النص الكامل لحلقة Exploring Philosophical Conundrums with José Zepeda

don't perfectly understand how those networks are organized, and how they're functioning. But chances are that the neurons and their networks there are probably doing something important. For my research topic, I set out to study the calretinin neurons of the nucleus accumbens, which up until this point, we've known nearly nothing about. It started with a very basic characterization of their physiological properties. That eventually transformed into different questions asking, “If we knock these neurons out, if we ablate them, will this affect any behaviors?” and so forth. The sort of eagle's eye view of what I do is, we like to call it microcircuit busting: trying to figure out how these networks function, how the neurons there are connected with each other, essentially. What is it that you're busting? It's as simple as neuron A connects to neuron B, which sends information to neuron C. But then you superimpose all these other neurons that are connected onto those, right? They might be modulating how information is flowing through the brain area. They might be recruited under very specific circumstances and so forth. A lot about their physiology can clue us in on to what function they might have in an awake, behaving animal. Essentially, it's taking a ground up, or a rudimentary sort of standpoint- like a fundamentalist standpoint. Interesting. And when you study addiction, do you study addiction as a whole or is there a type of addiction you're paying attention to? The brain isn't the best at discriminating between kinds of addiction. It turns out that eating addictions, gambling addictions, all sorts of addictions, they all converge on the same neural circuitry in terms of the manifestation of the pathophysiology. It's really hard to answer that question because I think that there are certain types of addictions, or use disorders, that we would like to tackle. But the interesting thing is that all of these different substances and behaviors- they all converge onto similar pathways. They tweak them a little bit differently. So, there's a lot to figure out before we can try to tackle one specific addiction. Very well said. Thank you. And then can you tell us a little bit about how it was that you got interested in this particular topic and niche area? I didn't know from the beginning that I wanted to be a neuroscientist in high school. I was obsessed with physics, theoretical physics specifically. I found it to be the most interesting thing. All of these philosophical conundrums that some of those questions would lead to would just keep me up at night. I left high school thinking that I wanted to do physics. And so, I enrolled at UMass Boston. Originally... well, I have to back up a little bit actually, because it kind of all goes back to a science fair that I participated in as a high schooler, where I presented my project on this thing called a double pendulum chaotic system. It sounds very fancy, but really all it is, is you have a pendulum. If you swing that pendulum, it has a very predictive motion. We can figure that motion out with very simple algebra. But if you were to stick a pendulum to the end of another pendulum, then once you introduce energy into that system, it actually becomes chaotic- the way that it starts to swing. And if you haven't seen it, I highly recommend searching it on YouTube. It's really fascinating. I read in a book on philosophical conundrums that it was impossible to repeat the swing of a double pendulum. I thought, okay, I want to test that. And so, we- me and one of my best friends from high school- we put together a double pendulum in my garage, and just started playing around with it. And pretty quickly realized that this was true, and it was pretty much impossible to replicate a swing at higher energy levels. Because if you imagine the double pendulum here, you can barely just nudge it, and it's essentially functioning like a single pendulum. It's at higher energy levels that it sort of transforms. There's beauty to that. I was presenting this project at a science fair, and I was approached by a professor at MIT, who I really hate that I didn't get their name down. I'm sure I wrote it down, but I've lost it. But they explained to me that they did their PhD, studying the brain as a chaotic system. That just got my mind going because I thought, wow, I hadn't really thought about this before- the fact that a neuron, a single neuron, can very much behave like a chaotic system. I began to think of the brain as this orchestra of double pendulums going on all at once and that inherent chaos may be leading to some of the illusion of free will- but that's another conversation. And so, I knew that I wanted to study physics and try to understand neurons. The language of neurons is biochemistry. So, I enrolled in biochemistry and a minor in physics- that eventually got dropped, because that was crazy that I was trying to take the hardest biology, chemistry, maths and physics courses all at the same time. But yeah, I guess throughout undergrad, I became more and more fascinated with the brain, the more that I learned about it, and eventually got to join a lab studying something called synaptic plasticity. Synaptic plasticity refers to the brain's ability to essentially rewire itself, to make and break connections between neurons. This is a process that's fundamental for, and required for, learning and memory, right? We have a set amount of neurons in our brain. Everybody knows this. You're born with a certain amount. You lose them. The question is, if we're not making new neurons, then how are we forming new memories, right? It turns out that the brain's solution to this is to essentially rewire- reconfigure- these networks. In that process is the encoding of a memory. I thought that process, or that mechanism, was fascinating. I began to think about how maybe that process could be applied to something like addiction where you have potentially these

ultra-strengthened connections that have become pathophysiological. Essentially, the brain doesn't have a lot that it can do to break down those connections once they've gone awry. The million-dollar question here is, which connections, why, and how? That path led me to Vanderbilt because now I wanted to study addiction. I can't get this visualization out of my mind now of a brain made up of double pendulums that are just going everywhere. That's quite the visual you gave us. Thank you. Tell us what discoveries have you made in your research since coming here? I told you a bit about my main thesis project looking at a specific population of neurons. A sort of side project, that I grew to love and enjoy, was one looking at something completely different- where we were looking at these ion channels that are expressed on neurons that their pores happen to dilate quite large when they're activated. It had been shown that larger molecules could flux through there that aren't able to get through other ion channels. We thought, well, what if we could use these ion channels to deliver specific drugs into cells? There's a thing we call membrane impermeant molecules- essentially just meaning that they can't cross the cellular membrane readily. Usually, not always, but usually, that'll be because of some positive charge on the molecule. Basically what we did is, we delivered a membrane impermeant molecule into cells in the brain in an awake, behaving animal. And this animal was essentially shut down. It's really interesting, actually, because the animals, what we did is, we injected them with cocaine. Turns out that when you inject an animal with cocaine, they run around a whole lot, and you can quantify that. When we delivered this molecule into these cells, this molecule specifically blocks action potentials. By doing that, we were able to prevent an increase in what we would call cocaine-induced hyperlocomotion. It didn't affect the basal movement, which is interesting. But it's a proof of principle sort of experiment. We can use these natively expressed ion channels as portals into these cells. I think it's exciting because, although this is a completely different field from my own, I think that certain therapeutic areas could really benefit from such a strategy- such as cancer, where one of the biggest issues that we have with chemotherapeutics is that they affect cells somewhat indiscriminately, right? You stop growth and people start losing hair and they start having all sorts of problems- digestive issues and the such. Imagine if we were able to maybe hijack some ion channel that's already expressed on these cancer cells and target them with a membrane impermeant molecule, then I think that could achieve something. I think that if we have better precision, we will have better outcomes. That's fascinating. It's clear in hearing you talk, that you are passionate about research, that you enjoy this. Tell us what has been something you've enjoyed

most about being in graduate school? I think that you have to really enjoy research, and science in general, to be able to make it through graduate school. Because science, as fun as it is to talk about, will often require tedious, repetitive, sometimes monotonous work and huge amounts of time dedicated to these experiments. Lots of trial and error, troubleshooting along the way. So, I think that, despite all the obstacles or barriers, if I may, in performing or doing science, there are sort of a lot of things that motivate you and inspire you. For me, one of those things is mentorship. I really enjoy talking about my science, explaining my science, and seeing somebody else get excited about it- transmitting that energy and really just paying it forward. I'm here because somebody looked out for me and helped me along the way. I think it's the right thing to do- to try to uplift other people and mentor them. That's something that I've greatly enjoyed. Well, I hope that there are people listening right now that are excited and energetic about starting science and listening to this conversation. You mentioned some of those challenges and obstacles. What would you say has been your greatest one? I think for me, one of my greatest obstacles has been organization outside of academic science. There's a lot of strict guidelines on what you're supposed to do. There are very clear criteria often for whether or not you're doing a good job. And with science, it's easy to get distracted with experiments- maybe trying to pursue one direction and then another because they're all interesting and you want to learn so many things. But, we only have so much time. I think that it's definitely a skill that I've developed through graduate school-stopping and planning my experiments, doing my analysis, making sure that I'm keeping up with it so that I have a clear picture of what I'm going to do next week and what I'm going to do three weeks from now. Because it's really easy to plan an experiment one and a half months out, but there may be other experiments that you can do before then that might tell you if that's even a good idea. You can save yourself a lot of time by just stopping, reading the literature, thinking about it, talking about your science. I feel like that's been one of my biggest struggles- stopping and really thinking about what the next best step is. I love that idea of almost prototyping an experiment to see if it's worth the time and investment. Could you give an example of a time that you've done that and decided, yeah, it's worth it or, oh, it's not worth my time? I really like the idea that, as scientists, we should be trying to prove ourselves wrong instead of trying to prove ourselves right. And so, I tend to approach science that way where I'm going to do as many experiments to prove this one thing wrong or that thing wrong. I can think of so many experiments that, I mean, they're negative data and they're not going to get published. I think that as graduate students, we end up publishing such a small percentage of all the experiments that we actually do because sometimes we're proving that our other result wasn't true, it wasn't real, or it was unreliable. I can think of countless experiments where that's happened. It sucks because you feel like you've wasted time. But I've come to learn that's just part of the process, especially when we're dealing with sort of the frontier of the unknown- where a lot of these experiments, you might be the first person in the world to ever do it. You can't be too hard on yourself. That is a reminder that, at some point, somebody needs to create the journal of failure and there will be multiple submissions in there. Think of all that we could learn, if people were publishing all of these things. It's definitely a movement that's gaining some traction, just maybe not quickly enough. Talk to us a little bit about the Vanderbilt resources that you've discovered- any that you've found to be particularly helpful? I've personally benefited a lot from Vanderbilt programming, from a lot of the resources here. One that stands out to me immediately is the ASPIRE Program. The ASPIRE Program offers so many different sort of resources, such as drop-in clinics for your CVs and your applications, they’ll even do mock interviews with you. One of the things that I got to do that I really appreciated and learned a lot from, was something called the ASPIRE Road Trip, or ASPIRE on the Road rather, where essentially the idea is that a select amount of students and postdocs travel to somewhere else in the country. For us, it was St. Louis., and essentially for two days, get to explore different biopharmaceutical, biotechnological companies, biomedicine companies in the area. That was extremely useful- getting to see the science outside of academia. We often forget that a huge chunk of science careers are in the private sector. It was really awesome to be able to network with people and learn about what they did and learn to think of science from a different standpoint. Oftentimes in the private sector you could see an end goal- something that you want to accomplish that's marketable. That requires a very different kind of thinking than what we do in academia. And so, getting to learn about all of that was amazing. It's one of those things where, sure, you could set those things up yourself, but they take time, and you're busy, and there's probably other priorities. Having something that's prepackaged, “Hey, sign up and come along,” probably made that experience a lot easier for you. Definitely. It's not easy to go to a private pharmaceutical company and just start chatting with someone there. What would you say that you've discovered about yourself through the PhD process? I feel like I've been empowered to be disciplined in different ways and be organized in different ways and really becoming an independent thinker. Which, I think is the goal of a PhD. Maybe

it's a very generic answer, but it's definitely a unique process and you learn a lot, like I said- way, way more than just the science and the minutiae. Earlier you talked a bit about mentorship and being able to give to others. Can you talk a little bit about your own mentor network- how that's been established throughout the years here? I've been blessed with excellent mentors all the way through undergrad into Vanderbilt. I've felt very supported within my program by various different PIs and also teaching faculty. All of this would have been impossible without them. But of course, I got to give a shout out to my own PI, who's been absolutely critical for my success within the graduate program. Brad's been the greatest. He's mentored me well, beyond just the science, because I feel like there are so many other aspects to becoming- one: a professional, and two: an academic. There are so many strategic ways that you can do things and so many things that you're not taught in textbooks. He'll sit down and go through an email with me. You know, is this proper? Is this coming out this way, that way? How should I ask for this? And he's always there to help with whatever I ask. I've learned and grown so much because of him. He also has an open door policy, which I absolutely love because I'm more the spontaneous-type scientist where I don't do super well with the scheduled weekly meeting. I just feel like it becomes more of a chore and maybe kills creativity a little. And so, being able to walk into his office when I come up with some crazy idea or whatever has been amazing. Great. As you think back on this journey, what's something that you wish you had known that would be helpful to others? I mentioned the organizational aspect, or requirement, for doing a PhD. I wish that if I could go back in time and tell myself or give myself any piece of advice, it would be, “Hey, Jose, schedule some time every week to catch up on all of your analysis, to plan out next week's experiments, to really reflect on what the experiments you already did are telling you.” I think that could have helped me avoid so many dead ends with certain experiments or efforts. Yeah, planning some reflection time. Exactly. Jose, you're an NSF GFRP recipient. Can you talk a little bit about your process in that and what it's allowed you to do here at Vanderbilt? I did get the GRFP. I remember when I got it, I couldn't believe it. It's an amazing scholarship, an amazing opportunity. I think that the funding provided by the GRFP allows one to really pursue a basic science proposal. Especially in my field and a lot of biomedical fields, that can become a lot harder to pursue in graduate school because the mechanisms might be very disease-oriented. This journey of trying to understand some neuron subpopulation, and their basic properties, and their basic wiring might not have been possible if I hadn't had the GRFP. I'd probably have to be working on some other project. And as much

as I care about addiction and finding cures and treatments for all of these different disorders, my heart lies in fundamental science, and let's figure out how this works for the sake of knowing how that works, without necessarily having to see some sort of endpoint or goal. I think that a lot of the best discoveries in science came from all these places that we wouldn't have expected them to come through. The green fluorescent protein that revolutionized all of biology, and that started with looking at some algae or something under a microscope. Then you have CRISPR which is now being used to edit genes in living animals, and that was coming from completely different basic biology. So, there's a need for fundamental science. I'm so grateful to the GRFP for allowing me to pursue my basic science research proposal. It sounds like it really gives a freedom for your creative license. Absolutely, absolutely. I mean, not to mention that there's some extra funding for other things. Also excellent programming. Through the GRFP, I was able to obtain something known as the NSF Intern, which is a funding opportunity for GRFP recipients so that they can go and perform research in a non-academic institution. I was able to work at Eli Lilly for three months because of that funding and got to learn a lot because, like I said, when you're in academia, you don't really know a lot about how science is being done in these private sectors. It's just a black box. I want to know what's going on over there. I want to see if there are opportunities for collaboration in the future. Because ultimately, I am interested in pursuing that fundamental neuroscience that will enable the development of treatments. Also, if I want to stay in academia, then I think it's going to be important that I'm able to mentor people on these jobs in the private sector as well. Jose, I know you're close to the dissertation defense. As that looms, what are you most excited about? I think that right now there's the light at the end of the tunnel and I'm crawling towards it. I'm a little bit tired. I think that as soon as I can graduate, I'm going to go to Mexico and enjoy a pina colada on the beach. I think that one of the things I'm most excited about, though, is my next research endeavor. I'm going to be joining a lab at the HHMI Janelia Research Campus, working with David Wong-Campos to develop cutting-edge microscopes with the ability of ultra-fast scanning, without compromising on spatial resolution. I know that sounds so different from everything that I've been talking about- the past, whatever. But it's relevant because photons are such a great vector for information. I didn't go into this, but I use a technique known as electrophysiology. I can stick an electrode into one cell at a time and learn all sorts of things about the electrical properties of that neuron, which is great- that's the language of neurons. They communicate through neurotransmitters, but also their main language

is electricity, essentially. Problem with that is that it's almost impossible to figure out where that electrical signal came from exactly, because you don't have that kind of spatial resolution. Whereas with light, you might be able to see some sensor light up on just a specific patch of the membrane and figure out where that input came from and really start to get at this neural code. The idea is that we'll use these microscopes to image voltage in neurons, and look at these sub-compartments, and try to figure out: how is it that these neurons are piecing all that information together to inform maybe just one single communication coming out from them? Because that process isn't fully understood. All of our models are incomplete. Well, and that sounds like a continuation of the basic science learning, and what could potentially expand from that. So, wish you well in that endeavor. Thank you. I'm really excited. Is there anything else that you'd like to share with the audience before we close? I think it's important to never forget the power that there is in communicating science, and in brainstorming, and in theory. Because I think that all great scientific achievements start at the whiteboard, or at some point in history, the chalkboard, or even further back maybe on some stone tablets- all of that was science communication. I think that the whiteboard, the chalkboard- whatever area where people are getting together and brainstorming- is really... The whiteboard is the most powerful instrument in the lab because that's where everything else is coming from. It's important not to get so carried away with the nitty gritty specifics about our specific projects. I feel like sometimes scientists feel like even other people in the lab, like their project is just a little bit too different from their’s. And so, they're not sharing ideas as often as they probably should be. I think a lot of the best science ideas are also coming from this sort of interdisciplinary space, where these different disciplines are communicating. And so, stay curious and creative, and both those things are going to help your science and help you be a happier scientist. That's great. Well, thank you so much for sharing your time and wisdom with us today. This has been another episode of Discovery Dialogues. Thanks so much for having me. Thanks for joining us on another episode of Discovery Dialogues. Stay connected with the Vanderbilt Graduate School as we continue exploring ideas, innovations, and people shaping our academic community. Until next time, keep asking questions. Stay curious and continue discovering what inspires you. This podcast was recorded in Studio 608 at Vanderbilt University, with the assistance of JT Spangler. This podcast is produced by Anna Thomas, Nick Hyer, and Stacey Satchell in the Graduate School at Vanderbilt University, with audio editing by Ben Hill. The perspectives shared in this episode are those of the speakers and do not necessarily represent the views of Vanderbilt University.