Gilman et al. used a similar multi-modal approach and found that gray matter density , shape , and volume of the left nucleus accumbens was significantly different for recreational users and controls. More specifically, in users, the left and right accumbens showed a negative relationship between shape and marijuana use frequency, such that the accumbens showed more inward deflections with more frequent recent use, as well as greater volume. However, in an attempt to replicate these findings, Weiland et al. found that the results of Gilman et al. held only when participants were not matched in terms of alcohol use. When matched for alcohol use, marijuana users and controls showed no significant effects in terms of cortical or sub-cortical morphometry. Moreover, when Weiland et al. examined the effect sizes observed in 11 prior studies, they found a net zero effect for the effects of marijuana on brain morphometry . Notably the results of the current study take into consideration the potentially confounding factors of alcohol and tobacco use as well as gender, age, and years of education. A recent paper using some of the same sample from the HCP examined how genetic vs. environmental factors might contribute to brain volume as a function of marijuana use. They found that marijuana use was associated with smaller volume of the left amygdala and right ventral striatum. However, their analyses suggested that the results for the amygdala are likely driven by shared genetic factors as compared to environmental factors as both marijuana user twins and their non-user twin showed reduced volume compared to concordant non-user twins. While the current study did not directly examine the role of genetics and shared environment in mediating the effects of marijuana on the brain, our analyses accounted for family structure. When accounting for family structure, we found no effects of marijuana on the amygdala, and a trend for a more inward deflection of the right accumbens, but no accompanying difference in volume. However, the analysis of Pagliaccio et al. trim bin tray was limited in sample size for certain sibships, in particular, monozygotic twins discordant for marijuana use.

With the upcoming complete HCP dataset consisting of 1200 participants, it will be important to update the analyses of Pagliaccio and colleagues, as well as adding sub-cortical shape as a measure, to see if a causal relationship arises with a larger sample .As the current study is not longitudinal but rather cross-sectional, it cannot speak to whether the use of marijuana causes changes in neural structures. Such an analysis will require longitudinal data as is to-be collected by the new national ABCD study funded by NIH. Nonetheless, the current study can provide hints as to potential effects of marijuana use due to its large sample size and because family structure was controlled for in the analyses. In addition, despite the 100-fold increase in the number of marijuana users from most studies and the high-quality of imaging data, the data on marijuana use history from the HCP is relatively sparse. Whereas alcohol and tobacco were assessed in terms of recent and past use, questions of marijuana use were restricted to self-report measures of the age of first use and the number of times used in the lifetime. As such, it was not possible to accurately examine the effects of duration of use or more specifically, the effects of time since last use. As noted in the Materials and methods Section, the age of first use and number of times used data was coarsely coded using relatively arbitrary ranges. In particular, the number of times used score presented widely different categories for participants to select, ranging from 1–5 times used to “more than 1000.” Moreover, no data was available regarding the recency of this use. Additionally, while tobacco and alcohol were controlled for using scores selected to best represent the impact of chronic, co-morbid substance use, it is possible that alternative metrics would change the representation of variance due to these substances. As it stands, the alcohol and tobacco use scores used in this presented significant co-variance with age of first use and times used, highlighting both the need to control for these factors and the importance of a data-set large enough to separate the effects of each variable.

While participants were excluded from the HCP for major psychiatric or neurological illness participants underwent a psychiatric screening as part of the SSAGA, and psychiatric symptoms were assessed with the NIH Toolbox and the Achenbach Adult Self-Report questionnaire . Examining the interactions of psychiatric symptoms and marijuana use was beyond the scope of the current study, but future studies should examine these effects. Previous studies have shown that adolescent marijuana use is co-morbid with a number of psychopathologies including childhood trauma , depression , attention deficit hyperactivity disorder , and psychosis . Furthermore, the HCP contains information about parental psychopathology. While much of the psychiatric information available in the HCP has less information than a targeted study of psychopathology, there is enough information for future studies to assess multivariate effects of marijuana use variables and co-morbid psychopathology and other substance use. Lastly, while the advanced imaging analyses used in this study provide powerful ways to non-invasively understand the anatomical changes occurring with a brain, they are limited in that they cannot speak to the mechanisms whereby marijuana use might influence brain structure. specifically, they cannot elucidate the microscopic changes responsible for the more macroscopic GM and WM impacts . For example, while shape changes of the accumbens and hippocampus might reflect inflammation, marijuana has been found to have anti-inflammatory properties . Macroscopic morphological changes could be caused by neuronal loss or changes in cytoarchitecture such as neuronal size, dendritic spine density, dendritic length, or synaptic protein levels . As such, morphometry studies can strongly inform where such changes are occurring, but cannot pinpoint the microscopic causes of these structural changes. It is important to note that the two major components of marijuana, Δ-9-tetrahydrocannabinol and cannabidiol , have opposite effects behaviorally, symptomatically, and in terms of functional activation of all of the regions-of-interest for the current study . With legalization of marijuana comes more accurate assays of THC and CBD concentrations, and thus, future research can and should focus on examining whether THC and CBD have dissociable effects on brain morphometry .Parkinson’s disease is the second most common neurodegenerative disorder worldwide, affecting 1e2% of the population over the age of 65.

The condition is characterised by the progressive loss of dopaminergic neurons from the substantia nigra pars compacta.There are a number of studies that link the development of PD with the exposure of certain pesticides such as rotenone.As a result, rotenone is commonly used to create in vivo and in vitro models to study the disease.H2O2 is a compound commonly used to model oxidative stress in vitro and in vivo. As mitochondrial dysfunction and oxidative stress are thought to contribute to cell death in PD, we aimed to assess the effects of both rotenone and H2O2 on SH-SY5Y neuroblastoma cells. The SH-SY5Y neuroblastoma cell line has been previously used to create a cellular model of PD.The cells share many biochemical and functional characteristics with mature dopaminergic neurons and have the ability to differentiate into adopaminergic phenotype. As tyrosine hydroxylase and dopamine seem to be central to the pathogenesis of PD and dopaminergic neurons are specifically targeted in the condition, we opted to utilise a cell line that had been transfected with human TH isoform 1.There were two main aims to be addressed in this study, firstly we aimed to compare the effects of rotenone and H2O2 treatment on cell viability and TH expression and once we had established these changes we would then assess the ability of a number of potentially neuroprotective compounds to protect against this toxicity. Cinnamon is a spice commonly used in food throughout the world. The spice has been demonstrated to have anti-diabetic and anti-inflammatory effects as well as some neuroprotective properties.For instance a previous study demonstrated that treatment with cinnamon prevented the development of PD like symptoms and pathology in 1-methyl-4-phenyl-1,2,3,6-tetra hydropyridine treated mice, however the effect of cinnamon on rotenone is yet to be investigated. Hemp seed and its oil have been used as both a food and medicine in China for at least 3000 years and hemp seed extracts have been found to demonstrate antioxidant and antiaging effects as well as improve cognitive impairment induced by chemicals in mice.In addition to all of this, epidemiological studies suggest societies that commonly use curcumin, cinnamon and hemp seed appear to demonstrate a lower incidence of PD and neurodegenerative disorders.We included the use of curcumin within our study as a positive control as this substance has been shown previously to provide protection against rotenone and H2O2 toxicity.Polygonum cuspidatum is widely distributed in the world and has been shown to possess antiviral, antimicrobial, anti-inflammatory,pollen trim tray neuroprotective, and cardioprotective properties,however these properties are yet to be investigated using a cellular model of PD.

In this study we assessed the effect of rotenone and H2O2 on SHSY5Y cell survival and TH protein expression. We also evaluated the protective effects of curcumin, cinnamaldehyde, and constituents isolated from hemp seed and polygonum cuspidatum .Methylthiazolyldiphenyl-tetrazolium bromide powder was used as a means to assess cell viability as previously described.Briefly, at the conclusion of the 24 h treatment duration, MTT was added to culture medium at a final concentration of 0.5 mg/ml and the plate was mixed gently for 1 min before 2 h incubation in a 37 C, 5% CO2 incubator. After the incubation, media was removed and 100 mL of DMSO was added to each well for 10e15 min while shaking. The intensity of the purple colour produced in each well was measured colourimetrically using a plate reader at 595 nm. The values of absorbance are expressed as a proportion of the controls.The Trypan Blue assay was used as another means of visually assessing cell viability to support the MTT assay findings. Cells were seeded into 12 well plates and treated as described above. When the treatment protocol had ended the media was removed from the wells and 30 mL of Trypan Blue was added and left for 30 s. After 30 s an image of the plate was taken using ‘Cell Pad’. While not quantitative this protocol provided a means of visually assessing the viability of cell cultures.Whole cell lysates were used for western blotting experiments to analyse total TH and Poly-ADP ribose-polymerase protein levels. Cells were seeded in a 24 well plate and treated as described above. At the end of the treatment protocol media was removed and 110 mL of 2% SDS stop buffer with inhibitors , 2% SDS, 2 mM EDTA, 1 mM Na orthovanadate, 1 mM Na fluoride, 10 mM Na pyrophosphate was added. The lysed cells were collected and heated for 10 min at 100 C. Samples were then frozen and stored at -20 C for later analysis. Samples were prepared for electrophoresis by dilution with sample buffer . Samples were run on an 8 or 10% SDS-polyacylamide gel and transferred to nitrocellulose membrane . To minimise non-specific binding membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.075% tween 20 for 1.5 h at room temperature. Membranes were then incubated with anti-PARP-1 or anti-total TH antibodies for 1 h at room temperature. Blots were washed and exposed to appropriate secondary antibody for 1 h at room temperature. Blots were then exposed to enhanced chemiluminescence detection reagent for 1 h and developed using a LAS 4000 imaging system . Later, membranes were washed and then immunoblotted with b-actin antibody as a marker of the total protein loaded per lane.Quantitation of tTH and PARP-1 were normalised relative to b-actin levels.This study demonstrates that rotenone and H2O2 have markedly different effects on SH-SY5Y cells. H2O2 treatment does not appear to have any significant effect on TH protein expression and the cell death induced by H2O2 can be prevented by a number of compounds tested. In contrast, rotenone treatment was associated with an increase in TH protein levels and its toxicity could not be prevented with any of the compounds.