A cannabinoid refers to any natural or synthetic compound that interacts with the CB1 and CB2 receptors. There are currently three different groups of cannabinoids: endogenous cannabinoids, phytocannabinoids and synthetic cannabinoids. The most common phytocannabinoid is delta-9-tetrahydrocannabinol (THC), which is the active component in the Cannabis sativa or marihuana plant1–3. Two examples of synthetic cannabinoids that are present in case reports from 2012 to 2018 are AB-FUBINACA and AB-PINACA4–7. THC and synthetic cannabinoids are commonly encountered drugs in forensic toxicology cases, therefore, being able to extract these compounds and their metabolites is imperative for toxicological interpretation. There are a variety of commercially available sample preparation techniques for these analytes. Companies such as UCT, Biotage, Millipore-Sigma, Tecan, and Thermo Fisher Scientific manufacture these products. The focus of this research was to evaluate these techniques for their cleanliness, efficiency and cost effectiveness. Sample preparation techniques are designed to remove the different components of the matrix and other prescription or illicit substances present in the sample that could interfere with the assay, increase the analyte recovery, extraction efficiency, decrease variability, and clean-up the sample to allow for less instrument downtime and longer column life8. This study focused on comparing a liquid-liquid extraction (LLE), solid phase extraction (SPE), and supported liquid extraction (SLE). The primary purpose of this study was to develop and validate the three above mentioned sample preparation techniques for the analysis of THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC (THCCOOH), AB-FUBINACA, AB-FUBINACA metabolite 3, and AB-PINACA in blood and urine. Parameters assessed followed Academy Standards Board (ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology, including recovery, suppression, and matrix effects. For urine and blood analysis, the calibration range was determined to be 1 ng/mL to 50 ng/mL for all three techniques. Urine recovery was highest for the LLE method, with all compounds having a recovery greater than 50%. The SLE method had the lowest LOQ results for urine, with 0.5 ng/mL for 11-hydroxy-THC and THCCOOH, 0.75 ng/mL for THC, AB-FUBINCA and AB-FUBINACA metabolite 3, and 1 ng/mL for AB-PINACA. Ion suppression was reduced using the SLE method for urine along with having the shortest sample preparation time of 1 hr for up to 48 samples. For blood analysis, the LLE method had the greatest recovery of all analytes. The LLE method also had reduced suppression and matrix effects compared to the SPE method. Sample preparation was shorter for the SPE method, consuming 2 hrs for an average sample batch, compared to 4 hrs for the LLE method, which included a 2 hr freezing step. In conclusion, for urine analysis, all three sample preparation techniques were acceptable for the analysis of THC, synthetic cannabinoids, and their metabolites, with the SLE method being the preferred method. For blood analysis a LLE and SPE method were developed and are adequate for the analysis of THC, synthetic cannabinoids, and their metabolites, with the LLE method being the preferred method.

Although cannabis is widely consumed by humans for the intoxicating effects that are mediated by delta-9 tetrahydrocannabinol (THC), pre-clinical models of THC self-administration have been difficult to establish. We hypothesized that THC may have reinforcement enhancing effects comparable to other drugs (e.g., nicotine and caffeine), which are also widely consumed by humans but difficult to establish as primary reinforcers in non-human animals. To investigate whether THC is a reinforcement enhancer, male (M, n=8) and female (F, n=8) rats were shaped to self-administer a reinforcing saccharin (SACC) solution (0.2% w/v) in standard operant chambers equipped with infrared beams to monitor locomotor activity. At baseline, we found a significant sex difference for active lever responses and reinforcers earned (F

Indiana University-Purdue University Indianapolis (IUPUI) / Although marijuana and alcohol are two of the most commonly used drugs in the United States, relatively little is understood about how these drugs interact to effect drug use, cognitive behaviors, and neurophysiological changes. Specific drug use patterns such as simultaneous use may produce differential effects for consumption and other behaviors in addition to unique neurobiological changes compared to singular drug use. In order to better understand the effects of simultaneous alcohol and marijuana (SAM) use, we used the selectively bred crossed High Alcohol Preferring mice to examine consummatory, cognitive, and neurobiological changes following chronic alcohol and THC self-administration. We hypothesized that SAM mice would consume more drug than animals exposed to either substance alone. We used an operant behavioral flexibility paradigm to assess cognitive impairments believing that drug-exposed animals would show deficits relative to Control animals, with SAM mice being the most impaired of all drug conditions. Finally, we assessed CB1 receptor changes in the dorsal striatum, as this region is critical for behavioral flexibility (Bissonette & Powell, 2012; Ragozzino, 2007), CB1 receptors are the primary target of THC and these receptors are involved in numerous alcohol related behaviors (Maldonado et al., 2006; Pava & Woodward, 2012). Contrary to our hypothesis, SAM animals did not consume higher levels of drug compared to mice exposed to only THC or alcohol. Interestingly, female THC consumption was robust when THC was consumed alone but was reduced when simultaneous access to alcohol was available. Surprisingly, although we speculated that drug-exposed mice would be impaired compared to Control animals, and that SAM animals would likely be more compromised than THC and alcohol for Reversal Learning and Attentional Set-Shifting respectively, behavioral flexibility deficits were absent in our paradigm. Finally, alterations to dorsal striatal CB1 receptor expression were observed following a Short Abstinence period. Despite an absence of cognitive behavioral effects, this research contributes to furthering our understanding of co-drug use for consummatory and neurobiological changes, both of which are critically necessary given the evolving landscape surrounding simultaneous alcohol and recreational marijuana use.

No description available.

Cannabis is widely consumed by humans for pharmacological effects that are mediated by THC, though there is little evidence that THC is a primary reinforcer in non-human animal models. We hypothesized that THC may have potent reinforcement enhancing effects, comparable to other drugs (e.g., nicotine and caffeine) which are also widely consumed by humans, but difficult to establish as primary reinforcers in non-humans. In three experiments with male and female rats saccharin (SACC) or a visual stimulus (VS) served as reinforcers for operant behavior. We explored several pharmacological parameters of THC on responding for SACC or VS, including THC dose, intervals between THC injections and testing, and intervals between test sessions. THC acts as a reinforcement enhancer for both SACC and a VS across a range of doses and intervals. Daily THC injections systematically reduced behavior, possibly due to accumulation of THC bioavailability.

Mammary gland (MG) is a dynamic organ that is essential for the production and secretion of breast milk. During pregnancy the MG undergoes a critical phase of remodeling, which is accompanied by the differentiation of mammary epithelial cells (MECs). During lactation, the MG requires high level of energy for proper folding of proteins in the secretory pathway, which takes place in the endoplasmic reticulum (ER). Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the differentiation of MECs. However, both THC and CBD have been reported to induce ER stress in various cell types, resulting in impacting cellular function. Furthermore, consequences of cannabis use in the perinatal period on breast milk composition have not been reported. Using the HC11 cell line, we investigated whether THC and CBD evoke ER stress in MECs resulting in the impaired cellular function. We also reported on the effects of cannabis use during pregnancy and lactation on the levels of macronutrients and bioactive factors in the breast milk of cannabis users. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP) and lipid synthesizing enzymes (FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, 10μM THC, 10μM CBD, and the combination of 10μM THC + 10μM CBD significantly induced the expression of ER stress genes in HC11 cells. Furthermore, in the milk of women who used cannabis during pregnancy and lactation, the levels of secretory immunoglobulin A (SIgA) were decreased, relative to non-users. Altogether, our findings indicate that cannabis use in the perinatal period may have implications on breast milk composition and infant health. / Thesis / Doctor of Philosophy (Medical Science) / Breast milk is crucial to the nutrition and immunity of the newborn. It is produced by the mammary gland (MG), which is composed of mammary epithelial cells (MEC) that play a key role in producing and secreting proteins and factors into the milk. During pregnancy, the MG undergoes remodeling which is accompanied by differentiation of the MECs (change from unspecialized to a specialized stage that allows the MECs to produce milk proteins and other factors). The disruption in MEC differentiation can result in altered milk production and composition. Cannabis is used by women during pregnancy and breastfeeding. In this study, we investigated the impact of cannabinoids on the MEC differentiation and examined the effect of cannabis use in the perinatal period on the breast milk composition in humans. We reported that cannabinoids reduced the differentiation of MECs and maternal cannabis use during breastfeeding reduced levels of an essential immune factor.

Background: Cannabis is the third most consumed drug worldwide. Thus, healthcare providers should be able to identify users who are in need for an intervention. This study aims to explore the relationship of acute, chronic, and early exposure (AE, CE, and EE) to cannabis with cognitive and behavioral harms (CBH), as a first step toward defining risky cannabis use criteria. Methods: Adults living in Spain who used cannabis at least once during the last year answered an online survey about cannabis use and health-related harms. Cannabis use was assessed in five dimensions: quantity on use days during the last 30 days (AE), frequency of use in the last month (AE), years of regular use (YRCU) (CE), age of first use (AOf) (EE), and age of onset of regular use (AOr) (EE). CBH indicators included validated instruments and custom-made items. Pearson correlations were calculated for continuous variables, and Student’s t-tests for independent samples were calculated for categorical variables. Effect sizes were calculated for each of the five dimensions of use (Cohen’s d or r Pearson correlation) and harm outcome. Classification and Regression Trees (CART) analyses were performed for those dependent variables (harms) significantly associated with at least two dimensions of cannabis use patterns. Lastly, logistic binary analyses were conducted for each harm outcome. Results: The mean age of participants was 26.2 years old [standard deviation (SD) 8.5]. Out of 2,124 respondents, 1,606 (75.6%) reported at least one harm outcome (mean 1.8 and SD 1.5). In our sample, using cannabis on 3 out of 4 days was associated with an 8-fold probability of scoring 4+ on the Severity Dependence Scale (OR 8.33, 95% CI 4.91–14.16, p < 0.001), which is indicative of a cannabis use disorder. Also, a start of regular cannabis use before the age of 25 combined with using cannabis at least once per month was associated with a higher probability of risky alcohol use (OR 1.33, 95% CI 1.12–1.57, p = 0.001). Besides, a start of regular cannabis use before the age of 18 combined with a period of regular use of at least 7.5 years was associated with a higher probability of reporting a motor vehicle accident (OR 1.81, 95% CI 1.41–2.32, p < 0.0001). Results were ambiguous regarding the role that age of first use and milligrams of THC per day of use might play regarding cannabis-related harms. Conclusions: The relationship among AE, CE, and EE with CBH indicators is a complex phenomenon that deserves further studies. The pattern of cannabis use should be carefully and widely evaluated—(not just including frequency but also other dimensions of pattern of use)—in research (preferably in longitudinal studies) to assess cannabis-related harms.

Adults exhibit significant differences in drug vulnerability, learning ability, and emotive processing than adolescents. Long-lasting synaptic changes, including long-term depression (LTD), can endure many hours and are believed to be key to encoding memories and persistent cognitive changes such as addiction. The ventral tegmental area (VTA) is the primary source of midbrain dopamine (DA) and is regulated by local inhibitory GABA neurons. GABA regulation can decrease, resulting in dis-inhibition of DA neurons and increased feelings of reward, learning, or salience attachment to memories. Endocannabinoids (eCBs) are signaling molecules that often result in synaptic plasticity changes, and the eCB system has shown pronounced cross-talk with opioid signaling and receptor pathways. We examined eCB mediated plasticity of excitatory inputs to GABA neurons within the VTA and how THC, opioids, and age influence this plasticity. We determined that direct CB1 receptor activation in the mouse results in reduced excitatory input activity to GABA, resulting in GABA depression. CB1-mediated GABA depression was lost following chronic exposure to THC, suggesting THC injection has already activated this pathway. Chronic THC occlusion of GABA LTD was then reversible following a week of drug withdrawal. Next, as adult animals tend to exhibit reduced emotive influence, learning, and drug affect when compared to adolescents we examined if LTD of VTA GABA neurons was present in adults. We determined that adult mice no longer undergo HFS-induced LTD. Interestingly, the eCB pathway is still active, as we determined that both CB1 receptor activation and mGluR5 activation still results in GABA depression in adults. We then determined that a greater electrical HFS could induce LTD of excitatory inputs to adult VTA GABA neurons, suggesting a change in adults leading to an increase in induction thresholds for GABA plasticity. Morphine was found to induce similar LTD of GABA neurons through the mu-opioid receptor in both adolescents and adults. This LTD is likely pre-synaptic, similar to THC induced LTD, and is potentially mediated through the same presynaptic pathways as CB1-dependent LTD as injection of either THC or morphine eliminates depression by the other. Chronic morphine injection eliminates HFS-induced LTD in adolescents but only results in a loss of LTD in 58% of adult experiments suggesting resistance to morphine exposure at this synapse in adults. In summary, THC and morphine appear to act pre-synaptically to induce GABA LTD within the VTA and occlude further LTD by the other drug. Additionally, development into adulthood significantly alters the propensity of GABA neurons to undergo plasticity and greater levels of stimulus are required to elicit lasting changes.

The purpose of this integrative literature review was to understand the effects of medical cannabis for chronic pain management in adults. Anecdotal reports suggest the use of medical marijuana as a pain management therapy could be an alternative to opioids and other medications which have long term consequences. Potential uses span the health care continuum, from prescribed outpatient symptom management, to acute care, extended care, home care, and hospice treatment settings. The methodology included a review and synthesis of relevant research articles from 2012 to 2018, written in the English language. The findings suggest medical cannabis has the potential of effectively managing chronic pain in older adults. Adverse effects, if present, are mild and resolve without intervention. Lower doses of medical cannabis were reported to be more effective in treating chronic pain compared to higher doses. Inconsistencies in the efficacy of THC were noted compared to CBD for managing neuropathic pain. Implication for nursing practice, policy, education, and recommendation for future research were discussed along with study limitations.

Background: Cannabis use is highly prevalent among adolescents in Germany, and chronic cannabis use (CCU) during this developmental stage raises significant public health concerns due to its potential impact on neurodevelopment. Adolescents with CCU are at a major risk of developing Cannabis use Disorder (CUD), particularly when co-occurring psychiatric disorders are present, which can negatively affect treatment outcomes for CUD. Among these co-occurring disorders, psychotic and depressive disorders have frequently been associated with adolescent CCU. Moreover, CCU in adolescence is associated with cognitive impairment, with impaired verbal learning ability being a common manifestation. These cognitive impairments and co-occurring disorders are rooted in underlying neurobiological alterations that may disrupt normal brain development during adolescence. The psychoactive effects of cannabis primarily stem from Δ⁹-trans-Tetrahydrocannabinol, and it is suspected that cannabis use affects the signaling of the endocannabinoid system (ECS). The ECS, known for its regulatory role in various physiological systems such as stress and immune responses, has been implicated in the neurobiological processes underlying certain psychiatric disorders. In recent years, there has been a growing interest in understanding the factors contributing to neurobiological changes associated with psychiatric disorders. Epigenetic processes, which describe the interplay between environmental factors and genetic regulation of phenotypic expression, have gained particular attention. DNA methylation (DNAm), the most extensively studied epigenetic mechanism, has emerged as a central focus in identifying epigenetic changes that underlie the neurobiological processes involved in psychiatric disorders. The present dissertation aims to investigate the psychological, neurobiological, and epigenetic correlates associated with CCU in adolescent outpatients with CUD. By exploring these dimensions, this work seeks to enhance our understanding of the multifaceted factors contributing to CCU and its impact on adolescent mental health. Methods: This thesis consists of five studies presented in chapters 2 to 6. Chapter 2 provides a description of the sample of adolescent outpatients who sought treatment at the SUD outpatient center. The chapter focuses on the prevalence of cannabis and other substance use, the occurrence of cannabis and other substance use disorders (SUDs), and the presence of co-occurring psychiatric disorders. In chapter 3, a study examines the association between cannabis use, additional 3,4-methylenedioxymethamphetamine (MDMA) use and attenuated psychotic symptoms (APS). Chapter 4 presents a study that investigates the relationship between CCU and DNAm. Additionally, it explores the potential mediating effects of DNAm on the association between CCU and impaired cognitive performance. The focus of chapter 5 is on the relationship between depressive symptoms and DNAm in target genes related to depression. The study also examines the association between DNAm of depression-related genes and CCU, while exploring the mediating effects of DNAm on the relationship between CCU and depressive symptoms. Finally, in chapter 6, a study analyzes site-specific DNAm to estimate the proportions of circulating white blood cell types in adolescents with CCU compared to non-using controls (NCU). The study also investigates the DNAm profiles of selected immune cell markers and determines their gene expression levels. Results: In a first step, the prevalence of cannabis use and CUD among treatment seeking adolescents with suspected SUD was examined. Cannabis use and CUD was highly prevalent in the sample. The results indicated no effects of gender or age on cannabis use and CUD prevalence rates. Further, co-occurring conduct disorders, attention deficit hyperactivity disorders and depressive disorder were also highly prevalent in this sample. In chapter 3, the results did not indicate a relationship between APS and cannabis use, which was contrary to the predictions. MDMA use additional to cannabis however was associated with increased APS. However, as described in Chapter 4, CCU was associated with impairment in a verbal learning task. Furthermore, six CpG sites were identified whose DNAm levels showed a significant mediating effect on the relationship between CCU and verbal learning impairment. The results presented in Chapter 5 indicate that CCU is associated with increased depressive symptoms, and both depressive symptoms and CCU are associated with DNAm of one key gene in the stress system. Moreover, the findings in the last chapter, Chapter 6, suggest that CCU has immune-modulatory effects and affects the proportion of B-cells in adolescents with CCU, potentially acting through the alteration of DNAm of related genes. Discussion: The studies conducted in this thesis have produced several significant findings. Firstly, it was observed that cannabis use and CUD are highly prevalent among adolescents with SUDs. While there was no cross-sectional association between cannabis use and increased APS, it was associated with higher levels of depressive symptoms, poorer performance in verbal learning tasks, and a higher prevalence of co-occurring psychiatric disorders. Additionally, it was found that adolescent CCU may have an impact on the epigenome through alterations in DNAm. Specifically, these alterations may affect the regulation of cognitive processes by influencing neurogenerative processes. Adolescent CCU may also interfere with the stress axis through DNAm-related changes. Furthermore, there is evidence to suggest that adolescent CCU may impair parts of the immune system. However, it is important to note that the studies presented in this thesis are based on cross-sectional data, and therefore, causal interpretations cannot be made. The relationships discussed, which imply causality, are speculative and require examination in longitudinal studies.:Statement for a publication-based dissertation List of Tables List of Figures Important Abbreviations Abstract 1. General Introduction 1.1 Prevalence of Cannabis Use and Cannabis Use Disorder (CUD) 1.2 CCU associated cognitive and behavioral changes 1.3 Neurobiological changes associated with CCU in adolescence 1.4. Epigenetic regulations and DNA methylation 1.5 Research Aims and Hypotheses 1.6 Sample Population and Measurements 2. Prevalence of cannabis use and CUD in an outpatient clinic 2.1 Background 2.2 Methods 2.3 Results 2.4 Discussion 2.5 Conclusion 2.6 Acknowledgements, Funding, Conflicts of Interest 3. Cannabis use and psychotic symptoms 3.1 Background 3.2 Methods 3.3 Results 3.4 Discussion 3.5 Conclusion 3.6 Acknowledgements, Funding, Conflicts of Interest 4. CCU, cognitive impairment and shared epigenetic associations 4.1 Background 4.2 Methods 4.3 Results 4.4 Discussion 4.5 Conclusion 4.6 Acknowledgements, Funding, Conflicts of Interest 5. CCU, depressive symptoms and shared epigenetic associations 5.1 Background 5.2 Methods 5.3 Results 5.4 Discussion 5.5 Conclusion 5.6 Acknowledgements, Funding, Conflicts of Interest 6. Immunological alterations associated with CCU 6.1 Background 6.2 Methods 6.3 Results 6.4 Discussion 6.5 Conclusion 6.6 Acknowledgements, Funding, Conflicts of Interest 7. General Discussion 7.1 Summary 7.2. Implications for DNAm mediated disruption of cognitive systems due to adolescent CCU 7.3 Implications for DNAm mediated disruption of immunological systems and its interactions with depression in chronic cannabis users 7.4 Neurobiological and epigenetic alteration may be affected by CCU 7.5. Clinical Implications 7.6. Limitations & Future Research 8. References 9. Appendices 9.1 Appendix A (chapter 2) 9.2 Appendix B (chapter 3) 9.3 Appendix C (chapter 4) 9.4 Appendix D (chapter 5) 9.5 Appendix E (chapter 6) 10. Erklärung gemäß der Promotionsordnung