Investigating the Role of Thiamine in Epileptic Activity in the Drosophila melanogaster

Samhita Vinay, Keertana Yalamanchili
Thomas Jefferson High School for Science and Technology

This paper was originally included in the 2020 print publication of the Teknos Science Journal.

Abstract

Epilepsy is a neurological disorder characterized by recurrent, unprovoked seizures. This disorder can occur at any age and affects more than 65 million individuals globally. Seizures involve abnormal and excessive neuronal activity and in humans can manifest as abnormal involuntary movements. Some of these epileptic behaviors can be modeled using Drosophila melanogaster due to its genetic similarities with humans. We used Bang-senseless (BS) mutant flies as they have been shown to exhibit seizure-like episodes due to a lower mechanical threshold for abnormal activity. This study investigated whether an increase in thiamine levels in the diet of Drosophila melanogaster would reduce epileptic behavior. Cost-effective webcam technology was used to record the seizure-like activity (SLA) in bang-senseless mutant flies that were fed either standard or thiamine-enriched media. These recordings were analyzed with the computer program ImageJ, where the movement of the flies was modelled as  distance, duration, and speed. Data were analyzed using three unpaired t-tests and a MANOVA test. We found that increased thiamine levels reduce SLA distance, but do not significantly affect SLA duration and speed. Thus, a thiamine enriched diet reduced the behavioral intensity of seizure-like activity, but not the SLA duration or speed.  

Introduction

Epilepsy is a disorder that is characterized by repeated, unprovoked seizures caused by abnormal and excessive neuronal activity in the brain [1]. In particular, the paleocortex and archicortex are the most common source of epileptic activities in humans [5]. On a cellular level, seizures may occur  due to ionic imbalances, such as a reduced K+ gradient in the neuron, blood-CSF barrier dysfunctions, as well as alterations in Glutamatergic and γ-aminobutyric acid (GABA)-ergic transmitters, which are examples of excitatory and inhibitory transmitters of the brain, respectively [14]. During a seizure, bursts of electrical activity in the brain occur, as the neurons in the brain become discharged [16].

Some epileptic behaviors , which primarily occur in humans, can be modeled using Drosophila melanogaster due to its genetic similarities with humans. For example,  Bang-senseless (BS) mutant flies have been shown to exhibit seizure-like episodes due to a lower mechanical threshold for abnormal activity. Electrophysiology setups from BS Drosophila suggest that there are six phases of seizures after the “Bang” ( or mechanical stimulation), including the initial seizure, paralysis, tonic-clonic phase, recovery seizure, refractory recovery and complete recovery (Figure 1) [10]. The initial seizure phase lasts for a few seconds and is represented by leg shaking, wing flapping, and muscle contractions. This phase is followed by paralysis, in which the flies remain inactive and dormant. In the tonic-clonic phase, the BS mutants undergo arbitrary movements [10]. The recovery seizure follows, where the flies experience seizures, but with lessened effects. Before the complete recovery phase, the BS mutants go through refractory recovery, where they become resistant to additional seizures [15].

In humans, generalized seizures can fall into several categories, including absence, generalized tonic-clonic (GTC), atonic, and myoclonic. Absence seizures are characterized by unresponsiveness and GTC seizures involve stiffening and jerking. Head drops are often associated with atonic seizures and brief movements characterize myoclonic seizures [16]. A few of the associated symptoms of epilepsy include fatigue, involuntary movements, temporary paralysis, and headaches. Although there is no permanent cure for epilepsy, there are several treatments that can suppress convulsive activity, including anti-epileptic medications, neurosurgery, and a ketogenic diet [3].

Drosophila melanogaster, commonly referred to as the fruit fly, is a model organism used in biological research. 75% of human diseases can be modeled with the Drosophila, as these diseases have a recognizable alternative within the genome of the Drosophila. These fruit flies are inexpensive, and both the wild type and mutant models are readily available for purchase [7]. One such mutant model used in this study was the bang-senseless (BS) mutant model, which has a lower mechanical seizure threshold. The lowered threshold is due to a missense mutation (the replacement of one nucleotide with another) in the paralytic Nav gene, which codes for a voltage-gated sodium channel protein [11]. This protein creates action potentials responsible for regulating locomotion in the fly [3]. As a result of the lowered threshold for seizures in the BS Drosophila, it is easier to induce seizure-like activity.

Prior research on BS Drosophila  has aimed to develop new anti-epileptic drugs. In one such study, three models of BS Drosophila (the technical knockout mutants, the easily shocked mutants, and the parabss mutants) were each fed a lipid-rich dietary supplement. These researchers found that the BS mutants displayed suppressed levels of seizure-like activity. Also, both the control and experimental groups exhibited similar glucose levels, locomotion, and triglyceride levels [12]. Another study found that metformin, a drug used to treat diabetes, caused BS Drosophila to demonstrate reduced seizure-like activity. This study used cost-effective webcam technology to record the seizure-like activity and converted the recordings into distances using  ImageJ’s Multitracker Plugin [17].

Thiamine, otherwise known as Vitamin B1, plays a vital role in the metabolism of lipids, amino acids, and glucose, and is a widely available dietary supplement [18]. Deficiencies in thiamine often cause decreased activity of a subset of enzymes, resulting in an altered brain metabolism [9]. These deficiencies have also been linked to several neurological disorders, including epilepsy. In fact, infants who were fed a formula lacking thiamine developed seizures in addition to severe motor and cognitive delays [8]. Furthermore , around 32% of patients with both a predisposition for seizures (similar to the BS Drosophila) and a thiamine deficiency also showed signs of epileptic behavior [6]. Based on these associations between thiamine levels and seizures , it was hypothesized that an increase in thiamine levels in the diet of the Drosophila melanogaster will reduce its epileptic behavior. In this study, fly movement was analyzed using ImageJ, a java-based image analysis program created by the National Institutes of Health. The software is often used for the imaging of neuronal and cellular movement. Users can either program commands to adjust images or use plugins, which are preprogrammed java classes for ease of analysis. Images can easily be imported through other recording software and modified to be in black and white [13]. A program, FIJI, was used within ImageJ. It provided several other plugins, including the TrackMate plugin. This plugin was used to assay the distance, duration, and speed of fly movement according to the ImageJ Trackmate guide [19].

Materials and Methods

Fly Cultures

Seven Drosophila melanogaster (BS mutants) from the Bloomington Drosophila stock center were cooled and transferred to the control vial and to the thiamine enriched vial. The control vial was filled with 15 mL of standard media mixed with 15 mL of water and 4 yeast pellets. The Thiamine enriched vial had 500 mg of thiamine nitrate added additionally. 

Procedural Setup

A Logitech Webcam (insert type) was placed to capture the images directed at a white surface under the  petri dish. Following this, each fly was placed into separate vials and closed with cotton plugs, where they remained for 15 minutes. The HandyAvi software was then opened, “capture” was clicked, and then “Time-Lapse images” was clicked. The Logitech 3550 webcam was selected, and the video frame size was set to “640 by 480”. The compression level was set to 0.1 sec/frame by selecting the “Intel IYUV codec” bottom. I Calibration was done using a dead fly. The  dead fly was  placed on the petri dish. Within “Video settings” and then “Device settings," the image was modified so that there is a bright white background with a black dot representing the fly. After the 20 minutes, a vial was vortexed for 10 seconds under the setting labeled “10” in order to induce seizure-like activity in the bang-senseless mutants. Following this, the dead fly on the petri dish was replaced with the vortexed fly, and we  recorded the fly’s activity until the jerky movements of the fly halted.

Data Analysis        

The FIJI ImageJ Platform was opened, and then "File," "Import," and "Import Sequence" were selected in that order to import the image stack. Next, "Type," and "8 bit" (under "Image") were selected to facilitate image analysis. Then, , the images were modified to only show the white background with one black dot to represent the fly. Finally, the TrackMate plug-in was selected to convert the fly's path into a distance, speed, and duration output. All the distances were added up in order to determine the total distance traveled by the fly. In addition, the sum of the duration of each track was taken. The average speed of the fly was also found by dividing the total distance by the total time.

Figure 4 shows the representative distances of seizure-like activity in two of the Drosophila bang senseless (BS) mutants fed with standard media or standard media with 500 mg of thiamine. According to Figure 4, feeding Drosophila BS mutants with thiamine reduces the seizure-like activity distance.The average distance (distance in pixels and is proportional to any standard measurement) travelled by  the control group was 392.913, and that of the thiamine-enriched group was 155.747. Furthermore, the data generated using the seizure assay was used to analyze the effect of thiamine on the duration and speed of seizure-like activity. The data indicated that the average SLA duration was 383.571 for the control group and 343.143 for the thiamine-enriched group, as illustrated in Figure 5. Lastly, and as shown in Figure 6, the average SLA speed was 1.322 for the control group and 0.671 for the group with 500 mg thiamine added. 

The SLA distance, duration, and speed were analyzed using unpaired t-tests. Table 1 shows that the average distance of the thiamine-fed group was 392.913 pixels, with a standard error of 35.392 pixels, which was higher than the average distance of the control group (flies fed with only standard meal) at 155.747 pixels, with a standard error of 13.991 pixels (p-value < 0.0001). In Table 2, the average duration of the 500 mg treatment group was 343.143 frames with a standard error of 69.151 frames. This data was slightly lower than the average duration of the control group, which was 383.571 frames, with a standard error of 100.572 frames (p-value 0.7462). The average speed of each of the flies was calculated by dividing the total track distance by the total track duration, Table 3 shows that the average speed of the 500 mg flies was 0.671 pixels/frame (SEM 0.538), and the average speed of the control group flies was 1.322 pixels/frame (SEM 0.683) (p-value = 0.0713).

A Multivariate (MANOVA) test was also conducted to determine if there was a statistically significant difference between the two groups with respect to the three dependent variables. A p-value of 0.001 was obtained, which suggested a significant difference between the control group and the experimental group.

Discussion and Conclusion

The purpose of this study was to determine whether elevated dietary thiamine levels can diminish the SLA of Drosophila BS mutants by analyzing distance, duration, and speed of the Drosophila. We found a significant relationship between higher levels of dietary thiamine and decreased distance of SLA. While increased dietary thiamine also decreased the  mean speed and duration of SLA, these findings were not  statistically significant. We hypothesize that the SLA duration data did not yield significant results since  recordings were taken until the SLA  ceased. This duration varied among the flies. In humans, poor intake of Vitamin B1 often leads to thiamine deficiency associated diseases, including certain types of epilepsy. Although thiamine deficiency has been shown to  increase seizure-like activity, the underlying mechanisms for this phenomenon are not completely understood. Thiamine helps metabolize glucose, amino acids, and lipids; therefore , its deficiency may cause molecular imbalances resulting in unusual brain activity [2]. Thus, increasing thiamine levels in the Drosophila medium may have equilibrated the concentrations of molecules and ions in the brain, lowering the intensity  of seizure-like activity.

However, our research did have some limitations. It is important to acknowledge that confirming SLA in the flies through an electrophysiology setup was not feasible, and only behavioral manifestations were studied. However, it was apparent that flies did undergo seizure-like activity since each of the established epileptic phases was observed in all of the vortexed flies [10]. Another major limitation in our study was the small number of flies used. In addition, adjustments to the experimental setup could be improved. For example, we were able to analyze only one fly at a time using the ImageJ platform. Using a different plugin within the ImageJ platform may have allowed tracking the movements of multiple flies simultaneously. To our knowledge, we conducted  one of the first studies to investigate the relationship between thiamine enriched diet and seizure-like activity in Drosophila melanogaster. This study demonstrated the potential value of using  thiamine to diminish seizures in Drosophila and potentially humans.  However, future research utilizing mammalian models and clinical testing will be essential before we can translate these results into a  treatment strategy for epileptic patients.

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