New Insights Into Dopamine with Raj Awatramani, PhD, and Daniel Dombeck, PhD
When most of us think about dopamine, we think about reward signals. But new research from Northwestern Medicine, published in Nature Neuroscience, has found a genetic subtype of dopamine neurons that do not respond to rewards at all, and instead, fire when the body moves.
Rajeshwar Awatramani, PhD, and Daniel Dombeck, PhD led this work, and in this episode they explain how the results could change the field of dopamine research and pave the way for new research possibilities, especially concerning Parkinson's disease, a condition marked by a loss of dopamine neurons and motor system challenges.
“It's a new way of thinking about Parkinson's disease that we're focusing on, that the disease is likely a result of the loss of these ‘pro-locomotor,’ if you will, dopaminergic neurons, rather than just a sort of loss of all dopaminergic neurons. And it has implications for the treatment of disease…”
– Rajeshwar Awatramani, PhD
- John Eccles Professor of Neurology
- Professor of Neurology (Movement Disorders) and Weinberg College of Arts and Sciences
“From a neuroscience perspective, there's been a lot of conflicting results in the field of dopamine recordings, a lot of data that didn't quite make sense… I think a lot of clarity is going to come from this way that we've found to dissect the dopamine neurons into different subtypes and different groups… This genetic means to access them is something that should be accessible to groups around the world.” – Daniel Dombeck, PhD
Dombeck and Awatramani, along with two graduate students who are lead authors on this study, have identified several different molecularly distinct dopaminergic neuron subtypes, including one motor responsive dopamine neuron that did not respond to rewards. The results offer a new way of thinking about Parkinson’s disease.
- Dombeck’s research is primarily based on developing new imaging and behavioral technologies to record neuron subtypes in mice as they behave. He began dopamine research when he unexpectedly focused his imaging research on dopamine neurons.
- Awatramani has been studying dopaminergic neurons for 18 years. He met Dombeck very fortuitously on a university shuttle, when they discovered how complementary their research is: Awatramani was interested in dopamine neuron diversity from the molecular angle, and Dombeck was interested in dopamine neuron heterogeneity from the functional angle.
- Using technology developed by Awatramani’s graduate student, Zachary Gaertner – called single nucleus RNA sequencing, which allowed the harvesting of large numbers of dopaminergic nuclei – their team found about 15 different molecularly distinct dopaminergic neuron subtypes.
- The team discovered a motor responsive dopamine neuron subtype that did not respond to unexpected rewards.
- This discovery sheds new light on Parkinson’s disease, which is characterized by a loss of dopamine neurons and motor system challenges. This new study reveals there are different types of dopaminergic neurons in the substantia nigra, and that Parkinson’s is likely a result of the loss of these “pro-locomotor” dopaminergic neurons specifically.
- The first authors of this study are, in fact, two graduate students from Awatramani and Dombeck’s labs – Maite Azcorra and Zachary Gaertner – who are credited with the lion’s share of the project.
- The hope is that these discoveries could lead to treating dopamine related diseases in a subtype specific manner, and potentially lead to greater clarity and consensus in the field at large about their influence on disease.
- Explore a previous study on this topic from Dombeck’s lab
- Lean more about Awatramani’s lab
- Go inside Dombeck’s lab
Recorded on November 8, 2023.
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Rajeshwar Awatramani, PhD, has nothing to disclose. Daniel Dombeck, PhD, has nothing to disclose. Course director, Robert Rosa, MD, has nothing to disclose. Planning committee member, Erin Spain, has nothing to disclose. Feinberg School of Medicine's CME Leadership and Staff have nothing to disclose: Clara J. Schroedl, MD, Medical Director of CME, Sheryl Corey, Manager of CME, Allison McCollum, Senior Program Coordinator, Katie Daley, Senior Program Coordinator, Trish Gougis, Senior RSS Coordinator, and Rhea Alexis Banks, Administrative Assistant 2.
Read the Full Transcript
[00:00:00] Erin Spain, MS: This is Breakthroughs, a podcast from Northwestern University Feinberg School of Medicine. I'm Erin Spain, host of the show. When most of us think about dopamine, we think about reward signals. But new research from Northwestern Medicine has found that there is one genetic subtype of dopamine neurons that do not respond to rewards at all, and instead fire when the body moves. These findings, recently published in Nature Neuroscience, not only offer fresh insights into brain function, but also pave the way for new research possibilities, especially concerning Parkinson's disease, a condition marked by a loss of dopamine neurons and motor system challenges. Here to discuss this research are two of the key Northwestern medicine investigators on the project. Raj Awatramani, The John Eccles Professor of Neurology, and Daniel Dombeck, Professor of Neurobiology at the Weinberg College of Arts and Sciences and Professor of Neurology and Neuroscience. Welcome to the show.
[00:01:18] Daniel Dombeck, PhD: Hi.
[00:01:18] Rajeshwar Awatramani, PhD: Hello.
[00:01:19] Erin Spain, MS: So, can you both tell me a little bit about your research, broadly, and how it relates to dopamine? Dr. Dombeck, do you want to start?
[00:01:27] Daniel Dombeck, PhD: Sure. A lot of my research is based on developing new technologies for recording from the brain, new imaging technologies, new behavioral technologies to record from mice and different neuron subtypes in their brains as they're behaving. We actually came to work in the dopamine system almost by accident, I'd say. We were, trying to pilot some studies, using a new imaging technique in the hippocampus, and things weren't quite working out how we were expecting, and so we thought we'd go to a brain region where things were better understood where we had labeled dopamine neurons and look at their activity patterns, and we should see something that was understood based on the literature. And when we looked there with our new imaging technique, we saw something really new and unexpected. And so this has been a deviation from the kind of traditional work in my lab. We weren't traditionally working in, you know, originally working in the dopamine system, but we came to it through the new technologies we were developing.
[00:02:19] Rajeshwar Awatramani, PhD: What about you, Dr. Awatramani? Yeah, I started studying this since I came to Northwestern about 18 years ago. I originally started studying the development of dopamine neurons. So maybe more than a decade ago, we identified a very unique origin of dopaminergic neurons in the embryo. So we defined that these neurons originated in a structure called the floor plate that was not thought to give rise to neurons. And not only did in the midbrain, the floor plate give rise to neurons, but they turned out to be dopaminergic neurons. And then during the course of the studies, I began to realize that these dopaminergic neurons, they don't just project to a single target. Some project to a region called the striatum, some project to the amygdala, some project to the cortex. And that made me wonder, they must have been different to begin with to get to those different targets, you know, the guidance cues and the mechanisms to arrive at those targets must have been coded differently during development. And so there were some fundamental distinctions between these neurons. And that led me to start thinking about dopamine neuron diversity. And then, of course, it's already known, at the same time, it was being realized that these dopamine neurons have various different functions and are involved in very different diseases, ranging from Parkinson's to drug addiction, which have nothing in common with each other. So that made me really start thinking more about dopamine neuron diversity.
[00:03:51] Erin Spain, MS: So here we have someone who's been studying this topic for 18 years. And someone who kind of stumbled upon it. How did you two come to collaborate together on this study we're going to discuss today?
[00:04:01] Rajeshwar Awatramani, PhD: So that was a very fortuitous circumstance. I happened to be taking the intercampus shuttle from Feinberg back to Evanston, and next to me was seated Dan. And I hadn't really, I may have met him once before, but only cursorily. The journey is about 40 minutes. And so during that time, I started, we started just chatting and seeing what each other's labs were up to. And then it turns out I was interested in dopamine neuron diversity from the molecular angle. And he was starting to gather preliminary data on dopamine neuron heterogeneity from the functional angle. And so by the end of that journey, we thought, Oh, my God, this could really come together nicely as a potential RO1. And so a few months later, that's what we did. We wrote an RO1, which eventually was successful.
[00:04:55] Erin Spain, MS: Now, this is the true definition of a happy accident, I guess, or the water cooler talk that really turns into something. How long ago was this bus ride?
[00:05:03] Daniel Dombeck, PhD: Yeah, I think this is 2016.
[00:05:03] Rajeshwar Awatramani, PhD: 2016. Yeah.
[00:05:06] Erin Spain, MS: Well, before we get into these latest results, just give me an overview of what was generally accepted knowledge of dopamine neurons prior to this study.
[00:05:17] Daniel Dombeck, PhD: So, you know, I think the, popular press view, like the sort of general public view of dopamine, when you think about dopamine, you usually think about, reward signals, you think about that hit of dopamine you get when, you know, you win your favorite video game or you eat a good piece of chocolate cake or something like that, right? I think that's what people often think about when they think about dopamine. But, we also know that when dopamine neurons die, people have trouble moving. This is exactly what happens in Parkinson's disease. And so this has really been sort of a mystery for, you know, why there's these differences. And the idea that dopamine neurons are reward related comes from recordings that were done by some scientists, Wolfram Schultz and colleagues back in the 1980s where they stuck electrodes down into the midbrain where these dopamine neurons reside in non-human primates. And I think they were originally expecting to see motor signals because it was certainly known at the time that, you know, about Parkinson's disease and the death of these neurons lead to motor problems. And what they found instead were predominantly reward related signals. When the monkeys received unexpected rewards, they saw big bursts of signaling in these neurons, and, you know, not very much motor related signaling. And so that was pretty confusing and it's led to what we often refer to as the dopamine paradox ever since, which is that if those neurons die. People have trouble moving, but when we record from those neurons, they seem to respond to reward. So where's that, you know, difference coming from? That's what, I'd say, a lot of what was known before our work.
[00:06:45] Erin Spain, MS: So let's talk a little bit about your work. You tagged, you and your team tagged neurons in the brains of genetically modified mouse models with fluorescent sensors to see which neurons control different specific functions. So tell me about this approach and what you were able to find.
[00:07:03] Daniel Dombeck, PhD: So we use a molecule called GCaMP. It's a genetically modified fluorescent protein that can be expressed through genetic means in specific neuron subtypes. I'll let Raj explain in a few minutes exactly how we get those different subtypes labeled and how we make those genetically modified mice. But, for the recording side of things, we label these different groups of dopamine neurons with this GCaMP molecule. And when neurons fire action potentials, calcium comes into the cells and this molecule binds to that calcium and it gives off a burst of light. And so we can then use optical tools, basically light as a readout for neural activity, and we can then stick optical fibers into the brain and record these bursts of light to know when the different neuron subtypes are actually active and responding to stimuli. And one of the things we've pioneered in my lab is this ability to do these recordings in behaving mice while they're behaving, running on a treadmill, for example, and receiving rewards. And so we can look at these responses of different dopamine neuron subtypes in relation to movement of animals and while they're receiving unexpected rewards. The two things that dopamine neurons, you know, are thought to be involved in and try to tease out when the neurons are active with respect to those different behaviors.
[00:08:23] Erin Spain, MS: Raj, tell me a little bit more about that and the role that your team played in identifying these genetic subtypes.
[00:08:30] Rajeshwar Awatramani, PhD: This started about a decade ago. And, you know, when I first started getting interested in the heterogeneity of dopamine neurons , and back then we used more crude techniques to separate out different types of dopamine neurons. Now, in this study, we use more advanced techniques, the technique pioneered by my graduate student, Zach Gartner. What he did is he developed a pipeline called single nucleus RNA sequencing, his pipeline allowed us to harvest large numbers of dopaminergic nuclei, which was previously not possible. And because he had such a large collection of dopaminergic nuclei, he could now use single nucleus transcriptomics provided by the core at Northwestern to group dopaminergic neurons based on their molecular signatures, based on their commonalities. You know, in this study, we found about 15 different molecularly distinct dopaminergic neuron subtypes. And then using that information, we could develop genetically targeted mice. I also direct the transgenic core at Northwestern, and so using the core facility, we were able to make a few different genetically targeted mice, which would allow access to only that subtype of dopaminergic neuron in an otherwise intact brain. And so using those genetically targeted mice, we could now access these subtypes and look at, for example, their projections, which we did in our lab. But then we handed these mice over to Dan's lab and he could introduce GCaMP viruses into these mice to study, again, that population in isolation..
[00:10:15] Erin Spain, MS: Well, tell me the results of this study. They were not what many would have expected. What did you find?
[00:10:22] Daniel Dombeck, PhD: Yeah, that's right. So several years ago, even before this study, we had found the unexpected finding that I was talking about earlier in our discussion here, that we found some dopamine neurons that were active when animals were running, and not so much when they were receiving reward, and that was when against, you know, The recordings that were made in the primates, for example, it was a bit of a surprise, but it fit in with, you know, what we know about dopamine's role in Parkinson's disease, for example. But after that, a lot of people kind of went back to the way of thinking that most dopamine neurons still respond to reward. Maybe there's a small subset that have some motor activity, but, by and large, dopamines are reward responsive neurons. And so it was really, you know, with these genetically modified mice that Raj's lab was developing that we could then really go in and ask, is there a defined subtype that is motor responsive and not reward responsive? Or is it true that almost all of the subtypes have this reward signal and maybe some of them have a small motor signal? And what we found was that, we found a specific subtype in the substantia nigra, part of the midbrain where dopamine neurons live, that makes up a pretty substantial fraction of the dopamine neurons there that was active when the animals were running. And did not seem to care at all when the animals were receiving rewards. So here was a motor responsive dopamine neuron subtype not responding to unexpected rewards, that is defined genetically, based on expression patterns that Raj's lab had found. And so we really isolated this subtype and that's a very important component of the study of what we found.
[00:11:54] Erin Spain, MS: This is something that people have been chasing really for decades. And what was the reaction of your peers in the scientific community when the study came out and that you were able to finally identify these specific neurons?
[00:12:07] Daniel Dombeck, PhD: It's very recent, the paper coming out. I think, we were, Raj and I were at a meeting recently and a lot of people were very interested. The scientific community is usually pretty slow to adopt big changes and new ways of thinking. And I think it's going to take some time for people to adopt, what we've found and sort of bring it into their way of thinking, but I think people are generally quite excited and, you know, someone, at one of these meetings recently said that we've sort of changed the field in a way that everyone is now going to have to go back and redo their experiments based on different subtypes. You can't just think of dopamine neurons as one homogenous group now. People are going to have to go back and look and ask, well, what dopamine subtype was being recorded for that type of experiment or this type of results, et cetera. And so that's the sort of change that's probably coming.
[00:12:52] Erin Spain, MS: Well, and let's talk a little more about that correlation to Parkinson's disease, which is characterized by a loss of dopamine neurons and motor system challenges. Raj, can you explain this to me, this correlation and what this could mean for people who are investigating this disease?
[00:13:07] Rajeshwar Awatramani, PhD: So very simply the medical textbook would tell you that the substantia nigra degenerates in Parkinson's disease, really leading to less dopamine in the striatum. That's sort of the dogma in the field for the last 50 years. Now, what this work is adding to it is that there's different types of dopaminergic neurons in the substantia nigra, and they have different properties, some of which are correlated to locomotion, and it turns out by the location of that population we can surmise based on other studies that we and others have published that those are the populations that the subtypes of dopaminergic neurons that degenerate during Parkinson's disease. And so it's a sort of new way of thinking about Parkinson's disease that we're focusing on, that the disease is likely a result of the loss of these pro-locomotor, if you will, dopaminergic neurons, rather than just a sort of loss of all dopaminergic neurons. And it has implications for the treatment of disease because what it says is that not all dopamine neurons are degenerating and the remaining ones when you treat patients with levodopa those remaining ones are intact and they may at least in part be the result of some of the unexpected or the adverse effects of levodopa.
[00:14:33] Daniel Dombeck, PhD: I was just going to add that. I focused mostly on the subtype that is motor correlated and not responding to reward, but there's other subtypes that we recorded from, and Raj was hinting at other subtypes that are more resilient in the disease and remain. And one of the interesting findings that we made was that those resilient dopamine neurons, those subtypes, some of them also had motor related signals. But they were more active when the animals were decelerating or stopping rather than accelerating or starting to move. And so one of the implications, potential implications, of that difference is that if it's the acceleration movement driving population that's dying first in the disease, then this resilient population is the one that might be involved in driving stopping movements, then there's sort of an imbalance that's leftover between the system. Instead of having one population that can drive movements, another that might be, you know, driving stopping signals, what you're left with then is this sort of deceleration, stopping related populations that might be, making the disease worse and worse as that imbalance progresses.
[00:15:41] Erin Spain, MS: Raj, could this be guiding your future research into Parkinson's disease, and could there be any implications for any other neurodegenerative diseases that affect motor function?
[00:15:52] Rajeshwar Awatramani, PhD: So first of all, it allows us to hone in on specific subtypes of dopaminergic neurons. And now we can begin to inquire what makes these different? Now we know they're transcriptomes, we know their genetic signatures, we can begin to start to think what makes these ones degenerate? So that's a clear implication. The second implication is we can start to query, Are these different subtypes parts of different circuits? Do they have different connectivities? Do they have different inputs and outputs? And that's something our team is actively investigating. As to the question about, different dopamine related diseases, yes, not neurodegenerative diseases, but there are many diseases associated with dopamine, one of which we're studying is opioid addiction, and we are trying to elucidate populations of dopaminergic neurons that may be particularly responsive to opioids, or that may be, driving the withdrawal symptoms, seen in opioid patients. And this is being done as part of a separate collaboration with the Center for Pain at Northwestern, driven by Vania Apkarian.
[00:17:02] Erin Spain, MS: In many ways this paper should be viewed as a starting point. Tell me about some of the different aspects that still need to be flushed out.
[00:17:09] Rajeshwar Awatramani, PhD: Our current recordings were bulk recordings, and Dan's lab and other members of our team are starting to look at this at more at a single axon or at a single cell level in terms of the function. Dan's lab is also looking into stimulating these neurons to see if you can drive locomotion and that's turning out to be quite interesting. So it's leading to a lot more new avenues of research.
[00:17:35] Daniel Dombeck, PhD: Just to add to that, all of our findings were made in mice, and the disease, the implications that it has for humans, it's still far away, right? And so there's a lot that needs to be done to try to translate these results to first, possibly to primates and then to into human primates. And so it's tempting to think of the direct connections between what we've done in the dopamine system in a mouse and try to make direct connections to human disease. But, a lot of work needs to be done to fill in the gaps and make sure that the mouse system is not just fundamentally different in some way, which is still a possibility.
[00:18:11] Erin Spain, MS: This all started with the bus ride to Evanston and R01, and then you were able to get more funding, NIH funding, foundation funding. Tell me about the support that you've had for this project and support that you're hoping to get going forward.
[00:18:26] Rajeshwar Awatramani, PhD: After the NIH grant, we had acquired quite a lot of interesting preliminary data. And so we decided to apply for the Aligning Science Across Parkinson's Disease grant, which is a large grant. It's a team grant. And so we formed a team at Northwestern and beyond. So there's a few investigators, including Mark Bevin, Lucia Perciadu, Jim Surmeier at Northwestern, and Tom Nasco at UCSD who form part of our team. And so we wrote this grant and even though we don't come into this field as Parkinson's disease experts, me being a developmental biologist and Dan being interested, you know, in the functionalities of dopamine neurons, but the foundation found this work very compelling and therefore they funded our grant and we're very excited to be part of this larger network of PD investigators. And when we go to those meetings, we hear about different aspects of this disease, which we are not studying, but it adds to our sort of understanding and it sort of helps us, put our work into the context of that larger field as well.
[00:19:35] Erin Spain, MS: I want to point out that the paper's first authors are both graduate students in both of your laboratories. Tell me about these extraordinary young people that you have working with you and the roles that they've played.
[00:19:47] Daniel Dombeck, PhD: I think it's pretty remarkable that they're both grad students. And if you look at the amount of work that went into this paper, it's an extraordinary amount of work. And the quality of the data, and the analysis that was done is just, you know, I think pretty stunning. So, Maite Azcorra was the grad student. She was joint between our two labs. She built the optical system to do these recordings, develop the techniques to isolate the signals, make sure they weren't movement artifacts. And she was piloting all of the methods to label the neurons with different types of viruses. And, it was great to see, you know, after what, four or five years of work, you know, I credit her and Zach, for the lion's share of what went into the paper and the discoveries that were made.
[00:20:30] Rajeshwar Awatramani, PhD: Zach Gaertner was an MD PhD student who joined my lab, I think in 2018 or 2019. And a few months after he joined the pandemic struck. And so he's sitting at home. and very frustrated that he didn't have a good glimpse into dopamine neuron heterogeneity, because the studies that had been published at that point, including one from our lab, had analyzed very few neurons. And so the first thing he did while he was sitting at home is he compiled all those studies into one data set and to increase the numbers. And that was moderately satisfactory. But then, as soon as he was allowed to come into the lab, he devised this new pipeline to extract larger numbers of dopaminergic neurons, and in this case nuclei that allowed him to gain a sort of much more granular view of the dopaminergic system. So yeah, between him and Maite, they did a fantastic job on this paper.
[00:21:28] Erin Spain, MS: As we wrap up today, what would you like to leave our listeners with? What do you want them to know about this discovery and what it could mean for the future?
[00:21:35] Rajeshwar Awatramani, PhD: My take is that there's a lot of different dopaminergic neuron subtypes. We've just started this exploration, and it's gonna get more and more sophisticated and interesting. And ultimately, I hope that we even might be able to, based on the transcriptome of these individual subtypes, maybe there's channels on the subtype that are specific to that subtype or receptors, and then we could start treating dopamine related diseases in a subtype specific manner. That would be a great outcome of this work.
[00:22:13] Daniel Dombeck, PhD: I'd say, from a neuroscience perspective, there's been a lot of conflicting results in the field of dopamine recordings, a lot of data that didn't quite make sense, you know, conflicting reports from different groups, things that should have looked similar, and I think a lot of clarity is going to come from this way that we've found to dissect the dopamine neurons into different subtypes and different groups that have different activity patterns and this genetic means to access them is something that should be accessible to groups around the world. And so having that way of dissecting the circuitry is hopefully going to lead to some clarity and more consensus in the field about what these neurons do and what they're, what diseases they're involved in and what activity patterns they're involved in.
[00:22:55] Erin Spain, MS: Well, thank you so much for coming today and explaining the results of this very exciting paper. And we look forward to the future and what's coming next.
Daniel Dombeck, PhD: My pleasure.
Erin Spain, MS: Thanks for listening and be sure to subscribe to this show on Apple Podcasts or wherever you listen to podcasts and rate and review us. Also for medical professionals, this episode of Breakthroughs is available for CME credit. Go to our website, feinberg.northwestern.edu and search CME.