A. meyeri was highly enriched in the saliva from chronic cannabis smokers compared to those of non-smokers, and oral enrichment of A. meyeri was associated with the age of first cannabis use. We further investigated if direct administration of this bacterium, in the absence of cannabis or the psychoactive THC component, could elicit alteration in the brain-immune axis in a mouse model. Long-term oral inoculation of A. meyeri bacterium to mice resulted in behavioral changes, macrophage infiltration into the brain, and increased Ab 42 protein production in the brain. Oral flora plays a role in maintaining oral health; however, environmental changes can result in dysbiosis. Prior results have shown tobacco smoking alter the oral micro-biome. Decreased abundance of Neisseria and Capnocytophaga and increased abundance of Streptococcus were found in tobacco smokers compared with those of non-smokers. Consistent with these findings, here we found that the abundance of Neisseria, Capnocytophaga, and Cardiobacterium genera was reduced and the abundance of Streptococcus was increased in cannabis smokers compared to non-smokers. In contrast, cannabis use was associated with increases in the genera, Actinomyces, Atopobium, Megasphaera, and Veillonella. A previous study demonstrated a strikingly similarity in the physical and chemical properties produced by cannabis and tobacco smoking, which contained large amounts of hydrocarbon and changed the acidity of saliva. Thus, Streptococcus and Actinomyces, acid-tolerant and facultative anaerobes, may preferentially grow in a smoking-mediated environment. In contrast, bacteria such as Neisseria sp. and Corynebacterium sp. were decreased in cannabis smokers, suggesting that smoking renders an unfavorable environment to facultative or strict anaerobes.

Although some oral micro-biome is shared between tobacco smokers and cannabis smokers, including increased Streptococcus and decreased Neisseria genus bacteria compared to non-smokers, A. meyeri was only increased in cannabis smokers. Moreover, the younger the age of first cannabis use, the more A. meyeri was orally enriched. Although the gut micro-biome has been shown to play a crucial role in the CNS activities via the bidirectional gut-brain axis,pollen trim tray strong connections between the oral micro-biome and the CNS have been reported as well. P. gingivalis, a key pathogen in chronic periodontitis was identified to contribute to Alzheimer’s disease. Besides P. gingivalis, other oral resident microbes were shown to associate with neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Moreover, oral bacteria Treponema and N. meningitidis can infect the brain through the trigeminal or olfactory nerve. The perturbations of intestinal microbiota resulted in the impairment of memory formation and cognition in the hippocampus. To test the effect of cannabis use-associated oral dysbiosis on CNS functions, we performed various behavioral tests in mice following bacteria oral inoculations but did not observe significant memory changes. Actinomyces are gram-positive facultatively anaerobic bacteria. Some Actinomyces species are commensal bacteria in the skin, oral, gut, and vagina of humans, but can also become opportunistic pathogens leading to infections in the dental cavity and other systemic sites. A. meyeri is associated with brain infection or dysfunction, but the causality and mechanisms of A. meyeri-mediated CNS dysfunction have never been reported. In this study, A. meyeri enrichment in the oral micro-biome was inversely correlated with the age of first cannabis use, which indicates that the longer duration of cannabis exposure, the more enrichment of A. meyeri in the oral cavity.

A previous study found younger age of first cannabis use was associated with decreased orbital prefrontal cortex volume. Another study found negative correlations between the age of initiation of cannabis use and altered thickness of the right superior frontal gyrus. Our study implies that the age of first cannabis use may be critical for particular oral micro-biome development and its potential impact on cognitive function. Given the many toxicant components found in cannabis smokers, it is not surprising that cannabis smoking notably alters the oral microbial ecology. Importantly, long-term repeated oral inoculation of A. meyeri, which mimicked cannabis exposure-increased oral A. meyeri in humans, resulted in the development of CNS abnormalities. Recent studies have found correlations between Actinomyces and Alzheimer’s disease. For example, brains from patients with Alzheimer’s disease have been reported to have strikingly large bacterialloads compared to controls. Actinobacteria, a phylum of Actinomyces, were exclusively detected in the post mortem brain samples from patients with Alzheimer’s disease compared with those of normal brains. Actinobacteria were also found enriched in the gut microbiota of patients with Alzheimer’s disease. Another study using 16S rDNA sequencing in the brain cell lysates further found Actinomycetales, Prevotella, Treponema, and Veillonella were exclusively present in the brain of patients with Alzheimer’s disease. In a previous study, oral micro-biome and resting-state functional magnetic resonance imaging scans were conducted in cannabis smokers; the enrichment of Actinomyces in the oral micro-biome was positively correlated with brain resting-state functional networks which are significantly perturbed with Alzheimer’s disease. Neuropathological hallmarks of Alzheimer’s disease include loss of neurons, progressive impairments in synaptic function, and deposition of amyloid plaques within the neuropil. Although mice do not readily develop amyloid plaques, our results show Ab 42 deposition was increased in the brain from A. meyeri-treated mice compared with controls, suggesting oral micro-biome-induced neuronal responses that have relevance to Alzheimer’s disease neuropathology. Previous studies have suggested that bacteria in the oral cavity were initially taken up by tissue macrophages which may facilitate CNS infection.

In the current study, A. meyeri treatment resulted in increased myeloid cell migration and phagocytosis in vitro and elevated macrophage infiltration into the mouse brain in vivo, compared with those of N. elongata treatment. The cytokines that differed in cannabis users and non-users and in A. meyeri-treated mice and control mice are related to monocyte/macrophage functions. The TNF super family cytokine promoted a compromised blood-brain barrier , and monocytes migrated across the BBB into the brain in response to MCP-1. Although it is not clear if macrophage infiltration results in CNS abnormalities in the setting of disease-associated immune perturbations, macrophage infifiltration into the brain has been demonstrated in the pathogenesis of several diseases. MIP-1 cytokines are induced in myeloid cells in response to bacterial endotoxins or membrane components. In the current study, A. meyeri administration increased plasma levels of MIP-1a in some mice. However, cannabis smoking altered oral micro-biome not limited to A. meyeri; thus, the decreased plasma levels of MIP-1a in cannabis users may stem from myeloid cell activation by other bacteria or by reduced total bacterial translocation due to cannabisreduced barrier permeability. In general, bacterial stimulation reduces phagocytosis and promotes proinflammatory cytokine production by myeloid cells. Unexpectedly, A. meyeri did not affect phagocytosis and did not induce proinflammatory cytokines but did increase myeloid cell infiltration and amyloid production in the brain. It is possible that A. meyeri maybe a new exposure to mice which induces the immune responses and CNS effect. However, there is no evidence on the causal link between a new bacterial exposure in the oral cavity and neuropathology in mice. Thus, we believe that A. meyeri is a unique oral bacterium that is linked to CNS function. We have tested novel object recognition in C57/B6 mice after 6- month exposure to A. meyeri, but did not find significant memory deficits. The reasons for the null finding are as follows: 1) more than 6- month exposure is necessary to see memory changes, 2) the nature of wildtype C57/B6 mice, and 3) the age of mice might play an important role with our mice being too young to detect any changes.

To date, there were few to no published studies measuring effects of a specific oral microbial dysbiosis pathobiont on behavior in wild type mice. In 2018, the study of P. gingivalis found that this pathobiont induces memory impairment in 13-month-old mice and not 2-month-old mice suggesting an age-related effect, but without enough age cross sections to determine when susceptibility occurred. Thus, we have refined our future strategy to analyze other neurological defects or pathological signs and started to conduct studies that use mice at different ages and include memory-related longitudinal measures, such as the Novel Object Recognition task that focuses on the hippocampus and prefrontal cortex memory functions, the Novel Tactile Recognition task that focuses on the hippocampus and parietal cortex memory functions, and finally the Water radial arm maze that focuses on spatial memory and cognitive flexibility. We have identified a rotarod and marble burying methods and will add these to the battery of tests in future studies.In 2018, Canada became the second country in the world to legalize adult recreational cannabis use , following its legalization for medical use in 2001 . Canada’s Cannabis Act dictates that cannabis policies should “keep cannabis out of the hands of youth”, “keep profits out of the pockets of criminals” and “protect public health and safety by allowing adults access to legal cannabis” .Earlier and more frequent adolescent cannabis use is associated with greater risk of harm to the developing brain and multiple adverse outcomes including impaired neurocognitive functioning, affective problems, suicidality, psychosis, cannabis dependence syndrome, and cannabis-related morbidity in later years . With the legalization of adult recreational cannabis use, however, adolescents may experience increased cannabis availability,grow tent increased social acceptance of cannabis, and confusing messages about whether cannabis use is safe . Evidence regarding the effects of adult cannabis legalization on adolescents is mixed. Some studies show that more permissive cannabis laws increase rates of adolescent cannabis use while others do not . Although research surrounding the impact of recreational cannabis legalization on youth in Canada is scarce, national survey data show a gradual increase in cannabis use among youth coinciding with increased public discourse on the topic .

The extent to which Canada’s shift towards more liberal cannabis policies, practices and culture will impact youth cannabis attitudes, intentions, and use are largely unknown. A key influence on youth cannabis attitudes, beliefs, expectancies, and intentions to use, is cannabis-related marketing . Though it is illegal to market cannabis products to youth in Canada, recent studies , and a long history of research on other age-restricted substances with abuse potential , demonstrate that companies ignore these laws and intentionally target their products to youth . Research on alcohol and tobacco marketing shows strong correlations between youth exposure to marketing and earlier initiation, and higher consumption among those already using . All told, exposure to cannabis marketing could similarly spur youth cannabis use . While emerging research suggests that cannabis marketing puts Canadian youth at risk , preliminary studies are limited because they use inexact measures such as general awareness of marketing and receptivity to marketing that rely on retrospective recall, which are subject to participant recall error and bias . Existing studies also describe marketing exposures in aggregate, obfuscating the context of individual exposures, such as when and where exposures occur, and other psychosocial factors which could influence their effects . In particular, existing research does not describe the channels through which cannabis marketing exposures occur, nor the ways in which federal marketing prohibitions are violated. Policymakers also need research that shows whether cannabis marketing of different types and through different channels has varying impacts on youth. Real-time, real-world assessment techniques such as Ecological Momentary Assessment  may be used to reduce bias and increase the reliability, accuracy, and acuity of information about adolescents’ exposures to cannabis marketing. In EMA protocols, participants use smartphone technology – that they already use throughout the day in multiple settings – to track a range of phenomena as they occur in participants’ natural environments. Previously, we created an EMA protocol for tracking youth exposure to alcohol and tobacco marketing . Middle- and high-school participants made electronic time-stamped recordings of tobacco and alcohol marketing exposures, demonstrating that exposures primarily occurred in the afternoon, at point-of-sale locations, and on days leading up to the weekend . To our knowledge, no research has similarly documented Canadian adolescent cannabis marketing exposures using an EMA approach. The goal of this pilot study was to assess the feasibility of a 9-day, smartphone-based EMA protocol to obtain a preliminary understanding of the frequency of Canadian adolescents’ exposures to cannabis marketing, their reactions to such exposures, and the context in which exposures occur in the real-world and in real-time.