The Effects of Modeling a Ketogenic Diet on the Neurological Development of Drosophila Melanogaster
Samantha Biseinere, Westfield State University
Abstract: The ketogenic diet has been used as a treatment for medication-resistant epilepsy in children since the 1920s. Due to the restrictive diet, the neurological development of the children placed on the diet is a concern. We gave Drosophila melanogaster food that was supplemented with beta-hydroxybutyrate (BHB), a ketone body created as a byproduct of the ketogenic diet, and analyzed their motor function, survival, and synapse staining. We hypothesized that BHB supplementation would result in increased motor function and altered brain anatomy, but that survival rate would not change. Interestingly, we found no significant difference in the motor function or survival rate of the Drosophila. There was a small increase in the amount of synapses in the brain; however, no statistical analyses were performed to confirm this finding due to small sample size. These results suggest that there may not be a strong effect of BHB supplementation on neurological development. Future research can implement a true ketogenic diet on Drosophila to determine if the results would be the same.
Introduction
The ketogenic diet (KD) is a high-fat, low-carbohydrate diet which is intended to induce ketosis by making the body use fat as the primary energy source. The reduction in carbohydrates leads to an increase in lipolysis which increases ketones, an acidic byproduct of lipolysis, in the blood (The Nutrition Source, n.d.), and has been used since the 1920s to help reduce epileptic seizures in children (Golabek et al., 2022). In more recent years this diet has been used to lose weight as low-carb diets have become more popular (The Nutrition Source, n.d.). The ketogenic diet is very restrictive and without proper supplementation can lead to deficiencies in different nutrients such as Vitamin D, selenium, copper, and magnesium (Armeno et al., 2019). Therefore, the neurological development of children at a young age who have been placed on this diet has become a concern. Previous research has looked at altered brain metabolic pathways, brain volume (Mayengbam et al., 2021), and body mass (Wojciech et al., 2021). However, very few studies have assessed motor functions, survival and brain volume using Drosophila melanogaster as a model organism. There are many unanswered questions regarding how the ketogenic diet affects children. This study aims to answer the question: When administered soon after eclosion, or emergence from pupil case, how does the ketogenic diet affect the neurological development of Drosophila melanogaster?
Past research studies have assessed the brain volume and metabolic difference in juvenile mice when given a ketogenic diet (KD) (Mayengbam et al., 2021). The experimental group, who had received the ketogenic diet, had a decreased brain volume and altered brain metabolic profiles and metabolic pathways compared to the control group who was fed a standard diet. There were various metabolic differences noted between the two groups, as there was a lower level of amino acids in the brain such as methionine. Further, the level of carnitine, an amino acid synthesized from methionine and lysine, was also lower in KD-fed mice. Lastly, there was an increase in glutamate in the brain, the precursor to GABA synthesis which was noted in other studies as well (Mayengbam et al., 2021).
Another study had similar results when assessing the effects of the ketogenic diet on pregnant mice and their offspring (Wojciech et al., 2021). The brain mass of the female mice who were pregnant and fed a ketogenic diet, decreased compared to the controls who were fed a standard diet. However, the long-term effects and whether the results can be reversed are unknown. Additionally, the offspring from the pregnant females fed the ketogenic diet had a significant decrease in body mass compared to the normal diet group. The offspring that were later switched to a normal diet began to restore their body weight quickly (Wojciech et al., 2021). Through all of the research reviewed, none have reported any decrease in survival rate of the mice fed the ketogenic diet compared to the groups fed a standard diet.
The ketogenic diet has shown neuroprotective effects on different neurological diseases such as in patients who suffer a traumatic brain injury (TBI) (reviewed in Barry et al., 2018). After a TBI, there are various metabolic changes and dysfunctions in the brain that can last for many days. There is an increase in glucose uptake within 8 days of the injury, followed by a period of glucose metabolic depression. Cerebral ketone metabolism has been shown to help with TBI recovery as the brain is able to bypass the issue of glucose metabolic depression that occurs early on. Additionally, ketone metabolism is also more efficient and decreases the production of free radicals in mitochondria and cytosol. After moderate injuries, adult rats fasting for 24 hours showed a decrease in oxidative stress, cortical tissue sparing and mitochondrial calcium loading (Prins & Matsumoto, 2014).
As mentioned above, the KD has shown great benefits to helping reduce seizures in children with medication resistant epilepsy. Epilepsy is a brain disorder where an individual may have seizures throughout their life (Center for Disease Control and Prevention ([CDC], 2020). A seizure is a random and temporary burst of energy in the brain that can cause involuntary changes in the body such as changes in movement or function (Kiriakopoulos, 2019). There are many medications that are used to control the number and severity of seizures an individual may have; however, for some individuals, medications alone do not work well to manage symptoms (Golabek et al., 2022). Researchers have found that most seizures are caused by issues with Υ-Aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter which reduces the excitability in the nervous system (Treiman, 2001). In ketosis there is an increase in glutamate in the brain, and due to the increase in ketone bodies, this glutamate can be more readily available to synthesize GABA (Yudkoff et al., 2008). Overall, GABA synthesis is increased in the presence of ketone bodies such as acetoacetate (Yudkoff et al., 2008). With increased presence of GABA in the brain, there is a decrease in seizures as the GABA is able to inhibit neuronal excitation, the main cause of a seizure to occur (Treiman, 2001). These anticonvulsant effects have been shown in individuals who are placed on the ketogenic diet (Freeman et al., 1998).
Past research has shown that the ketogenic diet can be effective at treating neurological issues such as epilepsy and even possibly protecting the brain against damage in TBIs. Studying the effect of the ketogenic diet on neurological development is important to provide a complete understanding of the safety and effects on the ketogenic diet for both healthy brains and different neurological disorders. Drosophila melanogaster, commonly known as fruit flies, are a model used in a wide range of different research topics. They are also able to model different diseases such as Alzheimer's (Giaimo, 2020) and traumatic brain injuries (Bloomer et al., 2021). Scientists have a great understanding of the genome of Drosophila melanogaster and have found that humans share over half of their genome with them (Giaimo, 2019). Additionally, they have a very short period of development compared to other organisms where after just 1 day there is a fully functional instar larvae and after an additional 10 days a full adult emerges (Crews, 2019). This makes them a great model system, as multiple different experiments and replicates can be performed under a relatively short period of time. In this study, beta hydroxybutyrate (BHB) was used to supplement the Drosophila medium. BHB is a ketone body that is naturally abundant in organisms who are actively using the ketogenic diet (Newman & Verdin, 2017). Supplementation of this ketone body can help to resemble similar effects to a true ketogenic diet (Stefan et al., 2021). We were able to test motor function by performing multiple replicates of the rapid iterative negative geotaxis (RING) assay, which is used to assess the ability of Drosophila to climb vertically in response to gravitational cues (Gargano et al., 2005). The survival of the Drosophila were monitored and brain dissections were performed to assess the survival rate and the amount of synapses in the brain by evaluating mean gray value, respectively.
The main purpose of this study is to assess the negative geotaxis, synapse staining, and survival rate of Drosophila melanogaster when given Beta-hydroxybutyrate (BHB). I hypothesize that Drosophila, who are fed a diet supplemented with BHB, will have an increase to their motor functions, specifically negative geotaxis; however, there will be no difference in the survival rate between the experimental and control groups, and there would be altered brain anatomy.
Methods
Set-up and Care
Over the course of the study, 4 experiments were performed using different groups of Drosophila melanogaster. A RING analysis was performed on all 4 experiments. In addition to the RING analysis, experiment 3 included measuring survival, and experiment 4 included measuring the mean gray value of the brain. Each experiment was set up and monitored in the same manner. The Drosophila melanogaster used were Oregon R wild type flies from Carolina Biological (Burlington, North Carolina) and maintained in vials in a cabinet with regulated twelve hours of light and twelve hours of darkness. Ambient temperature of the vials ranged from 70℉ - 79℉. Once flies were approximately 1 to 3 days post-eclosion, male flies were selected, as their mating habits and reproductive hormones would not affect the results. These male flies were then transferred to new vials. Drosophila were anesthetized by placing them in the freezer between 3 to 5 minutes. Both vials were set up with Carolina 4-24 instant Drosophila medium, yeast and netting in order to have the same environment for both sets of flies. The control vials contained a regular diet consisting of only instant Drosophila medium and distilled water. The experimental vials also contained instant Drosophila medium and 2 Mmol of Beta-Hydroxybutyrate (BHB, Sigma Aldrich, St. Louis, Missouri). Survival was monitored by recording the number of flies alive once to twice a week. Figure 1 illustrates the timeline for the experiments. A Log Rank Test was performed to determine the significance in survival of the Drosophila.
RING Analysis
The Rapid Iterative Negative Geotaxis (RING) analysis was performed before the diet was introduced and multiple times after between 4 to 5 days apart. The RING analysis is a method for testing the locomotor functions in Drosophila. The setup included a light box, vial, ruler, camera, tripod, and books to place vials on top of. The camera was set up 30 cm from the position of the vial. A picture of an empty vial with a ruler next to it was taken in order to have a scale for statistical analysis. Once that was done, the Drosophila were anesthetized for 3 minutes in the freezer. The flies were transferred to an empty vial and allowed to recover for 30 minutes at room temperature. Then they were placed in front of the camera. In rapid succession, the vial was tapped against the table, and a picture was taken after 3 seconds. The Drosophila were allowed to rest for 30 seconds, and using the same group of flies, the process was repeated 5 times for each RING analysis. These photos were uploaded to Image J and were assessed manually in order to record the positions of the Drosophila in the vials (Gargano et al., 2005). Statistical analysis of the RING using a two-way ANOVA was performed using SPSS.
Brain Dissections and Staining
Brain dissections were performed on the fruit flies 5 days after diet initiation. Once the brains were removed from the fruit fly they were placed in 4 percent Paraformaldehyde (PFA, Sigma Aldrich) for at least 30 minutes. This was repeated until there were 3 to 4 brains. The brains were then fixed utilizing a nutator and washing the brains using Phosphate buffer saline solution, 0.1% tween (PBT) 3 times. Immunostaining of the brains was performed using primary and secondary antibodies diluted in PBT and 5% normal goat serum (Fisher Scientific, Hampton, New Hampshire). The primary antibody used was mouse anti-nc82 (Developmental Studies Hybridoma Bank, Iowa City, Iowa) in order to label presynaptic active zones. The secondary antibody used was goat anti-mouse (ThermoFisher Scientific, Waltham, Massachusetts) in order to bind to the primary antibody. Immu-Mount (Fisher Scientific) was used for immunostaining; the procedure was adapted from Wu and Luo (2006) and Kelly et al. (2017). Finally, the brains were mounted on a glass coverslip and examined through an Olympus IX-73 inverted fluorescence microscope (Center Valley, Pennsylvania) and camera SP-27. They were analyzed using image J in order to determine the mean gray value (Brightness) of the brains.
Results
Survival
The flies in both groups were monitored over the course of the third experiment, and the survival rate was recorded. There were 11 flies in the control group, and there were 8 flies in the BHB group. After 33 days post-eclosion, all the flies were dead in both groups. In Figure 2, the survival rate was recorded and showed a decline over time in both the control and experimental group. On days 4, 9, and 14 post-eclosion the flies were rehoused in both groups in order to refresh the BHB in the experimental vial. Both groups had a similar trend downward, and there was no statistical difference in the survival rate for either group (p-value = 0.339).
Negative Geotaxis
For experiment 1, there were 6 male flies placed in each vial. A RING analysis was performed on the flies before the diet was started in order to have a reference for the second RING analysis that was performed 4 days later. This data is reflected in Figure 3. There was a significant difference between the control and the BHB group on day 3 (p< 0.05), but not on day 7 (p > 0.05). Additionally, there was no significant difference between day 3 or day 7 for either group (p= 0.865). Eight days post-eclosion, a majority of the flies were dead in the vials, and there was apparent mold, so the vials were discarded.
For experiment 2, there were 10 original flies that were divided equally into 2 new vials. Two RING analyses were performed on day 7 and day 14 post-eclosion and this data is reflected in Figure 3. There was a significant difference between day 7 and day 14 (p= 0.004); however, there was no significant difference between the control group and the BHB group on day 7 or on day 14. On day 6 there was 1 dead fly found in the control group and after 30 days post-eclosion, all flies were dead in both the control and the experimental group.
For the third experiment, the control vials contained 12 male flies and the experimental vial contained 8 males. Four RING analyses were performed in total at 7 days, 9 days, 14 days, and 19 days post-eclosion as seen in Figure 3. There was a significant difference between Day 7 and Day 9 (p =0.005), there was also a significant difference between Day 9 and Day 19 (p =0.001); however, no other differences in time were significant. Additionally, there was a significant difference between the control group on Day 9 and Day 14 (p < 0.05), but not on Day 7 or Day 19.
In the fourth experiment, the Drosophila in the experimental group were on the diet for 5 days before the brain dissections were performed. There were 11 flies in each group before the diet, 5 days after the diet was started, there were only 4 flies alive in the BHB group. There was no significant difference between either group (p =0.589) at any time point (p =0.092) as seen in Figure 3.
Brain Dissections and Staining
In experiment 4, there were 2 partial brains in each group; therefore, brain volume was not able to be assessed. However, the cell death, or brightness of the brain was measured as seen in Figure 4. Using Image J, the mean gray value was measured indicating the amount of synapses in the brain. The control group had an average mean gray value of 21.6995, and the BHB group had an average mean gray value of 25.691, which indicates the BHB group was brighter, as seen in Table 1. Due to the small sample size in each group, no statistical analysis was run.
Discussion
In the present study, there were 4 different experiments that analyzed the negative geotaxis of the Drosophila. There was no difference found in the Drosophila’s climbing ability when comparing their normal Drosophila medium and Drosophila medium with Beta-Hydroxybutyrate (BHB) by the end of the experiments. Further, the survival of the third experiment was recorded over the course of 33 days. Similar to the negative geotaxis, there was no difference in the survival rate between the two groups of Drosophila. This indicates that the diet supplemented with BHB did not have an effect on their overall survival. The brain dissections performed on experiment 4 showed a slight trend of increased mean gray value in the BHB group, which appears to show a higher concentration of synapses in the brain.
This study did not note any lasting differences in climbing ability of the Drosophila, this indicates that there is little to no effect on the neurological functions after the BHB had been introduced to the Drosophila. Further, the mean gray values indicate a potential trend that the Drosophila who were given the medium supplemented with BHB had an increase in synapses in the brain. Further research would need to be performed to validate this, as there was not enough data to perform statistical analyses. This is contrasted to other studies that did find a difference in neurological development when using a mice model. The diet had affected both the pregnant female and the neurological development of the offspring (Wojciech et al., 2021). The offspring that were given a ketogenic diet in utero and during lactation had a delayed response to most of the reflex tests performed, such as opening their eyes and the righting test. Further, Mayengbam et al. (2021) noted a change in the volumetric development and the metabolic profile of inbred juvenile mice. While there was this noted difference, further research was needed in order to determine whether these changes had consequences affecting the functional capabilities of the mice. This indicates while there can be changes to the brain, this may not correlate to changes in function of the animal. There are many differences between a Drosophila model and a mice model as well. The mice in the previous study were given the diet while still in utero; however, that is not possible with Drosophila. The Drosophila were given the BHB after eclosion and after they have developed, as this is the most compatible part of the life cycle of Drosophila as a model for the current study. Further, our study used BHB as an additive into their current food, rather than controlling a true ketogenic diet like in the previous study.
Survival rate was not noted in many other studies. When assessing survival rate for healthy Drosophila there was no difference between each group. In other studies, a difference was found when assessing the ketogenic diet’s effect on survival rate of a certain disease or trauma. Bloomer et al. (2021) found that the ketogenic diet reduced early mortality following a TBI. The Drosophila that were fed a ketogenic diet immediately after the injury had a higher chance of surviving, and they had a higher median lifespan (Bloomer et al., 2021). Another study looked at a mice model, and the effects of the ketogenic diet on survival of mice with systemic metastatic cancer. The mice fed a ketogenic diet had slowed tumor growth and an increase in mean survival of 56.7%, this increase in mean survival increased to 77.9% when the ketogenic diet was used with HBO2T (hyperbaric oxygen therapy) (Poff et al., 2013). These previous studies have shown a difference in survival rate with different neurological problems; however, none of them looked at healthy groups. Our present study found no difference in survival rate when comparing both healthy groups, and while supplementing their diet with BHB.
My original hypothesis was that the motor functions of the Drosophila would increase, the survival rate would stay the same, and there would be altered brain anatomy. Through the current data from this study, this hypothesis was not fully supported. The survival rate was measured, and as predicted, there was no difference between the two groups. This study helped to determine if there was any effect on the neurological development of Drosophila if the ketogenic diet was administered earlier in their life span. The results of the study show that when you supplement with BHB, which can mimic the effects of a ketogenic diet, there is no large effect on the neurological health of Drosophila. Future studies can assess brain volume and determine if there is a difference. A true ketogenic diet could also be used to determine if you would get the same results as using BHB. While there was no change seen in this study, actually analyzing any differences in brain metabolic profiles may provide a fuller picture of the changes in the brain. The length of the study may also provide clearer results, as in a mice model, the mice would be able to be on the ketogenic diet for a longer period of time generally starting in the womb.
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