Voxel-wise statistics were corrected at the cluster-level using Threshold-Free Cluster Enhancement to a Family-Wise Error rate of p b 0.05, with 5000 permutations. Nevertheless, as a further control for heritability, we conducted follow-up analyses by randomly selecting one user from each family such that there were no siblings within the sub-sample, resulting in 272 marijuana users. Identical analyses were performed using the full user sample and the unrelated sub-sample. These analyses were conducted using FSL’s randomise package, which performs permutation methods, without using exchange ability blocks. Following the PALM analyses, 5000 permutations were performed. Likewise, voxel wise results were corrected using Threshold-Free Cluster Enhancement to a FWE-corrected p b 0.05.We further processed the pre-processed diffusion images using FSL’s FDT toolbox and Tract-Based Spatial Statistics . Briefly, the diffusion tensor model was fit at each voxel yielding maps for fractional anisotropy , axial diffusivity , radial diffusivity , and mean diffusivity . These maps were then nonlinearly aligned to the FMRIB 1 mm FA template and skeletonized. This last step creates a skeleton which represents the centers of all fiber bundles that are generally common to all participants in the study. The skeleton was thresholded at FA b 0.3. The skeleton and nonlinear registration parameters were generated using the FA maps, and these transformations were subsequently applied to the other diffusion parameter maps. Voxel-wise statistics were then performed in this skeleton space.The effect of marijuana use on white matter was assessed with a linear parametric analysis across level of marijuana use. Participants reported a wide-range of levels of marijuana use in terms of age of first use as well as the number of times used over the lifetime, as shown in Fig. 1. Age of first use and times used were included as factors of interest and, as described above, all analyses included alcohol and tobacco use, age, gender, years of education, and Total Motion Index as co-variates. For a table of correlations between all co-variates see Table 1.Analyses of voxel wise gray matter morphometry were carried out with FSL-VBM an optimized VBM protocol carried out with FSL tools . First, structural images were brain-extracted and gray matter-segmented before being registered to the MNI 152 standard space using non-linear registration.
The resulting images were averaged and flipped along the x-axis to create a left-right symmetric,trimming tray study-specific gray matter template. Second, all native gray matter images were non-linearly registered to this study-specific template and “modulated” to correct for local expansion due to the non-linear component of the spatial transformation. The modulated gray matter images were then smoothed with an isotropic Gaussian kernel with a sigma of 3 mm. Finally, voxel wise GLM was applied using permutation-based non-parametric testing, correcting for multiple comparisons across space, using Threshold-Free Cluster Enhancement . Following Weiland et al. , we also performed a multivariate analysis on the effects of marijuana use on sub-cortical and cortical volumes and cortical thickness extracted with FreeSurfer.Rather than analyzing whether marijuana showed a multivariate effect across all 35 cortical regions contained in this table as did Weiland et al. , we chose an a priori approach, focusing on prefrontal regions and sub-cortical regions where marijuana has been shown to have significant effects . Regions of interest included 15 prefrontal cortical regions : medial and lateral orbitofrontal cortex, caudal anterior cingulate, caudal middle frontal, inferior frontal gyrus , rostral middle frontal, superior frontal, and frontal pole. Subcortical regions included nucleus accumbens, hippocampus, cerebellum cortex and white matter, thalamus, and amygdala. White matter volumes were included for the anterior and mid anterior corpus callosum.Shape analyses of sub-cortical ROIs were carried out using FSL’s FIRST tool . FIRST allows for a model-based segmentation and registration of anatomical images, where volumetric labels are parameterized as surface meshes.Vertex locations from each participant were projected onto the surface of the average shape , resulting in scalar projection values. This approach normalizes for brain size.
The same models of marijuana use generated for the white matter analyses were used here. Again, PALM was used to perform multi-level block permutation analyses. In order to identify which, if any, sub-region of the hippocampus, amygdala, or nucleus accumbens exhibited a shape difference, we referred to the Jülich histological atlas contained in FSL. Because FIRST only reports changes at the surface of the structure and does not contain information about which deep levels of a structure are impacted, these sub-region labels represent approximations.We then examined the effects of marijuana use on white matter diffusion parameters . The group comparison showed no significant effects, possibly suggesting that the frequency of marijuana use in the HCP sample is not severe enough to replicate previous studies, which largely focused on comparisons of non-users and daily marijuana users. In line with this possibility, there were no linear effects of the number of times used on white matter coherence in users. As shown in Fig. 2, age of first use had a positive association with FA as well as a negative association with RD, such that an earlier age of first use was associated with lower FA and greater RD in a large cluster of right hemisphere white matter. These tracts primarily subsisted of the Superior Longitudinal Fasciculus , Inferior Longitudinal Fasciculus , and Forceps Major and Minor. The SLF connects the prefrontal cortex and parietal cortex and is involved in executive functions , and the ILF connects the temporal and occipital cortices, has been shown to affected byadolescent marijuana abuse . The Forceps Major and Minor are extensions of the corpus callosum connecting the left and right occipital and frontal lobes, respectively. Thus, even though most of the effects on FA and RD were found in the right hemisphere, communication between the left and right hemispheres may be impacted by marijuana age of onset.When examining the subset of unrelated marijuana users, we confirmed the negative effect of an earlier age of first use on FA and RD in the SLF, as shown in Fig. 3. Increased RD has been related to demyelination in neurodegenerative diseases such as multiple sclerosis , suggestive of a neurotoxic effect with early age of first use of marijuana. These findings taken together, and in line with previous studies , suggest that an earlier age of onset is associated with decreased coherence of white matter. Moreover, these results suggest that marijuana use has an impact on the development of these white matter tracts during adolescence.We then investigated whether the shape of the hippocampus, amygdala, or accumbens was impacted by marijuana use. For the full sample of users, the results are shown in Fig. 4. There was a trend for a significant negative relationship between the number of times used and scalar values in the left amygdala .
Participants with a higher number of times used showed more inward deflection in the superficial group of the left amygdala. In the right nucleus accumbens, there was a significant positive relationship between age of first use and shape, such that participants with an earlier age of onset of use showed greater outward deflection. This finding suggests that a later age of first use is related to more outward deflection of the right anterior nucleus accumbens. In the left hippocampus cornu ammonis and posterior hippocampus subiculum, a greater number of times used was associated with greater inward deflection . The effects of the number of times used marijuana in the restricted sample closely mirrored those in the full sample. In the left superficial amygdala there was a trend for participants with a higher number of times used to show more inward deflection . Likewise, the same pattern was observed in the left hippocampus cornu ammonis . For age of first use, however, there was the opposite pattern , albeit in the left posterior nucleus accumbens , suggesting that a later age of first age is associated with a more inward deflection of the left posterior nucleus accumbens. This latter finding is similar to that observed by Gilman et al. who found that more frequent use was associated with an inward deflection of the nucleus accumbens. When comparing users and non-users,trim tray pollen significant effects on shape were discovered in the right amygdala and left hippocampus. There were two clusters in the right amygdala, one in the dorsal superficial group and another in the ventral laterobasal group, that showed more outward deflection in users compared to non-users. Similarly, in the left ventral anterior hippocampus , users showed more outward deflection compared to non-users. This region was more ventral and anterior to the cluster in the subiculum that showed a negative association with times used in users. These group results are somewhat surprising, as the correlational analyses within users showed inward deflections in more heavy users. However, the left and right nucleus accumbens also showed opposite patterns of inward and outward deflections with greater use, respectively. Thus, one possibility is that marijuana has differential effects in the left and right hemispheres. Nevertheless, Gilman et al. found more inward deflections for heavy users in both left and right nucleus accumbens and the right amygdala.Despite a trend of de-criminalization and softening societal views, the scientific literature on the effects of marijuana on the brain has not yet reached a consensus. Work with adolescents has consistently shown that heavy marijuana use leads to disruptions in the integrity of white matter . However, work investigating the effects of marijuana on the morphometry of gray matter and sub-cortical regions has yielded inconsistent findings, with several papers even providing strong evidence that heavy marijuana has no effects on brain morphometry . The current study took a broad look at this question using a multi-modal neuroimaging approach on data from almost 500 participants reporting varying levels of recreational marijuana use. These data from the HCP include the highest quality neuroimaging data publically available, with almost twice the resolution for structural images , and using state of the art diffusion imaging . This large dataset enabled us to investigate parametric associations between marijuana use and cortical and sub-cortical brain morphometry and white matter integrity. An advantage of this dataset is that it represents a representative community sample, with great variability in race,ethnicity, gender, education level, mental health symptoms, and drug use . Moreover, such ‘big data’ sets like the HCP are becoming more and more commonplace , and it will be important to mine these datasets to discover new insights about the functions and organization of the human brain. Importantly, this data set provided information on age of first use providing information on whether or not use occurred during early adolescence. Adolescence is a time of critical brain development for white matter tracts, with white matter volume increasing into young adulthood . Heavy marijuana use during this critical period may impact development , and specifically, a number of frontal and association white matter tracts show decreased white matter integrity with heavy adolescent use. Recently, Becker et al. found that over the course of two years, young adults with an adolescent onset of marijuana use showed reduced longitudinal development of FA in key frontal, central, and parietal white matter tracts. The current findings are largely consistent with those reports. We found that an earlier age of first use was associated with decreased FA and increased RD in long-range tracts including the forceps minor, Superior Longitudinal Fasciculus, and Inferior Longitudinal Fasciculus. Given that the average age of participants in the current study was 29.2, the effects of marijuana on white matter appear to be long lasting, persisting 10–20 years after the first use of marijuana. Future, studies should examine the effects of duration of use on the trajectory of brain development. With regards to shape, a recent meta-analysis has suggested that the sub-cortical structure most consistently implicated in showing an effect of marijuana use is the hippocampus . Consistent with that finding, we observed that anterior aspects of the hippocampus showed shape differences due to the number of times marijuana has been used. This finding is interesting as anterior and posterior sub-regions of the hippocampus have dissociable roles in memory, with the anterior portion being involved more in memory encoding and the posterior portion being involved more in memory retrieval ; however, an exploratory analysis revealed no connection between hippo-campal or amygdala shape and memory performance in these participants .