Study population

Twenty-four participants [17 healthy and 7 major depressive disorder (MDD)] between the age of 21 and 65 years were recruited for this study. Of the healthy subjects, one subject did not complete the second scan, and two subjects had unsuccessful scans. Subjects completed an informed consent process and were screened for study inclusion and exclusion criteria. For all participants, exclusion criteria consisted of medical conditions that contraindicate MRS or increase the risks of ketamine administration. A negative drug test, and for women, a negative pregnancy test and use of a medically accepted means of contraception were required. Healthy participants were excluded if they had a lifetime history of any psychiatric disorder, or if they had a first-degree relative with mood, anxiety, or psychotic disorder. MDD participants were included if they met the following study criteria: (1) current MDD as determined by a structured interview; (2) Montgomery-Åsberg Depression Rating Scale (MADRS) severity of 18 or more at baseline; (3) no substance abuse or dependence in the previous 12 months; (4) no lifetime history of psychosis or bipolar disorder; and (5) medication-free or on a stable (>4 weeks) antidepressant treatment. The MDD group was included in the current report to explore the possibility of differences between the two groups and to test the feasibility of conducting the 13C MRS pharmacoimaging paradigm in this population. The target sample for the MDD group is 18 subjects, which will be reported in the future once enrollment is completed.

Overview of study design

Two 13C MRS scans, separated by at least 1 week, were performed using a single-blind, placebo-controlled, fixed-order within-subject design (Fig. S2). On day 1, participants received normal saline and uniformly 13C-labeled glucose infused over 120 min as described previously [24]. Concurrently, 13C MRS of the frontal brain region was used to observe glutamate and glutamine 13C enrichments during the placebo infusion. On day 8+, healthy participants received a subanesthetic dose that has been commonly used in studies examining the psychotomimetic effects of the drug (0.23 mg/kg bolus followed by 0.58 mg/kg infusion over approximately 75 min). This dose was administered to ensure rapid and robust psychotomimetic effect [25, 26], which we believed would enhance statistical power to ascertain the relationship between glutamate cycling and the psychotomimetic symptoms during ketamine infusion. MDD patients received the dose most commonly used in depression clinical trials (0.5 mg/kg infused over 40 min) [1, 2], concurrently with otherwise identical 13C-glucose infusion and 13C MRS scans. The psychotomimetic effects of ketamine were assessed using the Clinician-Administered Dissociative States Scale (CADSS) and Brief Psychiatric Rating Scale (BPRS) (Fig. S2). The behavioral assessments in the MDD participants will be investigated in future reports once a larger cohort is enrolled.

13C MRS acquisition and processing

MRS data were acquired on a 4.0 T whole-body magnet interfaced to a Bruker AVANCE spectrometer (Bruker Instruments, Billerica, MA, USA). Subjects were placed supine in the magnet, with their head immobilized with foam. An RF probe consisting of one circular 13C coil (8.5 cm ∅) and two circular, quadrature-driven 1H RF coils (12.5 cm ∅) were used for acquisition of 13C MR spectra from the frontal lobe (Fig. 1a, b). Following tuning and acquisition of scout images, second-order shimming of the region of interest (ROI) was performed using phase mapping provided by Bruker.

13C MR spectra were acquired with a pulse-acquire sequence using an adiabatic 90° excitation pulse and optimized repetition time. Nuclear Overhauser enhancement (nOe) was achieved by applying 1H block pulses before the 13C excitation pulse. 1H decoupling during acquisition consists of pseudo noise decoupling as described by Li et al. [12], to decouple the long-range 1H–13C coupling of the carboxylic carbon positions. The pseudo noise decoupling pulse has a constant amplitude and the phase of each 1.2-ms unit pulse is randomly assigned to either 0° or 180°. Following the start of [U 13C]-glucose infusion, 6.5-min blocks of MR spectra were acquired for 120 min (Figs. 1c and S1). In the last 60 min of acquisition, we alternated 6.5-min blocks of acquisition using our standard parameters with acquisition without nOe and a 30 s delay in order to obtain a correction for nOe efficiency and saturation effects.

Spectral data were analyzed with −2/6 Hz Lorentzian-to-Gaussian conversion and 16-fold zero-filing followed by Fourier transformation. An Linear Combination (LC) model approach was used to fit the peak areas of the labeled carbon positions of glutamate C45 and glutamine C45 (Fig. 1c), which overlapped with aspartate C34. Due to the overlap, we analyzed the combined aspartate and glutamate peaks. However, it is unlikely that the surge in glutamine enrichment was affected by changes in aspartate during ketamine considering that the kinetics of aspartate labeling closely track that of its glutamate precursor [27], which was not shown to be affected by ketamine infusion. The amplitude of 13C-labeling over early time points (approximately 20 min) was averaged and normalized by labeling over the steady-state amplitude of the 13C enrichment time course (post-90 min). This procedure provided a normalized measure of 13C glutamate and 13C glutamine enrichment, which reflects the enrichment rate of these metabolites early during the infusion of placebo and ketamine. Cramer–Rao Lower Bounds were used to estimate the quality of the individual measurements, averaging 3% for glutamate and 11% for glutamine. To ensure that the observed 13C enrichment values are not affected by variability in input glucose enrichment, plasma fractional enrichment (FE) of 13C glucose was determined during placebo (early FE = 51.2%; steady state FE = 61.1%) and ketamine (early FE = 51.6%; steady state FE = 61.8%), which did not differ between sessions (p > 0.2). To provide an estimate of the variability of the 13C MRS measures between sessions, we computed the ratio of the mean over the standard deviation of each of the measures across all 21 subjects during the placebo scans. We found relatively low variability, with ratios of 7.3 for 13C glutamine enrichment and 11 for 13C glutamate enrichment. The ratios were comparable across healthy (7 for 13C glutamine and 11 for 13C glutamate) and depressed subjects (8 for 13C glutamine and 10 for 13C glutamate).

Statistical analyses

Before conducting each analysis, the distributions of outcome measure were examined. Estimates of variation are provided as standard error of mean (SEM). Considering that this is a first-in-human study, this study should be considered a first-level pilot study implementing a novel technique, rather than a confirmatory study. Effect sizes are provided as Cohen’s d. All tests are 2-tailed with significance set at p ≤ 0.05.

The percent change of 13C glutamate and 13C glutamine enrichment during ketamine compared to placebo was computed and represented the primary outcome measures. One sample t-tests examined whether the percent changes of each metabolite were statistically significant. Follow-up analysis used independent t-tests to determine whether the percent changes of each metabolite differed between the study groups. Spearman’s correlations examined the relationship between the metabolites enrichment and psychotomimetic effects of ketamine as determined by CADSS and BPRS in healthy participants.