In addition, our experiment found that cerebral organoids may be used to model neuroinflammation. This is an unexplored avenue of research and may warrant the use of organoids to study neurological diseases associated with neuroinflammation. Previous findings related to METH research on brain development were done with mice models and found that prenatal METH exposure in mice has led to a decrease in bodily growth, learning capabilities, and increased aggressive behavior. These observations are behavioral and cannot be observed through our experiment. In mice, METH has also been known to promote neuroinflammation and neurodegeneration, which is consistent with our results. Human brain organoid models show the same results, strengthening the claims that neuroinflammation and neurodegeneration are likely consequences of METH exposure.Alternatively, the neuroinflammation and neurodegeneration that we found may also be due to the lack of microglia in our cerebral organoids. Currently, organoids produced from human induced pluripotent stem cells lack microglia which are an integral part of the brain’s immune system. These cells play a key role in maintaining the brain’s homeostasis and development. Therefore, it is important to consider how microglia’s role is altered in METH treatment as well. Recently, there have been advancements in organoid research and it is possible that they may contain endogenous microglia or microglia may be added via a co-culture. It would be beneficial to test the effects of the same drugs with cerebral organoids that have microglia incorporated for a more holistic understanding of the effects of METH treatment on the developing brain. Moreover, as organoid research develops, it be may interesting to try the same experiment on whole-brain organoids that have been vascularized,cannabis growing supplies which may be a better model because it is even more similar to the developing brain. In THC, we saw that our organoids represented a diverse array of neural cell types found in the developing brain .

Due to the discrepancy in cell counts per organoid, we decided to focus on cell types that had more even counts in our data when comparing THC and control organoids. Thus, there were three clusters of interest GFAP+ astrocytes, proliferative neural progenitor cells, and neurons that had more even cell counts .When looking at neurons, we decided to focus on neural sub-types to see if THC treatment was causing any changes there. Clustering the neuronal population again revealed a population of glutamatergic, GABAergic, and an unidentified cluster of SOX11+ and MEIS2+ cells . Pathways related to neurogenesis were found to be up-regulated as a result of THC treatment with some notable genes including NEUROD2 and NEUROD6 . These genes are associated with neurogenesis and were up-regulated in THC glutamatergic neurons, suggesting that THC treatment is affecting this neuronal sub-type in some way, perhaps by leading to its preferential differentiation towards glutamatergic fate via NEUROD6. THC glutamatergic neurons also expressed CNR1 at a higher level than control organoids . Typically, CB1 expression has been shown to decrease as a result of chronic THC exposure, however, we see an increase in CNR1 especially in glutamatergic neurons further suggesting THC may be altering this neuronal subset. The cluster of cells that did not express some of the key glutamatergic markers, does express NEUROD2, NEUROD6, and CNR1 which may indicate that this population may also differentiate into glutamatergic neurons. These findings suggest that THC treatment may lead to differentiation into glutamatergic neurons in the developing brain. These results, however, are based on a small population of cells and would need to be functionally validated in vivo to confirm that this phenomenon does occur.

The field of single-cell RNA-sequencing analysis is constantly evolving. Recently, new tools and packages have been made to address the issue of ambient RNA. A new package by the name of DecontX has recently been used to remove the ambient RNA contamination before downstream analysis, so this may a novel way to address the problem of contamination that we faced in the THC dataset48. In the future, it may also be interesting to explore other single-cell analysis techniques to see if results differ. We use Chromium, a 10x single-cell RNA-sequencing technique, which sequences from just the 3’ end of RNA, however, there are many other methods such as Smartseq2 which provide full-length transcripts49. Full-length transcript may allow for increased sensitivity and a higher level of detail about each cell. This increased sensitivity may provide more information about more minute changes in gene expression. All in all, this study has provided valuable insights into the effects of METH and THC on the developing brain via cerebral organoids, a topic that is difficult to study otherwise. This model and the results of this experiment may be used to properly advise pregnant women about the effects of METH and THC use on fetal brain development. Substance use emerges and greatly increases across adolescence, and early substance use and more frequent use are risk factors for substance use disorders in adulthood . In 2018, the percentage of students who had used an illicit drug doubled from 8th to 10th grade, and nearly half of students reported using at least one substance by 12th grade. High substance use in adolescence has been reliably linked with substance use problems and addiction in adulthood . Converging evidence from cross-sectional and prospective studies has indicated that earlier use of alcohol, tobacco, and marijuana during adolescence is a risk factor for more problematic substance use and substance use dependence . Furthermore, youth who engage in substance use broadly have more problems with respect to academics, social function, psychopathology, and the legal system both throughout adolescence and during adulthood . Therefore, it is important to identify youth at heightened risk for substance use. Although stress and distress are consistent risk factors for substance use, few studies have examined whether stress reactivity is related to substance use among adolescents.

Adolescents show neurobiological development, and substances can influence one’s neurobiology by altering neural reward circuitry to promote substance dependence and addiction . This substance-induced neural rewiring may be most notable during adolescence, as adolescents experience neurodevelopment and already show increased reward sensitivity relative to children and adults . Excessive substance use can also reduce executive function and attentional control . These deficits may cause youth to struggle with academics,cannabis indoor growing and academic motivations and school engagement provide an important buffer for substance use . Substance use can also manifest in social changes which further promote dependence and risky behavior. For instance, substance use has been linked with increases in impulsivity and sensation seeking and greater involvement with deviant peers during adolescence . Because youth cannot legally acquire substances, youth may need to identify deviant peers to obtain access, and identification with deviant peers can promote further substance use . In this way, early substance use can prompt youth to engage with others who promote delinquent behaviors and poorer adjustment . Adolescents may use substances for varied reasons related to stress. For instance, common motives include to enhance their social and emotional well-being, to reduce distress, and to conform to peer pressure . Just as substances can influence emotion, emotional states can influence adolescents’ inclination to engage in substance use. For instance, substance use is higher on days when individuals experience higher levels of positive emotion as well as negative emotion . Similarly, daily experiences can also influence one’s proclivity for substance use. Daily stressors are generally followed by increased negative emotion and decreased positive emotion, and people turn to substances to mitigate these emotional responses. This may be particularly the case during adolescence, a period when youth experience more frequent stressors in varied domains as well as enhanced reactivity to stress relative to children and adults . Further, adolescents experience greater variability and intensity in emotion relative to adults, such that stressful experiences may be especially distressing . Heightened emotional responses with reduced capacity for regulation can contribute to increased psychopathology broadly during adolescence, including substance use . In addition to emotional responses, stress is followed physiological changes from biological systems that naturally respond to stress. Physiological responses often do not correspond to self-reported emotion, and profiles of psychological and physiological responses together or interactions between systems can impart unique information about adolescents’ experiences with stress . Two physiological systems that may aid in the identification of at-risk youth are the hypothalamic pituitary adrenal gland axis and the autonomic nervous system. Adolescents become more sensitive to social stress following pubertal onset, and the HPA axis is sensitive to social-evaluative stress . The two branches of the autonomic nervous system—the sympathetic nervous system and parasympathetic nervous system —jointly alert the body to threat, and they have also been found to relate to impulsivity and emotion regulation, traits relevant for substance use . People with blunted SNS activity tend to be more impulsive and people with blunted PNS reactivity have poorer mental health and self-regulation . Although blunted activity across all systems is linked substance use, limited work has examined whether individuals with dysregulation across systems are at greater risk for substance use. Limited work has assessed how stress responses reactivity can relate to substance use and behavioral problems concurrently and prospectively.

However, among youth with depression, conduct problems, or no psychiatric condition, Brenner and Beauchaine have found that greater increases in SNS activity in response to reward were associated with delinquency, both externalizing and internalizing problems, and anxiety and depressed mood . Moreover, they found that lower SNS reactivity to reward predicted greater alcohol use four and six years later among middle school children . Yet, no work has assessed links between psychophysiological responses and substance use in middle or late adolescence. Although substance use often begins in mid-adolescence and increases in frequency during late adolescence, limited work has examined associations between stress responses and substance use onset and frequency in adolescence. Early identification of at-risk youth is imperative as earlier substance use is linked with greater severity of use in adulthood . Therefore, this dissertation investigated how stress responses relate to lifetime substance use in middle adolescence among youth with high adversity backgrounds and frequency of substance use in late adolescence among a community sample across middle and late adolescence . Associations between emotional and cortisol reactivity to stress at age 14 and lifetime substance use at ages 14 and 16 were assessed in Study 1, as well as differences in associations by gender and poverty status. Associations between SNS and PNS reactivity to stress, as well as profiles of responses across the two systems, at age 14 and substance use at ages 14 and 16 will also be assessed in Study 2. In both studies, sensitivity analyses were limited to adolescents who had never used substances by age 14 to determine whether stress responses were related to use versus initiation specifically. These studies will highlight whether stress reactivity can prospectively predict substance use onset and have utility for identifying at-risk youth. In contrast, Study 3 assessed whether daily emotional reactivity, as opposed to acute reactivity, related to substance use count and frequency of alcohol and marijuana use in late adolescence, as well as whether associations differed by gender. By better understanding pathways that contribute to heightened predisposition for adolescent substance use and which groups of adolescents are at heightened risk for substance use, treatments can be tailored to address aspects of stress regulation among specific populations of at-risk youth. Substance use initiation greatly increases across adolescence . Youth with greater internalizing and externalizing problems tend to show high risk for substance use, and differences in the activation of to two key stress response systems—hypothalamic pituitary adrenal axis and emotion—have been related to both . However, limited research has examined whether differences in the biological and psychological responses to stress, with respect to changes in cortisol secretion and emotions following stressor onset and across a recovery period, relate to substance use among adolescents, especially those at heightened risk for substance use.