Blog 4: End of Year Report

Although my research was halted due to the pandemic, from my completed work I have preliminary data indicating that diet impacts overall microglia morphology during early development, a topic that has yet to be discussed in published research. I already presented this research at a conference in February and will present these findings once more for my honors thesis and UGR presentations. 

During my time conducting research, I found that I most enjoyed the ability to learn firsthand about neuroscience as a scientific field rather than a just subject, as I felt that I was able to be an active participant in the application of my learning. Additionally, I also enjoyed learning about the scientific method in a more personal rather than theoretical way, as researching revealed alot to me about how science is conducted as well as the immense amount of work it takes to conduct and publish a scientific study. Most of all however, I enjoyed the possibility of discovering novel findings that could not only add to my prior knowledge and understanding, but also the knowledge and understanding of others both in and outside of the neuroscience field. It was this possibility of discovery and the improvement it could make to people’s lives, that enabled me to push through any obstacles I faced in designing and conducting my experiments, and what drew me to research in general. 

I am very fortunate to have Dr. Sally Marik as my faculty mentor, as she taught me so much about the scientific process as well, from writing grants, to designing experiments and data analysis. I appreciate that she allowed me to design my own project, take initiative in conducting my experiments, and work independently while she provided guidance. I also deeply appreciate her support in encouraging me to present my research and her guidance on how scientific conferences function. I think my experience working with Dr. Marik has one that has definitely confirmed my interest in the scientific field, as doing research with her really exposed me to how science is conducted in the real world. Overall, I am very grateful for the learning experience I got from participating in the UGR program and will definitely use this learning experience in the future endeavors.

 

Blog 3: Effect of Diet on Microglia Dynamics in PTEN-ASD: Preliminary PTEN mutant results

 

During the past couple of months I have analyzed microglia dynamics of both wildtype and pten mutant fish fed 3 different diets (high fat, high glucose, and high protein (control). So far I found that there were no significant differences between area, nearest neighbor distance and perimeter of microglia in pten mutant fish. I also found that the wildtype fish that were fed a high-fat diet had on average microglia with a larger area than the other wildtype fish fed the other 2 diets. However, each group’s microglia had a similar perimeter. This indicates the microglia in the wildtype high fat diet are becoming more activated based on the change to a more rounded morphology. This finding fits with previous literature on the effect of a high fat diet on microglia as well. Additionally, the microglia of pten mutant fish had similar areas to those of wildtype fish, however their perimeter was significantly smaller, again indicating they are assuming a more rounded shape, which indicative of microglia activation. This is very interesting and brings me to question why this is the case. Nevertheless, I presented these findings at a poster session at 2 conferences: NEURON and Dyson Society of Fellows in which I received feedback on my results and what techniques I could use in the future. 

Moving forward, I will test both pten mutant and wildtype fish that haven’t been fed to see if it is the mutation itself causing this or if the diet is the leading cause of this difference in morphology. I also want to analyze microglia motility, however I have been having some challenges in stabilizing the fish under the microscope. I plan to image fish with my mentor to help with this. So far, I have learned more about not just diet and microglia, but also the role PTEN plays in neuronal insulin signalling, which is relevant to my project which looks at the brain. Overall, my research is helping me see how much diet plays a role in brain health and function.

Blog 2: Effect of Diet of Microglial Dynamics in PTEN-ASD: Wildtype Fish Preliminary Results

For my project so far I have analyzed microglia dynamics of wildtype fish that were fed 3 different diets (high fat, high glucose, and high protein (control). So far I have found differences between microglia dynamics (area and movement) of wildtype fish fed a high fat diet compared to the other two diets. I have spoken with my faculty mentor about the possible explanations behind these observed differences and we have came up with a few hypotheses that we are planning to test next semester. I have also been looking at scientific literature about microglia motility and the effects of a high fat diet on microglia as well, to help explain my preliminary results. I have also presented my preliminary findings at the monthly biology research group meeting we have here at Pace and I received some input from other professors on what we could do to get a better understanding of what our results mean. 

My results of my experiment have found that the microglia of wildtype fish fed a high fat diet had a significantly larger cell size as well as faster velocity. Additionally, I have found that microglia of fish in this condition moved greater distances and also moved for less time overall. Other research done on the effects of high fat diet have also found signs of microglial dystrophy, one of them being soma enlargement. While this helps validify my findings, I plan to analyze more fish (my current number of fish analyzed is 6) to see if this trend holds up. Interestingly, the research conducted on the effects of high fat diet on microglia did not analyze motility, which makes it difficult to verify my findings. However, I found that there was some research conducted on epilepsy’s effect on microglial motility in a mouse model, which could help give me some basis to compare my results, as epilepsy is characterized by neuroinflammation and microglia activation, which is similar to the effects of high fat diet on the brain. 

This study found that microglia in epilepsy model mice moved greater distances, while maintaining their velocity. This partially matches my findings, as I also found that microglia in the high-fat diet condition moved overall greater distances, however I also found that they moved at higher velocities. Again, I plan on analyzing more fish to see if this trend continues. Overall, after speaking my faculty mentor, we devised a few more tests to get a greater grasp on what are the effects of diet on microglia. 

Some of these include acridine orange staining (will help us analyze neuronal death to see if microglia are reacting to neuronal damage induced by certain diets), sholl analysis (will help us analyze differences microglial morphology in response to certain diets), annexin-v staining (will help us see if microglia are undergoing phagocytosis), cell counts (will help us see if microglia are proliferating in response to certain diets) and western blots (will help us see if microglia are producing pro-inflammatory cytokines in response to certain diets). Additionally, next semester we are getting a new microscope in the lab, which will help us get a total view of the zebrafish tectum, allowing us to analyze all of the microglia within it. 

In closing, my current hypothesis based on my results is that a high fat diet causes increased microglial activation, which can be evidenced by their enlarged cell bodies, and lower duration of movement, as both imply engulfment of cellular matter, which microglia are known to do in their activated state. However, further analysis using the methods I described above will give me more of a clue if this is what is indeed occurring.

 

Blog 1: Effect of Diet on Microglia Activation and Dynamics in PTEN-ASD

The title of my research project is “Effect of Diet on Microglia Activation and Dynamics in PTEN-ASD”. The purpose of this research is to analyze the impact of diet on the role of microglia in synaptic plasticity in a phosphatase and tensin homolog Autism Spectrum Disorder (PTEN-ASD). From this research, I expect to learn if microglia has a role in the synaptic abnormalities observed in individuals with PTEN-ASD and if dietary composition has an effect on both microglia function and the general brain anomalies observed in individuals with PTEN-ASD. Based on reviewing previous scientific studies, I hypothesize that microglia motility will be negatively impacted in my PTEN-ASD model which will lead to an increase in synaptic numbers. Furthermore, I hypothesize that diet will impact synaptic numbers in both WT and PTEN-ASD models via microglia motility and activation. 

Research Design (Methods):

To test this hypothesis, I will use the zebrafish as a model organism, as they are vertebrates, undergo rapid development, and are transparent early in life. Due to genome duplication they have two genes encoding PTEN: ptena and ptenb. These two genes have redundant function and removing either version yields a viable fertile zebrafish. Using fish that are lacking the ptena or ptenb will be my PTEN-ASD model, as using partial knockout of PTEN closely captures the reduced PTEN protein observed in PTEN-ASD.  I will quantify microglia dynamics in ptena-/- or ptenb-/- fish crossed with a transgenic macrophage/microglia reporter line (Tg(mpeg1:gfp)) and compare it to WT fish crossed with the reporter line Tg(mpeg1:gfp). At 5 days postfertilization (dpf) I will administer one of three diets to feed each the PTEN-ASD and WT fish: a high glucose diet, a high fat diet (egg yolk), and a high protein diet (control).  Following feeding, I will perform time-lapse imaging of the microglia and observe any changes in their dynamics and morphology. I currently have preliminary data showing that a high glucose diet in wild type zebrafish causes aggregation of microglia, as well as, a lack of interaction of microglia with retinal ganglion axons, something that was not observed in the control protein diet.  I believe this research will be beneficial in understanding and examining the role of diet on the activity of microglia in a PTEN-ASD model. I believe if a link to diet and microglia activity is found, this knowledge may be useful in making dietary recommendations for individuals with PTEN-ASD.