Emerging empirical data indicate converging evidence for neurochemical alterations in MJ users, with lower NAA being the most frequently observed alteration, apparent in six out of the eight published studies. Lower NAA levels were observed in frontal lobe regions of interest, including the DLPFC, ACC, inferior frontal gyrus and midfrontal GM. Thus, NAA may be a more sensitive marker reflecting MJ neurotoxic effects on neuronal viability. Notably, when frontal lobe regions of interest were included in the MRS study design, NAA alterations were observed in the youngest study participants. The second most common finding was lower mI, observed globally in MTL WM and regionally specific to the left thalamus, in the basal ganglia and in the ACC. Given the role of mI in inflammatory responses, observed lower mI levels are suggestive of MJ-related immunosuppression. Importantly, when age was included as a covariate in the analysis of mI levels in participants who were in their fourth decade of life, mI differences were no longer statistically significant in the basal ganglia, suggesting that age is an important factor when examining interactions between neurochemical levels and MJ use [49]. The one published study documenting evidence of alterations in frontal lobe GABA levels in adolescent MJ users suggests that GABAergic abnormalities may contribute to frontal dysfunctions associated with MJ use, however, it is difficult to discern whether or not this was a focal MJ-related alteration, as no other comparison brain regions were examined [47], Nonetheless, this is consistent with animal models demonstrating that cannabinoids modulate the GABA system via the CB1 receptor with some evidence that interactions between the GABA and cannabinoid systems are more pronounced in adolescent animals likely due in part to ongoing development of the GABA and cannabinoid systems during adolescence [50–56],

Relationships between metabolite levels, clinical and cognitive variables examined in some of these published studies were significant, indicating that metabolite levels may be useful predictors of altered functioning associated with MJ use. The most frequent relationships reported with metabolite levels were with duration or frequency of MJ use: with greater amounts of MJ use being associated with lower Cho, lower NAA and lower mI. Only two published studies, which examined the same cohort of MJ-dependent young men, reported significant correlations between impulsiveness and mood and global mI levels in MTL WM and in the left thalamus. Low mI levels, indicative of suppression of glial function and which may lead to alterations in neuronal function and efficiency, was associated with less impulse control. It is plausible that greater levels of impulsiveness led to the initiation of MJ use in the first place, necessitating the need to characterize antecedents, as well as consequences of MJ use. None of the other MRS studies published to date reported significant relationships between metabolite levels and behavioral data. It is possible that the relatively small sample sizes included in each of these studies, the largest of which included 24 HIV− MJ users, precluded the ability to detect significant differences or relationships due to a lack of statistical power [43]. Clearly brain region of interest and metabolites of interest, along with duration of MJ use, have emerged as important factors for identifying neurochemical correlates of MJ use.

It should also be noted that two of the published studies included MJ-users who were also using additional substances. One study reported findings from MA + MJ adolescent users relative to those using MA alone, thereby limiting conclusions regarding the impact of MJ on neurochemistry, although significant correlations with MJ use were reported [48]. The second study included recreational young adult drug users who were using ecstasy, alcohol, MJ and cocaine, with no MJ-only group or matched non-using comparison group, again limiting conclusions about MJ use alone [45]. Finally, the largest study published to date included MJ users who were both HIV+ and HIV−. Given that the MJ-only group was significantly younger than the other three groups, conclusions drawn from this study are also limited.

There are other methodological factors that should also be considered, including variables associated with drug consumption, co-morbid psychiatric conditions, and age and sex differences. For instance, period of abstinence from MJ use, this was not accounted for in any of the published studies. To the extent that washout period may play an important role in recovery of neurometabolite concentrations, empirical investigation of length of abstinence should be examined in future studies. Structured clinical interviews confirmed MJ dependence in three of the eight studies, whereas the five remaining studies included heavy users. Thus, it is important to consider the potential risk factors and effects of psychiatric comorbidity, especially given recent reports that MJ use is associated with earlier onset of psychosis and psychotic thinking and that lower NAA/tCr ratios in the DLPFC and associated neuropsychological deficits have been reported in patients with schizophrenia [57–60]. The age of the participants in the eight studies ranged from approximately 15 to 45 years, limiting comparisons across studies. However, given that the frontal cortex undergoes the most substantial changes during adolescence, and the onset of drug use often occurs during this period, age of testing in a very important variable to include in MRS investigations of MJ use [61]. Indeed, drug use during the adolescent period may alter the healthy development of the brain via neurotoxic effects of substances on neurochemistry, with polydrug use leading to even greater neurochemical alterations during this critical period of brain development [48]. Finally, there is a significant need for the investigation of sex differences in neurochemistry, underscored by the inclusion of only eight females collectively in MJ-only groups across each of the eight MRS/MRSI studies. In light of sex-specific findings from other non-MRS studies of MJ use, as well as reports on the role of the menstrual cycle phase and sex hormones on neurochemical metabolites identifying potential sex differences in MJ-related alterations of brain metabolite concentrations will significantly contribute to this growing literature [55,62,63],

There are challenges associated with MRS acquisition and quantification that are necessary to consider when interpreting MRS data. Increasing scanner field strength permits increased MR signal, which is field strength dependent, thereby increasing overall sensitivity. Only one published study acquired MRS data at 1.5 Tesla, whereas all other MRS data were acquired at relatively higher field strengths of 3.0 and 4.0 Tesla. The method chosen for spatial localization also can impact the quality of the data collected, with single voxel spectra typically having higher resolution than spectra acquired using MRSI. An advantage of MRSI, however, is the extraction of metabolite spectra from multiple adjacent voxels that can be selected from various regions of interest and investigated during post-processing of MRS data. The choice of spatial localization approach is dependent, therefore, on the a priori hypotheses of the study. It is also important to empirically quantify tissue contributions in the voxel or voxels of interest, as previous studies have shown that proton metabolite concentrations vary with tissue type [64]. Another important factor to consider is the selection of an unbiased reference standard with which to derive absolute metabolite concentrations, rather than assuming that the usual internal references (i.e., Cr, water) are unchanged between populations. As such, the majority of MJ studies conducted to date have utilized either Cr or unsuppressed water as a denominator for determining metabolite ratios, with the exception of the study by Chang and colleagues, who determined absolute concentrations. Cr has typically been considered a relatively stable in vivo metabolite peak, which is typically maintained at constant levels in healthy tissue, and therefore routinely used for determining metabolite ratios. The investigation by Chang and colleagues showed that Cr levels varied between subject populations, thereby reducing the likelihood that Cr is an unbiased denominator. The unsuppressed peak arising from water is also routinely used as an internal reference for calculating metabolite ratios, as used by Prescot et al. [32] group, but also is complicated due to the need to discriminate between water in tissue versus water in CSF, which can differ in measurement up to 10–40% [65,66]. Notably, although each of these studies used varying acquisition methods, different field strengths, and examined a variety of regions of interest, there remains converging evidence for an overall trend for MJ-related disruptions in NAA and mI concentrations.