Tag Archives: neuroscience

2018 Summer of Science – Out of Breath

Research Project

My research project this summer has focused on evaluating lung function in patients with cystic fibrosis (CF), a disease that causes excessive mucus build-up in the lungs and digestive organs. Symptoms include substantial breathing difficulty and exercise intolerance, and patients with CF undergo hours of treatment per day that involve medication, chest physiotherapy, and exercise. One important medication is albuterol, a bronchodilator that ensures the delivery of antibiotics, steroids, and other inhaled treatments to airway tissues.

To assess lung function, these patients regularly do breathing tests where, after taking in a full breath, they breathe out as hard and fast as possible. I have been using a mathematical measure, called “slope ratio”, to evaluate these breathing tests and investigate the impact of albuterol and/or exercise on lung function. Lower slope ratios indicate improved airway function, and we hypothesized that albuterol and exercise would decrease slope ratios. My research may aid understanding of how albuterol and exercise affect the lung, which might eventually lead to better treatment strategies for lung disease.

Patients with CF performed the above-mentioned breathing tests during three separate visits: 1) after inhaling albuterol, 2) after exercise, and 3) after both albuterol and exercise. Following this data collection, research has been heavily data-based: the data from just nine patients took weeks to fully analyze. However, developing a conceptual understanding of “slope ratio” kept me engaged; I also developed my skills with writing code (i.e. macros in Excel) to streamline my data analysis, which was a fun learning experience. The results we obtained from our slope ratio analyses closely matched our research hypotheses, were quite interesting to interpret and made logical sense regarding the effects of albuterol and moderate-intensity exercise. Briefly, we found that albuterol decreased slope ratios significantly, suggesting albuterol improves airflow and drug delivery in previously congested airways.

Realities of Research

Mentally, scientists have to remain vigilant; when they encounter contradictions to their prior knowledge, they critically re-evaluate their hypotheses and conceptual understanding. I enjoyed interpreting results and discussing hemoglobin/slope ratio concepts with post-docs in the lab, both one-on-one and in daunting lab meetings. And while it was difficult to work with seemingly-endless data, learning how to write macros helped me to be productive, learn a new coding language, and keep myself engaged.

 

Winston Guo is a junior and Neuroscience major at the University of Minnesota- Twin Cities in Minneapolis, MN. He is a 2018 Undergraduate Research Excellence Fellowship (UGREF) recipient, and is working in Dr. Michael Joyner’s Human Integrative Physiology Lab at the Mayo Clinic in Rochester, MN. Winston’s fellowship is funded by the APS. After graduation, Winston hopes to attend medical school and eventually become a practicing physician.
Multisensory Control of Blood Pressure

When we go from a lying down position to standing up, our blood tends to pool in our legs and has to fight against the force of gravity to travel our brain. Overcoming the force of gravity is the greatest challenge the cardiovascular system faces. Luckily for us, our body is able to detect when such changes are to occur and can effectively change blood pressure to compensate for the change in posture. The body has many different sensory inputs that contribute to homeostatic regulation of blood pressure during these postural changes. The vestibular system, baroreceptors, chemoreceptors, and higher order brain centers (hypothalamus and cerebral cortex) are all examples of the body’s many sensory inputs (Yates et al. 2014). If these homeostatic responses perform inadequately, then conditions such as orthostatic hypotension can be the result (Yates et al. 2017). Orthostatic hypotension results from a drop in blood pressure due to a change in posture, where insufficient perfusion of blood with the brain occurs. My research project this summer focused on the cardiovascular responses that are necessary to maintain normal biological homeostasis during anticipated imposed postural changes. Homeostasis is defined as the tendency of biological systems to maintain relatively constant conditions in the internal environment while continuously interacting with and adjusting to change originating within or outside of the system (The Free Dictionary, n.d.).

In this study, my lab investigated the activity of neurons in the RVLM (rostral ventrolateral medulla), located in the brainstem, in awake adult female cats. There has been significant research to support the claim that neurons in the RVLM play in important role in blood pressure regulation in both animals and humans (Yates et al. 2017). RVLM neurons are necessary for cardiovascular responses to maintain homeostasis during postural movements. The neurons in the RVLM were identified by their changes in firing rate correlated with the cardiac cycle and their location. The experiments in the lab involved recording the activity of neurons in the RVLM during imposed 40º head-up tilts that were preceded by a light cue. The light cue occurred 10 seconds before the 40º tilt, and the heart rate and firing rate of neurons were determined for 5 second time periods before and during the tilts.

Our hypothesis is that in neurons that exemplify cardiac related activity there is an increase in neuronal activity prior to the onset of the 40º imposed tilt, in the time interval after the light stimulus as compared to before the light. This would indicate that when the animal is able to anticipate when the change in posture is about to occur, the animal’s cardiovascular response system will increase heart rate and blood pressure to prepare for the postural change. This would be an example of a feedforward mechanism rather than a feedback. In a feedback mechanism, the animal would have already undergone the change in posture and body positon in space before the cardiovascular response system could compensate heart rate and blood pressure.  This research project will ultimately further our knowledge of cardiovascular homeostasis and may lead to new treatment options for orthostatic hypotension and also a treatment for cognitive mechanisms that can cause changes in blood pressure such as stress and anxiety.

Figure 1: An example of the data that was collected and analyzed during one of the tilt table experiments. The x-axis shows the change in time as the table went from a nose down position, to a 40º head-up tilt. The neuronal firing was isolated along with the heart rate through the use of an EKG. The sun cartoon indicates when the light stimulus was shown with the five second time intervals appropriately labeled before and during the head-up tilt. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

 

Realities of Research

I was extremely grateful to take part in this research opportunity this summer as it really allowed me to grow as an undergraduate researcher. The reality of research is that you get out of it what you put in. If you’re motivated and passionate about what you are doing, then I believe that anyone will be satisfied with the outcome of their experience. I became enamored with learning about physiology after completing Dr. Yates Honors Human Physiology class. After completing his course, I was eager to investigate what research was like on the physiological spectrum. Over a course of 10 weeks, I completed dozens of experiments and analyzed large amounts of data. We performed single-unit in vivo recordings using electrophysiological techniques and procedures. When experiments ran to completion and a respectable amount of usable data was collected, the day was considered successful. Another reality of research is that not every experiment is going to go as planned. There were experiments where either the animal wasn’t cooperating on the table, an electrode bent, or we just simply couldn’t isolate any good neurons to record from. When problems like these would occur, we would simply end the experiment and try again the next day. Because there are a great deal of experiments left to perform and more data analysis to complete, there are no final results or conclusions yet to be made. However, we do predict that the firing rate of the cardiac-related neurons will increase after the light stimulus is given, prior to the onset of the tilt.

Figure 2: The tilt table that is used in all of the single-unit in vivo recordings. During an actual experiment, the curtains would be drawn and the lights would be dimmed to allow for the light stimulus to appropriately work. The machines in the back are used to find and isolate a neuron in the RVLM and to change the position of the table. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.

 

Life of a Scientist

I sincerely enjoyed my daily routine as a scientist in the Yates lab. I was able to set my own schedule and I developed meaningful friendships with everyone I worked with. It was a very-low stress working environment and most days I would follow the same routine. A huge part of my project this year was analyzing the data collected from the experiments. When I wasn’t running any experiments I would be on the computer analyzing large amounts of data. Analyzing data is something that a lot of undergraduate students have trouble with, mostly because it takes a lot of time to learn and pick up. I was very surprised by how quickly I was able to pick up the skill and it allowed the lab to make huge amounts of progress throughout the summer. My fondest memory of my time in the lab didn’t actually happen in the lab. On a Friday afternoon after work, everyone in the lab went out to a Pirates game at PNC Park in Pittsburgh. It was my first one and it was really fun to see everyone outside of the lab doing something together. Overall, I had a great summer and I’m very thankful that APS gave me the opportunity to see what full-time research was actually like. This summer experience was very rewarding and has motivated me to pursue my continued interest in biomedical research.

 

References

  1. Yates, B. J., P. S. Bolton, and V. G. Macefield. 2014. Vestibulo‐sympathetic responses. Compr. Physiol. 4:851–887.
  2. Patel, N.M., Baker, E.A.G., Wittman, S.R., Engstrom, I.C., Bourdages, G.H., McCall, A.A., Miller, D.M. and Yates, B.J. Cardiovascular adjustments during anticipated postural changes. Physiol. Rep. 6(1), e13554, 2018
  3. TheFreeDictionary.com. (2018). Homeostasis (Biology). [online] Available at: https://medical-dictionary.thefreedictionary.com/Homeostasis+(Biology)
John Bielanin is a rising senior majoring in Neuroscience and minoring in Chemistry, with a certificate in Conceptual Foundations of Medicine at the University of Pittsburgh in Pittsburgh, PA. He is a 2018 Undergraduate Summer Research Fellow working in Dr. Bill Yates’ lab in UPMC’s Department of Otolaryngology at the University of Pittsburgh. John’s Undergraduate Summer Research Fellowship (UGSRF) is funded by the American Physiological Society. Outside of school and work, John enjoys reading, writing music, and spending time outdoors. After graduation, John plans on applying to medical school while continuing to pursue research opportunities in his gap year.
Learning to Become a Researcher

When people or animals feel threatened, their sympathetic nervous system, a.k.a. ‘fight-or-flight’ system, releases chemicals that increase their blood pressure and heart rate to prepare for fighting or fleeing danger.  Unfortunately, when someone is obese or eats a chronically high-fat diet, their fight-or-flight system can be in an almost permanent state of overdrive.  This can place too much strain on the heart and blood vessels, potentially leading to hypertension (high blood pressure) and subsequent cardiovascular disease such as a heart attack or stroke.  My research project for the summer was to identify specific pathways in the mouse brain that influence the fight-or-flight response.  More specifically, I aimed to determine how inhibition of the dorsomedial hypothalamus (an area of the brain) by neuropeptide-Y (a brain-specific chemical messenger) leads to decreased activity in the fight-or-flight system.  By determining how various chemicals and pathways in the body and brain influence the fight-or-flight system, we may be able to find new treatments for people who have hypertension, hopefully increasing their longevity by decreasing their risk for serious conditions like heart attack or stroke.

 

Working in a research lab is simply amazing.  There is an almost endless amount of techniques, equipment, and software available to learn how to use.  This summer I have learned how to perform immunohistochemistry, how to use a confocal microscope, and how to utilize different analysis software programs to interpret results from fluorescent images.  If time permits, I may even learn how to perform microinjection surgery on a mouse and how to use RNAscope to complement my immunohistochemistry experiments.

 

Two things that surprised me about working in a research lab were how time-consuming experiments can be, and how expensive research supplies are.  For instance, it takes a minimum of sixteen days post-injection before the mouse brains are ready for me to begin processing them.  The brains must then be frozen, sectioned, immunohistochemically treated, mounted onto slides, then imaged, all of which adds up to around thirty hours of processing for a set of three or four brains.  Additionally, much of the processing utilizes expensive solutions and equipment, such as the $400 primary antibody used in the immunohistochemistry, or the fluorescent microscope which costs around $55/day to use for imaging.  This experience helped me to realize the importance of organization, precision, and time-management when conducting an experiment, since any mistake could result in hundreds of dollars wasted and countless hours lost.  Thankfully the experiments I’ve conducted so far this summer have turned out great, and I look forward to starting my next large batch of experiments next week.

 

The day-to-day life of a scientist is highly variable based on my experience this summer.  During any one week I might complete a variety of different tasks based on the needs of my research project as well as the needs of my lab colleagues. While there are general deadlines to be met for certain things and some experiments that require assistance from others, for the most part I am free to schedule which tasks I will be working on for any given day.  One downside to working in research is that since certain equipment is too expensive for each lab to have one of their own, it must be purchased and shared by the whole department.  For instance, the fluorescent microscope that I use is a very popular tool for the type of research done in our department, so you must make a reservation in order to use it.  Unfortunately, if your imaging is taking longer than expected and you didn’t reserve enough time on the microscope to finish, you could end up waiting an entire week before another reservation is available.  Thankfully, with careful planning, this problem can usually be avoided.

 

Overall, working in research as part of a team with the members of my lab has been wonderful.  Each person has their own unique background in research, and since I’m the most junior member of the lab there is a wealth of knowledge I can learn from each of them.  I truly appreciate how much each of my lab colleagues is willing to teach me what they know, provide answers to my questions, and give me guidance for not only my research project, but for my education and career goals as well.

 

Alyssa Bonillas is a senior at Portland State University in Portland, OR, majoring in both Biology and Psychology.  She is a Hearst Fellow working in Dr. Virginia Brooks’ lab at the Oregon Health & Science University in Portland, OR.  Alyssa’s fellowship is funded by APS through a grant from the Hearst Foundation.  After graduation, Alyssa plans to further her education by completing an MD/PhD program, and continuing on to become a physician-scientist at an academic research institution.
My Summer of Scientific Research

Over the summer I have been working at Emory University with Dr. Tansey and other lab members looking for potential causes of Parkinson’s disease (PD). Parkinson’s disease is an illness that damages important parts of the brain and nervous system. This can cause a loss in efficient body movement. We believe that a specific protein, something that the body naturally makes, called LRRK2 may play a role in this disease because there is an increased amount found in PD patients compared to healthy controls. Therefore, we have studied this protein by trying to pin point its location in the body and learning how it causes other PD symptoms such as inflammation. This research will not only provide answers for PD, but can be used to learn more about other neurodegenerative diseases. The goal is to one day cure such debilitating illnesses for everyone who is unfortunate to develop them.

Realities of Research

One of the biggest things I have learned about working in a lab is that your plan of action can suddenly change, while the goal stays the same. However, there are many times where you must go back to the “drawing board” and erase or insert something new. To me, going back and finding out that you have to try something new is a good thing. You are improving on your research and hopefully it will bring you closer to significant results. I also had other learning opportunities such as improving on specific lab techniques. During my BCA assay (Bicinchoninic Acid assay), which is a protocol used to standardize amounts of proteins in a sample, I had to perfect my pipetting skills for the assay to work. I practiced for hours to get the exact amount of solution every time I withdrew from a tube before I actually ran the real assay. This summer, my project started off bumpy because of an experiment not going as planned. I was taking tissue sections of kidney to stain and investigate for the colocalization of LRRK2 with the primary regulator NFAT (Nuclear Factor of Activated T-cells) and pNFAT (NFAT promoter) status in the nucleus. We were looking at these specific cells because they are involved with the immune system which LRRK2 seems to help regulate. However, after my staining protocol, the kidney cells would essentially combust. After many days of trial and error, we decided that there was not enough knowledge of kidney staining in our lab, and instead of spending time and resources to figure it out we moved on and started looking at a new location. There were plenty of times where we had to re-evaluate our plan, and as I said before I only see them as learning opportunities. Sometimes research is not always going to be straight forward where you make a hypothesis, go through an experiment, and at the end collect an answer. It can take a lot of time and creative thinking to get where you need to go.

Damaged kidney cells.

Life of a Scientist

Being a part of a lab is great for many reasons. One reason is that I prefer the type of work style that it offers. Those aspects include being able to have flexibility within your schedule and not having to be constantly micromanaged. Yet, I am still offered enough guidance to steer me on the right track. I love that every day is another day pushing towards a goal that you most likely had a hand in setting. I am also surrounded by people who all want to see each other succeed. Although everyone in the lab may be working on their own personal agendas, people are constantly helping others with their projects whether it’s by lending their skills for a certain assay or giving an extra hand to make timely experiments go by faster. I would go on to say that the entire science community within your field begins to feel more familiar as your work progresses, as well. I value this idea of a connected community within my workspace. Overall, my experiences in my lab this summer were positive and resourceful. Of course, I have witnessed complications occurring in the lab such as assays not going as planned or having to re-due them because of minor mistakes. Human error constantly occurs and I have learned that you can only work with it and make sure to try as hard as possible to get accurate, significant results. Knowing that I could make a career into doing what I love, conducting science and answering important questions that benefit humanity, gives me the motivation of becoming a neurologist in the future.

Chayla Vazquez is a rising junior at Emory University in Atlanta, GA where she is majoring in Neuroscience and Behavioral Biology, with a minor in Ethics. She is working at Emory over the summer with Dr. Malu Tansey as an Undergraduate Summer Research Fellow (UGSRF) funded by the American Physiological Society. Chayla strives to become a biomedical scientist and utilize her skills in research to help people who struggle with cognitive defects.
Stressed Rats and a Stressed Undergraduate

Chronic stress leads to a greater likelihood of the development of many conditions including post-traumatic stress disorder (PTSD), anxiety, irritable bowel syndrome (IBS), functional dyspepsia and other gastrointestinal (GI) dysfunction. This indicates a likely rearrangement of neural pathways and regulation, although the mechanisms of how this happens are not yet known. As an APS Undergraduate Summer Research Fellow, I worked for ten weeks under my research mentor Dr. R. Alberto Travagli studying the neurochemical oxytocin’s role in stress adaptation. My project focused on the regulation of oxytocin signals between the brain and GI tract under conditions of chronic stress in rats. In other words, I studied whether oxytocin has a different effect on the brain and gut of rats after they have been stressed.

Following a 5-day stress treatment on each rat, oxytocin was microinjected in the dorsal vagal complex (i.e. the brain area that directly signals the GI tract). The response to these injections on gastric tone and motility in two areas of the stomach were then recorded and analyzed. The research is still ongoing, but we hope to answer a few questions: How does the regulation of oxytocin change after stress adaptation? Does oxytocin work through different neural pathways after homotypic stress (i.e. same stress each day) or heterotypic stress (i.e. different stress each day)? Since females have a greater likelihood of developing GI disorders, do sex/estrogen levels affect the regulation of oxytocin under stress? Although we are still collecting data, I am very excited to see the results when completed and honored to participate in this research!

What surprised you most about working in the lab?

Upon starting this project, I was surprised by how much skill is required to complete the tasks at hand. Although the technology we use to inject oxytocin and record the gastric response is quite advanced, it can easily be faulted by a human mistake. For example, if I did not suture the sensors tight enough to the stomach, the responses were difficult to read and interpret. There was a huge learning curve to carrying out the research day-to-day and then it was another challenge to ensure I was as consistent as possible between animals. Additionally, I was surprised by how much my project changed between the beginning and end of the summer. For example, early on we injected a new pathway-blocker out of curiosity, expecting it to have little to no effect on oxytocin injections. Surprisingly, however, in one treatment group it seems to be blocking the effects of oxytocin. After, we used that pathway-blocker for every animal and its effects may be crucial to our final conclusions.

I am very grateful to the American Physiological Society for providing me this opportunity because it has made me realize how challenging a career as a basic research scientist is! This summer has exposed me to how exhausting, long, and physically demanding lab research can be. But, I love the big-picture parts of research; designing the experiments, analyzing the results, and adjusting when results are not as predicted. It is amazing to work on research that could be part of a bigger solution (i.e. understanding of anxiety/IBS/colonic pain), especially when you can collaborate with other researchers and pool data to come to even more conclusions within each study. However, I will admit that lab research is grueling work and, like the rats, I was a little stressed at times! I look forward to next year’s summer project so that I can experience translational or clinical research and gain a more holistic view of the research world.

Julia Zimmerman is a sophomore majoring in Neuroscience at Swarthmore College in Swarthmore, PA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. R. Alberto Travagli’s lab at Pennsylvania State University College of Medicine in Hershey, PA, funded by the APS. After graduation, Julia intends to pursue work as an MD-MPH, or MD-PhD, bridging basic research into the clinical world.