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