This is the second in a three-part series of interviews with young investigators who were recognized for their work at the 8-11 May 2013 Annual Scientific Meeting of the American Pain Society in New Orleans, Louisiana, US. See Part 1 and Part 3.
Reza Sharif-Naeini, PhD, is an assistant professor in the Department of Physiology and Cell Information Systems group at McGill University, Montreal, Canada. He received his PhD in physiology at McGill University in 2007 under the mentorship of Charles Bourque and completed postdoctoral fellowships at the Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France, and at the University of California, San Francisco, US.
While at UCSF, Sharif-Naeini began a pharmacogenetic project investigating the role of inhibitory interneurons in neuropathic pain, and he continues that research today in his own lab. He received the 2013 Rita Allen Foundation Award in Pain to support ongoing work on the pharmacogenetic project. The award, a research grant in the amount of $50,000 annually for up to three years, is given to young investigators who show “persuasive evidence of distinguished achievement or extraordinary promise in basic science research in pain,” according to the APS. (See Part 3 of this interview series, which features a conversation with Rebecca Seal, the other 2013 recipient of the Rita Allen Foundation Award in Pain).
Neil Andrews, PRF co-executive editor, caught up with Sharif-Naeini at the APS Annual Scientific Meeting to learn about what spurred the young investigator’s interest in pain and how his work on the inhibitory interneurons, and a new experimental approach to alleviating neuropathic pain, is progressing. The following is an edited transcript of their conversation.
PRF: How did you come to focus on pain?
RSN: I actually started out with an interest in biochemistry, but while doing internships in biochemistry labs, I realized that it was not for me at all—I could not just watch yeast grow overnight and be excited about it! But after taking a class on the nervous system as an undergraduate at the University of Montreal, I realized that neurobiology was what I wanted to study. The following summer, I did an internship at the Institut de Recherches Cliniques de Montréal (IRCM) in the lab of Terence Coderre, who works on metabotropic glutamate receptors. I worked on a project investigating the development of opioid tolerance, and I really enjoyed it.
I went on to do a master’s degree with James Henry at McGill University, where I performed in-vivo recordings from dorsal horn neurons in rodent models of chronic pain. I then worked with another professor at McGill, Charles Bourque, who works on the hypothalamus and the mechanisms that regulate fluid balance and the generation of thirst; those processes involve neurons that are intrinsically osmosensitive due to the presence of mechanosensitive ion channels. I had a great time studying this with Charles.
As a postdoc, I wanted to look at the channels themselves, so I joined the lab of Eric Honoré at the Institut de Pharmacologie Moléculaire et Cellulaire. This is an institute that had cloned many of the ion channels we know today. My work there wasn’t related to pain but was rather in a cardiovascular context, studying a disease called autosomal dominant polycystic kidney disease. It was a great experience, but as that work was progressing, I kept thinking it would be nice to study these channels in sensory neurons and try to understand how they contribute to pain. To re-immerse myself in pain research. I did a second postdoc in Allan Basbaum’s lab [at UCSF], where it seemed like there would be plenty of opportunities for me to develop my own ideas.
What project did you work on while you were in Allan’s lab?
I went there with the idea of studying mechanosensitive channels in pain. I started getting some preliminary data, but then the work they were doing in the lab on neuronal networks in the spinal cord and how these can be modulated during chronic pain was so exciting that it influenced my whole approach to pain. There was a young scientist there, Joao Braz, who was attempting to restore the inhibitory tone in the dorsal horn of the spinal cord [by transplanting embryonic GABAergic cortical interneuron precursors into the spinal cord of mice with injury-induced neuropathic pain] in hopes that this approach might alleviate chronic pain. The transplant experiment worked beautifully [see PRF related news story].
But then I thought that if we could find a way to raise the activity of the existing inhibitory interneurons in the spinal cord, and if that alleviated pain, perhaps we wouldn’t even need a transplant. That led to my pharmacogenetic project, which I continued in my own lab.
What questions are you addressing with that project?
The first question is to understand how these inhibitory interneurons contribute to information processing in the spinal cord. Are they modality specific—do they only control mechanical information? Do they control heat pain information? [For more on the specialized roles of spinal interneurons in pain (and itch), see PRF related news story.]
Second, if we increase their activity, does that alleviate the mechanical allodynia observed in neuropathic pain? There is evidence that there are inhibitory neurons in the spinal cord that under normal conditions tonically prevent touch information from activating pain circuits. But in conditions with neuropathic pain, this level of inhibition may decrease, either because the interneurons that are responsible for inhibiting this connection die, or because they reduce their release of GABA [gamma-aminobutyric acid], for instance, or glycine.
Finally, we want to know, with which particular cells do the interneurons communicate—what neurons are presynaptic, and what is the target of the interneurons?
We know that the dorsal horn is extremely complex. We know very little about the subsets of interneurons in the dorsal horn and the nature of their connections with other cells; we know very little under normal conditions, and we know even less under chronic pain conditions. We have now identified markers that are specific to different subsets of interneurons, but there are many more for which we don’t have markers yet.
What is your experimental approach to address these questions?
The first step we took was to determine whether the dorsal horn interneurons die or not in the setting of neuropathic pain. Our preliminary evidence indicates that they don’t die, but it could be that their activity is reduced.
The next step is to get these dorsal horn interneurons to express, in a naive animal, a molecular switch that we can turn on to increase their activity and see whether the processing of sensory information is altered or not. Then we use the spared nerve injury model of neuropathic pain that Shannon Shields developed in Allan’s lab to see whether increasing interneuron activity alleviates mechanical allodynia.
How does the switch work?
Our approach uses a method developed by Bryan Roth’s lab at the University of North Carolina [Chapel Hill, US]. What they did was take a G protein-coupled receptor, mutate the binding site for its endogenous agonist, and create another binding site for an exogenous ligand that doesn’t exist in mammals [Alexander et al., 2009]. That ligand can cross the blood-brain barrier, so you can inject the ligand peripherally—subcutaneously or intraperitoneally—and it still activates its receptor.
We use transgenic mice that express the Cre recombinase enzyme in subsets of interneurons, and then we inject a virus that expresses the mutated G protein-coupled receptor only when Cre recombinase is present. Then we give an agonist and it activates the receptor in the inhibitory interneurons that we are interested in, and we observe the effect in vivo. This is a new approach to restoring inhibitory function in neuropathic pain.
Because we inject the virus only in the spinal cord, when we give the agonist we are only activating spinal cord inhibitory interneurons. That is important because those same inhibitory interneurons are also present in the cerebellum, the hippocampus, and many other sites.
At this early stage of research, how are you finding the work—are the experiments difficult to perform?
This is a complex project. The main experiment is an easy one: We test our ligand in a mouse with neuropathic pain. But there are so many controls we need, to make sure that we are doing things properly. All the experiments are done blind—the experimenter is blind to whether the ligand or a saline solution is injected. The experimenter is also blind to the genotype—wild-type or transgenic—of the animal, as well as to the type of virus we inject—one that expresses the receptor or one that expresses only a green fluorescent protein. Then, when we inject the virus, we always have to go back after euthanizing the animals to verify that our receptor really was expressed only in the Cre-expressing neurons—that is an essential part of this study.
We are also doing a lot of validation on the receptor itself, and all of the validation is performed in vitro, before we actually inject the virus into the animal, to verify that we do, in fact, turn on the interneurons. If we culture the interneurons and have the receptor expressed in them, do we clearly see an increase in activity—do we see an increase in GABA release, for example? Thus, there are many levels of control, and they involve culturing dorsal horn neurons, doing in-vitro electrophysiology and calcium imaging studies, and then slice electrophysiology as well—taking spinal cord slices from the animals and recording from the virus-infected interneurons to verify that their activity is increased. Only then can we confidently say that what we see in the behavioral experiment is really due to all the things that we’re proposing.
In the end, the behavioral experiment, which is the one that definitely conveys the message, is really the icing. The core of the study comes from the electrophysiological validation that the experiment does, in fact, work in vitro.
What is it like, as a young investigator, to have your own lab and to really get it going with all of this work?
It is very exciting, but it is also a learning experience. As one of my colleagues put it, it is baptism by fire because you have to learn a lot of things in a very short amount of time—managing the lab, doing the administrative work, and teaching.
Starting as a new investigator, it is scary at times, but then you take a step back and realize that your training has prepared you for this, and you have to remember that this is what you are good at and it will go well.
Thank you so much for speaking with the PRF.
Thank you for giving me this chance to talk about my work.
