This report focuses on spectroscopy findings.In addition to the K-SADS-PL,the Personal Experience Inventory was used to further assess alcohol and marijuana use in both the MJU group and in the healthy controls.Briefly,the PEI consists of two main sections,one focused on patterns and severity of substance use,and the other focused on psychosocial consequences of use.In most cases,participants endorse items from the inventory using a four-point Likert response format.Different versions of the PEI have been developed for adolescents versus adults.Participants younger than 18 years of age received the adolescent version and participants older than 18 years of age received the adult version; both versions were computer administered.All MJU participants received the adult version.Scoring was implemented to create comparable metrics across the two versions.Finally,an in-house questionnaire based on guidelines provided by the National Institute on Alcohol Abuse and Alcoholism was implemented to assess detailed daily,weekly,yearly and lifetime use patterns of alcohol and marijuana in the sample,considering frequency and amount of use.The MR spectroscopy voxel was positioned in the right basal ganglia using the T1-weighted image.The caudate and putamen were the primary regions of interest.The voxel was positioned in the following way: left/right—the voxel was positioned so that it was as medial as possible,without containing any portion of the lateral ventricle,anterior/posterior—the voxel was positioned as anterior as possible in the caudate,without entering the anterior horn of the lateral ventricle,superior/inferior—the voxel was positioned such that the inferior portion of the voxel was as close as possible to the most inferior aspect of the putamen,and such that the superior portion of the voxel was approximately 3 mm inferior to the most superior aspect of the caudate.Fig.1 illustrates the voxel placement in a typical subject.confirmation of consistent voxel placement across subjects was achieved by segmenting and parcellating the T1-weighted image.
A high-resolution structural scan was acquired to position the voxel during data acquisition and to determine the tissue composition of the voxel through segmentation.The T1-weighted scan was processed using the standard Free Surfer pipeline for tissue segmentation and anatomical parcellation.Further details related to the Free Surfer processing can be found online,and in one of our previous publications.In-house software was used to compute the transformation matrix from the scanner coordinates to the FreeSurfer-processed T1-weighted image.A mask representing the spectroscopy voxel in the anatomical image space was then created using tools from the FMRIB Software Library,planting racks which was subsequently segmented and parcellated using the Free Surfer anatomical information.Thus,each T1-weighted voxel within the spectroscopy volume,was classified as either white matter,gray matter,cerebrospinal fluid,or non-brain,and was further parcellated into subcortical and cortical structures.This was done to confirm a consistent voxel placement across all subjects and to determine the basic tissue composition within the voxel.Further details of the voxel composition can be found in the results section below.Data were analyzed with the Statistical Package for the Social Sciences,version 19.Data were examined for normality in order to ensure appropriateness of parametric statistics.Univariate analyses of covariance were used to test group effects between the MJU individuals and the controls,with age and alcohol use entered as co-variates.Group and sex were both entered as between-subjects variables.Alcohol use frequency over the past 12-month period summarized by the PEI was used in the above model as the alcohol use co-variate.Two-way interaction effects between group and sex,when present,were examined further by running the model separately in males and females,or by examining sex effects within MJU individuals and controls.Finally,significant effects were re-evaluated by matching the MJU and control samples by age to verify that patterns remained significant with more stringent control over developmental differences that might otherwise impact the findings.There was no group by sex interaction for age or for IQ.
Marijuana users were college students of middle to high-middle socioeconomic backgrounds and most were free of a non-substance DSM-IV Axis I diagnosis.None were psychotic.Nearly all met DSM-IV diagnostic criteria for marijuana abuse or dependence.Use of other recreational drugs within the MJU group was limited,with no participants meeting DSM-IV criteria for abuse or dependence.One subject met diagnostic criteria for current alcohol dependence,and a small proportion met criteria for alcohol abuse.Compared to controls,alcohol use over the past twelve months use was found to be significantly higher in the MJU group,F = 43.93,p b 0.001.Marijuana users on average had a PEI score of 3.7,which corresponds to endorsing use of alcohol between 21 and 100 times in the previous 12 months.Controls on average had a PEI score of 1.5,which corresponds to endorsing use of alcohol between 1 and 20 times in the previous 12 months.When the sample is restricted to include only individuals aged 17 and higher,the difference in alcohol use remains significant but the mean value for control participants is slightly higher at 1.9.Thus,the amount of alcohol use endorsed over the past twelve months was entered as a covariate in analyses comparing metabolite concentrations between groups.The marijuana users reported that their age of first use of marijuana was 15.2 ± 1.2 years,and also reported smoking 9.8 hits per day during the past year.In addition,supplemental analyses were conducted to verify that female users did not differ from male users in their self-reported patterns of use,age of use onset,use of alcohol,or symptoms of psychopathology.Findings are presented in Table 3.The only group difference to emerge was that female users reported fewer symptoms overall of alcohol abuse/ dependence than did males.Otherwise,they did not significantly differ in variables that would suggest an increased frequency or duration of marijuana use,use of other substances,or presence of concomitant psychopathology.The spectroscopy voxel was consistently placed in the same anatomical location,centered in striatum,in both marijuana users and controls.
The voxel was primarily composed of gray matter,as determined by the Free Surfer parcellation procedure.The majority of the voxel composition was statistically similar between groups,with the exception of the pars opercularis,which accounted for less than 1% of the total voxel composition.The remaining 2% of the voxel composition was relatively variable.Moreover,these additional regions always represented very small amounts of tissue,and were not represented in all subjects.This study examined a cohort of college-aged heavy marijuana users and a control group of non-using young-adults.Using MR-spectroscopy,it was shown that females,but not males,who used marijuana heavily starting in mid-adolescence and persisting for several years have lower levels of glutamate and glutamine in the dorsal striatum when compared to controls,even after accounting for age and alcohol use.Similarly,female but not male users differ from controls in their estimated concentrations of myo-inositol,demonstrating higher levels than controls.These patterns are interpreted as pathological in the female users given that male users had comparable levels to controls of both sexes.Female users did not differ from male users in their overall rates of self-reported marijuana use,in their concomitant level of alcohol use,in their numbers of symptoms of marijuana dependence,or presence of other conditions that might impact brain metabolism.These findings have broad parallels in the extant literature,both in relation to the overall patterns observed but also in relation to sex differences.Decreased glutamate/glutamine concentrations have been reported in two other MRS studies of marijuana users,one that focused on the basal ganglia and one that targeted the anterior cingulate cortex.First,in an older cohort of marijuana users than is described in the current study,Chang et al.reported lower glutamate levels in the basal ganglia,suggesting that heavy marijuana use during young adulthood as well as later in life is associated with disruptions in glutamate signaling as has been shown for other drugs of abuse.Recently,Prescot et al.reported lower glutamate concentrations in the anterior cingulate cortex,which was nonetheless strongest when females were eliminated from the analysis.Interpretation of the current findings is complicated by poor resolution of the glutamate versus glutamine signal.Glutamate is present in all cell types with the largest pools evident in glutamatergic neurons; smaller pools are evident in GABA-ergic neurons and astroglia.
Upon release,astroglia convert glutamate to glutamine,which in turn is transferred back to the neuron for conversion once again to glutamate.Glutamine is primarily located in astroglia.Thus,low glutamate levels would be difficult to ascribe to a particular neuronal process.In contrast,if glutamine levels are low,then glial dysfunction may be present,a finding that would be consistent with white matter aberrations in marijuana users.Others have not reported specific metabolic disruptions in female marijuana users; indeed,within young samples,marijuana is more commonly used in males.Although it has been recognized that females are at an increased risk for some behavioral consequences of drug use such as sexual risk-taking and an increased risk of depression and anxiety following a pattern of daily marijuana use,sub irrigation cannabis there are relatively few human studies of brain-based sex differences associated with marijuana.McQueeny et al.showed adolescent girls had larger amygdalae and increased internalizing symptoms when compared to both control and marijuana using boys.Moreover,certain behavioral problems have also been linked to prenatal marijuana exposure in girls,but not in boys.Recent neuroimaging work suggests that young female users may be vulnerable to marijuana-induced alterations in brain volume,given suggestions of greater prefrontal cortex volumes and relatively poorer levels of executive function.Alcohol is similarly disruptive to females’ cognitive function and regional brain morphology,and it has long been recognized that females are more vulnerable to psychomotor sensitization with psychostimulant exposure.Preclinical data are somewhat stronger and indicate that female adolescents are particularly vulnerable to the effects of long-term THC administration on the CB1 receptor system in multiple brain regions,including the prefrontal cortex,striatum,and periaqueductal gray.A recent study of THC in mid-adolescent rats during the period of drug administration and following abstinence indicated greater sensitization of THC-induced locomotor depression in females versus males.Moreover,high doses resulted in increased anxiety-like behaviors during THC administration,particularly in females,although a general tendency is for females to experience greater anxiolytic effects of the drug.Glutamate is critically important in the neuroplasticity that accompanies the transition from drug use to abuse.Under conditions of extreme trauma or stress,its release is associated with neurotoxicity and cell death.Endocannabinoids block glutamate release under such conditions,which could lead to neuroprotection.However,the concomitant observation of high mIns levels argues against this interpretation.Given that mIns is considered to be a glial marker,high levels would be associated with gliosis as well as white matter injury as occurs in the context of neural injury.High mIns concentrations have been observed in early dementia,in frank Alzheimer’s disease,as well as in abstinent methamphetamine users,although this latter observation was in the frontal lobes.This pattern is intriguing given that deficits in learning and memory represent one of the robust areas of reported cognitive dysfunction in marijuana users.Although our data analyses do not suggest that female marijuana users in this sample are more vulnerable to cognitive impairments,this is a relatively young and high functioning sample.It may be that frank behavioral deficits will emerge more strongly in females over time as chronicity of use progresses.We hypothesize,too,that we may have observed altered NAA levels had we also measured frontal concentrations of each metabolite.Even though our statistical analyses do not show any significant effect of alcohol,it is important to consider the possibility of an underlying biological interaction between the two substances.Male marijuana users in this study had the highest levels of alcohol use,but did not show significant neurochemical alterations relative to controls.
Females showed the greatest apparent impact of marijuana use on Glx and mIns,but in the context of lower levels of alcohol use.These findings could suggest a neuroprotective effect in individuals who use both marijuana and alochol,as described by others.Alternatively,previous work has shown greater levels of Glx in the anterior cingulate of chronic alcohol users relative to controls.Considering this,taken together with the findings of the present study,it is possible use of the two substances together may drive metabolite concentrations to “normal” levels via opposing processes,as has also been suggested by others in the context of brain morphology.Differences in metabolic function in heavier versus lighter alcohol users can also impact the conversion of acetate into glutamate.It is possible,then,that the male marijuana users in this study who were heavier alcohol users as compared to females,demonstrated differences in glutamate metabolism,contributing to the observed sex difference.However this assertion is only speculative.While our data do not fully support these conclusions,the issue of alcohol use in the context of marijuana use requires careful examination in future studies.Sex but not group-related effects were also observed in total choline estimated concentrations.Independent of marijuana use,males showed higher estimated concentrations of tCho compared to females.