Abstract

Seasonal affective disorder (SAD) impacts around 10 million Americans, causing a decline in emotional well-being and productivity. This project aims to scientifically establish the use of probiotics as potential remedies for depressive symptoms incurred by seasonal changes. While previous studies have shown a link between the state of the gut microbiome and neurological conditions as a result of the gut-brain axis, research is sparse on the investigation of seasonal depression. This is notable because inducing SAD differs from other mental illnesses as it is based on outside factors such as shorter days and extended periods of darkness. To expand upon SAD research and provide a possible method for its alleviation, this study introduces probiotics to the gut microbiome of Drosophila with seasonally-induced depressive symptoms and records whether an improvement in motivation and behavior can be seen. The process began with Drosophila being subjected to conditions simulating the effects of seasonal changes. After this period, acquired depressive symptoms were measured on behavioral assays along with an PCR-derived analysis of the gut microbiome. After the administration of probiotics, results indicate Lactobacillus-acidophilus and Lactobacillus-rhamnosus lead to an improvement on the gap-climbing and locomotive assay, due to growth of bacterial diversity and health in the microbiome. The findings directly correlate the improvement of the state of the gut microbiome to improved results on the behavioral assays. This conclusion suggests that introducing these probiotics into the human diet could have a similar positive impact on the microbiome, potentially counteracting seasonal depressive symptoms and improving overall mood and behavior.

Background

Seasonal depression, also known as seasonal affective disorder (SAD), is a subtype of depression or bipolar disorder that begins and ends around the same time each year. This condition is due to the change in seasons, with most symptoms beginning in the fall and lasting throughout the winter months. Seasonal depression can still occur in the summer or spring, although this is less common. In a given year, 5% of the U.S population experiences seasonal depression (Cotterell, 2010). The degree of severity can vary, but it’s clear that those susceptible will be impacted both in a mental and emotional sense. While generally not taken seriously due to its relationship with seasonal changes, SAD remains classified as a form of Major Depressive Disorder (MDD) with the potential to turn into year-round depression, depending on the genetic susceptibility of a person. Harrowingly, The World Health Organization (WHO) predicts that depression may become the second leading burden of global disease, outranking cardiovascular disease, from its current fourth rank by 2030.

When one consumes food, it passes through the digestive system. In various areas of the digestive system, most commonly the small intestine and large intestine, the gut microbiome can be found, an ecosystem holding trillions of bacteria along with viruses and fungi. All these organisms work symbiotically with the host and regulate a multitude of important functions necessary for the host’s survival. Because of the connection between the Enteric Nervous System (ENS), located in the gastrointestinal system, and the Central Nervous System (CNS), these two systems communicate and rely on one another through the vagus nerve. In fact, there has been a large amount of research establishing a link between the state of the gut microbiome and neurological conditions. According to Limbana et al, patients diagnosed with mental conditions, including depression, have demonstrated gut microbiome dysbiosis, an imbalance in bacterial composition such as the loss of beneficial bacteria and diversity of microflora (Limbana et al.,2020).

Procedure

Drosophila must first be induced into a depressive state through circadian misalignment. This was done with two methods. First, flies were exposed to varying periods of light and dark to simulate the reduced daylight during transitions to the winter season. To achieve an effective simulation, the control and test groups were placed into respective buckets with lanterns attached to a self-timer. The control group was on a continuous 12/12 light-dark cycle. The test group began on a 12/12 cycle as well, but light hours were manually decreased by 2 hours every two days. This resulted in the test group experiencing drastic shifts in light exposure, from 12 hours, to 10, to 8, all the way down to 2 hours. The next goal was for the flies to undergo melatonin overproduction, an effect humans also sustain when experiencing seasonal depression. Excess melatonin in drosophila was achieved through administering a liquid melatonin solution into the feed.

To assess for SAD, two tests were performed, a motivational test and a test for anhedonia, the loss of pleasure. The flies completed behavioral assays that measured climbing rate and walking activity. For the first behavioral assay, 30 flies from each group were placed on a gap-climbing paradigm. Each individual fly must climb over a 4.5 mm (about 0.18 in) gap, a difficult distance for the size of the fly, to reach a gratifying sucrose reward at the other end. The overall number of climbing attempts (characterized by leg over head movement) in comparison to the control was measured.

The second behavioral assay, the stop-for-sweet paradigm, focused on evaluating anhedonia in the flies. The locomotive assay entails watching for the number of stops a fly makes in their walking activity to taste a pleasing reward, in this case sucrose. Each individual fly was placed inside of a petri dish with a small drop of a 30% sucrose solution and given 8 minutes for observation.

Along with behavioral tests, this study also involves a gut microbiome analysis for each fly group to see if the conditions they were subjected to have an effect on gut microbiota diversity and composition. To conduct this analysis, around 30-50 flies were transferred to separate vials and fecal samples were taken as a whole. Then, DNA from these samples was isolated and amplified through PCR (polymerase chain reaction). The enzyme polymerase exponentially replicates a segment of DNA so that there is sufficient quantity. This quantity is then shipped to an external laboratory, Primordium Labs, so that they can conduct long-DNA sequencing to determine how many different bacterial species exist within the fly group.

After the first round of behavioral testing and gut microbiome analysis, probiotics Lactobacillus acidophilus and Lactobacillus rhamnosus were administered to the drosophila. A 5% concentration of bacteria was applied to the feed and given a period of three days before the next round of microbiome analysis.