STEM I

Obsessive-compulsive disorder (OCD) is a chronic mental health disorder characterized by repeated and unwanted thoughts (obsessions) and actions (compulsions). OCD is also often comorbid with other mental health disorders such as anxiety, depression, and eating disorders. The current treatments include therapies and medication; however, they lack widespread efficacy among patients with OCD. The most common medications are antidepressants, normally a kind of selective serotonin reuptake inhibitor (SSRI). SSRIs aim to address commonly observed imbalances of serotonin in patients with mental health disorders. Likewise, patients with OCD have been observed to have excess glutamate in their brain, which could be a target for potential treatments. One way to address this imbalance is through a pathway in the human body called the gut-brain axis (GBA). Recent studies have shown that the level of diversity of bacteria within the gut is correlated to areas of both mental and physical health, where greater diversity is linked to positive improvements. The goal of this project is to utilize probiotics to increase gut diversity to understand their effects on OCD. To accomplish this goal, an established C. elegans model for OCD was used along with various types of probiotics. Behavior was observed through periods of locomotion with and without stimulation. Differences in these behaviors were analyzed and contribute to the understanding of OCD and the gut-brain axis. Keywords: Obsessive-compulsive disorder, antidepressants, SSRIs, glutamate, probiotics

Obsessive-compulsive disorder (OCD) is a chronic and debilitating mental health disorder, affecting 1 in 40 adults globally at some point in their lifetime (International OCD Foundation, 2024). It is a mental health disorder characterized by repeated and unwanted thoughts (obsessions) and actions (compulsions) (Pittenger, 2015). Obsessions are often distressing and cause discomfort, causing an individual to alleviate that discomfort through their compulsions (Pittenger, 2015). Both obsessions and compulsions can significantly affect one’s daily routine and overall quality of life. For many individuals, OCD is also comorbid with mental health disorders like anxiety, depression, and eating disorders (Breteler et al., 2021; Roefs et al., 2022).

Current treatments for OCD include therapy, oftentimes cognitive behavioral therapy (CBT) or exposure and response prevention therapy (ERP), and medication, usually an antidepressant. (National Institute of Mental Health, n.d.; Lissemore et al., 2018). These current treatments are effective in around 40 to 60 percent of patients; however, this percentage leaves many people without proper care (Lissemore et al., 2018). Another downside to the current treatments for OCD is the lack of accessibility and flexibility. Qualified therapists are in short supply, with around 60 percent not having openings for new patients (Stringer, 2024). When it comes to antidepressants, as with most medications, side effects must be considered before using them as a form of treatment. For many people, the severity of withdrawal from antidepressants, potentially leading to worsening symptoms or suicidal ideation, is a deterrent (Palmer et al., 2023). These factors lead to around one third of adults in the United States lacking access to proper mental health care (Stringer, 2024).

Mental health disorders such as anxiety, depression, and OCD have been linked to abnormal levels of certain chemicals in the brain, such as decreased levels of serotonin (Jones et al., 2020; Lissemore et al., 2018). These abnormalities serve as the basis for the previously mentioned treatments for health disorders. For example, a common form of antidepressant used as treatment today is a selective serotonin reuptake inhibitor (SSRI) which functions by keeping serotonin in the synaptic space and therefore increasing the overall amount in the brain (Jones et al., 2020). Antidepressants have been shown to reduce the severity of depressive symptoms; however, they also have downsides, such as their extreme withdrawals (Jones et al., 2020; Palmer et al., 2023). Brain imaging and testing of cerebrospinal fluid (CSF) in OCD patients have also shown increased levels of glutamate in the brain (Pittenger, 2015). Reasons for the high levels could be rooted in glutamate reuptake in the brain, meaning that it is not effectively transported out of the synaptic space by transport proteins (Pittenger, 2015). However, before ineffective glutamate transportation can be attributed to OCD, a significantly greater amount of research is needed due to varying results of studies in the field. For example, two previous studies connected the glutamate transport protein EAAT3 to OCD, yet this protein is not responsible for most of the glutamate transport done in the brain (Arnold et al., 2006; Danbolt, 2001; Dickel et al., 2006; Pittenger, 2015). Another set of proteins that may also be connected to OCD are the SAPAP and DLGAP proteins (involved in glutamate signaling regulation in mice and humans) based on their ability to cause repetitive behaviors; however, a lack of statistical significance means that neither of these proteins can be attributed to OCD without more information (Pittenger, 2015; Welch et al., 2007).

A major signaling pathway in the body that has recently shown promise in the treatment of mental health disorders is the gut-brain axis (GBA). The GBA relays information and connects the health of the gut and brain (Akram et al., 2023; Clapp et al., 2017). At the root of this pathway lies the vast number of bacterial species that make up the human gut microbiota (Akram et al., 2023; Clapp et al., 2017). Connections have been shown between the level of diversity in gut microbiota, obesity, and mental health, where a more diverse gut is linked to improvements in the other two areas, such as a lower body fat percentage and antidepressant effects (Akram et al., 2023; Beilharz et al., 2017; Clapp et al., 2017). Through the targeted administration of probiotics in particular, studies have shown that gut diversity can be increased along with other benefits, such as a reduction in cortisol levels (Clapp et al., 2017). With further research on their efficacy, probiotics have the potential to be a beneficial form of treatment due to their accessible nature. They can be found in common fermented foods such as yogurt and kombucha (Clevland Clinic, 2024). These foods contain probiotic genera Lactobacillus and Bifidobacterium, two types of probiotics that have been shown to aid in immune function and digestion support (Dempsey & Corr, 2022; O’Callaghan & Van Sinderen, 2016). In addition, probiotics have been shown to reduce levels of glutamate in the brain. This connection means that probiotics could be useful in glutamate modulation, potentially influencing OCD.

Commonly used model organisms for OCD that have been used in previous studies include rats and mice, as they can display repetitive behaviors characteristic of OCD, including repetitive grooming, checking, and circling (Beilharz et al., 2017; Chamberlain & Ahmari, 2021). However, C. elegans is another model organism that can be useful to understand the effects of probiotics on OCD. Because their normal diet is bacteria, they can be easily fed different probiotic strains to understand the effects on mental health. There is also an established C. elegans model with a knockout mutation for a glutamate transport protein (GLT-1) that will cause the C. elegans to mimic the repetitive behaviors of OCD through frequent and spontaneous reversals (Katz et al., 2019). These behaviors were observed during both regular locomotion in the exploration phase and through mechanosensation as an escape behavior (Katz et al., 2019). This strain could be particularly useful for modeling OCD if it is in fact connected to glutamate transport in the brain. Its use as a model organism will allow for further understanding of the connections along the GBA and how this pathway can be utilized for treatment of mental health disorders in the future.

The materials used in this project include wild-type (N2) and an OCD model (OS11091) C. elegans with a GLT-1 knockout mutation (Katz et al., 2019) Both strains were obtained from the Caenorhabditis Genetic Center (CGC) at the University of Minnesota. Standard Nematode Growth Media (NGM) plates were prepared and seeded with E. Coli OP50 and probiotic bacteria L. acidophilus and L. bulgaricus that were cultured overnight at 28° C. These two strains of probiotic bacteria were identified through bacteria morphology. Additionally, an autoclave and centrifuge were used to create NGM plates and synchronize worms. The C. elegans were manipulated using a worm pick and observed through a stereo microscope. Data collection and analyses involved a microscope camera, ImageJ with a wrMTrck plugin, and manually counting worm behaviors (Pedersen, 2011).

The independent variables in this project were the type of bacteria administered to the C. elegans and the duration of this administration. The wild-type C. elegans (N2) were separated into groups that were fed diets of standard E. Coli OP50, L. acidophilus, and L. bulgaricus. These three groups were further separated into a group that was fed the non-standard probiotic bacteria from the egg stage of their life cycle to the young adult stage and a group that was fed E. Coli OP50 from the egg stage to L4 stage and then transferred onto plates with the probiotic bacteria for 24 hours to develop to the young adult stage. These experimental groups allowed for the observation of the effects of different probiotics that were introduced at different stages of life which can aid in the understanding of the environmental and genetic factors as potential causes for OCD. Based on previous studies, there are possible links between genetic factors such as glutamate transport genes and OCD as well as environmental factors such as diet (Akram et al., 2023; Clapp et al., 2017; Pittenger, 2015).

The locomotion assay was conducted to understand how the behaviors of the C. elegans change with the administration of probiotics in both the wild-type and OCD model. When conducting the locomotion assay, worms were observed on NGM plates with a thin layer of their respective bacteria. Body bends and reversal events were counted over a period of 30 seconds for each individual worm. Analysis of these behaviors provides insight of the effects of probiotics on the baseline behavior of the C. elegans without stimulation.

The mechanosensation assay was performed to assess the response of the C. elegans to a stimulus. This process involved a gentle touch sensation to the anterior side of the worms (Chalfie et al., 2005-2018). A thin eyebrow hair fixed to the end of a toothpick was used to accomplish this. The body bends and reversal events were counted over a period of 20 seconds before the gentle touch stimulus and over an additional period of 20 seconds directly following the stimulus. The difference of the body bends per second and reversal events per second were calculated to determine the response of the C. elegans to the stimulus. These observations provide insight on their response to gentle touch and how probiotics have the ability to influence this response.

The results of the ANOVA showed that there was a statistically significant difference between the behavior of the C. elegans with different treatments. The wild-type C. elegans that were treated with different types of probiotic bacteria from the egg to young adult stage had varying rates of body bends and reversals (Figure 1). The C. elegans treated with probiotics had an overall higher rate of body bends (p < 0.001) (Figure 1a) and lower rates of reversals (Figure 1b). There was no significant difference between the two types of probiotics. The C. elegans that were treated with probiotics for 24 hours also had significant variability in the rates of body bends (p < 0.01) (Figure 3a) and reversals (p < 0.05) (Figure 3b). These results show that probiotics have an influence on C. elegans behavior. Additionally, the duration of probiotics also has an influence on worm behavior. There was a significant difference in the body bends of the C. elegans treated with probiotics for the duration of their life and for 24 hours for both the L. acidophilus (p < 0.00001) (Figure 4a) and L. bulgaricus (p < 0.00001) (Figure 4c). The results of the gentle touch mechanosensation assay conducted on the C. elegans treated with probiotics were not significant. The stimulation of the C. elegans posed a significant challenge with the eyebrow hair due to the need to accurately make contact with the anterior side of the worm.

The goal of this project was to further increase knowledge and understanding of the GBA and mental health disorders through the use of C. elegans as a model organism. Although not all results were significant and expected, they provide significant insight to achieve the goal of this project. Overall trends in the data show that when treated with probiotics, the C. elegans displayed higher rates of body bends which indicate increased activity (Zhang & Chen, 2023). At the same time, they often displayed higher rates of reversals when left untreated, which is another indicator of increased activity (Zhao et al., 2003). These results appear to contradict each other; however, they are two different measurements and therefore may be affected by probiotics differently. Research done by Katz et al. in 2019 focused on reversal rates as one of the primary behaviors indicative of OCD. This information supports many of the results achieved in this study that showed probiotics contributed to lower reversal rates, although results were inconsistent and need to be replicated. Additionally, probiotics and increased gut diversity have been linked to improvements in physical health, which supports the increased activity of the C. elegans (Akram et al., 2023; Beilharz et al., 2017; Clapp et al., 2017).

The future steps of this project include replication and expansion of results. Additional trials with different types of probiotics should be conducted. The use of the GLT-1 knockout strain will also allow more accurate modeling and testing of the effects of probiotics on OCD (Katz et al., 2019). It is important to ensure accuracy of results, which means that a software or machine learning model should be used to analyze worm behavior in place of manual tabulation (Zhang & Chen, 2023). The implementation of software to analyze worm behavior through videos will eliminate possible biases and errors in results (Zhang & Chen, 2023). The use of magnetic resonance spectroscopy (MRS) will also allow levels of neurotransmitters to be measured with the treated C. elegans (Katz et al., 2019). Glutamate and serotonin are believed to play significant roles in mental health disorders overall, but particularly OCD (Jones et al., 2020; Lissemore et al., 2018; Pittenger, 2015).

The results of this experiment ultimately increase the current understanding of the GBA and mental health disorders. Significant results were achieved that showed the influence of probiotics on C. elegans behavior. Although not a true OCD model, the results on wild-type worms provide support for their use as treatment. With further testing on a GLT-1 knockout strain, an increased understanding can be achieved on the true effects of probiotics on the disorder. Probiotics and treatments that interact with the GBA may also be applicable to other mental health disorders, such as depression. All individuals deserve access to proper mental health treatment. Many factors such as access to therapy, expenses, and individual needs can pose significant challenges to individuals receiving care; however, affordable and accessible probiotics are a promising solution. The hope is that this project can be one step along the way to making mental health treatment available to all.