Elisa Guma and Kevin Sitek
Using a variety of methods to map circuits in the primate brain
Takafumi Minamimoto is team leader of the Neural Systems and Circuits Research Group and deputy director of the Department of Functional Brain Imaging at the National Institutes for Quantum Science and Technology in Chiba, Japan. His research focuses on the interaction between motivation, emotion, and memory in the brain of non-human primates.
To address these questions, Dr. Minamimoto uses a range of methods including neuroimaging with functional MRI and PET as well as chemogenetic techniques such as Designer Receptors Activated by Designer Drugs (DREADDs), which are a class of proteins that allow scientists to control neural activity in awake, freely moving animals.
In this interview, Elisa Guma and Kevin Sitek talked with Dr. Minamimoto about his research program, the path he took to get there, and what we can expect from his 2023 Keynote address.
Read on to learn more!
Elisa Guma (EG): Thank you so much for joining us today, Dr. Minamimoto. To get the audience familiar with your work, would you mind giving us a little bit of background about yourself and telling us how you got into science and the current research that you're doing?
Takafumi Minamimoto (TM): My work uses non-human primates and macaques as a model for humans. I first started by doing monkey neurophysiology, putting electrodes in their brains and recording neural signals. During my postdoctoral training at the National Institutes of Health (NIH) I became interested in using non-invasive imaging technology, such as positron emission tomography (PET) and MRI, to study the brain. Next, I wanted to combine these imaging techniques with genetic or neuronal modifications using technologies like optogenetics and chemogenetics. These methods were well established in small animals like rodents, but not in non-human primates due to technical limitations.
Using these chemogenetic approaches in non-human primates is technically challenging. We tried to streamline the process by using PET imaging to visualize the expression of our chemogenetic receptors, rather than having to perform invasive, costly, and time consuming histology. This allows us to combine this technology with other neuroimaging techniques like functional MRI, which allows us to map the neural circuit and also manipulate those circuits.
EG: That's quite an impressive range of techniques and skills that you've acquired over the years going from in vivo electrophysiology, all the way up to PET imaging and back. You're covering multiple scales of investigation of how the brain works. How do you think about integrating these techniques to try to understand brain anatomy and function? Do you have some ideas about how they work together?
TM: A major goal of mine is to understand brain wide function using nonhuman primates as a model as the complexity and size of the brain is more similar to the human brain than other small animals. This can help us translate information from the rodent to the human as well.
Kevin Sitek (KS): That's really incredible to be able to think on all of these scales at the same time, and choose the best methods for any particular question. Can you maybe give us a sneak peek of what you're going to be presenting at the OHBM annual meeting? Is it going to be more methodologically focused? Or are you going to focus on a few specific circuits?
TM: I'm going to talk about the chemogenetic technology, DREADDs, that we recently developed in the non-human primate. [DREADD receptors can be introduced into specific brain regions through a range of gene transfer strategies, giving researchers a unique ability to manipulate certain brain circuits or cell populations and observe their effects on freely behaving animals.] I’ll cover what it is, how this works, and some of the challenges we faced when applying this specifically to non-human primates. I’ll provide some examples of how we have successfully applied this technology to manipulate specific circuits or specific cell types in the non-human primate brain to modulate higher brain function. I will also discuss the greater clinical applications of this technology.
Finally, I'd like to talk a little bit about the future of data collection, how we use this technology to understand human brain function and functional connectivity. If we manipulate the nonhuman primate functional connectivity with chemogenetics, we can see how it affects behavior.
KS: That's really exciting. I think that's going to be really well appreciated and inspiring for the members of our organization for human brain mapping. Looking beyond this next meeting, now that you have these good set of methodologies for investigating specific circuits, where do you see your research going over the next five or 10 years?
TM: I have several directions going on. One direction is to advance this chemogenetic technology further. I would like to be able to modify specific neural or cell type circuitry, such as dopamine or serotonin to understand how changing the function of these neurotransmitter systems and their circuits affects brain function. Additionally, I’d like to understand the biology underlying functional connectivity of the brains, such as the default mode network. In my earlier work I focused on electrophysiological recording where I set up experiments to understand how stimulation of one region affected another region. These measure faster reactions in the brain and are easier to analyze and correlate with the environment. Now, however, I’m more interested in understanding how small changes in mood, for example, affect brain function.
EG: That sounds like a very important question to be tackling. How do you think you would look at changes in mood using non-human primates, or would you look at humans? Do you have some ideas as to how to unpack this question?
TM: This is technically challenging. It’s difficult to ask a monkey, “How do you feel?”. Instead, I have been working on understanding motivated behaviours in this species because often, if we are motivated to perform a task, we are feeling good. One way to do this is to record animal behavior and perform video based behavioral analysis to measure body movement and facial expression, using behavioral tracking tools such as DeepLabCut. This can help us infer aspects of mood or internal state in non-human primates.
EG: It sounds like the models you have are really useful for being able to dissect circuits and identify mechanisms with closer relevance to humans, given how we're more evolutionarily similar to nonhuman primates than other common animal models like mice or rats.
I am curious to hear a little bit more about how you generate these models of depression in the non-human primates. Do you have other models of psychiatric conditions, too?
TM: We have tried to manipulate the dopaminergic and serotonergic systems in the nonhuman primate given their relevance to human neuropsychiatric conditions. However, they are not “complete” models. We have also used a model of hypothyroidism in the macaque monkeys. We have previously shown that animals with hypothyroidism have low motivation, symptoms of fatigue and depression, as well as decreased dopamine release in certain brain areas. Studying these animals could help us understand certain behaviors associated with decreased motivation as well as brain function using MRI and PET imaging.
EG: I also wanted to circle back to chemogenetics, which is a huge innovation in the nonhuman primate. I'm curious to know what some of the bigger challenges were that you faced in getting it to work.
TM: Thank you. The first major technical difficulty was in determining whether the injected virus was targeting the correct brain region or neural population. This technique had been developed in rodents, so there were no viruses that worked for non-human primates. We had to develop these specific viruses. Determining whether these viruses worked was a painstaking process involving injecting a monkey, waiting for the virus to express, then removing the brain, cutting tissue, and staining it to see if the virus was transfected. This process can take several months or years, and it can be very costly. To avoid this, we thought of using PET imaging to help us determine if the viral vector actually worked in vivo, rather than having to perform all that postmortem validation. So, we developed a PET ligand, [11C]CLZ, that would specifically bind to the receptors we were transfecting with our viral vectors and allow us to visualize the DREADD expression in vivo. This allowed us to examine the spatial extent of the receptors, which are critical to optimizing the technology.
In addition, we also faced several obstacles with the dose of clozapine-N-oxide (CNO), which is the molecule required to activate the DREADDs. We had to administer very large doses of CNO since the monkeys have a much larger body mass than rodents. This is very costly.
Finally, we had to wait a long time for the DREADD receptors to activate after we injected them. So, we developed a new DREADD agonist named Deschloroclozapine (DCC) which gets into the brain very easily and can be activated with high specificity. These innovations have improved the efficiency of DREADD technology in nonhuman primates.
With these technological advances in place, we can now try to manipulate brain circuits, or one brain area by injecting the viral vector and visualizing the effects.
EG: It sounds like you had some significant challenges that you overcame with some very creative methods. I think that's a good lesson for early career scientists in thinking how to innovate around potential roadblocks. I was just wondering if there's anything else that you wish you knew as an early career scientist or some piece of advice that you have for younger scientists about how they can succeed in this field?
TM: I always tried to be a researcher who was working on something different from what others were doing. Being unique is very important. At the time when I was starting my training there were many people doing non-human primate research, especially in Japan, so I was worried about how to move my career forward. I decided to chase the new technologies, which is very challenging and can be a bit of a gamble. But it can also work out, and it is sometimes both important and essential. I was so lucky to have many collaborators, friends, and good teammates.
KS: Yeah, that's great advice, finding the right team and finding the right questions so that you can tackle a unique problem.
EG: I was just thinking about your comments on trying to make yourself unique and identifying or answering questions in ways that people haven't thought of. Do you think that there were particular pivotal moments along your career that really brought you towards the unique path that you're currently on, or was it more of a gradual build over time?
TM: Good question. When I started my career as a university student, I was conducting neurophysiology experiments. I trained monkeys every day—stuck electrodes in their brains and tried to pharmacologically manipulate their neural activity to understand brain circuitry. I also used immunohistochemistry and histology. Experiencing several techniques as a PhD student opened my mind to try using other techniques. Then when I joined Barry Richmond at NIH as a postdoc, I worked on projects using completely different methods and experienced a different culture of doing things. It is common for each researcher to be training one monkey at a time, so one researcher might train one monkey for several years. But we used three set-ups and tested more than 10 monkeys per day. Compared to human neuroimaging or rodent research, nonhuman primate studies may take a long time, but we can still design experiments to collect lots of data. This opened my mind in a different way as well.
EG: It sounds like you are one to try to solve a problem creatively rather than get stuck on it. I think another good piece of advice that you shared at the beginning of this comment was that you got to try lots of different techniques, and often that can open your mind to applying them in a novel way or in a novel species and whatnot. I think there's lots of good advice for junior trainees and what you're sharing with us today.
KS: One issue that I became aware of through a blog post by one of our OHBM members, Hiromasa Takemura, was the lack of visibility of Japanese neuroscientists and OHBM. Despite the strong research culture in Japanese institutions, Japanese members are underrepresented in organizations like OHBM, and those members may prefer to present posters over oral presentations. This is not only very unfortunate for those members but also for the larger community. I think there's a lot that we could gain from richer interactions between members across the globe. As an international organization, this is very important to us. I’m wondering if you have any ideas about how we can better develop cross-linguistic presentations or conferences in a better way?
TM: Yeah, that's a good question. I think one way to reach more Japanese researchers could be to better advertise to Japanese neuroimagers and neuroscientists specifically. For example, updates could be posted on Japanese neuroscience websites. Another way could be to organize a joint symposium with Japanese researchers either in Japanese or at international conferences. We Japanese people tend to not go abroad as much as other groups. Perhaps providing more information in Japanese could be a good way to reach more researchers.
EG: Maybe to close out our time together today, I was wondering if there was anything else you'd want to share with the OHBM audience about your science or your path or anything you'd like to leave this with.
TM: I think that with all of the neuroimaging technologies available to us, it can be overwhelming for early career researchers. Even one paper may include lots of different technologies that are difficult to understand without lots of background knowledge. Even if it may be challenging, do not worry or get discouraged by the situation. Try to understand one method really well. This can give you good intuition about how the brain works in one way, which you can then expand upon by using different methods later on. So, here, for example, with human brain mapping we mainly use neuroimaging as a core technology to understand the brain. But there are a number of technologies that can help us understand the amazing human brain and complex neural circuits and function. So please, open your mind to other methods. I started working in neurophysiology, so my basic idea of how the brain works comes from the neuronal spike. Only afterward did I start using neuroimaging, but I can always try to connect the dots between neurophysiology and neuroimaging and keep updating my intuition. Research is very fun, so enjoy what you are doing! If you don't enjoy it, then you're doing the wrong thing.
EG: I think the advice you share—about not being overwhelmed or scared by not knowing something—is also very important to hear as scientists because we're often trying things for the first time that no one else has done and will probably fail several times before we figure something out. So thank you for sharing that advice with everyone.