Tag Archives: research

Getting Salty About Hypertension

About 30% of the world’s population has hypertension due to a recent surge of salt in our diets (1). According to the American Heart Association, the average American eats more than 3,400 milligrams of sodium daily, while the ideal limit is 1,500 mg per day for most adults (2). So, how does your body know when you have ingested salt? Your brain has specialized cells that monitor the amount of sodium (and other ions or substances). Some of these cells are located in the Organum Ventral Lamina Terminus (OVLT). OVLT is one of a few special section of the brain that lacks a complete blood brain barrier that allows substances in the blood to pass more freely from the bloodstream to the brain. So, this section of the brain essentially has the equivalent of “x-ray vision”; it can “see” the particles in the blood, such as salt, and send messages throughout the body to initiate responses. One of these responses may be to increase blood pressure, which is known as salt-sensitive hypertension. Salt-sensitive hypertension refers to an increase in arterial blood pressure to an increase of salt intake.

 

Figure 1. Rat diagram with optogenetic set-up.

My project utilized a new innovative tool known as optogenetics to inhibit or “turn off” OVLT neurons during dehydration and acute NaCl loads. Optogenetics works by shining a light at different wavelengths at a region of the brain where a light activated or inhibited ion channel was injected via a virally-mediated delivery system. In my experiments, water intake, urine output, and blood pressure (via telemetry) were measured in rats dehydrated overnight or infused with a concentrated NaCl solution with or without the laser on to inhibit OVLT neurons. I found that when these neurons were inhibited, they would decrease the volume of water intake of the rats. Upon further investigation, if OVLT continues to prove to be a major contributor to salt-sensitive hypertension, we hope more methods will be developed and implemented in humans suffering from this disease.

 

Figure 2. Leica Vibratome used to take serial sections of brain to determine injection location. Credit: Dr. Sean D. Stocker’s Lab, University of Pittsburgh Medical Center

Research is a very rewarding occupation. Although experiments can be long and time sensitive, the outcome is worthwhile. As long as you are careful and perform experiments honestly, any result can be used to learn. Further, I was amazed by the techniques I used to complete my experiments. The science and technology behind them is very interesting and innovative. The use of optogenitics is relatively new and entails complex surgeries and sciences. Unfortunately, with complexity comes consequences, and I have hit some bumps in the road. The technology used on the rats was very fragile and could not be fixed if broken. In addition, since it is not possible to alter salt concentrations without altering other aspects of the rat, we had to test the secondary stimuli to see what affect they had on the rats’ behavior. I am not finished collected data yet for this summer, but the results appear promising. In addition, I still need to test more animals and determine if the injected virus hit OVLT in order to define my conclusions.

 

Upon arriving, I was most surprised by the dynamic of the lab. All lab members are working on separate projects, but with related goals. Work is mostly independent, yet the goal is interdependent.  Working with a lab team was a fulfilling experience and helped me gain a better understanding of my project. I was able to learn new things and make better sense of my project through communicating with my team members. The best part of my research is the technology and innovation associated with it. One of my favorites is the way we analyzed water intake by applying basic principles of circuits. During experiments, the rats are in cages with metal floor, plastic walls, and water bottles with metal spouts. A wire connected the floor to the spout of the water bottle, creating an incomplete circuit. When the rat takes a drink, it completes the circuit because it is touching both the metal spout and floor. This causes a voltage to occur, which is recorded on a computer application. This data is used to determine when the rats were drinking, and it can approximate water intake because duration of time at the water bottle spout correlates with the amount of water the rats ingested. In contrary, the worst part of my research was the delicacy of the animals. It is very disheartening when the head caps fall off the rats and no longer can be used for experiments. Another incommodious aspect was the time sensitivity of experiments. I have to give the rats time between experiments, so some days were much slower than others. All in all, the positive aspects of research have outweighed the negative, thus far. It has further peaked my interest in a future of biomedical research, and I would recommend anyone interested in the sciences with a love of problem solving to engage in research!

References

  1. Choi HY, Park HC, Ha SK. Salt Sensitivity and Hypertension: A Paradigm Shift from Kidney Malfunction to Vascular Endothelial Dysfunction. Electrolytes & Blood Pressure 13:7-16, 2015 [14 July 2017].
  2. How Much Sodium Should I Eat Per Day? [Online]. American Heart Association. https://sodiumbreakup.heart.org/how_much_sodium_should_i_eat?utm_source=SRI&utm_medium=HeartOrg&utm_term=Website&utm_content=SodiumAndSalt&utm_campaign=SodiumBreakup [14 July 2017].
Rachel Maile is a junior majoring in Biological Systems Engineering and minoring in Biomedical Engineering at Virginia Polytechnic Institute and State University (Virginia Tech) in Blacksburg, VA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Sean D. Stocker’s lab at the University of Pittsburgh Medical Center in Pittsburgh, PA. Rachel’s fellowship is funded by the APS and has enabled her to further her appreciation for research. After graduate, Rachel plans to perform biomedical research, using her engineering background and physiological expertise.
The Life of a Summer Student Researcher

Childhood obesity has become a major health issue in the United States recently. Research studies show that childhood obesity is associated with an increased risk of declined renal function, which is defined as renal injury. Since renal hyperfiltration, which is when the kidneys are working more to than needed, this leads to excessive amounts of protein to be produced in the urine (proteinuria). This condition is typically associated with obesity. The current study will determine if we prevent the renal hyperfiltration, can we decrease proteinuria and/or renal injury?

Working on ground breaking research and being in an environment that is focused and effective was eye-opening for me. I enjoyed being around scientists that had similar mindsets and were able to help me develop a scientific mind of my own. It surprised me that everyone was so open and welcoming to a new unexperienced college student. But it made me more comfortable, especially when some of my experiment’s failed, but they were very helpful and encouraging throughout the entire process. During my time in the lab the initial study I worked on was not successful, so I was give the study I have now and the results that were collected were what my research host expected and it was a successful study that will be continued.

Being a scientists is like riding a roller coaster every day that you enter into the lab. Some days you are so busy with experiments that you forget to eat lunch. But then other days are very slow, because you are waiting on data or results. It is a great environment to learn not only about science but about yourself, how you manage time, how you interact with people, and how well you work alone. I think the best part about being a scientist is that every day is a new challenge because every day you are working on something that could possibly change the world. I think that worst part is that even if you do everything correctly the data can still not turn out how you want it. But overall I really enjoyed the team aspect of working in a lab. Everyone in the lab helps everyone on their projects and vice versa, because everyone wants to see the lab/experiments succeed because it could have a positive impact on the world.

Alyssa Pennington is a senior majoring in Chemistry at Jackson State University. Alyssa is working in Dr. Jan Michael Williams lab at the University of Mississippi Medical Center in Jackson, MS. Alyssa is a second year Short-Term Education Program for Underrepresented Persons (STRIDE) Fellow which is funded by the APS and a grant from the National Heart, Lung and Blood Institute (Grant # 1 R25 Hl115473-01). After graduation Alyssa plans to pursue a career in medicine and research.
Investigating the Role of Piezo2 in Serotonin Release

At the mention of serotonin, people commonly think of its role as regulating anxiety, mood, and happiness in the brain. However, 95% of the body’s serotonin is produced by enterochromaffin (EC) cells present in the tissue lining of the gut (1) before being taken up by platelets that circulate it throughout the body (2). The EC cell releases serotonin in response to mechanical and chemical stimulation, and this release differs depending on whether the body has been fed or is fasted (3). Gut-derived serotonin is important for regulating:

  • Stretch and contraction of the gut
  • Gut sensation
  • The ability for platelets to clot blood
  • Bone development
  • Blood pressure
  • Metabolism (1)

There are still many questions regarding how the EC cell releases serotonin. Recent studies from Dr. Beyder’s laboratory of Mayo Clinic discovered that a mechanosensitive ion channel called Piezo2 is present in human and mouse EC cells, and that Piezo2 is important for serotonin release in EC cell models after a mechanical force like pressure is applied (4, 5). In the broad scheme of things, understanding the role of Piezo2 in EC cell serotonin release and in regulation of blood serotonin levels could lead to future drug developments for gastrointestinal diseases.

A visual representation of serotonin release from EC cells and platelet uptake. Reference: Modified from Mawe & Hoffman. Nature Rev Gastro & Hep. 2013. (1)

Dr. Beyder’s laboratory created a mouse model with the Piezo2 gene knocked out of the gastrointestinal tissue lining the gut. These knockout mice can be compared to the control mice that do not have the Piezo2 gene removed to see how Piezo2 affects the physiology of the gut. My aims for my ten week research fellowship were:

  • To determine the role of Piezo2 in mechanosensitive EC cell serotonin release from primary EC cells from fed and fasted epithelial Piezo2 knockout and control mice
  • To determine the role of Piezo2 in systemic serotonin levels in fed and fasted epithelial Piezo2 knockout and control mice

As is the case with most research, everything did not turn out as planned!

In the first few weeks of my research project, I had already faced unexpected complications. I was planning to collect cells from mouse colon and place them on a special plate with the proper nutrients and environment to help them grow into what is called a primary culture. However, we were not able to take pictures to show that the primary cultures were present since we did not have a microscope lens that could fit the wells in the plate. With the limited time I had for the project, my team decided to focus on measuring whole gut serotonin instead. This highlights how quickly a research project can change and the many approaches a scientist can take to solve a problem. There were also times I would try a new technique to yield better results, but would ultimately go back to the original technique. Overall, the general research process was to look back to see how an experiment was done in the past, and then complete a series of trial and error cycles from there to optimize the protocol for my specific project.

After collecting and preparing samples, I ran a test called an enzyme-linked immunosorbent assay (ELISA) that quantifies the amount of serotonin in a sample.

Amid the journal clubs and experiments, this summer I learned about the collaborative efforts required to be successful as a scientist. I shared equipment and bench space with other researchers in the lab, so we had to coordinate a schedule so that everyone could complete their experiments. I enjoyed getting to know the other members of the lab, many with diverse backgrounds and specialties. Every week the department had a lab meeting where someone from each lab would take turns presenting research updates. It was a great opportunity to learn about other projects that related to the enteric nervous system and new adjustments made to fix problems. That being said, life as a researcher has its challenges. There were days I would spend hours preparing an experiment, only to made a mistake that could have been prevented if I would have asked the right person. If I had to give any advice from my summer experience, I would say don’t be afraid to ask for help – especially if you are feeling discouraged! Everyone in a lab develops his or her own expertise in something, which makes it impossible to know everything. But one of the beauties of science is that there is still a lot for us to learn, and sometimes the most frustrating failures can bring unexpected discoveries.

References

  1. Mawe GM, Hoffman JM. Serotonin signalling in the gut–functions, dysfunctions and therapeutic targets. Nature Reviews: Gastroenterology and Hepatology 10: 473-86, 2013; 10.1038/nrgastro.2013.105.
  2. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu Rev Med. 60: 355-66, 2009; 10.1146/annurev.med.60.042307.110802.
  3. Sumara G, Sumara O, Kim JK, Karsenty G. Gut-derived serotonin is a multifunctional determinant to fasting adaptation. Cell Metab. 16: 588-600, 2012; 10.1016/j.cmet.2012.09.014.
  4. Wang F, Knutson K, Alcaino C, Linden DR, Gibbons SJ, Kashyap PK, Grover M, Oeckler R, Gottlieb PA, Li HJ, Leiter AB, Farrugia G, Beyder A. Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces. J Physiol. 595: 79-91, 2016; 10.1113/JP272718 PMCID: PMC27392819.
  5. Alcaino C, Knutson K, Gottlieb PA, Farrugia G, Beyder A. Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4. Channels. 11: 245-253, 2017; 10.1080/19336950.2017.1279370.
Alecia Alto attends the University of Minnesota Rochester in Rochester, MN. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) doing research in Dr. Arthur Beyder’s lab at Mayo Clinic in Rochester, MN.  After graduating with a bachelor or science in health sciences, she is considering a career as a physician scientist.

 

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.
A Summer with Gryllus firmus
adult male sand field cricket

Credit: Paul M. Choate, University of Florida.

This past summer, I was awarded an American Physiological Society Integrative Organismal Systems Physiology (APS IOSP) fellowship, which enabled me to perform 10 weeks of intense research at my home institution, Penn State University. During those 10 weeks, I got to delve deeper into what it means to be a true research scientist. I also learned the importance of networking, and being truthful in my own work.

During my summer fellowship, I worked in an entomology lab under my research mentor Dr. Ruud Schilder studying Gryllus firmus, a sand field cricket usually found throughout the southeastern U.S. In nature, this species usually exists as either a long winged or short winged morph1. My research project entailed the use of a respirometer setup (shown below), a device that can be used for studying metabolic rate in small animals to examine whether metabolic rates differ significantly during development of these two morphs. In other words, are long-winged morphs more energetically costly to produce than short-winged morphs? Our research is still ongoing, and I am extremely excited awaiting to see the results when completed.  Understanding metabolic rate in the cricket community is particularly important because it can tell us a lot about their reproductive power as well as specific differences in their energetic pursuits.

lab setup

Respirometer setup. Credit: Avril C, Schilder Research Lab

When we look at smaller animals in comparison to larger animals, it appears that the smaller animals’ tissues are more active than that of their larger counter parts1. What we are trying to figure out is if this general rule applies to size difference during cricket development as well (ontogeny). In other words, my research will hopefully lead to an answer of the following the questions: Are the tissues of smaller more immature crickets more active than fully matured crickets? Does metabolic rate vary across the two different morphs significantly, across age, or both?

I cannot thank the American Physiological Society enough for allowing me this opportunity. I hope other STEM students will take the initiative to do a program like this sometime throughout their undergraduate career. In our endless evolving world, we need more research scientists to unlock the key and take leadership. In closing, I want to leave readers with two questions:

  1. How important is scientific research in today’s society?
  2. In 10 years, do you see the research scientist profession growing significantly? Why or Why not?

 

References:

  1. Zera, Anthony J., Jeffry Sall, and Kimberly Grudzinski. “Flight Muscle Polymorphism in the Cricket Gryllus Firmus: Muscle Characteristics and Their Influence on the Evolution of Flightlessness.” Physiol Biochem Zool Physiological and Biochemical Zoology 70.5 (1997): 519-29. Web.

 

Avril Cooper

 

 

 

 

Avril Cooper is a senior majoring in Biology at Penn State University. After graduation, she plans on pursuing a master’s degree in medical science and eventually going on to medical school.