The key finding of the present study is that acamprosate, given to alcohol dependent subjects on initiation of abstinence, markedly suppressed MRS measures of central glutamate over 4 weeks of treatment. Advances in spectrum acquisition and analysis allowed us to obtain a glutamate signal mostly uncontaminated by glutamine. Because steady state plasma concentrations of acamprosate were reached already on day two, the delayed onset of acamprosate action is unlikely to be explained by pharmacokinetics. Interpretation of the MRS measures on day 4 of acamprosate treatment may be complicated, because severity of alcoholism, acute withdrawal, benzodiazepines and acamprosate may all have an impact on the ACC glutamate concentration at this time point. For two reasons, we do not believe that these are major limitations. First, measures of dependence severity, withdrawal intensity, or benzodiazepine use did not contribute to the results when included in the analysis. Secondly and more importantly, no effect of acamprosate was observed at the early timepoint, when the impact of these confounds might be expected. Instead, the acamprosate effect was observed at a time when subjects had been free of both withdrawal symptoms and benzodiazepine for about 3 weeks. Furthermore, analyses of other brain metabolites, such as NAA and choline, suggest that the acamprosate effect is specific, and not driven by changes in e.g. creatine levels.
Limited data are available on MRS measures of glutamate in alcoholics during withdrawal and early protracted abstinence. One prior study, using a combined glutamate + glutamine measure, did not find changes over time in alcoholics, and also did not find a difference between patients and controls after controlling for tissue composition 19
. This paper did find an influence of smoking status on the combined MRS measure. We therefore controlled for this as a potential confound in our study, but did not find any influence of this factor. Because all but 5 of our subjects were smokers, we did not have adequate power to assess an independent influence of smoking, something that was not an objective of the study. Another paper reported serial MRS scans in alcoholics during early abstinence, but did not provide measures of glutamate or glutamine, which were stated to be too hard to resolve 35
Although important methodological differences exist, a picture of acamprosate action emerges from our findings that is in general agreement with available animal data 3, 4, 36
. When alcohol abstinence is initiated in alcohol dependent individuals, brain levels of glutamate show a tendency to rise in placebo treated subjects, but are suppressed by acamprosate treatment. These data were obtained from the cingulate cortex, and it remains to be established whether other brain areas are similarly affected. Nevertheless, to the extent a persistent rise in glutamate contributes to craving and relapse in alcoholism as has been commonly hypothesized, our data support the notion that acamprosate may exert its therapeutic effect by counteracting this pathophysiological process. It is important to note that our data do not directly address the question whether elevated levels of brain glutamate are present in alcohol dependent patients compared to healthy subjects. The spectroscopic method used to determine central glutamate relies on a ratio vs. creatine. Our control data with NAA and choline ratios make it unlikely that acamprosate treatment per se
would confound the glutamate measure by affecting creatine levels. It remains unknown, however, whether alcohol dependence might influence the spectroscopy results, e.g. through structural changes, in ways that would differentially affect the glutamate and the choline signal, making a comparison between alcoholics and normals difficult to interpret.
A hyperglutamatergic state has also been implicated in the hyperexcitability of alcohol withdrawal 3, 4, 36
. An ability of acamprosate to suppress central glutamate might therefore also be expected to suppress acute alcohol withdrawal symptoms. However, in agreement with a previous study 37
, we did not find any acamprosate effect on acute withdrawal. These findings are consistent with the delayed nature of the acamprosate effect on central glutamate levels observed in our study. Acute withdrawal symptoms subside within 3 – 5 days, while no effect of acamprosate on MRS measures of central glutamate was found after 4 days. Acamprosate was inactive at this timepoint despite steady state plasma levels that were ultimately effective. Pharmacokinetics could nevertheless be relevant for the lack of acamprosate effect on acute withdrawal, because it is not known whether brain concentrations are in immediate equilibrium with the plasma compartment. Alternatively, the delayed onset of acamprosate action may reflect a slow, possibly indirect pharmacodynamic mechanism. Finally, our evaluation of sleep quality with the PSQI also failed to show a significant effect of acamprosate. This is in contrast to a previous study, in which acamprosate had a beneficial effect on polysomnographic measures of sleep architecture. The delayed nature of acamprosate actions may be equally critical in this case, since the previous study initiated acamprosate treatment eight days before the onset of withdrawal 38
We found that CSF levels of glutamate were not correlated with central glutamate as measured by MRS, and were unaffected by acamprosate treatment. In contrast, CSF glutamate levels in protracted withdrawal were strongly correlated with severity of alcohol dependence. Two mutually non-exclusive mechanisms may account for these observations. First, increased CSF levels of glutamate are observed in both stroke and trauma, where they are caused by an efflux of cytosolic glutamate from neurons and astrocytes 39
. Significant loss of gray matter occurs over time in alcoholism 30
, and animal models have directly demonstrated cell death following a period of intoxication 40, 41
. Thus, an efflux of cytosolic glutamate from damaged cells, similar to that observed with trauma and stroke, might occur following a period of intoxication. Secondly, a gradient of glutamate is normally maintained between blood and the CSF compartment by an energy dependent transport mechanism across the choroid plexus endothelium (CPE), protecting the nervous system from high (appr. 0.2mM) plasma glutamate concentrations. Thiamine deficiency resulting from heavy alcohol use results in impaired ability of CPE cells to maintain the blood – CSF glutamate gradient 42
. If the degree of this impairment increases with the severity of alcohol dependence, a correlation of the type that was observed would be expected. Although we provided standard clinical thiamine supplementation, it has been suggested that impairment of CPE dependent transport in chronic alcohol dependence may become lasting.
Both the mechanisms discussed here point to sources of CSF glutamate other than the transmitter pool. The observation that CSF glutamate was unaffected by our pharmacological intervention is also consistent with this notion. In contrast, the pool of glutamate reflected by MRS was uncorrelated with CSF levels, and was sensitive to acamprosate. This indicates that it originates from a pool distinct from that contributing to glutamate in CSF, and one that is likely to be more closely related to neurotransmission. Finally, our data leave unresolved the question why CSF levels of glutamate in protracted, but not in early abstinence were strongly correlated with alcohol dependence severity. It may be speculated that CSF levels in early withdrawal are mostly influenced by state-related factors with high individual variability, such as e.g. nutritional status. Following a month in the highly standardized environment of the inpatient care unit, state-related factors may play a lesser role, while remaining variance is to a higher extent accounted for by trait-like factors, such as alcoholism severity.
Finally, up-regulation of hypothalamic-pituitary-adrenal (HPA)-axis reactivity, measured by dex-CRH responses, has been reported in the first weeks following initiation of alcohol abstinence 43
, and may offer a window on central CRH activity, which is suggested by animal studies to be up-regulated following a prolonged history of brain alcohol exposure 44
. We therefore explored whether a modulation of dex-CRH test responses by acamprosate would indicate that glutamatergic and CRH-related neuroadaptations in alcoholism are related to each other. However, in agreement with another recent study 45
, we found no effect of acamprosate on the dex-CRH response, neither in early nor in protracted abstinence. Probes more directly tapping into central CRH function, such as positron emission tomography (PET) ligands for central CRH receptors, may be needed to address a possible relationship between neuroadaptations that encompass glutamate and CRH systems in alcoholism.
In conclusion, we find that 1
H-MRS spectroscopy is a valuable, non-invasive translational tool to study measures of glutamate function in alcoholism. Although it cannot be excluded that our finding reflect a lowering of glutamate by acamprosate in a compartment not directly relevant to neurotransmission, this interpretation is made less likely by the concordance between our finding and available animal data 5, 14
. MRS offers an attractive surrogate marker for early human evaluation of candidate therapeutics that target the glutamatergic system.