How peppers and peppermint identified sensory receptors for temperature and pain

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00:00:06.19 Hi my name's David Julius, I'm in the Physiology Department
00:00:09.21 at UC San Francisco. And I'd like to take a few minutes to tell you
00:00:13.19 about how we have used spices to understand mechanisms
00:00:17.14 for temperature and pain sensation. And I thought I'd begin
00:00:20.09 just by telling you a little bit about things that have interested me
00:00:23.28 and what sort of inspired me to work in this area.
00:00:26.22 So for many years, I've had an interest in natural products
00:00:30.28 and how these can be used to explore physiology and neuropharmacology.
00:00:35.05 And to wonder how people have discovered the use of plants
00:00:39.12 to folk medicine and other ritualistic behaviors. How the
00:00:43.08 active ingredients were identified and how they've been used
00:00:45.22 in biology to explore signaling pathways. And I just put
00:00:50.01 this slide together to show you some of my heroes in this area.
00:00:53.01 And how they've used natural products from things from opium poppies,
00:00:57.15 psilocybin, or marijuana to understand key signaling pathways
00:01:01.20 in the brain, like opiate receptors, serotonin receptors,
00:01:04.28 and cannabinoid receptors. And to illustrate the poewr of natural products
00:01:10.01 for identifying endongenous signaling components in a system.
00:01:14.22 The other thing that I've been interested in for many years
00:01:16.29 like many people in science, is to understand sensory systems.
00:01:21.12 And I think for many of us, including myself, this fascination
00:01:25.06 really comes from the idea that sensory systems really determine
00:01:29.00 how we perceive our world. The kinds of frequencies of light we can see,
00:01:32.17 the sounds -- the frequencies of sound we can hear, the
00:01:36.07 things that we can taste, are really determined entirely
00:01:40.22 by the sorts of receptors that we have in our sensory system and how
00:01:44.04 that information is integrated in our neural circuits. And
00:01:47.25 what's also interesting is that different animals see the world
00:01:50.18 in different ways, because they have different receptors.
00:01:52.24 A rattlesnake, for example, can see a rodent on a moonless night.
00:01:58.13 Because it can detect its body heat, and so I think this raises
00:02:02.14 fascinating questions about evolution and about how biophysics has
00:02:06.03 tuned sensory systems to enable us to live in the environment
00:02:09.01 that we need to live in. Some years ago, we decided to merge
00:02:14.25 these interests and to study an aspect of sensory systems that
00:02:20.08 at the time was less well understand, than say chemosensor
00:02:24.08 systems like olfaction, taste, and certainly vision.
00:02:27.02 And that is to understand somatosensation, or sensations of touch,
00:02:30.11 in particular, to understand this in relationship to how we experience
00:02:34.17 pain. And the aspect of pain sensation that we focused on
00:02:38.08 is nociception, which is the initial act of detecting noxious stimulants. So
00:02:42.17 nociception is to pain, for example, what phototransduction is to vision.
00:02:47.10 And we've been interested in this subclass of sensory nerve
00:02:50.10 fibers called C and A-delta fibers, the so-called nociceptors
00:02:53.06 that are the subset of somatosensory neurons that carry
00:02:56.24 out the bulk of detection of noxious stimuli that contribute to pain
00:03:00.25 sensation. And our goal was really to identify the molecules and
00:03:04.18 mechanisms that enable these nerve fibers to detect
00:03:07.12 things like environmental stimuli, changes in temperature
00:03:10.24 or pressure, or noxious chemicals, or even endogenous
00:03:14.11 things that are produced during inflammation. Endogenous
00:03:17.15 chemicals like prostaglandins and other things that generate
00:03:20.28 or contribute to pain sensation. And the place where this all
00:03:24.28 comes together for me is really that natural products have played such a
00:03:29.18 key role in trying to understand how these nerve fibers work.
00:03:32.18 And probably the best example is given by pungent agents from
00:03:38.00 capsicum peppers, or hot chili peppers. And back in the 70s, Jansco and
00:03:43.12 colleagues in Hungary showed that the pungent ingredient in hot chili
00:03:47.06 peppers, mainly capsaicin, illicits a sensation of burning pain.
00:03:52.14 By serving as a specific chemical activator of these nerve fibers that are
00:03:56.08 involved in pain reception, mainly the nociceptors. And some years
00:04:00.03 later, Bevan, Rang, and others in London carried out some biophysical studies
00:04:05.06 to suggest that what capsaicin does when it interacts with nerve fibers
00:04:08.08 in your mouth or anywhere else in your somatotopic surface,
00:04:11.15 is to depolarize the nerve fiber and to allow sodium, calcium ions
00:04:15.27 to flow in. And the main questions that we're still unresolved
00:04:19.16 by this was, does capsaicin interact with a specific site, a so-called
00:04:24.01 vanilloid site? Because capsaicin is a vanilloid compound,
00:04:27.05 like other pungent agents in peppers. Is there a specific receptor
00:04:31.29 for capsaicin? If so, what does it look like? And what might its normal
00:04:36.08 physiologic function be in the body? What role does it serve
00:04:39.14 in our endogenous ability to sense pain? And we, like many other
00:04:43.05 people in the field, realized identifying or addressing these questions
00:04:48.17 could lead to some key insights into pain sensation, because
00:04:51.19 sensitivity to capsaicin was a major functional hallmark
00:04:55.18 or characteristic of these nerve fibers that are involved in
00:04:59.14 making this sensation. In many ways, this was to the nociceptor
00:05:02.03 what the T-cell receptor was to lymphocytes. And so we set out
00:05:07.06 to do this, but the problem was that nobody really knew what
00:05:09.12 this receptor might look like, even if it existed. And so we decided
00:05:12.18 to take sort of a structure-blind identity-blind approach to doing
00:05:17.12 this. By just exploiting some functional attributes. And the idea
00:05:21.07 was that, if the capsaicin receptor does exist, you could make a cDNA library
00:05:26.05 from sensory nerve ganglia, and introduce those clones into
00:05:29.21 a non-neural cell that's normally capsaicin-insensitive,
00:05:32.13 such as a fibroblast. And then if you expose these to capsaicin
00:05:37.09 and that allowed calcium to flow into the cell or somehow be
00:05:41.04 released from extracellular stores, you could then detect this
00:05:43.27 using calcium-sensitive dyes, such as those developed by Roger Tsien and
00:05:48.00 all those like Indo or Fura. This has inherent risks, the advantage
00:05:53.02 is that you make no predispositions about what this receptor,
00:05:55.27 mythical receptor, might look like. But the risk is that if the
00:05:59.09 receptor is encoded by multiple gene products, the ability
00:06:03.03 to transfer a single gene to a fibroblast and recapitulate this activity
00:06:07.05 would be very difficult, if not impossible. But you know, you can't do
00:06:10.04 science without luck and some risk, and that's kind of what we're all
00:06:13.20 in this business for. In some ways, to do something new and exciting.
00:06:16.05 And so we tried this and low and behold, we ended up by this method
00:06:21.05 of identifying a single cDNA that encoded for a capsaicin receptor,
00:06:24.28 which we called the vanilloid receptor. And this is work done by
00:06:28.01 a wonderful postdoc in my lab at the time, Mike Caterina,
00:06:30.17 who now has his own group at Johns Hopkins University.
00:06:33.04 And we could show that this cloned gene encoded a functional
00:06:37.17 receptor, because if you introduce it into these cells, and use
00:06:40.21 patch clamp electrophysiology to look at what's happening,
00:06:43.24 we could show that when you put on capsaicin into these
00:06:46.11 cells, in fact you induce inward current flow of sodium and calcium
00:06:49.21 ions into the cell. And so this told us that the TRPV1 that
00:06:53.22 we had identified, this gene, is in fact a vanilloid activated channel.
00:06:57.05 But Makoto Tominaga and Mike Caterina and others in my lab then
00:07:00.25 addressed really the most interesting question, which is what
00:07:03.13 else does this receptor respond to and what might its normal
00:07:06.26 physiological role be in our ability to sense noxious stimuli?
00:07:11.12 And we tried to activate this receptor with all kinds of things
00:07:15.05 that we knew generated pain psychophysically or activated
00:07:19.06 sensory neurons in culture or nerve fibers. And we went through a whole
00:07:22.14 gamut of things, neuropeptides, other neurotransmitters.
00:07:25.06 And it was only after some time that we realized, gee these
00:07:27.27 nerve fibers detect changes in our environment, as well as
00:07:30.16 changes internally that relate to pain sensation. And we should try
00:07:34.02 some of these things. And the one that we tried that worked was
00:07:38.20 heat. And in retrospect, this seems obvious because you describe
00:07:42.15 a chili pepper often as a hot chili pepper. And this is a very nice
00:07:46.26 parsimonious explanation of that. But this wasn't the first thing that
00:07:50.03 we thought about, but you know, shortly after failing to look at it
00:07:53.17 -- to find results in the other stimuli, it occurred to us that this might
00:07:56.21 be the case. And it's really a beautiful result, in that you see that
00:08:00.08 the channel, when expressed in this case, in a frog oocyte, is silent
00:08:04.06 until you heat the solution, the external solution up, and then the channel
00:08:07.21 opens, and you see this large ionic current go through the cell
00:08:10.13 with a threshold that's very consistent with the sensitivity
00:08:14.07 of heat-sensitive nociceptors to heat. And in fact, is very close
00:08:18.01 to the threshold that most of us would describe as the demarcation
00:08:21.07 point between determining if something is innocuously warm
00:08:24.09 and noxiously hot. So this has led us to propose that TRPV1
00:08:27.21 is a little molecular thermometer that sits at the membrane, and
00:08:30.07 at least contributes, maybe doesn't underlie all, but contributes
00:08:33.06 to our ability to detect ambient temperature in the warm and the hot
00:08:37.02 range. Now we all know that we can sense temperatures over a
00:08:40.08 wide range, including cold, not just hot. And how might this
00:08:43.26 work? We thought that if we could figure this out, we could maybe
00:08:46.09 get some larger view of how temperature sensation works.
00:08:49.05 And ask if our ability to sense temperatures of different degrees
00:08:53.29 is due to a sort of similar, unifying molecular concept or
00:08:58.29 represents different molecular mechanisms. And again, we wanted
00:09:03.11 to take an approach that was not biased. We didn't want
00:09:05.10 to look for channels and genes that were similar to TRPV1,
00:09:08.02 but instead, step back and ask how is cold sensed
00:09:11.13 from just a functional point of view. And again, the great way
00:09:14.07 here, we turned to natural products. Because we all know
00:09:16.14 that cooling agents that are derived from mint leaves or eucalyptol,
00:09:21.04 produces a sensation of cooling. And Hensel and Zotterman
00:09:24.01 in the '50s in Sweden showed that this was the case in neurophysiological
00:09:27.18 recordings of sensory nerve fibers. So we played the same game
00:09:30.25 that we played with capsaicin to ask if we could identify
00:09:33.25 a menthol receptor. And the question was, is there a menthol
00:09:37.25 receptor? If so, does it respond to cold? And is it related to TRPV1?
00:09:42.23 Or is this a totally independent molecular mechanism?
00:09:45.00 And so we did, meaning David McKemy, a postdoc in my lab
00:09:49.02 at the time, and Werner Neuhausser, ended up identifying
00:09:52.03 a clone and a molecule called TRPM8, that when expressed
00:09:56.17 in fibroblasts, renders them sensitive to menthol and
00:09:59.25 other cooling agents. And as you can tell from the name, TRPM8,
00:10:03.05 it belongs to the same family of so-called TRP ion channels,
00:10:06.07 as does TRPV1, it's a molecular cousin. And the question is,
00:10:12.06 does it respond to cold? And in fact, again, when you express this
00:10:15.03 in a frog oocyte or some other cells, you can see that the channel is
00:10:18.12 quiet at warm temperatures, but then opens when you cool the
00:10:21.26 cell below about 26 degrees. And so this led to this idea that
00:10:25.18 there is some unifying concept of how we sense changes in
00:10:29.16 ambient temperature, and TRP channels serve as, or at least contribute
00:10:34.25 to our ability to detect changes in ambient temperature over a pretty
00:10:39.21 wide range. And they act as little molecular thermometers in
00:10:42.23 membrane that initiate the polarization of the neural fiber
00:10:47.19 in response to changes in temperature. And the best way really to test this,
00:10:50.29 of course, is in a living animal with genetics. Using genetics.
00:10:54.07 And I think the clearest demonstration of this really comes
00:10:57.07 from knock outs of TRPM8, which show a very beautiful phenotype
00:11:00.06 in terms of deficits and thermosensitivities. So we made a mouse
00:11:04.09 that lacks this channel, this is work done by these wonderful
00:11:07.24 people in my lab, Jans Siemens, Diana Bautista, and Sven Jordt, who now
00:11:10.21 all have their own labs in Heidelberg, and UC Berkeley, and
00:11:14.15 Duke University, respectively. And they set up a very nice
00:11:18.05 simple paradigm to look and see if deletion of this gene has a
00:11:22.06 phenotype associated with it, with temperature sensitivity. So
00:11:25.03 here you do this very simple test, where you take in this case, a wild type
00:11:28.00 mouse. And you ask if it can discriminate between a warm
00:11:31.12 platform and one that's somewhat cooler. Namely 20 and 30 degrees.
00:11:35.04 And you can see without any training, this animal avoids the cool
00:11:38.24 side and likes to stay on the nice, warm, cozy side. As is true for most
00:11:42.05 of us. And he'll stay on this side all day long. Now
00:11:46.04 if instead, you play this game with a mouse that, in which the gene
00:11:50.13 for the menthol receptor is knocked out, TRPM8 deficient mouse,
00:11:53.20 you'll see that the animal cannot discriminate between these two
00:11:56.29 platforms. And will wander, will spend equal time on each
00:11:59.17 of them. In fact, it won't be able to discriminate between platforms
00:12:03.00 even when the cool side goes down to very low temperatures,
00:12:05.27 to about 10 degrees. Below that, the animal can detect changes,
00:12:09.11 can detect the preference for the warm side, which may be due
00:12:13.04 to other processes that contribute to cold sensation. But in any case,
00:12:16.12 I think this validates the idea that TRP channels, and in particular,
00:12:19.18 TRPM8, play a really important role as a molecular detector
00:12:23.04 of ambient temperature. So through these studies, we've
00:12:26.29 been able to identify key elements that enable cells, that
00:12:30.20 really enable cells to specify their sensitivity to environmental stimuli.
00:12:34.09 And as in other sensory systems, the great things about doing this,
00:12:38.08 identifying genes and molecules that specify the sensitivity
00:12:42.00 of cells and nerve fibers to stimuli, is that you can use them as
00:12:47.18 molecular probes to try and understand the cellular and circuit
00:12:51.11 logic, as to how this information is conveyed to the nervous system
00:12:55.12 and ultimately, to your brain. So that you can discriminate different
00:12:58.02 percepts, in this case, heat from cold. And what these markers,
00:13:01.28 antibodies to these channels now tell us, is that the cold and
00:13:05.02 hot receptors are really segregated. They're expressed by
00:13:09.08 distinct subpopulations of sensory nerve fibers. Which tells us
00:13:14.13 at least, at the offset that the information as it comes into the spinal
00:13:19.03 chord, at least at the beginning, is segregated. And that information about cold
00:13:22.28 and information about heat, is conveyed by two so-called labeled lines.
00:13:26.24 Separate lines of sensory nerve fibers. And this at least begins to give
00:13:29.29 us some idea as to how behavioral specificity is dictated
00:13:34.01 by the circuitry in at least a peripheral aspect of the system.
00:13:40.07 So, we've used natural products to identify some important
00:13:43.16 players in pain sensation, some of which I haven't had time to
00:13:46.02 tell you about today. And all of this work has really come
00:13:49.14 about through just sort of curiosity driven research, which by the way
00:13:53.00 was funded by you, the taxpayers, via the NIH. And we've just
00:13:57.20 been curious about how the system works and allows us to
00:14:02.03 appreciate the world around us. But as so often the case, curiosity
00:14:05.22 driven research can have very practical and important
00:14:09.22 therapeutics aspects. And all these channels are now novel
00:14:13.17 targets through the development, hopefully, of new types of
00:14:16.09 analgesic drugs that we can use to treat a variety of peripheral pain
00:14:19.24 syndromes. And that's work that's in progress by a number of
00:14:22.20 pharmaceutical and academic labs. But hopefully, our goal
00:14:27.18 to understand how this system works, and the curiosity-based
00:14:30.27 level, will also have some practical outcome that will benefit
00:14:34.03 the patients who suffer from common pain syndromes.
00:14:36.16 And finally, I'd like to say that, my lab is small. There are maybe
00:14:39.20 about -- or relatively small, anywhere between 7 and 9 people
00:14:42.16 at any given time. And so the way that we cover the waterfront,
00:14:46.23 I'm a big believer in trying to hit a problem from multiple
00:14:49.18 different angles, and in my lab, we try to do this starting from
00:14:52.25 molecular biology through biophysics, through animal behavior
00:14:56.21 and anatomy, and physiology, and ultimately structural
00:14:59.23 biology. And the way that we do this is to work with other fantastic
00:15:03.01 scientists, through close collaborations, some at my own institutions,
00:15:07.02 some other places in the world. And this has really allowed us to
00:15:11.20 expand our scientific horizons, which is something I think every
00:15:14.11 scientist is trying to do. And to work outside the comfort
00:15:17.05 zone, and to stretch yourself. And of course, all these people
00:15:21.02 are now great friends. And this is a wonderful way to meet people
00:15:24.20 and have life-long friends. And also enlarge the sphere of people
00:15:30.15 who focus on what your lab can interact with. And so, I'm a big believer
00:15:34.02 in collaborative science. And we've had a fantastic time working
00:15:38.10 on these projects within my lab and with friends around the world.
00:15:42.13 Thank you very much.

This Talk

Speaker: David Julius

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: January 2016

Talk Overview

How do we perceive our surroundings, the things we smell, hear or even feel? Dr. David Julius explains how his lab used natural products, such as capsaicin from chili peppers and menthol from peppermint, to identify sensory receptors that detect both temperature and pain.

Speaker Bio

David Julius

Dr. David Julius is a Professor in the Department of Physiology at the University of California, San Francisco. His laboratory was the first to identify the sensory receptors involved in detecting both temperature and the natural heating and cooling agents found in foods such as chili peppers and mint. Julius was elected to the National… Continue Reading