In GAD patients treated with the glutamate-modulating agent riluzole for 8 weeks, a strong relationship was found between changes in anxiety symptoms and changes in hippocampal NAA from baseline to endpoint. A significant group-by-time interaction was evident, signifying that the pattern of change in hippocampal NAA across the three assessment points (Baseline, 24 hours, Week 8) differed for responders, non-responders, and non-anxious healthy volunteers. In most patients who responded to riluzole (7 of 9), hippocampal NAA increased from baseline to study endpoint (+17.0% mean increase), while in all non-responders (5 of 5), hippocampal NAA remained stable or decreased (−15.6% mean decrease). A similar proportion of healthy volunteers displayed increased hippocampal NAA as the riluzole responders.
The acute effect of riluzole on NAA concentrations was consistent with the overall trajectory of increase (responders) or decrease (non-responders) over the course of the study. However, the relative acute change in NAA did not differ significantly between responders and non-responders, and significant relationships between NAA and symptoms did not emerge until study endpoint. This distinction between riluzole's acute and chronic effects suggests that riluzole's anxiolytic properties might be dependent on longer-term processes associated with enhanced neuronal viability and neuroplasticity. However, given that acute trends mimicked chronic effects, rapid restoration of mitochondrial function in extant neurons may also be associated with symptom alleviation, as suggested previously in ALS (20
Riluzole's effect on hippocampal NAA in responders is consistent with the view that neuroplasticity-enhancing therapies may benefit subgroups of patients with GAD and mood disorders. Modulation of the glutamatergic system for stress-related mood disorders may confer neuroprotection (42
) and enhance neuroplasticity (e.g., 43
), which encapsulates a range of neural processes (e.g., dendritic function, axonal sprouting, synaptic remodeling, long-term potentiation) that support the brain's ability to perceive, adapt to, and respond to internal and external stimuli (44
). Riluzole's effect on neural and behavioral plasticity in hippocampus is mediated in part by its role in AMPA receptor trafficking, a process implicated in the regulation of activity-dependent synaptic strength and postsynaptic receptor responsiveness (18
). Riluzole administered chronically and at therapeutically relevant concentrations in cultured hippocampal neurons enhanced surface expression of the AMPA receptor subunits GluR1 and GluR2 (18
). Although we did not find baseline evidence of impaired neuroplasticity, GAD patients who responded to riluzole after 8 weeks showed a robust increase in hippocampal NAA, suggesting that this putative marker of neuroplasticity may signify a surrogate endpoint for clinical response. Long-tem outcome data are needed to test whether hippocampal NAA increases during treatment can predict sustained clinical benefit. Conversely, decreased hippocampal NAA in riluzole non-responders may reflect illness-associated impairments in neuronal viability and/or mitochondrial function. It is noteworthy that the 2 GAD non-responders at Week 8 with the greatest decreases in hippocampal NAA from baseline (>25%) had NAA concentrations below the range for healthy volunteers at any time point, representing a pathological NAA deficit. No baseline clinical, demographic, or MRSI variables distinguished riluzole responders from non-responders, and baseline NAA did not predict change in anxiety, nor did baseline anxiety measures predict change in NAA. Additional studies are thus necessary to determine the value of hippocampal NAA as a biomarker of clinical anxiety.
In interpreting the neurobiological significance of riluzole's impact on hippocampal NAA, several salient issues regarding NAA merit discussion. First, it is now accepted that NAA is present in immature oligodendrocytes and is not neuron-specific (45
). As riluzole has been demonstrated to modulate extracellular glutamate levels through glial reuptake mechanisms (14
), increased hippocampal NAA may reflect increased non-neuronal activity. Second, genetic variation in the regulation of synaptic glutamate concentrations has been found to impact NAA concentrations (46
) while polymorphisms in neurotrophic factors contribute to individual differences in hippocampal volume (47
). Neuroimaging investigations that assess genetic moderators of hippocampal plasticity such as the brain-derived neurotrophic factor Val66Met polymorphism (47
) would enable further scrutiny of the relationship between riluzole response, hippocampal NAA, and neuroplasticity. Third, while the NAA resonance peak in MRSI consists predominantly of NAA (20
), there are contributions of up to 25% from other N
-acetyl compounds, including the dipeptide N
-acetylaspartylglutamate (NAAG) (48
). Thus, it is possible, although unlikely, that the increased hippocampal NAA in GAD responders with chronic riluzole administration may reflect increased NAAG with normal NAA, a pattern observed in normal-appearing white matter in multiple sclerosis (49
). Finally, studies designed to measure the glutamate resonance (which includes glutamate and glutamine), using either high-field 1
H MRS or appropriate spectral editing techniques, could also advance our understanding of the effects of riluzole on this metabolic pathway. Notably, glutamate-glutamine contamination of the NAA peak has been observed at short echo times (TE = 35 ms) (50
) but not at long TEs, as were used in this study.
The lack of baseline group differences in hippocampal NAA concentrations is consistent with the only previous published MRSI study in GAD (29
). Increased NAA/Cr ratios were found in right DLPFC of 15 GAD patients compared to healthy controls, although the use of ratio analyses hinders direct comparability between studies. In major depression, most studies have failed to detect cortical NAA abnormalities (51
), although in PTSD, hippocampal NAA reductions have been reported even in the absence of volumetric reductions (52
). Our correlational results at study endpoint add to an emerging literature relating NAA concentrations to anxiety variables, though the directionality and regional localization of the findings have been inconsistent across studies. In a non-clinical sample, NAA concentrations in the orbital frontal cortices were positively related to a composite measure of state and trait anxiety (55
), while in social phobics, NAA/Cr in the ACC was found to be elevated and positively related to symptom severity (32
). These divergent findings underscore the need for additional research, using consistent metabolite measures and ROIs, in discrete patient populations.
This study is the first to examine the neurochemical effects of riluzole in a clinically anxious patient group. We studied a well-characterized GAD sample with no substance abuse comorbidity or concurrent major depressive episodes. Collecting 1
H-MRSI data at three time points allowed us to differentiate between the acute and chronic effects of riluzole on metabolite measures, and the use of a reliable multi-slice multi-voxel spectroscopic acquisition (38
) with absolute quantitation mitigated limitations of ratio analyses of MRSI data. We employed an absolute quantitation scheme for NAA rather than report NAA/Cr ratios due NAA/Cr's poor association with absolute levels of NAA (23
), and its relatively low test-retest reliability reported in medial temporal lobe (22
). In our report, we found low variability of hippocampal NAA for healthy volunteers (CV = 4.1%), which compares favorably to the CV (> 10%) using the same acquisition H-MRSI acquisition procedure reporting hippocampal NAA/Cr ratios (56
Nevertheless, several methodological limitations are noted. First, we cannot determine whether the changes in hippocampal NAA reflected a specific mechanism of riluzole or epiphenomenal symptom improvement, due to lack of placebo control. This concern is particularly relevant for GAD clinical trials, which are associated with high placebo responsivity (40
). Secondly, the relatively small sample size of healthy comparison subjects and riluzole non-responders likely limited the power to detect significant differences in hippocampal (and prefrontal cortical) neurochemistry at any time point. Finally, the confounding effects of partial volume averaging cannot be ruled out, since tissue segmentation was not performed due to lack of volumetric MRI data on the subjects at each of the three time points. However, even after taking into account a 40% broadening due to PSF, our voxels were still sufficiently small to be contained within the hippocampal ROIs, thereby minimizing tissue heterogeneity. Furthermore, since this study compared within-subject NAA changes over time, with each subject effectively serving as his or her own control, the possibility that the variability in NAA over these 3 time points would be due to significant partial volume effects appears remote.
In conclusion, we have identified hippocampal metabolic correlates of anxiolytic response to the glutamate-modulating agent riluzole in GAD. We suggest that riluzole might be efficacious for GAD (and subtypes of mood disorders) in part due to reduced glutamate excitotoxicity and enhancement of hippocampal neuroplasticity. Further investigation of neuroimaging biomarkers of response remains an important goal for development of novel treatments for these conditions.