Glutamate is involved in excitatory neurotransmission and metabolic processes related to brain function. Previous studies using proton functional magnetic resonance spectroscopy (1H fMRS) have demonstrated elevated cortical glutamate levels by 2–4% during visual and motor stimulation, relative to periods of no stimulation. Here, we extended this approach to working memory cognitive task performance, which has been consistently associated with dorsolateral prefrontal cortex (dlPFC) activation. Sixteen healthy adult volunteers completed a continuous visual fixation “rest” task followed by a letter 2-back working memory task during 1H fMRS acquisition of the left dlPFC, which encompassed Brodmann areas 45 and 46 over a 4.5-cm3 volume. Using a 100% automated fitting procedure integrated with LCModel, raw spectra were eddy current-, phase-, and shift-corrected prior to quantification resulting in a 32s temporal resolution or 8 averages per spectra. Task compliance was high (95 ± 11% correct) and the mean Cramer-Rao Lower Bound of glutamate was 6.9 ± 0.9%. Relative to continuous passive visual fixation, left dlPFC glutamate levels were significantly higher by 2.7% (0.32 mmol/kg wet weight) during letter 2-back performance. Elevated dlPFC glutamate levels reflect increased metabolic activity and excitatory neurotransmission driven by working memory-related cognitive demands. These results provide the first in vivo demonstration of elevated dlPFC glutamate levels during working memory.

fMRI has provided vibrant characterization of regional and network responses associated with associative learning and memory; however, their relationship to functional neurochemistry is unclear. Here, we introduce a novel application of in vivo proton functional magnetic resonance spectroscopy (1H fMRS) to investigate the dynamics of hippocampal glutamate during paired-associated learning and memory in healthy young adults. We show that the temporal dynamics of glutamate differed significantly during processes of memory consolidation and retrieval. Moreover, learning proficiency was predictive of the temporal dynamics of glutamate such that fast learners were characterized by a significant increase in glutamate levels early in learning, whereas this increase was only observed later in slow learners. The observed functional dynamics of glutamate provides a novel in vivo marker of brain function. Previously demonstrated N-methyl-D-aspartate (NMDA) receptor mediated synaptic plasticity during associative memory formation may be expressed in glutamate dynamics, which the novel application of 1H MRS is sensitive to. The novel application of 1H fMRS can provide highly innovative vistas for characterizing brain function in vivo, with significant implications for studying glutamatergic neurotransmission in health and disorders such as schizophrenia.

Proton functional magnetic resonance spectroscopy (H fMRS) is a noninvasive neuroimaging technique capable of detecting dynamic changes in glutamate related to task-related demands at a temporal resolution under 1 min. Several recent H fMRS studies demonstrated elevated steady-state levels of glutamate of 2% or greater during different 'task-active' conditions, relative to a 'non-task-active' control condition. However, the 'control' condition from these studies does vary with respect to the degree of constraining behavior, which may lead to different glutamate levels or variability between 'control' conditions. The purpose of thisH fMRS study was to compare the steady-state levels and variability of glutamate in the left dorsolateral prefrontal cortex (dlPFC) of 16 healthy adults across four different putative 'non-task-active' conditions: relaxed with eyes closed, passive visual fixation crosshair, visual flashing checkerboard, and finger tapping. Results showed significantly lower glutamate levels during the passive visual fixation crosshair than the visual flashing checkerboard and the finger tapping conditions. Moreover, glutamate was significantly less variable during the passive visual fixation crosshair and the visual flashing checkerboard than the relaxed eyes closed condition. Of the four conditions, the passive visual fixation crosshair condition demonstrated the lowest and least variable glutamate levels potentially reflecting the least dlPFC engagement, but greatest behavioral constraint. These results emphasize the importance of selecting a proper 'control' condition to reflect accurately a 'non-task-active' steady-state level of glutamate with minimal variability during H MRS investigations.

Working memory processes are associated with the dorsolateral prefrontal cortex (dlPFC). Prior research using proton functional magnetic resonance spectroscopy (1 H fMRS) observed significant dlPFC glutamate modulation during letter 2-back performance, indicative of working memorydriven increase in excitatory neural activity. Acute stress has been shown to impair working memory performance. Herein, we quantified dlPFC glutamate modulation during working memory under placebo (oral lactose) and acute stress conditions (oral yohimbine 54mg + hydrocortisone 10mg). Using a double-blind, randomized crossover design, participants (N=19) completed a letter 2-back task during left dlPFC 1 H fMRS acquisition (Brodmann areas 45/46; 4.5cm 3). An automated fitting procedure integrated with LCModel was used to quantify glutamate levels. Working memory-induced glutamate modulation was calculated as percentage change in glutamate levels from passive visual fixation to 2-back levels. Results indicated acute stress significantly attenuated working memory-induced glutamate modulation and impaired 2-back response accuracy, relative to placebo levels. Follow-up analyses indicated 2-back performance significantly modulated glutamate levels relative to passive visual fixation during placebo but not acute stress. Biomarkers, including blood pressure and saliva cortisol, confirmed that yohimbine + hydrocortisone dosing elicited a significant physiological stress response. These findings support a priori hypotheses and demonstrate that acute stress impairs dlPFC function and excitatory activity. This study highlights a neurobiological mechanism through which acute stress may contribute to *

Background Single-voxel proton magnetic resonance spectroscopy (1H MRS) is a powerful technique for studying in vivo neurochemistry, but has an often-overlooked source of error variance: inconsistent voxel placement between scans. We developed and evaluated an Automated Voxel Placement (AVP) procedure for accurate and reliable 1H MRS voxel prescription. AVP is a suite of Linux-based programs that facilitate automated template-driven single-voxel coregistration. Methods Three studies were conducted to evaluate AVP for prescription of one voxel: left dorsolateral prefrontal cortex. First, we evaluated how robust AVP was to ‘extreme’ subject head positions/angulations within the scanner head coil. Second, subjects (N = 13) were recruited and underwent MR scans. Manual voxel prescription (n = 5) was contrasted with AVP (n = 8). A subset of AVP subjects (n = 4) completed a second scan. Third, ongoing data collection (n = 16; recruited for a separate study) helped evaluate AVP. Voxel placement accuracy was quantified as 3D geometric voxel overlap percentage between each subject’s voxel and the template voxel. Reliability was quantified as 3D geometric voxel overlap percentage across subjects at each time point and within subjects who completed two scans. Results Results demonstrated that AVP was robust to ‘extreme’ head positions (97.5% - 97.9% overlap with the template voxel). AVP was significantly more accurate (baseline and follow-up: 96.2% ± 3.0% and 97.6% ± 1.4% overlap) than manual voxel placement (67.7% ± 22.8% overlap; ps<.05). AVP was reliable within- (97.9%) and between-subjects (94.2% and 97.2% overlap; baseline and follow-up; respectively). Finally, ongoing data collection indicates AVP is accurate (96.0%). Conclusion These pilot studies demonstrated that AVP was feasible, accurate, and reliable method for automated single voxel coregistration.