First and foremost the opportunities to glimpse what research is like in BSURF were plentiful. We heard talks from revolutionary scientists and trailblazers, read countless papers, and presented our work in a variety of methods. These activities revealed examples of the larger picture of life as a scientist: asking questions and discovering answers. This picture of science has only made research more compelling and exciting.
I cannot begin to express my gratitude for Bel, my mentor for the summer. Her exuberance and brilliance will forever baffle me, and I was fortunate enough to observe both her techniques and thought processes in working to answer her hypothesis. With immense help from Bel, I have acquired some technical lab skills as well as some insight into how to communicate science. BSURF has afforded us with invaluable practice in communicating our work, from talking with fellow BSURFers, giving a brief pitch which one might do in an interview, to creating and presenting a scientific poster. Apart from revealing the shortcomings of Powerpoint, creating and then presenting a poster was a very real yet very minuscule experience of moving science forward, that ended up looking like a few members of the Eroglu lab congregating around my poster to exchange ideas.
In these eight weeks I have found research to involve slow unsuccessful projects. Since I don’t know enough to formulate questions or work on finding answers, I only experienced a microcosm of scientific research. The big picture of what it means to do research is what is the most meaningful and what I want to do. BSURF has cemented this goal in my mind and I am excited to keep working towards it.
Over the past few weeks of faculty talks, we have learned about innovative research on organisms from archaea to song birds. One particularly fascinating creature, studied by Dr. Sheila Patek, is the mantis shrimp. These crustaceans have club-like appendages which they launch at prey with astounding speed and an acceleration of up to 10, 400 g’s. In fact, the force of their punches is strong enough to create cavitation bubbles in the water. With their collapse, a second explosive force hits nearby prey. From such a small creature, attacking with this magnitude of force and power seams unbelievable and even physically impossible.
With vigor, Patek discussed first what drew her into the field of mantis shrimp study and then the challenges and technologies required to unravel the mechanism for these punches. She discovered how the mantis shrimp wind up its exoskeleton as a kind of spring in order to propel its appendage forward. However, there are drawbacks to generating this much force. Not only does it take time to wind up its exoskeleton, but the shrimp cannot adjust its aim after striking as the motion is too fast for it to register its own movement.
Beyond being a fascinating biological mechanism, the shrimps skeletal spring has important engineering implications many of which Patek has worked on. Not only can mantis shrimp be a model for breaking strong materials, but their eyes can possibly be used to detect cancer. These shrimp have many useful applications; however, when Patek begun her research into shrimp she wasn’t doing it for the purpose of finding an application to help humans. She was driven by curiosity and awe to trying to understand these amazing shrimp. Later, she eventually made a compelling case to a US senator for why her research is useful and important. It was very illuminating to hear her speak about some her doubts about the relevance of her research. While people argue that studying almost everything will have human applications, sometimes it is very unclear what those might be if there are any. Others say that studying something solely for the sake of learning about it is inherently valuable. I often wrestle with these kinds of questions, and it was immensely helpful to hear someone as accomplished as Dr. Patek speak about her experience with research and with finding value in what she is doing.
CTNND2 is an astrocyte-enriched Autism risk gene that encodes for the protein delta-catenin. Preliminary experiments revealed that knockdown of delta-catenin in astrocytes impairs astrocyte survival and decreases astrocyte processes both in vitro and in vivo. Astrocyte morphology coincides with synapse formation during cortical development. Delta-catenin was assumed to be neuron-specific and has been implicated in modulating cadherin-based homophilic interactions between pre and post-synapses. We hypothesis that delta-catenin stabilizes astrocyte-neuron adhesion signalling via cadherin molecules in order to control astrocyte and synapse development. We have verified the presence of endogenous Ctnnd2 mRNA in both cortical and hippocampal astrocytes through fluorescence in situ hybridisation and detail our attempts to visualize N-cadherin through immunohistochemistry.
At this stage of my project, I am staining brain sections with Fluorescence In Situ Hybridization (FISH) and then imaging them. The FISH procedure to stain the mRNA of interest requires one overnight incubation in order for the probes to bind to the mRNA. First, I block and wash the sections. I use salt solutions and buffers to poke holes in the membranes of the cells allowing the probes to enter. I pipette the probes onto the sections with a hybridization buffer and then allow the slides to incubate. The next day I wash the samples and either fix them or proceed with antibody staining.
The next step is imaging the cells. I have signed up to use the microscope for a few hours each day in order to take pictures of the astrocytes. So far I have had difficulties with visualizing the mRNA. One problem is that the neuron specific antibodies block clear visualization of the mRNA. If anyone has any thoughts on how to troubleshoot this problem, let me know. To mitigate the second problem which is unspecific binding of the probes, I performed FISH without using the probes so we could have a control slide. I am working on using this control to be sure that the fluorescent spots I am seeing are really mRNA.
When I am not working on my project, I work on various things for my mentor. Shoutout to Bel my amazing mentor for the summer. She has taught me procedures unrelated to my project from bacterial miniprep to co-culturing astrocytes and neurons. She makes sure I understand how the buffers and reagents work, as well as the overall biological systems the lab is studying. She has also worked with me on every step of my project, from perfusing the mice to harvesting and sectioning their brains to what I am working on now.
I have been thinking about my project from one direction. As I am looking at a specific gene linked with autism, I know the process of how the gene is translated into mRNA then into a protein. After that the protein possibly stabilizes a cell adhesion molecule. The big picture goal would be to understand how this gene affects behavior. Knowing that when this gene is silenced, I could map the cellular processes which lead to behavioral abnormalities. Unfortunately, this thinking has not lead very far as we do not even know what cell adhesion molecule is stabilized by the protein. One of the many positive outcomes of listening to my talented peers present their chalk talks was it made me think about my project from a different direction.
Alissa Kong’s research project is to analyze how visual manipulations affect spatial memory. When you move, your brain is aware of what you are doing. This cognitive ability is known as path integration. You also visually take in your surroundings; your brain is able to integrate this sensory stimuli into path integration. The Gong lab is focused on the role of place cells in this process. Place cells are hippocampal neurons which have been implicated in spatial memory. Alissa’s lab is using virtual reality to test the changes in how place cells fire in response to visual stimuli.
In the hippocampus of mice, the Gong lab first injects a virus which transmits a protein into the place cells. The protein will bind to calcium and fluoresce, which allows for the mapping of when these cells fire. A 1-photon microscope attached to the mouse records the fluorescence data. These mice are then placed on a treadmill with a virtual reality display in front of them. The mice are divided into experimental and control groups. The control group mice see a normal track which they “virtually” run across. The experimental group runs across a track that has been altered in an unnatural way. This variable is designed to test how the brain uses visual information to correct path integration. During these tests, the 1-photon microscope records the fluorescence outputs from the path cells. Alissa has hypothesized that path cells will fire when the visual manipulation occurs indicating that these cells correct physical sensory information.
Alissa’s project is trying to understand one interesting ability of the brain by taking a video of the cells in a live mice. By imaging the firing of these cells, you can then begin to understand the mechanism that allows the cells to fire. The design of Alissa’s experiment made me think about the multitude of different angles to approach my research question and the very cool technologies that make these methods possible.
Dr. Cagla Eroglu PI, runs an organized and efficient lab very unlike Daenerys season 8. Also debatably unlike the Mother of Dragons, Cagla, Mother of Astrocytes, enjoys working with her lab members. Outside of weekly meetings, She schedules one-on-one meetings with each of her mentees in order to keep updated on their research and to offer guidance. Cagla often posts literature relevant to folks projects on the lab’s communication app along with a picture of a moose or possibly some ducks. Working with students is really meaningful to Cagla. She learns a lot from them and she gives sage advice and wisdom in order to help them. Cagla recognizes that we really do not know much about mechanisms of the brain and biology processes. She is adamant that one of the most important things she does is to help her students in how to interpret the data they receive, whether that means using existing theories or challenging the current beliefs as they may very well be incorrect. This philosophy has manifested in a lab of individuals who are open minded and thoughtful about their work.
I had the opportunity to interview this incredible individual to learn to hear her talk passionately about what I had observed in her interactions with lab members. Originally from Turkey, Cagla’s mother was a scientist so she grew up running experiments and doing scientisty things. In her close proximity to research, she held the misconception that most people wanted to do science. Realizing that was not the case heightened her interest in STEM, so she continued on that path in her undergraduate studies. She felt the all too familiar pressure to become either a doctor or an engineer and decided to major in chemical engineering. This intense training provided her with a strong background in math, physics, and chemistry. All of these subjects, she says, have helped support her research endeavors and her training also taught her not to be afraid to apply new procedures, instruments, and technology to her experiments if they might provide better results.
Cagla really wanted to be in biology, so after graduating with a degree in chemical engineering, she pursued a Master’s degree in biology. As she had only learned intro level biology in Undergrad, she had to work hard to learn all of the biological processes. Despite the challenges in entering the field of biology, she was impassioned and determined to continue learning. She was fortunate enough to enter a PhD program in Germany for international students. The labs in Germany collaborated often sharing equipment and ideas. Cagla emphasized the importance of this environment, as it engenders greater exchange of ideas between people. Cagla worked on her PhD on the glutamate receptors on neurons. Her work on cellular receptors lead her to glial cells. She wanted to understand cell-cell connections and glial-neuron interactions were of particular interest to her. In order to pursue this line of study, she entered a lab at Stanford University for her Post-Doctoral Fellowship. This lab was well established allowing her to conduct new and exciting research on astrocytes that launched her career. Fortunately, this lead her to Duke where the Eroglu lab is now known for its work demonstrating how astrocytes secrete factors that cause synapse formation and maturation.
Delta-catenin is a protein that was believed to be neuron specific but spoiler alert it is in astrocytes as well! In the catinen-cadherin complex, delta-catenin is important in cell adhesion by cadherins between pre and post synaptic neurons. Literature on delta-catenin has shown that it can coordinate changes in perisynaptic processes (Arikkath, J., et al). Recently delta-catenin was discovered in astrocytes posing the question of whether it is important for neuron-astrocyte interactions by the same or similar mechanism?
Astrocytes are a type of glial cell that regulates neural functions. Astrocyte morphogenesis is correlated with synaptogenesis or the uptake and removal of synapses (Strogsdill, J.A., et al). Astrocyte release factors have been shown to affect synaptic processes such as glutamate uptake and ion homeostasis. I am working on answering the question of whether direct interaction between astrocytes and neurons affects astrocyte morphogenesis or, in other words, astrocyte complexity?
CTNND2 is the gene that encodes delta-catenin in both neurons and astrocytes. Over-expression in astrocytes results in stickier astrocytes with more adhesions and less complexity. This summer I am working on experiments to knockdown CTNND2 and then test the effects on cell adhesion. One set of tests involves entering a mRNA knockdown of CTNND2 into astrocytes alone to see the resulting effects on astrocyte morphology compared with wild type astrocytes. Preliminary results have showed a decrease in astrocyte complexity which supports my hypothesis. Knocking down CTNND2 in neuron culture will also provide insights. My project is to run a series of combination co culture experiments: wild type astrocytes with CTNND2 knockdown neurons, CTNND2 knockdown astrocytes with wild type neurons, and CTNND2 knockdown astrocytes with CTNND2 knockdown neurons. These permutations will provide some data on the importance of cadherin and delta-catenin in cell interactions.
The next set of experiments I am working on are done in order to image the effects of knocking down CTNND2 in the brain of mice. With a technique called Post Natal Astrocyte Labeling by Electroporation (PALE) astrocytes can be visualized with a confocal microscope to analyze their complexity. One experiment is to genetically introduce a flox into the genome to knockdown CTNND2 mice. This allows for the researcher to choose which cells they don’t want to express the floxed gene. Another aspect of the project is to knockdown the gene in the anterior cingulate cortex (ACC) of the brain which is associated with autonomic processes as well as attention, decision making, and other thinking processes. The ACC is linked to autism in several ways, so visualizing the function of delta-catenin in astrocyte morphology in this region could provide some insight into how the mutation of certain genes leads to cognitive abnormalities. This project fits within the larger goal of the lab which is to understand the interactions of cells in the brain. How cells pass information back and forth is the first small step in understanding how people retain information, make decisions, and have such complex cognitive abilities.
My familiarity with the anatomy of the brain is extremely limited as is my knowledge of biology in general. During my first week when I was learning to use the cryostat to take coronal slices of a mouse brain, my mentor Bel was explaining that the ACC region of the brain is widest between when the ventricle and hippocampus become visible. On a napkin Bel drew the hippocampus as it would appear in a coronal section of the brain, and thus began a serious of educational events where a member of the Eroglu lab would explain something on a napkin.
This summer, I hope to take advantage of the positive energetic learning atmosphere of the Eroglu lab recalling and building off of the wealth of information available to me. By the end of the summer, I expect to be able to understand my project clearly so that I can explain it on a poster or even a napkin. Furthermore, I hope to discover how I can contribute to a lab now with very little experience. I hope to figure out in my niche in the lab, so I can contribute as much as possible in two months. From the lab’s PI Dr. Cagla Eroglu to the Postdoctoral researcher Dr. Krissy Sakers who drew a diagram of Sholl’s analysis on a napkin, I hope to establish positive relationships between the brilliant members of the lab, so they trust me to do certain procedures and speak up when I do not know something.
Bsurf has established an incredible community of individuals outside of lab. The opportunities to hear from professors, researchers, and my fellow Bsurfers will communicate different exciting research avenues within the field of biology. In eight weeks, I can glimpse into what life as a researcher is like and become inspired by the passionate researchers working at the forefront of the field.
The hippocampus, Sholl’s analysis, and an astrocyte as shown in a microscope.