes, participants were told that the study capsules could contain one of several classes of drugs (stimulants/appetite suppressants, sedatives/tranquilizers, anti-depressants, and placebo). Participants consumed a standardized meal 2 h prior to the sessions. Breath alcohol level and urine toxicology screening confirmed participants were drug and alcohol free. Cardiovascular status was evaluated using an Invivo Precess MRI Patient Monitoring System (Soma Technology, Bloomfield, CT) within the scanner and an Omron wrist blood pressure monitor (Omron, Lake Forest, IL) outside the scanner (Fig. 1). Participants were in verbal contact with the experimenter at all times during the experiment.

Fig. 1 Experimental design. Timing of MRI test sessions on days 4–6. Sessions were 5.5 h in duration (340 min total). X-axis denotes time relative to administration of the blinded study capsule at time 0 (black arrow). Participants entered the scanner 90 min after administration of the study capsule (vertical dashed black line). MR spectroscopy was conducted 140–150 min post-capsule (gray shading). Physiological data were collected at half hour intervals for internal validity (open arrows). Mood data were collected at half hour intervals outside the scanner to assess subjective drug effects (see Materials and methods for details). Participant arrival and departure times are indicated (gray arrows) Full size image

Monetary compensation

Participants were paid $220 for completing the study. Prorated payment was provided to those not completing all three MRI test sessions ($70/session).

Study drugs and dosing

Study drugs were d-amphetamine sulfate (AMP, 20 mg oral), methamphetamine hydrochloride (MA, 20 mg oral; Desoxyn®), and placebo (PBO).

Rationale

Dopamine and norepinephrine transporters are sensitive to AMP [42, 43]. MA differs from AMP in its greater impact at the serotonin transporter [2].

Dose

A 20 mg oral dose was used for both drugs. This dose is within the range of doses recommended for ADHD, reliably increases subjective measures of stimulant effects [44, 45], is equipotent across AMP and MA [46], and is well studied in healthy volunteers [47, 48].

Encapsulation

AMP and MA were individually compounded with dextrose filler and placed in separate opaque, colored gelatin capsules. PBO capsules contained only dextrose.

Order

Order of administration was randomized and counterbalanced, with six permutations of drug order across participants (PBO-AMP-MA; PBO-MA-AMP; AMP-PBO-MA; AMP-MA-PBO; MA-PBO-AMP; and MA-AMP-PBO).

Timing

MRS was conducted 140–150 min after administration of the capsules on each test session (see Fig. 1).

Structural imaging

A Siemens 3T TIM Trio system (Siemens Medical Solutions, New York, NY) was used for MR data collection. Whole-brain T 1 -weighted images (MPRAGE sequence) were acquired in the sagittal plane at the beginning of each MRI session (resolution = 1 mm × 1 mm × 1 mm; TR = 1900 ms; TI = 900 ms, TE = 2.98 ms; flip angle = 9°; FOV = 256 × 256). The structural T 1 images were processed using SPM12 [49] and Gannet Coregister was used to reconstruct the voxel [50, 51].

Spectroscopy

Spectra were acquired in the dACC using single voxel, point-resolved spectroscopy (PRESS). Voxel size was 15 × 15 × 10 mm (10 mm in SI direction) and was placed on the T 1 -weighted anatomical image using the three-plane reconstructions. The voxel was placed on the dACC immediately anterior and parallel to the corpus callosum (see Fig. 2a). On sagittal view, the midline slice where the corpus callosum was most distinct was selected and the voxel was placed anterior and superior to the corpus callosum. The voxel was rotated to be tangential to the corpus callosum. In coronal, sagittal, and axial views, the voxel was aligned anterior and parallel to the corpus callosum such that the posterior edge of the voxel was in line with the horns of the lateral ventricles. In coronal view, the voxel was right lateralized to capture connectivity with the salience network [52] and minimize CSF contamination from the longitudinal fissure. Voxel placement was confirmed by post hoc voxel reconstruction in subject-specific anatomical space. Reconstructed MRS voxels were checked to confirm agreement with voxel placement protocol for individual anatomy, using subject-specific anatomical landmarks (above). During the MRS sessions, standard first-order auto shimming was implemented, line width checked and shimming manually adjusted to minimize the free water line width. Prior to acquisition of spectral data, non-water-suppressed data sets were obtained to provide for eddy current compensation and to provide a water reference. Repetition time (TR) was 3000 ms, echo time (TE) was 30 ms, with 128 averages taken for a total acquisition time of 6 m 24 s for water-suppressed data. The TR of 3000 ms reduced differential T 1 effects to less than 12% and the T 2 differentiation was minimized by the echo time of 30 ms [53]. Spectra were processed and metabolites quantified using LCModel [54]. All of the data received zero- and first-order phase correction as part of the regular LCModel process, done automatically by LCModel with no user intervention. Data were compensated for differential T 1 effects according to individual metabolite T 1 values at 3 Tesla [53, 55, 56]. Only metabolites with Cramer-Rao lower bound (CRLB) less than or equal to 20% were evaluated. Example spectra using this procedure are in Fig. 2b.

Fig. 2 MRS Voxel and Example Spectra. a Voxel placement in dorsal anterior cingulate cortex (dACC). Left: sagittal; middle: coronal; right: axial views, respectively. b Example MRS spectra with labeled peaks. The solid dark gray curve overlay is the fitted spectrum from LCModel and the raw data shown in light gray, with the baseline below. The upper panel is a plot of the residuals, which in the case of a proper fit appears as noise. Labeled peaks: Cr creatine, Ins myo-inositol, Cho choline, Glx glutamate and glutamine, NAA N-acetyl-aspartate Full size image

1H MRS measures

Primary measures

Metabolites reliably assessed by 1H MRS include Glx, a composite measure of Glu and Gln primarily comprised of Glu [23]; tNAA, a combined measure of N-acetyl-aspartate and N-acetylaspartyl-glutamic acid; tCr, a combined measure of creatine and phosphocreatine; Cho, a measure of choline-containing compounds glycerophosphocholine and phosphocholine (GPC + PCh); and Ins, a measure of myo-inositol. Reliability for these metabolites is well established [54, 57, 58]. All metabolites were quantified relative to water in institutional units (i.u.) per standard practice [59]. Ratio-based measures were considered but decided against, to avoid interpretive confounds should metabolites change simultaneously, see refs. [22, 60, 61]. Metabolites were corrected for partial volume effects using the formula [*1/(1 − fCSF)], where fCSF is the voxel volume fraction of cerebrospinal fluid therefore providing a measure of metabolite concentration in tissue within the voxel (per methods of refs. [62,63,64]). Voxel placement reliability was assessed using dice coefficients [65, 66].

Subcomponent measures of Glx

Glu and Gln were evaluated separately to provide information on the specificity of drug findings with Glx. LCModel analyzes Glu and Gln using the entire in vivo spectrum in relation to the complete model spectra produced by metabolites at known concentrations in vitro, and provides good discrimination between Glu and Gln when FWHM are on the order of 0.05 ppm [54]. The acquired spectra were uniformly well resolved with an average FWHM of 0.06 ppm and a modal FWHM of 0.05 ppm. Mean (±SD) CRLB uncertainty was 5(±1)% for Glx, 5(±1)% for Glu, and 13(±2)% for Gln.

Mood responses

Subjective responses were evaluated using self-reports on the Drug Effects Questionnaire (DEQ) [67]. Participants indicated on 100-mm lines their response to DEQ items “I LIKE the effects I am feeling right now,” rated from “dislike” to “like very much” (DEQ “Like Drug”), and “I am HIGH,” rated from “not at all” to “very much” (DEQ “Feel High”). Responses were assessed at half hour intervals outside the scanner, for eight time points of assessment on each session (details in Fig. 1).

Quality control procedures

Quality control was implemented in three steps: (a) All MRS spectra were visually inspected for quality by an MRS data acquisition and analysis expert (co-author Woods). (b) Using co-registration tools from the Gannet analysis package [51], voxels were reconstructed in each anatomical scan and segmented using SPM12 [50] to allow for quantification of the proportion of white matter, gray matter, and CSF in the voxel. Data were corrected for partial volume effects using the formula [*1/(1 − fCSF)], per established methods [62,63,64]. (c) Data for individual metabolites were excluded, where the CRLB exceeded 20% SD. These procedures indicated that the majority of the data were of high quality: from a total of 73 acquired scans in 26 participants, 7 scans were excluded for lack of water reference files, and CRLB(Gln) cutoffs were exceeded in 3 instances. Thus, 66 scans in 24 participants were available for analysis, with additional exclusions for Gln (63 scans).

Statistical analysis

Drug effects

Linear mixed models with random intercepts were used to evaluate drug effects on each CSF-corrected metabolite individually. Separate analyses examined differences between placebo and AMP, placebo and MA, and AMP and MA. Beta coefficients indicate the model-predicted difference in the metabolite in the active drug condition compared to PBO, with positive beta coefficients indicating higher metabolite levels under drug. Linear mixed models were chosen because the estimation method (i.e., full maximum likelihood estimation) is able to handle missing data without excluding cases listwise, thus making full use of the available data. Effect sizes were calculated using Cohen’s d for repeated measures (d RM ) for within-subjects effects [68]. Bonferroni correction for seven tests of drug effects on Glx, Glu, Ins, Cho, tCr, tNAA, and Gln was conducted to control family-wise Type I error rate at α = .05 (adjusted α = .0071).

Manipulation checks

Sympathomimetic drug activity across the protocol, effectiveness of counterbalancing, reliability of voxel placement across test sessions, %voxel overlap, structural composition within the voxel, LCModel fit characteristics, gender, and order effects on metabolites were evaluated using repeated measures drug × time ANOVA models, Pearson χ2 tests, paired t-tests, linear mixed-effects models, and generalized estimation equation (GEE) models (details in Supplementary Methods).

Relationship of Glx, Glu, and Gln

The relationship between Glx, Glu, and Gln was assessed using bivariate correlations.

Mood correlates

Time-series mood data were reduced to a single metric in each participant, with Time-to-Peak “Like Drug” ratings to AMP and Peak “Feel High” ratings to MA meeting quality control criteria for analysis (details in Suppl. Methods). Delta (Δ) scores quantified drug-induced changes in mood and metabolites relative to PBO in each participant (Mood: ΔTime-to-Peak DEQ “Like Drug”[AMP-PBO]; ΔPeak DEQ “Feel High”[MA-PBO]; Metabolites: ΔGlu[AMP-PBO], ΔGlx[AMP-PBO], ΔtCr[AMP-PBO]; ΔGlu[MA-PBO], ΔGlx[MA-PBO]). Positive Δ scores indicate a longer duration of peak drug liking on AMP compared to PBO (ΔTime-to-Peak DEQ “Like Drug”[AMP-PBO]) and a larger drug high on MA compared to PBO (ΔPeak DEQ “Feel High”[MA-PBO]), and negative scores indicate the inverse. Relationships between drug effects on mood and drug effects on metabolites were evaluated using bivariate correlation. There were three tests of AMP effects (ΔGlu[AMP-PBO], ΔGlx[AMP-PBO], ΔtCr[AMP-PBO] with ΔTime-to-Peak DEQ “Like Drug”[AMP-PBO]) and two tests of MA effects (ΔGlu[MA-PBO], ΔGlx[MA-PBO] with ΔPeak DEQ “Feel High”[MA-PBO]). Effect magnitude was estimated by R2 and reliability-corrected R2 (details in Suppl. Methods).

Statistical power

Power analyses for within-subjects effects were conducted in G*Power 3.1 using an alpha of .05 [69, 70]. The final sample of 24 had high power (1 − β = .96) to detect large effects (d = .80), adequate power (1 − β = .80) to detect medium effects (d ≥ .60), and low power (1 − β = .16) to detect small effects (d = .20).