Author Archives: Martín Acosta

Happy Accidents

Happy accidents are those moments of sheer luck and coincidence in life that we’ve learned to cherish whenever they should pop up. We don’t really think about them though. We never take the time to reflect on them, to think where we might be in life should they have never occurred. Dr.Mohamed Noor made me think critically about happy accidents and for that reason I’d like to talk about what I gathered from his talk.

Dr.Mohamed’s talk woke me up from a deep sleep. Literally. I was face-down on the table when he walked in rambling about evolution and genetics. He went on to give a riveting talk about his path he followed as a scientist, the theory of evolution in the context of today’s society, and his own work. As a group we discussed possible solutions for communicating the theory of evolution to lay audiences in a way that they could be receptive to. He played us some example recordings of fly courtship ‘songs’ that were part of some of the work hs has done in speciation. However, the part about his talk that I really resonated with was the happy accident that occurred during the research project he was undertaking during graduate school.

Dr.Noor told us about amazing data he was able to produce during a graduate project that put him in the spotlight as an evolutionary biologist. He discussed it with humility and humor as he described how the amazing results were almost all sheer luck, a happy accident if you will. I found it interesting that Dr.Noor was so open about this. Where would he be if it hadn’t been for this happy accident. Would I be calling him ‘Dr.’ today? If someone had not sent me a snapchat of the Duke application two days before it was due, would I even be here today? I don’t have the answers to these questions or how my life would be different today but I do know it doesn’t matter. I’m so grateful for having been able to follow the path that I was set on by a trail of happy accidents. However, even if I hadn’t been ended up at Duke and at this program I would still know my future lies in science. I would have come to this realization in some other way in some alternate timeline. The paths we follow and the lives we live can seem so haphazard and random but what we can control is what we feel and know on the inside. No happy accident can change that.

Draft of Abstract

Below you will find the first draft of the abstract that summarizes my summer research project in the Kuhn lab. Please keep in mind that this abstract is very preliminary especially regarding result interpretation and conclusions. Enjoy with a grain of salt.

 

The neurobiology of depression remains largely unknown and has not been studied in respect to sex-differences. Rapid tryptophan depletion (RTD) is a method used to lower levels of endogenous tryptophan (TRP) and serotonin (5-HT) and was used in this study to explore possible sex differences in the way rats react to tryptophan depletion. This study administered one of three treatments to 36 rats: 1) RTD, 2) a balanced amino acid treatment (BAL), or 3) None. Plasma and brain samples were collected and later analyzed using high performance liquid chromatography (HPLC) to determine concentrations of TRP and 5-HT. While RTD had similar depletion effects in the plasma of both females and males, there were significant differences in how TRP and 5-HT levels shifted in the hippocampal brain region of males and females. Compared to female controls, RTD females experienced significantly lowered levels of 5-HT and TRP, more so than RTD males compared to male controls. This suggest that sex-mediated differences in the serotonergic systems of rats is responsible for the different responses to tryptophan depletion. Further work must explore this neurobiological distinction between male and female rats and clinical research should focus on determining whether a similar difference exists in humans.

WC: 200

Memories of Cultural Primatology

When Georgia started presenting her chalk talk on that particularly humid Tuesday morning, I felt myself leave the room for just a few seconds. I was no longer in our room in the LSRC but on the ground level of Bell Tower house in a small projection room with eleven other students and one red-haired professor. It took only a microsecond for me to realize what was going on: I was having a flashback to my first semester Writing 101 class, “Can chimps have culture?”. A class I ultimately got stuck with due to scheduling difficulties and grew to despise, it was an experience I had tried to obliterate from memory yet I was here, dreaming about it. But just like that, I was back to reality and listening to Georgia talk about environmentally-induced stress in yellow baboons. Her chalk talk brought up some old memories and it made me think critically about how I view science outside of my own.

I grew to hate Writing 101 not because of my professor or her teaching quality but the students and the course content. It tore me to shreds and killed me internally every single time I had to force myself to read a research paper about chimpanzees, gorillas, and a number of assorted monkeys and their propensity for culture. I lost more and more hope for my intellectual future as a Blue Devil when I would have to sit in a room full of closed mouths in the middle of a class discussion we were supposed to have. But more than anything it was the primates. I just couldn’t stand reading about them. All the research began to blend together and paper after paper seemed to be talking about the exact same thing. I thought they would never be a part of my life again but they came back with Georgia.

She was describing her research project: analyzing the glucocortisol levels in the fecal matter of yellow baboons in different environmental contexts to assess stress levels. She was talking about primates again and I really thought my eyes were going to roll to the back of my head and not come back until she was finished. But just the opposite was happening: I was interested. I cared about how food conditions and hierarchy could affect yellow baboons in such a way as to induce stress. I was engaged and even asked how this research might pertain to humans: due to the fact that the two species share more than 90% of their DNA, understanding stress responses in yellow baboons might allow us to understand them more in humans. At the end of it I realized that other than that memory of Writing 101 at the beginning, I had managed to pay attention to the whole talk.

Why was that? Why was I suddenly engaged and interested in an area of research that I despised just a few months ago? I didn’t know it but the answer was simple: I had been engaged effectively in this research for the first time. While my professor had taken us through stacks of research articles in the study of primates, I had always felt detached from the research. I knew about all these studies and their impacts but I was struggling to see the “So what?” in what I was studying. Georgia changed that for me. She had contextualized interesting research in primates for me and made me understand that it was a real, living scientific field with important implications in the real world. I couldn’t feel detached from this research anymore because it was staring me in the face. Someone was finally talking about it with excitement in their voice and vigor in their conviction. For this, I thank Georgia. In the future, I now know that I can avoid the pitfall of detaching and even resenting a field of scientific research just because I think it isn’t interesting. It’s a matter of going to people that know how to communicate their science and have them show you just why their work is interesting, a lesson for all.

Syringe. Clean. Aspirate. Inject.

In four actions, that is essentially a day at work for me. Let me clarify, that’s just the series of actions you go through when you’re running samples through High Performance Liquid Chromatography (HPLC). I’m essentially a lab assistant and in a day of work I help around lab, clean, and run samples through HPLC. I’ve also helped in lab with an unexpectedly biological aspect: the sacrifice and dissection of rats and rat brains, respectively. I want to talk about the two main contributors to my day in lab but also discuss how I’m still growing by working in lab.

HPLC is an analytical chemistry technique used to separate analytes of interest in a solution and analyze them. Paired with an electrochemical detection (ED) system and computer software it can visualize the presence of your analytes as peaks on a chromatogram and estimate concentrations. This technique is simply column chromatography performed under pressure. Simple column chromatography is a column with some sort of “stationary phase” particles through which a liquid mobile phase is passed through at the same time a sample is in the column. A combination of the interactions of the analytes with the stationary and mobile phases will determine the order in which the analytes are eluted, or released. HPLC is this in essence except in order to achieve effective detection of little analyte, it is run under pressure. The applied pressure pumps the mobile phase through the column of stationary phase and a sample is ejected in. This is the most tedious part of the day for me. The apparatus just runs for hours when the pump is turned on and it is just a matter of constantly taking a syringe, cleaning it, aspirating sample, and injecting it. Though tedious, it is a necessary process. Further, the interesting components of HPLC is the ED system.

The ED system works through an electrochemical cell with working, auxiliary, and reference electrodes. As analytes pass through the column they diffuse through into the cell and undergo an electron transfer reaction, an oxidation or reduction reaction, electrons are transferred and detected by the working electrode, and the signal appears as a peak on a chromatogram through a computer program. Almost exclusively because of the electron absorbing properties of the working electrode, an oxidative reaction is the preferred electrochemical reaction to occur after diffusion of analyte into the electrochemical cell. HPLC conditions such as the pH of the mobile phase and the temperature used to run the apparatus are tailored to favor oxidative reactions. The peaks are further processed using computer programming to determine concentration of analytes by comparison with a pre-made standard curve. This final step wraps up of the process of HPLC paired with an ED system. What is left to be discussed is the animal handling part of my work before moving onto what I have gained from these experiences.

A total of three times, I have helped in the process of harvesting blood and brain samples from male and female rats. We begin by weighing the rats and performing one of three treatments on them: 1) gavaging them with the RTD (Rapid Tryptophan Depletion) amino acid (AA) solution, 2) the BAL (balanced AA solution with all essential AAs including tryptophan), or 3) treating them with neither solution. We euthanize the rats in a bell jar to the point that they will not suffer during the sacrifice. The brain is extracted from the cranium and dissected for predetermined brain parts. The blood is also centrifuged for the extraction of plasma and the brain is stored before being sonicated later and centrifuged for analysis. Both the plasma and brain samples are run by me and my lab supervisor, Zack Reavis, through HPLC for sample analysis.

These two processes are essentially tedious yet necessary for the continuation of progress in lab. Sometimes it does become quite drab and it really starts to feel like I’m working a glorified 9-5 job. However, I quickly correct myself. I cannot take for granted just how much I have learned in lab, how many problems I’ve encountered that I’ve had to actually think about to solve. My PI was so particular about designing a project that would somehow allow me to encounter a problem essentially chemical in nature for me to solve and actually do some chemistry! I helped adjust a protocol such that it was tailored for our needs during sample analysis: adjusting the pH of the mobile phase such that the eluted analytes came at a rate that would produce a clean chromatogram with nicely separated peaks. I need to appreciate that even though I am completing a project here that will allow me to experiment to answer a question I’m mostly here this summer to learn what it is like to work in a lab environment, learn some lab techniques, and just get a feel for the life of lab work. This is exactly what I have gotten to do and I’m getting quite a good feeling about what it’s like working in the lab. I’m beginning to think this is something I might want to do for the rest of my life.

What’s in a Rat Brain Smoothie?

Even though my summer research project can be summarized as simply running a seemingly endless amount of HPLC samples, such a description would not do service to the lab I’m a part of. What I will do is part of a larger project with a much wider scope.  My summer project falls under the umbrella of a Tryptophan depletion study conducted in rat models which is a continuation of work done by my lab in the pas

Of particular interest in the etiology of depression and related affective disorders is the role of the serotonergic neurotransmitter system, the system responsible for the transport of serotonin/5-hydroxytryptamine (5-HT). Even though the exact underpinnings of serotonin’s role in mental disease are unknown, it has been confirmed to be involved with the onset of depression. The theory that depression involves a serotonin deficiency is still widely circulated but study results have sometimes produced contradictory and confounding results. Other neurotransmitter systems have been speculated in playing a role in affective disorder including the dopaminergic system. However, the serotonin deficiency theory is the most accepted and makes up the basis of Tryptophan depletion as a method of studying depression.

Due to its clinical significance, effective methods of studying depression in animal models have been the object of scrutiny in the past. One of the more relevant methods involves implementing a social defeat model: a small male mouse is put in an enclosure with a bigger male mouse which then proceeds to “beat up” the smaller mouse. This method leads to depressive behavior in the small mouse but such a model is not effective in female mice and thus excludes the gender that suffers the most from depression. To correct this problem, rapid tryptophan depletion (RTD) has been suggested as a method to induce depressive behavior in both male and female animals to study it.

Based on the serotonin deficiency theory of depression, RTD involves feeding animals (rats in our lab’s study) a mixture of several large neutral amino acids (LNAAs) which reduces the level of endogenous tryptophan (TRP) in the animal and subsequently the levels of 5-HT. TRP depletion relies on the fact that only a limited amount of LNAAs can pass through the blood brain barrier (BBB). The ingested LNAAs do two things to lower endogenous TRP and 5-HT levels: they stimulate protein synthesis and compete with endogenous TRP for entrance into the brain through the BBB. Because TRP is the amino acid precursor to 5-HT, reduced TRP levels theoretically lead to reduced 5-HT synthesis and depressive behavior.

My lab has done work with RTD in the past and right now they are focusing on studying possible differences of RTD between adolescents and adults. I have joined the lab just as they are in the middle of another RTD study and I will contribute the most in two different was: HPLC analysis and rat sacrifice and dissection. High performance liquid chromatography (HPLC) is an advanced analytical chemistry technique used to detect the presence of certain analytes in a sample. It relies on an electrode system to detect analytes as they pass through the column of the apparatus. Analytes take the form of different peaks in an HPLC program and the area is indicative of the concentration. My summer work deals mostly with the analysis of tryptophan, serotonin, dopamine, and the respective metabolites of these neurotransmitters.

Running samples through the HPLC apparatus makes of the bulk of my work but I also help in harvesting blood and brain samples from the rats used in the study. On my first day of brain dissection, my PhD supervisor even said I was a natural for rat brain dissection and that I knew how to chop a rat head just perfectly! What I think is hilarious, however, about analyzing rat brains using HPLC is that one must “homogenize” them. Rat brain matter on its own is too large for the apparatus so we liquefy them and essentially turn them into rat brain smoothies.

I hope to have analyzed enough samples to complete an interesting poster at the end of the summer. Even though this project has elements that might not align exactly with the research I want to do in the future, I hope to continue learning and enjoying this project like I already am.

“I wanted to be a volcanist”: Confessions of a Neuropharmacologist

When I sat down to interview my mentor, Dr.Cynthia Kuhn, the last thing I expected was for her to tell me she once aspired to be a volcanist. This accomplished neuropharmacologist was telling me that she had never really imagined she’d be at Duke with her own lab, it just wasn’t what she’d imagined for herself. Yet, here she was. In the span of thirty minutes, Dr.Kuhn went on to divulge the details of her unique path in science and dispense some truly heartfelt advice.  I’d like to share her story and exactly what I took away from the interview.

Dr.Kuhn’s interests in pharmacology can be traced as far back to her high school career. It began with an amazing physiology teacher that had imbued her with a love of the human brain. She started at Stanford with hopes of majoring in geology, only to be told that women were barred from the major, settling for biology instead. A biology major with interests in drugs and the brain, she took a pharmacology course that explored drugs’ effect on the brain in the context of the Swinging 60s (Dr.Kuhn made sure to specify she did not partake in the fun of the times). On the cusp of completing her Biology degree a year early, Dr.Kuhn knew she wanted to continue exploring pharmacology in her graduate studies.

Intent on continuing at Stanford, she set off on what turned out to be a wild goose chase. After bouncing back and forth between offices, she was finally told that Stanford was not taking any female applicants for their PhD programs. A beam of hope came through when someone finally recommended her to the doctorate program in pharmacology at Duke. It was March and the application was due in December; the situation was dire to say the least. However, Duke had a new training grant that required more people than they could supply so when Dr.Kuhn gave them a call, they were happy to take her on. She was taken under the wing of Dr.Saul Schanberg and completed her PhD in pharmacology at Duke with a post-doc at UNC-Chapel Hill. After her postdoctoral work, she landed a faculty position at Duke and fought her way to build the empire that is the Kuhn Lab today. If the success she found throughout her career wasn’t impressive enough, she also managed to come out on top in an age where science wasn’t too welcoming of women.

For the most part, she has studied the influences of sex and developmental stage on the underlying neuropharmacological and biological mechanisms of psychiatric disease. Her recent work has focused specifically on studying adolescents and this will include the project that she put me on for the summer. Her work, she states, has a public conscious to it. She does it knowing that it will have extremely important implication for the betterment of humankind and that the work itself and science as a whole must be communicated to the public, especially kids. To me, she perfectly encapsulated what it is to be a scientist: a human being capable of logic and reason that studies the natural world with an intent to enrich and better the state of humankind through their work.

When I asked her about mentors and the process of getting one, she gave me an interesting answer: there’s no secret to it. Dr.Kuhn told me that mentors are meant to be organic relationships. It’s not about who can go to the most networking mixers or talk to the most important people in a field. Rather, a mentor relationship comes about when one finds that they have a profound intellectual connection, “synergy” as described by Dr.Kuhn, with a superior that just happens to be interested in the same scientific material. Networking has never been my forte and I lack the social skills for it but what Dr.Kuhn told me really resonated. I don’t need to look for a mentor; I need to feel strongly about my science, communicate it well, and hope that someone will connect with me one day.

All in all, I learned one large thing from my interview with Dr.Kuhn: the path of a scientist is inconsistent and bendy to the point that it might induce nausea. A lot of the steps that she made forward sometimes hinged on saying the right thing at the exact moment or taking advantage of unconventional opportunities but the dedication with which she moved forward was consistent and I still see it burn within her. The only conclusion that I can draw is that as long I keep working and pushing for what I want I can’t fail. In addition, however, I have picked up a myriad of little lessons. The most important one definitely had to be recognizing that as a scientist, I’m doing my work with the hopes of bettering humankind and enriching it by communicating my work. This is the role that I want to keep alive in my work within the sciences. It’ll be a privilege and honor to keep learning from one of the most down-to-earth scientists at Duke.

High Performance what now?

After just one week in the Kuhn lab I can now officially say I am an *expert* in high performance liquid chromatography (HPLC) protocols. Well, not really. Actually, I can’t because I’m not. But that sure is one of my many goals and expectations for the summer. So, let me just start out by talking about exactly what I hope to learn, gain, and do this summer

When I first found out I was admitted into BSURF, I had a literal meltdown in the middle of my common room and my friend snapped me out of it by saying “Martín, it’s not as if all the success in the world is going to come from this.” I thought about it then and I thought about it on the car ride to Duke West Campus. I realized he was right. I needed to have realistic expectations about this summer, the summer of my first significant research experience. So naturally I made list of goals to accomplish or at least start on by the end of the summer.

From a completely technical and academic perspective, I want to become fluent in some important techniques used in pharmacology laboratories, learn more about the field of pharmacology and topics relevant to my project, and familiarize myself with the environment and expectations inherent to the lab workplace. I’m so happy to say that I’ve already had a start on all of these goals in my first week. From the mound of research paper to a neuropharmacology text, Dr.Kuhn has already given me a place from where to dive nose first into her field. She has elucidated on to me that my summer project will revolve around the HPLC analysis of the tryptophan levels in rat brains (more on that later….). My helpful lab supervisor, Zack, has already managed to train me on the HPLC apparatus and has even guided me through my first run all by myself! He even introduced me to one of the most important lessons of the professional lab environment: always check your email in the morning. The lesson took place as I awkwardly waited in lab for a half-hour for him to show up only to realize he wouldn’t be there.

Apart from the work I’ll do in the lab, I’m also interested in what growth I’ll make over the summer, as a person and as a member of the scientific community. To my surprise, we’re actually running a small experiment in lab entirely concerning a question I asked. A total accident if I do say so myself but exciting nonetheless. In the future, however, I don’t want my questions to be accidents. I want to reach the level of maturity and knowledge in my field where I’m able to propose important questions that might be used to kickstart major projects and contribute to scientific progress. Within that realm of personal development, I want to become more ambitious and goal-oriented. There is a reason this is my first research experience: I didn’t really look for lab work my freshmen year. Most of it was out of fear and naivety but also laziness. After getting a taste of lab work, I know I don’t want to stop anytime soon. There might be some major hoops to jump through in the future just to continue research but I hope I have to the constitution to continue what I’m beginning to think is something I want to do for the rest of my life.

There is so much to do in a summer that has just started and I could not be more excited for what this summer holds for me.

My biggest disappointment in lab each morning is going to the fridge to get what looks like a pint of ice cream but is actually just a container filled with HPLC standards.